Exp. Eye Res. (1979) 29,109-121
Gamma-Glutamyl Transpeptidase of Bovine Ciliary Body : Purification and Properties NOVEEN
D. DAS AND HITOSHI
SHICHI
L&oratory
of I’ision Research, National Eye Institute. Xational Institutes of Health, U.S. Department of Health, Education and TYe@e. Bethesda, Md 20205, U.S.A.
(Received 9 November 1978> Xrw York) Gamma-glutamyl transpeptidase activity in the bovine ciliary body is six to ten times higher than those in the iris, retina and pigment epithelium. The enzyme activity of the bovine lens is very low. When the ciliary body homogenate is fractionated, the activity is largely found in the microsomal fraction. After solubilization with 0.5% Emulphogene BC-720, the enzyme has been purified 32 times by fractionation with ammonium sulfate and ion exchange chromatography on epichlorohydrin triethanolamine cellulose. The apparent K,, of the ciliary body enzyme for L-y-glutamyl-p-nitroanilide is determined to be 1.3 mM when glycylglycine is used as the y-glutamyl acceptor. The activity is inhibited markedly by Ca2+ (150 m&r), Mg2f (150 IIIM), Na2+ (100 mM) and maleate (60 mM). Of the 23 amino acids tested, glycylglycine, L-glutamine and L-methionine are especially good acceptors. A comparison of the ciliary body enzyme with bovine kidney enzyme indicates that the two enzymes are similar in their solubility in ammonium sulfate, behavior on epichlorohydrin triethanolamine cellulose, activity toward different amino acid acceptors, pH optima, and apparent K, values for y-glutamyl-p-nitroanilide. However, they are different in the electrophoretic mobilities in polyacrylamide gel; the ciliary body enzyme migrates faster toward the anode than the kidney enzyme. After treatment of bhe enzymes with neuraminidase, however, they migrate in electrophoresis at essentially identical mobilities. It. is therefore concluded that the difference in electrophoretic behavior between ciliary enzyme and kidney enzyme is attributed to different amounts of sialic acid present. Key words: Gamma-glutamyl transpeptidase; bovine ocular tissues; ciliary body; kidney: neuraminidase, digestion; sialic acid cont,ent.
1. Introduction The enzyme? y-glutamyl transpeptidase (E.C.2.3.2.2.), catalyzes transfer of the yylutarnyl group of glutathione (and of certain other y-glutamyl compounds) to a suitable amino acid acceptor to form y-glutamyl amino acids or hydrolysis of glutathionc to glutamic acid and cysteinylglycine. In either reaction, the enzyme is involve (l:os,~ et al., 1973). Thus. the ciliary I~~tly opitheliunr is one of thr t,issuc< t,hat sho\v t II{, highest y-GT activity in the body. Whatever the physiological role of y-G’l’ ttt:ty Iw. the high level of the cnzymic activity in the cilia,r,v tmdy suggests its flmctiotial a cellular I’(TIC li tr importance for this tissuca. As a prelinrinarv .I step to eluciclating y-GT. wc have attempted in t)his work t,o purify ant1 characterize y-GT f’rotr1 I~ocitrc~ cilmry l)otlr.
2. Materials and Methods L-y-Glutamyl-pnitroanilide, glycylglycine and amino acids were purchased from Sigma acrylamide, bisacrylamide, N,N,N’.N’Chemical Co., MO. ECTEOLA-cellulose, tetramethylethylenediamine, Coomassie blue and other reagents for electrophoresis were purchased from Bio-Rad, CA. Emulphogene BC-720 was a generous gift from GAF Corporation, NY. All other reagents used were of analytical grade. DL-3-;lnlillo-l-ohlortl5-methylhexan-2-one was kindly provided by Dr Peter Kador.
