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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 229, No. 1, February 15, pp. 237-245, 1984

Kinetic, Chromatographic and Electrophoretic Studies on Glucose- Phosphorylating Enzymes of Rat Intestinal Mucosa’

MARIA LILA VERA,2 MARIA LUZ CARDENAS, AND HERMANN NIEMEYER3

Departamento de Biologia, Fwultad de Ciencias Bckicas y Farmakuuticas, Universidad de Chile, Casilla 653, Santiago, Chile

Received July 19, 1983, and in revised form October 3, 1983

The number and nature of glucose-phosphorylating enzymes of rat intestinal mucosa were investigated by chromatographic, electrophoretic, and kinetic methods. Three fractions with glucose-phosphorylating activity were obtained from the supernatant fluid of mucosa homogenate by means of DEAE-cellulose chromatography, corresponding to hexokinases A and B (EC 2.‘7.1.1.), and N-acetyl-D-glucosamine kinase (EC 2.7.1.59). Although the latter uses N-acetylglucosamine as the main substrate, it is also able to phosphorylate glucose. Electrophoresis in polyacrylamide and in cellulose acetate gels showed the same three enzyme activities. None of these procedures revealed the presence of either hexokinase D (“glucokinase”) or hexokinase C in the intestinal mucosa. In the sediment fractions hexokinase A and B, but not N-acetylglucosamine kinase, were found. The K, values for glucose of partially purified hexokinases A and B were 0.025 and 0.174 mM, respectively, and their substrate specificity was the same as that of hexokinases A or B from other tissues. Partially purified N-acetylglucosamine kinase showed hyperbolic saturation functions for N-acetylglucosamine and ATP, with Km values of 0.021 and 0.38 mM, respectively. This enzyme also phosphorylated glucose, mannose, fructose, 2-deoxyglucose, and glucosamine. The dependence of velocity on glucose concentrations was complex, mimicking negative cooperativity. The molecular weight of both hexokinases A and B was 98,000 and that of N-acetylglucosamine kinase was 59,000. The kinetic properties, as well as the chromatographic and electrophoretic mobilities, of N-acetylglucosamine kinase may serve to confuse it with hexokinase D, and thus several criteria should be applied for correct identification.

Rat liver contains four hexokinase isozymes (ATP:D-hexose-6-phosphotransfer- ase, EC 2.7.1.1) which were separated by DEAE-cellulose chromatography and were named A, B, C, and D, according to their order of elution (1). When separated by

’ Supported by grants from the University of Chile (B-174-783), the Multinational Program of Biochem- istry from The Organization of the American States, and the Regional Program for Graduate Training in Biological Sciences, PNUD/UNESCO.

* Permanent address: Departamento de Ciencias Bioquimicas y Fisiol6gicas, Universidad de Antofa- gasta.

3 To whom correspondence should be addressed.

starch gel electrophoresis the isozymes were designated as I, II, III, and IV, respectively, depending on their anodal mobility (2). Three of the hexokinases (A, B, and C) display high affinity for glucose and are often referred to as low-K, hexokinases or simply hexokinases; the fourth (hexokinase D) was found to exhibit a high Km for glucose and is commonly called “glucokinase” (EC 2.7.1.2). This discovery was soon followed by the search of several other tissues in various mammalian species for their content of hexokinase isozymes and by the study of the main kinetic properties of the isozymes (4-6). In the case of the rat intestinal mucosa, several reports

237 0003-9861/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

238 VERA, CARDENAS, AND NIEMEYER

differ with respect to the number and nature of isozymes present (3, 7-11). Of spe- cial interest is the reported presence in this tissue of a high-K, glucose isozyme (10, ll), since it has been shown in this laboratory that phosphorylation of glucose by N-acetyl-D-glucosamine kinase (ATP:S- acetamido-2-deoxy-D-glucose-6-phospho- transferase (EC 2.7.1.59) may mimic that of “glucokinase” (12, 13). The confusion arises because isozyme identification either in tissue extracts or in electrophoretic strips has usually been based on parallel assays at high and low glucose concentrations (14). Although useful as a prelimi- nary, tentative diagnostic tool, this procedure is susceptible to errors and must be checked by alternative procedures (15).

