The localization of galactosyltransferases in polyacrylamide gels by a coupled enzyme assay

ANALYTICAL BIOCHEMISTRY 102. 441-449 (1980)

The Localization of Gaiactosyltransferases in Polyacrylamide Gels by a Coupled Enzyme Assay

MICHAEL PIERCE, RICHARD D. CUMMINGS, AND STEPHEN ROTH

Departmenf of Biology, The McCollum-Pratt Institute. The Johns Hopkins University, Baltimore, Maryland 21218

Received August 9, 1979

A coupled enzyme assay for GlcNAc’:UDP-galactose galactosyltransferase has been developed that allows this enzyme to be assayed spectrophotometrically and in nondenaturing polyacrylamide gels. Utilizing three, intermediate enzymes, galactosyltransferase activity has been coupled to the production of NADH with a stoichiometry of 2 mol of NADH produced for each mol of galactose transferred to GlcNAc. The enzyme reactions coupled to the production of UDP by galactolyl- transferase can be summarized as follows:

ATP Glc- I-PO, 2NAD UDP - UTP - UDPGlc - UDPGA + ZNADH.

The activities of partly purified bovine milk galactosyltransferase and galactosyltransferase in dialyzed fetal calf serum have been determined spectrophotometrically by measuring NADH production at 340 nm. The reaction is dependent on N-acetylglucosamine, UDP-galactose, and Mn2+. For both enzyme sources, activities calculated from NADH production are similar to those determined from assays that use radioactive sugar nucleotide substrates. Both galactosyltransferase activities have been localized on 7.5% nondenaturing polyacrylamide gels after electrophoresis by incubating the gel with an agarose indicator gel containing the coupled enzyme system. Enzyme activity is marked by NADH fluorescence, which is dependent on the presence of N-acetylglucosamine in the indicator gel. The intensity of fluorescence increases with increasing galactosyltransferase activity applied to the gel.

The glycosyltransferases catalyze the transfer of sugars from sugar nucleotide donors to glycoprotein, glycolipid, and polysaccharide acceptors, as well as to simple sugars (1). Soluble forms of these enzymes have been found in milk and serum (2,3) and membrane-bound forms have been localized to Golgi, endoplasmic reticulum, and plasma membranes (4-6). In addition, glycosyltransferases present on the surfaces of some cell types may be involved in cell- cell or cell-substrate recognition (7).

In almost all cases, glycosyltransferase activities are measured by incubating a

* Abbreviations used: GlcNAc, N-acetylglucosamine- (2-acetamido-2-deoxy-D-glucose); PPO, 2,5-diphenyloxazole; PGPOP, 1,4-bis[2-(5phenyloxazolyl)]benzene; INT, p-iodonitrotetrazolium.

sugar nucleotide, radiolabeled in the sugar moiety, with enzyme, cation, if necessary, and acceptor. The labeled, glycosylated acceptor is then separated from the unused substate and its breakdown products. Previously, if any of these enzymes were subjected to nondenaturing, polyacrylamide gel electrophoresis, enzyme activity could be detected only by slicing the gel, eluting the protein, and analyzing each fraction by assay with radioactive substrate. This means of localization is tedious and cannot be used effectively to analyze samples separated in two dimensions on polyacrylamide gels. Efficient localization of these glycosyltransferases would not only allow rapid detection of enzyme patterns separated from crude samples, but would aid in the ultimate purification of these enzymes.

441 OOO3-2697/80/040441-O9$02.OO/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

442 PIERCE.CUMMINGS,ANDROTH

Generally, only coupled enzyme assay techniques allow the detection of individual enzyme activities in supports such as polyacrylamide. Although a coupled enzyme assay for galactosyltransferase has been developed previously (8), it measures a decrease in absorbance of NADH, and thus its utility for resolving enzyme activities on polyacrylamide gels and determining activities of crude enzyme preparations is marginal.

