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Glutathione; Metabolism and function via the γ-glutamyl cycle
Pergamon Preas
Life Sciences Vol . 15, pp . 177-190 Printed in the U.S .A .
MINIRSVIL~41
GLUTATHIONE ; h1ETABOLISM AND FUNCTION VIA THE r-GLUTAMYL CYCLE Alton Meister Department of Biochemistry Comell University Medical College 1300 York Avenue, New York, N .Y . 10021
. ~umma~ The intracellular synthesis of glutathione and the broakdown of this ubiquitous S tripeptide aro linked by a series of enryme-catalyzed reactions which have been collectively termined the y-glutamyl cycle . Earlier work on the Y-glutamyl cycle has been reviewed (1,2); this miniroview wmmarizss the background of this aroa, the major previous findings, recent developments, and evidence that the r-glutamyl cycle functions in amino acid transport . Int roduc tion : the r-glutamyl cycle consists of six enzyme-catalyzed reactions (Figure 1) :
(AminoAcid) AA~
glutathione j'-glu-cysH-gly
~-giy
AA
5-oxôproline ATP
a
glycine
1 %glu-cysH c y:t~~ j~ ADP+P ; glutamate
4
ATP
ADP+P;
Fig . 1 : The Y-Glutamyl Cyele : 1 .Y~luiamyl tranapeptidass, 2 . y-glutamyl eyelotransfera:e, 3 . dlpeptidase, 4 . 5-oxoprolinase, 5 . r-glutamyl-cysteins synthetase, thions synthetase . 177
6 . gluta-
Glutathione and the y-Glutamyl Cycle
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The synthesis of glutathione from glutamate, cysteine, and glycine is catalyzed by the successive actions of 7-glutamyl-cysteine and glutathione synthetases (for a review, see (3)) .
A quantitatively major pathway of glutathione degradation is catalyzed by y -
glutanyl tronspeptidase, a membrane-bound enzyme that catalyzes the transfer of the yglutamyl moiety of glutathione (or of certain other 7-glutamyl compounds) to amino acids (or to certain peptides) to form 7-glutamyl amino acids and cysteinyl-glycine . latter dipeptide is enrymatically split to cysteine and glycine .
The
The y-glutamyl amino
acids, which are formed in the amino acid-dependent breakdown of glutathione, are converted to the corresponding free amino acids and 5-oxoproline (pyroglutamate, pyrrolidone carboxylate) by the action of y-glutamyl cyclotransferose . Conversion of the y-glutamyl deriwtives of virtually all of the protein amino acids to 5-oxoproline and free amino acids occurs in the presence of r-glutamyl trornpeptidase and r-glutamyl cyclotransferase (1) . 5-Oxoproline is converted to glutamate in an ATP-dependent reaction catalyud by 5-oxoprol inase . Them is now much evidence for the existence of the 7-glutamyl cycle in a number of different mammalian cells .
It has been suggested that the 7-glutamyl cycle functions in
the transport of amino acids across cell membranes (1,4) . A scheme has been proposed (1) according to which (a) the amino acid to be transported binds noncovalently to o cell membrane site, ro) a group on the membrane-bound y-glutamyl tronspeptidase interacts with the y-glutamyl moiety of intracellular glutathione (or of other y-glutamyl compounds) to yield a y-glutamyl enryme, (c) there is attack of the amino acid nitrogen atom on the y -carbon atom of the y-glutamyl enzyme ~ yield o y-glutamyl amino acid .Formation ofthe 7 -9lutamyl amino acid is associated with removal of the amino acid from its membrane binding site and movement of the amino acid moiety into the cell perhaps through a space in the membrane; this may be facilitated by a conformation change in the membrane . The amino acid is roleased from its y-glutamyl carrier within the cell .
Thus, transpeptidase
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and cyclotronsferase oatalysse the "translocation" and "roleass" steps of transport, rsspsctively .The ATP-dependent dscyclimtion of 5-oxoprolins and the synthesis of glutathione aro energy-requiring rocovery steps needed for resynthesis of the carier procursor . Bac
round : The possibility that the action of r-glutamyl transpeptidase might be involved
in amino acid transport was first suggested about 1950-1954 by several investigators (5-9), and this idea has been mentioned again by others (10-1~ . That r-glutamyl tronspeptidase is bound ro particles in tissue homogenates (18), and histochsmicol studies (16, 19-23) which indicate that the enzyme is bound to cell membranes are consistent with this view . However, this idea has apparently not been mentioned or cornidered in reviews by investigators interostsd in amino acid transport. It has also been wggested that the tronspeptidass may function in protein rynthesis (24-2n, collagen formation (1~, and in the transport and degradation of peptides (1,4) . The hypothesis of the 7-glutamyl cycle was stimulated by two key observations in our laboratory : (a) It was found that rot kidney exhibits
very high y-glutamyl-cysteine and
glutathione rynthstass activities . (b) 5-Oxoprolins was found to be rapidly metabolized by intact animals and tissue slices, and the major initial product formed from 5-oxoproline was found to be glutamate (28) .
the two reactions inwlved in the synthesis of glutathione wero
elucidated by Bloch and collaborators (29) . The studies on glutathione synthesis in our laboratory led to isolation of highly purified and apparontly homogeneous proparotians of glutathione rynthetase (30) and r-glutamyl-cysteine rynthetase (31), and to studies which showed that these roactions involve intermediate formation of acyl phosphatss(30-33) .ihs finding in kidney of very high activities of these enzymes seemed significant to us since this tissue, which is also the most active in catalyzing tronspsptidatlon of glutathione, maintains a glutathione concentration of about 3-5 mM .
ihat animals metabolize injected
[14C] 5-oxoproline rapidly was shown in studies on mice in our laboratory (34) and independently in experiments on rots (35) . Thus, it became evident that a pathway exists for
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the utilization of 5-oxoproline formed by the action of 7-glutamyl cyclotransferase . Earlier work had suggested that 5-oxoproline wuld be metabolized by intact animals; however, conflicting results were observed in different laboratories . It is notable that Sroxopraline was found to serve in place of glutamate in the rynthesis of glutathione by liver slices (36) . However, it is now clear that 5-ow~proline is not used directly for glutathione synthesis, nor is it used as wch for protein rynthesis (37); the N-terminal 5roxoproline residues of certain peptides and proteins are formed by cyclization of N-terminal glutamate (or of o glutamate derivative) during or after protein rynthesis . Utilization of 5roxoproline inwlves prior conversion of 5~xoproline to glutamate, a reaction catalyzed by 5-oxoprothe discovery of 5-oxoprolinase (38,39) was of importance because it showed that
lipase .
there is a link between the enzymes that utilize glutathione and those which catalyze its rynthesis, thus making it possible to visualize a cycle (Fig . 1) . Function of the y-Glutamyl Cycle in Various Tiswes : The kidney is very active in amino acid transport and the presence of high activities of the Y-glutamyl cycle enzymes in this organ is consistent with the proposed role of the cycle in the renal handling of amino acids . It is notable that small amounts of a number of Y-glutamyl amino acids have been found in normal human urine (40) .