For the reasons stated above we decided to reinvestigate the presence of glucose- phosphorylating enzymes in rat intestinal mucosa. Ion-exchange chromatography, polyacrylamide gel or cellulose acetate electrophoresis, molecular sieve exclusion, and affinity chromatography were used to separate the enzymes, which were further characterized by kinetic analysis and substrate specificity studies. The results show that only hexokinases A and B are present in the intestinal mucosa and that the enzyme exhibiting a high Km for glucose- phosphorylating activity is not hexokinase D but N-acetyl-o-glucosamine kinase. The last conclusion agrees with recently pub- lished data indicating that the putative hexokinase D present in several extrahe- patic tissues indeed corresponds to N-acetyl-D-glucosamine kinase (12, 13,16). Pre- liminary accounts of this work have been reported (17).

EXPERIMENTAL PROCEDURES

Materials. Sephadex G-100 and G-200, CH-Sephar- ose 4B, and Blue Dextran 2000 were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden), DL-

[‘“C&eucine was purchased from New England Nu- clear (Boston, Mass.). Cellulose acetate membranes were from Chemetron. Substrates, coenzymes, auxiliary enzymes, protein markers, DEAE-cellulose, and other reagents and biochemicals were products of Sigma Chemical Company (St. Louis, MO.).

Animals and preparation of tissue extracts. Male adult rats were obtained from the Instituto de Nu-

tricion y Tecnologia de Alimentos, Universidad de Chile, and used when they were approximately 2 months old and weighed between 180 and 200 g. They were maintained on commercial rat food. Rats were decapitated and, after exsanguination, the portion of the intestine extending from the pilorus to the il- eocecal valve was quickly removed and immersed in cold 150 mM KCI, 100 mM glucose. The intestines were carefully washed and then kept for a while in the same solution until all the rats were processed. The intestines were everted over filter paper and the mucosa was separated from the muscularis by gently scraping with a slide glass with sintered borders. The mucosa was rapidly weighed and homogenized in 4 vol (20% w/v) of 10 mM phosphate buffer, pH 7.0, containing 1 mM EDTA, 5 mM 2-ME,4 and 100 mM

glucose, using a Potter-Elvejhem homogenizer provided with a Teflon pestle. After centrifugation at 105,OOOg in a Spinco ultracentrifuge for 60 min, the supernatant fluid (S,) was removed by aspiration with syringe and needle. When the particulate fraction was to be used, homogenates were incubated with 0.5% Triton X-100 for 30 min followed by centrifugation at 105,OOOg for 60 min. The supernatant fluid was examined for glucose-phosphorylating enzymes.

Enzyme assays. Glucose-phosphorylating activity was measured through the production of glucose 6- phosphate (method A) or ADP (method B), using auxiliary enzymes. In method A the phosphorylation was coupled to glucose-6-phosphate dehydrogenase and the rate of reduction of NADP was measured at 340 nm in a Gilford spectrophotometer 2400 provided with a thermospacer maintained at 30°C. The assay medium was that of Cardenas et al. (18). The same reaction mixture with ATP omitted acted as a blank. When fructose was used as the substrate, 1 U of glucose-6-phosphate isomerase was added as a second auxiliary enzyme. In method B, ADP formation was followed through coupling the glucose-phosphorylating activity to pyruvate kinase and lactate dehydrogenase and measurement of NADH oxidation at 340 nm (19). Essentially the same buffer solution as in method A was used and the reaction medium contained 1 U of both auxiliary enzymes. A similar system with the sugar omitted acted as a blank. The reaction was initiated by addition of the hexose substrate. Commercial ATP contained about 2% ADP, which was converted to ATP during a preincubation of about 5 min in the absence of the hexose substrate. This method was used to measure N-acetylglucosamine kinase with iV-acetylglucosamine or other substrates, and also hexokinase with all substrates, excluding glucose and fructose. One unit of enzyme (TJ) corre-

* Abbreviations used: 2-ME, 2-mercaptoethanol; EDC, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide; DTT, dithiothreitol.

GLUCOSE-PHOSPHORYLATING ENZYMES OF INTESTINAL MUCOSA 239

sponds to the amount of enzyme that transforms 1 rmol of substrate in 1 min at 30°C under the conditions described.