In this report a coupled enzyme assay is described for GlcNAc:UDP-galactose galactosyltransferase in which 2 mol of NADH are produced for each mole of galactose transferred from UDP-galactose to acceptor. This assay is sensitive, inexpensive, and reproducible for the two, soluble galactosyltransferases studied. Using this coupled enzyme assay system, galactosyltransferase activities are localized in nondenaturing polyacrylamide gels by observing the production of fluorescent NADH.

EXPERIMENTAL PROCEDURES

Materials. UDP-galactose, UDP-glucose, UDP, ATP, P-NAD, /3-NADH, diethylbarbituric acid, imidazole, N-acetylglucosamine, a-D-glucose l-phosphate, cacodylic acid, bovine milk galactosyltransferase, p-nitrophenyl-N-acetyl-/3-o-glucosaminide, nucleoside-5’-diphosphate kinase (grade III), uridine-5’-diphosphate glucose pyrophosphorylase (type X), uridine-5’-diphosphate glucose dehydrogenase (type VI), bovine serum albumin, and agarose (low-gelling, Type VII) were purchased from Sigma Chemical Company. Fetal calf serum was obtained from GIBCO. Acrylamide, N,N’- methylene-bisacrylamide, TEMED, and am- monium persulfate were obtained from Bio- Rad Laboratories. Uridine diphospho-D-16- 3H]galactose (13.2 Ci/mmol) was purchased from AmershamSearle. Imidazole was purified by boiling 15 g of imidazole in 70 ml benzene with 5 g of decolorizing carbon for 3 min. The mixture was filtered on Whatman

No. 1 filter paper, and white, crystalline needles formed as the filtrate cooled to room temperature. The crystals were filtered and washed with cold benzene, and the benzene was removed by vacuum desiccation.

Spectrophotometric assay for galactosyl- transferuse. The bovine milk galactosyltransferase, coupling enzymes, and cofactors were dissolved in 100 mM cacodylate, pH 7.5, and stored at -10°C before use. Fetal calf serum, dialyzed against the same cacodylate buffer, was stored as above. One unit of enzyme activity was defined as 1 pmol of product per minute at the optimal assay conditions for each enzyme. The spectrophotometric assay consisted of the following components (prewarmed to 37°C) added in the order given: 0.5 ml 20 mM MnCI,, 0.28 ml cacodylate buffer, 0.01 ml nucleoside-5’diphosphate kinase (1000 units/ ml), 0.01 ml UDP-glucose pyrophosphorylase (25 units/ml) 0.01 ml UDP-glucose dehydrogenase (2 units/ml), 0.067 ml NAD (10 mg/ml), 0.05 ml ATP (10 mg/ml), 0.05 ml glucose l-phosphate (10 mg/ml), 0.02 ml 5 mM UDP-galactose, 0.02 ml 1 M N-acetylglucosamine. The galactosyltransferase sample was added such that the final volume in the cuvette was 1.07 ml. After inverting the mixture several times, the absorbance at 340 nm was recorded continuously in a Beckman DU spectrophotometer with a Honeywell recorder. The cuvette chamber was warmed to 37°C by a circulating water bath. To calculate nanomoles of UDP produced, a molar extinction coefficient for NADH of 5850 was used, which corrected for the deviation from 1 cm3 in the assay. The rate of NADH production was normally calculated from the linear portion of the recorder tracing during the first 30 min of incubation. Endogenous values, which were negligible in these assays, were determined by omitting N-acetylglucosamine.

Though managanese ion was found to not interfere with the rates of the coupled enzymes, it did preclude the use of Tris and phosphate buffers because of precipitate

GALACTOSYLTRANSFERASE LOCALIZATION IN POLYACRYLAMIDE GELS 443

formation. The assay was adjusted to pH 7.5, which is near optimal for the galactosyltransferase and two of the coupled enzymes, though below optimal for UDP-glucose dehydrogenase (optimal at pH 8.7).

