THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 251, No. 8, Issue of April 25, pp. 2271-2278, 1976

Printed in U.S.A.

Studies of Human Kidney y-Glutamyl Transpeptidase

PURIFICATION AND STRUCTURAL, KINETIC, AND IMMUNOLOGICAL PROPERTIES*

(Received for publication, June 16, 1975)

STEVEN P. MILLER, YOGESH C. AWASTHI, AND SATISH K. SRIVASTAVA

From the Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77550

y-Glutamyl transpeptidase, present in various mammalian tissues, transfers the y-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37” followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used

throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on

polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The K, for y-glutamyl transpeptidase using the artificial substrate, y-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of y-glutamyl transpeptidase when y-glutamyl-p-nitroanilide was used as the substrate.

Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified y-glutamyl transpeptidase had no phosphate- independent glutaminase activity. The ratio of y-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of y-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney.

y-Glutamyl transpeptidase transfers the glutamyl moiety from GSH and other y-glutamyl peptides to a variety of amino acids and peptides (1). The product of this reaction is a y-glutamyl peptide. This enzyme was first demonstrated by

Hanes et al. in sheep kidney (2). Since then, y-glutamyl transpeptidase has been partially purified from mammalian

and plant tissues, and from bacteria (3-11). Some properties of the partially purified enzyme from hog, beef, and human kidney have been studied (3-5). The level of y-g!utamyl transpeptidase in the serum has been used as an index of hepatic function (12); and this enzyme may also be involved in the formation of mercapturic acids (13).

y-Glutamyl transpeptidase has been suggested to play an important role in the transport of amino acids into the cell (14-16). The presence of a high concentration of y-glutamyl

transpeptidase in the brush border cells of the proximal convoluted tubules of kidney, apical portion of the intestinal epithelium, choroid plexus, and several other tissues (1, 17-19)

* This work was supported in part by Grants EY01677, GM 21655, and 1 T32, GM07204 from the National Institutes of Health.

in which a higher amino acid transport rate would be antici- pated, appear to support this hypothesis. The distribution of y-glutamyl transpeptidase and the presence of y-glut&my1

cyclotransferase, 5-oxoprolinase, y-glutamylcysteine synthe- tase, and glutathione synthetase led Meister (20, 21) to propose the y-glutamyl cycle for the transport of amino acids in the kidney and other tissues.

Based on the localization of y-glutamyl transpeptidase and phosphate-independent glutaminase in the brush border mem- brane of the proximal tubules of rat kidney and the co-purifica- tion of these enzymes, it has been suggested that both of these reactions are catalyzed by the same enzyme (22, 23). Using purified y-glutamyl transpeptidase from rat kidney, it has been shown that maleate blocks the transpeptidation reaction and increases the phosphate-independent glutaminase activity

(22, 23). Curthoys and Kuhlenschmidt (22) suggested that in the presence of a higher concentration of amino acids, the phosphate-independent glutaminase activity is blocked, and the enzyme primarily functions as y-glutamyl transpeptidase.

We have purified y-glutamyl transpeptidase from human

2271

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2272 Human Kidney y-Glutamyl Transpeptidase

kidney cortex and have studied its structural, kinetic, and immunological properties. The apparently homogeneous en- zyme had a transpeptidase activity of about 200 units/mg of protein. The y-glutamyl transpeptidase activity of our final preparation was not inhibited by 60 mM maleat,e, and even in the presence of maleate no detectable phosphate-independent glutaminase activity was observed.

MATERIALS AND METHODS

r-Glutamyl-p-nitroanilide, concanavalin A insolubilized on beaded A&rose, and DEAE-cellulose (medium mesh) were purchased from Sigma Chemical Co. DEAE-cellulose (DE52) was Durchased from Whatman. Sephadex G-200 and all the columns used-were purchased from Pharmacia Chemical Co., Uppsala, Sweden. The electrophoresis column, 110 ml, and Ampholine were purchased from LKB-Produkter AB. Bromma-A, Sweden. Ouchterlonv agar ael double immunodiffu- sion plates were purchased from Hyland Laboratories, Costa Mesa, Calif. Some of the plates were soaked overnight in 0.1 M Tris-HCl, DH 8.0, containing 1%.sodium deoxycholate. AC the other reagents ;sed were of analytical grade. Human kidneys obtained postmortem were stored at 4’ for a period of up to 15 days. The cortex was then removed and stored at -20” for up to 2 months.