Histochemical studies (19-21) and studies on isolated kidney
brush border fractions (41) indicate that the transpeptidase (about 1 .5% of the membrane protein (41)) is localized in the proximal convoluted tubule, the region which is thought to be involved in renal amino acid reabsorption . The enzymes of the y-glutamyl cycle are found also in many other mammalian tissues . The transpeptidase is localized in the epithelial cells of the small intestinal mucosa; much less activity is present in stomach and colon epithelium (16) . The finding of high activities of the y-glutamyl cycle enrymes in the choroid plexus (42) and the ciliary body (43) support the hypothesis that the r-glutamyl cycle is involved in amino acid transport in these locations . It is generally believed that the choroid plexus functions in traropor
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Glutathione and the yGlutamyl Cycle
18 1
phenomena at the blood-cerobrospinal fluid barrier, and it is notable that the epithelium of the choroid plexus is similar to that of the kidney tubule . lhero appears to be an analogy between the formation of urine by the nephron and the secrotion of cerobrospinal fluid by the choroid plexus . The activities of the r-glutamyl cycle enrymes are much higher in the choroid plexus than in other regions of the brain, and histochemical studies indicate that the y-glutamyl tronspeptidase is localized in the apical portions of the epithelial cells of the choroid plexus, which aro probably the sites at which transport occurs . It is relevant to note that the concentrations of amino acids in the cerobrospinal fluid are much lower than those of the blood plasma . Consistent with the function of the r-glutamyl cycle in brain is the obserwtion that a number of y-glutamyl amino acids have been found in borin (44-46) . Other regions of the brain also exhibit the activities of the 7-glutamyl cycle and it has been proposed that the cycle may play a role in intracellular amino acid transport associated with synaptic transmission (4~ . The ciliary body of the eye also contains high concentrations of the enzymes of the r-glutamyl cycle, and all of these, except for 5-oxoprolinase, ara prosent in the lens (43) . The tronspeptidase is localized in the basal portions of the epithelial cells of the ciliary body; the histochemical and enzyme studies suggest that the y-glutamyl cycle may function in the transport of amino acids across the blood-aqueous humor barrier . It is of interest that the development of cataroch from a variety of causes is associated with a decreased concentration of glutathione in the lens . While the function of lens glutathione is not yet understood, it is known that lea: glutathlone is continually synthesized from and degraded ro its constituent amino acids . A high percentage of patients with congenital ocular cataracts also exhibit aminoaciduria (e .g ., congenital galactosemia); it has been suggested fhat decreased amino acid transport across both the ronal tubule and the ciliary epithelium may bs aaoeiated with a similar enyeme defect inwlving the y -glutamyl cycle (43) .
Glutathione and the y-Glutamyl Cycle
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The available data indicate that glutathione is present and is synthesized in virtually all cells; Y-glutamyl tronspeptidase, r-glutamyl cyclotransferase, and 5-oxoprolinase are present at least to some extent in most mammalian tissues . these cansiderotions suggest that the r-glutamyl cycle functions in many cells . It is not proposed that the cycle accounts for all amino acid transport; there are undoubtedly a number of different amino acid transport systems and the relative function of the Y-glutamyl cycle may vary considerably in different cells . The mature erythrocyte contains all of the cycle enrymes except for 5-oxoprolinase ; these activities are of sufficient magnitude to account for most of the observed erythrocyte glutathione turnover (48) . Certain tumors (e .g ., Morris rot hepatomas and rat kidney tumors) exhibit substantial activity of the Y-glutamyl cycle enzymes (49); the reported involvement of glutathione in hepatic carcinogenesis (50-52) is interesting and requires additional study . Studies on the enzymes of the Y-glutamyl cycle in prokaryotes are currently sparse . However, r-glutamyl trnnspeptidase has been found in several bacteria (53-55) ; glutathione occurs in bacteria and bacterial 5-oxoprolinase has been purified and studied (56) . Evidence for the function of the Y-glutamyl cycle in vivo has come from studies in which mice were injected with L-2-imidazolidone-4-carboxylate, a competitive inhibitor of 5-oxoprolinase (57) . When the inhibitor is administered to mice there is markedly de creased utilization of 5-oxoproline; under these circumstances, 5roxoproline acwmulates in a number of tissues (brain, kidney, liver, eyel and unusually large amounts of 5roxoproline are excreted in the urine .
In studies in which the inhibitor was given together with
one of several L-amino acids, significantly more 5-oxoproline accumulated and vras excreted (58,59) . These findings on the augmentation of 5roxoproline accumulation in the presence of L-amino acids offer strong evidence for the function of the y-glutamyl cycle in vivo and also support the conclusion that 5-oxoproline is a quantitatively significant metabolite of glutathione .
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Glutethione end the y-Glytemyl Cycle
18 3
Inborn Metabolic - Emxs Involvin~the Y-Glutamyl Cycle : Thus far, two types of inborn errors have been found in man in which them are specific enrymatic lesions of the yglutanyl cycle .
In one type, the patients (two siblings) exhibit marked deficiency of
erythrocyte r-glutamyl-tysteine synthetase activity and of erythrocyte glutathione in association with hemolytic anemia (60) . They have central nervous system disease (mental rotardation, psychosis, spinocerebellar degeneration), and also decreased glutathione levels in their leukocytes and muscles indicating that they probably have a rather generalized deficienty of glutathione . It is of interest in relation to the proposal that the Y-glutamyl cycle function : in amino acid transport that these patients exhibit generalized aminoaciduria (1,61) . The second type of inbom error is associated with 5-oxoprolinuria; three patients have thus far been observed . the first of these to be reported, a 19 year old mentally rotarded boy, excroted 25-35 g of 5-oxoproline per day (62) . He was at first thought to have a defect in the urea cycle (62), but later a block in the y-glutamyl cycle was wggested (63) . When this patient was given an intravenous infusion of a mixture of amino acids, there was a large incroase in the urinary excrotion of 5-oxoprolins . When the patient was given [14C] 5-oxoproline he excroted very little ~C02 in his expirod broatfi (in marked contrast to a normal control) . While this finding suggests a block of 5-oxoprolin~e, it would be expected that the administered [~C] 5-oxoproline would be greatly diluted by the high blood plasma concentration of 5-oxoproline (50 mg per 100 ml or 3 .9 mM (64)) ; this value was initially reported erroneously to be 5 m8 per 100 ml (62,63,65) . Furthermore, the cultured skin fibroblaats from this patient have considerable 5roxoproltnase activity (66) . Two additional patients (sisters, now 3 and 0 .25 yeah old) with 5-oxoprolinuria wero wbsequently discovered ; both have blood plasma 5-oxoproline eoneentratior~ of about 4-5 mM (67,68) . Studies in which one of these patients was given [ 14C] 5-oxoprollne led to the conclusion that about 25% of the amount of 5-oxoproline formed is exeroted, the remainder
184
Glutathione and the Y-Glutamyl Cycle
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being metabolized (6~ . Similar conclusions may now be drawn from the studies on the 19 year old patient (64); the peripheral leukocytes of this patient exhibit
5-oxoprolinase
activity . The studies on these patients do not indicate a deficiency of 5-oxoprolinase, but rather that there is an overproduction of 5-oxoproline associated with a deficiency of glutathione synthetase as previously suggested (1) . Recent work in our laboroiory on erythrocytes, fibroblasts, and placenta from these patients shows that they have a marked deficiency of glutathione synthetase, and that their 5-oxoprolinuria is secondary to this enryme defect (69) . A block in glutathione synthesis would lead to 5-oxoprolinuria if under these circumstances much more than normal amounts of r-glutamyl-cysteine were formed and converted to 5-oxoproline by the action of y-glutamyl cyclotransferase . Such overproduction of 5oxoproline would exceed the capacity of 5-oxoprolinase to convert it to glutamate . YGlutamyl-cysteine is a good substrate for r-glutamyl transpeptidase and could serve in place of glutathione in tronspeptidation reactions with amino acids . Thus, a modified Yglutamyl cycle (requiring only 4 enzymes) in which 7=glutamyl-cysteine partiçipates in tronspeptidation may operate in such patients (Fig . 2) . Y-Glutamyl-cysteine may normally be protected from the action of the cyclotransferose (I), but with decreased glutathione synthetase, the unprotected dipeptide would serve as substrate for the cyclotronsferose leading to a futile cycle of 7-glutamyl-cysteine synthesis followed by its wnversion to 5roxoproline and cysteine . Larger amounts of Y-glutamyl-cysteine would need to be produced for transpeptidation . Normally, glutathione may regulate, diroctly ôr indirectly, Y-glutamyl-cysteine synthesis; availability of free cysteine may also play a role . Discussion : Questions still remain about the role of glutathione and the way in which its synthesis is regulated . Earlier work showed that the liver is very active in its nthesis and degradation; however, the en matic ca city of the kidne to nthesize e to now avai a e suggest t at a cyc otrons erase as a ower a finny or Yglutanyl-cysteine than does glutathione synthetase .