Native polyacrylamide gel eiectrophore- sis. The buffer system for polyacrylamide gel consisted of diethylbarbituric acid as the weak acid and imidazole as the weak base and was modified from Richards et al. (9). This buffer system was chosen because the pH of the separating gel was in the neutral range and because Mn2+ does not precipitate in this buffer as it does in Tris at pH values greater than 7.4. Separation gels (100 x 140 x 1.5 mm) contained 7.5% acrylamide, while stacking gels contained 5% acrylamide. Separation gel buffer solution (4x) was prepared by adding imidazole to 10 ml of 1 N

HCl and 20 ml of water until the pH reached 7.8 and then diluting the solution to 50 ml. Stacking gel solution was prepared similarly except that imidazole was added to bring the pH to 5.8. Electrode buffer solution contained 5.52 g diethylbarbituric acid and imidazole to pH 7.0 in a total volume of 1 liter. Protein samples were mixed with separation buffer (4x), 50% glycerol, and bromophenol blue. Seventy-five microliters of this solution was loaded into sample wells. During sample stacking 50 V were applied to the gel; voltage during separation was 100 V. Electrophoresis was stopped when bromophenol blue reached the bottom of the gel (approximately 2 h).

Agarose gel overlay technique for detection of galactosyltransferase. A 3% solution of agarose in 100 mM cacodylate, pH 7.5, was prepared by gently heating the mixture over an alcohol flame, and then incubating at 37°C until ready for use. A solution of coupling enzymes and cofactors was mixed with the 3% agarose solution at 37°C to give a final agarose mixture of 2%. The final concentration of coupling enzymes and cofactors was as described for the spectrophotometric assay, except that the concentrations of NAD, glucose l-phosphate,

ATP, and UDP-galactose were lo-fold higher and the concentrations of N-acetylglucosamine and MnC& were 2.5-fold higher. These concentrations were found to be optimal after varying the concentrations of coupling enzymes and cofactors in an experiment with a constant amount of galactosyltransferase in polyacrylamide slabs. The glass plates were separated from the acrylamide slab gel allowing the gel to remain attached to one plate. The slab gel was blocked off at the ends and sides with glass tubing coated with Cello-Seal, and the 2% agarose solution of coupling enzymes and cofactors was poured over the gel surface. The agarose was allowed to harden at room temperature for 5 min and the indicator gel-slab gel bilayer was sealed with Saran Wrap and aluminum foil in a humidified atmosphere and incubated at 37°C for up to 15 h. The gel was viewed and photographed on a long-wavelength ultraviolet light box (Ultra-Violet Products, Inc.) using Polaroid film (Type 665, positive/nega- tive Land film). A lens from a pair of ultraviolet light protective spectacles was used as a filter.

Radioactive assay for galactosyltransferase. Assays were done as described under Spectrophotometric assay for galactosyltransferase in identical volumes except that UDP-[3H]galactose (7 cpm/pmol, 100 PM final concentration) and p-nitrophenol- N-acetyl-P-D-glucosaminide (1.5 mM) were included. The latter compound was necessary to inhibit a contaminating glycosidase in the UDP-glucose dehydrogenase as described in Results and Discussion. At times up to 40 min, 50-~1 samples were removed from the incubation mixture and mixed with 10 ~1 of 250 mM EDTA in 100 mM cacodylate (pH 7.5) to stop the reaction. Fifty microliters of this solution was spotted on Whatman 3 MM paper and electrophoresed in 1% sodium tetraborate at 44 V/cm for 50 min. The origins, containing the radioactive transferase product N-acetyllactosamine, were cut out and counted in a toluene-based,

444 PIERCE, CUMMINGS, AND ROTH

2,5diphenyloxazole (PPO)/l ,4-bis [2-(5phen- Protein assay. Protein was assayed by the yloxazolyl)]benzene (POPOP) fluor at 6% method of Lowry et al. (II), using counting efficiency for tritium. Endogenous crystalline bovine serum albumin as a activity (obtained by omitting N-acetylglu- standard. cosamine from the assay) was negligible in all experiments with both fetal calf serum RESULTS AND DISCUSSION and bovine milk galactosyltransferase. In other radioactivity assays, the cofactors and The coupled enzyme assay for galactosyl- coupling enzymes were omitted, and the transferases as shown below depends on the enzyme was assayed at the above concentra- production of UDP from UDP-galactose tions of UDP-13H]galactose, MnCl,, ATP, during transfer of the galactose to an and N-acetylglucosamine. acceptor.

