THE .JOURNAL OF BIOLOGICAL CHEMISTRY VoI. 258, No. 17. Issue of September 10, pp. 1Oi74-10778,1983 Printed in U . S . A.

Co-purification and Characterization of UDP-glucose 4-Epimerase and UDP-N-acetylglucosamine 4-Epimerase from Porcine Submaxillary Glands*

(Received for publication, May 16, 1983)

Friedrich PillerS, Mary H. Hanlon, and Robert L. Hill From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

UDP-glucose 4-epimerase and UDP-N-acetylgluco- samine 4-epimerase have been co-purified about 9,000-fold from porcine submaxillary glands by affin- ity chromatography on UDP-hexanolamine-agarose. The homogeneous epimerase has apparent M, = 88,000 and contains two subunit species with apparent M, = 37,000 and 35,000, respectively. The two subunits, however, are indistinguishable as judged by peptide mapping.

The purified enzyme catalyzes equally well the re- versible reactions UDP-glucose + UDP-galactose and UDP-N-acetylglucosamine + UDP-N-acetylgalac- tosamine. At saturating substrate concentrations, the ratio of the rate of the former reaction to that of the latter is 1.13 in the forward direction and 0.44 in the backward direction. Both reactions have the same K , = 0.38 and the same dependence on pH. Moreover, both activities are lost at about the same rate by heat dena- turation of the epimerase or reaction with N-ethyl- maleimide. Kinetic analysis reveals that the reactants for one reaction are competitive inhibitors of the other reaction, with the Ki values of the inhibitors essentially identical with their K,,, values as substrates. Taken together, these studies suggest that UDP-glucose 4- epimerase and UDP-N-acetylglucosamine 4-epimerase activities reside in a single enzyme.

Galactose and N-acetylgalactosamine, two monosaccha- rides commonly found in various glycoconjugates, are incor- porated into oligosaccharide groups of glycoconjugates by the action of specific glycosyltransferases that utilize UDP-ga- lactose or UDP-N-acetylgalactosamine as donor substrates (1). UDP-galactose and UDP-N-acetylgalactosamine are syn- thesized by enzymes that catalyze epimerization a t carbon 4 of UDP-glucose and UDP-N-acetylglucosamine, respectively (2). Thus, UDP-glucose 4-epimerase (EC 5.1.3.2) catalyzes Reaction 1.

UDP-Glc e UDP-Gal (1)

The enzyme requires NAD+ as an essential cofactor, and is thought to act by formation of a 4-keto intermediate with

* This work was supported by Grant GM-25766 from the National Institute of General Medical Science, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Supported by the Max Kade Fund of the Austrian National Science Foundation, New York. Present address, Centre National de Transfusion Sanguine, Paris, France.

concomitant reduction of NAD+ to NADH, followed by re- duction of the intermediate to the epimeric species and reox- idation of NADH to NAD’ (3). UDP-N-acetylglucosamine 4- epimerase (EC 5.1.3.7), which catalyzes Reaction 2,

UDP-GlcNAc $ UDP-GalNAc (2)

has not been as thoroughly studied as UDP-glucose 4-epimer- ase, but is thought to act by a similar mechanism.

The UDP-glucose 4-epimerases from Escherichia coli and yeast (4,5) have been highly purified and have been found to be devoid of UDP-N-acetylglucosamine 4-epimerase activity. Similarly, partial separation of the two activities in Bacillus subtilus has been reported (6). Many mammalian tissues contain both epimerase activities (7-11); however, separation of the two activities has not been reported. Indeed, a partially purified UDP-glucose 4-epimerase from rat liver, the most thoroughly studied mammalian epimerase, catalyzes Reaction 2 as well as Reaction 1 (12).

Characterization of the UDP-N-acetylglucosamine 4-epi- merase has been hindered, in part, by the lack of a simple, rapid, and sensitive assay of its activity. With the recent development of such an assay ( l l ) , i t has been possible to study both epimerases and examine their properties. Thus, we report here that both epimerase activities from porcine submaxillary glands appear to reside in a single enzyme, and catalyze equally well Reactions 1 and 2.