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Glutathia~ne and the y-Glutasryrl Cycle
185
ADP+P ; 5-oxoproline ~
ÂTP
Fig . 2: Modified r-Glutamyl Cycle (see the text and Legend for Fig . I) . and degrade glutathione is much higher than that of liver, and recent studies indicate that the overall turnover time of kidney glutathione is only a fraction of that of liver (70) . Transport of amino acids via the r-glutamyl cycle would appear to require more energy than might be thought minimally necessary, but, as discused elsewhero (1), such an "expensive" transport system seems not unroawnabls .
the high energy requiroment may
reflect the need for high efficiency, and it is notable that other biological processes (e .g ., synthesis of protein, glycogen, urea) also requiro much energy .
Less energy would be
required if Y-glutamyl-cysteine wero normally to function in transpeptidation; other ramifications and modificattona of the cycle aro conceivable, including, for example, pathways involving wccessivs transpeptidation roactions and special transport systems for glycine and cysteine inwlving glutathione and Y-glutamyl-cystsine synthetases, respectively . The theory that the r-glutarryl cycle functions in amino acid trornport does not explain the apparont requiroment of Na+ for transport :inca none of the cycle enzymes seem
186
Glutathione and the Y- Glut ~Y 1
to specifically require Na+ .
cle
Vol . I5, No . 2
However, results on the Na+-dependence of amino acid
transport are inconsistent, and definite conclusions do not yet seem possible (71) . It is possible that the cycle is involved in tronslocation rather than in recognition (1), and that Na+ is involved in the latter phenomenon . The cycle does not account in detail for all of the various specificity phenomena that have been observed in amino acid transport . Although wch amino acids as glutamine and methionine are good substrates of the transpeptidose, a-aminoisobutyrote and praline are not, and it would thus appear that the imino acids and a-substituted amino acids could not be transported by the cycle . The significance of the finding that glycyl-glycine and certain other peptides are good acceptor substrates for the tronspeptidase is not yet clear . the peptides may interact with the enryme by virtue of their similarity to cysteinyl-glycine; however, one must con eider also the interesting possibility that the transpeptidase functions in the transport, secretion, or metabolism of certain peptides . It is notable that histochemical studies inditote localization of the tronspeptidase in a variety of epithelial cells, e .g ., renal tubules, ciliary body, choroid plexus, seminal vesicles, epididymis, pancreatic ducts, bile ducts, small intestine, bronchi, bronchioles, fallopian tubes; this finding is consistent with a transport or secretary function . However, histochemical studies also indicate various intracellular (including neuronal) and capillary localizations; the significance of these observations may become evident after further histochemical and biochemical study . In conclusion, the Y-glutamyl cycle accounts for the synthesis of glutathione and for a significant fraction of its utilization . Several lines of evidence indicate that the cycle functions in the kidney and in other mammalian tiswes . These studies also demonstrate that free 5roxoproline is a quantitatively significant metabolite of glutathione . the proposed function of the cycle in amino acid transport is supported by a number of observations and considerations . the glutathione molecule has two interesting structural features, a wlfhydryl group and a y-glutamyl linkage; many studies on glutathione have centered on its sulfhydryl
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Glutathione and the y-Glutamyl Cycle
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group. The findings and ideas reviewed hero may serve to stimulate further interost in the biological function of the Y-glutomyl moiety of this tripeptide which occurs in substantial concentrations in virtually all cells . References 1 . A . Moister, Science 180, 33-39 (1973) . 2. A . Moister, in Glutathione (eds . L . Flohé et al) pp . 57-69, 1973 Georg Thiem Verlag, Stuttgart. 3. A . Moister in The Enrymes(3rd edition), 10, 671-697 (1974) . 4. M. Orlowski and A . Moister, Proc . Natl . Acad . Sci . U .S ., 67, 1248-1255 (1970) . 5. F .J .R . Hird, The y -Glutamyl Transpeptidation Reaction, Doctoral diuertation, Cambridge University, England, (1950) . b. F. Binkley, Nature 167, 888-889 (1951) . 7. F. Binkley,in Symposium on Glutathione (eds . Colowick et al). Academic Press,lnc ., New York, N .Y . p.160 (1954) . 8. P. H . Springell, Amino Acid Metabolism with Special Reference to Peptide Bond Transfer, Doctoral Thesis, Melbourne University, Australia, (1953) . 9. E .G . Ball, O. Cooper and E .C . Clorke, Biol . Bull ., 105, 369-370 (1953) . 10 . W .E . Knox in The Enzymes (2nd Edition), 2, 253-294, Academic Press, Inc ., New York, (1960) . 11 . M. Orlowski and A . Szewczuk, Acta Biochimica Polonica 8, 189-200 (1961) . 12 . A. Szewczuk and T. Bawrwwski, Biochem. Zsit . 338, 317-329 (1963) . 13 . M. Orlawski, Arch . Immunol . Ther . Exp . 11, 1-61 (19163) . 14 . A. Moister, Biochemistry of the Amino Acids (2nd Edition) 1, 473-482 (1965) . 15 . F . Kokot, J. Kuska and H . Grrybek, Arch . Immunol . Thsrop . Exp. 13, 549-556 (1965) .