UDP-galactose + acceptor galactosyltransferase

l acceptor-galactose + UDP

UDP + ATP nucleoside-5’-diphosphate kinase

> UTP + ADP

UTP + glucose-l-P UDP-glucose pyrophosphorylase

z UDP-glucose + PPi

UDP-glucose + 2NAD+ UDP-glucose dehydrogenase

> UDP-glucuronic acid + 2NADH

Following phosphorylation of the UDP to UTP, a two-step reaction sequence produces 1 mol of UDP-glucuronate and 2 mol of

0 20 40 60 60 100

Time (minutes 1

FIG. 1. Typical spectrophotometric assay of galactosyltransferase using the coupled enzyme system described under Experimental Procedures with continuous monitoring of NADH absorbance at 340 nm. (A) Assay of bovine milk galactosyltransferase (0.04 pg) in the coupled enzyme system. At times indicated the following additions were made: 1, bovine milk galactosyltransferase; 2, UDP-galactose: 3, N-acetylglucosamine. (B) Assay of two samples of fetal calf serum (1.5 mg each) in the coupled enzyme system. At times indicated the following additions were made: 1, fetal calf serum; 2, UDP-galactose; 3, N-acetylglucosamine. (-) N-acetylghtcosamine added; (- - -) N- acetylglucosamine not added.

NADH per mole of UDP. Since ATP is present at millimolar levels, it competitively inhibits any pyrophosphorylases or phos- phatases that could degrade UDP-galactose, UDP, or UTP (12). The concentrations of enzymes and cofactors were empirically determined to provide efficient coupling of the reactions.

The spectrophotometric assay measures NADH production at 340 nm and can detect as little as 1-2 nmol of UDP produced per hour. Also, of course, it provides continuous monitoring of the reaction progress. Figure 1A shows a typical assay of bovine milk galactosyltransferase using N-acetylglucosamine as acceptor. After initial stabilization of the absorbance, it is clear that continuous NADH production is dependent on the addition of N-acetylglucosamine. A similar experiment for dialyzed fetal calf serum is shown in Fig. lI3, and again indicates the absence of NADH production in the absence of added acceptor. (Without dialysis, serum has an endogenous NADH production that is not dependent on UDP-galactose


although, by subtraction, the activity of the galactosyltransferase can be determined.) Additionally, cr-lactalbumin, the modifier protein of galactosyltransferase which inhibits the transfer of galactose to N- acetylglucosamine (13), causes an 85% reduction in the rate of NADH production, when added at 2 mg/ml (data not shown).

The coupled enzyme reaction is dependent on the protein concentration of the galactosyltransferase as illustrated in Fig. 2. Above these protein concentrations for both fetal calf serum and milk galactosyltransferases, UDP-galactose, and the coupled enzymes become 4imiting. In the coupled enzyme reaction mixture described under Experimental Procedures, an absolute rate of 12 nmol/min of NADH production occurs after adding 200 nmol of UDP to the mixture. This limitation may result from the low specific activity of UDP-glucose dehydrogenase since this enzyme, in the presence of the usual concentrations of NAD, Mn2+, and 200 nmol of UDP-glucose shows a maximal rate of 1 l- 12 nmol/min of NADH production.

Table 1 compares the rate of 13H]galactose transfer to N-acetylglucosamine and the

8.

-0 0.05 0.10 0.15 0 I .o 2.0 3.0

Protein Ipq/ml) Protein (rnghl)

FIG. 2. Effect of galactosyltransferase protein concentration on the production of NADH absorbance at 340 nm. All assays were done in duplicate for 1 h as described under Experimental Procedures, and the variation was less than 10%. (A) bovine milk galactosyltransferase; (B) fetal calf serum.

