Electrophoretic,Kinetic,and ... - Clinical Chemistry

CLIN. CHEM. 26/11,

1523-1527

(1980)

Electrophoretic,Kinetic,and ImmunoinhibitionPropertiesof y-Glutamyltransferasefrom VariousTissuesCompared Leslie M. Shaw, Lorette Petersen-Archer, Jack W. London,1 and Elizabeth Marsh y-Glutamyltransferase (EC 2.3.2.2) from human liver, kidney, pancreas, and duodenum migrated in acrylamide gels (65 g/L) as a single band with the following decreasing order of electrophoretic mobility: liver > pancreas > kidney > duodenum. The initial velocity kinetic constants of pancreatic and duodenal y-glutamyftransferase and of the enzyme in human serum were determined and compared with those we previously established for the enzyme from human kidney, liver, and serum. The greatest differences were in the glycylglycine competitive-inhibition constants: kidney ‘y-glutamyltransferase was the most strongly inhibited and pancreatic enzyme the second most strongly inhibited by high concentrations of glycylglycine, with the liver and duodenal isoenzymes only slightly inhibited and the enzyme in serum not inhibited over thern concentration range (0-150 mmol/L) of glycylglycine used. Differences between the other kinetic constants of these isoenzymes were much smaller. Human liver y-glutamyltransferase was obtained in a highly prifled form by a six-step procedure that included papain digestion of the original homogenate. Rabbit antisera raised against this preparation inhibited liver, kidney, and pancreatic y-glutamyltransferase activity equally well (78, 76, and 78% inhibition, respectively), but inhibited the hog-kidney enzyme only slightly (1%). We conclude that the polypeptide portions of the isoenzyme molecules are structurally similar but that the carbohydrate moieties differ significantly in structure and topography. Addftlonal Keyphrases: lsoenzymes inhibition by glycylglycine purification of ‘y-glutamyltransferase from human liver enzyme kinetics #{149}

#{149}

From several recent studies of the structure of y-glutamyltransferase [GGT; (5-glutamyl)-peptide:amino-acid 5 glutamyltransferase, EC 2.3.2.2] prepared from animal and human tissues, we know the following: (a) GGT is a glycoprotein enzyme (1-10); (b) the enzyme molecule is composed of two nonidentical subunits (2, 11, 12); (c) the relative molecular mass of the human liver enzyme (90 000-120 000) is greater than that of the kidney isoenzyme (84 000) (2, 7, 12); (d) the lectin-binding properties (and therefore carbohydrate structure) of GGT from the human tissues liver, kidney, pancreas, and duodenum differ significantly (9); (e) human liver and kidney GGT maybe immunologically identical (13), as may be rat hepatoma and rat kidney GGT (8). Most of these studies do not deal with the question of whether there are truly isoenzymic forms of GGT within a given species. However, major clues point to the existence of isoenzymic forms of GGT within a species, based on studies in our laboratory comparing the carbohydrate structure of GGT isolated from four human organs (9) and on the findings of Tsuchida et al. (8) of dif-

William Pepper Laboratory, Departments of Pathology and 1Radiology, Hospital of the University of Pennsylvania, Philadelphia, PA 19104. Received March 28, 1980; accepted May 28, 1980.

ferences in carbohydrate structure between rat hepatoma GGT and the rat kidney enzyme. Here, to more rigorously define human tissue isoenzymes of GGT, we compare the initial velocity kinetic properties of GGT prepared from pancreas, duodenum, and human serum with those of kidney and liver GGT previously determined by us. We also compare the electrophoretic properties of GGT prepared from human liver, kidney, pancreas, and duodenum and the inhibition by antiserum against highly purified liver GGT of the catalytic activity of the liver, kidney, and pancreatic enzymes.

Materials and Methods Materials Sephadex G-200 was obtained from Pharmacia Fine Chemicals, Piscataway, NJ 08854; diethylaminoethyl-cellusose (DE-52) from Whatman Inc., Clifton, NJ 07014; concanavalin A-agarose, glutathione, polyethylene glycol (M 600), methyl-a-D-mannopyranoside, papain (type IV, crystallized from papaya latex), and glycylglycine from Sigma Chemical Co., St. Louis, MO 63178; “Hydroxylapatite Bio Gel” from Bio-Rad, Rockville Centre, NY 11570; and L‘-glutamyl-3-carboxy-4-nitroanilide from Boehringer Mannheim Biochemicals, Indianapolis, IN 46250. Normal liver, kidney, pancreas, and duodenum tissues were obtained at autopsy less than 18 h postmortem and frozen at -30 #{176}C immediately.