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16 . E. Greenberg, E .E . Wollneger, G .A . Fleisher and G .W . Engstrom, Clin . Chim . Acta, 16, 79-89 (1967) . 17 . M. Orlowski, P. G . Richman and A . Meister, Biochemistry 8, 1048-1055 (1969) . 18 . F.J .R . Hird and P. H . Springall, Biochim. Biophys . Acta 15, 31-37 (1954) . 19 . Z . Albert, M. Orlowski and A . Szewczuk, Nature 191, 767-768 (1961) . 20 . G. G . Glenner and J .E . Folk, Nature 192, 338-339 (1961) . 21 . G. G . Glenner, J .E . Folk and P.J . McMillan, J . Histochem. Cytochem . 10, 481489 (1962) . 22 . Z. Albert, Z . Rzucidlo and H . Starzyk, Acta Histochem . 37, 74-79 (1970) . 23 . Z . Albert, J . Orlowska, M. Orlowski and A . Szewczuk, Acta Histochem . 18, 7889 (1964) . 24 . C .S . Hones, F .J .R . Hird and F .A . Isherwood, Nature 166, 288-292 (1950) . 25 . C .S . Hones, F .J .R . Hird and F .A . Isherwood, Biochem . J ., 51, 25-35 (1952) . 26 . J .S . Fruton, R . B . Johnston and M. Fried, J . Biol . Chem . 190, 39-53 (1951) . 27 . H. Waelsch, Symposium on Phosphorus Metabolism (ed . McEln~y, W. D. and Glass, B.) . Johns Hopkins Press, Baltimore, Md . 2, 109-128 (1952) . 28 . P. Van Der Werf, unpublished data, 1969 ; cited in Ref (4) . 29 . J .E . Spoke and K . Bloch, in Symposium on Glutathtone, (ads . S . Colowick et al) Academic Press, Inc. pp . 129-141 (1954) . 30 . E .D . Mooz and A . Meister, Biochemistry b, 1722-1734 (1967) . 31 . M. Orlowski and A . Meister, J . Biol . Chem . 246, 7095-7105 (1971) . 32 . J .S . Nishimura, E .A . Dodd and A . Meister, Fed. Proc ., 22, 536 (1963); J . Biol . Chem, 238, PC1179-1180 (1963) . 33 . J .S . Nishimura, E .A . Dodd and A . Meister, J . Biol . Chem . 239, 2553-2558 (1964) . 34 . P. Richman, unpublished work, 1969 ; cited in Ref. (4) . 35 . M. Ramakrishna, P. R. Krishnaswamy and D.R . Rao, Bioehern . J . 118, 895-897 (1970) .
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36 . A .E . Braurutein, G.A . Shamshikova and A .L . bffe, Blokhimiya 13, 95-100 (1948) . 37 . E.A . Rush and J .L . Starr, Biochem. Biophys. Acta 199, 41-55 (1970) . 38 . P. Van Der Warf, M. C>rlowski and A . Meister, Proc . Amer . Soc . Biol . Chem ., Fed. Proc . 30, 933 (1971) . 39 . P. Van Der Werf, M. C>rlowski and A . Meister, Proc . Natl . Acad . Sci . U .S . 68, 2982-2985 (1971) . 40 . D.L . Buchanan, E .E . Haley and R .T . Morkiw, Biochemistry 1, 612-620 (1962) . 41 . H . Glosunan and D . M. Neville, Jr ., FEBS Letters 19, 340-344 (1972) . 42 . S .S . Tate, L . L . Ross and A . Meister, Proc . Natl . Acad . Sci . U .S . 70, 1447-1449 (1973) . 43 . L. L. Ross, L. Barber, 5 .5 . Tate and A . Meister, Proc . Natl . Acad . Sci . U .S . 70, 1447-1449 (1973) . 44 . A . Kanazawa, Y . Kakimoto, T. Nakajima, and J . Sano, Biochim . Biophys . Acta, 111, 90-95 (1965) . 45 . Y . Kakimoto, T. Nakajima, M. Kanazawa, M. Takesada and J . Sano, Biochim . Biophys . Acta 93, 333-338 (1964) . 46 . K .L . Reichslt, J . Neurochem., 17, 19-25 (1970) . 47 . A . Meister, in Proc . Am . Assoc . Res . Nerv . Msnt . Dis ., Symposium on Brain Dysfunction in Metabolic Disorder; Raven Press, N .Y . (1974) In Press. 48 . A .G . Pblekar, S .S . Tats and A . Meister, Pros . Natl . Acad . Sci . U .S ., 71 , 293. 297 .(1974) . 49 . R .V . Krishna, 1973, unpublished studies in this laboratory . 50 . W .J .P . Neish and A. Rylett, Blochern . Phannacol ., 121 893-903 (1963) . 51 . W.J .P . Neish, H. M. Davies and P.M . Reeve, Biochem. Pharmacol ., 13, 1291-1303 (1964) .
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the y-Glutamyl
Cycle
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52 . S. Fiala, A .E . Fiala and B . Dixon, J . Natl . Cancer Inst ., 48, 1393-1401 (1972) . 53 . P. S. Talalay, Doctoral Dissertation, Cambridge University (1953) . 54 . P.S . Talalay, Nature 174, 516-517 (1954) . 55 . R. Malbauer and N . Grossowicz, J . Gen . Microbial . 41, 185-194 (1965) . 56 . P. Van Der Werf and A . Meister, Biochem. Biophys. Res . Comm . 56, 90-95 (1974) . 57 . P. Van Der Werf, M. Orlowski and A . Meister, Proc . Natl . Acad . Sci . U .S ., 68, 2982-2985 (1971) . 58 . P. Van Der Werf, R.A . Stephani and A . Meister, Fed. Proc ., 32, 1%9 (1973) . 59 . P. Van Der Werf, R.A . Stephani and A. Meister, Proc . Natl . Acad . Sci . U .S . 71, 1026-1029 (1974) . . 60 . P.N . Konrad, F . Richards, W .N . Valentine and D .E . Paglia, New Eng . J . Med ., 286, 557 (1972) . 61 . F . Richards, unpublished data; personal communication (1972) . 62 . E . Jellum, T. Kluge, H .C . Barrosen, O . Stokke and L. Eldjarn, Scand . J . Clan . Lab . Invest ., 26, 327-335 (1970) . 63 . L. Eldjam, E . Jellum and O . Stokke, Clin . Chim . Acta 40, 46}-476 (1972) . 64 . L. Eldjarn, E . Jellum and O. Stokke in Inborn Errors of Metabolism (Hommes, F.A . and Van Den Berg, C .J ., editors), pp " 255-268; Academic Press, New York (1973) . 65 . L. Eldjarn, O . Stokke and E . Jellum in Stem, J ., and Toothill, C. (editors), Soc . for the Study of Inbom Errorsa of Metabolism, pp . 113-120 (1972) . 66 . J .H . Stromure and L. Eldjarn, Scand . J . Clan . Lab . Invest . 29, 335-342 (1972) . 67 . L. Hagenfeldt, A. Larsson and R. Zetterstrom, Acta Paediat . Scand ._62, 1-8(1973) . 68 . A . Larason, personal communications, 1973, 1974 . 69 . V .P . Wollner, R. Sekuro, A. Meister, A . Larsson, Proc .NatI .Acad .Sei .,71,(1974) . 70 . R. Sekura and A. Meister, Proc . Natl . Acad . Sci . U .S ., 71, (1974) . 71 . H . N . Christensen, C. ds Cespedes, M. E . Handlogten and R . Ranguist, Biochim. Biophys. Acta 300, 487-522 (1973) .
Life Sciences Vol . 15, pp . 177-190 Printed in the U.S .A .