Other determinations. Protein concentration was determined from the absorbance at 280 and 260 nm. KC1 concentrations were estimated by conductimetry.

DEAE-cellulose chrmatography. The equilibrating solution was 10 mM potassium phosphate buffer, pH 7.0, containing 5 mM Z-ME and 1 mM EDTA. To separate protein fractions a linear gradient of 0 to 0.5 M KC1 in the same buffer was applied. As a routine all tubes were assayed in parallel at 0.5 and 100 mM glucose.

Sephudezjltration. This method was used as a pre- parative procedure during enzyme purification and for the determination of molecular weights according to Andrews (20). Sephadex G-100 and G-200 were prepared for chromatography following the instruc- tions given by the manufacturers. The columns were equilibrated and eluted at 4°C with 10 mM potassium phosphate buffer, pH 7.0, containing 100 mM KCl, 5 mM 2-ME, and 1 mM EDTA. Blue Dextran 2000 was used to measure the column void volume (V,) and [‘*C]leucine (50,000 cpm) to determine the total volume accessible to solvent (Vi). Cytochrome c (M, = 12,500), ovalbumin (M, = 45,000), bovine serum albumin (M, = 68,000), and yeast alcohol dehydrogenase (Af, = 141,000) were used as marker proteins. For the estimation of the molecular weight of the enzymes through their elution volumes (V,), the distribution coefficient [& = (V, - Vd( Vi - V,)] was plotted against the molecular weights of the standard proteins used.

Afinity chromatography. Sepharose-N-(6-amino- hexanoyl)-2-amino-2-deoxy-D-glucopyranose (Se- pharose-glucosamine) was prepared by coupling CH-Sepharose 4B and D(+)-glUCOSamine, through the action of EDC, as described in detail by the manufacturers (Pharmacia Fine Chemicals). Details on the protocol used are given in Fig. 3.

Partial purQicatim of glucose-phosphorylating enzymes. A partial purification of the two hexokinases and of N-acetylglucosamine kinase of rat intestinal mucosa was accomplished by the sequential appli- cation of DEAE-cellulose chromatography and gel filtrations as described. The DEAE-cellulose fraction containing the enzyme of interest was treated with 65% saturated ammonium sulfate to concentrate the enzyme before gel filtration. Sephadex G-206 was used for hexokinase A and B, and Sephadex G-100 for N- acetylglucosamine kinase. Affinity chromatography on Sepharose-glucosamine was used after the Se- phadex step to further purify hexokinase B. The specific activities of hexokinase A, hexokinase B, and N- acetylglucosamine kinase were about 0.4, 3.3, and 0.4 units/mg protein, respectively.

Ekctrophvre&. Polyacrylamide gel electrophoresis was conducted according to Grossman and Potter (21). The electrode solution was a Tris-barbital buffer, pH

7.8, containing 50 mM glucose and 5 mM DTT or 2- ME as protecting agents, plus 4 mM EDTA and 5 mM Mg2S04. The electrophoresis was run in 7% polyacrylamide for 3.5 h at 4°C with 3 mA applied to each gel. For detection of enzyme activity the procedure was that of Katzen et al (3), with minor modifications.

Cellulose acetate membrane electrophoresis was conducted according to Sato et al. (22). The procedure was performed at 4°C during 3.5 h at a fixed voltage of 200 V (13 to 16 mA). The electrophoresis buffer contained 50 mM sodium barbital, pH 8.6,10 mM glucose, 2 mM 2-ME, and 5 mM EDTA. Staining of enzyme activities was performed with slight modifications of the procedure of Sato et a/. (22).