Procedures Enzyme assay. All assays of enzyme activity were done at 30#{176}C with a Stasar III spectrophotometer (Gilford, Oberlin, OH 44074), as described elsewhere (14). The standard assay included imal reagent concentrations (per liter) of 4 mmol of y-glutamyl-3-carboxy-4-nitroanilide, 100 mmol of glycylglycine, and 100 mmol of tris(hydroxymethyl)aminomethane HC1 (Tris HC1), pH 8.25. The standard assay was used in all steps of the isolation of liver GGT and in determining enzyme activity in the immunoinhibition studies. Enzyme purification. GGT was prepared from normal liver, kidney, pancreas, and duodenum according to the trypsinization procedure previously described (7). Highly purified GGT from liver was prepared as follows: 1. Homogenization: 100 g of frozen liver was thawed and homogenized at 4#{176}C in a Waring Blendor with three volumes of Tris HC1 (50 mmol/L, pH 7.5; hereafter referred to as “the Tris buffer”). 2. Papain digestion: To the homogenate we added 2 g of papain. After incubation for 3 h at 37 #{176}C, this mixture was maintained at 4#{176}C for 14 h, then centrifuged at 15 000 X g for 40 min. The supernatant fluid was dialyzed against three 18-L changes of the Tris buffer during 36 h. 3. DE-52 chromatography: DE-52 was prepared as described by the manufacturer (Whatman circular no. 1L2). The equilibration buffer was the Tris buffer. After loading the dialyzed supernatant fluid from Step 2 onto the DE-52 column (2.5 X 95 cm) and washing it with 1400 mL of equilibration buffer, we eluted the enzyme as a single peak by using a linear gradient established between 750 mL of the Tris buffer and .

.

CLINICAL CHEMISTRY,

Vol.26, No. 11, 1980

1523

FIg. 1. Acrylarnkie-gel electrophoresis of (left to rigs’,t)duodenum (0), kidney, (K), liver (L), and pancreatic (P) GGT The enzymes were prepared by the trypsln-solubllizatlon procedure described in Materials and Methods. Approximately 10 mU of each enzyme, each In a volume of 10 ML was electrophoresed In 65 gIL gels prepared in Tris . HCI (0.3 moIIL, pH 9.0) by the method of Davis (19). The gels in this figure were all run at the same time and stained for GGT activity as described In the text

750 mL of the same buffer containing 0.2 mol of NaCl per liter. The flow rate throughout this step was 80 mL/h. The fractions containing enzyme activity were combined and dialyzed against three changes of 18 L of the Tris buffer. 4. Heat denaturation and hydroxylapatite chromatography: To the dialysate from Step 3 we added glutathione to a final concentration of 0.02 mol/L. This mixture was incubated at 57 #{176}C for 1 h, then cooled to 4 #{176}C and applied to a column of hydroxylapatite (5 X 6.5 cm, equilibrated with the Tris buffer). Next, the column was rinsed with 870 mL of the Trig buffer. To elute enzyme from the column we used Tris buffer containing 0.1 mol of phosphate buffer per liter, pH 7.5; the flow rate throughout this step was 150 mL/h. 5. and 6. Affinity chromatography and gel filtration chromatography: Affinity chromatography on concanavalin ASepharose-4B of the enzyme peak eluted from the hydroxylapatite column was performed as we previously described for human liver GGT (7). The enzyme was eluted from the affinity column with 0.2 mol/L methyl-a-D-mannopyranoside in the Trig buffer. The enzyme eluted was dialyzed against three changes of 18 L of the Tris buffer for 36 h, then concentrated to a final volume of 5 mL in the dialysis bags by surrounding them with polyethylene glycol. The concentrated enzyme fraction was chromatographed on a 2.5 X 95 cm column of Sephadex G-200 that had been equilibrated with the Tris buffer, as previously described (7). 1524