rate of NADH production in the coupled enzyme assay. N-Acetyllactosamine synthesis was measured by the radioactivity assay employing UDP-13H]galactose and electrophoretic isolation. The radioactivity assays described in Table 1 included in the coupled enzyme mixture, p-nitrophenyl-N- acetyl-/3-o-ghicosaminide. We found that N- acetyllactosamine was hydrolyzed by a glycosidase contaminating the UDP-glucose dehydrogenase. After assaying the ability of a variety of p-nitrophenyl-glycosides to

TABLE 1

MEASUREMENT OF GALACTOSYLTRANSFERASE SPECIFIC ACTIVITY IN THE

SPECTROPHOTOMETRIC AND RADIOACTIVITY ASSAYS

Enzyme source

Spectrophotometric assay”

OD/ nmol UDP produced/ mg.h mg.h

Radioactivity assay” nmol PHIGal transferred/

mg.h

Bovine milk galactosyltransferase

Fetal calf serum 3.77 x IO” 3.23 x 10” 3.24 x IO”

0.122 10.4 10.3

fl Assays were done as described under Experimental Procedures using 0.07 pg of bovine milk galactosyltransferase or 1.5 mg fetal calf serum protein per milliliter of assay. The assay was continuously monitored for absorbance at 340 nm for 40 min. The nanomoles of UDP produced was calculated by assuming that 2 nmol of NADH were produced for each nanomole of UDP.

’ Assays were done as above except that UDP-[“H]galactose (7 cpm/pmol, 100 pM final concentration) and

1.5 mMP-nitrophenyl-N-acetyl-/3-o-glucosaminide were included. Instead of monitoring the absorbance at 340 nm, 50-~1 samples were removed at times during the initial 40 min of incubation and assayed for N-[“Hlacetyllactos- amine synthesis as described under Experimental Procedures. All assays were done in duplicate and the variation was less than 10%.

446 PIERCE,CUMMINGS,ANDROTH

inhibit N-acetyllactosamine hydrolysis, it was found that p-nitrophenyl-GlcNAc was the only effective inhibitor. The glycosidase has unusual substrate specificity, and does not hydrolyze p-nitrophenyl-P-o-galactoside. In neither the radioactivity nor spectrophotometric assay does p-nitrophenyl-GlcNAc serve as a galactosyl acceptor. The contaminating glycosidase has no effect on the spectrophotometric coupled enzyme assay, since UDP, not N-acetyllactosamine, production is measured. By including p- nitrophenyl-GlcNAc, the synthesis of N- ]3H]acetyllactosamine by both fetal calf serum and milk galactosyltransferases is the same in the coupled enzyme mixture as in the direct radioactivity assays in the absence of coupled enzymes. The rates of NADH production and the synthesis of N- acetyllactosamine were determined and a specific activity for each of the assay methods was calculated. As shown in Table 1, the specific activities calculated from both assays are comparable.

The spectrophotometric assay is useful with galactosyltransferases that produce at least l-2 nmol of UDP per hour. Preliminary results with assays conducted in a Perkin- Elmer recording spectrofluorometer indicate that the standard assay is at least lo-fold more sensitive if NADH fluorescence at 460 nm is monitored instead of absorbance at 340 nm. This modification would also make the assay of particulate enzymes, which generally have low activity, feasible. Since 0.1% Triton X-100 has no effect on the coupled enzymes, it may be possible to assay solubilized membrane-bound enzymes.

A potentially important use of this coupled enzyme assay is for making transferase activities visible on gels and, perhaps, in situ. Previous techniques for detecting transferases in gels involved slicing gels, eluting enzymes from the slices, and assaying for activity by the standard radioactivity assays (14). This would not be practical, however, if multidimensional gel

analyses were desired. Therefore, we adapted the spectrophotometric assay conditions to an agarose gel overlay to detect galactosyltransferase activities in native polyacrylamide gels.

Two samples of fetal calf serum containing 1.25 x lo-* and 2.5 x 10e4 units of galactosyltransferase activity, respectively, were subjected to electrophoresis into a 7.5% polyacrylamide gel, pH 7.8, buffered with imidazole and diethyl barbituric acid (9). After removing a glass support plate from one surface of the gel, a 2% agarose indicator gel containing the coupled enzyme system used in the spectrophotometric assay was cast over the polyacrylamide gel. After a 3-h incubation at 37°C the gels were viewed using a long-wavelength ultraviolet light source. NADH fluorescence was clearly visible over a small area of the gel, indicating the location of the galactosyltransferase in the polyacrylamide gel, shown in Fig. 3A. NADH fluorescence increased with increasing amounts of galactosyltransferase activity applied to the gel.