RESULTS

DEAE-cellulose chrornat~aphy pattern. Only two peaks with glucose-phosphorylating activity were observed in the S1 fraction and the sediment from the intestinal mucosa homogenates when 0.5 or 100 mM glucose concentrations were used for enzyme assay. An illustrative pattern is shown in Fig. 1. The first and the second peak of activity were identified as hexokinase A and hexokinase B, respectively. The elution profile from the S, fraction presented a shoulder on the right limb of the peak at 100 mM glucose that increased markedly when the assay was performed at 400 mM glucose (Fig. 1). This shoulder corresponds to the glucose-phosphorylating activity of N-acetylglucosamine kinase, as evidenced with the specific assay. The apices of hexokinases A and B eluted at 55 and 115 mM KCl, respectively, which agrees with previous experience in liver from mammals (23, 24). The amount of hexokinase B varied between 80 and 90% of the total phosphorylating activity in several preparations. In the Triton X-loo-treated sediment only hexokinases A and B were found; there was no N-acetylglucosamine kinase.

Several variations in the DEAE-cellulose chromatographic procedure were tried in an attempt to detect the presence of hexokinases C and D. For instance, the appli- cation of a shallower than usual concentration gradient (larger volumes, and from 0 to 0.4 M KCl) allowed a better separation of N-acetylglucosamine kinase from hexokinase B. Under these conditions careful

240 VERA, CARDENAS,

,,o-’ P.

Effluent volume. ml

FIG. 1. DEAE-cellulose ion exchange chromatography of hexokinases A and B and N-acetylglucosamine kinase. The extract of intestinal mucosa from a single rat was prepared as described under Exper- imental Procedures; column size, 0.6 X 6 cm; volume of each effluent fraction, 0.9 ml. N-acetylglucosamine kinase (A) was assayed with 2 mM N-acetylglucosamine by measuring ADP production (method B), and glucose-phosphorylating activity was measured by method A (glucose 6-phosphate formation), with variable concentrations of glucose: l , 0.5; 0, 100; and 0,400 mM glucose. (A) supernatant fraction. (B) sediment fraction after treatment with Triton X-100 as indicated under Experimental procedures.

exploration for glucose-phosphorylating activity in all fractions at 100 and 0.5 mM concentrations of glucose again did not reveal the presence of either hexokinase C or D (not shown). In a few cases the chromatography was performed in the presence of 5% (v/v) glycerol as a protecting agent with the same results.

Filtration on Sephadex. Gel filtration on Sephadex was used for the determination of the molecular weight of the enzymes using marker proteins (20), as shown in Fig. 2. The molecular weight of hexokinase A and B was 98,000 t_ 1200 for six preparations (two of hexokinase A and four of hexokinase B). The filtration of N-acetylglucosamine kinase on Sephadex G-100, assayed with N-acetylglucosamine as sub-

AND NIEMEYER

strate, gave a single symmetrical peak with an elution coefficient corresponding to a molecular weight of 56,000 and 62,000 for two different preparations (Fig. 2).

Afinity chromatography on Sepharose- glucosamine. This procedure was applied to a fraction enriched in hexokinase B activity obtained after the Sephadex step of purification. This isozyme is retained at KC1 concentration not higher than 25 mM. When retained at 20 mM KC1 it was eluted in a major peak by the addition of 100 mM glucose (Fig. 3). Further increase to 50 mM KC1 permitted the recovery of a small fraction of glucose-phosphorylating activity that corresponded to the same enzyme as judged by kinetic properties. An increase of KC1 up to 300 mM did not elute any fraction that could correspond to small, un- detected contaminations with other glu- case-phosphorylating enzymes accompa-

.- - 2 5 10

I l-.

Molecular weight x 1O-4

FIG. 2. Molecular weight determination of hexokinases A and B and N-acetylglucosamine kinase. (A) Hexokinases: Filtration was performed in a Sephadex G-200 column (2 x 48 cm); sample size, 1 ml; fraction size, 1 ml; V,, 53 ml; r/,, 115.5 ml. (B) N-Acetylglu- cosamine kinase: Filtration was conducted in a Se- phadex G-100 column (3 X 63 cm); sample size, 1 ml; fraction size, 2 ml; V,, 152 ml; v, 444 ml. Marker proteins were 1, cytochrome c; 2, ovalbumin; 3, serum albumin; 4, yeast alcohol dehydrogenase. Blue Dextran 2000 and proteins were located by their absorbance at the appropriate wavelength. Alcohol dehydrogenase was assayed in the presence of 220 mM ethanol and 0.5 mM NAD. [‘%jLeucine was determined in a standard toluene scintillation mixture plus Triton X-100. Columns were equilibrated and eluted as described under Experimental Procedures.


