Specificity of y -Glutamyl Cyclotrsnsfersse A P ~ L ~ N ASZEWCZUK' RY AND GEORGE E. CONNELE
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Department of Bbclaemistry, Universify of akpronfo, Torottto, Ontario M5S IAS Received October 15, 1974 Szewczuk, A. & Connell, 6. E. (1975) Specificity of r-Glutamyl Cyclotransferase. CQPLJ. Biochern. 53, 706-712 Using the phthaloyl method, 18 r-~-glutamylpeptides labelled with '46 in the N-terminal position have k e n synthesized. The products evere isolated by simple procedures using a Bowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified 7glutamyl cyclotransferase and determination of the resulting 2-pyfrolidone-5-carboxylicacid (5oxoproline). Evidence was obtained that y-glutamyl cyclotransferase acts only on the L-form of yglutamyl substrates; the enzyme could, therefore, be used for preparation of y-~-glutamyl peptides from their racemic mixtures. The specificity of y-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as r-~-glutamyl--NH-CHR---COOH. The amino acid linked to the y-glutamyl group must be in the L configuration. Szewczuk, A. & Connell, G. E. (1975) Specificity of y-Glutamyl Cyclotransferase. Cara. 9 Biockem. 53, 786-91 2 Utilisant la mCthode au phthaloyle, nous avons synthCtisC les 18 r-~-glutamyl peptides marquis au I4C en position N-terminale. Les produits sont isolCs par des techniques simples faisant appel ti une colonne de Do~vex-1ou h I'Clectrophorbe B haut voltage. Les peptides synthktiques contiennent des impuretCs mineures correspondant aux isomkres D-glutamyle. La proportion des D-isomkres est determinee a l'aide de la glutamique dkcarboxylase ou par une nouvelle mCthode de digestion en prCsence de la r-glutamyl cyclotransfCrase et dktermination du 5-oxoproline obtenu. I1 est dt5montrC que la y-glutarnyl cyclotransfCrase agit seulement sur la forme L des substrats 7-glutamyle; l'enzyme peut done servir a prdparer les peptides 7-Dglutamyle A partir de leurs mClanges rackmiques. Nous avons examine Ba spCcificitC de la r-glutamyl cyclotransfCrase utilisant une preparation enzymatique purifiCe du foie de porc et des extraits tissulaires du rat et de l'homme. La structure de base des substrats peut Ctre reprCsentee c o m e Ctant le y-~-glutamyl-NH---CMR> 45 42 44 25 42 23
Ratio G1u:Phe 1.00 : 1.04 Glu:Gly2.00:0.98 Glu:Ala2.00:1.00 G1u:Ala 3.00 : 1 .O1 Glu:Leu2.00:1.02 Glu :Gly :Cys 2.00:0.99:0.88 Glu:Ala1.00:2.15 G1u:Ala 1 .OO : 1.94 G1u:Ala 1 .OO : 3 -25
[14G]Glu/total Glu
R,P
1.00 : 1.05 1.Q(B:2.10 1.00:2.04 1.00 : 3.20 1.00:2.69
0.43 0.06 0.10 0 .O8 0.295
1.00:I.08 l.00:1.08 1.OO : 1.04 1 .OO : 1.09
0.00 0.15 0.15 0.625
Relative electrophoretic migration1
*Purification on Dowex-i colun~n:C, washed with 500 ml of 0.05 IWacetic acid and product eluted with 0.2 1Cf acid; D, wasbled with 500 mi of 0.2 M acetic acid and eluted with 1 M acid; E, washed with 500 ml of 1 1Lf acetic acid and eluted with 3 1Cf acid, r-di-Glu-GSSG finally purified by paper high voltage electrophoresis at p H 3.6; F, washed with 0.1 Macetic acid and eluted with 0.4 2Cf acetic acid; G , purified only by paper high voltage :c:lcctrsphoresis. ?Descending chromatography on Whatman No. 1 paper in a solvent composed of rr-butanol :pyriJine:water, I :I : I . $Electrophoretic migration relative to Orange G (1.00).
