Rat liver dihydroxyacetone-phosphate acyltransferase and their contribution to glycerolipid synthesis

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc. Vol. 259, No. 14, Issue of July 25, pp. 9064-9075,1984

Printed in U.S.A.

Rat Liver Dihydroxyacetone-phosphate Acyltransferases and Their Contribution to Glycerolipid Synthesis*

(Received for publication, December 14,1983)

Peter E. DeclercqSQ, Henk P. Haagsmanll, Paul Van Veldhovent, Luc J. Debeers, Lambert M. G. Van GoldeB, and Guy P. Mannaertst 11 From the PAfdeling Farmakologie, Katholieke Universiteit Leuuen, Campus Gasthuisberg, B-3000 kuven, Belgium and the Yhboratory of Veterinary Biochemistry, 3508 TD Utrecht, The Netherlands

Differential and isopycnic centrifugation of rat liver homogenates showed that, besides its established localization in peroxisomes and endoplasmic reticulum, dihydroxyacetone-phosphate acyltransferase is also present in mitochondria. The three activities differed in a number of properties (pH optimum, palmitoyl-CoA and dihydroxyacetone-phosphate dependence, and sensitivity toward N-ethylmaleimide) and are therefore likely associated with three distinct proteins. Glycerol 3-phosphate (5 mM) did not inhibit peroxisomal dihydroxyacetone-phosphate acyltransferase but inhibited the extraperoxisomal activities virtually completely.

Peroxisomal dihydroxyacetone-phosphate acyltransferase was located at the inner aspect of the peroxisomal membrane, but the enzyme was not latent. Purified microsomes, from which intact peroxisomes had been removed, were still contaminated with peroxisomal membranes as deduced from the presence of two dihydroxyacetone-phosphate acyltransferase activities: a glycerol 3-phosphate-resistant activity with properties similar to those of peroxisomal dihydroxyacetone-phosphate acyltransferase and a glycerol 3- phosphate-sensitive “true” microsomal dihydroxyacetone-phosphate acyltransferase. We propose that, assayed in the presence of 5mM glycerol 3-phosphate, dihydroxyacetone-phosphate acyltransferase can be used as a marker enzyme for peroxisomal membranes. Such a marker enzyme has not hitherto been available.

The differential effect of 5 mM glycerol 3-phosphate on peroxisomal and extraperoxisomal dihydroxyacetone-phosphate acyltransferases enabled us to deter- mine the relative contribution of these activities to overall dihydroxyacetone-phosphate acylation in whole liver homogenates. At near-physiological pH and at near-physiological concentrations of unbound palmitoyl-CoA and of dihydroxyacetone-phosphate plus glycerol 3-phosphate, peroxisomes contributed 50-7590. The remaining percentage was mostly accounted for by the microsomal enzyme.

At near-physiological concentrations of glycerol 3- phosphate plus dihydroxyacetone-phosphate, glycerol- phosphate acyltransferase contributed 93% and dihy-

* This work was supported by grants from the Belgian “Fonds voor Geneeskundig Wetenschappelijk Onderzoek,” the “Onderzoeksfonds van de Katholieke Universiteit Leuven,” the Netherlands Foundation for Chemical Research (SON), and the Netherlands Organization for the Advancement of Pure Research (ZWO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Aspirant of the Belgian “Nationaal Fonds voor Wetenschappelijk Onderzoek.”

1) To whom correspondence should be addressed.

droxyacetone-phosphate acyltransferase 7% to overall glycerolipid synthesis in homogenates. This suggests that the dihydroxyacetone-phosphate pathway is of minor quantitative importance in overall hepatic glycerolipid synthesis but that its main function lies in the synthesis of ether lipids, which have acyldihydroxyacetone-phosphate as obligatory precursor,

Peroxisomes, which appear to be responsible for the major share of hepatic dihydroxyacetone-phosphate acylation, are not equipped, however, with all enzymes needed to synthesize complex ether lipids. The enzymes cholinephosphotransferase, ethanolaminephosphotransferase, and diacylglycerol acyltransferase, that catalyze the terminal reactions involved in the synthesis of complex ether lipids, are absent from peroxisomes. Consequently, intermediates are probably transported to the endoplasmic reticulum for further metabolism.

The first intermediate committed to glycerolipid synthesis is lysophosphatidate. The classical view holds that this intermediate arises from the acylation of glycerol-3-P‘ by glycerol- P acyltransferase, which is located on the cytoplasmic side of the endoplasmic reticulum (1) and on the inner surface of the outer mitochondrial membrane (2). The microsomal (endoplasmic reticulum) and mitochondrial enzymes have different properties and are therefore most likely different proteins (for review see Ref. 3).

Mainly through the work of Hajra (see Ref. 4) it has become clear that lysophosphatidate can also be synthesized in two steps by an alternative route: acylation of dihydroxyacetone- P to acyldihydroxyacetone-P, catalyzed by dihydroxyacetone- P acyltransferase, and reduction of acyldihydroxyacetone-P to lysophosphatidate, catalyzed by acyldihydroxyacetone-P reductase. In liver, dihydroxyacetone-P acyltransferase appears to be located in peroxisomes (5) and microsomes (6).

The abbreviations used are: glycerol-3-P, sn-glycerol-3-phosphate; dihydroxyacetone-P, dihydroxyacetone-phosphate; dihydroxyacetone-P acyltransferase, dihydroxyacetone-phosphate acyltransferase; glycerol-P acyltransferase, glycerol-phosphate acyltransferase; :-ratio, molar [palmitoyl-CoA]:[albumin] ratio or molar [fatty acid]: [albumin] ratio; NEM, N-ethylmaleimide; EGTA, ethylene glycol bis(0-aminoethyl ether)-N,N,N’,N’-tetraacetic acid Mops, 34N- morpho1ino)propanesulfonic acid; TPCK, l-chloro-3-tosylamido-4- phenyl-2-butanone; TLCK, l-chloro-3-tosylamido-7-amino-2-hep- tanone; PMSF, phenylmethylsulfonyl fluoride. Enzyme numbers: dihydroxyacetone-P acyltransferase, EC 2.3.1.42; glycerol-P acyltransferase, EC 2.3.1.15; ethanolaminephosphotransferase, EC 2.7.8.1; cholinephosphotransferase, EC 2.7.8.2; diacylglycerol acyltransferase, EC 2.3.1.20.

9064

Rat Liver Dihydroxyacetone-phosphate Acyltransferase 9065

From cell fractionation studies, performed with rat and guinea pig liver, Hajra and his co-workers (4, 5) concluded that at least 90% of the hepatic dihydroxyacetone-P acyltransferase activity was peroxisomal. The authors studied the main properties of the enzyme in crude peroxisomal fractions from guinea pig liver (7). However, the experiments of Schlossman and Bell (8) performed on crude microsomal fractions from different rat tissues, including liver and brain, suggest that the endoplasmic reticulum has a considerable share in overall dihydroxyacetone-P acylation. These authors also concluded that microsomal dihydroxyacetone-P acyltransferase and glycerol-P acyltransferase are dual catalytic functions of a single enzyme (8). As far as brain is concerned, this conclusion seems to disagree with the results of Hajra and Burke (9) who described a dihydroxyacetone-P acyltransferase in rat brain microsomal fractions that was distinct from glycerol-P acyltransferase and had properties reminiscent of those of the peroxisomal enzyme from guinea pig liver. It is possible that, like liver, brain and other extrahepatic tissues may contain two dihydroxyacetone-P acyltransferase activities: one associated with the endoplasmic reticulum and also displaying glycerol-P acyltransferase activity and another located in microperoxisomes, which may sediment with the microsomal fraction during centrifugation. The confusion regarding intracellular localization, characteristics, and contribution to overall dihydroxyacetone-]? acylation of the separate dihydroxyacetone-P acyltransferase activities emphasizes the need for a comprehensive study in which the different dihydroxyacetone-]? acyltransferase activities from one tissue from a single animal species are investigated simultaneously and compared.

The importance of the dihydroxyacetone-P pathway for hepatic glycerolipid synthesis is much debated. Studies with specifically labeled radioactive precursors of glycerol-3-P and dihydroxyacetone-P and with specifically labeled radioactive reduced pyridine nucleotides in liver homogenates, liver slices, and isolated liver cells have produced conflicting results, possibly as a consequence of isotope discrimination and isotope exchange effects (for review see Ref. 3). The acylation of dihydroxyacetone-P is generally accepted as an obligatory step in ether lipid synthesis (for reviews on the synthesis of ether glycerolipids, see Refs. 10 and 11). However, its contribution to hepatic triacylglycerol synthesis has been designated as important (12-14) or as unimportant (8,15-17).

The purpose of the present study is 5-fold. 1) To reinves- tigate the subcellular localization of the various dihydroxyacetone-P acyltransferase activities and to compare their main properties in livers from a single species (rat). 2) Since most enzymes involved in glycerolipid synthesis are membrane-bound, to investigate whether peroxisomal dihydroxyacetone-P acyltransferase is also a membrane-bound enzyme and whether, assayed under the proper conditions, it can be used as a marker enzyme for the peroxisomal membrane. Such marker enzyme is presently not available. 3) To deter- mine the contribution of the separate enzyme activities to overall dihydroxyacetone-P acylation under a variety of assay conditions, including physiological concentrations of dihydroxyacetone-P and glycerol-3-P, which inhibits the microsomal enzyme. 4) To estimate the contributions of the glycerol-3-P and dihydroxyacetone-P pathways to overall glycerolipid synthesis in homogenates, incubated in the presence of physiological concentrations of both glycerol-3-P and dihydroxyacetone-P. 5) To investigate whether peroxisomes contain the enzymes that catalyze the terminal reactions involved in the synthesis of phosphatidycholines, phosphatidyletha- nolamines, triacylglycerols, and their 1-alk(en)yl analogues.

EXPERIMENTAL PROCEDURES AND RESULTS AND DISCUSSION^

Our studies confirm the presence of separate dihydroxyacetone-P acyltransferase activities in peroxisomes and microsomes but we also detected a third distinct activity that was associated with mitochondria?

Peroxisomal Dihydroxyacetone-P Acyltransferase pH Profile and Kinetic Properties-The properties of per-

oxisomal dihydroxyacetone-P acyltransferase were studied in peroxisomes that were highly purified by a combination of differential and isopycnic centrifugation. Fig. 2a shows an example of a pH profile. Six experiments performed on different batches of peroxisomes indicated that the optimum was situated in the pH range of 5.7-6. This acidic pH optimum is in agreement with the observations made by Jones and Hajra (7) for the peroxisomal enzyme from guinea pig liver. These authors further described a shift in pH optimum for the guinea pig liver enzyme, from 5.5 to 7.5, after incubation of crude peroxisomal fractions with detergents. We did not observe this phenomenon with the rat liver enzyme. Fig. 2a

FIG. 2. pH profile and kinetic properties of peroxisomal dihydroxyacetone-P acyltransferase. Peroxisomes were purified by differential and isopycnic centrifugation and dihydroxyacetone-P acyltransferase was measured as a function of the pH (a), molar [palmitoyl-CoA]:[albumin] ratio ( b ) , and dihydroxyacetone-P (DHAP) concentration (c). Dihydroxyacetone-P concentration was 0.94 mM (a, b) and the 5-ratio was 0.65 (a, c). In the experiments described in a, dihydroxyacetone-]? was generated in the presence of 5 mM acetate, pH 7.5 (preincubation), and at the start of the incubation the pH was adjusted to the desired values by addition of the following buffers (final concentration, 45 mM): acetate, pH 4.8 and 5.4; 2-(N-morpholino)ethanesulfonic acid, pH 5.7, 6.0, 6.5, and 6.9; Tris-HC1, pH 7.2, 7.5,8.0, and 8.5. Dihydroxyacetone-P acyltransferase activity is expressed as nanomoles of dihydroxyacetone-P incorporated into lipid per min and per mg of peroxisomal protein. The curves of a and c are means for 2 determinations. The inset of c represents the linear transformation of the dihydroxyacetone-P curve according to the method of Eadie-Hofstee; correlation coefficients. 0.97 (high affinity component) and 0.99 (low affinity component). Closed symbols, absence of glycerol-3-P open symbols, presence of 5 mM glycerol-3-P.

