Neurochemical Research (1) 251-259 (1976)

SUBCELLULAR AND REGIONAL L O C A L I Z A T I O N OF y - G L U T A M Y L T R A N S P E P T I D A S E IN SHEEP B R A I N EDWARD REYES AND ERIC E. PRATHER The Department of Pharmacology The University of New Mexico School of Medicine Albuquerque, New Mexico 87131 The University of Wyoming School o f Pha~wmcy Laramie, Wyoming 82071

Accepted February 17, 1976

Studies of the subcellular distribution of y-glutamyl transpeptidase from sheep brain by discontinuous sucrose density gradient centrifugation showed that 40% of the transpeptidase activity associated with the mitochondrial-synaptosomal fraction was localized with the synaptosomal-enriched fraction. The microsomal fraction was found to have the highest specific activity when y-glutarnyl p nitroanalide was used as substrate. This activity, however, represented only 5% o f the total y-glutamyl transpeptidase activity. Approximately 90% of the total enzyme activity was apparently associated with the fraction containing cell debris and membrane fragments. The 160,000g supernatant fluid (soluble supernatant fraction) represented the least total activity, with only 1.2% recovery; however, this fraction contained two apparent forms of the enzyme. One form had a high K,~ and the other a low K,,, for the substrate, y-glutamyl p-nitroanilide. It was observed that the enzyme y-glutamyl transpeptidase was not evenly distributed in all areas of brain when the homogenate was used as the enzyme source. The brain region with the highest enzyme activity was the thalamus, which was able to form 1.10 /zmol p-nitroanaline/min/g wet brain tissue. The cortex was found to have the lowest activity. The 40,000g supernatant fluid from each region, however, exhibited only slight distribution differences.

INTRODUCTION y-Glutamyl transpeptidase (2.3.2.1.) a predominantly membrane-bound enzyme, has been isolated and studied in preparations of liver, kidney, 251 9 1976 Plenum PublishingCorporation,227 West 17th Street, New York, N.Y. I0011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying,microfilming,recording,or otherwise,without written permissionof the publisher.

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pancreatic tissue, brain choroid plexus, and cerebral cortex (1-5). This enzyme catalyzes the metabolism of glutathione (GSH) and has therefore been integrated into the y-glutamyl cycle. Because of the high concentration of the enzyme in the brush border of the proximal convoluted tubules of the kidney, it has been postulated that the enzyme is involved in amino acid transport in the kidney (6). There is additional evidence to suggest that the enzyme is involved in movements of amino acids in the central nervous system (CNS) (7). Enzymes of the y-glutamyl cycle have been implicated in mental retardation and other symptoms of degenerative diseases in the CNS (5). Although several studies have demonstrated high concentrations of G S H in all regions of the brain, little is known about the regional and subcellular localization of the major enzyme involved in its metabolism, y-glutamyl transpeptidase (8-10). Szewczuk (11) has shown that although most of the y-glutamyl transpeptidase activity can be associated with membranes, approximately 10% of the enzyme activity can be ascribed to a soluble fraction. This study was undertaken in an effort to establish the regional and subcellular localization of y-glutamyl transpeptidase in sheep brain. A secondary goal of the project was to determine whether the soluble form of the enzyme present in liver and kidney is likewise present in the brain. We wished to further determine whether both enzyme forms have the same kinetic characteristics toward the substrate, y-glutamyl pnitroanilide.

EXPERIMENTAL PROCEDURE

Materials All chemicals used were of the highest quality that could be obtained from commercial sources. Sigma Chemical Co. supplied L-y-glutamyl p-nitroanilide, glycylglycine (free base), Tris (hydroxymethyl) aminomethane, 5-hydroxytryptamine, pyruvic acid as the sodium salt, and/3-nicotinamide adenine dinucleotide, reduce form (NADH). Amersham/ Searle supplied 5-hydroxy[side chain-2-14C] tryptamine creatinine sulphate.