TABLE3. Stereospecific action of GCT and stereoanalysis Percentage of -,-Glu form assayed by two methods PGA liberated from peptide by long digestion with an excess of GCT and determined by two methods Substrate labelled on N-terminal -,-Glu
Optical method* at 205 nm
Radioactivity" determination
GABA formed by digestion of acid hydrolyzate with L-glutamic decarboxylase and determined by two methods Amino acid"fadioactivityt analysis determination
r-Glm-Ala -,-D,L-Glu-Ala -,-D-Glu- Ala y-~-GIu-Alal (-,-G1u)rAla -,-Glu-Gly PCA liberated by GCT from -,-Glu-Ala PGA liberated by GCT from r-Glu-Gly [=c~G~u *Calculated for total amount of substrate used for enzyme digestion. ?The s u m of GABA plus residual GBu was taken as 100%. $Commercial preparation, not radioactive.
con~poundsusing GCT has also been developed. The details of these methods have been described above. Distribution of GCT Activity in Human cind Rat Tissile GCT activity has been found in several human and animal tissues (6-9). In the present studies: 11 tissues of man and rat were tested for the
enzyme activity. To avoid interference by GGTP, which may cause transformation of the substrate, the homogenates were centrifuged at 100 000 X g and the supernatant was used. This procedure removes more than 90y0 of total GGTP, which is mainly bound to sedirnented cell particulates (1, 3, 679) In man (Table 4) the highest activity measured was found in human brain. with (?-G~U)~-ANA
710
CAN. J. BHWHEM.
TABLE 4. GCT activity in human and rat tissues determined with (-y-Glu)?-ANAsubstrate
Mean activity in tissue extracts (rnU/rng protein)
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Tissue
Human
Rat
Brain Kidney Liver Pancreas Spleen Lung Testes Ovaries Intestine Heart muscle Skeletal muscle Red blood cells Serum *Calculated per 1 ml of packed cells or 1 rnl of serum.
Almost identical activity (about 160 mU / mg protein) was found in cerebral cortex (frontal and temporal lobes), in the hypoplaysis? hypothaIarnus, thalamus, corpus callosum, pons, and medulla oblongata. Lower activity was found in the occipital lobe (31 rnU,/mg) and cerebellum. In human kidney, liver, intestine, and testes, the emynae activity wzs about 40y0 of that in brain, and in other tissues 15-25y6 of that in brain. Wed cells in man have activity about 1000-fold higher than serum when equal volumes of packed cells and serum are compared. In rat tissues (Table 4) the highest GCT activity measured with ( ~ - G ~ U ) ~ - Awas N Afound in the kidney. Much lower activity was observed in pancreas and liver; other tissues, inclaading brain, have extremely low activity. Rat blood cells have only 2.5-fold higher activity than serum.
VOL. 53,
1975
TABLE 5. Activity sf GCT with various pepaides relative to (-y-Glu)X-ANA
Substrate
Pig liver enzyme
Human tissues*
(-y-Glu)2-ANA (-y-G1u)s-ANA -y-Glu-Gly (-y-Glu)z-Giy y-Glu-Ala (-y-Gl~)~-Ala (-y-Gl~)~-Ala -y-D-Glu-Ala (-y=Gl~)~Lera -y-Glu-Met -y-Glu-Gln -y-di-Glsa-GSSG y-Glu(Ala)z -y-Glu(Ala)a *List of human tissues presented in Table 4. tActivity observed only in experiments with kidney, pancreas, Lung, and intestine.
tested was similar to that of the pig liver enzyme, except that PCA was slowly liberated from y - C l ~ - ( A l a )and ~ y - G l ~ - ( A l a )by ~ extracts of kidney, pancreas, lung, and intestine. This was probably caused by hydrolysis of the Ala-Ala bond by a carboxypeptidase present in these tissues, yielding 7-Glu-Ala, which is a substrate for GCT. This latter peptide was identified in reaction mixtures after incubation of y-Gluand y-Glu-(Ala), with extracts s f kidney, pancreas, lung, and intestine. The relative activities of the rat tissues were quite unlike those observed with hun~antissues and with the pig liver enzyme. ( ~ - G ~ U ) ~ - A N was among the poorer substrates for many rat tissues. The best were y-Glu-Met and y-Glu-Gln. There was no pattern common to all tissues, and for any particular substrate the relative activities among tissues frequently varied from twofold to fourfold.