Portions of this paper (including “Experimental Procedures,” part of “Results and Discussion,” Tables 1-111, VI, and VI11 and Figs. 1,8, and 9) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 83M-3535, cite the authors, and include a check or money order for $4.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

Although dihydroxyacetone-P acyltransferase was originally described in crude mitochondrial fractions from liver, it was later concluded that its activity in mitochondrial fractions was caused by peroxisomal contamination ( 5 ) .

9066 Rat Liver Dihydroxyacetone-phasphte Acyltransferase

also shows the pH profile of peroxisomal dihydroxyacetone- P acyltransferase measured in the presence of 5 mM glycerol- 3-P, which strongly inhibits microsomal and mitochondrial dihydroxyacetone-P acyltransferase (see below). The only major effect of glycerol-3-P is an obvious stimulation at low pH values, causing a slight shift of the optimum to an even more acidic region. The mechanism underlying this phenomenon is not clear. At higher pH values, glycerol-3-P has a negligibly small inhibitory effect on dihydroxyacetone-P acyltransferase activity (see below).

Acyl-CoA dependence was studied at pH 7.5 at a dihydroxyacetone-P concentration of 0.9 mM by adding decreasing amounts of albumin, which strongly binds acyl-CoA esters, to a constant concentration of palmitoyl-CoA in the assay mixtures.' Fig. 2b shows that a plateau was rapidly reached. Half- maximal activity was attained at a low i-ratio of approximately 0.04.

Fig. 2c represents the effect of increasing dihydroxyacetone- P concentrations on peroxisomal dihydroxyacetone-P acyltransferase activity, studied at pH 7.5 and at a i-radio of 0.65. Linear transformation of the data according to Eadie (36) and Hofstee (37) revealed a break in the plot at a dihydroxyacetone-P concentration of approximately 0.44 mM. This may be an indication for negative cooperativity or it may suggest the existence of two enzymes with different K,, one enzyme in two different membrane environments (see below), or two activation states. Jones and Hajra (7) also described a break in Lineweaver-Burk plots constructed for dihydroxyacetone- P acyltransferase activity, measured as a function of the dihydroxyacetone-P concentration in crude peroxisomal fractions from guinea pig liver. At least in our own experiments, the possible presence of two different enzymes cannot be explained by contamination of the purified peroxisomal fraction with extraperoxisomal dihydroxyacetone-P acyltransferase. Measurement of the activities of dihydroxyacetone-P acyltransferase and various marker enzymes for endoplasmic reticulum (glucose-6-phosphatase, esterase) and for mitochondria (glutamate dehydrogenase, monoamine oxidase) in highly purified peroxisomal, microsomal, and mitochondrial fractions from the same liver revealed that, at various dihydroxyacetone-P concentrations, less than 5% of the dihydroxyacetone-P acyltransferase activity in purified peroxisomes could be explained by contamination. Another possible espla-

'In the present study, acyl-CoA dependence was studied as a function of the molar [palmitoyl-CoAJ:[albumin] ratio. Albumin strongly binds acyl-CoA esters. The addition of albumin to reaction mixtures containing excess palmitoyl-CoA makes it possible to reach extremely low concentrations of unbound palmitoyl-CoA without risk of palmitoyl-CoA depletion during enzymatic conversion of this substrate. In addition, in the presence of albumin, the resulting low concentrations of unbound palmitoyl-CoA are less likely to be influ- enced by binding of the acyl-CoA ester to variable amounts of homogenate or organelle pro te i and membranes, added to the ~y mixtures. For fatty acid:albumin mixtures it has been demonstrated that within certain limits the concentration of unbound fatty acid at any given i-ratio is nearly independent of the total fatty acid or albumin concentration but mainly dependent on the i-ratio (for references, see Ref. 22). Likewise, in the present experiments, enzyme activities did not change when assayed at different palmitoyl-CoA (and albumin) concentrations, but at identical i-ratios. The concentration of unbound palmitoy1-CoA, which is available for enzymatic conversion, increases almost linearly with increasing i-ratios until a i-ratio of 3 is reached. At this ratio the unbound palmitoyl-CoA concentration is approximately 1 pM (22). However, since the concentrations of unbound palmitoyl-CoA are not accurately known in the present experiments, apparent K, values were not calculated. The intracellular concentration of unbound acyl-CoA is not known, but as discussed further, it is probably lower than the unbound palmitoyl-CoA concentration at a i-ratio of 0.65.

nation for the break in the Eadie-Hofstee plot is that part of the enzyme shows only limited access for its substrate. La- tency was never detected, however (see below). If one assumes the presence of two enzymes, or the occurrence of one enzyme in two activation states, then the apparent K, for dihydroxyacetone-P of both activities is merely approximated, but not accurately represented by the absolute values of the slopes of the two straight lines in the Eadie-Hofstee plot (K, app: 0.06 and 0.30 mM).

When dihydroxyacetone-P dependence was studied at pH 5.7, there was an approximately 1.6-fold increase in V,, and, interestingly, there was also an increase in K, to 0.61 mM (Fig. 3a). No break in the Eadie-Hofstee plot was observed, suggesting that the protonation state of the enzyme or ita immediate membrane environment may be related to the unusual kinetics observed at higher and more physiological pH.

Schlossman and Bell (8) reported that rat liver microsomal dihydroxyacetone-P acyltransferase was strongly inhibited by NEM and by glycerol-3-P. Therefore, the effects of both agents on peroxisomal dihydroxyacetone-P acyltransferase were investigated. After pretreatment for 15 min of a purified peroxisomal fraction with 10 mM NEM, dihydroxyacetone-P acyltransferase was slightly stimulated when measured at pH 7.5 (30 f 4% stimulation, n = 6); at pH 5.7 no significant stimulation was observed (4 f 5% stimulation, n = 3). Per- oxisomal dihydroxyacetone-P acyltransferase was only slightly or not inhibited by glycerol-3-P (see also Fig. 2a). When this metabolite was ueed at a concentration of 5 mM, the average inhibition observed at pH 7.5 was 5.2 f 3.0% (n = 9). Disruption of the peroxisomes by sonication or by treatment with detergent did not alter the effect of NEM or glycerol-3-P.

SuborganeUur Localization-In order to investigate the sub- organellar localization of dihydroxyacetone-P acyltransferase, a sample of purified peroxisomes, suspended in hypotonic medium, was sonicated, layered over a discontinuous sucrose gradient, and centrifuged. Table IV shows that the bulk of

FIG. 3. Dependence of peroxisomal and microsomal dihydroxyacetone-P acyltransferase on the dibydrosyaoetoae-P concentration at pH 6.7. Peroxisomes and microsomes were purified by differential and isopycnic centrifugation and dihydroxyacetone-P acyltransferase was measured as a function of the dihydroxyacetone-P (DHAP) concentration at a i-ratio of 0.66. At the start of the incubation, the pH was adjusted to 6.7 as described in the legend to Fig. 2. Dihydroxyacetone-P acyltransferase activity is expressed as nanomoles of dihydroxyacetone-P incorporated into lipid per min and per mg of peroxisomal (a) or microsomal (b) protein. Closed symbols, absence of glycerol-3-P; open symbols, presence of 5 mM glycerol-3-P. The insets represent the linear transformation of the curves according to the method of Eadie-Hofstee. The apparent K, was 0.61 mM (peroxisomes) and 0.51 mM (microsomes). correlation coefficients (r): 0.99 (a) and 0.88 (b).


TABLE IV Association of peroxisomal dihydroxyacetone-P acyltransferase with

the peroxisomal membrane Peroxisomes were subfractionated by centrifugation through a

discontinuous sucrose gradient. Dihydroxyacetone-P acyltransferase was determined on the different fractions. Phospholipids, catalase, and urate oxidase were measured as markers for the peroxisomal membrane, matrix, and cores, respectively. Results are expressed as Dercentaees of t o t a l activity (or total amount).

Sample layer 81.0 0.0 8.9 0.9 24.8 Interface between dense 17.5 54.4 85.2 97.8 64.5