MINIRSVIL~41
GLUTATHIONE ; h1ETABOLISM AND FUNCTION VIA THE r-GLUTAMYL CYCLE Alton Meister Department of Biochemistry Comell University Medical College 1300 York Avenue, New York, N .Y . 10021
. ~umma~ The intracellular synthesis of glutathione and the broakdown of this ubiquitous S tripeptide aro linked by a series of enryme-catalyzed reactions which have been collectively termined the y-glutamyl cycle . Earlier work on the Y-glutamyl cycle has been reviewed (1,2); this miniroview wmmarizss the background of this aroa, the major previous findings, recent developments, and evidence that the r-glutamyl cycle functions in amino acid transport . Int roduc tion : the r-glutamyl cycle consists of six enzyme-catalyzed reactions (Figure 1) :
(AminoAcid) AA~
glutathione j'-glu-cysH-gly
~-giy
AA
5-oxôproline ATP
a
glycine
1 %glu-cysH c y:t~~ j~ ADP+P ; glutamate
4
ATP
ADP+P;
Fig . 1 : The Y-Glutamyl Cyele : 1 .Y~luiamyl tranapeptidass, 2 . y-glutamyl eyelotransfera:e, 3 . dlpeptidase, 4 . 5-oxoprolinase, 5 . r-glutamyl-cysteins synthetase, thions synthetase . 177
6 . gluta-
Glutathione and the y-Glutamyl Cycle
178
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The synthesis of glutathione from glutamate, cysteine, and glycine is catalyzed by the successive actions of 7-glutamyl-cysteine and glutathione synthetases (for a review, see (3)) .
A quantitatively major pathway of glutathione degradation is catalyzed by y -
glutanyl tronspeptidase, a membrane-bound enzyme that catalyzes the transfer of the yglutamyl moiety of glutathione (or of certain other 7-glutamyl compounds) to amino acids (or to certain peptides) to form 7-glutamyl amino acids and cysteinyl-glycine . latter dipeptide is enrymatically split to cysteine and glycine .
The
The y-glutamyl amino
acids, which are formed in the amino acid-dependent breakdown of glutathione, are converted to the corresponding free amino acids and 5-oxoproline (pyroglutamate, pyrrolidone carboxylate) by the action of y-glutamyl cyclotransferose . Conversion of the y-glutamyl deriwtives of virtually all of the protein amino acids to 5-oxoproline and free amino acids occurs in the presence of r-glutamyl trornpeptidase and r-glutamyl cyclotransferase (1) . 5-Oxoproline is converted to glutamate in an ATP-dependent reaction catalyud by 5-oxoprol inase . Them is now much evidence for the existence of the 7-glutamyl cycle in a number of different mammalian cells .
It has been suggested that the 7-glutamyl cycle functions in
the transport of amino acids across cell membranes (1,4) . A scheme has been proposed (1) according to which (a) the amino acid to be transported binds noncovalently to o cell membrane site, ro) a group on the membrane-bound y-glutamyl tronspeptidase interacts with the y-glutamyl moiety of intracellular glutathione (or of other y-glutamyl compounds) to yield a y-glutamyl enryme, (c) there is attack of the amino acid nitrogen atom on the y -carbon atom of the y-glutamyl enzyme ~ yield o y-glutamyl amino acid .Formation ofthe 7 -9lutamyl amino acid is associated with removal of the amino acid from its membrane binding site and movement of the amino acid moiety into the cell perhaps through a space in the membrane; this may be facilitated by a conformation change in the membrane . The amino acid is roleased from its y-glutamyl carrier within the cell .
Thus, transpeptidase
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179
and cyclotronsferase oatalysse the "translocation" and "roleass" steps of transport, rsspsctively .The ATP-dependent dscyclimtion of 5-oxoprolins and the synthesis of glutathione aro energy-requiring rocovery steps needed for resynthesis of the carier procursor . Bac
round : The possibility that the action of r-glutamyl transpeptidase might be involved
in amino acid transport was first suggested about 1950-1954 by several investigators (5-9), and this idea has been mentioned again by others (10-1~ . That r-glutamyl tronspeptidase is bound ro particles in tissue homogenates (18), and histochsmicol studies (16, 19-23) which indicate that the enzyme is bound to cell membranes are consistent with this view . However, this idea has apparently not been mentioned or cornidered in reviews by investigators interostsd in amino acid transport. It has also been wggested that the tronspeptidass may function in protein rynthesis (24-2n, collagen formation (1~, and in the transport and degradation of peptides (1,4) . The hypothesis of the 7-glutamyl cycle was stimulated by two key observations in our laboratory : (a) It was found that rot kidney exhibits
very high y-glutamyl-cysteine and
glutathione rynthstass activities . (b) 5-Oxoprolins was found to be rapidly metabolized by intact animals and tissue slices, and the major initial product formed from 5-oxoproline was found to be glutamate (28) .
the two reactions inwlved in the synthesis of glutathione wero
elucidated by Bloch and collaborators (29) . The studies on glutathione synthesis in our laboratory led to isolation of highly purified and apparontly homogeneous proparotians of glutathione rynthetase (30) and r-glutamyl-cysteine rynthetase (31), and to studies which showed that these roactions involve intermediate formation of acyl phosphatss(30-33) .ihs finding in kidney of very high activities of these enzymes seemed significant to us since this tissue, which is also the most active in catalyzing tronspsptidatlon of glutathione, maintains a glutathione concentration of about 3-5 mM .
ihat animals metabolize injected
[14C] 5-oxoproline rapidly was shown in studies on mice in our laboratory (34) and independently in experiments on rots (35) . Thus, it became evident that a pathway exists for
Glutathione and the y-Glutamyl Cycle
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the utilization of 5-oxoproline formed by the action of 7-glutamyl cyclotransferase . Earlier work had suggested that 5-oxoproline wuld be metabolized by intact animals; however, conflicting results were observed in different laboratories . It is notable that Sroxopraline was found to serve in place of glutamate in the rynthesis of glutathione by liver slices (36) . However, it is now clear that 5-ow~proline is not used directly for glutathione synthesis, nor is it used as wch for protein rynthesis (37); the N-terminal 5roxoproline residues of certain peptides and proteins are formed by cyclization of N-terminal glutamate (or of o glutamate derivative) during or after protein rynthesis . Utilization of 5roxoproline inwlves prior conversion of 5~xoproline to glutamate, a reaction catalyzed by 5-oxoprothe discovery of 5-oxoprolinase (38,39) was of importance because it showed that
lipase .
there is a link between the enzymes that utilize glutathione and those which catalyze its rynthesis, thus making it possible to visualize a cycle (Fig . 1) . Function of the y-Glutamyl Cycle in Various Tiswes : The kidney is very active in amino acid transport and the presence of high activities of the Y-glutamyl cycle enzymes in this organ is consistent with the proposed role of the cycle in the renal handling of amino acids . It is notable that small amounts of a number of Y-glutamyl amino acids have been found in normal human urine (40) .