Spect~ckfysj' GCT The relative activity s f the pig liver enzyme and of various human tissues was tested against Discussion 22 different y-Glu peptides. The results obtained We have shown that, in the method of King are presented in Table 5. The highest activity of pig liver G@Twas noted when it was tested with and Kidd for synthesis of y-Glu peptides, the ('-G~U)~-ANA. The enzyme activity measured crude product consists of approximately one against other y-Glu peptides was less than 3076 tenth a-peptide relative to y-Glu peptide; the s f the activity with (y-Gl~a)~-Ab!A.Only a trace y-peptide, however, can be easily purified on a of PCA was formed whew y-Glu-Val, y-Glu-Glu, Dowex-1 column, The only disadvantage of this 7-Glu-Phe, and y-Glu-Leu were used. Pig liver procedure is a small amount of racemizatisw, GCT did not act on GSSG, Gln, y-Glu(Ala)n, which probably takes place during synthesis of y G h ~ ( A l a ) ~y-Glu-D-Ala, , and y-Glu-(~-Ala)~.phthaloyl-L-glmtarnic anhydride. Evidence has been presented that GCT puriThe specificity of GCT in all human tissues
SZEWCZUM AND CONNELL: y-GLUTAMYL CYCLBTRANSFERASE
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fied from pig liver is rigorously stereospecific for the L form of the r-glutamyl substrate and, therefore can be used in a simple method of determination of the proportion of L-isomer in a r-glutamyl peptide. This method is simpler and more precise than the enzymatic method using L-glutaanic acid decarboxylase. GCT has been used also to prepare 7-D-Glu peptides from their racemic ~nixtures. The series of 7 - ~ - [ ~ 4 e ] G lcompounds u prepared synthetically made it possible to study the specificity of GCT in crude tissue extracts. The results obtained provided evidence that the specificity of the enzynae in all human tissues is practically the same. The pattern of specificity of the human enzyme is similar to that of pig liver. In the case of the rat enzyme, differences in specificity were observed among the various tissues. The high activity which was observed in human brain with many of the peptides was not seen in the rat. There are some characteristic features which are common t o the enzyme from human and rat tissues and the pig liver enzyme. They do not attack e-glutamine and the following 7-Glu peptides: GSSG, r - G l ~ ( A l a ) ~or, 7-Glu(Ala),. It has been observed that the enzyme does not attack 7-Glu-ANA (9) or 7-Glu-1~-nitroanilide (4). Based om these observations, we postulate that the enzyme requires the presence of a free a-carboxyl group on the amino acid linked to the glutamyl residue. Peptides lacking a free carboxyl group at this site are susceptible to attack by GGTP. The latter enzyme can transfer 7-Glu from one molecule to another, resulting in synthesis of (7-Glu)z peptides, which are then subject to attack by GCT. Thus, the formation of PCA can result froin coupled action by GGTP and GCT, as was postulated earlier (9). It is known that GCF.is a ubiquitous enzyme but its physiological role remains obscure. PGA occupies the N-terminal position in a variety of proteins and peptides (15). It has been found also in small pegtides produced by the hypothalamus, which control the release of hormones from the hypophysis (16, 17) and are inactivated by enzymatic removal of terminal PCA (18, 19). There is evidence from several laboratories (20, 21) that there is no enzymic system which incorporates PCB directly into proteins. In living organisms or in tissue slices, PCA is rapidly metabolized t o glutamic acid, which may then be
71 1
used for synthesis of N-terminal PCB in proteins (22). An enzyme which catalyses this conversion has been found in kidney and several other tissues of the rat, and named 5-oxoprolinase by Meister and his colleagues (23). According to these investigators, 5-oxoprolinase is blocked or reduced in a mentally retarded patient whose daily urinary excretion of PCA is very large (24). PCA produced by GCT is, therefore, probably not used directly by the organis111 but transforn~edto glutamic acid. The most significant role of the enzyme may be not to form PCA but t o remove di- and tri-glutamyl co~ngounds,which may be formed in reactions catalyzed by GGTP but which would be broken down only very slowly by that enzyme. The authors are grateful to Mr. Wagner of the coroner's office, Toronto, for assistance in obtaining autopsy material. The authors wish to express their appreciation to Mr. Arnold Duckworth for assistance with enzyme activity determinations, to Mr. C . K. Yu for assistance with amino acid analysis determinations, and to Dr. Theo Hofmdann for helpful discussions. This work was supported by the Medical Research Council of Canada.