Procedure Subcellular and Regional Distribution of y-Glutamyl Transpeptidase. In studies concerning the subcellular distribution of 3,-glutamyl transpeptidase, sheep brains were placed, following decapitation, in cold (4~ isolation media [0.32 M sucrose, 4 mM Tris (hydroxymethyl) aminomethane, 1 mM E D T A (disodium ethylenedinitrilotetraacetate) adjusted to pH 7.4]. The brains were homogenized in sufficient isolation media to yield a 20% (wt/vol) suspension. Homogenization was accomplished in a cold glass homogenizer

y-GLUTAMYL TRANSPEPTIDASE IN SHEEP BRAIN

253

with a tightly fitting Teflon pestle (clearance 0.004-0.006'). The homogenate was centrifuged at l l00g for 10 min at 2~ and the supernatant fluid was combined with the supernatant fluid similarly obtained after homogenization of the pellet in half the original volume of isolation media. The combined supernatant fractions were then centrifuged at 20,000g for 20 min. The resultant supernatant fluid was again centrifuged at 160,000g for 60 rain to yield the soluble supernatant enzyme fraction. The pellet from the 20,000g centrifugation (crude synaptosomal-mitochondrial fraction) was washed twice by resuspending the pellet in the original volume of isolation media and centrifuging the suspension at 20,000g for 20 rain. The final crude synaptosomal-mitochondrial pellet was suspended in isolation media (15 mg/ml) and layered over a discontinuous sucrose gradient, and various fractions were obtained by the method of DeRobertis et al. (12) and Whittaker et al. (13). The sucrose gradient preparation was centrifuged at 102,000g for 45 min in a Beckman SW 27 rotor. Various fractions were collected from the sucrose gradient by means of a Jshaped pipette, y-Glutamyl transpeptidase activity was determined in each fraction. Lactic dehydrogenase activity was also determined as a synaptosomal marker for individual fractions, as described by Coyle and Axe~rod (14). Monoamine oxidase activity was determined as a mitochondrial marker for each fraction (15). In studies concerning the regional distribution of y-glutamyl transpeptidase, sheep brains were placed in cold (4~ isolation media immediately after decapitation, and the various regions were separated within 1 h. Each brain region was homogenized as described above, y-Glutamyl transpeptidase activity was determined for each region for both the homogenate and the 40,000g supernatant fluid. Enzyme Assays. Sheep brain y-glutamyl transpeptidase activity was determined spectrophotometrically by following the increase in absorbance ofp-nitroaniline at 405 nm at 37~ with a Beckman Model 25 kinetics system spectrophotometer (16,17). A typical reaction mixture consisted of protein (0.15-1.5 nag); 5.3 mM L-y-glutamyl p-nitroanilide, dissolved in dilute HCI; 110.5 mM glycylglycine; and 92 mM Tris (hydroxymethyl) aminoraethane, pH 8.5, in a total volume of 2.0 ml at 37~ as described by Rosalki and Tarlow (17). The reaction was initiated by the addition of L-y-glutamyl p-nitroanilide to the reaction mixture. All graphs and tables indicate which concentration of substrate was used. Lactic dehydrogenase activity was determined spectrophotometrically by following the decrease in absorbance of N A D H at 340 nm. The reaction mixture consisted of protein (0.03-0.06 rag); 0.07 mM N A D H ; 0.3 mM sodium pyruvate; and 0.1 M sodium phosphate, pH 7.4, in a total volume of 3.0 ml at 25~ (14). Monoamine oxidase activity was determined by the method of Robinson et al. (15). The reaction mixture consisted of protein (0.03-0.06 mg); 1 mM 5-hydroxytryptamine; 0,043 /xM 5-hydroxy [side chain-2-14C] tryptamine; and 0.1 M sodium phosphate, pH 7.4, in a total volume of 0.5 ml at 37~ The reaction mixture was allowed to incubate for 30 rain. Protein concentrations in homogenates and supernatant fractions were determined by the biuret method (18). Bovine serum albumin was used as the standard for protein determinations.

RESULTS y-Glutamyl

transpeptidase

activity was observed

i n all a r e a s o f s h e e p

b r a i n t h a t w e r e e x a m i n e d , a s s h o w n i n T a b l e I. I n t h e b r a i n h o m o g e n a t e , a c t i v i t y r a n g e d f r o m 1.10 t o 0.01 / x m o l p - n i t r o a n i l i n e f o r m e d / m i n / g

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REYES A N D P R A T H E R

TABLE I DISTRIBUTION OF "y-GLUTAMYL TRANSPEPTIDASE ACTIVITY IN VARIOUS AREAS OF SHEEP BRAIN a 3'-Glutamyl transpeptidase activity (/xmol p-nitroaniline formed/rain per g tissue) Area

Homogenate

Supernatant (40K • g)

1.10 0.71 0.27 0.12 0.06 0.06 0.01

0.11 0.06 0.10 0.08 0.12 0.08 0.08

Thalamus Medulla Caudate b Midbrain-hypothalamus Pons Cerebellum Cortex

a Procedures for dissection of the various areas are described in the text. Assays for activity were performed using L-y-glutamyl p-nitroanilide as substrate (5.4 AM). b Head of the caudate nucleus.

wet tissue, with the thalamus having the highest activity and the cortex the lowest. When the 40,000g supernatant fluid was used as the enzyme source, only slight distributional differences were observed. This distribution order was different from the order observed when the homogenate was used as the enzyme source.