EXPERIMENTAL PROCEDURES

Materials

Porcine submaxillary glands were obtained at a local slaughter- house, chilled on ice, and used immediately for preparation of the epimerase. All 14C-labeled compounds were from New England Nu- clear. UDP-Glc, UDP-Gal, UDP-GlcNAc, hexanediamine, N-ethyl- maleimide, UDP, NAD+, NADP+, a-methyl-D-galactoside, and V8 protease (Staphylococcus a u r e u ) were from Sigma. UDP-GalNAc (11) and UDP-hexanolamine-Sepharose 4B (13) were prepared as described earlier. Lectin I from Griffonia simplicifolia seeds (Calbi- ochem-Behring) was prepared by published methods (14). Lectin I and hexanolamine were coupled to CNBr-activated Sepharose 4B as described earlier for preparation of other adsorbents (15). The result- ing adsorbents contained the following amounts of ligand/ml of packed adsorbent: 3 mg of lectin I, 17 pmol of hexanediamine, and 14 pmol of UDP-hexanolamine. The buffers employed had the follow- ing compositions: Buffer A, 50 mM Tris-HC1, pH 7.6, 25 mM potas- sium chloride, 1 mM EDTA, and 350 mM sucrose; Buffer B, 50 mM Tris-HC1, pH 7.6, 25 mM potassium chloride, 1 mM EDTA, and 10 mM magnesium chloride; Buffer C, 50 mM Tris-HC1, pH 7.6, 25 mM potassium chloride, 1 mM EDTA, 10 mM magnesium chloride, 0.1% Triton X-100, and 0.05 mM NAD+; Buffer D, 10 mM sodium dihydro- gen phosphate, pH 7.2, 150 mM sodium chloride, 0.1 mM magnesium chloride, 0.1 mM calcium chloride, and 0.1 mM manganese chloride; Buffer E, 100 mM Tris-HC1, pH 8.5, 350 mM sucrose, 0.1 mM NAD’, and 1 mg/ml of bovine serum albumin (Sigma).

10774

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UDP-glucose and UDP-N-acetylglucosamine 4-Epimerase 10775

Enzyme Assays UDP-Glc and UDP-GlcNAc 4-epimerase activities were measured

by modification of the method previously described (11). A reaction mixture (30 pl) containing 3 pmol of Tris-HC1, pH 8.5,O.OOl pmol of NAD+, 0.003 pmol of either UDP-['4C]Glc, UDP-["CIGal, UDP-["C] GalNAc, or UDP-['4C]GlcNAc (-10,000 cpm/nmol), and up to 20 microunits of epimerase was incubated at 37 "C for 15 min. Ice-cold Buffer D (75 pl) containing 1 mM UDP was added to stop the reaction, and the mixture was applied to a column (0.5 X 5 cm) of lectin I- Sepharose adsorbent. The column was washed with Buffer D (1 ml) and the eluate discarded. The column was then washed with Buffer D (2 ml), the eluate containing either UDP-[14C]Glc or UDP-["C] GlcNAc was collected in a scintillation vial and mixed with 2.5 ml of scintillation fluid (Aquasol), and the radioactivity was counted. UDP- Gal or UDP-GalNAc is then specifically eluted from the column with 25 mM a-methyl-o-galactoside (0.2 ml) followed by 1.8 ml of Buffer D, and the eluate was collected and counted as above. One unit of epimerase activity is defined as the amount of enzyme that catalyzes formation of 1 pmol of product/min under the conditions of the assay. Activity may be calculated from the decrease in substrate concentra- tions or from the increase in product concentrations. The latter measurement is more accurate, but both usually give essentially the same activity values. The lectin-agarose columns are regenerated by washing with 5 ml of Buffer D. The elution profiles of columns that

judged by calibration with UDP-[14C]GlcNAc and UDP-['4C]Gal. had been used routinely remained unchanged for over 6 months as

Protein concentrations were determined by the Amido schwarz method (16).

