ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 330, No. 2, June 15, pp. 409–413, 1996Article No. 0269

Vitamin B6 Deficiency Accelerates Metabolic Turnoverof Cystathionase in Rat Liver

Atsushi Sato,* Michiko Nishioka,* Shiro Awata,† Kazuko Nakayama,† Mitsuko Okada,‡Saburou Horiuchi,§ Nobuhiko Okabe,Ø Toshihiro Sassa,Ø

Tatsuzo Oka,Ø and Yasuo NatoriØ,1

*Department of Food and Nutrition, Kochi Women’s University, Kochi 780, Japan; †Department of NutritionalBiochemistry, Kochi Gakuen College, Kochi 780, Japan; ‡Faculty of Health and Living Science,Naruto University of Education, Naruto 772, Japan; §Department of Biochemistry,Iwate Medical University, Morioka 020, Japan; and ØDepartment of Nutrition,School of Medicine, University of Tokushima, Tokushima 770, Japan

Received August 14, 1995, and in revised form March 18, 1996

nine, catalyzing the cleavage of cystathionine to cys-Although most of cystathionase was found to exist teine. Rat cystathionase is composed of four 43,000-Da

as an inactive apoenzyme in the liver of vitamin B6- subunits and contains 4 mol pyridoxal 5*-phosphatedeficient rats, the concentrations of the immunoreac- (PLP2) as coenzyme (1).tive enzyme protein were virtually the same for con- An earlier report (2) from our laboratory has showntrol and vitamin B6-deficient livers. Under vitamin B6 that, in the liver of vitamin B6-deficient rats, the enzymedeficiency, however, the rate of synthesis of cystathio- activity of cytosolic aspartate aminotransferase (ECnase, measured by incorporation of labeled amino acid 2.6.1.1) is decreased but the concentration of the enzymeinto the immunoprecipitated enzyme, was increased protein remains at the control level. Further study onseveralfold due to an increased level of cystathionase the metabolic turnover of aspartate aminotransferasemRNA. Western blot analysis of lysosomal proteins has indicated that both the rate of synthesis and the rateshowed that the amount of cystathionase in the lyso- of degradation of the enzyme protein are increased in thesomes from the liver of vitamin B6-deficient rats was liver of vitamin B6-deficient rats (3).also increased severalfold. This observation suggests

In the present communication, we will show that syn-that lysosomes specifically recognize the apocystathio-thesis and degradation of cystathionase are affectednase for sequestration in preference to the holoen-in a manner similar to aspartate aminotransferase inzyme. The present study provides the molecular basisvitamin B6 deficiency and will provide the molecularfor dual roles of vitamin B6 in controlling the metabolicmechanisms for these metabolic alterations.turnover of cystathionase; it regulates synthesis of the

enzyme by modulating the expression of cystathionaseMATERIALS AND METHODSgene, and it regulates degradation of the enzyme by

different susceptibilities of apo- and holoenzymes to Animals. Male Wistar rats, weighing about 50 g, were given alysosomal proteolysis. q 1996 Academic Press, Inc. 70% casein diet, with or without 0.029% pyridoxine, ad libitum for

4 weeks. The composition of the diet has been described previouslyKey Words: cystathionase; vitamin B6 deficiency; pro-(4). Animals were killed by a blow to the head and decapitated beforetein turnover; lysosome; proteolysis.isolation of the liver.

Enzyme assay. The liver was homogenized in 3 vol of ice-cold 0.01M potassium phosphate buffer (pH 7.5) containing 0.15 M KCl and0.5 mM EDTA. The homogenate was centrifuged at 10,000g for 10Cystathionase (EC 4.4.1.1, cystathionine g-lyase) is min and then the supernatant was centrifuged at 100,000g for 1 h

the final enzyme in the transsulfuration pathway that to obtain the cytosolic fraction. The activity of cystathionase wasassayed by a modification of the method of Matsuo and Greenberg (5).in mammalian cells synthesizes cysteine from methio-

2 Abbreviations used: PLP, pyridoxal 5*-phosphate; ELISA, en-1 To whom correspondence should be addressed. Fax: 81-886-33-7082. zyme-linked immunosorbent assay; NP-40, Nonidet P-40.

4090003-9861/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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410 SATO ET AL.