y-GT activity was assayed according to the method of Szasz (1969). The reaction mixture (1 ml) contained 90 pmol Tris HCI, 0.5 pmol glycylglycine, 0.12 pm01 y-glutamyl-l/nitroanilide, and 0.26 pmol MgCl,. The reaction was started by the addition of 25 ~1 enzytne and the optical density increase at 405 nm was followed with a Cary I4 recording spectrophotometer. Protein was determined by the method of Lowry, Rosebrough, Farr and Randall (1951) using bovine serum albumin as standard. Cytochrome c ox&se and NADPH-cytochrome c oxidoreductase activities were assayed essentially following the methods described previously (Shichi and Uritani, 1964; Shichi, 1969). Fivepg of antimycill A (dissolved in ethanol) was included in the reaction mixture for NADPH-rvt,ochrolllr c oxidoreductase assay. Antimycin A acts as an inhibitor between cvtoc;hrotrre h and cytochrome c1 in the mitochondrial electron transport (Crane, 1977). Therefore, NADPHcytochrome c oxidoreductase activity assayed in the presence of antimycine A serves as a-marker for endoplasmic reticulum (Hodges and Leonard, 1974). Purijcatiou
of diary
body y-&T
Approximately 500 bovine ciliary bodies were frozen, thawed and macerated for 3 min at 3°C in 500 ml of prechilled 1 mM-Tris buffer, pH 8.25, containing 0.25 sr-sucrose in a large Waring Blender. The homogenate was quickly passed through a double layer of cheese cloth. The extract was centrifuged for 14 mm at 17OXg. The supernatant \vas saturated with solid ammonium sulfate to 70yb (43.6 g added per 100 ml) at pH 8 in an ice bath and centrifuged at 27 000 x g for 45 min. The sediment was homogenized at SC with a glass homogenizer in (I.1 M-Tris buffer, pH 8.25, containing 0.15 &r-KC1 illrd centrifuged at 122 000 xg for 60 min. The microsomal pellet thus obtained was washecl in 1 m&r-Tris buffer, pH 8.25, containing 0.50/:, Emulphogene and stirred at 4°C for 24 hr. The microsomal ext’ract was centrifuged at 122 000 x g for 61) min. The supernatant (60 ml) was saturated with solid ammonium sulfate to 700/” at pH 8 as described above and centrifuged at 54 000 xg for 60 min. The pellet formed on the surface of supernatant~ was collected, dissolved in 1 nix-Tris buffer, pH 8.25, containing 0.5’l,o Emulphogene, and dialyzed overnight at 4°C against three changes of 2 liters of 1 m&r-Tris buffer, pH 8.25, containing (j.5”; Emulphogene. The dialyzed protein was then centrifuged at 122 OO( 1~j~g for 30 min and the supernutant (23 ml) removed and frozen. An ECTEOLA-cellulose
CILIARY
BODY
y-GLUTAMYL
TRARSPEPTIDASE
111
column (45 x 1.5 cm) was thoroughly washed at 4°C with 1 mnn-Tris buffer, pH 8.25, containing 0.5% Emulphogene. The equilibrated column showed a flow rate of 24.6 ml/hi and a void volume of 24.0 ml. Five ml of the dialyzed ciliary body enzyme were applied to the column and washed with 160 ml of the equilibrating buffer to collect 40 fractions. The enzyme was then eluted from the column with 400 ml of a linear NaCl gradient’ (( b-O.5 M) in 1 mnr-Tris buffer, pH 