CLINICAL CHEMISTRY, Vol. 26, No. 11, 1980

Protein determinations by the method of Bradford (15) were performedto follow the purification of liver GGT; human albumin was the standard. Kinetic studies. On the basis of the mathematical model we had previously developed for the “ping-pong bi-bi” mechanisms for both the autotransfer and transpeptidation reactions catalyzed by GGT, we determined the kinetic constants for purified GGT from pancreas and duodenum and for GGT in a pool of human sera from patients with obstructive liver disease (14, 16). The data base for calculating the kinetic constants consisted of GGT activity measurements over a y-glutamyl-3-carboxy-4-nitroanilide concentration range of 0.25 to 10 mmol/L and glycylglycine concentration range of 0 to 150 mmol/L. Kinetic constants were derived by the nonlinear-regression fit of the mathematical model to the kinetic data by the method of Fletcher and Powell (17) for minimizing the deviation between calculated and observed enzyme velocities. From the Hessian Matrix (18) of this minimization procedure, we calculated 95% confidence limits for the kinetic constants, to indicate how well the constants were defined. Acrylamide gel electrophoresis. GGT preparations were electrophoresed in acrylamide (65 gIL), according to the method of Davis (19). The gels were stained for GGT activity by an adaptation (7) of the histochemical GGT staining method of Rutenburg et al. (20). Staining of gels for carbohydrate was by the method of Zacharius et al. (21); for protein we stained with Coomassie Brilliant Blue R-250. Antiserum production. Anti-GGT serum was obtained by immunizing two rabbits by the protocol described by Livingston (22). We mixed 100 g of liver GGT (purified by the papain procedure outlined above) in 1 mL of the Tris buffer with 1 mL of Freund’s complete adjuvant and injected this mixture into each animal subcutaneously. Four weeks later we mixed 50 g of the GGT preparation with Freund’s incomplete adjuvant and injected it into each animal subcutaneously. Two weeks after that, the rabbits were bled and the serum was obtained. The rabbit antiserum was mixed with sodium aside (final concentration of the azide, 20 mg/L) and stored at 4 #{176}C.

Results Acrylamide

Gel Electrophoresis

The acrylamide gel electrophoretic analysis of the trypsin-solubilized preparations of GGT from liver, kidney, pancreas, and duodenum is displayed in Figure 1. According to these data, GGT from each tissue migrates as a single band, in the descending order liver> pancreatic> kidney > duodenal GGT.

Initial Velocity

Kinetic

Constants

In previous studies we developed a mathematical model and kinetic constants for GGT prepared from normal human liver and kidney and for GGT in pathological human sera and from hog kidney (7, 14, 16). In the present work we determined the initial velocity kinetic constants for GGT prepared from human pancreas and duodenum and for GGT in human serum. These new data are summarized as follows: Values and 95% confidence limits for pancreatic GGT of 5.9 ± 1.00,0.70 ± 0.04, 10.4 ± 4.5, 307 ± 63, and 72.9 ± 27.6 were obtained for the constants KMA (glycylglycine), KM’ (y-glutamyl-3-carboxy-4-nitroanilide), KMDA (autotransfer), K (glycylglycine competitive inhibition constant), and K1” (7-glutamyl-3carboxy-4-nitroanilide non-competitive inhibition constant), respectively. For duodenal GGT the mean values (and 95% confidence limits) we obtained were 5.2 ± 0.90, 0.91 ± 0.10, 9.17 ± 3.8, 618 ± 323, and 56.1 ± 18.0, respectively; for serum GGT the first three values were 9.6 ± 1.1,0.98 ± 0.08, and 19.2

Table 1. Purification Scheme for Human Liver y-Glutamyltransf erase Total acty.,

Homogenate Papain extract Chromatography on DE-52 Heat denaturation and chromatography on hydroxylapatite Concanavalin-A affinity chromatography Sephadex G-200 chromatography

± 6.4, with no glycyiglycine or ‘y-glutamyl-3-carboxy-4-nitroanilide substrate inhibition occurring. When we compare these data with the kinetic constants for human kidney and liver we previously reported (7), the major differences among the human isoenzymes are differences in inhibition by the substrate glycyiglycine. Thus, as we previously noted, the competitive inhibition constant K11t is 7.4-fold lower for the kidney enzyme (K1’ = 92± 19) than that for liver GGT (K1A = 678 ± 188), reflecting the significantly greater inhibition of the kidney enzyme by high concentrations of glycylglycine. In comparing the values of K for pancreas (307 ± 63) and duodenum (618 ± 323) GGT with that for kidney, we note that the kidney K1A value is 3.3- and 6.7-fold lower than the former two, respectively. Furthermore, we confirmed in this work something that we observed previously (14): that GGT in human serum is not inhibited by glycylglycine in concentrations ranging from 0 to 150 mmol/L. Much smaller differences between the isoenzymes were observed for the other kinetic constants. It should be pointed out that the procedure for preparing pancreatic and duodenal GGT, which involves proteolytic digestion, differs from the procedure we previously used (no proteolytic digestion) to prepare the liver and kidney enzymes (7). We do not believe that these differences in preparative procedure would alter the GGT kinetic constants so as to make invalid our comparisons of the duodenal and pancreatic isoenzymes with the liver and kidney isoenzymes. Huseby (12) compared the kinetic constants of human liver GGT that had