Glc 100 yc1 20

i +

y 20 <Cl 50 KC1100 KCISW KCIZM

t 160 r 2.L

E I 5 ? E 120. i

Effluent volume. ml

FIG. 3. Affinity chromatography of hexokinase B on Sepharose-glucosamine. Hexokinase B (0.76 U) from a Sephadex G-100 column was applied to a column (1 X 4 cm) of Sepharose-glucosamine, which was equilibrated and washed with a buffer solution containing 25 mM Tris-HCl, pH 8.0, 15 mM KCI, 5 mM 2- ME, and 1 mM EDTA. The enzyme was eluted by including 100 mM glucose in the developing buffer (at the arrow). Increasing the concentration of KC1 to 50 mM eluted a small, retained portion of hexokinase B.

nying hexokinase B through the previous purification steps.

Polyacrylumide electrophoretic pattern. An illustrative pattern for rat liver hexokinase D (GK) and for N-acetylglucosamine kinase (NK) from intestinal mucosa is shown in Fig. 4a. The mobility relative to the tracking dye (R,) for both purified enzymes were very similar, 0.528 and 0.485, respectively. When a mixture of the enzymes was electrophoresed, a single, wider band was obtained with a R, value of 0.50. The pattern of an S1 fraction displaying three bands is shown in Fig. 4b. Hexokinase A corresponded to the upper band with less mobility, and hexokinase B to the mid- dle band. The lower band could be interpreted as corresponding to either hexokinase D or to N-acetylglucosamine kinase, since it was stained only when glucose at a high concentration (100 mM) was used. Although the mobility resembled that of N-acetylglucosamine kinase, the method did not permit a clear distinction between both enzymes. Lack of staining when ATP was omitted ruled out the possibility that some of the bands could correspond to glucose dehydrogenase. Incubation in a staining mixture at low glucose concentration

(0.5 mM) did not reveal the presence of glu- case-inhibited hexokinase C.

Electrophoresis on cellulose acetate membrane. With this procedure the supernatant fraction of intestinal mucosa again showed three bands with glucose-phosphorylating activity when 200 mM glucose was used for staining (Fig. 5). With 0.5 mM glucose as substrate the fast migrating band was no longer visible and no band that could be interpreted as hexokinase C appeared (Fig. 5a). No activity was visible in the absence of ATP (Fig. 5a). The fast band migrated at the same position as purified N-acetylglucosamine kinase and faster than purified hexokinase D in a parallel experiment (Fig. 5b). Furthermore, when purified liver hexokinase D was added to the intestinal mucosa extract, an intense extra band appeared, which migrated as hexokinase D (Fig. 5~).

Kinetic properties. The saturation functions for glucose of hexokinases A and B gave apparent K,,, values that were 0.025 and 0.174 mM, respectively. Both hexoki-

GK NK NK

G+K

a

S, NK S, -ATP

0

0

b FIG. 4. Polyacrylamide electrophoretic patterns of

intestinal mucosa extract, hexokinase D, and N-acetylglucosamine kinase. The gels shown were stained with 100 mM glucose. GK, highly purified rat liver hexokinase D. NK, N-acetylglucosamine kinase from rat intestinal mucosa at the Sephadex step of purification. S,, high-speed supernatant fluid from rat intestinal mucosa. Electrophoresis details as described under Experimental Procedures.


ST S1 NK Sj S1 NK NK GK S, NK S1 GK -ATP +GK

FIG. 5. Electrophoresis patterns on cellulose acetate membranes of intestinal mucosa extract, hexokinase D, and N-acetylglucosamine. S,, supernatant fluid after centrifuging the intestinal mucosa homogenate of a single rat at 105,OOOg. NK, intestinal N-acetylglucosamine kinase at the Sephadex step of purification. GK, highly purified rat liver hexokinase D. S, + GK, supernatant fluid from homogenate to which purified hexokinase D was added. S, - ATP, the same as S, with ATP omitted for staining. A 24 aliquot of sample was applied in each case. Experiments a and b correspond to a simultaneous run. The procedure followed was as indicated under Experimental Procedures. Staining was done with 200 mM glucose, with the exception of S:, which was stained in the presence of 0.5 mM glucose.

nases phosphorylate mannose, fructose, and 2-deoxyglucose, but do not act on N- acetylglucosamine, glucosamine, galactose, and pentoses (Table I). The saturation functions of N-acetylglucosamine kinase were hyperbolic for both N-acetylglucosamine (Fig. 6) and ATP (not shown), and the apparent Km values were 0.021 and 0.38 mM, respectively.