and light sucrose layers

Bottom 1.5 45.6 5.9 1.3 10.7 % recoverv 91 72 98 79 96

~~~ ~~ ~

DHAPAT, dihydroxyacetone-P acyltransferase.

TABLE V Association ofperoxisomal dihydroxyacetone-P acyltransferase with

the inner aspect of the peroxisomal membrane Purified peroxisomes (intact and sonicated) were treated with

trypsin for 15 min at 30 “C, the proteolytic activity was quenched by the addition of an equivalent amount of soybean trypsin inhibitor and dihydroxyacetone-P acyltransferase was measured. In controls, trypsin and trypsin inhibitor were added simultaneously at the end of the 15-min preincubation period. Free (nonlatent) and total catalase were determined at the end of the incubations for dihydroxyacetone-€’ acyltransferase. The ratio of trypsin to peroxisomal protein was varied from 70 to 700 Fg/mg. Results were nearly identical and are therefore not reported separately. Results are means * S.E. for 5 experiments. Free catalase is expressed as percentage of total catalase and dihydroxyacetone-P acyltransferase activity is expressed as per- ceniage of the activity measured in intact, nontrypsinized peroxisomes. Trypsin did not inactivate catalase activity either in intact or in sonicated oeroxisomes.

Catalaae DHAPAT”

Intact Sonicated Intact Sonicated % free % control

Peroxisomes

Controls 22.5 +- 5.2 85.4 f 5.9 100.0 91.1 f 6.2 Tmsinized 29.8 +- 7.6 NDb 67.3 +- 5.4 9.2 f 2.1

DHAPAT, dihydroxyacetone-P acyltransferase. ND, not determined.

catalase, a soluble matrix enzyme, remained in the sample layer, whereas urate oxidase (cores) was collected partly at the interface of the light and dense sucrose layers and partly at the bottom of the tube. The bulk of phospholipids (membranes) and almost all dihydroxyacetone-P acyltransferase were recovered at the interface of the sucrose layers, indicating that peroxisomal dihydroxyacetone-P acyltransferase is a membrane-bound enzyme.

In a next series of experiments, peroxisomes were treated with trypsin, a proteinase that cannot penetrate the peroxisomal membrane. Free (nonlatent) catalase, expressed as a percentage of total catalase and determined at the end of the incubation for dihydroxyacetone-P acyltransferase, was taken as an index for the damage to the peroxisomes. Table V shows that, in the presence of trypsin, peroxisomes were partly damaged and approximately 33% of dihydroxyacetone-P acyltransferase activity became inactivated. However, when sonicated peroxisomes, which were almost completely disrupted, were incubated with trypsin, almost all dihydroxyacetone-P acyltransferase was inactivated. These results indicate that the catalytic site, or at least essential domains of the enzyme, are located at the inner aspect of the peroxisomal membrane. Our results are in agreement with those of Jones and Hajra

(7) who observed an inactivation of dihydroxyacetone-P acyltransferase by trypsin when crude peroxisomal fractions from guinea pig liver were disrupted with detergent. The partial inactivation by trypsin of dihydroxyacetone-P acyltransferase in “intact” peroxisomes is most probably the result of the presence of damaged organelles, as can be deduced from the presence of nonlatent catalase. It cannot be excluded, however, that a smaller part of the enzyme, or a less essential domain, is located at the cytoplasmic side of the peroxisomal membrane.

Since dihydroxyacetone-P acyltransferase appears to be located at the inner aspect of the peroxisomal membrane, we investigated whether the enzyme would show latency. Soni- cation or repeated freeze-thawings of the peroxisomes or addition of 0.1% Brij 35, Lubrol PX, or Tween 20 to the assay mixtures for dihydroxyacetone-P acyltransferase did not affect enzyme activity, although catalase latency was completely abolished by these treatments. These results indicate that the enzyme is not latent and that rat liver peroxisomes are most likely permeable for dihydroxyacetone-P and acyl-CoA. This conclusion is in agreement with our recently published direct permeability measurements on purified intact rat liver peroxisomes, which revealed that the organelles are permeable to various small uncharged or charged molecules, including NAD+ (23). Addition of 0.1% sodium dodecyl sulfate, deoxycholate, or Triton X-100 to the assay mixtures almost completely abolished dihydroxyacetone-P acyltransferase activity. On the contrary, Jones and Hajra (7) reported a 2- to 3- fold stimulation of the enzyme activity, measured at pH 7.5 in crude peroxisomal fractions from guinea pig liver, by the addition of various detergents including Triton X-100 and deoxycholate. This stimulation was accompanied by a partial solubilization of the enzyme and a shift in pH optimum from pH 5.5 to 7.5 (see above). We were able to solubilize peroxisomal dihydroxyacetone-P acyltransferase by a combination of Triton X-100 and high salt concentration treatment (Table VI).

Microsomal Dihydroxyacetone-P Acyltransferase Contamination of Microsomal Fractions with Peroxisomal

Membranes-Our crude microsomal preparations (P-fractions) contained approximately 60, 9, and 24% of total homogenate glucose-6-phosphatase, catalase, and urate oxidase, respectively (Table 11), indicating a significant contamination with peroxisomes. Microsomes were therefore further purified on a sucrose gradient.

Fig. 4a shows a pH profile of dihydroxyacetone-P acyltransferase, measured in purified microsomes. An optimum around pH 7 was observed but the profile also displayed a shoulder in the acidic region, exactly where the pH optimum of peroxisomal dihydroxyacetone-P acyltransferase can be found, suggesting that even purified microsomes, which contain very little catalase or urate oxidase (Table II), may still be seriously contaminated by peroxisomal membranes. Schlossman and Bell (8) reported that microsomal dihydroxyacetone-P acyltransferase was competitively inhibited by glycerol-3-P. We therefore studied the pH profile in the presence of 5 mM glycerol-3-P. Fig. 4a demonstrates that it closely resembled the pH profile of peroxisomal dihydroxyacetone-P acyltransferase (Fig. 2a), especially that obtained in the presence of glycerol-3-P. This strongly suggests that glycerol-3-P-insensitive activity in microsomes was due to contamination by peroxisomal membranes and that the glycerol-3-P-sensitive activity represents the “true” microsomal enzyme. This impression is strengthened even more by the marked stimulation by glycerol-3-P of the activity at low pH values, a

Rat Liver Dihydroxyacetone-phosphate Acyltransferase

5 6 1 6

ib)

i

pH

FIG. 4. pH profiles of microsomal dihydroxyacetone-P acyltransferase. Microsomes were purified by differential and isopycnic centrifugation and dihydroxyacetone-P acyltransferase was measured at a dihydroxyacetone-P concentration of 0.94 mM and a i-ratio of 0.65. The pH of the incubation mixtures was adjusted as described in the legend to Fig. 2. a, untreated microsomes; b, trypsin-treated microsomes (22 pg of trypsin per mg of microsomal protein). 0, dihydroxyacetone-P acyltransferase activity in the absence of glycerol-3-P; 0, dihydroxyacetone-P acyltransferase activity in the presence of 5 mM glycerol-3-P A, difference between the dihydroxyacetone-P acyltransferase activities in the absence and presence of 5 mM glycerol-3-P (glycerol-3-P-sensitive dihydroxyacetone-P acyltransferase). Dihydroxyacetone-P acyltransferase activity is expressed as nanomoles of dihydroxyacetone-P incorporated into lipid per min and per mg of microsomal protein.

phenomenon also observed for peroxisomal dihydroxyacetone-P acyltransferase. The glycerol-3-P-sensitive microsomal dihydroxyacetone-P acyltransferase activity displayed an optimum around pH 7. A shoulder at acidic pH was no longer observed.

Additional evidence for the presence of peroxisomal membranes in microsomes was obtained from studies with trypsinized microsomes and from studies on the dihydroxyacetone- P dependence of dihydroxyacetone-P acyltransferase at low pH. As shown above, the peroxisomal enzyme is largely trypsin-insensitive. The pH profile, obtained in trypsinized microsomes, again closely resembled the pH profile of peroxisomal dihydroxyacetone-P acyltransferase and the inhibition by glycerol-3-P was dramatically reduced after treatment with the proteinase (Fig. 46). At pH 5.7, the optimal pH for the peroxisomal enzyme, the true microsomal enzyme is likely to show little activity (see the pH profile of glycerol-3-P-sensitive microsomal dihydroxyacetone-P acyltransferase, Fig. 4a). Fig. 3 shows that the dihydroxyacetone-P dependence of dihydroxyacetone-P acyltransferase in purified peroxisomes and microsomes a t pH 5.7 was quite similar. I t is also apparent from Fig. 3b that the microsomal activity, measured at pH 5.7, is not affected by 5 mM glycerol-3-P, which supports its peroxisomal origin.

Another indication for the presence of peroxisomal dihydroxyacetone-P acyltransferase activity in microsomes comes from experments with NEM: when microsomes were treated with NEM, glycerol-3-P-insensitive dihydroxyacetone-P acyltransferase, measured at , pH 7.5, was not inhibited, but slightly stimulated (22 & 12% stimulation, n = 3), again indicating its peroxisomal origin. Finally, the palmitoyl-CoA dependence of the glycerol-3-P-insensitive microsomal activity and of peroxisomal dihydroxyacetone-P acyltransferase were very similar (see below).

The above results indicate that microsomal frackions, pre- pared in the conventional way, are contaminated with peroxisomal membranes. These could not be removed by further purification of the microsomes on sucrose (or Percoll) gradients, which eliminated most of the intact peroxisomes (and, possibly, naked peroxisomal cores) as shown by the almost complete disappearance of catalase and urate oxidase (Table 11). The membranes are probably present as closed vesicles or “empty” peroxisomes, since glycerol-3-P-insensitive dihydroxyacetone-P acyltransferase was resistant to trypsin. The occurrence of “empty” peroxisomes in microsomal fractions is not too surprising, however, since 30-40% of catalase, a peroxisomal matrix enzyme, is found in soluble form after homogenization of the liver, reflecting the extreme lability of the organelles. Liver homogenates also contain naked peroxisomal cores, presumably originating from disrupted peroxisomes. Part of these cores appears to sediment with the microsomal fraction, as suggested by the enrichment of urate oxidase, relative to sedimentable catalase, in the P-fraction (Table 11).