In the assay of basal activity (activity of holoenzyme), the reaction TABLE Imixture, in a final volume of 1.0 ml, contained 32 mM D,L-homoserine, Effect of Vitamin B6 Deficiency and Supplementation on7.5 mM mercaptoethanol, 7.0 mM EDTA, 0.1 M potassium phosphate

Activity and Concentration of Cystathionase in Rat Liverbuffer (pH 7.5) and the cytosolic fraction as an enzyme source. Forthe assay of stimulated activity (combined activities of apo- and holo-enzyme), 0.05 mM PLP was added to the reaction mixture used in Enzyme activity

(units/g of liver)the assay of basal activity. One unit of the enzyme activity was Enzymedefined as the activity forming 1 mmol of a-ketobutyric acid per 1 h. concentration

/PLP 0PLP (mg/g of liver)Enzyme-linked immunosorbent assay. Enzyme-linked immuno-sorbent assay (ELISA) for cystathionase was carried out as described

Control 8.09 { 0.33 7.13 { 0.57 0.61 { 0.06previously (6).0B6 9.72 { 1.07 0.87 { 0.33 0.60 { 0.10Isotope-labeling experiments. Control and vitamin B6-deficient

rats were fasted overnight and 100 mCi L-[4,5-3H]leucine/100 g bodyNote. Experimental details are described under Materials andwt (32 Ci/mol, Daiichi Pure Chemicals Co., Tokyo, Japan) was in-

Methods. Rats were fed on a diet with (control) or without (0B6)jected intraperitoneally. The animals were killed 4 h after the injec-pyridoxine for 4 weeks. Enzyme activities were assayed in the pres-tion of the isotope.ence (/PLP) or absence (0PLP) of PLP in the reaction mixture. DataThe liver was removed after extensive perfusion with cold salineare given as means { SD of four separate determinations.solution and the cytosolic fraction was prepared as described above

for the enzyme assay. A portion (0.5 ml) of the cytosolic fraction wasused as the source of total cytosolic proteins and the radioactivityincorporated into the protein was counted in an Aloka LSC-3500liquid scintillation counter as described previously (7). To another schedule (13). The livers from the control and vitaminportion (3.0 ml) of the cytosolic fraction, 0.6 ml of antiserum against B6-deficient rats were homogenized and cystathionasecystathionase (6) was added and left overnight in a cold room. The activity in the cytosolic fraction was assayed. As shownimmunoreaction solution was then mixed with 0.4 ml of 10% protein

in Table I, the enzyme activities in the control rat liv-A suspension (Pansorbin Cells, Calbiochem-Novabiochem, La Jolla,ers, assayed with or without addition of PLP to theCA) and the immunocomplex was precipitated by incubation for 2 h

at 47C. The precipitate was collected by centrifugation, washed twice reaction mixture, were essentially the same. However,with 50 mM Tris buffer (pH 8.0) containing 500 mM NaCl and 1.0% the activity in the vitamin B6-deficient rat livers, as-NP-40, and finally treated with 60 mM Tris buffer (pH 6.8) containing sayed without addition of PLP, was only 9% of the ac-2% SDS, 10% glycerol, and 100 mM dithiothreitol at 907C for 10 min.

tivity measured with added PLP. This result indicatesAfter centrifugation, the solubilized immunocomplex was counted inthat, whereas most of cystathionase exists as holoen-a scintillation counter as above.zyme in the liver of control rats, the majority (90%) ofNorthern blot hybridization. Total RNA was isolated from the

livers and subjected to Northern blot hybridization using 32P-labeled the enzyme exists as apoenzyme in the liver of vitaminprobe as described previously (8). The oligonucleotide probe (GCA- B6-deficient rats. The result also indicates that, sinceGCGATCACACC), corresponding to nucleotides 327 to 341 of rat the PLP-supplemented activities are essentially thecystathionase cDNA (9), was synthesized on an Applied Biosystems

same for both control and vitamin B6-deficient livers,synthesizer, and the oligonucleotide was labeled with 32P at the 5*end by T4 polynucleotide kinase. The autoradiographs were densito- the concentrations of the enzyme protein may be simi-metrically scanned in order to determine mRNA levels quantita- lar. That this is in fact the case was shown by enzyme-tively. linked immunosorbent assay; both control and vitamin

Preparation of rat liver lysosomes. Highly purified lysosomes B6-deficient livers were found to contain approximatelywere isolated from the livers by the method developed in our labora-