The saturation function of N-acetylglucosamine kinase for glucose was atypical, generally mimicing a negative cooperativity. In fact, the double-reciprocal plot showed a curve with a downwards con- cavity (Fig. 7), which does not allow the estimation of a Km value. It is worthwhile to point out that the use of a narrow range of glucose concentrations to study the saturation function would give misleading results. In the inset of Fig. 7 three subsets of experimental data were selected to draw straight lines by the least-squares method in the double-reciprocal graph. Depending on which set of points was considered, different values of Km for glucose could be estimated. Thus, glucose concentrations between 4 and 16 mM gave a Km value of 1 mM. For glucose concentrations between

20 and 67 mM, the K, would be about 20 mM, and for a range between 100 and 400 mM glucose the estimated Km would be about 280 mM. The possibility of multiple forms of the enzyme, as an explanation for these results, is not supported by our chromatographic results or from those of others (12, 13, 16, 27). N-Acetylglucosamine kinase seems to phosphorylate mannose, fructose, 2-deoxyglucose, and glucosamine with about the same low efficiency as glucose, when studied at a constant 100 mM concentration. The saturation functions for these substrates were not studied. NO phosphorylation of galactose, xylose, and arabinose was observed.

DISCUSSION

The chromatographic evidence reported above indicates that hexokinase activity from the mucosa of rat intestine is dis- tributed between the supernatant fraction and the sediment obtained by centrifugation at 105,OOOg, in agreement with previous reports (8-11). The relative proportion of the isozymes was somewhat variable, but B always predominated over A,


TABLE I

SUBSTRATE SPECIFICITYOFHEXOKINASES A AND B, ANDoFN-ACETYL-D-GLUCOSAMINE KINASE

FROMRATINTESTINALMUCOSA'

Substrate concentrations (mM)

Substrate

Hexo- Hexo- GlcNAc kinase kinase kinase

A B 10 100 10 100

D(+)-Glucose 1.00 1.00 0.03 0.04 GlcNAc 6 b 1.00 1.00 D(+)-Mannose 0.88 1.17 0.02 0.07 D(f)-Fructose 1.19 1.82" 0.02 0.03 2-Deoxyglucose 1.02 2.53 0.03 0.11 D(+)-Xyhe b 0.07 b b

L(+)-Arabinose b b b b

D(+)-Galactose b b b b

D(+)-Glucosamine b b - 0.04

a Partially purified enzymes were used and activities were measured by method B unless specified, using fixed concentrations of glucidic substrates (10 or 100 mM, as indicated) and ATP (5 mM). Relative activities of hexokinases and of N-acetylglucosamine kinase (GlcNAc kinase) refer to glucose and N-acetylglucosamine (GlcNAc), respectively.

b Relative activity < 0.01. ‘Fructose 6-phosphate formed was measured in a

coupled system with phosphoglucose isomerase and glucose-6-phosphate dehydrogenase (method A).

the latter being 10 to 20% of total glucose- phosphorylating activity in both supernatant fluid and sediment. The variability in the proportion of the hexokinases may be explained by the partial loss of hexokinase B during the procedure, due to its well-known instability. In the experiments on cellulose acetate membranes the band of hexokinase B was much more intense that that of hexokinase A (slowest migrating enzyme), which agrees with these results. However, in polyacrylamide gels isozyme B stained less than A, again sug- gesting a partial inactivation of hexokinase B.