Samples of fetal calf serum, 2.5 X low4 units, and bovine milk galactosyltransferase, 10m3 units, were applied to separate lanes (Fig. 3B, lanes 3 and 4) of an identical polyacrylamide gel, subjected to electrophoresis and covered with the indicator gel. NADH fluorescence was visible after 3 h and the position of the fluorescence differed between the two samples, demonstrating different electrophoretic mobilities for the two enzymes. To test whether fluorescence was dependent on the presence of the acceptor, N-acetylglucosamine, samples identical to those applied to lanes 3 and 4 were applied to lanes 1 and 2 of the same polyacrylamide gel, shown in Fig. 3B. The indicator gel cast over lanes 1 and 2 after electrophoresis did not contain N-acetylglucosamine. No fluorescence was observed over these lanes even when incubations lasted 15 h. Figure 3B thus demonstrates that for both enzyme sources, NADH


FIG. 3. Localization of galactosyltransferase activities in polyacrylamide gels. Samples were separated in 7.5% polyacrylamide gels, pH 7.8 as described under Experimental Procedures. In each case, electrophoresis was stopped when bromophenol blue reached the end of the gel. Sections of gel or entire gel was then overlayed either with a 2% agarose indicator gel containing the coupled enzyme mixture or stained with Coomassie blue. To visualize galactosyltransferase activity, gels were incubated 3 h at 37”C, viewed over long-wavelength ultraviolet light, and photographed. (A) Fluorescent NADH areas produced by reaction of fetal calf serum galactosyltransferase and coupled enzymes. Lane 1,2.5 x 10mJ units of galactosyltransferase activity applied before electrophoresis; lane 2, I.25 x 1O-4 units applied. (B) Fluorescent NADH areas produced by reaction of either bovine milk galactosyltransferase (lanes 2 and 4, lo-” units each) or fetal calf serum galactosyltransferase (lanes 1 and 3,2.5 x 10V4 units each) and coupled enzymes. Indicator gel over lanes 3 and 4 contained the acceptor, N-acetylglucosamine, whereas that over lanes 1 and 2 did not. (C) Lane 1, Coomassie blue staining pattern of identical sample of fetal calf serum applied to lane 2. Lane 2, fluorescent NADH area produced by reaction of 2.5 x lo-’ units of fetal calf serum galactosyltransferase and coupled enzymes.

fluorescence is dependent on the presence of enzyme acceptor.

The fetal calf serum galactosyltransferase localized by the indicator gel was separated from the majority of the proteins in the serum as detected by Coomassie blue staining. Two samples of serum, 2.5 x 10e4 units each, were applied to two adjacent lanes of a polyacrylamide gel and subjected

to electrophoresis. The gel was then sliced to separate the lanes and one (Fig. 3C, lane 2) was overlayed with indicator gel to visualize enzymatic activity whereas the other lane (Fig. 3C, lane 1) was stained with Coomassie blue. The results shown in Fig. 3C suggest that this electrophoretic system separated galactosyltransferase from most of the fetal calf serum proteins.

448 PIERCE, CUMMINGS, AND ROTH

Using the indicator gels described in this report, as little as lob4 units of galactosyltransferase activity is detectable by NADH fluorescence. After about 12 h of incubation, the area and intensity of fluorescence becomes constant. After gels have been viewed and photographed, the indicator gel can be easily separated and removed from the surface of the polyacrylamide gel, which may allow additional staining of the gel or excision and elution of enzyme. However, we have no data concerning the degree of cross-contamination, which may cause difficulties in subsequent treatment, between the two gels.