I. Conneli, G. E. & Hanes, C. S. (1956) Nature 177, 377-378 2. Adamson, E. D., Connell, G. E. & Szetvczuk, A. (1970) in MethOSa,~pim Enzymology (Colowick, S . P . & Kaplan, N. O., eds), vol. 19, pp. 789-797 3. Adarnson, E. D., Szewczuk, A. & Connell, G. E. (1971) Can. J . Biochem. 49, 218-226 4. Orlowski, M., Richman, P . G. & Meister, A. (1969) Biochemistry 8, 1048-1055 5 . Bodnaryk, W. P. & McGirr, L. (1973) Biochint. Biophys. Acta 315, 352-362 6 . Cliffe, G. E. & Waley, S. G. (1968) Biochem. J. 79, 118-128 7. Kakimsto, Y., Kanazawa, A. & Sano, I. (1967) Biochim. Bicaphy..s. Actc~132, 472-480 8. Orlowski, M. & Meister, A. (1973) /. Biol. Ckem. 248, 2836-2844 9. ConneIl, G. E. & Szewczuk, A. (1967) Clin. Chim. Acts 17, 423-430 10. Lowry. 0.H., Rosebrough, N. J., Farr, A. L. 22 Randall, It. J . (1951) J. Biol. Clzem. 193, 265-275 11. Wajjar, V. A. & Fisher, J. (1954) J . Biol. Chem. 206, 215-219 12. King, F. E. & Kidd, B. A. A. (1949) J . Chem. Soc. 3315-3319 13. Sheehan, J. C . & Bolhsfer, W. A. (1950) J. Am. Chem. Soc. 71, 2469-2472 14. King, F. E., Clark-Levis, J. W. & Wade, W. (1957) J . Chesn. Soc. 886-$94 15. Daolittle, R. I;. & Armentrout, R. W. (1968) Biochemistry 7, 516-521 16. Rush, E. A., McLaughlin, C. L.& Solomon A. (1971) Cancer 84s. 31, 1134-1139
m www.nrcresearchpress.com b For personal use only.
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CAN. 9. BIOCHEM. VOL. 53, 1975
17. Baba, Y . , Matsuo, H. & Schally, A. V. (1971) Bio- 2 1 . chern. Biopi~ys.Res. Commun. 44, 459-463 18. Fellows, R. & Munge, A. (1971) Fed. Proc. 30? 1899 22. 19. Schally, A. V., Arimura, A., Baba, Y., Nair, T. M. G., Matsuo, W., Redding, T. M. & Bekljuk, %. (1971) 23. Biochem. Biop61~:~. Res. Comn?iin. 43, 393-399 20. Folkes, K., Enzrnaatn, F., Boler, J., Bovers, C . Y. & 24. Schally, A. V. (1969) Biockem. Biopiays. Rg.6. C~~mnarur. 37, 123-126
Baglioni, C. (1970) Biockenz. 5iopizjk)v. Wes. Cornmsata. 38, 212-219 Ramstkrishna, M., Krishnaswamy, P. 8.& Rajgopal, R. D. (1970) Biochem. J. 118, 895-897 Van Der Werf, P., Orlowski, M. & Meister A. (I 971) Proc. Natl. A c d . Sci. U.S. 68, 2982-2985 JeBl~ern,E., Kluge, T., Borrensen, H. C., Stskke, 0 . Bs Edjarn, E. (1970) Sclrtci. J. CIira. Lab. brtvest. 26. 327-335
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Department of Bbclaemistry, Universify of akpronfo, Torottto, Ontario M5S IAS Received October 15, 1974 Szewczuk, A. & Connell, 6. E. (1975) Specificity of r-Glutamyl Cyclotransferase. CQPLJ. Biochern. 53, 706-712 Using the phthaloyl method, 18 r-~-glutamylpeptides labelled with '46 in the N-terminal position have k e n synthesized. The products evere isolated by simple procedures using a Bowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified 7glutamyl cyclotransferase and determination of the resulting 2-pyfrolidone-5-carboxylicacid (5oxoproline). Evidence was obtained that y-glutamyl cyclotransferase acts only on the L-form of yglutamyl substrates; the enzyme could, therefore, be used for preparation of y-~-glutamyl peptides from their racemic mixtures. The specificity of y-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as r-~-glutamyl--NH-CHR---COOH. The amino acid linked to the y-glutamyl group must be in the L configuration. Szewczuk, A. & Connell, G. E. (1975) Specificity of y-Glutamyl Cyclotransferase. Cara. 9 Biockem. 