y-Glutamyl transpeptidase activity was determined using y-glu- tamvl-p-nitroanilide as described bv Szasz (24). The l-ml svstem used for the assay consisted of 50 I&I Ammediol (Z-amino-2.methyl- 1,3-propandiol hydrochloride) buffer, pH 8.5: 19.8 rn~ alvcvlalvcine: ” ” .,” 9.9 rni magnesiim chloride; and 3.96.mM y-glutamyl-pynitroanilide. The enzyme reaction was started by the addition of the enzyme, and the formation of p-nitroaniline was measured by following the increase in optical density at 405 nm using a Gilford 2400.2 recording spectrophotometer. One unit of enzyme releases 1 rmol of p-nitroani- line/min at 37” from y-glutamyl-p-nitroanilide. Unless otherwise specified, all the determinations of y-glutamyl transpeptidase activity were performed using y-glutamyl-p-nitroanilide. The y-glutamyl transpeptidase was also determined using GSH as substrate as de- scribed by Palekar et al. (25). GSH was determined by using the sulfosalicylic method (26). Protein was determined by the method of Lowry et al. (27) using crystalline bovine serum albumin as the stan- dard. Analytical disc electrophoresis was performed according to the method of Davis (28). The gels were stained for the enzyme activity by suspending them in 4.4 rnM y-glutamyl-p-nitroanilide and 22 mM glycylglycine, pH 8.5. The yellow color of the reaction product, p-nitroaniline, diffuses very rapidly. The gels were stained for protein with 0.5% Amido black in 7% acetic acid for 15 min.

Electrofocusing was carried out as described earlier (29) using pH 4 to 6 Ampholine. Amino acid analysis was performed by single column methodology using a Beckman automatic amino acid analyzer model 119. For the determination of subunit structure, sodium dodecyl sulfate-urea-&mercaptoethanolpolracrylamide disc electrophoresis was carried out as described previously (30).

Antibodies against the purified preparation of y-glutamyl transpep- tidase were produced in rabbits by injecting into the foot pad 50 Kg of enzyme in 0.5 ml of 10 mM phosphate buffer mixed with 0.5 ml of Freund’s Adjuvant (complete). A similar booster injection followed 15 days later. The blood was drawn from the ear vein and the serum was separated and incubated at 56” for 1 hour.

RESULTS

Purification and Structural Properties-Five hundred grams of kidney cortex was homogenized with 4 volumes of 80 mM magnesium chloride containing 0.75 mM sodium hydroxide in a

Waring Blendor. The temperature of the homogenate was brought to 37” in a water bath with periodic stirring and maintained at this temperature for 2 hours. After the incuba-

tion, the homogenate was brought rapidly to 4” in an ice bath. The homogenate was then centrifuged at 13,000 x g for 30 min in a Sorvall RC-3 centrifuge at 4”. Unless otherwise specified all further steps were performed at 4”. The supernatant was

discarded, and the sediment was suspended in 1.5 liters of 0.1 M

Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate.

The suspension was homogenized using a Sorvall Omni-Mixer

at 8,000 rpm for 15 min. The homogenate was then stirred overnight at 4’ and centrifuged at 13,000 x g for 45 min. The pellet was discarded and the supernatant was dialyzed exten- sively against demineralized water.

The dialyzed supernatant was mixed with DEAE-cellulose (bed volume, 200 ml) equilibrated with 10 mM phosphate buffer, pH 7.0. After stirring at room temperature for about 2 hours, the suspension was filtered through a Buchner funnel. The DEAE-cellulose cake was washed with 10 mM phosphate buffer, pH 7.0, and the enzyme was extracted from the DEAE-cake with 10 mM phosphate buffer, pH 6.0, containing 300 mM NaCl and 0.5% Triton X-100. The enzyme solution was dialyzed against 10 mM phosphate buffer, pH 8.5, and passed through a DEAE-cellulose (DE52) column (2.5 x 50 cm) with a flow rate of about 40 ml/hour. The column was then washed

with 2 liters of 10 mM phosphate buffer, pH 8.5, and the enzyme was eluted from the column with a l-liter gradient of linearly increasing NaCl from 0 to 200 mM in 10 mM phosphate buffer containing 0.5% Triton X-100 and a simultaneously decreasing pH from 7.0 to 6.0 (Fig. 1). The fractions containing the enzyme activity’ were pooled and concentrated to 40 ml in