Lactose synthase (17), A+ blood group N-acetylgalactosaminyl- transferase (18), and polypeptide N-acetylgalactosaminyltransferase (19) activities were assayed by published methods. UDP-GlcNAc dehydrogenase, UDP-Glc dehydrogenase, and UDP-GlcNAc 2-epi- merase activities were assayed in reaction mixtures (100 yl) contain-

of UDP-[14C]Glc or UDP-["CIGlcNAc (-20,000 cpm/nmol), 5 pmol ing submaxillary gland extract (50 pl, 3 pg of protein/ml), 0.006 pmol

of Tris-HC1, pH 7.5, 1.25 pmol of potassium chloride, 0.05 pmol of EDTA, 1 pmol of magnesium chloride, and 0.01 pmol of NAD+. For the dehydrogenase assays, the reaction mixtures were applied to columns (0.5 X 3 cm) of Dowex 1x2 (C1- cycle) and the nucleotide sugars were recovered in the eluate hydrolyzed in 1 N HCl for 30 min at 100 "C in sealed tubes. After removal of the HCl, the monosac- charides in the hydrolysate were identified by paper chromatography (Whatman No. 3") in ethyl acetate:pyridine:acetic acid:water (5:5:1:3, v/v). For the UDP-GlcNAc 2-epimerase assay, the reaction mixture was directly analyzed by paper chromatography in 1 M ammonium acetate, pH 3.8, 95% ethanol ( 2 5 , v/v) to detect the free N-acetylmannosamine produced. UDP-G1c:galactose-1-phosphate uridyltransferase activity was assayed according to the published procedure (20).

Purification of the Epimerase Step I : Submaxillary Gland Extract (21)"All procedures were

performed at 4 "C unless otherwise noted. Fresh glands (500 g) were trimmed of fat and connective tissue and homogenized in Buffer A (750 ml) in a Waring blender (4 X 30 s at highest speed). The homogenate was stirred for 3 h at 0 "C and centrifuged for 100 min at 35,000 X g, and the supernatant was separated from the fatty layer and then lyophilized. The resulting powder was stored at -20 "C for over a year with no loss in epimerase activity. The powder ( 5 g) was suspended in Buffer B (200 ml) containing 0.03 mmol of NAD+, and then centrifuged for 30 min at 25,000 X g.

Step 2: Hexanediamine-Sepharose Adsorption-Sufficient Triton X-100 was added to the solution from Step 1 to give a final concen- tration of 0.5%, and the solutions was then stirred with hexanedi- amine-Sepharose 4B (100 ml, settled volume) suspended in a total volume of 400 ml of Buffer B. The suspension was gently shaken by rotation (16 h), and the hexanediamine-Sepharose was removed by filtration on a sintered glass funnel. The Sepharose was washed on the filter with Buffer C (three 100-ml washes), poured into a column (2.6 X 19 cm) with the aid of Buffer C, and washed with 3 volumes of Buffer C. The epimerase was eluted from the column with a linear gradient made of 4 volumes each of Buffer C and Buffer C containing 1 M sodium chloride at a flow rate of 25 ml/h. Fractions (5 ml) of the eluate containing the enzyme were pooled, made 0.05 mM in NAD+, and dialyzed against Buffer B (20 volumes) for 4 to 6 h.

step 3: Affinity Chromatography on UDP-hexanolamine-Sepha- rose-The solution from Step 2 was applied to a column (0.8 X 6 cm)

of UDP-hexanolamine-Sepharose at a flow rate of 15 ml/h, and the column was then eluted successively with 4 volumes of Buffer C, 3 volumes of Buffer C containing 1 M NaCl, and 3 volumes of Buffer B containing 0.05 mM NAD'. The epimerase was eluted at a flow rate of 1 ml/h with a linear gradient formed with 4 volumes of Buffer B containing 0.1 mM NAD+ and 4 volumes of the same buffer containing 200 p~ UDP-GlcNAc.

RESULTS

Purifzcation of the Epimerase-Table I summarizes the purification of the epimerase. The preparation of a lyophilized extract of the glands (Step 1) aids considerably in the purifi- cation. First, considerable amounts of insoluble matter that may interfere with subsequent steps are removed. In addition, the two epimerase activities in extracts of the powder are as high as those in extracts of the gland. However, several other activities that utilize UDP-Glc, UDP-Gal, UDP-GlcNAc, or UDP-GalNAc as substrates are absent in extracts of the powder, including lactose synthase (17), A+ blood group N - acetylgalactosaminyltransferase (18), polypeptide N-acetyl- galactosaminyltransferase (19), UDP-Glc dehydrogenase, UDP-GlcNAc dehydrogenase, and UDP-GlcNAc 2-epimer- ase. UDP-G1c:galactose-1-phosphate uridyltransferase (20) was present in the extract of the powder, but was absent after Step 2.