0.6 mg cystathionase protein/g liver (Table I).tory (10).Western blot analysis. Electrophoresis of the lysosomal proteins

Increase in Synthesis of Cystathionase in Vitamin B6and Western blot analysis of intralysosomal cystathionase and thetotal cytosolic proteins were carried out as described previously (11), Deficiencyusing antisera against cystathionase (6) and rat liver total cytosolic

We next examined the influence of vitamin B6 defi-proteins (12).ciency on the synthesis rate of cystathionase in theliver. Whereas the incorporation of [3H]leucine into theRESULTStotal cytosolic proteins was the same for the control

Activity and Concentration of Cystathionase in the and vitamin B6-deficient rats, the incorporation intoLiver of Vitamin B6-Deficient Rats the immunoprecipitated cystathionase in the liver of

vitamin B6-deficient rats was about fourfold higherRats were fed on a 70% casein diet, with or withoutpyridoxine ad libitum for 4 weeks. The weight gains than that in the liver of control rats (Table II). We

cannot determine the absolute rate of protein synthesiswere about 7 g/day for the control rats and 3 g/day forthe rats on the pyridoxine-deficient diet. A previous by measuring the incorporation of labeled amino acid

into proteins because of the difficulty in the assessmentreport from our laboratory has shown that the concen-tration of PLP in the liver of rats fed on the vitamin of the specific radioactivity of the precursor amino acid

at the site of protein synthesis. However, the data pre-B6-deficient diet was reduced to approximately 15% ofthat in control rats at the end of a similar feeding sented in Table II indicate that the rate of synthesis of

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411VITAMIN B6 AND METABOLIC TURNOVER OF CYSTATHIONASE

TABLE II intralysosomal cystathionase (Fig. 2A). The blottingshowed a major 43-kDa band, corresponding to unde-Effect of Vitamin B6 Deficiency and Supplementation on

Incorporation of Radioactive Leucine into Liver Cytosolic graded cystathionase subunit, and a minor 36-kDaProteins and Cystathionase band probably representing a degradative intermedi-

ate. Cystathionase level in the lysosomes from vitaminTotal cytosolic B6-deficient rat liver, estimated by densitometric trac-

protein (A) Cystathionase ings, was fivefold higher than that in control lysosomes.(dpm 1 1005/g (B) (dpm 1 B/A In contrast, the Western blotting of the lysosomal pro-liver) 1003/g liver) (%)

teins from vitamin B6-deficient rat liver, using antise-rum against total cytosolic proteins, showed essentiallyControl 3.32 { 1.15 1.83 { 0.92 0.55

0B6 3.37 { 0.57 6.87 { 0.73 2.04 the same pattern as the control liver (Fig. 2B). Thisresult indicates that vitamin B6 deficiency specifically

Note. Rats were fed on a diet with (control) or without (0B6) pyri- enhances the lysosomal sequestration of cystathionasedoxine for 4 weeks and each rat received an injection of 100 mCi[3H]-without affecting the sequestration of total cytosolicleucine/100 g body wt. Four hours after the injection, animals wereproteins in the liver.killed, and cytosolic proteins and cystathionase were prepared from

the liver as described under Materials and Methods. The values rep-resent means { SD of four rats.

DISCUSSION

The present study has demonstrated that althoughmost of cystathionase exists as an inactive apoenzymecystathionase, relative to the rate of synthesis of totalin the liver of vitamin B6-deficient rats, the concentra-cytosolic proteins, in the liver of vitamin B6-deficienttions of the immunoreactive enzyme protein are virtu-rats is fourfold higher than that in the liver of control

rats.

Increase in Cystathionase mRNA in Vitamin B6

Deficiency

Total RNA isolated from the livers was subjected toNorthern blot analysis using an endlabeled syntheticoligonucleotide, corresponding to nucleotides 327 to341 of rat cystathionase cDNA (9), as a probe (Fig.1). The size of the hybridizing band relative to RNAstandards was found to be about 2.0 kb, in agreementwith the published size of cystathionase mRNA (9).Cystathionase mRNA concentration in the liver of vita-min B6-deficient rats, estimated by densitometric trac-ing, was fivefold higher than that in control rats. Thisobservation suggests that the increased rate of synthe-sis of cystathionase in vitamin B6 deficiency, shown inTable II, is largely due to the increased level of cystathi-onase mRNA.