an Amicon ultrafiltration cell with a PM-10 membrane and dialyzed against 10 mM phosphate buffer, pH 7.4, containing 50 mM NaCl and 0.05% Triton X-100. The dialyzed enzyme was filtered through an ascending Sephadex G-200 column (5 x 85 cm), pre-equilibrated with the dialyzing buffer, at a flow rate of 64 ml/hour. The enzyme was eluted immediately after the void volume. The fractions containing enzyme activity were pooled and dialyzed against 10 mM phosphate buffer, pH 6.0. The dialyzed enzyme was absorbed at a rate of about 8 ml/hour on a column (1 x 28 cm) of concanavalin A insolubi- lized on beaded Agarose equilibrated with 10 mM phosphate buffer, pH 6.0. The column was then washed with 50 ml of the equilibrating buffer, and the enzyme was eluted with 200 ml of 10 mM phosphate buffer, pH 6.0, containing 0.5 M Lu-methyl- glucoside, 0.5% Triton X-100, and chloride salts, 2 mM each, of magnesium, manganese, and calcium. The fractions contain- ing enzyme activity were pooled and dialyzed against‘distilled water. The dialyzed enzyme preparation was used in all the immunological, structural, and kinetic studies.

The purification steps are summarized in Table I. r-Glu- tamvl transpeptidase was purified about 740.fold to a specific

activity of 200 units/mg of protein using y-glutamyl-p-ni- troanilide and 364 units/mg of protein using GSH as substrate. The purified enzyme was found to lose about 50% of its activity in 2 months at 4”.

Molecular Weight Determination-The molecular weight of the enzyme was determined by Sephadex G-200 gel filtration using cytochrome c, aldolase. glutathione peroxidase purified from red cells as described previously (31), and blue dextran as standards. The Sephadex G-200 column (5 x 90 cm) was equilibrated with 10 mM potassium phosphate buffer, pH 7.4, containing 50 mM NaCl and 0.05% Triton X-100. The flow rate was adjusted to about 60 ml/hour. The cytochrome c and blue dextran were determined spectrophotometrically at 415 nm and 540 nm, respectively. The aldolase activity was deter-

mined by the method of Beutler (32), and glutathione peroxi- dase activity was determined as described by Awasthi et al. (31). y-Glutamyl transpeptidase was eluted in two peaks (Fig. 2). The specific activity of y-glutamyl transpeptidase in both

’ Aliquots from the main enzyme activity peak and tailing fractions were subjected to rechromatography on DEAE-cellulose (DE52) for molecular aggregation studies (Fig. 4).

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Human Kidney y-Glutamyl Transpeptidase 2273

FIG. 1. DE52 column chromatography of y-glutamyl transpepti- dase. The experimental conditions are described in the text.

TABLE I

Purification of y-glutamyl transpeptidase from human kidney

The purification steps are described in the text

Homogenization Incubation at 37” Sodium deoxy-

cholate extract DEAE-cellulose

(batchwise) DE52 Sephadex G-200 Concanavalin A

51,988 51,168

4,592

956

II 55 12.0

T c

1

?nzyme s&it>

units

Specific activit)

Purili- cation Yield

unitslmg protern

0.27 0.34 6.61

fold o/

13,992 17,490 30,340

1 24

100 125 217

11,900 12.5 46 85

5,520 71.6 265 39 4,445 80.8 299 32 2,405 200.4 741 17

the peaks was the same. The major peak of enzyme activity and blue dextran were eluted simultaneously, indicating that the molecular weight of the aggregated enzyme is greater than the exclusion limit of Sephadex G-200. The apparent molecu- lar weight of the minor enzyme activity peak was found to be

about 90,000 (Fig. 2). Polyacrylamide Disc Electrophoresis-The final preparation

of y-glutamyl transpeptidase did not move on polyacrylamide disc electrophoresis in the absence of a detergent. However, in a 5% gel containing 0.02% Triton X-100, the enzyme separated into three protein bands (Fig. 3A), all of which were found to be enzymatically active when stained with y-glutamyl-p-nitroani-

lide (Fig. 3B). Amino Acid Composition of y-Glutamyl Transpep-

tidase-The amino acid composition of the purified enzyme was determined by hydrolyzing the protein at 101” with 5.5 N HCl of a constant boiling point for 24, 56, and 76 hours. The amino acids were determined by using an automatic amino acid analyzer. The results are presented in Table II. Glucosa- mine was determined to be about 7% of the total hexosamine and amino acid residues, indicating that y-glutamyl transpep- tidase is a glycoprotein.