Our results also indicate that dihydroxyacetone-P acyltransferase, measured a t low pH or measured in the presence of a high concentration of glycerol-3-P, can be used as a marker enzyme for the peroxisomal membrane. Such a peroxisomal membrane marker enzyme has not hitherto been available.

The inhibition of the “true” microsomal dihydroxyacetone- P acyltransferase by 5 mM glycerol-3-P appears to be essen- tially complete. The ratio of dihydroxyacetone-P acyltransferase activity at pH 5.7 (optimum for the peroxisomal enzyme) uers’sus that at pH 7.5 (optimum for the microsomal enzyme) was 1.29 0.06 (n = 9) for purified peroxisomes and 1.51 t 0.21 ( n = 4) for microsomes in the presence of 5 mM glycerol-3-P. If significant residual activity of the “true” microsomal enzyme had persisted in the presence of glycerol-3- P, the latter ratio would have been considerably smaller than the former. Contamination with peroxisomes or peroxisomal membranes was not investigated in earlier studies on microsomal dihydroxyacetone-P acyltransferase. However, Bates and Saggerson (28) suspected that the microsomal preparations used in their studies were seriously contaminated with nonmicrosomal dihydroxyacetone-P acyltransferase.

Kinetic Properties-Fig. 5a shows the palmitoyl-CoA dependence of microsomal dihydroxyacetone-P acyltransferase, measured at pH 7.5 in the presence and absence of 5 mM glycerol-3-P. In the absence of glycerol-3-P, half-maximal activity was reached at a Y-ratio of approximately 0.30 and, in the presence of glycerol-3-P, at a &ratio of approximately 0.06. The latter low ratio is again remarkably similar to that observed for the peroxisomal enzyme (Fig. 2b). Half-maximal activity for the glycerol-3-P-sensitive dihydroxyacetone-P acyltransferase was attained at a Y-ratio of 0.6-0.7, indicating that the affinity for palmitoyl-CoA of the “true” microsomal enzyme is considerably lower than that of the peroxisomal enzyme. Next, the dihydroxyacetone-P dependence was studied in the absence and presence of 5 mM glycerol-3-P (Fig. 5b). Linear transformation of the data according to the method of Eadie-Hofstee revealed a low and high affinity component, both in the absence and presence of glycerol-3-P. When the data obtained for the glycerol-3-P-sensitive activity were linearly transformed, the values approached a straight line with a slope of -0.34, suggesting an apparent K,,, of 0.34 mM for the “true” microsomal enzyme. It is clear, however, that an accurate kinetic study requires further purification of the enzyme. At pH 7.5, glycerol-3-P (5 mM) inhibited total microsomal activity by 68 f 3% (n = 7). Because of the


i o 5

io 4

, + " " A 3 ""_J

. "" o* -0- - 1::

n I 0.5 1 I*

1 3 0.1 0.5 1 2 [palmltoyl-CoA] /[album1111 [OHAP] , mil

FIG. 5. Dependence of the microsomal dihydroxyacetone-P acyltransferase on the molar [palmitoyl-CoA]:[albumin] ratio and on the dihydroxyacetone-P concentration. Microsomes were purified by differential and isopycnic centrifugation and dihydroxyacetone-P acyltransferase was measured as a function of the ?- ratio (a) and the dihydroxyacetone-P (DHAP) concentration (b), in the absence (0) and presence (0) of 5 n i ~ $lycerol-3-P. The triangles (A) represent the differences between the-.activities in the absence and presence of glycerol-3-P (glycerol-3-P-sensitive dihydroxyacetone-P acyltransferase). The dihydroxyacetone-P concentration was 0.94 mM (a) and the ?-ratio was 0.65 (b). Dihydroxyacetone-P acyltransferase activity is expressed as nanomoles of dihydroxyacetone- P incorporated into lipid per min and per mg of microsomal protein. The inset of b represents the linear transformations of the dihydroxyacetone-P curves according to the method of Eadie-Hofstee. Corre- lation coefficients (r): 0.99 (low affinity component) for total dihydroxyacetone-P acyltransferase (0); 0.92 (high affinity component) and 0.99 (low affinity component) for glycerol-3-P-resistant dihydroxyacetone-P acyltransferase (0); 0.96 for glycerol-3-P-sensitive dihydroxyacetone-P acyltransferase (A). The results of b are means for 2 experiments.

complex kinetic properties of microsomal dihydroxyacetone- P acyltransferase activity, the type of inhibition by glycerol- 3-P was not studied. Treatment with NEM suppressed total microsomal activity at pH 7.5 by 53 & 5% (n = 3). As already mentioned, NEM did not inhibit, but slightly stimulated, the glycerol-3-P-insensitive component. Schlossman and Bell (8) suggested that microsomal dihydroxyacetone-P acyltransferase and glycerol-P acyltransferase might be dual catalytic functions of a single enzyme. Although our expermients were not designed to confirm this hypothesis, and certainly do not prove it, our observations (inhibition of the true microsomal dihydroxyacetone-P acyltransferase by glycerol-3-P and by NEM, same pH profile and palmitoyl-CoA dependence as the microsomal glycerol-P acyltransferase, data not shown) are not in disagreement.

Mitochondrial Dihydroxyacetone-P Acyltransferase

In the course of our studies, we began to suspect that a small amount of a separate glycerol-3-P-inhibitable dihydroxyacetone-P acyltransferase was associated with mitochondria. Crude mitochondrial fractions were therefore further purified on self-generating Percoll gradients. Fig. 6 shows the distribution of marker enzymes and of total, glycerol-3-P-sensitive, and glycerol-3-P-resistant dihydroxyacetone-]? acyltransferase activities. A major amount of the glycerol-3-P-sensitive activity was indeed associated with the mitochondrial fractions at the bottom of the gradient, whereas the remainder was recovered in the microsomal fractions at the top of the gradient. The distribution of the glycerol-3-P-resistant activ-

t I I L I I

n

10

5 0 1 L tn

- (g)

50 -

v r 1 5 10 1 5 10

BOTTOM FRACTION NUMBER TOP

FIG. 6. Distribution of dihydroxyacetone-P acyltransferases and marker enzymes after subfractionation of a subcellular fraction enriched in mitochondria. An "fraction, enriched in mitochondria, was subfractionated by isopycnic centrifugation in a self-generating Percoll gradient. The different fractions of the gradient were analyzed for: a, total dihydroxyacetone-]? acyltransferase activity; b, glucose-6-phosphatase activity; c, glycerol-3-P-resistant dihydroxyacetone-P acyltransferase activity (measured in the presence of 5 mM glycerol-3-P); d , glycerol-3-P-sensitive dihydroxyacetone-P acyltransferase activity (difference between total and glycerol-3-P-resistant activities); e, urate oxidase activity; f, monoamine oxidase activity; g, catalase activity; h, glutamate dehydrogenase activity. Results are expressed as percentages of total recovered activities present in each fraction versus cumulative fraction volume.

ity paralleled the distribution of urate oxidase, indicating its peroxisomal origin?

Fig. 7a shows that dihydroxyacetone-P acyltransferase, measured in purified mitochondria, has a broad optimum around pH 7-8. Glycerol-3-P strongly inhibited the enzyme, whereas NEM had only a slight inhibitory effect. The pH profile as well as the differential effects of glycerol-3-P and NEM confirm that mitochondrial dihydroxyacetone-P acyltransferase is distinct from the microsomal and peroxisomal enzymes. The mitochondrial enzyme had a very high affinity

' In the Percoll gradients used for the purification of mitochondria, but to a lesser extent also in those used for the purification of peroxisomes, a small density shift in the distributions of catalase uersus urate oxidase was frequently observed. Glycerol-3-P-resistant (peroxisomal) dihydroxyacetone-P acyltransferase generally followed the distribution of urate oxidase, suggesting that in rat liver, peroxisomal dihydroxyacetone-P acyltransferase is enriched in core-containing peroxisomes. Urate oxidase recovered at the bottom of the gradient represents naked cores.

9070 Rat Liver Dihydroxyacetone-phosphate Acyltransferase

E < - la1 ~ ib) it1

% E -

2% < $ I o h-, B - 0 5 O E g k

c c w

-

&," - v o < E * c - ::

0 1 - - I - *

5 6 7 8 0 1 0.5 pn [palmttoyl-CoA]/[albumln] [MAP]. mtl

FIG. 7. pH profile and kinetic properties of mitochondrial dihydroxyacetone-P acyltransferase. Mitochondria were purified by differential and isopycnic centrifugation and dihydroxyacetone-P acyltransferase was measured as a function of the pH (a), V - ratio (b), and dihydroxyacetone-P (DHAP) concentration (c ) . The dihydroxyacetone-P concentration was 0.94 mM for a and 0.56 mM for b; the 5-ratio was 0.65 (a and c). In the experiments of a, the pH was adjusted as described in the legend to Fig. 2. Dihydroxyacetone- P acyltransferase activity is expressed as nanomoles of dihydroxyacetone-P incorporated into lipid per min and per mg of mitochondrial protein. The curves of c are means for 2 expermients. 0, absence of glycerol-3-P; 0, presence of 5 mM glycerol-3-P; 0, mitochondria pretreated with NEM.

for palmitoyl-CoA (Fig. 76), but, surprisingly, its affinity for dihydroxyacetone-P appeared to be very low in comparison with the other dihydroxyacetone-P acyltransferase activities (Fig. 7c). The enzyme was not stimulated by sonication, which released 60-70% of glutamate dehydrogenase, a soluble matrix enzyme, indicating that mitochondrial dihydroxyacetone-P acyltransferase is not latent; the acyltransferase itself was not released, suggesting that the enzyme is membrane-bound.

Its low affinity for dihydroxyacetone-P, the strong inhibition of its activity by glycerol-3-P, its high affinity for palmitoyl-CoA, and its relative insensitivity toward NEM, as well as its pH profile, leave open the possibility that mitochondrial dihydroxyacetone-P acyltransferase is a side activity of mitochondrial glycerol-P acyltransferase.