Galactosyltransferase activity has also been detected in polyacrylamide gels after electrophoresis by coupling NADH production to the reduction of p-iodonitrotetrazo- hum violet which, when reduced, forms a colored, insoluble formazan. Diaphorase was used to catalyze dye reduction since phenazine methosulfate, the most com- monly used catalyst (lo), was ineffective in the presence of millimolar levels of MnZ+. Enzyme activity could be seen in this manner; at present, however, sensitivity is less than that obtained with NADH fluorescence (data not shown). This may be due to the fact that diaphorase is somewhat inhibited by 10 mM MnZ+. No attempt was made to quantitate the difference in sensitivity between INT reduction by diaphorase and NADH fluorescence as a means of visualizing the enzymes in the indicator gel methods.

Galactosyltransferases that use acceptors other than N-acetylglucosamine should be assayable using both spectrophotometric and indicator gel methods. In addition, UDP- N-acetylglucosaminyl- and UDP-N-acetyl- galactosaminyltransferases, as well as UDP- glucuronyltransferases could also be assayed by these coupled enzyme methods. Since the coupled enzyme assay is active in a number of nonionic detergents, it may be possible to separate and visualize membrane-

bound galactosyltransferases as well as soluble ones.

This coupled enzyme assay could also be used to identify directly isozymes of galactosyltransferases present in normal and pathologic tissues or fluids. The latter possibility is particularly interesting in light of reports of the appearance of a galactosyltransferase isozyme in sera from individuals with malignant disorders (14~95). Finally, of course, if NADH can be coupled efficiently to the precipitation of an electron-opaque dye, then the subcellular localization of many glycosyltransferases could be determined directly.

ACKNOWLEDGMENTS

We wish to thank Dr. Robert Dottin, Rick Manrow, and Barbara Fishel for advice and helpful discussion and Dorothy Regula for assistance in the preparation of this manuscript. This work was supported by a research grant from the National Cancer Institute of the Public Health Service. S.R. is a recipient of a research career development award from the National Institute of Child Health and Human Development. This is manuscript No. 1046 from the McCollum-Pratt Institute.

REFERENCES

1. Roseman, S. (1970) Chem. Whys. Lipids 5, 270- 297.

2. Turco, S. J., and Heath, E. C. (1976) Arch. Biochem. Biophys. 176, 352-357.

3. Brew, K., Vanaman, T. C., and Hill, R. L. (1968) Proc. Nat. Acad. Sci. USA 59, 491-497.

4. Morre, D. J. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 130-148, Academic Press.

5. Cummings, R. D., Cebula, T. A., and Roth, S. (1979) J. Biol. Chem. 254, 1233-1240.

6. Weiser, M. M., Neumeier, M. M., Quaroni, A., and Kirsh, K. (1978) J. Cel[ Biol. 77, 722-734.

7. Pierce, M., Turley, E. A., and Roth, S. (1980) in International Review of Cytology (Bourne, G. H., and Danielli, J. F., eds.), Vol. 65, in press.

8. Fitzgerald, D. K., Brodbeck, U., Kiyosawa, I., Mawal, R., Calvin, B., and Ebner, K. E. (1970) J. Biol. Chem. 245, 2103-2108.

9. Richards, E. Cr., Coll, J. A., and Gratzer, W. B. (1975) Anal. Biochem. 12, 452-471.

10. Bergmeyer, H. U., and Berm, E. (1974)in Methods


of Enzymatic Analysis (Bergmeyer, H. U., ed.) 13. Morrison, J. F., and Ebner, K. E. (197l)J. Biol. 2, 579-582. Chem. 246, 3992-3998.

11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and 14. Weiser, M. M., Podolsky, D. K., and Isselbacher, Randall, R. J. (1951) J. Biol. Chem. 193, 26% K. J. (1976) Proc. Nat. Acad. Sci. USA 13, 275. 1319- 1322.

12. Patt, L. M., and Grimes, W. J. (1975) Biochem. 15. Podolsky, D. K., and Weiser, M. M. (1979)5. Biol. Biophys. Res. Commun. 67, 483-490. Chem. 254, 3983-3990.

Documents

The localization of galactosyltransferases in polyacrylamide gels by a coupled enzyme assay