Only hexokinases A and B were found in the intestinal mucosa. The careful search for hexokinase D during the chromatographic and electrophoretic analyses did not reveal the presence of this isozyme. However, a glucose-phosphorylating ac-

tivity with slightly lower chromatographic mobility than hexokinase D was identified as N-acetyl-D-glucosamine kinase. The electrophoretic migration on polyacrylamide gel was very similar for both enzymes, but it was quite different when the support was a membrane of cellulose acetate. These observations suggest that the reported high-K, glucose-phosphorylating activity of intestinal mucosa (10, 11) may correspond to N-acetylglucosamine kinase. Furthermore, using a monospecific rabbit antiserum against hexokinase D, we have also been unable to detect traces of this isozyme (C. Toro, X. Espinosa, and H. Nie- meyer, unpublished work). Our results agree with those of others who have identified as N-acetylglucosamine kinase the glucose-phosphorylating activity with high Km for glucose in intestinal mucosa (16) and other tissues (12,13,16). The evidence accumulated strongly suggests that hexokinase D is mainly in the liver, where it would accomplish the function of variable uptake of glucose consequent to changing concentrations in the portal vein. At least one of the multiple forms of “glucokinase” described in the liver indeed corresponds to N-acetylglucosamine kinase (13, 16). Very recently a hexokinase of high K, for glucose, similar to the liver isozyme D in several kinetic properties, has been identified in the pancreatic Islets of Langer- hans (25).

[N-Acetylglucosamlne] mM

FIG. 6. Hyperbolic saturation function of N-acetylglucosamine kinase with N-acetylglucosamine as substrate. The enzyme was at the Sephadex step of purification (3 mu; sp act 0.39 U/mg protein). Con- centration of MgATP was 5 mM. The apparent Km was 0.021 mM.


IO - 0

FIG. 7. Complex saturation function of N-acetylglucosamine kinase with glucose. The enzyme was at the Sephadex step of purification (90 mU; sp act 0.39 U/mg protein). Method B was used for assay. Con- centration of MgATP was 5 mM. Inset: Lines were drawn with three arbitrarily chosen subsets of experimental points considered as independent of each other. 0, Range of glucose concentrations, 4 to 16 mM. 0, Range of glucose concentrations, 20 to 67 mM. 0, Glucose concentrations ranging from 100 to 400 mM.

We have not recognized the presence of isozyme C, even in analytical preparations in which the mucosa extract of one single animal was examined by DEAE-cellulose or by various electrophoretic procedures. Our observations agree with some reports (3, 8) but not with others (9-11). The reasons for the discrepancy were not further investigated.

The K, values of hexokinases A and B were in the range reported for the isozymes from rat intestinal mucosa (8) and several other tissues (1,3,5,23,24). The substrate specificity was the same reported for hexokinases from all mammalian tissues so far investigated, i.e., they phosphorylate mannose, 2-deoxyglucose, and fructose, the latter with higher maximal velocity than glucose (1, 4-6, 15, 23, 24). In the case of hexokinase A the value for fructose phosphorylation was underestimated since the K, for this substrate is 3-5 mM (1, 3, 5, 23). As in other tissues, the intestinal isozymes did not act on N-acetylglucosamine, galactose and pentoses.

The saturation function of N-acetylglucosamine kinase for N-acetylglucosamine was hyperbolic and the Km value of 0.021

mM was similar to or slightly lower than those reported for the enzyme from other tissues (13, 26, 27). We have not observed the non-Michaelian kinetics reported for rat liver and kidney enzymes (27). Since the Km value is so small, it is important to be sure that linear activities versus time at the lowest activities be recorded, oth- erwise untrue cooperativity may be observed. The atypical saturation function of N-acetylglucosamine kinase with glucose (Fig. 7) has been also observed in this laboratory in semipurified enzymes from spleen, human placenta, and rat kidney (12) and in homogeneous N-acetylglucosamine kinase from bovine spleen (28). In con- tradistinction, work from another laboratory has shown hyperbolic saturation functions with glucose of N-acetylglucosamine kinases from rat liver, kidney, and intestinal mucosa, with Km values of 600, 400, and 200 mM, respectively (16,27). The discrepancy may lie in the range of concentrations used.

N-Acetylglucosamine kinase appears to phosphorylate several hexoses and deriv- atives, including glucosamine, with about the same low efficiency at subsaturating concentrations of substrates (10 and 100 mM). Our experience is similar to that of others for enzymes from different tissues (16, 27).