The slow linear increase of the mitochondrial activity at increasing dihydroxyacetone-P concentrations might suggest that dihydroxyacetone-P was reduced to glycerol-3-P by the mitochondrial FAD-linked glycerol-3-P dehydrogenase and that mitochondrial glycerol-P acyltransferase, which is much more active than mitochondrial dihydroxyacetone-P acyltransferase, was actually being measured in our experiments. The following evidence rules out this possibility. Addition of rotenone (1-2 pg/mg of mitochondrial protein) or 5 mM acetoacetate, which should prevent the reduction of FAD by electrons originating from NADH, did not affect dihydroxyacetone-P acyltransferase activity. Furthermore, when the enzyme was measured at a dihydroxyacetone-P concentration of 0.9 mM, the highest glycerol-3-P concentration found in the assay mixtures was approximately 1 pM. A glycerol-3-P concentration of 20-30 ~ L M was required to obtain comparable glycerol-P acyltransferase activity, determined under similar assay conditions and on the same mitochondrial preparation.

Contribution of Peroxisomal and Extraperoxisomal Dihydroxyacetone-P Acyltransferase to Overall

Dihydroxyacetone-P Acylation Assuming that the mitochondrial and microsomal dihy-

droxyacetone-P acyltransferase activities are completely inhibited by 5 mM glycerol-3-P and that the peroxisomal enzyme is not affected, we determined the contribution of the peroxisomal and extraperoxisomal enzymes to overall dihydroxy-

acetone-P acylation by measuring dihydroxyacetone-P acyltransferase in liver homogenates in the absence and presence of 5 mM glycerol-3-P. Part A of Table VI1 shows the results obtained when dihydroxyacetone-P acyltransferase was assayed under the conditions used in the preceding experiments. It is apparent that, at pH 7.5, the peroxisomal and extraperoxisomal enzymes comprise approximately one-third and two-thirds, respectively, of total dihydroxyacetone-P acyltransferase activity. The contribution of the peroxisomal and extraperoxisomal enzymes was also estimated by comparing activities in liver homogenates at pH 5.7 and 7.5 (bottom lines of Table VIIA). Assuming that the peroxisomal enzyme is 1.3 times more active at pH 5.7 than at pH 7.5 (see above) and that the microsomal and mitochondrial enzymes are 10 times less active at pH 5.7 than at pH 7.5, contributions, quite similar to those estimated from the inhibitory effect of glycerol-3-P, could be calculated.

We were particularly interested in determining the contribution of the peroxisomal and extraperoxisomal components under assay conditions that mimic the physiological situation with regard to dihydroxyacetone-P, glycerol-3-P, and acyl- CoA concentrations. The dihydroxyacetone-P and glycerol-3- P contents of freeze-clamped livers were measured and the in vivo concentrations of the phosphate esters were calculated (Table VIII). A dihydroxyacetone-P concentration of approximately 0.14 mM and a glycerol-3-P concentration of approximately 0.5 mM were found (fed state). The intracellular concentration of unbound acyl-CoA is not known but, from the linear increase in esterification rates in isolated hepato- cytes with increasing palmitate concentrations up to 1 mM (38) and from the fact that a k-ratio of 0.65 is equal to or

TABLE VI1 Contribution of peroxisomal and extreperoxisomal dihydroxyacetone- P acyltransferases to overall dihydroxyacetone-P acylation in whole

liver homogenntes Dihydroxyacetone-P acyltransferase was measured in whole liver

homogenates under a variety of experimental conditions. The first part (A) of the table applies to the assay conditions commonly used in the course of this study; the second part (B) is a survey of the results obtained at conditions that mimic the in uiuo situation (see text). Dihydroxyacetone-P acyltransferase activities are means -C S.E. for the number of experiments indicated in parentheses and are expressed as nanomoles of dihydroxyacetone-P incorporated per min and per g of liver in lipids. As described in detail in the text, the % contribution of peroxisomal and extraperoxisomal dihydroxyacetone- P acyltransferases was calculated from the inhibitory effect of 5 mM glycerol-3-P and from the comparison of dihydroxyacetone-P acyltransferase activity at pH 7.5 and 5.7 (bottom lines of part A).

Assay conditions' DHAPAT

Contribution

Extra- pH

Peroxi- [3-GP] i-ratio [DHAP) somal peroxisomal

A. 7.5 0.65 7.5 0.65

0.65 7.5 0.65 7.5

0.65 5.7 0.65 7.5

B. 7.5 0.65 7.5 0.65 7.5 0.65 7.5 0.11 7.5 0.11 7.5 0.11

mM

0.56 0.56 0.94 0.94 0.94 0.94

0.14 0.14 0.14 0.14 0.14 0.14

mM nmnl/min/g liver % ?&

65.6 zt 6.0 (5 )

60.8 39.2 85.3 zt 4.6 (3)

67.7 (15.3 + 52.4)b 32.3 89.2 zt 3.5 (5)

67.3 32.7

48.7 f 3.3 (3) 89.4 10.6

5.0

0 100 28.8 f 2.9 (5 ) 5.0

0 100 21.4 f 2.1 (5)

0.5 5.0

0.5 5.0

25.8 f 1.6 (6)

8.3 f 0.3 (3) 100 74.0 11.2 zt 0.5 (3) 56.3 14.7 f 0.5 (3)

100 9.4 k 0.5 (6) 51.4 18.3 & 0.9 (6) 36.4 63.6 (6.5 + 517.1)~

48.6 0

43.7 (11.4 + 32.3)b

1 26do The abbreviations used are; DHAP, dihydroxyacetone-P 3-GP,

Contribution of mitochondria uers'sus microsomes: see text. glycerol-3-P DHAPAT, dihydroxyacetone-P acyltransferase.


markedly higher than the ratios at which half-maximal activities are reached for the various cellular glycerol-P acyltransferase and dihydroxyacetone-P acyltransferase activities, it can be deduced that the intracellular unbound acyl-CoA concentration is lower than the concentration of unbound palmitoyl-CoA at a i-ratio of 0.65. In a recent communication, McGarry and Foster (39) suggested that a “physiological” condition, with respect to unbound palmitoyl-CoA, may be approximated at i-ratios of 0.3 or less. In a second series of experiments, we therefore measured dihydroxyacetone-P acyltransferase in whole homogenates at near-physiological pH (7.5), near-physiological concentrations of dihydroxyacetone-P (0.14 mM), and glycerol-3-P (0.5 mM) and at i-ratios of 0.65 and 0.11, which probably result in unbound palmitoyl- CoA concentrations that comprise the physiological concentrations in the cell. The results are given in part B of Table VII. At a ;-ratio of 0.65, which was also used in the experiments summarized in part A of the table, the contribution of peroxisomal dihydroxyacetone-P acyltransferase to overall dihydroxyacetone-P acylation at a physiological dihydroxyacetone-P concentration was approximately 36%, which does not differ significantly from the results obtained at higher dihydroxyacetone-P concentrations. However, when dihydroxyacetone-P acyltransferase was assayed at a i-ratio of 0.11, the share of the peroxisomal enzyme was increased to approximately 56%, most probably as a result of the high affinity of the peroxisomal enzyme for palmitoyl-CoA (see above). When we included a physiological concentration (0.5 mM) of glycerol-3-P, which partly inhibits microsomal and mitochondrial dihydroxyacetone-P acyltransferase, the relative importance of the peroxisomal enzyme rose even more: contributions of approximately 51 and 74% were found, at i- ratios of 0.65 and 0.11, respectively.

The contribution of the mitochondrial enzyme to overall dihydroxyacetone-P acylation was estimated from measurements of dihydroxyacetone-P acyltransferase at pH 7.5 and of marker enzymes for mitochondria (monoamine oxidase, glutamate dehydrogenase) in liver homogenates and in purified mitochondrial fractions. It was calculated that, at a i- ratio of 0.65 and at dihydroxyacetone-P concentrations of 0.14 mM and 0.94 mM, the mitochondria contributed 6 and 15% to overall dihydroxyacetone-P acylation, respectively, thereby bringing the contribution of the microsomal enzyme to 57 and 52%, respectively. At a i-ratio of 0.11 and a dihydroxyacetone-P concentration of 0.14 mM, the share of the mitochondrial enzyme, which has a high affinity for palmitoyl-CoA, amounted to approximately 11%, leaving a microsomal contribution of 32%. It can be concluded that, under near-physiological conditions, peroxisomal dihydroxyacetone- P acyltransferase may account for as much as 50-75% of overall dihydroxyacetone-P acylation; the extraperoxisomal dihydroxyacetone-P acyltransferase activity appears to be mainly, but not exclusively, microsomal.

Contribution of Dihydroxyacetone-P Acyltransferase and Glycerol-P Acyltransferase to Overall Glycerolipid Synthesis Although it has been established that the dihydroxyace-

tone-P pathway is an obligatory route for ether lipid synthesis, its importance in overall hepatic glycerolipid synthesis re- mains unclear. Surprisingly, no experiments have been reported in which glycerol-P acyltransferase and dihydroxyacetone-P acyltransferase were measured in the presence of a combination of physiological concentrations of glycerol-3-P and dihydroxyacetone-P. Table IX shows measurements of dihydroxyacetone-P acyltransferase and glycerol-P acyltransferase in liver homogenates from fed rats, at a i-ratio of 0.65,

and in the presence of physiological concentrations of the combination of both substrates. Comparison of lines 1 and 3 shows that addition of unlabeled glycerol-3-P (0.5 mM) inhibited dihydroxyacetone-P acyltransferase activity, measured at 0.14 mM dihydroxyacetone-P, by approximately 33%, which agrees with previous results (Table VII). Glycerol-P acyltransferase activity was hardly affected by addition of unlabeled dihydroxyacetone-P (approximately 7% inhibition, compare lines 2 and 4). When both labeled glycerol-3-P and dihydroxyacetone-P were used simultaneously as substrates, the resulting sum of dihydroxyacetone-P acyltransferase and glycerol-P acyltransferase (line 5) was almost equal to the sum of the separate activities (lines 3 and 4). The table further reveals that in the presence of the substrate combination, the dihydroxyacetone-P pathway contributed only 7% to overall acylation. Nearly identical results were obtained at a i-ratio of 0.11 and with liver homogenates from starved rats (data not shown), The results strongly suggest that the dihydroxyacetone-P pathway is only of minor quantitative importance in hepatic triacylglycerol and phospholipid synthesis, but that its main function lies in initiating the synthesis of ether lipids.

Role of Peroxisomes in the Synthesis of Ether Lipids As the first step in the synthesis of alkyl glycerolipids is