Histochemical studies (19-21) and studies on isolated kidney
brush border fractions (41) indicate that the transpeptidase (about 1 .5% of the membrane protein (41)) is localized in the proximal convoluted tubule, the region which is thought to be involved in renal amino acid reabsorption . The enzymes of the y-glutamyl cycle are found also in many other mammalian tissues . The transpeptidase is localized in the epithelial cells of the small intestinal mucosa; much less activity is present in stomach and colon epithelium (16) . The finding of high activities of the y-glutamyl cycle enrymes in the choroid plexus (42) and the ciliary body (43) support the hypothesis that the r-glutamyl cycle is involved in amino acid transport in these locations . It is generally believed that the choroid plexus functions in traropor
Vol. 15, No . 2
Glutathione and the yGlutamyl Cycle
18 1
phenomena at the blood-cerobrospinal fluid barrier, and it is notable that the epithelium of the choroid plexus is similar to that of the kidney tubule . lhero appears to be an analogy between the formation of urine by the nephron and the secrotion of cerobrospinal fluid by the choroid plexus . The activities of the r-glutamyl cycle enrymes are much higher in the choroid plexus than in other regions of the brain, and histochemical studies indicate that the y-glutamyl tronspeptidase is localized in the apical portions of the epithelial cells of the choroid plexus, which aro probably the sites at which transport occurs . It is relevant to note that the concentrations of amino acids in the cerobrospinal fluid are much lower than those of the blood plasma . Consistent with the function of the r-glutamyl cycle in brain is the obserwtion that a number of y-glutamyl amino acids have been found in borin (44-46) . Other regions of the brain also exhibit the activities of the 7-glutamyl cycle and it has been proposed that the cycle may play a role in intracellular amino acid transport associated with synaptic transmission (4~ . The ciliary body of the eye also contains high concentrations of the enzymes of the r-glutamyl cycle, and all of these, except for 5-oxoprolinase, ara prosent in the lens (43) . The tronspeptidase is localized in the basal portions of the epithelial cells of the ciliary body; the histochemical and enzyme studies suggest that the y-glutamyl cycle may function in the transport of amino acids across the blood-aqueous humor barrier . It is of interest that the development of cataroch from a variety of causes is associated with a decreased concentration of glutathione in the lens . While the function of lens glutathione is not yet understood, it is known that lea: glutathlone is continually synthesized from and degraded ro its constituent amino acids . A high percentage of patients with congenital ocular cataracts also exhibit aminoaciduria (e .g ., congenital galactosemia); it has been suggested fhat decreased amino acid transport across both the ronal tubule and the ciliary epithelium may bs aaoeiated with a similar enyeme defect inwlving the y -glutamyl cycle (43) .
Glutathione and the y-Glutamyl Cycle
18 2
Vol . 15, No . 2
The available data indicate that glutathione is present and is synthesized in virtually all cells; Y-glutamyl tronspeptidase, r-glutamyl cyclotransferase, and 5-oxoprolinase are present at least to some extent in most mammalian tissues . these cansiderotions suggest that the r-glutamyl cycle functions in many cells . It is not proposed that the cycle accounts for all amino acid transport; there are undoubtedly a number of different amino acid transport systems and the relative function of the Y-glutamyl cycle may vary considerably in different cells . The mature erythrocyte contains all of the cycle enrymes except for 5-oxoprolinase ; these activities are of sufficient magnitude to account for most of the observed erythrocyte glutathione turnover (48) . Certain tumors (e .g ., Morris rot hepatomas and rat kidney tumors) exhibit substantial activity of the Y-glutamyl cycle enzymes (49); the reported involvement of glutathione in hepatic carcinogenesis (50-52) is interesting and requires additional study . Studies on the enzymes of the Y-glutamyl cycle in prokaryotes are currently sparse . However, r-glutamyl trnnspeptidase has been found in several bacteria (53-55) ; glutathione occurs in bacteria and bacterial 5-oxoprolinase has been purified and studied (56) . Evidence for the function of the Y-glutamyl cycle in vivo has come from studies in which mice were injected with L-2-imidazolidone-4-carboxylate, a competitive inhibitor of 5-oxoprolinase (57) . When the inhibitor is administered to mice there is markedly de creased utilization of 5-oxoproline; under these circumstances, 5roxoproline acwmulates in a number of tissues (brain, kidney, liver, eyel and unusually large amounts of 5roxoproline are excreted in the urine .
In studies in which the inhibitor was given together with
one of several L-amino acids, significantly more 5-oxoproline accumulated and vras excreted (58,59) . These findings on the augmentation of 5roxoproline accumulation in the presence of L-amino acids offer strong evidence for the function of the y-glutamyl cycle in vivo and also support the conclusion that 5-oxoproline is a quantitatively significant metabolite of glutathione .
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Glutethione end the y-Glytemyl Cycle
18 3
Inborn Metabolic - Emxs Involvin~the Y-Glutamyl Cycle : Thus far, two types of inborn errors have been found in man in which them are specific enrymatic lesions of the yglutanyl cycle .
In one type, the patients (two siblings) exhibit marked deficiency of
erythrocyte r-glutamyl-tysteine synthetase activity and of erythrocyte glutathione in association with hemolytic anemia (60) . They have central nervous system disease (mental rotardation, psychosis, spinocerebellar degeneration), and also decreased glutathione levels in their leukocytes and muscles indicating that they probably have a rather generalized deficienty of glutathione . It is of interest in relation to the proposal that the Y-glutamyl cycle function : in amino acid transport that these patients exhibit generalized aminoaciduria (1,61) . The second type of inbom error is associated with 5-oxoprolinuria; three patients have thus far been observed . the first of these to be reported, a 19 year old mentally rotarded boy, excroted 25-35 g of 5-oxoproline per day (62) . He was at first thought to have a defect in the urea cycle (62), but later a block in the y-glutamyl cycle was wggested (63) . When this patient was given an intravenous infusion of a mixture of amino acids, there was a large incroase in the urinary excrotion of 5-oxoprolins . When the patient was given [14C] 5-oxoproline he excroted very little ~C02 in his expirod broatfi (in marked contrast to a normal control) . While this finding suggests a block of 5-oxoprolin~e, it would be expected that the administered [~C] 5-oxoproline would be greatly diluted by the high blood plasma concentration of 5-oxoproline (50 mg per 100 ml or 3 .9 mM (64)) ; this value was initially reported erroneously to be 5 m8 per 100 ml (62,63,65) . Furthermore, the cultured skin fibroblaats from this patient have considerable 5roxoproltnase activity (66) . Two additional patients (sisters, now 3 and 0 .25 yeah old) with 5-oxoprolinuria wero wbsequently discovered ; both have blood plasma 5-oxoproline eoneentratior~ of about 4-5 mM (67,68) . Studies in which one of these patients was given [ 14C] 5-oxoprollne led to the conclusion that about 25% of the amount of 5-oxoproline formed is exeroted, the remainder
184
Glutathione and the Y-Glutamyl Cycle
Vol. 15, No . 2
being metabolized (6~ . Similar conclusions may now be drawn from the studies on the 19 year old patient (64); the peripheral leukocytes of this patient exhibit
5-oxoprolinase
activity . The studies on these patients do not indicate a deficiency of 5-oxoprolinase, but rather that there is an overproduction of 5-oxoproline associated with a deficiency of glutathione synthetase as previously suggested (1) . Recent work in our laboroiory on erythrocytes, fibroblasts, and placenta from these patients shows that they have a marked deficiency of glutathione synthetase, and that their 5-oxoprolinuria is secondary to this enryme defect (69) . A block in glutathione synthesis would lead to 5-oxoprolinuria if under these circumstances much more than normal amounts of r-glutamyl-cysteine were formed and converted to 5-oxoproline by the action of y-glutamyl cyclotransferase . Such overproduction of 5oxoproline would exceed the capacity of 5-oxoprolinase to convert it to glutamate . YGlutamyl-cysteine is a good substrate for r-glutamyl transpeptidase and could serve in place of glutathione in tronspeptidation reactions with amino acids . Thus, a modified Yglutamyl cycle (requiring only 4 enzymes) in which 7=glutamyl-cysteine partiçipates in tronspeptidation may operate in such patients (Fig . 2) . Y-Glutamyl-cysteine may normally be protected from the action of the cyclotransferose (I), but with decreased glutathione synthetase, the unprotected dipeptide would serve as substrate for the cyclotronsferose leading to a futile cycle of 7-glutamyl-cysteine synthesis followed by its wnversion to 5roxoproline and cysteine . Larger amounts of Y-glutamyl-cysteine would need to be produced for transpeptidation . Normally, glutathione may regulate, diroctly ôr indirectly, Y-glutamyl-cysteine synthesis; availability of free cysteine may also play a role . Discussion : Questions still remain about the role of glutathione and the way in which its synthesis is regulated . Earlier work showed that the liver is very active in its nthesis and degradation; however, the en matic ca city of the kidne to nthesize e to now avai a e suggest t at a cyc otrons erase as a ower a finny or Yglutanyl-cysteine than does glutathione synthetase .