Adsorption to and elution of the epimerase from hexane- diamine-Sepharose did not give much purification of the epimerase. This step was required, however, for effective purification of the enzyme on UDP-hexanolamine-Sepharose in Step 3. Attempts to directly purify the enzymes in extracts of the powder by affinity chromatography required more UDP-hexanolamine-Sepharose adsorbent than that needed in Step 3 and gave epimerase in low yields (3 t o 5%).

Fig. 1 shows the elution profile of the protein and the epimerase activity at Step 3. The vast majority of the protein emerges unretarded as the solution from Step 2 is applied and the column washed with buffer. Although the epimerase is not eluted by 1 M sodium chloride, it will not bind the affinity adsorbent at sodium chloride concentrations above 0.1 M. It was also found that Triton X-100 enhances the binding of the epimerase to the affinity adsorbent as well as to hexanedi- amine-Sepharose. The epimerase, however, is irreversibly in- activated in Triton X-100 solutions within hours unless NAD+ (0.05 mM) is present to stabilize the enzyme. UDP, as well as substrates, also stabilize the activity. Neither Triton X-100 nor NAD' affect the elution of the epimerase from the affinity adsorbent, and the elution profile of the enzyme activity is identical if either UDP or UDP-Glc instead of UDP-GlcNAc is used as a specific eluting agent. In a 0 to 200 p M linear gradient of these eluting agents, the epimerase emerges as a sharp peak at about 20 h M eluting agent.

As shown in Table I, the ratio of the UDP-glucose 4- epimerase activity to the UDP-N-acetylglucosamine 4-epi- merase activity was 1.6 at each of the three steps of the purification procedure. Both activities were purified about 1300-fold, although this may be a minimum value since the Amido schwarz method used for determining protein concen- tration is insensitive to mucin, a major protein contaminant of Step 1. Because most methods for determining protein concentration do not accurately measure mucin concentra- tions, dry weight measurements on the protein content of the solution obtained at Step 1 suggest that the overall purifica- tion of the epimerase activity is likely nearer 9000-fold, or about seven times greater than observed (Table I).

The enzyme from Step 3 was stored in 50% aqueous glycerol containing 0.05 mM NAD' and either 0.01 mM UDP-Glc or UDP-GlcNAc at -20 "C. No loss of activity was observed

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10776 UDP-glucose and UDP-N-acetylglucosamine 4-Epimerase

TABLE I Purification of the UDP-glucose 4-epimerase and UDP- N-acetyighcosamine 4-epimerase octioities from porcine

submaxillarv glands

Step

Soecific activitv Total Total Ratio of

Volume protein activity UDP-Clc C I ~ N A ~ ities UDP- two activ- Purification epimerase epimerase

ml mR units % units/mg -fold

1. Extract of lyophilized powder 200 540 15.6 100 0.048 0.029 1.65 1 2. Adsorption on hexanediamine-sepha- 45 72 10.1 64.7 0.224 0.14 1.60 4.8

3. Affinity chromatography on UDP-hex- 2 0.1 1 4.1 26.3 59.7 37.3 1.60 1286 rose

anolamine-Sepharose

1

0.4 jrr( 4

ELUTION VOLUME I mi)

FIG. 1 . Affinity chromatography of the epimerase activities on UDP-hexanolamine-Sepharose (Step 3). The dialyzed eluate from hexanediamine-Sepharose 4B was applied to a column (0.8 X 6 cm) of UDP-hexanolamine-Sepharose 4B as described under "Exper- imental Procedures." At arrow A, the column was washed with 1 M NaCl in Buffer C; at orrow R, the column was washed with Buffer C without Triton X-100; and at arrow C, the UDP-GlcNAc gradient was started. Fractions were monitored for protein (0) and UDP- GlcNAc 4-epimerase (0) activity.

under these storage conditions for up to 5 months. Purity of the Epimerase-The epimerase (Step 3) on elec-

trophoresis in polyacrylamide gels (12%) in the presence of sodium dodecyl sulfate and e-mercaptoethanol (22) gave the pattern shown in Fig. 2. More than 95% of the protein was present in two species with apparent M , = 35,000 and 37,000, respectively. In the absence of mercaptoethanol, two species with slightly lower molecular weights were observed (Fig. 2). Both species appear to have similar stmctures as judged by gel electrophoresis of V8 protease digests (23, 24) of each separate species or both species together (data not shown). This result is consistent with other data given below that indicate that the two species are contained in one enzyme with both epimerase activities.