the formation of acyldihydroxyacetone-P and as dihydroxyacetone-P acyltransferase at near-physiological substrate concentrations is mainly active in peroxisomes, it was of interest to investigate whether peroxisomes contain other enzymes required for the synthesis of complex ether lipids. Fig. 9 (in Miniprint) shows the distribution of diacylglycerol acyltransferase, ethanolaminephosphotransferase, cholinephosphotransferase, and peroxisomal and microsomal marker enzymes after subfractionation by isopycnic centrifugation of an L- fraction, enriched in peroxisomes. Glucose-6-phosphatase displayed a bimodal distribution, indicating the presence of two populations of microsomal vesicles of different density. The distribution of diacylglycerol acyltransferase, ethanolaminephosphotransferase, and cholinephosphotransferase coin- cided with the distribution of the microsomal vesicles of low density and no activity was observed in microsomes of high density, suggesting that all three lipid-synthesizing enzymes are similarly, but unevenly, distributed in the endoplasmic reticulum. Localization of these enzymes in the endoplasmic reticulum has been reported before (40). No indication was found for the presence of significant diacylglycerol acyltransferase, ethanolaminephosphotransferase, or cholinephosphotransferase in peroxisomes. Addition of variable amounts of microsomes to a fixed amount of purified peroxisomes (according to the method of McMurray (41)) revealed no intrinsic peroxisomal activities of these enzymes either.

It could be argued that peroxisomes contain cholinephosphotransferase and ethanolaminephosphotransferase that specifically use l-alk-l‘-enyl-2-acyl-sn-glycerols as substrates. Therefore, enzyme activities were also determined using alkenylacylglycerols as substrates. Table X shows that peroxisomes are not capable of synthesizing choline or ethanolamine plasmalogens from alkenylacylglycerols. Indeed, the activities of cholinephosphotransferase and ethanolaminephosphotransferase can be totally ascribed to microsomal contamination. Compared to “egg”-diacylglycerol 1-alk-1’- enyl-2-acyl-sn-glycerol was a better substrate for cholinephosphotransferase and ethanolaminephosphotransferase.

The results indicate that peroxisomes are devoid of enzymes that catalyze the terminal reactions in the synthesis of complex ether lipids. This implies that intermediates in the synthesis of plasmalogens have to be transported to the endo-


TABLE IX Relative importance of dihydroxyacetone-P acyltransferase and glycerol-P acyltransferase

Dihydroxyacetone-P acyltransferase was measured at a dihydroxyacetone-P concentration of 0.14 mM and glycerol-P acyltransferase at a glycerol-3-P concentration of 0.5 mM. Enzymatic measurements of glycerol-3-P and dihydroxyacetone-P showed that there was no interconversion of these two substrates. The i-ratio was 0.65. The contributions of dihydroxyacetone-P acyltransferase and glycerol-P acyltransferase, in the presence of physiological concentrations of both dihydroxyacetone-P and glycerol-3-P, were calculated from the sum of dihydroxyacetone- P acyltransferase activity in the presence of glycerol-3-P (line 3) plus glycerol-P acyltransferase activity in the presence of dihydroxyacetone-P (line 4). Results are means k S.E. for 3 experiments and are expressed as nanomoles of glycerol-3-P/dihydroxyacetone-P incorporated, per min and per g of liver.

Enzyme % -

Measured Labeled activity” substrate

Addition unlabeled activity contribution

nmol/minjg liuer

DHAP, 0.14 mM 29.3 f 0.6 285.1 % 2.5

19.7 k 0.2 6.9 266.4 2 3.5 93.1 292.8 2 9.3

DHAPAT GPAT DHAPAT DHAP, 0.14 mM 3-GP, 0.5 mM GPAT DHAPAT + GPAT DHAP, 0.14 mM

3-GP, 0.5 mM

3-GP, 0.5 mM DHAP, 0.14 mM

+3-GP, 0.5 mM

DHAP, dihydroxyacetone-P 3-GP, glycerol-3-P. The abbreviations used are: DHAPAT, dihydroxyacetone-P acyltransferase; GPAT, glycerol-P acyltransferase;

TABLE X Cholinephosphotransferase and ethamlaminephosphotransferase

activities in microsomes and peroxisomes Purified microsomes and peroxisomes were assayed for choline-

phosphotransferase and ethanolamine phosphotransferase activities. Enzymes were measured with 1,2-diacyl-sn-glycerols or 1-alk-1’-enyl- 2-acyl-sn-glycerols as substrates. The specific activity of glucose-6- phosphatase in peroxisomes was 20.4% of that in microsomes. This . . experiment was repeated twice with similar results. I_

Cholinephospho- Ethanolaminephos- transferase photransferase

Diacyl- Alkenyl- Diacyl- Alkenyl- glycerol acylglycerol glycerol acylgIycero1 nmol/min/ng protein nmolfmin/mg protein

Microsomes 3.16 8.06 0.59 4.31 Peroxisomes 0.36 0.78 0.08 0.26 Peroxisomal activity as 11.4 9.6 12.8 6.0

% of microsomal activity

plasmic reticulum for further metabolism. Lipid transport proteins have been described in rat liver (42). However, it has not been investigated whether these proteins are capable of transporting intermediates in the synthesis of ether lipids.

Conclusions

The present study establishes the presence of dihydroxyacetone-P acyltransferase activity in peroxisomes, microsomes (endoplasmic reticulum), and mitochondria. The three activities differ among each other in kinetic properties, pH dependence, and their sensitivity toward inhibitors such as glycerol-3-P and NEM, indicating that they are catalytic functions of three separate enzyme proteins.

Although our experiments were not designed to test the hypothesis of Schlossman and Bell (8) that microsomal dihydroxyacetone-P acyltransferase and microsomal glycerol-P acyltransferase are dual catalytic functions of a single enzyme protein, our observations are not in disagreement. The properties of mitrochondrial dihydroxyacetone-P acyltransferase do not rule out a similar hypothesis for mitochondrial dihydroxyacetone-P acyltransferase and glycerol-P acyltransferase. Peroxisomes do not contain significant glycerol-P acyl-

transferase activity: Peroxisomal dihydroxyacetone-P acyltransferase is most likely located at the inner aspect of the peroxisomal membrane and, when assayed under the appro- priate conditions, it can be used as a marker enzyme for peroxisomal membranes. Microsomal fractions are contaminated with peroxisomal membranes to a much greater extent than expected from the presence of catalase, a marker for the peroxisomal matrix.

The contribution of the separate dihydroxyacetone-P acyltransferase activities to overall dihydroxyacetone-P acylation strongly depends on the assay conditions. Under conditions that are likely to prevail in vivo, peroxisomes appear to be responsible for 50-75% of total dihydroxyacetone-P acylation. The remaining percentage is probably mainly accounted for by microsomes.

Acyldihydroxyacetone-P has been established as an obligatory intermediate in ether lipid biosynthesis. Thus, three separate subcellular organelles have the potential of initiating the synthesis of ether lipids. Although the major site for dihydroxyacetone-P acylation appears to be the peroxisome, this organelle lacks the enzymes required to complete the synthesis of complex ether lipids. This does not discount peroxisomal dihydroxyacetone-P acyltransferase as the initiating enzyme in the synthesis of alkyl glycerolipids and plasmalogens. Intermediates in the biosynthesis of ether lipids may be transported to the endoplasmic reticulum where further metabolism is possible. Interesting in this respect is that both peroxisomes and plasmalogens are lacking in tissues from infants suffering from the Zellweger syndrome (43).

In the presence of physiological concentrations of dihydroxyacetone-P and glycerol-3-P, dihydroxyacetone-P acylation and glycerol-3-P acylation contribute 7 and 93%, respectively, to total acylation in liver homogenates. This strongly suggests that in the intact hepatocyte by far the greatest part of triacylglycerols and phospholipids (that do not contain ether bonds) originates from the acylation of glycerol-3-P.

Acknowledgments-We thank Dr. W. Stalmans for helpful discus-

When we measured glycerol-P acyltransferase in purified peroxisomes, we detected a very low activity that could not be completely explained by the presence of contaminating microsomes and mitochondria. This possible peroxisomal glycerol-P acyltransferase was not studied further since it could apparently account for only 1-2% of overall hepatic glycerol-3-P acylation.


sions; L. Govaert, G. Vandebroeck, and J. M. van den Heuvel for expert technical assistance; M. Bareau for dedicated secretarial help.

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Hohorst, H. J. (1970) in Methoden der Enzymatischen A d y s e (Bergmeyer,

Bucher, T., and Hohorst, H. J. (1970) in Methoden der Enzymatischen H. U., ed) Vol. 2, pp. 1379-1383, Verlag-Chemie, Weinheim

Analyse (Bergmeyer, H. U., ed) Vol. 2, pp. 1282-1288, Verlag-Chemie, Weinheim

Physiol. 270,399-412

Eadie, G. S. (1942) J. Biol. Chem. 146,85-93 Hofstee, B. H. J. (1952) Science 116,329-331 Debeer, L. J., Declercq, P. E., and Mannaerts, G. P. (1981) FEBS Lett.

124.31-34 39. McGark, J. D., andFoster, D. W. (1982) Biochem. J. 208,527-528 40. Van Golde, L. M. G., Fleischer, B., and Fleischer, S. (1971) Biochim.

42. Wirtz, K. W. A. (1982) in Lpid-Protein Interactions (Jost, P. C., and 41. McMurray, W. C. (1974) Bwch,em. Bwphys. Res. Commun. 58,467-474

43. Heymans, H. S. A., Schutgens, R. B. H., Tan, R., van den Bosch, H., and

Biophys. Acta 249,318-330

Griffith, 0. H., eds) Vol. 1, pp. 151-231, Wiley-Interscience, New York

Borst, P. (1983) Nature (Lord.) 306.69-70

c o n c e n t r a t i o n by d e s t r o y i n g one O f t h e r e a c t a n t s o r t h a t . d u r i n g d i h y d r o x y - a c e t o n e - ) c o n s u m p t i o n , e q u i l i b r i u m c o u l d n o t b e m a i n t a i n e d a s d r e s u l t o f ~ n h i b i t i o n O f a l d o l a s e or t r i o s e - p h o s p h a t e i s m e r a r e . A d d i t l a n O f R I P , a s t r o n g i n h 7 b i t o r o f f T U E t ~ l e - l . 6 - b i ~ p h o P p h d t d ~ e . c o m p l e t e l y D r e v e n t e d t h e d e c r e a s e i n d i h y d r o x y a c e t o n e - P m n ~ e n t r d t i o n ~n t h e presence O f CPude tissue . *._".

t h r e i t o l : 0 . 1 I , i / v . e t h a n o i ; d e n s i t y i.25 i per.ml It 0'C)~a.d 1 . 5 m1 O f li h t L 1 5 X u/w)I I U C ~ O S ~ . containing d i t h i o t h r e i t o l a n d e t h a n o l ( d e n s i t y 1 . 8 0 9 p e r mI a t 0 ° C ) . C e n t r i f u g a t i o n was per formed i n a Beckman S Y 5 0 . 1 r o t o r a t 84 000 PIY f o r 4 h a t 4 - c . The m a t r i x O m t e i n r ( c a t a l a s e 1 r e m a i n e d i n t h e sample layer, t h e membranes ( p h o r p h o l i p i d j we?e r e i o v e r e d a i t h e i n t e r - f a c e O f t h e l i g h t and dense suclose l a y e r s a n d t h e corer ( u r a t e o x i d a s e ) Y e l e f o u n d p a r t l y a t t h e i n t e r f a c e and p a r t l y a t t h e b o t t o m O f t h e g r a d i e n t .