8.25, containing 0.5 yk Emulphogene. Fractions containing y-GT (fractions 82-91, 4 ml/fraction) were pooled, dialyzed and concentrated. Kidne? enzyme was extracted from bovine kidney microsomes with O.So,bEmulphogene and purified on ECTEOLA-cellulose in a similar manner. In addition, the enzyme was purified by gel filtration on Sepharose 413 and by preparative gel electrophoresis. B detailed account & the purification of kidney enzyme will appear elsewhere. Gel electrophoresis
Polyacrylamide gel electrophoresis of y-GT from bovine ciliary body and kidney was performed by the method of Fairbanks, Steck and Wallach (1971) using a 57; resolving gel containing 0.5% Emulphogene. The electrophoresis buffer was 0.4 M-Tris containing 02 r+sodium acetate, 0.02 M-EDTA sodium salt, and U.5?; Emulphogene, pH 7.5, PAS (Periodic Acid Schiff) staining of gels for carbohydrate was done according to the method of Herbert a,nd Strobbel (1974). One hundred ~1 of y-GT (1.0 mg/ml protein) in 1 mnr-Tris buffer, pH 8.25, containing 0.5% Emulphogene was applied to the gel and electrophoresetl at 6 mA per gel (6 x 100 mm) for 20 hr. The lower buffer reservoir was cooled with circulating water and constantly stirred. Duplicate gels were removed and immediately frozen in test tubes in dry ice. One set of gels was stained for protein with Coomassie blue, and the other set stained for carbohydrate with P-4s. The frozen gels were thawed at room temperature, measured and immediately sliced into 1 mm sections. Each slice was in cubated with 1.0 ml of 10 mM glycylglycine in u.1 M-Tris buffer, pH 8.25 for 12-14 hr. For activity measurements, a 0.5 ml-aliquot of the incubation mixture was added to 0.5 1111 of’ 2.36 mu y-glutamyl-I/-nitroanilide conta.ining 5.2 mM-MgCl,, in 0.1 St-Tris buffers pH 8.2.;,.
TrentmelLt with neuraminidase y-(:T (U&M? mg) was incubated for 48 hr at 25°C with neuraminidase (Sigma) and :I drop of toluene. The mixture was dialyzed overnight at 3°C against 1 m&r-Tris buffer, pH 8.5 containing 0.5 “/b Emulphogene and centrifuged. The supernatant was collectetl and ZIpplied to So/, polyacrylamide gel containing 0.5O/, Emulphogene at pH 7.-j. TIIP acti+ of neuraminidase was tested with mucin as substrate.
3. Results
Distribution of y-GT in various tissues of the bovine eye was studied on crutlc tissue homogenates. The ciliary body showed the highest specific activity. followecl by the iris, retina, pigment epithelium and lens (Table I). Subcellular fractionation of the ciliary body homogenates indicated :I close association of the enzyme activit 17 with the microsornal fraction (Table II). XSDPH-cytochrome c oxidored~tctitsc~ activity, a marker enzrnm for microsomes, was highest in the fraction. The mitt,v chondrial fraction which was monitored by cytochrome c oxidase activity cxhihitcstl il low y-GT activity that could be attriljuted to microsomal contamination. The as::ociat,ion of y-GT wit’h liver microsomes has been reported (Teschke, Brand an(l dtrohmeyer, 1977).