53, 786-91 2 Utilisant la mCthode au phthaloyle, nous avons synthCtisC les 18 r-~-glutamyl peptides marquis au I4C en position N-terminale. Les produits sont isolCs par des techniques simples faisant appel ti une colonne de Do~vex-1ou h I'Clectrophorbe B haut voltage. Les peptides synthktiques contiennent des impuretCs mineures correspondant aux isomkres D-glutamyle. La proportion des D-isomkres est determinee a l'aide de la glutamique dkcarboxylase ou par une nouvelle mCthode de digestion en prCsence de la r-glutamyl cyclotransfCrase et dktermination du 5-oxoproline obtenu. I1 est dt5montrC que la y-glutarnyl cyclotransfCrase agit seulement sur la forme L des substrats 7-glutamyle; l'enzyme peut done servir a prdparer les peptides 7-Dglutamyle A partir de leurs mClanges rackmiques. Nous avons examine Ba spCcificitC de la r-glutamyl cyclotransfCrase utilisant une preparation enzymatique purifiCe du foie de porc et des extraits tissulaires du rat et de l'homme. La structure de base des substrats peut Ctre reprCsentee c o m e Ctant le y-~-glutamyl-NH---CMR> 45 42 44 25 42 23
Ratio G1u:Phe 1.00 : 1.04 Glu:Gly2.00:0.98 Glu:Ala2.00:1.00 G1u:Ala 3.00 : 1 .O1 Glu:Leu2.00:1.02 Glu :Gly :Cys 2.00:0.99:0.88 Glu:Ala1.00:2.15 G1u:Ala 1 .OO : 1.94 G1u:Ala 1 .OO : 3 -25
[14G]Glu/total Glu
R,P
1.00 : 1.05 1.Q(B:2.10 1.00:2.04 1.00 : 3.20 1.00:2.69
0.43 0.06 0.10 0 .O8 0.295
1.00:I.08 l.00:1.08 1.OO : 1.04 1 .OO : 1.09
0.00 0.15 0.15 0.625
Relative electrophoretic migration1
*Purification on Dowex-i colun~n:C, washed with 500 ml of 0.05 IWacetic acid and product eluted with 0.2 1Cf acid; D, wasbled with 500 mi of 0.2 M acetic acid and eluted with 1 M acid; E, washed with 500 ml of 1 1Lf acetic acid and eluted with 3 1Cf acid, r-di-Glu-GSSG finally purified by paper high voltage electrophoresis at p H 3.6; F, washed with 0.1 Macetic acid and eluted with 0.4 2Cf acetic acid; G , purified only by paper high voltage :c:lcctrsphoresis. ?Descending chromatography on Whatman No. 1 paper in a solvent composed of rr-butanol :pyriJine:water, I :I : I . $Electrophoretic migration relative to Orange G (1.00).
TABLE3. Stereospecific action of GCT and stereoanalysis Percentage of -,-Glu form assayed by two methods PGA liberated from peptide by long digestion with an excess of GCT and determined by two methods Substrate labelled on N-terminal -,-Glu
Optical method* at 205 nm
Radioactivity" determination
GABA formed by digestion of acid hydrolyzate with L-glutamic decarboxylase and determined by two methods Amino acid"fadioactivityt analysis determination
r-Glm-Ala -,-D,L-Glu-Ala -,-D-Glu- Ala y-~-GIu-Alal (-,-G1u)rAla -,-Glu-Gly PCA liberated by GCT from -,-Glu-Ala PGA liberated by GCT from r-Glu-Gly [=c~G~u *Calculated for total amount of substrate used for enzyme digestion. ?The s u m of GABA plus residual GBu was taken as 100%. $Commercial preparation, not radioactive.
con~poundsusing GCT has also been developed. The details of these methods have been described above. Distribution of GCT Activity in Human cind Rat Tissile GCT activity has been found in several human and animal tissues (6-9). In the present studies: 11 tissues of man and rat were tested for the
enzyme activity. To avoid interference by GGTP, which may cause transformation of the substrate, the homogenates were centrifuged at 100 000 X g and the supernatant was used. This procedure removes more than 90y0 of total GGTP, which is mainly bound to sedirnented cell particulates (1, 3, 679) In man (Table 4) the highest activity measured was found in human brain. with (?-G~U)~-ANA
710
CAN. J. BHWHEM.