T A B L E II SUBCELLULAR LOCALIZATION OF T-GLUTAMYL TRANSPEPTIDASE ACTIVITY IN SHEEP BRAIN a

Fraction Homogenate 1,100g Supernatant fluid 20,000g Supernatant fluid Mitochondrial-synaptasomal Microsomal 160,000g Supernatant fluid

p-Nitroanilineforming capacity b Recovery (~mol formed) (%) 832.71 81.62 31.99 57.51 41.42 10.05

I00 9.30 3.84 6.91 4.97 1.21

Total protein (rag)

Recovery (%)

103,700 7,760 4,380 1,800 504 3,320

100 7.48 4.22 1.74 0.49 3.20

a The various fractions were obtained by modification of the method of Whittaker, as described in the text. Aliquots of the various fractions were assayed for enzyme activity as described in the text, using L-y-glutamyl p-nitroanilide as substrate (5.4 AM). b Results are the means of 2 separate experiments.

T-GLUTAMYL TRANSPEPTIDASE IN SHEEP BRAIN

255

The subcellular distribution of y-glutamyl transpeptidase activity from whole sheep brain is shown in Table If. Approximately 90% of the total enzyme activity was found to be associated with the pellet obtained from centrifugation at l l00g. The supernatant fluid was found to have a specific activity of 0.003 ~mol p-nitroaniline formed/rain/rag protein. This fraction, however, represented only 9.3% of the total y-glutamyl transpeptidase activity recovered. The highest specific activity was associated with the microsomal fraction, which was able to utilize 0.082 izmol y-glutamyl p-nitroaniline/min/mg protein. The twice-washed curde mitochondrial-synaptosomal--enriched fraction represented 6.9% of the total enzyme activity. Discontinuous sucrose gradient centrifugation of the crude mitochondrial-synaptosomal-enriched preparation showed that 45% of the enzyme activity in this preparation was recovered in the fraction having the highest lactic dehydrogenase activity. As shown in Fig. 1, the fraction with the highest monoamine oxidase activity was found to have 8% of the recovered transpeptidase activity.

40

[]

GGTP

[ ] MAO IAJ 0

L I0 I0

FRACTION

SUCROSE

[]

LDH

2 A

E

B

0.8M

C

D

I.oM

E

F

1.2M

1.4M

FIG. 1. Distribution of enzyme activities in fractions of a crude sheep brain mitochondrialsynaptosomal preparation after separation on a discontinuous sucrose gradient. The crude mitochondrial-synaptosomal preparation, as described in the text, was separated into fractions A - F (see lower portion of figure) by centrifugation through a discontinuous (0.8-1.4 M) sucrose density gradient. The activities of y-glutamyl transpeptidase (GGTP), occluded lactic dehydrogenase (LDH), and monoamine oxidase (MAO) were measured in each fraction. Activity is expressed as a percentage of total activity recovered from the crude mitochondrial-synaptosomal fraction.

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REYES A N D P R A T H E R

1.0 1IV

O.5

~...~---~-"~

1;

2;

3b

4'0

100 / [ ~-GLUTAMYLp-NITROANILIDE],mM

FIG. 2. Formation ofp-nitroaniline by 3,-glutamyl transpeptidase from the soluble supernatant fraction of sheep brain. 7-Glutamyl transpeptidase from the soluble supematant fraction was assayed in 110.5 mM glycylglycine, 92 mM Tris ~hydroxymethyl) aminomethane, pH 8.5, and various concentrations of L-7-glutamyl p-nitroanilide. Results are presented as a double-reciprocal plot. The ordinate, 1/v represents /zmol p-nitroaniline formed/rain/rag protein.

The microsomal and soluble supernatant fractions were examined for 7-glutamyl transpeptidase activity over a 400-fold concentration range of L-y-glutamylp-nitroanilide and a constant glycylglycine concentration of 110.5 mM. The double-reciprocal Lineweaver-Burk plot obtained by using the soluble supernatant fraction as the enzyme source had a distinct biphasic shape, as seen in Fig. 2. Apparent Km values for 7-

12-

14-.

o

o

lO.~ v

[S]