Subunit Molecular Weight-For the determination of the molecular weight of y-glutamyl transpeptidase subunits, puri- fied human a chain globin. ribonuclease. aldolase. and bovine serum albumin were used as standards. Upon sodium dodecyl sulfate-urea-p-mercaptoethanol-polyacrylamide disc electro- phoresis the purified enzyme dissociated into one major and two minor protein bands corresponding to molecular weights of about 90,000, 57,000, and 21,000, respectively.

L

n

J

500 1000

Volume Through Column lml)

FIG. 2. Sephadex G-200 gel filtration of y-glutamyl transpeptidase fhr molecular weight determination. The enzyme purified from kidney was passed through a column (5 x 90 cm) by upward flow. The column was equilibrated with 10 rnM phosphate buffer, pH 7.4, containing 50 rnM sodium chloride and 0.05% Triton X-100. The upper figure shows that the minor peak of y-glutamyl transpeptidase was eluted from the column with the same volume as glutathione peroxidase, which has a molecular weight of about 90,000. The arrows indicate the elution of two enzyme peaks.

Aggr’egation of y-Glutamyl Transpeptidase-In the purifica- tion as well as in the molecular weight determination most of the enzyme was eluted with the void volume in Sephadex G-200 gel filtration. This indicated a very high molecular weight. possibly a molecular aggregation. Molecular aggrega- tion is further indicated by serological studies presented below and by rechromatography of the major enzyme peak fraction. (A). and the tailing fraction. (B). from DE52 column chroma- tography (Fig. 1). The tailing fraction separated into at least two peaks (Fig. 4).

Serological Studies-Serological studies were performed by incubating the reaction mixture, total volume 0.4 ml, consist- ing of 100 ~1 of purified enzyme (about 20 milliunits), 20 mM phosphate buffer, pH 7.0, and varying concentration of anti- serum at 4” for 24 hours. No inhibition of enzyme activity was observed after incubation. However after centrifugation of the reaction mixture at 48,000 x g for 1 hour, about 10 ~1 of antiserum was found to precipitate 19 milliunits of y-glutamyl transpeptidase activity.

In double immunodiffusion studies, the center well con- tained about 8 ~1 of antiserum (Fig. 5). Two precipitin lines were observed when 8 ~1 of the pure enzyme, with or without deoxycholate, was applied in the outer wells of the double immunodiffusion plates. The inner precipitin lines show iden- tity with the crude kidney extract (Fig. 5A). However, in the

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2274 Human Kidney y-Glutamyl Transpeptidase

FIG. 3. Polyacrylamide disc electrophoresis of y-glutamyl transpep- tidase. Polyacrylamide disc electrophoresis was performed by the method of Davis (28) using a 5% resolving gel containing 0.02% Triton X-100 and a 2.5% concentrating gel containing 0.04% Triton X-100. About 30 c(g of protein was applied in each gel and the electrophoresis was carried out for 4 hours at 2 mA per gel. The gels were stained for the enzyme activity and for protein as described in the text. Due to the presence of Triton X-100 in the gels, the background color was very high after staining with Amido black and destaining with acetic acid. Very diffusable yellow bands observed after staining the gels for the enzyme activity were difficult to photograph. The gels A and B are from two different polyacrylamide disc electrophoresis runs. A, stained fo+ protein; B, stained for the enzyme activity, indicated by arrows.

TABLE II

Amino acid composition of human kidney y-glutamyl transpeptidase

Values are presented taking all amino acids determined as 100.

Amino acid

Aspartic acid” Threonineb Serineb Glutamic acid” Proline” Glycine” Alanine” Half-cystine Valinec MethionineO Isoleucinec Leucine” Tyrosine’ Phenylalanine” Lysine” Histidineb Arginine” Tryptophand

Residue

70

9.47 1.74 6.98 8.87 5.83 6.70 7.52 0.98 5.08 0.25 4.35

10.39 3.26 5.15 5.71 7.71 3.95

N.D.

“Values are averages of hydrolysis for 56 and 76 hours. *Values extrapolated to zero time hydrolysis. c Values of hydrolysis for 76 hours. dN.D., not determined.

plate soaked in deoxycholate prior to application of the enzyme and the antiserum (Fig. 5B), single precipitin lines against crude kidney extract and purified enzyme show identity.

Kinetic and Other Studies-The isoelectric point of y-glu- tamvl transpeptidase was found to be about 4.5 (Fig. 6). The optimum pH for the activity of y-glutamyl transpeptidase using y-glutamyl-p-nitroanilide as substrate was found to be about 8.2.