r-C:T specific activity* (nmol p-nitroaniline formed/mg prot./mi n)
Tissues from which microsomes hare been prepared
4.05 f0.30 0.69 +WO5 043 & 044 043 +woa (b005~-0~002
CXliary body Iris Retina Pigment epithelium-choroid Len5
* The mean+range
for four measurements. II
TABLE
Subcellular distribution
of y-GT in the bocine diary Antimycin A-insensitive NADPH-cytochrome c oxidoreductase* (nmol cyt. e reduced/ mg prot./min)
Cytochrome c oxidase* (nmol cyt. c oxidized/ mg prot,./min)
Fraction
156.3&84 841 f5.6 1.9io.2
Nucleus Mitochondria Microsomes Supernatant
* The meankrange
body
y-GT* (pm01 p-nitroaniline formed/mg prot./min)
lo* ” 930+ 8 1400+63 2to+ 5
6.68&081 15.63+ 1.35 2.37 @.I9
for four determinations. III
TABLE
Effect of detergents on diary
Detergent
No addition Sodium deoxyoholate Triton X-100 Tween 80 Emulphogene BC-720 Digitonin Ammonyx LO Cetyltrimethylammonium bromide
* The mean*range
body microsmal y-GT
Final cont. (96)
Activity CCJo)
0.10 0.25 0.25 0.25 0.01 045 0.05
100 151+ 8 152& 6 156&t-12 170+15 160+ 6 1601 4 179&l”
0.05
156+
for three determinations,
5
CILIARY
BODY
y-GLUTANYL
TRANSPEPTII)ASE
11x
Efect of deterpats on microsomal y-GT y-GT activity could be solubilized from the ciliary microsomes by non-ionic (Triton, Tween, Emulphogene, digitonin and Ammonyx), anionic (Deoxycholate) and cationic (cetyltrimethyl ammonium bromide) detergents (Table III). The activity was enhanced as much as lSOo/o by the addition of detergent to the microsomed. Thr enzyme was stable for a month in these detergents at -20°C‘. Pu~rijictrtion
of ciliwy
y-GT
Tht, y-GT act’1~-‘t1 #y solubilized from ciliary body microsomes could be recovered almost completely by 70$/b saturation with ammonium sulfate. Since high concentrations of ammonium sulfate were inhibit,ory to the enzyme. it was necessary t,o remove the salt by dialysis before the activity was a.ssayed. Substantial amounts of
Froctmn number F’rc:. 1. ECITEOLA-cellulose chromatography of ciliary body y-GT. Forty fractious wwo collrctrd h? was then effected with elution with 1 mar-Tris buffer, pH 8.25, containing 0.546 Emulphoge ne. Elution 400 ‘111I 0 f a linear NnC’l gradient (O-0.5 nx) in the above buffer t,o collwt additional 100 fractions. T.ZlSLB
IV
Volume Procedure
(ml)
Pwztionation of miwosomes 0.5’$, Emulphopene extraction (NH,)&O, fractionation (O-70% saturation) EC’TEOLA-cellulose chromatography
* One unit ofactivity
is defined
Total
units*
l’rotrk (mgjml)
Yield I:nits/tq
(“J
195
3937
1 I .25
63
6363
540
18.71
100
104
23
6.505
11.0
“5.71
lo”
14.3
41
7330
x.1
57.67
115
32.1
as the activity
of enzyme
1 .cio
Puriticatioll
1.0
that releases 1 nmolp-nitrosniline
per minute.
K. T). UAS
114
ANI)
H. S HIC’H 1
contaminating proteins were removed l)y further purification on an E( “1’E( )I,.4 cellulose column (Fig. 1). The purification steps are summarize(1 in Table IV. hpprt~mmately 32fold purification was achieved over the microsomes with a recover>- 01 nearIy lOOg/,. The apparent h’,, vaIues of purified ciliary y-GT and kidney y-Gl’ for L-y-glutamyl-p-nitroanilide were determined to be 1.3 mM and 16 nnx, respectively. when glycylglycine was used as the y-glutamyl acceptor. The apparent Knl for the cultured human lens enzyme for L-y-glutamyl-p-nitroanilide was reported to be 0645 mM (Miller, Arya and Srivastava, 1976). Inhibitors Neither transpeptidation nor hydrolysis was inhibited by 1 mw-p-chloromercuribenzoate (Table V). Szewczuk and Baranowski (1963) reported inhibition of bovine kidney enzyme by mercurial ion. Iodoacetamide at 10 mM