TABLE 4. GCT activity in human and rat tissues determined with (-y-Glu)?-ANAsubstrate
Mean activity in tissue extracts (rnU/rng protein)
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Tissue
Human
Rat
Brain Kidney Liver Pancreas Spleen Lung Testes Ovaries Intestine Heart muscle Skeletal muscle Red blood cells Serum *Calculated per 1 ml of packed cells or 1 rnl of serum.
Almost identical activity (about 160 mU / mg protein) was found in cerebral cortex (frontal and temporal lobes), in the hypoplaysis? hypothaIarnus, thalamus, corpus callosum, pons, and medulla oblongata. Lower activity was found in the occipital lobe (31 rnU,/mg) and cerebellum. In human kidney, liver, intestine, and testes, the emynae activity wzs about 40y0 of that in brain, and in other tissues 15-25y6 of that in brain. Wed cells in man have activity about 1000-fold higher than serum when equal volumes of packed cells and serum are compared. In rat tissues (Table 4) the highest GCT activity measured with ( ~ - G ~ U ) ~ - Awas N Afound in the kidney. Much lower activity was observed in pancreas and liver; other tissues, inclaading brain, have extremely low activity. Rat blood cells have only 2.5-fold higher activity than serum.
VOL. 53,
1975
TABLE 5. Activity sf GCT with various pepaides relative to (-y-Glu)X-ANA
Substrate
Pig liver enzyme
Human tissues*
(-y-Glu)2-ANA (-y-G1u)s-ANA -y-Glu-Gly (-y-Glu)z-Giy y-Glu-Ala (-y-Gl~)~-Ala (-y-Gl~)~-Ala -y-D-Glu-Ala (-y=Gl~)~Lera -y-Glu-Met -y-Glu-Gln -y-di-Glsa-GSSG y-Glu(Ala)z -y-Glu(Ala)a *List of human tissues presented in Table 4. tActivity observed only in experiments with kidney, pancreas, Lung, and intestine.
tested was similar to that of the pig liver enzyme, except that PCA was slowly liberated from y - C l ~ - ( A l a )and ~ y - G l ~ - ( A l a )by ~ extracts of kidney, pancreas, lung, and intestine. This was probably caused by hydrolysis of the Ala-Ala bond by a carboxypeptidase present in these tissues, yielding 7-Glu-Ala, which is a substrate for GCT. This latter peptide was identified in reaction mixtures after incubation of y-Gluand y-Glu-(Ala), with extracts s f kidney, pancreas, lung, and intestine. The relative activities of the rat tissues were quite unlike those observed with hun~antissues and with the pig liver enzyme. ( ~ - G ~ U ) ~ - A N was among the poorer substrates for many rat tissues. The best were y-Glu-Met and y-Glu-Gln. There was no pattern common to all tissues, and for any particular substrate the relative activities among tissues frequently varied from twofold to fourfold.