040 100 5 E / z / z” I= A

/ /

/ 20

~

/ 50 /

/ /

/

/ 20 W 60 60 100

Volume Through Column (ml) FIG. 4. Rechromatography on a DE52 column of Fractions A and B,

from Fig. 1. Fractions A and B, as marked by parentheses in Fig. 1, were dialyzed against 10 mM phosphate buffer, pH 8.5, and equal amounts of enzyme activity (300 units) from both fractions were passed through separate DE52 columns (1 x 10 cm) at a flow rate of about 15 ml/hour. The enzyme was eluted with an increasing sodium chloride gradient from 0 to 200 mM and a decreasing pH gradient from 7.0 to 6.0 in 10 rnM phosphate buffer containing 0.5% Triton X-100. A, re- chromatography of Fraction A from Fig. 1 and B, rechromatography of Fraction B from Fig. 1.

K, for y-Glutamyl-p-nitroanilide-The K, of y-glutamyl transpeptidase for y-glutamyl-p-nitroanilide was found to be about 0.8 mM (Fig. 7). When glycylglycine was used as the acceptor amino acid and y-glutamyl-p-nitroanilide as the

substrate, GSH and GSSG were found to competitively inhibit the enzyme activity (Fig. 7).

Acceptor Amino Acids-The activity of human kidney y-glutamyl transpeptidase toward various acceptor amino acids was determined by using y-glutamyl-p-nitroanilide. In a l.O-ml system, 4 mM y-glutamyl-p-nitroanilide, 50 mM Am- mediol-HCl buffer, pH 8.0, and 20 mM amino acid were incubated for 10 min at 37’. Twenty microliters of enzyme was added to start the reaction.

Because y-glutamyl-p-nitroanilide acts also as an acceptor, a significant amount of y-glutamyl transpeptidase activity was observed even in the absence of an acceptor amino acid. In the presence of most of the amino acids, the enzyme activity was either unaltered or slightly increased (Table III). Glutamine and glycylglycine, however, increased the enzyme activity by 4- and lo-fold, respectively.

Effect of Monoualent Cations-In a total volume of 0.98 ml, the monovalent cations were pre-incubated with 4 mM y-glu- tamyl-p-nitroanilide, 50 mM Ammediol-HCl buffer, pH 8.1, and 20 mM glycylglycine for 5 min. Twenty microliters of enzyme solution was added to start the reaction. The monovalent cations such as sodium, potassium, and lithium at concentra- tions up to 300 mM, had no significant effect on the enzyme activity. However, ammonium ions had a slight inhibitory effect (35%) on the enzyme activity.

Effect of Divalent Cations-The enzyme was incubated with divalent cations (Mg’+, Mn*+ and Ca2+) in 0.5 ml of 50 mM Ammediol-HCl buffer, pH 8.04, for 5 min at 37”. Ten micro- liters of 0.25 M EDTA, pH 8.7, was then added where specified and ‘incubated for another 5 min. The enzyme reaction was started by the addition of 0.5 ml of the substrate mixture. The final concentration of y-glutamyl-p-nitroanilide was 2.2 mM

and that of glycylglycine and the cations was 10 mM. The

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Human Kidney y-Glutamyl Transpeptidase 2275

Fro 5. Double immunodiffusion studies in the presence and ab- sence of sodium deoxycholate. Plate A was used without soaking in deoxycholate and Plate B was soaked for 6 hours in 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate. The center well contained about 8 ~1 of anti-y-glutamyl transpeptidase antiserum. The outer wells contained about 8 ~1 of: 1, crude kidney extract prepared in 1% sodium deoxycholate; 2, purified y-glutamyl transpeptidase; and 3, purified y-glutamyl transpeptidase in 1% sodium deoxycholate. The slides were incubated overnight at room temperature.

50 loo VOLUME FROM COLUMN (ml)

FE. 6. Isoelectric point of y-glutamyl transpeptidase. The isoelec- tric point of y-glutamyl transpeptidase was determined using an isoelectric focusing column (110 ml) and Ampholine from pH 4 to 6. The electrofocusing was carried out for the first 2 hours at 300 volts, for the next 2 hours at 400 volts, and for the final 48 hours at 500 volts.

divalent cations, Mg2+, Mn2+, and Cal+ at a concentration of 10 mM did not significantly affect the enzyme activity. Preincubation of the enzyme with 2.5 mM EDTA had no effect on the enzyme activity.