Fig. 2. Acrylamide-gel electrophoresis of human liver ‘y-glutamyltransferase pLrified by the papain procedure described in the text About 10 ig of enzyme protein ma volume of 10 L was electrophoresed. The gel on the left was stained for protein with Coomassle BrIlliant Blue R-250, that on the right for carbohydrate by the procedure of Zacharlus et al. (21). These gels were run at the same time

U

Total protein, mg

Spec. acty., kU/g

679 645 441 392 289 240

19 546 5500 861 247 9.6 1.7

0.0347 0.117 0.667 1.59 30.2 142

Purification (n-fold) -

3.37 19.2 45.8 870 4092

Yl.id,

% -

95 65 58 43 35

been partly purified without the use of proteolytic enzymes with human liver GGT purified with use of a papain digestion step. The constants obtained were the same, within experimental error. Furthermore, some investigators have observed that treating GGT preparations from a wide variety of human and other mammalian tissues with papain (3, 12), bromelain (2), or trypsin (7, 11) produces no effect on catalytic activity. If kinetic constants were significantly altered by proteolytic treatment, catalytic activity would be expected to be altered.

Immunoinhibition

of GGT Catalytic

Activity

Using the immunization procedure described in Materials we developed rabbit antiserum to liver GGT purified to apparent homogeneity by the purification scheme summarized in Table 1. Overall purification was 4092-fold, yield was 35%, and specific activity of the enzyme was 142 kU/g. Aliquots of this preparation electrophoresed in acrylamide gels showed only one band when stained for either protein or carbohydrate (Figure 2). When the rabbit antiserum was added to an aliquot of purified liver GGT, a precipitate formed almost immediately. There was only a single precipitin line when the anti-GGT serum was incubated for 24 h at 25 #{176}C with the purified enzyme in an Ouchterlony double-immunodiffusion plate. We tested the effect of the anti-GGT serum on GGT catalytic activity by incubating for 19 h at 37 #{176}C increasing volumes of antiserum with 3#{174}-tL aliquots of a human serum (440 U/L) from a patient with obstructive liver disease; as illustrated in Figure 3, the antiserum inhibited catalytic activity. Each reaction mixture was then centrifuged for 20 mm at 2000 X g and the supernate tested for activity. GGT activity in the supernate was somewhat less than in the reaction mixtures before centrifugation (Figure 3), indicating that a portion of the antibody-GGT complex in the precipitates retained GGT catalytic activity. Similar results were obtained by Szewczuk et al. (13) with antiserum to GGT from human kidney and GGT from the kidney. Serum from unimmunized rabbits did not alter human liver GGT activity when tested with the same procedure and liver GGT source as above. We tested the effect of incubating anti-liver GGT serum on catalytic activity of GGT preparations from several sources, including human liver, kidney, pancreas, and serum, and hog kidney. The antiserum inhibited GGT catalytic activity from each of the human tissues to about the same extent (78,76, 78, and 76%, respectively) but did not inhibit the hog kidney enzyme (1%). Szewczuk et al. (13), using experimental conditions very similar to ours, obtained 77% inhibition of kidney GGT and 57% inhibition of liver GGT activity by anti-human kidney GGT serum; only 8% of bovine kidney GGT was inhibited. and Methods,

Discussion Little detailed information is available in the literature regarding the definition of isoenzymes of GGT in human tissues. Numerous studies of GGT in sera from patients with CLINICALCHEMISTRY,Vol. 26, No. 11, 1980

1525

>.. 4-

>

.4-.

C.)