M a r k e r e n z y m e s . p r o t e l n , p h o s h o l i p i d : M a r k e r enzymes (g lu tamate dehydro - g e n a s e : m 1 t o ~ h o n d r 1 a I m a t r l x y carnitine p a l n i t a y l t r a n r f e r a r e : m i t o c h o n - d r i a l i n n e r membrane; monoamine oxidase : m i tochondr ia l Ou te r membrane ; ~ I u c o ~ ~ - L - P ~ o ~ o ~ ~ ~ ~ I ~ , e s t e r a s e : endoDlasmlc reticulum: c a t a l a s e : ~ e ~ o x i - i o m a l m a t r 1 1 ; u r a t e o x l d a l e : p e r o x i r o i a l c a r e r ) w e l e d i t e r n i n e d a s d e r c r l b e d P r e v i o u I l y ( 2 1 - 2 3 ) . T o t a l c a t a l a s e a c t i v i t y w a s measured a f t e r p r e t r e a t m e n t O f t h e enzyme s o u ~ c e w ~ t h 10 mg o f T r i t o n X-100 p e r m l f o p 2 n i n t o d i s r u p t

measured I n 0.25 i4 Sucrose i n t h e a b s e n c e o f d e t e r g e n t . t he pe rox~ro rna l membranes . F r e e ( n o n - l a t e n t ) c a t a l a s e r e f e r s t o t h e a c t i v i t y

Y l o t e i n was de te rm ined by a m o d i f i c a t i o n ( 2 4 ) o f the method O f Lowry e t a l .

a c c o r d 7 n g t o t h e m e t h o d O f S l i g h a n d D y e r ( 2 6 ) a n d d e t e r m i n e d 1s d e s c r i b e d ( 2 5 ) w l t h b o v i n e r e r u n a l b u m i n I S s t a n d a r d . P h o s p h o l i p i d r were e x t r a c t e d

b y S t e w a r t ( 2 7 ) .


T a b l e I : C o m p o s i t i o n o f t h e L - f r a c t i o n a n d t h e p u r i f i e d p e r o x i s o m a l f r a c t i o n .

R e s u l t s (means + S E M f o r t h e n u m b e r O f e x p e r i m e n t s i n d i c a t e d i n p a r e n t h e s e s ) ~ T F e x p r e s s e d a s p e r c e n t a g e s O f t o t a l h o m o g e n a t e a c t i v i t y a n d r e l a t i v e s p e c i f i c a c t i v i t i e s . R e l a t i v e s p e c i f i c a c t i v i t y ( 1 0 ) i s d e f i n e d a s t h e p e r c e n t a g e O f t o t a l a c t i v i t y p r e s e n t i n a p a r t i c u l a r f r a c t i o n d i v i d e d b y t h e c o r r e r p o n d i n g p e r - c e n t a g e O f t o t a l p r o t e i n ; i t i n d i c a t e s t h e p u r i f i c a t i o n f a c t o r from t h e h o m o g e n a t e on a p r o t e i n b a s i s .

Enzyme L - f r a c t i o n P u r i f i e d p e r o x i r o m e r L - f r a c t i o n P u r l f l e d peraxrromer

X o f t o t a l h o m o g e n a t e R e l a t i v e s p e c i f i c a c t i v i t y 1 C a t a l a s e 3 0 . 7 2 + 1 .15 ( 2 4 ) 6.58 + 0 . 4 1 ( 2 5 ) 5 . 8 0 + 0.36 ( 2 4 ) 19.66 + 0 .95 (25 ) S e d i m e n t a b l e c a t a l a s e 4 8 . 1 4 5 1 . 4 3 ( 2 1 ) 1 0 . 4 9 7 0 . 6 5 ( 2 1 8 . 9 5 T 0 . 5 7 ( 2 1 ) 3 0 . 6 5 7 1 . 5 5 ( 2 1 ) U r a t e O x i d a s e 4 8 . 9 3 + 1 . 9 0 20 8 . 8 3 7 0 .59 (211 9 .45 T 0 . 5 1 20) 2 7 . 4 6 7 2 . 5 6 ( 2 1 ) G l u c o s e - 6 - p h 0 6 p h ~ t a s e 1 0 . 3 5 5 0.68 1 2 2 1 0 . 2 6 7 0.06 (23 1.87 I o . 1 1 122) 0.80 T 0 . 1 7 ( 2 3 ) E s t e r a s e A c i d p h o s p h a t a s e 50 .09 + 1 . 5 1 ( 9 ) :;I;; ((J 1 0 . 5 4 + 0 . 9 6 ( 9 ) 2 . 5 5 'i 0 . 3 2 ( 9 )

(2) 0.60 7 0.07 ( 3 )

G l u t a m a t e d e h y d r o g e n a s e 8 . 1 3 7 0 . 9 1 ( 9 ) P r o t e i n 5 . 7 0 5 0 . 3 7 ( 2 4 ) 0 . 3 5 i 0.02 (25)

1 . 7 0 5 0.24 ( 9 ) 0.60 5 0 . 0 7 ( I O )

8.57 - ( 2 ) 0.20 7 0 . 0 6 (3) 1 . 2 8

T a b l e 11 : C o m p o r i t l o n o f t h e P - f r a c t i o n a n d t h e p u r l f l e d m i c r o s o m a l f r a c t i o n .

F o r e x p l a n a t i o n : see T a b l e I .

i Enzyme I o f t o t a l h o m o g e n a t e P - f r a c t i o n P u r l f i e d m i c r o s o m e s P - f r a c t i o n P u r i f i e d m ~ c r o $ o m e s

R e l a t i v e s p e c i f i c a c t l v t t y

G l ~ ~ o ~ e - 6 - p h 0 l p h . t d s e 6 1 . 2 6 + 1 . 5 0 ( 1 2 ) 2 7 . 9 3 + 2 . 7 7 ( 1 4 ) 2 . 9 0 + 0 . 0 6 ( 1 2 ) 3 . 1 1 + 0 . 2 7 ( 1 4 ) E s t e r a s e C a t a l a s e 9.08 + 0 . 8 9 ( 1 2 ) 0 . 8 5 + 0 . 1 7 ( 1 1 ) 0.43 + 0.04 ( 1 2 ) 0 . 1 0 + 0 . 0 1 ( 1 1 ) S e d i m e n t a b l e c a t a l a s e 1 3 . 8 5 T 1.47 ( 1 2 ) 1 . 2 5 T 0 . 2 9 ( l o ) 0.65 3 0.06 ( 1 2 ) 0 . 1 5 i 0.02 ( 1 0 ) U p a t e o x r d a r e 23 e9 J 2 . 1 0 ( 9 ) 2.01 c 0.42 (IO) 1 . 1 2 + 0 .08 ( 9 ) 0 . 2 1 z 0.04 ( 1 0 , G l u t a m a t e d e h y d r o g e n a s e 3 . 1 3 5 0 . 3 2 ( 5 ) 1 . 1 5 ( 1 ) 0 . 1 9 5 0.03 ( 5 ) 0 . 1 5 ( 1 ) P r o t e i n 2 1 . 1 6 5 0 . 3 1 (12) 7 . 7 2 + 0 .16 1141

5 9 . 2 0 - ( 2 ) 3 4 . 6 0 - ( 2 ) 2 . 9 2 ( 2 ) 4 . 5 1 -

G l u t a m a t e d e h y d r o g e n a s e 49.45 + 2.36 ( 5 ) G l u c o l e - 5 - p h o s p h . t d s e 1 2 . 8 0 5 0 . 6 8 ( 5 ) C a t a l a s e S e d i m e n t a b l e c a t a l a s e 2 1 98 T 1 . 9 4 ( 5 )

A c i d P h o I p h d t d s e U r a t e O x l d d r e 1 6 . 9 3 T 0 . 8 2 ( I )

P r a t e ? " 13 76 7 0 53 ( 3 ) 17 85 T 1.05 ( 5 )

1 4 . 0 0 T 0 . 9 7 ( 5 1

1 llnitlal [ FRUCTOSE-1,6-BlSPHOSPHATE]. mMI'I*

2 3

F i g . 1 : Oependence o f d i h y d r o x y a c e t o n e - P c o n c e n t r a t i o n i n t h e d i h y d r o x y - a c e t o n e - P a c y l t r a n s f e r a s e a s s a y on t h e i n i t i a l C o n c e n t r a t i o n o f f r U C t O s e - 1 6 - b i s p h o s p h a t e . P ; e i n c u b a t i o n r w e r e p e r f o r m e d f o r 30 ra in a s d e s c r i b e d . I n c u b a t i o n s were

s t o p p e d a f t e r 1 5 m i n . b y t h e a d d l t i o n o f HClO4. O ihydroxyacetone-P IDHAP) s t a r t e d b y t h e a d d ~ t i o n o f h o m o g e n a t e ( 2 . 5 mg o f t o t a l l i v e ? p r o t e i n ] a n d

was measured on t h e H C l O I e x t r a c t s .

C h o l i n e p h o r h o t r a n s f e r a s e a n d e t h . n 0 l d . i n e p h ~ l p h o t l a n r f a r a r e : C h o l i n e p h o r - p h o t r a n l f e r t r e a n d e t h ~ n 0 l a . i n e p h ~ l p h 0 t r a n r f e r a r e were a l l a y e d w i t h Tween 20 e m u l s i o n s o f 1 . 2 - 6 n - d i ~ c y l g l y c e r ~ l s o r 1-~1k-l'-enyl-2-acyl-~n-glycerolr I s

d e g r a d a t i o n o f e 9 g - p h o r p h a t i d y l c h a l i n c a n d e t h a n o l a m i n e p l a s m a l o g e n . res- s u b s t r a t e s . T h e s e s u b s t r a t e s were p r e p a r e d b y p h o s p h o l i p a s e C ( f r o m 8.ceke.A)

p e r t i v e l y . E t h a n o l a m i n e plasmalogen was a g i f t f r o m Or. F . P a l t a u f ( T e c h - n i r c h e U n i v e r r i t l t G r a z A u s t r i a ) . A r r a y s ( 5 0 - 1 0 0 y g p r o t e i n ) were p e r f o r m e d a t 37'C i n 0 . 3 m l n e d i u ; o f t h e f o l l o w i n g ~ o m p o ~ i t i o n : 50 mM T r i s - H C I p H 8.5 1 0 mM Y g C l Z 0 . 1 nM CDP- 2 - 1 4 c e t h a n o l a m i n e o r 0.1 mM C D P - l N I - 1 4 C i c h o i i n r ( s p e c i f i t r a d i o a c t i v i t y : IO c i l n o l ) . 3 m~ 1 . z - ~ n - d i ~ c y 1 g l y c e r o l o r 1-alk-1'-enyl-an-glycerol a n d Tween 20 (0.06 mgln l ) . A s l a y s *em t e r m i n a t e d

m o n i t o r e d b y t h e f i l t e r d i s k m e t h o d i n t r o d u c e d b y G o l d f i n e (32). The f i l t e r a f t e r 10 m i " i n c u b a t i o n . T h e f o r m a t i o n o f r a d i o a c t i v e p h o s p h o l i p i d was

d i r k s were t r a n s f e r r e d t o s c i n t i l l a i i e n v i a l s a n d a r r a y e d f o r r a d i o a c t i v i t y .

D e t e r m i n a t i o n O f g l y c e r o l - 3 - P a n d d i h y d r o x y a c e t o n e - P : For t h e d e t e r m i n a t i o n 3 q l v c e r a l - 3 - P and d i h y d r o x y a c e t o n e - P i n l i v e r s er Y I V D . r a t s were anaer-

z 9 . ~ 4 + 1.92 ( 6 ) 2 . 8 2 + 2 4 ( 5 ) b 6 3 + o.44 ( 6 ) j 0 . 7 8 T 0 . 1 1 ( 6 ) 0 . 7 2 T 0 . 0 2 ( 5 ) 0 . 1 2 T 0 . 0 2 ( 6 ) 0 . 1 2 5 0 04 ( 5 ) 0 . 7 8 5 0 03 ( 5 ) 0 . 0 2 T 0.01 ( 6 ) , 0 . 1 8 T 0 07 ( 5 ) 1 . 2 5 T 0 15 ( 5 ) 0 . 0 1 T 0 . 0 1 ( 5 ) I 0 . 8 1 F 0 . 1 1 ( 4 ) 0.88 T 0 . 0 4 ( I ) 0 . 1 3 T 0 . 0 6 ( 4 ) 1 . 0 3 + 0 . 1 5 ( 3 ) 0.75 i 3 08 ( 3 ) 0 I 5 0 03 ( 3 ) 6 . 5 9 F 0 . 5 9 ( 6 ) 1

t;on w i t 6 i K I o 4 a n d n e u t r a I i ; a t i o n o f t h e e x t r a c t . b y a f l u o r i m e t r i c a d a p - t a t l o n O f t h e m e t h o d s r e f e r r e d t o above.

M a t e r i a l s : A l l r a d i o a c t i v e p r o d u c t s were f r o m t h e R a d i o c h e m i c a l C e n t r e . Amerrham. Bucks., U . K . ; t r y p s i n ( t y p e I l l . f r o m b o v i n e p a n c r e a s ) . t r y p s i n i n - h i b l t o r ( t y p e 1 - 5 , f r o m r a y b e a n ) a n d u n l a b e l l e d g l y c e r o l - 3 - P f r o m S i g m a Chemica l Co . . S t . Louis, Mo, U.S.A.; d i t h l o t h r e i t o l a n d p a l n i t o y l - C a A f r o m

enzymes a n d coenzymes used i n t h e Y a r i o u s d e t e r m i n a t i o n s f r o m B a e h r i n g e r GmbH. P - L B i o C h e m i C a l S . M i l w a u k e e , Y l . U.S.A.; u n l a b e l l e d fructore-l.6-birphorphate.

Mannheim. Bermany; TPCK, TLCK and PMSF f r o m S e r v a , H e i d e l b e r g . G e r m a n y .

T a b l e V I : S o l u b i l i z a t i o n O f p e r o x i r o m a l d i h y d r o x y a c e t o n e - P a c y l t r a n r f e r a s e .

P u r i f i e d p e r o x i l a m e r were r e r u r p e n d e d i n 1 0 mH p y r o p h o s p h a t e b u f f e r . pH 8. 0 . 1 mM d l t h i o t h f e i t o l a n d 0 . 1 Z ( v / v ) e t h a n o l . a n d s o n i c a t e d . w h i c h r e l e a s e d t h e s o l u b l e m a t r i x p r o t e i n s . M e m b r a n e s a n d c o r e s were r e d i m e n t e d b y c e n - t r l f u g a t T o n a t 1 0 0 000 9 f o r 40 nin. The p e l l e t uas s u c C e I S i v e l y t r e a t e d - e a c h t r e a t m e n t w a s f o l l o w e d b y C e n t r i f u g a t i o n a n d t h e s u p e r n a t a n t s were k e p t f o r t h e d e t e r m r n a t i o n o f d i h y d r o x y a c e t o n e - P a c y l t r d n r f e r a s e - w i t h 0 .1 X and 1 % T r l t a n X - 1 0 0 a n d 1 X T r i t o n X - 1 0 0 p l u s I H NaCl i n 10 mH M O P S . pH 7.2, 0 . 1 mN d i t h l o t h r e i t a l . 0 . 1 9 ( v l v ) e t h a n o l . I n e a c h i n s t a n c e , c o n t a c t t i m e w i t h d e t e r g e n t or h i g h s a l t C o n c e n t r a t i o n w a s 40 m i n . S u p e l n d t d n t o were I u f f i c i e n t l y d i l u t e d t o a v o i d t h e i n h i b i t o r y e f f e c t o f T r i t o n X-100 on d l - h y d r o x y a c e t o n e - P a c y l t r a n s f e r a s e . R e s u l t s a r e means f o r 2 e x p e r i m e n t s a n d a r e e x p r e s s e d a s p e r c e n t a g e o f r e c o v e r e d a c t i v i t y or amount .