Effect of Sulfhydryl Blocking Reagents-The effect of sulf- hydryl blocking reagents on the activity of y-glutamyl trans- peptidase was determined by incubation of the enzyme with a sulfhydryl blocking reagent in 0.5 ml of 50 mM Ammediol, pH 8.04 for 5 min at 37”. The enzyme reaction was started by the addition of 0.5 ml of substrate mixture. The final concentra- tions of Ammediol, y-glutamyl-p-nitroanilide and glycylgly- tine were 50 mM, 2.2 mM, and 11 mM, respectively. p-Hydrox- ymercuribenzoate, N-ethylmaleimide, and iodoacetate did not inhibit the enzyme activity up to a concentration of 1 mM.

However, higher concentrations (10 mM) inhibited the enzyme activity by about 30%.

Studies on Identity of y-Glutamyl Transpeptidase with

- Enzyme Alone

15. . 5OpMGSH x - IWpMGSH .e.... 2034,AGSH H( 4066pMGSH

/

15. O--O Enzyme AMne

b--d IWrMGSSG - 2034uMGSSG x--x 4066rMGSSG

I r-Glutamyl p-N~f,oan,l,de (mM)

FIG. 7. Inhibition of y-glutamyl transpeptidase activity by GSH and GSSG using y-glutamyl-p-nitroanilide as substrate. The reaction mixture contained in a final volume of 1 ml 50 rnM Ammediol-HCl buffer, pH 8.0, and 20 mM glycylglycine. The concentration of y-glutamyl-p-nitroanilide was varied from 0.22 to 1.1 rnw

Phosphate-independent Glutaminase-y-Glutamyl transpep- tidase and phosphate-independent glutaminase activity have been reported to be exhibited by an apparently single protein (22, 23). Maleate has been shown by various investigators to stimulate the phosphate-independent glutaminase activity and supress the y-glutamyl transpeptidase activit,y of the enzyme (22, 23, 33). Likewise, amino acids have been shown to inhibit the glutaminase activity and activate the transpepti- dase activity. In view of these findings, the effect of both maleate and methionine on the purified enzyme was studied. Because glycylglycine was found to be a better acceptor for the y-glutamyl moiety, methionine was omitted from the reaction mixture for the determination of transpeptidase (Table IV).

Papain has been used by different investigators for the solubilization of y-glutamyl transpeptidase. Therefore, to determine the effect of papain on the hydrolysis of y-glutamyl transpeptidase, 50 pg of purified enzyme was incubated with

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2276 Human Kidney y-Glutamyl Transpeptidase

TABLE III TABLE IV

Effect of various amino acids on actiuit 2

of human kidney y-glutamyl transpeptl ase

Phosphate-independent glutaminase actiuity of purified y-glutamyl transpeptidase and effect of papain treatment

In a 0.285ml system, 50 pg of purified y-glutamyl transpeptidase was incubated with 50 pg of soluble papain (1.0 unit), 0.1 mM P-mercaptoethanol, 1.0 mM cysteine, and 0.2 mM EDTA for 30 min at 37”. Papain was omitted from the control samples. After incubation the samples were immediately cooled to 4” and assayed for y-glutamyl transpeptidase as described in the text and phosphate-independent glutaminase as described by Curthoys and Weiss (34). Wherever specified the concentration of maleate and methionine was 60 rnM and 20 mhq respectively.

L-Amino Acid Enzyme Activity

None Glycylglycine Asparagine Arginine Histidine Lysine Glutamine Methionine Cysteine Leucine Isoleucine Tryptophan Phenylalanine Alanine Glycine Serine Threonine Valine Proline Hydroxyproline Aspartic acid Glutamic acid a-Aminoisobutyric acid

% of control

100 1,060

160 96

150 94

415 120

94 160 102

91 143 124 94 89 84 87 92 89

155 126 91

3.0 mg of papain (0.3 units) insolubilized on carboxymethylcel- lulose in the presence of 0.07 mM P-mercaptoethanol, 1.0 mM cysteine, and 0.2 mM EDTA for 90 min at 37” in a Dubnoff shaker (100 oscillations per min). Papain was omitted from the

control samples. The samples were subsequently centrifuged, and the supernatant was subjected to polyacrylamide disc electrophoresis (28). Treatment of the enzyme with papain had no effect on the total enzyme activity nor on the mobility of the enzyme on polyacrylamide disc electrophoresis. This indicates that papain does not cleave y-glutamyl transpeptidase. Incu- bation of purified y-glutamyl transpeptidase with soluble papain had no significant effect on the enzyme activity (Table IV). The purified y-glutamyl transpeptidase did not show any detectable phosphate-independent glutaminase activity, even

in the presence of maleate (Table IV). We have used various methods for the extraction of y-glu-