Vol . 15, No . 2
Glutathia~ne and the y-Glutasryrl Cycle
185
ADP+P ; 5-oxoproline ~
ÂTP
Fig . 2: Modified r-Glutamyl Cycle (see the text and Legend for Fig . I) . and degrade glutathione is much higher than that of liver, and recent studies indicate that the overall turnover time of kidney glutathione is only a fraction of that of liver (70) . Transport of amino acids via the r-glutamyl cycle would appear to require more energy than might be thought minimally necessary, but, as discused elsewhero (1), such an "expensive" transport system seems not unroawnabls .
the high energy requiroment may
reflect the need for high efficiency, and it is notable that other biological processes (e .g ., synthesis of protein, glycogen, urea) also requiro much energy .
Less energy would be
required if Y-glutamyl-cysteine wero normally to function in transpeptidation; other ramifications and modificattona of the cycle aro conceivable, including, for example, pathways involving wccessivs transpeptidation roactions and special transport systems for glycine and cysteine inwlving glutathione and Y-glutamyl-cystsine synthetases, respectively . The theory that the r-glutarryl cycle functions in amino acid trornport does not explain the apparont requiroment of Na+ for transport :inca none of the cycle enzymes seem
186
Glutathione and the Y- Glut ~Y 1
to specifically require Na+ .
cle
Vol . I5, No . 2
However, results on the Na+-dependence of amino acid
transport are inconsistent, and definite conclusions do not yet seem possible (71) . It is possible that the cycle is involved in tronslocation rather than in recognition (1), and that Na+ is involved in the latter phenomenon . The cycle does not account in detail for all of the various specificity phenomena that have been observed in amino acid transport . Although wch amino acids as glutamine and methionine are good substrates of the transpeptidose, a-aminoisobutyrote and praline are not, and it would thus appear that the imino acids and a-substituted amino acids could not be transported by the cycle . The significance of the finding that glycyl-glycine and certain other peptides are good acceptor substrates for the tronspeptidase is not yet clear . the peptides may interact with the enryme by virtue of their similarity to cysteinyl-glycine; however, one must con eider also the interesting possibility that the transpeptidase functions in the transport, secretion, or metabolism of certain peptides . It is notable that histochemical studies inditote localization of the tronspeptidase in a variety of epithelial cells, e .g ., renal tubules, ciliary body, choroid plexus, seminal vesicles, epididymis, pancreatic ducts, bile ducts, small intestine, bronchi, bronchioles, fallopian tubes; this finding is consistent with a transport or secretary function . However, histochemical studies also indicate various intracellular (including neuronal) and capillary localizations; the significance of these observations may become evident after further histochemical and biochemical study . In conclusion, the Y-glutamyl cycle accounts for the synthesis of glutathione and for a significant fraction of its utilization . Several lines of evidence indicate that the cycle functions in the kidney and in other mammalian tiswes . These studies also demonstrate that free 5roxoproline is a quantitatively significant metabolite of glutathione . the proposed function of the cycle in amino acid transport is supported by a number of observations and considerations . the glutathione molecule has two interesting structural features, a wlfhydryl group and a y-glutamyl linkage; many studies on glutathione have centered on its sulfhydryl
Vol . 15, No . 2
Glutathione and the y-Glutamyl Cycle
187
group. The findings and ideas reviewed hero may serve to stimulate further interost in the biological function of the Y-glutomyl moiety of this tripeptide which occurs in substantial concentrations in virtually all cells . References 1 . A . Moister, Science 180, 33-39 (1973) . 2. A . Moister, in Glutathione (eds . L . Flohé et al) pp . 57-69, 1973 Georg Thiem Verlag, Stuttgart. 3. A . Moister in The Enrymes(3rd edition), 10, 671-697 (1974) . 4. M. Orlowski and A . Moister, Proc . Natl . Acad . Sci . U .S ., 67, 1248-1255 (1970) . 5. F .J .R . Hird, The y -Glutamyl Transpeptidation Reaction, Doctoral diuertation, Cambridge University, England, (1950) . b. F. Binkley, Nature 167, 888-889 (1951) . 7. F. Binkley,in Symposium on Glutathione (eds . Colowick et al). Academic Press,lnc ., New York, N .Y . p.160 (1954) . 8. P. H . Springell, Amino Acid Metabolism with Special Reference to Peptide Bond Transfer, Doctoral Thesis, Melbourne University, Australia, (1953) . 9. E .G . Ball, O. Cooper and E .C . Clorke, Biol . Bull ., 105, 369-370 (1953) . 10 . W .E . Knox in The Enzymes (2nd Edition), 2, 253-294, Academic Press, Inc ., New York, (1960) . 11 . M. Orlowski and A . Szewczuk, Acta Biochimica Polonica 8, 189-200 (1961) . 12 . A. Szewczuk and T. Bawrwwski, Biochem. Zsit . 338, 317-329 (1963) . 13 . M. Orlawski, Arch . Immunol . Ther . Exp . 11, 1-61 (19163) . 14 . A. Moister, Biochemistry of the Amino Acids (2nd Edition) 1, 473-482 (1965) . 15 . F . Kokot, J. Kuska and H . Grrybek, Arch . Immunol . Thsrop . Exp. 13, 549-556 (1965) .