inhibited 420,; of the activity of bovine lens y-GT (Rathbun and Wicker, 1973). Rat hepatoma enzyme (Taniguchi, 1974) and human lens enzyme (Miller, Arya and Srivastava, 1976) were not inhibited by sulfhydryl inhibitors. At 150 mM, Ca2+ was more inhibitory than Mg2+. High concentrations of Na+ inhibited transpeptidase activity. Sheep kidney enzyme was reported to be activated by Ca2+, Mg2+, NaT and K+ (Zelazo and Orlowski, 1976). Maleate (60 mM) was a potent inhibitor of transpeptidation (83%); it also inhibited hydrolysis (ca. 40%). With rat kidney enzyme, maleate inhibited transpeptidation and stimulated hydrolysis (Tate and Meister, 19i4a). Amino-chloromethylhesan-‘3one, a leucine analog, inhibited neither transpeptidation nor hydrolysis. TABLE
V
Effect of cations ad inhibitors o’n c&q Final concentration Cm@
Inhibitor
Ei+ Na+ NC&+ Na+ Ca2+ Mg*’ PCMH Maleate n,L-3-amino-1.chloro-5met,hylhexsn-Z-one
* The activity
without
Relative
activity
Transpeptidation
10 10
100* 108 9s 82 4’
50 100 150 150
37 64
0.1 60
118 li 100
5.5
inhibitor
body y-GT
was arbitrarily
(76)
Hydrolysis
100* 114 103 -100 111 109 AS AXI)
H. SHIC’HI
Shaw et al.: 1978; Zelazo and Orlo~ski, 197’6) or by affinity cllronr~ttogral,tl~ OII ii concanavalin Aagarose column (Niller, dwasthi and Srivast#ava. 1976 ; Shw vt ;ll., 1978). These chromatographic substances proved to bc not effective for t.he l)urification of ciliary body enzyme. Tate and Meister (1975) reported that, rat kitlne? colun~.r~. \VIJ enzyme was denatured while chromatographin, (I on a lIlL4l!-cellulose ‘9 times t)- the l)roce(lurc have purified the microsomal enzyme activity about 3, involving chromatography on ECTEOLA-cellulose (Table IV). This is basecl WI initial homogenate; purification is only 10 times from the initial Emulphogene extract. The use of Emulphogene BC 720 and ECTEOLA-cellulose was facilitated by our previous experience with the rod membrane protein rhodopsin (Shichi~ Le\vis. Irreverre and Stone? 1969). The partially purified enzyme is a glycoprotein as is evident by its staining with PAS (Fig. 3) and catalyzes both transpepticlasc and hyclrolase reactions. The pH optimum ( = 8.2) of the ciliary body enzyme for transprl)ti~latiorl is in good agreement with those reported for the enzyme from human liver (Shaw. 1978), human lens (Miller, drya and Srivastava, 1976) and bovine lens (Rathbutl and Wicker, 1973) but somewhat lower than those for bovine kidney (Szewczuk mtl Baranowski, 1963), sheep kidney (Zelazo and Orlolvski, 1976) and rat lN:patOtlliL (Taniguchi? 1974). The apparent K,L value ( = 1.3 mM) of the ciliary hotly enzyitie for y-glutamyl-p-nitroanilide is within a range of the reported values 0.7-6 1llM for the enzyme from other tissues (Miller, Arya and Srivastava, 1976 ; Miller, Awastjhi and Srivastava. 1976; Taniguchi, 1974; Szewczuck and Bara,nowski> 1963). In WIItrast to the transpeptidase reaction, hydrolysis of y-glutamyl-I)-nitroanilidc did not’ show a distinct pH optimum over a range from pH 4 to 12 (Fig. 3). Zelazo ant1 Orlowski (1976) found that the rate of hydrolysis of glutathione by sheep kidney enzyme was essentially constant between pH 7.3 and 912. y-GT catalysis was explained by the ping-pong mechanism in which y-glutamyl-enzyme complex is formed as ml inbermediate (London, Shaw, Fetterolf and Garfinkel, 1976; Karkowsky and Orlowski, 1978). It is therefore possible that the formation as well as hydrolysis of the intcrmediate, but not the reactivity of amino acid acceptor, is insensitive to the pH. Partially purified