tamyl transpeptidase and phosphate-independent glutaminase

from human kidney cortex homogenate (Table V). The ratio of y-glutamyl transpeptidase activity to phosphate-independent glutaminase activity, determined in the presence of maleate, was found to be about 8.6 in the kidney supernatant without

treatment of the homogenate with sodium deoxycholate. How- ever. treatment of the homogenate with sodium deoxycholate or papain (as described in Table V) increases this ratio in the

supernatant to about 22 (Table V, Samples 1, 2, and 3). The increased solubilization of y-glutamyl transpeptidase without a concomitant increase in the activity of phosphate-independ- ent glutaminase indicates that these two enzyme activities are not exhibited by the same protein. The ratio of these two enzyme activities is further increased to about 40 when the

pellet is extracted with sodium deoxycholate (Table V, Sam- ple 5). No significant inactivation of either of the enzymes was observed when various methods, as presented in Table V, were used for the extraction of the enzyme. The addition or

I Phosphate-independent y-Glutamyl glutaminase actlwty transpeptidase

activity

Sample ma, a,

wit1 -̂

With ‘eate With Without Without Id maleate maleate lout and and

methi- methl- :“,“d’zF VJtk& With

maleate m&hi- Onlne onlne omne CmlM!

units/ml

No papain <0.02 <0.02 <0.02 <0.02 9.50 9 25 Papain <0.02 <0.02 <0.02 <0.02 9.97 8.91

omission of maleate and/or methionine did not significantly affect the phosphate-independent glutaminase activity.

DISCUSSION

y-Glutamyl transpeptidase transfers the y-glutamyl moiety from glutathione and other y-glutamyl peptides to a variety of acceptor amino acids. Recently, Meister (20, 21) has impli- cated this enzyme in the transport of amino acids into the cells of various tissues, especially kidney. Several attempts in the past have been made by various investigators (3-7, 9, 22, 23) to

purify y-glutamyl transpeptidase from mammalian tissues. During the purification of y-glutamyl transpeptidase from rat and human kidney, proteolytic enzymes were used for its solubilization (5, 22, 23); however, in the present studies

sodium deoxycholate was used. We have now purified this enzyme to an apparent homogeneity from human kidney cortex. The separation of purified y-glutamyl transpeptidase on polyacrylamide disc electrophoresis into three protein bands which stain for the enzyme activity indicates that the final preparation is apparently homogeneous. The slower moving bands probably represent aggregated forms of the enzyme. A single precipitin line observed against the homogenate in double immunodiffusion studies in the presence of a detergent (in the gel as well as in the enzyme preparation) further supports the homogeneity of the final preparation, and the

appearance of more than one precipitin line in double immuno- diffusion studies in the absence of a detergent indicates a molecular aggregation. Although it is unlikely, the possibility of deoxycholate preventing the precipitation of certain antigen. antibody complexes may also be considered.

y-Glutamyl transpeptidase was found to be sparingly soluble in the absence of a detergent such as sodium deoxycholate or Triton X-100. A strong tendency of the protein towards aggregation is evident by the two enzymatically active peaks obtained in Sephadex G-200 gel filtration. The major peak is

eluted from Sephadex G-200 immediately after the void volume, indicating a molecular weight larger than the exclu- sion limit of Sephadex G-200. The apparent molecular weight of y-glutamyl transpeptidase that appeared to be deaggregated

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Human Kidney y-Glutamyl Transpeptidase 2277

TABLE V

Different methods for solubilization of y-glutamyl transpeptidase and

phosphate-independent glutaminase from human kidney cortex

A 20% homogenate of human kidney cortex was prepared in 80 IIIM MgCl, containing 0.75 rn~ NaOH using a Sorvall Omni-Mixer at 8,000 rpm for 6 min. Ten-milliliter aliquots were treated as described below. The supernatant samples designated from 1 to 7 were used for the enzyme assays. All the centrifugations were carried out at 10,000 x g for 30 min at 4”. Wherever papain was used, 0.07 rn~ P-mercapto- ethanol, 1.0 rn~ cysteine, and 0.2 rn~ EDTA were incorporated in the reaction mixture. 1. Centrifugation, and separation of supernatant; 2. addition of 10% sodium deoxycholate, pH 8.0, to a final concentration of IYo, rehomogenization, stirring overnight at 4”, centrifugation, and separation of supernatant; 3. addition of papain (20 unit&g of protein) to a final concentration of 0.25 mg/ml of homogenate, incubation at 37” for 3 hours, and separation of supernatant; 4. incubation at 37” for 2.25 hours and separation of supernatant; 5. the pellet from Sample 4 was resuspended in 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate to make up to 10 ml, mixed overnight at 4”, and rehomogenized, and the supernatant was sepa- rated; 6. an aliquot from Sample 2 was incubated at 37” for 3 hours with 0.25 mg of papain/ml of sample; 7. an aliquot from Sample 5 was incubated at 37” for 3 hours with 0.25 mg of papain/ml of sample. The concentration of maleate when used was 60 mu; the concentration of methionine when used was 20 mu.