The molecular weight and Stokes radius of the native enzyme (Step 3) were estimated by gel filtration on Sephadex G-200 (superfine) as shown in Fig. 3. Both epimerase activities emerged as a single peak coincident with the protein and corresponde4 to a species with M , = 88,000 and a Stokes radius of 35 A.

Enzymatic Properties of the Epimerase-The activity of both epimerase reactions was essentially identical between pH 6 and 11, with a sharp optimum at pH 8.5 (Fig. 44). Moreover, both activities showed essentially the same rates of inactivation by heat denaturation a t 46 "C (Fig. 4B) or by reaction with N-ethylmaleimide a t 25 "C (Fig. 4C). NAD+ stabilizes the enzyme to heat denaturation, since about 5% of each activity remained in the absence of NAD+ under the same conditions as in Fig. 4B that gave only 50% loss of

" 7 7 , 0 0 0

" 68.0 o o

0 - 45,000

- 2 4,O 0 0

-

1 2 3 FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electro-

phoresis of the purified epimerase activities. Samples were made 50% in acetone and allowed to stand 12 to 14 h at -20 "C, the resulting precipitate was collected by centrifugation at 27,000 X g for 30 min, and the supernatant was discarded. The air-dried precipitate was redissolved in sample buffer for 5 min at 100 "C. Lane I , plus 2% p-mercaptoethanol. Lane 2, no P-mercaptoethanol. Electrophoresis was performed in 12% polyacrylamide gels as described earlier (22). and the gels were stained with Coomassie blue. Lane 3, molecular weight standards were human transferrin, bovine serum albumin, ovalbumin, and trypsinogen.

activity. NADP+, however, was totally ineffective as a cofactor and both epimerase activities showed an absolute requirement for NAD+ as cofactor. A variety of metals, cations, and anions did not enhance either activity, including Mn", Mg'+, Co2+, Zn", Fe2+, Sn2+, Cuz+, Cd", H P , NH:, Na+, K', C1-, SO:-, HPO:-, and acetate.

Table I1 lists the kinetic parameters for the two activities of the epimerase. The K, values estimated from double recip- rocal plots for UDP-Glc, UDP-Gal, and UDP-GlcNAc are nearly identical and about one-half the value of K,,, for UDP- GalNAc. The Vmax values for UDP-Glc and UDP-GlcNAc are nearly identical and about 2 to 5 times lower than the Vmex values for UDP-Gal and UDP-GalNAc. These kinetic param- eters were measured a t initial concentrations of substrates under conditions such that the formation of product was negligible. Thus, at saturating substrate concentrations, ini- tial velocity measurements revealed that the ratio of the two epimerase activities with UDP-Glc and UDP-GlcNAc as sub-

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UDP-glucose and UDP-N-acetylglucosamine 4-Epimerase 10777

TABLE I1 Kinetic parameters of the epimerase activities

FRACTION NUMBER

FIG. 3. Chromatography of the purified epimerase on Seph- adex G-200. The epimerase (0.6 ml, 0.1 mg of protein) was applied to a column (1.6 X 40 cm) of Sephadex G-200 (superfine) equilibrated in 50 mM Tris-HC1, pH 7.5, containing 150 mM NaCl and 0.05 mM NAD+. The flow rate was 1.32 ml/h, and fractions (0.825 ml) were monitored for protein (A) and UDP-Glc (0) and UDP-GlcNAc 4- epimerase (0) activities.