Increase in Lysosomal Sequestration of Cystathionasein Vitamin B6 Deficiency

The steady-state concentration of an enzyme proteinin a tissue is determined by the ratio between the rateconstant for synthesis and the rate constant for degra-dation. Since the concentration of cystathionase is un-changed in vitamin B6 deficiency in spite of an increase FIG. 1. Effect of vitamin B6 deficiency on the level of cystathionasein the rate of synthesis, one would expect that the rate mRNA in rat liver. The procedures for isolation and Northern blot

hybridization are described under Materials and Methods. (A) North-of degradation of cystathionase is also increased in vi-ern blot analysis of total RNA from the livers of control (con) andtamin B6 deficiency. Inasmuch as lysosomes are consid-vitamin B6-deficient (0B6) rats with oligonucleotide probe. The gelered to be the main site of intracellular protein degra- shown is representative of four independent experiments. (B) Ethid-

dation (12), we isolated highly purified lysosomes from ium bromide staining of ribosomal RNA as internal control of theamount of RNA loaded.the livers and performed a Western blot analysis of

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412 SATO ET AL.

aminotransferase gene contains several sequencesshowing homology to glucocorticoid-responsive ele-ments (16) and the recent study from our laboratoryhas shown that PLP modulates the expression of aspar-tate aminotransferase gene by inactivating the bindingactivity of glucocorticoid receptor to glucocorticoid-re-sponsive elements (15). Since the structural informa-tion of cystathionase gene is not available to date, wecannot elaborate the molecular mechanism for the en-hanced expression of cystathionase gene in vitamin B6

deficiency. However, it probably involves PLP-depen-dent inactivation of some tissue-specific transcriptionalfactors in a fashion similar to aspartate aminotransfer-ase gene (15) or albumin gene (8).

Since the concentration of cystathionase enzyme pro-tein is unchanged under vitamin B6 deficiency in spiteof severalfold increase in the rate of enzyme synthesis,the rate of degradation of the enzyme must likewise beincreased severalfold. We have now found that seques-tration of cystathionase into lysosomes is increasedfivefold in the liver of vitamin B6-deficient rats, indicat-ing that lysosomes are the major site of intracellulardegradation of cystathionase. We have previously ob-served a similar increase in the lysosomal sequestra-

FIG. 2. Western blot analysis of intralysosomal cystathionase and tion of aspartate aminotransferase in vitamin B6-defi-total cytosolic proteins in the livers of control and vitamin B6-defi-cient rat liver and proposed that lysosomes specificallycient rats. (A) Lysosomal proteins (5.0 mg) from the livers of therecognize the apoenzyme for sequestration (11). Thecontrol (lanes 2–5) and vitamin B6-deficient (lanes 6–9) rats were

subjected to SDS–polyacrylamide gel electrophoresis and immu- apocystathionase may likewise be more vulnerable tonoblotted with antiserum against cystathionase. Lane 1 represents lysosomal uptake than the holoenzyme. Another exam-the authentic cystathionase. (B) Lysosomal proteins from the livers ple is that tryptophan-2,3-dioxygenase in rat liver isof the control (lanes 1–4) and vitamin B6-deficient (lanes 5–8) rats

stabilized by the administration of its substrate, trypto-were similarly immunoblotted with antiserum against total cytosolicproteins. phan, by means of a decreased sequestration into lyso-

somes (17). In all of these cases, the rate of intracellulardegradation of enzymes is modulated by the binding ofthe enzyme proteins to their coenzyme or substrate.ally the same for control and vitamin B6-deficient liv-The ligand-bound enzymes are less vulnerable to lyso-ers. Under vitamin B6 deficiency, however, the rate ofsomal sequestration than the ligand-free enzymes. Pre-synthesis of cystathionase is increased severalfold duesumably, the dissociation of the ligands may bringto an increased level of cystathionase mRNA. The rateabout a conformational change of the enzyme proteinsof lysosomal degradation of cystathionase is also in-in such a manner as to expose certain domain for lyso-creased severalfold.somal recognition. The in vitro system demonstratingEssentially the same picture has been previously ob-the ‘‘ligand-modulated lysosomal proteolysis’’ will beserved in the case of cytosolic aspartate aminotransfer-reported elsewhere.ase in vitamin B6-deficient livers; most of aspartate

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