ciliary body y-GT and the enzyme purified front kidney are similar in their solubility in ammonium sulfate. behavior on ECTEOLA-cellulose. activity toward different amino acid acceptors, pH optima, and apparent li,, values for However, they are different in their elcctropll(~r(?tic y-glutamyl-p-nitroanilide. mobilities in polyacrylamide gel (Figs 2 and 3). Emulphogene is a non-ionic detergent, and will have little effect on the mobility of the enzyme protein. Therefore the different electrophoretic behavior of the two enzymes must be attributed to a difference in the surface charge. The ciliary body enzyme which shows a greater mobilit,y toward the anode than the kidney enzyme is obviously more negatively charged. The multiple forms of y-GT have been reported in rat liver (Kottgen, Lindinger and Reutter, 1977; Kottgen, Reutter and Gerok, 1978) rat kidney (Goldman ant1 Scgal, 1977) and rat adenocarcinoma (Jaken and Mason, 1978). The separation of the two molecular forms of y-GT in the regenerating rat liver on a concanavalin A-Sepharose colu~nn suggests that a difference in the carbohydrate moiety of enzyme is responsible for the multiple forms (Kottgen et al., 1977. 1978). PAS staining of the carbohydrate moiety of enzyme in a polyacrylamide gel indicates that the ciliary bodv y-GT migrates faster toward the anode than the kidney enzyme. After treatment of the native enzymes with neuraminidase, however, they migrate in gel electrophoresis at essentially identical mobilities. The smaller mobilities after the enzymic treatment indicate the removal of negative charges (sialic acid residues) from the enzyme. It is therefore
CILIARY
BODY
y-GLUTAMYL
TRANSPEPTIDASE
119
evident that the difference in electrophoretic behavior between ciliary enzyme and kidney enzyme is associated with a difference in the content of sialic acid. y-GT isolated from rat adenocarcinoma was shown to be more heavily sialylated than that from the normal tissue (Jaken and Mason, 19’78). Although the proposed role of y-GT in the y-glutamyl cycle for amino acid transport is a very stimulating idea (Meister, 1973), not all evidence seems to be compatible with the hypothesis. Inhibition by 6-diazo-5-ox-n-norleucine of y-GT associated with the rat ascites tumor cell membrane does not affect transport of amino acids into the cell (Inoue? Horiuchi and Morino, 1977). Cultured skin fibroblasts from a patient with hereditary glutathionemia and glutathionuria lacks y-GT activity, while serum concentrations of individual amino acids and renal resorption of the amino acids are normal (Schulman, Goodman, Mace, Patrick, Tietze and Butler, 1975). A recent study on pat,ients with 5-oxoprolinuria indicates that neither changes in the concentrations of Sree amino acids nor accumulation of y-glutamplcysteine occur in the erythrocytes of patients (Hagenfeldt. Larsson and Andersson, 1978). The cc-chloromethyl ketone analog of leucine. DL-3-amino-l-chloro-5-methylhexan-g-one reportedly inhibits the t,ransport of neutral amino acids in ascites X37 tumor cells (Lewis, Hes, Yip and Gazer, 1977). The inability of the chloromethyl ketone to inhibit the ciliary body y-GT does not support the proposed role of y-GT in amino acid transport in the ciliary body. However, the effect of the compound on the amino acid transport @em of the ciliary body is yet, to be investigated. ACKKOWLEDGMENTS