3 Lu o l d , , , , , - x , + ( , ( 4 0 6 7 8 9 IO I 1 5 10 15 IO 20 30

pH TIME 1 mm) TIME (nun)

FIG. 4. The effects of pH, heat, and N-ethylmaleimide on epimerase activity. A , the effect of pH on epimerase activities. The purified epimerase (15 pg/ml) in 25 mM Tris-HC1, pH 7.5, 25 mM potassium chloride, 5 mM magnesium chloride, 0.05 M NAD', and 0.1 mg of bovine serum albumin was diluted 1:80 in buffer solutions of varying pH. Tris maleate (0.1 M) buffers were used for pH 6.0 to 8.5 and glycine-NAOH buffers were used for pH 8.5 to 11.0; each buffer contained 0.1 mM NAD+. Aliquots (20 p l ) of the buffered enzyme solutions were added to solutions (10 p l ) of 0.3 mM UDP-Glc (0) or 0.3 mM UDP-GlcNAc (O), and their activity was measured as in the standard assay ("Experimental Procedures") after 15 min at 37 "C. B, thermal denaturation of the epimerase activities. The purified epimerase (0.06 unit/ml) in 50 mM Tris-HC1, pH 7.5, containing 25 mM potassium chloride, 5 mM magnesium chloride, 0.1 mM NAD', and 1 mg/ml of bovine serum albumin was incubated at 46 "C, and the activity of aliquots was measured with time by the standard assay ("Experimental Procedures") with UDP-Glc (0) and UDP-GlcNAc (0) as substrates. C, inactivation of the purified epimerase with N - ethylmaleimide. The epimerase (3.75 pg/ml) in 5 mM Tris maleate, pH 7.0, containing 5 p~ NAD+ was reacted with 2.5 p~ N-ethylmal- eimide at 25 "C. Aliquots were removed with time and diluted 1:25 in Buffer E at 0 "C, and the activity was measured with time in the standard assay ("Experimental Procedures") with UDP-Glc (0) and UDP-GlcNAc (0) as substrates.

strates was 1.13. In contrast, with UDP-Gal or UDP-GalNAc as substrates, the ratio of the two activities was 0.44. In the standard assays ("Experimental Procedures"), the back reac- tion due to product formation is not negligible and the ratio of the UDP-Glc to UDP-GlcNAc epimerase activities is 1.6.

Both epimerase reactions were found by direct measure- ment to give a K,, = 0.38, a value in close accord with those calculated from the Haldane equation relating V , and K,,, to Keq (25). By this means, the UDP-GlcNAc epimerase reaction was established to have a Keg = 0.37 and the UPD-Glc epimerase reaction to have a Keg = 0.41.

Fig. 5 shows graphical analysis of the inhibition of one epimerase activity by reactants for the other epimerase activ- ity. Each reactant for one activity inhibited the other activity competitively, and the calculated K, values (Table 11) were

I Substrates Kinetic parameter

UDP-Glc I UDP-Gal 1 $gic I K , (microunits)"

31 f 5 22.5 f 3.5 26 f 3 20% 4 Kt (pMIb 714 f 52.5 117 k 2.4 315 f 25.2 132 % 2.8 V,,,(pmol/min/mg)" 36 f 5.1 16 f 3.4 20 2 1.8 20 f 0.71

a Mean values from four separate experiments. Mean values from two separate experiments at UDP concentra-

tions from 5 to 60 p~ and substrate concentrations of 25 and 50 p ~ .

I O C + /

I I I I I I I I I I I I I I I I -60-40-200 20 40 60 -60-40-200 20 40 60

INHIBITOR CONCENTRATION [ p M ]

FIG. 5. Inhibition of one epimerase activity by reactants of the other epimerase. Purified epimerase (10 pl, 0.0375 pglml) in Buffer E was mixed with 10 pl each of substrate and inhibitor, and the activity was measured as in the standard assay after incubation for 6 min at 37 "C. In A and B, UDP-Glc a t 0.025 and 0.05 m M was the substrate, and in C and D, UDP-GlcNAc at the same concentra- tions was the substrate. The inhibitors were UDP-GlcNAc ( A ) , UDP- GalNAc ( B ) , UDP-Glc (C), and UDP-Gal ( D l .

close to the corresponding K, values for the reactants. UDP was also a competitive inhibitor with Kt = 26 pM for both epimerase activities (data not shown). In contrast, neither glucose, galactose, N-acetylglucosamine, nor N-acetylgalac- tosamine inhibited either reaction at concentrations up to 50 mM .