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16 . E. Greenberg, E .E . Wollneger, G .A . Fleisher and G .W . Engstrom, Clin . Chim . Acta, 16, 79-89 (1967) . 17 . M. Orlowski, P. G . Richman and A . Meister, Biochemistry 8, 1048-1055 (1969) . 18 . F.J .R . Hird and P. H . Springall, Biochim. Biophys . Acta 15, 31-37 (1954) . 19 . Z . Albert, M. Orlowski and A . Szewczuk, Nature 191, 767-768 (1961) . 20 . G. G . Glenner and J .E . Folk, Nature 192, 338-339 (1961) . 21 . G. G . Glenner, J .E . Folk and P.J . McMillan, J . Histochem. Cytochem . 10, 481489 (1962) . 22 . Z. Albert, Z . Rzucidlo and H . Starzyk, Acta Histochem . 37, 74-79 (1970) . 23 . Z . Albert, J . Orlowska, M. Orlowski and A . Szewczuk, Acta Histochem . 18, 7889 (1964) . 24 . C .S . Hones, F .J .R . Hird and F .A . Isherwood, Nature 166, 288-292 (1950) . 25 . C .S . Hones, F .J .R . Hird and F .A . Isherwood, Biochem . J ., 51, 25-35 (1952) . 26 . J .S . Fruton, R . B . Johnston and M. Fried, J . Biol . Chem . 190, 39-53 (1951) . 27 . H. Waelsch, Symposium on Phosphorus Metabolism (ed . McEln~y, W. D. and Glass, B.) . Johns Hopkins Press, Baltimore, Md . 2, 109-128 (1952) . 28 . P. Van Der Werf, unpublished data, 1969 ; cited in Ref (4) . 29 . J .E . Spoke and K . Bloch, in Symposium on Glutathtone, (ads . S . Colowick et al) Academic Press, Inc. pp . 129-141 (1954) . 30 . E .D . Mooz and A . Meister, Biochemistry b, 1722-1734 (1967) . 31 . M. Orlowski and A . Meister, J . Biol . Chem . 246, 7095-7105 (1971) . 32 . J .S . Nishimura, E .A . Dodd and A . Meister, Fed. Proc ., 22, 536 (1963); J . Biol . Chem, 238, PC1179-1180 (1963) . 33 . J .S . Nishimura, E .A . Dodd and A . Meister, J . Biol . Chem . 239, 2553-2558 (1964) . 34 . P. Richman, unpublished work, 1969 ; cited in Ref. (4) . 35 . M. Ramakrishna, P. R. Krishnaswamy and D.R . Rao, Bioehern . J . 118, 895-897 (1970) .
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Glutathione and tha y-Glutamyl GYcle
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36 . A .E . Braurutein, G.A . Shamshikova and A .L . bffe, Blokhimiya 13, 95-100 (1948) . 37 . E.A . Rush and J .L . Starr, Biochem. Biophys. Acta 199, 41-55 (1970) . 38 . P. Van Der Warf, M. C>rlowski and A . Meister, Proc . Amer . Soc . Biol . Chem ., Fed. Proc . 30, 933 (1971) . 39 . P. Van Der Werf, M. C>rlowski and A . Meister, Proc . Natl . Acad . Sci . U .S . 68, 2982-2985 (1971) . 40 . D.L . Buchanan, E .E . Haley and R .T . Morkiw, Biochemistry 1, 612-620 (1962) . 41 . H . Glosunan and D . M. Neville, Jr ., FEBS Letters 19, 340-344 (1972) . 42 . S .S . Tate, L . L . Ross and A . Meister, Proc . Natl . Acad . Sci . U .S . 70, 1447-1449 (1973) . 43 . L. L. Ross, L. Barber, 5 .5 . Tate and A . Meister, Proc . Natl . Acad . Sci . U .S . 70, 1447-1449 (1973) . 44 . A . Kanazawa, Y . Kakimoto, T. Nakajima, and J . Sano, Biochim . Biophys . Acta, 111, 90-95 (1965) . 45 . Y . Kakimoto, T. Nakajima, M. Kanazawa, M. Takesada and J . Sano, Biochim . Biophys . Acta 93, 333-338 (1964) . 46 . K .L . Reichslt, J . Neurochem., 17, 19-25 (1970) . 47 . A . Meister, in Proc . Am . Assoc . Res . Nerv . Msnt . Dis ., Symposium on Brain Dysfunction in Metabolic Disorder; Raven Press, N .Y . (1974) In Press. 48 . A .G . Pblekar, S .S . Tats and A . Meister, Pros . Natl . Acad . Sci . U .S ., 71 , 293. 297 .(1974) . 49 . R .V . Krishna, 1973, unpublished studies in this laboratory . 50 . W .J .P . Neish and A. Rylett, Blochern . Phannacol ., 121 893-903 (1963) . 51 . W.J .P . Neish, H. M. Davies and P.M . Reeve, Biochem. Pharmacol ., 13, 1291-1303 (1964) .
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and
the y-Glutamyl
Cycle
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52 . S. Fiala, A .E . Fiala and B . Dixon, J . Natl . Cancer Inst ., 48, 1393-1401 (1972) . 53 . P. S. Talalay, Doctoral Dissertation, Cambridge University (1953) . 54 . P.S . Talalay, Nature 174, 516-517 (1954) . 55 . R. Malbauer and N . Grossowicz, J . Gen . Microbial . 41, 185-194 (1965) . 56 . P. Van Der Werf and A . Meister, Biochem. Biophys. Res . Comm . 56, 90-95 (1974) . 57 . P. Van Der Werf, M. Orlowski and A . Meister, Proc . Natl . Acad . Sci . U .S ., 68, 2982-2985 (1971) . 58 . P. Van Der Werf, R.A . Stephani and A . Meister, Fed. Proc ., 32, 1%9 (1973) . 59 . P. Van Der Werf, R.A . Stephani and A. Meister, Proc . Natl . Acad . Sci . U .S . 71, 1026-1029 (1974) . . 60 . P.N . Konrad, F . Richards, W .N . Valentine and D .E . Paglia, New Eng . J . Med ., 286, 557 (1972) . 61 . F . Richards, unpublished data; personal communication (1972) . 62 . E . Jellum, T. Kluge, H .C . Barrosen, O . Stokke and L. Eldjarn, Scand . J . Clan . Lab . Invest ., 26, 327-335 (1970) . 63 . L. Eldjam, E . Jellum and O . Stokke, Clin . Chim . Acta 40, 46}-476 (1972) . 64 . L. Eldjarn, E . Jellum and O. Stokke in Inborn Errors of Metabolism (Hommes, F.A . and Van Den Berg, C .J ., editors), pp " 255-268; Academic Press, New York (1973) . 65 . L. Eldjarn, O . Stokke and E . Jellum in Stem, J ., and Toothill, C. (editors), Soc . for the Study of Inbom Errorsa of Metabolism, pp . 113-120 (1972) . 66 . J .H . Stromure and L. Eldjarn, Scand . J . Clan . Lab . Invest . 29, 335-342 (1972) . 67 . L. Hagenfeldt, A. Larsson and R. Zetterstrom, Acta Paediat . Scand ._62, 1-8(1973) . 68 . A . Larason, personal communications, 1973, 1974 . 69 . V .P . Wollner, R. Sekuro, A. Meister, A . Larsson, Proc .NatI .Acad .Sei .,71,(1974) . 70 . R. Sekura and A. Meister, Proc . Natl . Acad . Sci . U .S ., 71, (1974) . 71 . H . N . Christensen, C. ds Cespedes, M. E . Handlogten and R . Ranguist, Biochim. Biophys. Acta 300, 487-522 (1973) .