We thank Dr Suguru Fukushi for his help in staining glycoproteins with PAS. DL-3amino-l-chloro-5-methylhexan-&one was kindly provided by Dr Peter Rador. We also thank Mrs Joyce McIntyre for expert typing. REFERENCES Chasseaud, L. F. (1976). Conjugation with glutathione and mercapturic acid excretion. In Glutathione-Metabolism and Function (Eds Arias, J. M. and Jakoby, W. B.). Pp. 77-114. Raven Press, New York. Crane, F. L. (1977). Hydroquinone dehydrogenases. Ann. Rev. Biochem. 46,439-69. Fairbanks, G., Steck, T. L. and Wallach, D. F. H. (1971). Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-17. Fodor. P., Miller, A. and Waelsch, H. (1953). Q uantitative aspects of enzymatic cleavage of glutathione. J. Biol. Chem. 202,551-65. Goldmann, D. R. and Segal, S. (1977). y-Glutamyl transpeptidase activity in newborn rat kidney brush border. Enzyme 22,301-11. Hagenfeldt, L., Larsson, A. and Andersson, R. (1978). The y-glutamyl cycle and amino acid transport. New Eng. J. Mea. 29$4587-90. Herbert, J. P. and Strobbel, B. (1974). Double staining techniques for proteins and glycoproteins. LKB Application Note # 151 (June 12) 1-7. Hodges, T. K. and Leonard, R. T. (1974). Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. Methods in Enzymology Vol. XXXII. (Eds Fleischer, S. and Packer, L.). Pp. 392-406. Academic Press, New York. Inoue, M.. Horiuchi, S. and Morino, Y. (1977). y-Glutamyl transpeptidase in rat ascites tumor cell LY-5. Lack of functional correlation of its catalytic activity with the amino acid transport. Eur. J. B&hem. 78,609-15. Jaken, S. and Mason, M. (1978). Differences in the isoelectric focusing patterns of y-glutamyl transpeptidase from normal and cancerous rat mammary tissue. Proc. Nat. Acud. Sci. U.S.A. 75. 175063.
1’0
A-. T). I)AS
AND
H. SHI(‘H[
Karkowsky, A.M. and Orlowski, M. (1978). y-(<amyl transpeptidase. Determination ot’spe~iti~~lt!~ in the presence of multiple amino acid acceptors. J. Biol. phem. 253, 1574-81. Kinoshita, *J. H. and Ball, E. G. (1953). A transpept,idation reaction het,wrrn glutathionr* :111rt arginine. J. Riol. Chern. 200, 609-I 7. Kiittgen. E., Reutter, W. and Gerok, II:. (1978). Induction and “sllperinduct.ion” of aialylatioll of’ membrane-bound y-glutamyltransferase during liver regeneration. Eur. J. EZiochrm. 82, 279-84.
KBttgen, E., Lindinger, G. and Reutter, W. (1977). Concanavalin 8-Separose afl?init,y chromatography for routine microanalysis of y-glut,amyl--transpeptidase. CZinBn Chimictr i-l&c 80, 2214. Lewis, N. J. Hes, J., Yip, P. and Cazer, F. D. (1977). Synthesis of 2-14C-3-amino-l:chloro-5methylhexan-2-one hydrochloride. J. Labelled Comp. Radiopharmaceut. 13, 487-90. London, J. W.. Shaw, L. M., Fetterolf, D. and Garfinkel, D. (1976). Det,ermination of the merhsnism and kinetic constants for hog kidney y-glutamyltransferase. Hiochem. J. 157, 609-17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265-75. Meister, A. (1973). On the enzymology of amino acid transport. ScierLce 180, 30-39. Meister. A. and Tate, S. (1976). Glutathione and related y-glutamyl compounds: Biosynthesis and utilization. Ann Rev. Biochem. 45, 559-604. Miller, S. P., Arya, D. V. and Srivastava, S. K. (1976). Studies of y-glutamyl transpeptidaac in human ocular tissues. Exp. Eye Res. 22, 329-334. Miller, S. I’., Awasthi, Y. C. and Srivastava, S. K. (1976). Studies of human kidney y-glntamyl transpeptidase. Purification and struct,ural. kinetic and immunological properties. J. Biol. Chem.
251,
2271-8.
Orlowski, M. and Bfeist,er, A. (1970). The y-glatamyl cycle: A possible transport syst,em for amino acids. Proc. Xat. Ad. Sci. U.S.A. 67, 1248-55. Rathbun, W. B. and \Vicker, K. (1973). Bovine iensy-glutamyl transpept,idase. fZxp. &e Hr.