Kinetic analysis (data not shown) also revealed that NAD+ had a K,,, = 0.9 to 1.5 p~ with all four substrates involved in the two epimerase reactions.

DISCUSSION

The following observations reported here indicate that UDP-glucose 4-epimerase and UDP-N-acetylglucosamine 4- epimerase are very likely the same enzyme in porcine tissues. 1) The two epimerase activities co-purify from 1300- to 9000- fold (Table I). Thus, the ratio of the UDP-g1ucose:UDP-N- acetylglucosamine activities in each of the three steps is 1.6:l. 2) The activities were not separated from one another on elution from the UDP-hexanolamine affinity adsorbent by specific elution with either UDP-glucose, UDP-N-acetylglu- cosamine, or UDP. 3) Although two proteins with slightly different molecular weights were observed on gel electropho- retic analysis of the enzyme (Fig. a) , peptide maps of each were indistinguishable. 4) Both epimerase activities eluted in exactly the same position on gel filtration. 5 ) The activity profile from pH 6 to 11 as well as the rates of inactivation by heat and N-ethylmaleimide were identical for both activities, and NAD' or substrates protected against inactivation to the same extent. 6) Each activity was totally dependent on NAD+ as a cofactor, with NADP+ and several anions and cations incapable of supporting either activity. 7) The reactants for one epimerase activity were competitive inhibitors of the

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10778 UDP-glucose and UDP-N-acetylglucosamine 4-Epimerase

reaction for the other activity, and gave K, values in close accord with their K,,, values as substrates. Moreover, UDP was also a competitive inhibitor with a K, = 26 p~ for both reactions, in contrast to glucose, galactose, N-acetylglucosa- mine, and N-acetylgalactosamine, which were not inhibitors at concentrations up to 50 mM. Although no one of these observations conclusively indicates that the two epimerase activities are catalyzed by an identical enzyme, together they strongly support this conclusion.

In contrast to the mammalian epimerase studied here, those from E. coli (4) and yeast (5) apparently cannot catalyze both reactions, and separate enzymes are needed for the two epi- merase reactions. Partially purified UDP-glucose 4-epimerase from two other mammalian sources (7-11) that were studied earlier were not assayed to determine whether UDP-N-ace- tylglucosamine is a substrate.

There is some similarity between the structural properties of the porcine and the bacterial and yeast epimerases. The UDP-glucose 4-epimerase from E. coli and yeast are both dimers of identical subunits with a native M, = 80,000 and 125,000, respectively. The porcine enzyme also appears to have structurally similar subunits with M , - 88,000. The reasons for the slight differences in molecular weight of the two subunits of the epimerase studied here are unknown, since they appear to give indistinguishable peptide maps. Moreover, the sum of their molecular weights (72,000) is somewhat lower than that expected based on the observed M , = 88,000 obtained by gel filtration. Perhaps more accurate molecular weight estimates by other methods would clarify this discrepancy.

Finally, as reported earlier (11), the availability of partially or highly purified epimerase described here, along with meth- ods for separating UDP-N-acetylglucosamine from UDP-N-

1.

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20. 21.

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69.536-544 acetylgalactosamine, Offers a Simple quick means for prepar- 22. Laemmli, U. K. (1970) Nature (Lo&.) 227, 680-685 ing UDP-N-acetylgalactosamine, which is not easily obtained 23. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, commercially. U. K. (1977) J. Biol. Chem. 2 5 2 , 1102-1106

24. Morrissey, J . H. (1981) Anal. Biochem. 1 1 7 , 307-310 Acknowledgment-We thank Mark A. Lehrman for his help in 25. Segal, I. H. (1975) in Enzyme Kinetics pp. 35, Wiley-Interscience,

performing the peptide maps of the epimerase. New York

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F Piller, M H Hanlon and R L HillUDP-N-acetylglucosamine 4-epimerase from porcine submaxillary glands.

Co-purification and characterization of UDP-glucose 4-epimerase and

1983, 258:10774-10778.J. Biol. Chem. 

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