Phosphate-Independent glutaminase

Sam- de

Rlth maleate

and without methi- onine

With Without WIthout maleate maleate maleate

and and and with methi- methi~ methi- OllllltZ (mine onine

GluLyl trans-

peptidase

units/ml

1 0.36 0.35 0.31 0.33 3.15

2 0.28 0.27 0.26 0.26 7.00

3 0.46 0.43 0.43 0.46 9.90

4 0.37 0.31 0.37 0.39 2.47

5 0.19 0.19 0.18 0.20 7.64

6 0.27 0.26 0.29 0.31 7.80

I 0.30 0.26 0.30 0.27 8.00

in the presence of Triton X-100 was found to be about 90,000 by Sephadex G-200 gel filtration. Because the subunit molecular weight using sodium dodecyl sulfate-urea-p-mercaptoethanol- polyacrylamide disc electrophoresis was found to be 21,000 and 57.000, it appears that the subunits are heterogeneous. How- ever, the possibility that the enzyme is a tetramer of subunits having a molecular weight of about 21,000, and the possibility of the protein band corresponding to a molecular weight of 57,000 representing a trimer cannot be ruled out.

The competitive inhibition of y-glutamyl transpeptidase by GSH or GSSG when y-glutamyl-p-nitroanilide was used as substrate indicates that this enzyme uses the same catalytic site for the artificial substrate and for reduced and oxidized glutathione. Richter (5) also had demonstrated a significant inhibition by GSH and GSSG when y-glutamyl-p-nitroanilide

was used as substrate. Elce (35) has reported a significantly high K, for methionine, 2 to 4 mM, using [l’C]GSH as substrate. This would indicate that it is unlikely that y-glu-

tamyl transpeptidase plays a major role in the transport of amino acids into the kidney cell.

Magnesium has been used by various investigators (4-6, 24) in the determination of y-glutamyl transpeptidase, and Richter (5) has reported a 25% increase in the enzyme activity in the presence of 5 mM Mgz+ or EDTA. However, we have

failed to find any significant effect of this cation or EDTA on the activity of the purified enzyme preparation. Using kidney homogenate from various mammalian species, Orlowski et al. (36) have reported a significant (80 to 125%) increase in the activity of y-glutamyl transpeptidase by monovalent cations. Their pure enzyme preparation from hog kidney, having a specific activity of 18 units/mg of protein, was still significantly activated by Na+, K+, Cs+ and Li’. We, however, found no significant effect of the monovalent cations, such as sodium

and potassium on the activity of purified y-glutamyl transpep- tidase from human kidney. Ammonium ions, on the other hand, decreased the enzyme activity slightly.

The enzyme did not appear to require sulfhydryl groups for the activity because 1 mM N-ethylmaleimide, p-hydroxymer- curibenzoate, and 10 mM iodoacetate had no significant effect on the enzyme activity. These results are in agreement with Richter (5).

The absence of any significant amount of phosphate-

independent glutaminase activity in the purified preparation of y-glutamyl transpeptidase in the presence or absence of maleate (Table IV) suggests that glutaminase and transpepti- dase activities are not exhibited by the same protein in human kidney cortex as suggested by Curthoys and Kuhlenschmidt (22) and Tate and Meister (23) for the rat kidney enzyme. The separation of y-glutamyl transpeptidase and phosphate- independent glutaminase activity after extraction of the human kidney homogenate with papain and sodium deoxycho- late (Table V) also indicates that these enzyme activities are exhibited by different proteins. It is possible that human kidney y-glutamyl transpeptidase is different from rat kidney

enzyme. Richter (5) had shown earlier that human kidney enzyme is very unstable in the presence of butanol, whereas, under similar conditions, hog and beef kidney enzyme are more stable.

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S P Miller, Y C Awasthi and S K Srivastavastructural, kinetic and immunological properties.

Studies of human kidney gamma-glutamyl transpeptidase. Purification and

1976, 251:2271-2278.J. Biol. Chem. 

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