ACKNOWLEDGMENT

We wish to thank Dr. Tito Ureta for kindly re- viewing the manuscript and for helpful discussions.

REFERENCES

1. GONZ~~LEZ, C., URETA, T., SANCHEZ, R., AND NIE- MEYER, H. (1964) B&hem. Biophys. Res. Cmn- mun 16, 347-352.

2. KATZEN, H. M., SODERMAN, D. D., AND NITOWSKY, H. M. (1965) Biochem. Biophya Res. Commun.

19, 377-382. 3. KATZEN, H. M., AND SCHIMKE, R. T. (1965) Proc.

Natl. Acad. Sci. USA 54, 1218-1225. 4. GROSSBARD, L., WEKSLER, M., AND SCHIMKE,

R. T. (1966) Biochem. Biophys. Res. Commun 24, 32-38.

5. GROSSBARD, L., AND SCHIMKE, R. T. (1966) J. Biol. Chem. 241, 3546-3560.


6. GONZALEZ, C., URETA, T., BABUL, J., RABAJILLE, 17. VERA, M. L., CARDENAS, M. L., ANI) NIF:MEYER, II.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

E., AND NIEMEYER, H. (1967) Biochemistry 6, 460-468.

STIFEL, F. B., ROSENSWEIG, N. S., ZAKIM, D., AND HERMAN, R. H. (1968) Biochim. Biophys. Acta 170, 221-227.

SRIVASTAVA, L. M., SHAKESPEARE, P., AND H~BSCHER, G. (1968) B&hem. J. 199,35-42.

WEISER, M. M., QUILL, H., AND ISSELBACHER, K. J. (1971) J. Biol Chem. 246, 2331-2337.

ANDERSON, J. W., AND TYRREL, J. B. (1973) Gas- troenterology 65, 69-76.

ANDERSON, J. W., AND KING, P. (1975) B&hem. Med. 12, l-11.

DAVAGNINO, J., AND URETA, T. (1978) Arch. Biol. Med. Exp 11,12.

DAVAGNINO, J., AND URETA, T. (1980) J. Biol CXem 255, 2633-2636.

VIRUELA, E., SALAS, M., AND SOLS, A. (1963) J. Biol Chews. 238, 1175-1177.

NIEMEYER, H., AND URETA, T. (1972) in Molecular Basis of Biological Activity (Gaede, K., Ho- reeker, B., and Whelan, W., eds.), pp. 221-272, Academic Press, New York.

ALLEN, M. B., BROCKELBANK, J. L., AND WALKER, D. G. (1980) Biochim Biophys. Acta 614, 35% 366.

(1978) Arch. Biol. Med. Ex71. 11, R-10. 18. CARDENAS, M. L., RABAJILLE, E., AND NIEMEYER,

H. (1978) Arch. Biochem. Biophys. 190,142-148. 19. KORNRERG, A., AND PRICER, W. E. (1951) J. Bid.

Chem 193, 481-495. 20. ANDREW& P. (1964) Biochem. J. 91, 223-233. 21. GROSSMAN, S. H., AND POTTER, V. R. (1974) Anul.

Biochem. 59, 54-62. 22. SATO, S., MATSUSHIMA, T., AND SUGIMURA, T. (1969)

Cancer Res. 29, 1437-1446. 23. URETA, T., GONZ~~LEZ, C., AND NIEMEYER, H. (1971)

Camp. B&hem. Physiol. B 40, 81-91.

24. URETA, T., RADOJKOVI~, J., ZEPEDA, S., AND Gurxk, V. (1981) C<m~p B&hem. Physiol. B 70, 225- 236.

25. MEGLASSON, M. D., BURCH, P. T., BERNEK, D. K. NAJAFI, H., V~GIN, A. P., AND MATSCHINSKY, F. M. (1983) Proc. Natl. Acad. Sci. USA 80,85- 89.

26. GINDZIE~~SKI, A., GLOWACKA, D., AND ZWIERZ, K. (1974) Eur. J Biochem. 43, 155-160.

27. ALLEN, M. B., AND WALKER, D. G. (1980) Biochem. J. 185, 577-582.

28. DAVAGNINO, J. (1979) Thesis. Faculty of Sciences, University of Chile.

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