8

6 4 o

2" 0

~o ~ ~ . , . . , ~ . ~ . . . _ g

10

1,5

V FIG. 3. Formation ofp-nitroaniline by 7-glutamyl transpeptidase from the soluble supernarant fraction of sheep brain. 7-Glutamyl transpeptidase from the soluble supernatant fraction was assayed in 110.5 mM glycylglycine, 92 mM Tris (hydroxymethyl) aminomethane, pH 8.5, and various concentrations of L-y-glutamyl p-nitroanilide. Results are presented as an Eadie-Scatchard plot, v/[s] versus v, in which v represents p.mol pnitroaniline formed/mirdmg protein.

y-GLUTAMYL TRANSPEPTIDASE IN SHEEP BRAIN

257

glutamyl p-nitroanilide of 5.8 mM and 0.38 mM were obtained by extrapolation of the linear portions of the curve. The Eadie-Scatchard plot shown in Fig. 3 also exhibits a distinct biphasic shape. These effects are assumed to be due to the presence of two or more enzymes with different affinities for y-glutamyl p-nitroanilide in the same enzyme preparation. Examination of the microsomal preparation over the same substrate concentration range yielded a linear Lineweaver-Burk plot with an apparent K,~ value of 2.2 raM.

DISCUSSION The studies on the regional distribution of y-glutamyl transpeptidase provide evidence that the operation of the y-glutamyl cycle is not limited to any one specific region of the brain, as shown by the ubiquitous distribution of the enzyme. The subcellular distribution of y-glutamyl transpeptidase activity shown in Table II indicates that the largest amount of activity is found associated with the cell debris and nuclear material, and the least amount of activity with the soluble supernatant fraction, which represents soluble celt components. These data are in agreement with reports showing that the enzyme is membrane-bound in brain and other tissues (1,7). They are also consistent with the proposed function of the enzyme in coupling amino acid uptake to G S H metabolism (6). By use of lactic dehydrogenase as a synaptosomal enzyme marker, it was possible to associate the high y-glutamyl transpeptidase activity with the synaptosomal-enriched fraction (14). This finding also seems consistent with the enzymes' function in amino acid transport across membranes. Examination of Figs. 2 and 3 shows that the Lineweaver-Burk plot and the Eadie-Scatchard plot of the supernatant enzyme preparation had a distinct biphasic shape indicative of the presence of two enzymes with different affinities for ~/-glutamyl-p-nitroanilide. The microsomal preparation, however, contained only one enzyme form, as indicated by a linear Lineweaver-Burk plot. The values found with either preparation were substantially higher than those reported b3) other investigators for kidney (1). The difference may be attributed to the use of higher substrate concentrations in this study, as opposed to the procedure used by other investigators (1). The apparent presence of two ),-glutamyl transpeptidases in the CNS suggests the possibility for two physiological roles for the enzyme. The

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low-K,, form may function in the transport of amino acids across membranes where very low substrate concentrations would allow the enzyme to function at a maximum rate, whereas the high-Kin form may function in detoxification phenomena. This study raises the possibility that ~/-glutamyl transpeptidase may have at least one isozyme, or that negative cooperativity in this system may exist. Certainly, further studies are needed in this area.

ACKNOWLEDGMENT

This work was supported in part by a grant from The University of Wyoming Research Coordination Committee, The School of Pharmacy of the University of Wyoming, and by MBS program grant 081-39.

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12. DEROBERTIS, E., PELLEGRINADE DRAKDI, A., ARNAIZ, G. R., mad ZIEHER, L. M. (1965) Synaptic vesicles from the rat hypothalamus. Isolation and norepinephrine content. Life Sci. 4, 193-201. 13. WHITTAICER, V. P., MICHAELSON, I. A., and KIRKLANO, R. J. A. (1964) The separation of synaptic vesicles from nerve-ending particles (synaptosomes). Biochem. J. 90, 293-303. 14. COYLE, J. T., and AXELROO, J. (1972) Dopamine fl-hydroxylase in the rat brain: developmental characteristics. J. Neurochem. 19, 449--459. 15. ROBINSON, D. S., LOVENBERT, W., KEISER, H., and SJOEIDSNAS,A. (1968) Effects of drugs on human blood platelet and plasma amine oxidase activity in vitro and in vivo. Biochem. Pharmacol. 17, 109-119. 16. SKINNER, J. E. (1971) Neuroscience--A Laboratory Manual, W. B. Saunders, Co., Philadelphia, Pa. 17. ROSALKL S. B.,and TARLOW, D. (1974) Optimized determination of y-glutamyltransferase by reaction-rate analysis. Clin. Chem. 20, 1121-1124. 18. GARr~ALL, A. G., BARDAIVrLL, G. J., and DAVID, M. M. (1949) Determination of serum protein by means of the biuret reaction. J. Biol. Chem. 177, 751-766.