0) > 4-

Cs

Anti

GGT Serum

FIg. 3. Effect of increasing volumes serum on catalytic activity

(ji)

of anti-y-glutamyltransferase

To 0.3-n.. allquots of a human serum (GOT actIvity, 440 U/L) from a patient with obstructIve lIver dIsease were added the IndIcated volumes of rabbIt antI-GOT serum. Each reactIon mixtire was brought to a total final volume of 0.6 mL with 9 g/L MaCI solution and incubated at 37#{176}C for 19 h. The catalytic activities (S) In each reaction mixture were then determined by the standard assay for GOT, after which the reactIon mixtures were centrIfugedfor 20 mm at 2000 X gand the catalytic activities In each supernate (0) agaIn determined

liver diseases show as many as seven different electrophoretic zones of GGT activity, leading one to the conclusion that many “isoenzymes” of GGT are present in the diseased liver (23-25). On the other hand, Friese et al. (26) have shown that an extra GGT electrophoretic zone appeared at the origin of cellulose acetate strips that was artifactually produced by the interaction of chylomicrons with GGT, thus raising the question of the possible artifactual nature of GGT “isoenzymes” in patients’ sera. The proposal that GGT tends to produce artifactual isoenzymic forms was supported by the observations of Miller et al. (27) that the inclusion of the nonionic detergent Triton X-100 in the buffer solutions containing the enzyme, as well as in the elution and electrophoretic procedures, eliminated the tendency of purified human kidney GGT to form aggregates. Meister et al. (28) and others (3) have shown that when GGT is released from the membrane matrix of a number of mammalian tissues by proteolytic digestion, highly purified enzyme that does not produce aggregates in the absence of Triton X-100 may be obtained in good yield. When such GGT preparations are subjected to acrylamide gel electrophoresis, one zone of GGT activity is obtained. We have shown that GGT prepared from liver, kidney, pancreas, and duodenum by using a proteolytic digestion step migrates as one zone of GGT activity in acrylamide gels. The electrophoretic mobilities of GGT from these four tissues differ from one another in order of decreasing electrophoretic mobility: liver> pancreas> kidney > duodenum. These data are consistent with the findings of others that only one electrophoretic band of GGT activity is obtained from a given mammalian organ (2, 6, 7, 11, 28), although electrophoretic microheterogeneity is observed when the GGT preparations are analyzed by isoelectric focusing (2, 4, 8). Our electrophoretic data show that there are structural differences among GGT molecules from different organs. This finding raises the important question of what accounts for these differences. There are at least three possibilities: (a) the charge densities of each of the four GGT preparations are the same but molecular mass differences account for the observed differences in electrophoretic mobilities in acrylamide gel; (b) the charge densities of each GGT preparation are different and the molecular masses are the same; or (c) both the charge 1526

CLINICAL CHEMISTRY, Vol. 26, No. 11. 1980

densities and molecular masses differ from one another. Current evidence indicates the last possibility to be the most likely. We have found that the order of electrophoretic mobility of these same four GGT preparations was unchanged in agarose, a medium with much less molecular sieving properties than acrylamide (unpublished data). Furthermore, the relative molecular mass of human kidney GGT prepared by proteolytic digestion is 86 000 (2), whereas that prepared from human liver by proteolytic digestion is between 90000 and 120 000 (7, 12). Thus, because it has a lower molecular mass than the liver enzyme, kidney GGT would be expected to migrate faster in acrylamide gel than the liver enzyme if their charge densities were the same. The reverse is true. We therefore conclude that charge density differences are a major determining factor accounting for the observed differences in electrophoretic mobilities of the GGT isoenzymes. Our immunoinhibition data indicate the immunologic similarity of GGT from human liver, kidney, and pancreas. Szewczuk et al. (13) prepared rabbit antiserum to human kidney GGT, using highly purified kidney GGT as the antigen. They found that the kidney antiserum inhibited kidney GGT activity by 77% and the liver activity by 57%, showing a high degree of cross reactivity. The possible immunological identity of GGT from different organs in a mammalian species is strongly supported by the findings of Tsuchida et al. (8). These investigators developed rabbit antisera to highly purified rat kidney GGT and GGT obtained in a high state of purity from Yoshida ascites hepatoma cells. The anti-kidney serum inhibited 60% of the kidney and hepatoma GGT catalytic activities. Immunological identity of GGT from rat kidney and hepatoma was further demonstrated by Ouchterlony double-diffusion analysis in which one precipitate line was obtained between anti-kidney GGT serum and both the kidney and hepatoma GGT lines fused smoothly. In similar fashion the single lines obtained between anti-hepatoma GGT serum and the two antigens also fused. Thus there are differences in electrophoretic mobility of GGT from different organs in a given species, but the GGT preparations are immunologically identical. Then what accounts for the differences obtained by electrophoretic analysis? We believe the major differences in molecular structure of GGT that would explain the observed electrophoretic behavior are in the topography and structure of the carbohydrate moieties in the GGT molecule. Thus, we have shown that-for GGT from liver, kidney, pancreas, and duodenum-the lectin binding properties differ significantly (9). Indeed, Tsuchida et al. (8) found that rat hepatoma GGT had a faster electrophoretic mobility than the kidney isoenzyme but that after treatment with neuraminidase (EC 3.2.1.18) to remove terminal sialic acid residues their electrophoretic mobilities were comparable. The conclusion that GGT preparations from different organs within a mammalian species are immunologically identical but differ significantly in carbohydrate constitution is compatible with the findings of others (29-31) that antibodies against glycoproteins are specific to the protein portions and not to the carbohydrate portions, except for anti-blood-group antibodies (32). Based on our detailed initial-velocity kinetic studies the kinetic constants were somewhat different for GGT prepared from liver, kidney, pancreas, and duodenum, with the most significant difference (7.4-fold) occurring between the kidney and liver acceptor (glycylglycine) competitive inhibitive constants. This finding might be considered contradictory to the observed cross reactivity as determined by immunoinhibition of kidney and liver GGT with antisera to liver and kidney GGT, respectively, because the immunoinhibition data imply a similar structure at or near the active site of each enzyme. Little is known of the possible effect of carbohydrate moieties on the interaction of substrates with the active site