Spect~ckfysj' GCT The relative activity s f the pig liver enzyme and of various human tissues was tested against Discussion 22 different y-Glu peptides. The results obtained We have shown that, in the method of King are presented in Table 5. The highest activity of pig liver G@Twas noted when it was tested with and Kidd for synthesis of y-Glu peptides, the ('-G~U)~-ANA. The enzyme activity measured crude product consists of approximately one against other y-Glu peptides was less than 3076 tenth a-peptide relative to y-Glu peptide; the s f the activity with (y-Gl~a)~-Ab!A.Only a trace y-peptide, however, can be easily purified on a of PCA was formed whew y-Glu-Val, y-Glu-Glu, Dowex-1 column, The only disadvantage of this 7-Glu-Phe, and y-Glu-Leu were used. Pig liver procedure is a small amount of racemizatisw, GCT did not act on GSSG, Gln, y-Glu(Ala)n, which probably takes place during synthesis of y G h ~ ( A l a ) ~y-Glu-D-Ala, , and y-Glu-(~-Ala)~.phthaloyl-L-glmtarnic anhydride. Evidence has been presented that GCT puriThe specificity of GCT in all human tissues
SZEWCZUM AND CONNELL: y-GLUTAMYL CYCLBTRANSFERASE
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fied from pig liver is rigorously stereospecific for the L form of the r-glutamyl substrate and, therefore can be used in a simple method of determination of the proportion of L-isomer in a r-glutamyl peptide. This method is simpler and more precise than the enzymatic method using L-glutaanic acid decarboxylase. GCT has been used also to prepare 7-D-Glu peptides from their racemic ~nixtures. The series of 7 - ~ - [ ~ 4 e ] G lcompounds u prepared synthetically made it possible to study the specificity of GCT in crude tissue extracts. The results obtained provided evidence that the specificity of the enzynae in all human tissues is practically the same. The pattern of specificity of the human enzyme is similar to that of pig liver. In the case of the rat enzyme, differences in specificity were observed among the various tissues. The high activity which was observed in human brain with many of the peptides was not seen in the rat. There are some characteristic features which are common t o the enzyme from human and rat tissues and the pig liver enzyme. They do not attack e-glutamine and the following 7-Glu peptides: GSSG, r - G l ~ ( A l a ) ~or, 7-Glu(Ala),. It has been observed that the enzyme does not attack 7-Glu-ANA (9) or 7-Glu-1~-nitroanilide (4). Based om these observations, we postulate that the enzyme requires the presence of a free a-carboxyl group on the amino acid linked to the glutamyl residue. Peptides lacking a free carboxyl group at this site are susceptible to attack by GGTP. The latter enzyme can transfer 7-Glu from one molecule to another, resulting in synthesis of (7-Glu)z peptides, which are then subject to attack by GCT. Thus, the formation of PCA can result froin coupled action by GGTP and GCT, as was postulated earlier (9). It is known that GCF.is a ubiquitous enzyme but its physiological role remains obscure. PGA occupies the N-terminal position in a variety of proteins and peptides (15). It has been found also in small pegtides produced by the hypothalamus, which control the release of hormones from the hypophysis (16, 17) and are inactivated by enzymatic removal of terminal PCA (18, 19). There is evidence from several laboratories (20, 21) that there is no enzymic system which incorporates PCB directly into proteins. In living organisms or in tissue slices, PCA is rapidly metabolized t o glutamic acid, which may then be
71 1
used for synthesis of N-terminal PCB in proteins (22). An enzyme which catalyses this conversion has been found in kidney and several other tissues of the rat, and named 5-oxoprolinase by Meister and his colleagues (23). According to these investigators, 5-oxoprolinase is blocked or reduced in a mentally retarded patient whose daily urinary excretion of PCA is very large (24). PCA produced by GCT is, therefore, probably not used directly by the organis111 but transforn~edto glutamic acid. The most significant role of the enzyme may be not to form PCA but t o remove di- and tri-glutamyl co~ngounds,which may be formed in reactions catalyzed by GGTP but which would be broken down only very slowly by that enzyme. The authors are grateful to Mr. Wagner of the coroner's office, Toronto, for assistance in obtaining autopsy material. The authors wish to express their appreciation to Mr. Arnold Duckworth for assistance with enzyme activity determinations, to Mr. C . K. Yu for assistance with amino acid analysis determinations, and to Dr. Theo Hofmdann for helpful discussions. This work was supported by the Medical Research Council of Canada.
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m www.nrcresearchpress.com b For personal use only.
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17. Baba, Y . , Matsuo, H. & Schally, A. V. (1971) Bio- 2 1 . chern. Biopi~ys.Res. Commun. 44, 459-463 18. Fellows, R. & Munge, A. (1971) Fed. Proc. 30? 1899 22. 19. Schally, A. V., Arimura, A., Baba, Y., Nair, T. M. G., Matsuo, W., Redding, T. M. & Bekljuk, %. (1971) 23. Biochem. Biop61~:~. Res. Comn?iin. 43, 393-399 20. Folkes, K., Enzrnaatn, F., Boler, J., Bovers, C . Y. & 24. Schally, A. V. (1969) Biockem. Biopiays. Rg.6. C~~mnarur. 37, 123-126
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