RESULTS AN0 OlSCUSSlON

Phorphollpid O i h y d r o x y a c e t o n e - Q a c y l -

4 r e c o v e r e d

t r a n s f e r a s e

S Y P e r " a t a " t

a f t e r 0 . 1 % T r i t o n a f t e r sonication 1 0 . 2

5 . 7 a f t e r 1 P T T l t o n 3 4 . 2 a f t e r 1 I T r i t o n 2 5 . 3

11.8 I 8 . 2

3 7 . 1 p l u s 1 H NaCl

F l n a l p e l l e t Recovery 122

8 . 1

means was e r t a b l l r h e d b y S t u d e n t ' s t - t e s t . L i n e a r t r a n s f 0 m a t i o n o f c u r v e s S t a t i s t i c a l a n d m a t h e m a t i c a l a n a l y s i s : S i g n i f i c a n c e o f d i f f e r e n c e s b e t w e e n

r e l a t i n g eszyme a c t i v i t y t o s u b s t r a t e c o n c e n t r a t i o n was P e r f o r m e d a c c o r d i n g t o t h e t e c h n i q u e o f E a d i e a n d H o f s t e e ( 3 6 , 3 7 ) . The Km f o r t h e s u b s t r a t e i s r e o r e r e n t e d b y t h e a b s o l u t e v a l u e o f t h e s l o o e O f t h e I t r a i a h t l i n e ; t h e m a i i n a l v e l o c j t y b y i t s i n t e r c e p t v l t h t h e Y l a x i s .


p r o c e d u r e s w e ~ e c a r r i e d o u t i n t h e presence o f t h e lame p r o t e i n a s e i n h i b l t o r s 5 ) The e x c e l l e n t r e c o v e ~ i e s o f g l y c e r o l - 3 - P s e n s i t i v e a n d i n s e n s i t i v e d i - h y d r o x y a c e t o n e - P a c y l t r a n s f e r a s e a f t e r c e l l f r a c t i a n a t l o n (92.4 + 2 . 9 I and 98.8 + 3.1 I r e s p e c t i v e l y . n = 15) a l s o c o n f i r m e d t h a t s i g n i f i c a a t c h a n g e r

6 ) The g l y c e r o l - 3 - P i n s e n s i t i v i t y o f p e m X i s O m a l d i h y d r o x y a c e t o n e - P a c y l - i n g l y c e r o l - 3 - P s e n s l t i v i t y were n o t b e i n g p r o d u c e d d u r i n g p u r i f i c a t i o n .

t r a n s f e r a s e was n o t t h e r e s u l t O f a l i m i t e d p e r m e a b i l i t y O f t h e peToxIsOma1 membrane t o o l v c e r o l - 3 - P . s i n c e t h e en2v.e was n o t i n h i b i t e d a f t e r d i r r u p - t i o n o f t h e ie ;ox i roner by son ica t ion a; d e t e r g e n t t r e a t m e n t .

S u b c e l l u l a r d i s t r i b u t i o n o f i n t a c t , c a t a l a s e - c o n t a i n i n g p e r o r i r o m e r a n d o f e rox isomal membranes : F i g . 8 r e p r e s e n t s t h e d i r t r l b u t l a n O f marker enzymes

f n d O f d t h y d r o x y a c e t o n e - P a c y l t r a n s f e r a s e among s u b c e l l u l a r f r a c t i o n s . p r e p a r e d b y d i f f e r e n t i a l c e n t r i f u g a t i o n . The r e l a t i v e s p e c i f i c a c t i v i t y o f d i h y d r o x y a c e t o n e - P a c y l t r a n r f e r a r e . m e a s u r e d a t pH 7.5. was h i g h e s t i n t h e L - f r a c t i o n . w h l c h i s m o s t l y e n r i c h e d i n p e r a x i r o n e r and l y ~ o s o m e ~ . However ,

L - f r a c t i o n O f s e d i m e n t a b l e C a t a l a s e . w h i c h r e f l e c t s t h e d i s t r i b u t i o n o f i t w a s s t i l l c o n s i d e r a b l y l o v e r t h a n the r e l a t i v e s p e c i f i c a c t i v i t y i n t h e

i n t a c t . c a t a l a s e c o n t a i n i n g p e r o x i s o n e s , i n d i c a t i n g t h e presence O f d l h y d r o x y

S u b c e l l u l a r d i s t r i b u t i o n o f i n t a c t , c a t a l a s e - c o n t a i n i n g p e r o r i r o m e r a n d o f e rox isomal membranes : F i g . 8 r e p r e s e n t s t h e d i r t r l b u t l a n O f marker enzymes

f n d O f d t h y d r o x y a c e t o n e - P a c y l t r a n s f e r a s e among s u b c e l l u l a r f r a c t i o n s . o r e o a r e d b v d i f f e r e n t i a l c e n t r i f u a a t i o n . The r e l a t i v e I o e C i f i C a c t i v i t y o f i i h j . d roxya ;e tonc -P acy l t ran r fe ra r ; . measured a t pH 7 . 5 : ~ ~ h i g h e s t i n - t h e L - f r a c t i o n . w h l c h i s m o s t l y e n r i c h e d i n p e r a x i r o n e r and l y ~ o s o m e ~ . However ,

L - f r a c t i o n O f s e d i m e n t a b l e C a t a l a s e . w h i c h r e f l e c t s t h e d i s t r i b u t i o n o f i t w a s s t i l l c o n s i d e r a b l y l o v e r t h a n the r e l a t i v e s p e c i f i c a c t i v i t y i n t h e

i n t a c t . c a t a l a s e c o n t a i n i n g p e r o x i s o n e s , i n d i c a t i n g t h e presence O f d l h y d r o x y a c e t o n e - ) a c y l t r a n s f e r a s e i n O t h e r o r g a n e l l e s s u c h a s m i c r o s o m e s a n d n i t o -

o r a t pn 7 . 5 i n t h e presence o f 5 mu g l y c e r o l - 3 - P . i t s r e l a t i v e s p e c i f i c c h c n d r i a . When d i h y d r o x y a c e t o n e - P a c y l t r a n s f e r a s e was measured a t pH 5.7,

a c t i v i t y i n L - f r a c t i o n s was a p p r a x i m a t e l y d o u b l e d . b u t i t s t i l l d i d n o t

AI d i s c u s s e d a b o v e , t h e d i s t r i b u t i o n O f d i h y d r o x y a c e t o n e - P a C Y l t r a n S f @ r a S * . r e a c h t h a t o f r e d i m e n t a b l e c a t a l a s e a c t i v i t y .

measured under t h e l a t t e r t w o c o n d i t i o n s . s h o u l d e s s e n t i a l l y r e f l e c t t h e d i s t r i b u t i o n O f Q l a p p a r e n t o c c u r r e n c e ?rox i rana l membraner . Because o f t h e

_y1

D 1w % OF TOTAL PROTEIN

F i g . 8 : S u b c e l l u l a r d i s t r l b u t l o n o f d i h y d r o x y a c e t o n e - P d c y l t r a n r f e r a l e a n d m a r k e r e n z y m e s a f t e r d l f f e r e n t i a l c e n t r i f u g a t l o n o f 1 l i v e r homogenate. L i v e r h o m o g e n a t e r were f r a c t i o n a t e d i n t o a n u c l e a r ( N ) . h e a v y m i t o c h o n d r i a l

d t h y d r o x y a c e t o n e - P d ~ y l t r d n $ f e ~ d ~ e and a number O f marker enzymes were ( W ) . l i g h t m i t o c h o n d r i a l ( t ) . m i c r o s o m a I ( P ) a n d s o l u b l e ( 5 ) f r a c t i o n and

measured on t h e d i f f e r e n t f T a C t i o n $ . R e s u l t s a r e e x p r e s s e d a s r e l a t l v e s p e c i f i c a c t l v l t l e s v e r s u s c ~ r n ~ l a t ~ v e p e r c e n t a g e o f t o t a l r e c o v e r e d p r o t e l n .

a r e r e r u l t S f r o m one e x p e r i m e n t ; panels k en 1 a r e r e s u l t s f r o m a n o t h e r e x - R e l a t i v e S p e C l f i C d c t i v l t y 1 s d e f l n e d i n t h e l e g e n d t o T a b l e I . P a n e l s a - ~

p e r i m e n t . ( a ) G I u c o r e - 6 - p h o ~ p h a t l ~ e ; ( a ) e s t e r a s e : ( c ) d l h y d r o x y a c e t o n e - P a c y l t r a n r f e r a r e . pH 7.5; ( d ) d i h y d r o x y a c e t o n e - P a c y l t r a n r f e r a r e . p H 5 . 7 ; ( e ) C a t a l a s e ; ( f ) r e d i m e n t a b l e C a t a l a s e ; ( g ) a c i d p h o s p h a t a s e : ( k ) d l h y d w x y - a c e t o n e - P a c y l t r a n s f e r a s e . pH 7.5. i n t h e a b s e n c e ( s o l ~ d l l n e ) and presence (dashed I r n e ) O f 5 mW g l y c e r o l - 3 - P . ( 1 ) s e d i m e n t a b l e C a t a l a s e .

o f e m p t y p e r o x i r o n a l v e s i c l e s u h i c h h a v e l o s f t h e i r m a t r i x e n z y m e s d u r l n g

f e r e n t f r o m t h o s e o f i n t a c t . c a t a l a s e - c o n t a i n i n g p e r o r i s o n e s . t h e s u b c e l l u l a r homogen iza t ion and wh ich thc r ; fo re seem t o have masses and d e n s i t i e s d t f -

d i s t r i b u t i o n p a t t e r n s o f marker enzymes f o l t h e p e r o x i s o m a l m a t P 1 x a n d t h e p e r o r i r o n a l m e m b r a n e d o n o t e n t i r e l y C o i n c i d e . F o r i n s t a n c e , m i c r o r o n a l f r a c - t i o n s c o n t a i n e d a p p r o x i m a t e l y 30 I O f t h e l i v e r ' s g l y c e r o l - 3 - P r e s i s t a n t ( p e r o x i s o m a l ) d i h y d r o x y a c e t o n e - P a c y l t r a n r f e r a r e b u t o n l y 14 I o f i t s s e d i m e n - t a b l e c a t s l a r e a c t i v i t y ( s e e T a b l e 11).

T a b l e v l l l : c o n t e n t s and c o n c e n t r a t i o n s o f d i h y d r o x y a c e t o n e - P a n d g l y c e r o l - 3 - P i n f r e e z e - c l a m p e d 1 1 v e r s . Hepatic d i h y d r o x y a c e t o n e - P a n d g l y c e r o l - 3 - P c o n t e n t s were d e t e r m i n e d i n

c e n t r a t i o n s b y d i v i d i n g b y t h e number o f parenchymal ce l l s per g o f l i v e r and f r e e z e - c i a m p e d l i v e r s a n d t h e mean values were c o n v e r t e d t o i n t r a c e l l u l a r con-

b y t h e space o f p a r e n c h y m a l c e l l s (21). C o n t e n t s are means : SLH f o r 9 e x p e r i m e n t s .

D i h y d r o x y a c e t o n e - P G l y c e r o l - 3 - P

n m o l r l g o f l i v e r m M n n o l s l g o f l i v e r m I I Fed s t a t e 4 7 . 8 + 3.5 0 . 1 4 2 174.7 + 2 2 . 3 0.520

S t a r v e d s t a t e 72.8 5 15.0 0.188 177.2 i 19.9 0 . 4 5 8

F i g . 9 : D i s t r i b u t i o n O f d i a c y l g l y c e r o l aCyltranrfePale. ethdnoldmineph0spho- t r a n s f e r a s e . ~ h o l i n e p h 0 ~ p h o t r l n 9 f e r l ~ e and m a r k e r enzymes a f t e v I u b f l a c t i o n a - t i o n o f t h e l i g h t m i t o c h o n d r i a l ( L ) f r a c t i o n b y i s o p y c n i c c e n t r i f u g a t i o n i n s e l f - g e n e r a t i n g P e r c o l l g r a d i e n t s . ( a ) G l ~ c o l e - 6 . p h o l p h . t l l e ; ( b ) d i a c y l - g l y c e r o l a c y l t r a n r f e r a r e ; ( c ] e t h a n o l a ~ r n e p h o s p h a t r a n r f e r a r e ; ( d ) c h o l i n e - p h o r p h a t r a n r f e r a r e ; ( e ) u r a t e o r i d a r e ; ( f ) C a t a l a s e . U r a t e O x i d a s e PeCOYePed a t t h e b o t t o m O f t h e g r a d i e n t r e p r e s e n t s n a k e d c o r e s . D i a c y l g l y c e r o l a c y l - t r a n s f e r a s e . e t h a n o l a ~ i n e p h a r p h o t r a n r f e r a r e and Cholinephorphotranrferare were measured w i t h d i a c y l g l y c e r o l I I s u b s t v a t e .

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