of glycoprotein

enzymes. We propose that although the carbohydrate moieties of the GGT isoenzymes do not elicit antibodies, they can interact with substrates and the active site to influence substrate binding and in that way influence kinetic constants. This possibility is supported by the recent finding of Komoda et al. (33) that the terminal sialic #{225}id residues of human alkaline phosphatase (EC 3.1.3.1) significantly alter the substrate inhibition produced by high concentrations of p-nitrophenyl phosphate (33). According to their data the strong inhibition of intact human liver alkaline phosphatase by high concentrations of p-nitrophenyl phosphate does not occur with the same preparation of enzyme after removal of terminal sialic acid residues by neuraminidase-catalyzed digestion. Thus it appears that the terminal sialic acid residues in liver alkaline phosphatase confer a conformation in the enzyme molecule that favors inhibition of catalytic

activity

by high substrate

concentration.

References 1. Takahashi, S., Pollack, J., and Seifter, S., Purification of y-glutamyltransferase of rat kidney by affinity chromatography with concanavalin A conjugated with Sepharose 4B. Biochim. Biophys. Acta 371,71 (1974). 2. Tate, S. S., and Ross, M. E., Human kidney ‘y-glutamyltranspeptidase. J. Biol. Chem. 252,6042 (1977). 3. Hughey, R. P., and Curthoys, N. P., Comparison of the size and physical properties of ‘y-glutamyltranspeptidase purified from rat kidney following solubilization with papain or with Triton X-100. J. Biol. Chem. 251,7863 (1976). 4. Jaken, S., and Mason, M., Differences in the isoelectric focusing patterns of ‘-glutamyltranspeptidase from normal and cancerous rat mammary tissue. Proc. Nati. Acad. Sci. USA 75, 1750 (1978). 5. K#{246}ttgen, E., Reutter, W., and Gerok, W., Two different 7-glu-

tamyltransferases during development of liver and small intestine: Fetal (sialo-) and an adult (asialo-) glycoprotein. Biochem. Biophys. Re8. Commun.

72,61 (1976).

6. Taniguchi, N., Purification and some properties of -glutamyltranspeptidase from azo dye-induced hepatoma. J. Biochem. 75,473 (1974). 7. Shaw, L. M., London, J. W., and Petersen, L. E., Isolation of 7-glutamyltransferase from human liver, and comparison with the enzyme from human kidney. Clin. Chem. 24,905 (1978). 8. Tsuchida, S., Hoehino, K., Sato, T., et al., Purification of y-glutamyltransfersse from rat hepatomas and hyperplastic hepatic nodules, and comparison with the enzyme from rat kidney. Cancer Res. 39,4200 (1979). 9. Shaw, L. M., and Petersen-Archer, L., Interaction of y-glutamyltransferase from human tissues with insolubilized lectins. Clin. Biochem. 12, 256 (1979). 10. Scherberich, J. E., Kleeman, B., and Mandorf, W., Isolation of kidney brush border ‘y-gluthmyltranspeptidase from urine by specific antibody gel chromatography. Clin. Chim. Acta 93,73(1975). 11. Zelazo, P., and Orlowski, M., y-Glutamyltranspeptidase of sheep kidney cortex. Isolation, catalytic properties and dissociation into two polypeptide chains. Eur. J. Biochem. 61, 147 (19’75). 12. Huseby, N.-E., Purification and some properties of y-glutamyltransferase from human liver. Biochim. Biophys. Acta 483, 46 (1977). 13. Szewczuk, A., Milnerowciz, H., and Sobiech, K. A., Reaction of antibody with y-glutamyltranspeptidase. I. Isolation of the human

enzyme and inhibition of its activity by antiserum. Arch. Immunol. Titer. Exp. 25,589 (1977). 14. Shaw, L. M., London, J. W., Fetteroif, D., and Garfinkel, D., 7-Glutamyltransferase: Kinetic properties and assay conditions when 7-glutamyl-4-nitroanilide and its 3-carboxy derivative are used as donor substrates. Clin. Chem. 23,79 (1977). 15. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Riochem. 72, 248(1976). 16. London, J. W., Shaw, L. M., Fetterolf, D., and Garfinkel, D., Determination of the mechanism and kinetic constants for hog kidney y-glutamyltransferase. Biochem. J. 157,609 (1976). 17. Fletcher, R., and Powell, M. J. D., A rapid descent method for minimization. Comput. J. 6, 163 (1963). 18. Davidon, W. C., Variable metric method for minimization. A. E. C. Res. and Dev. Report, ANL-5990 (rev.), U.S. Dept. of Commerce, Washington, DC, 1959. 19. Davis, B. M., Disc electrophoresis-il. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121,404 (1964). 20. Rutenburg, A. M., Kim, H., Fiachbein, J. W., et aL, Histochemical and ultrastructural demonstration of y-glutamyltranspeptidase activity. J. Histochem. Cytochem. 17, 517 (1969). 21. Zacharius, R., Zell, T., Morrison, J., et al., Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30,148 (1969). 22. Livingston, D. M., Immunoaffinity chromatography of proteins. Methods Enzymol. 34 (Part B), 723 (1974). 23. Jacyszyn, K., and Laursen, T., A method for determination of the heterogeneity of y-glutainyltranspeptidase. Clin. Chim. Acta 19,345 (1968). 24. Patel, S., and O’Gorman, P., Demonstration of serum 7-glutamyltranspeptidase isoenzymes using Cellogel electrophoresis. Clin. Chim. Acta 49, 11 (1973). 25. Rutenburg, A. M., Smith, E. E., and Fischbein, J. W., Electrophoretic mobilities of serum y-glutamyltranspeptidase, and its clinical application in hepatobiliary disease. J. Lab. Clin. Med. 69, 504 (1967).

26. Friese, J., Magerstedt, P., and Schmidt,E., The electrophoretic pattern of y-glutamyltransferase in serum and its alteration by chylomicrons. J. Clin. Chem. Clin. Biochem. 14, 589 (1976). 27. Miller, S. P., Awasthi, Y. C., and Srivastava, S. K., Studies of human kidney ‘y-glutamyltranspeptidaae. Purification and structural, kinetic and immunological properties. J. Biol. Chem. 251, 2271 (1976). 28. Tate, S. S., and Meister, A., Identity of maleate-stimulated glutaminase with 7-glutamyltranspeptidase in rat kidney. J. Riot Chem. 250,4619 (1975). 29. Bergmann, F. H., Levine, L., and Spiro, R. G., Fetuin: Immunochemistry and quantitative estimation in serum. Biochim. Biophys. Acta 58,41 (1962). 30. Miller, F., Glycopeptides of human immunoglobulins II. Contribution to the antigenicity of the heavy chain. Immunochemistry 8,99(1971).

31. Kaplan, M., and Schlamowitz, M., Investigation of the antigenicity of the carbohydrate moiety of chicken ovalbumin. Immunochemistry 9,737 (1972). 32. Watkins, W. M., Blood group substances. Science 152, 172 (1966). 33. Komoda, T., and Sakagishi, Y., Partial purification of human intestinal alkaline phosphatase with affinity chromatography. Biochim. Biophys. Acta 445,645 (1976).

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