Exp. Eye Res. (1990) 50. 689-694
Some
Aspects
of Dr Kinoshita’s Contributions Lens Protein Chemistry ABRAHAM
Biochemistry
to
SPECTOR
and Molecular Biology Laboratqry, Department of Ophtha/mo/ogy, College and Surgeons, Columbia University, New York, NY 70032, U.S.A.
of Physicians
A review of some of Dr Kinoshita’s contributions to our understanding of lens protein and glutathione biochemistry is presented. Particular emphasis is placed on Dr Kinoshita’s work involved with the relationship of carbohydrate metabolism and the maintenance of reduced glutathione. the question of the biological function of glutathione in the lens, the effect of oxidative stress provided by diamide and azoester on glutathione, membrane pump function and protein and also ascorbate and H,O, effects on Na+, K+-ATPase. The importance of oxidative stress was recognized early by Dr Kinoshita and he has continued to make significant contributions in this area as illustrated by his work with Dr Zigler on posterior subcapsular cataracts and with Drs Garland and Zigler on mixed function oxidation. It is concluded that Dr Kinoshita’s overall contributions in the areas mentioned above have been broad and of considerable importance. Key words: glutathione: lensprotein; oxidative stress: cataract. discussion omits aspects of his work. The choice is based, in part, upon the interests of the writer as well asthe assumption that it was Dr Kinoshita’s interest in glutathione that significantly influenced his decision to accept Dr David Cogan’sinvitation to join the Howe Laboratory of Harvard Medical School, for it was already known in the early 1950s that the lens had a high glutathione concentration. At the time Dr Kinoshita was doing his graduate work, there was considerable interest in transpeptidation and transamidation reactions. Working with kidney extracts, Dr Kinoshita isolated and purified an enzyme from kidney which was shown to catalyze the reaction (Kinoshita and Ball, 1952 1:
Dr Kinoshita’s work in lens carbohydrate chemistry, the development of the osmotic hypothesis to explain sugar-induced cataract, the demonstration of the pivotal role of aldose reductase in that process, and finally, the development of aldosereductase inhibitors which were shown initially to delay and, with more effective drugs, prevent sugar cataracts, is considered his most important contribution to eye research. But Dr Kinoshita’s contributions extend far beyond his seminal work in carbohydrate chemistry and diabetic cataract. He has made fundamental contributions to almost every aspect of lens biochemistry. In reviewing his contributions to peptide and protein chemistry, it quickly became apparent that in the short time available, it was not possibleto review adequately this large body of work and show its impact on the present viewpoint. Thus, from his contributions to ,8- and ycrystallin structure, a-crystallin aggregation, aspects of the molecular biology of the crystallins, membrane chemistry, the effect of cataract development on protein structure and the impact of oxidative stress upon lens protein and glutathione (GSH), only the last subject is considered, and even in this case, the
i F-” (332
1
yGlu-Cys-Gly
+ Arg -+ Glu-Arg
+ Cys-Gly.
Further work demonstrated the general reaction catalyzed by the enzyme is in Scheme 1. While this enzyme was only present in the kidney and pancreas and did not appear to use glutamine as a substrate, it anticipated an important class of enzymes discovered by Waelsch, the trans-
ii F- R’ (332
+R’-
I
CH2
+R
(332
I WJAH‘OH
W
I PH‘COOH
R = Cys- Gly, Gly R’ = aminoacid
Scheme1. ‘)0144835/90/060689+06
$03.00/O
0 1990 AcademicPressLimited
690
A. SPECTOR
““ii”
R-NH2
+
T
C=O
e
Y=O NH I R
+
NH3
Scheme 2.
glutaminases (Waelsch. 1962 1. These enzymes are activated by Ca”+ and catalyze the general reaction shown in Scheme 2, where the amino acid utilized by the enzyme encompassesa broad spectrum of compounds including lysine sometimespresent in peptide linkage and the amide represents glutamine, in this example, in peptide linkage. Recently, work from Lorand’s and Bloemendal’s laboratories (Lorand et al., 1981; Berbers et al., 1983) has suggested that this enzyme may be activated by increasing Ca”+ concentrations found in cataract formation and specifically induce cross-linking between P-crystallin peptides shown in Scheme 3. It is interesting to note that the addition of amines will inhibit the reaction. But, it was with GSH, itself that Dr Kinoshita was fascinated. Thus, in the late 195Os, he published a number of papers concerned with GSH that developed into a general interest in oxidation of the lens. He was, at that time, evaluating the relative contributions to glucose metabolism of the glycolytic pathway, Krebs cycle, and the hexose monophosphate shunt. In the course of this work, Dr Kinoshita recognized that the unusually active shunt activity in the lens would result in the production of a significant amount of NADPH which could be utilized to reduce oxidized glutathione (GSSG).Anticipating current concepts, he was able to show that the presence of oxidized glutathione stimulated hexosemonophosphateactivity almost twofold (Kinoshita and Masarat, 19 57). Recent work has shown that H,O, causes rapid oxidation of GSH (Spector et al., 198 7) and has confirmed the stimulation of shunt activity (Giblin, McCready and Reddy, 1982). With characteristic care, Dr Kinoshita demonstrated that there was sufficient GSH reductase activity to reduce the oxidized glutathione. Indeed examining a number of tissues an interesting correlation between glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the shunt pathway, and GSHreductase activities was found. Where the activity of the G6Pd was high, GSHreductase activity was also high and where one enzyme had a low activity so did the other. Thus, the activities of the two enzymes appeared to be linked.
-Lys-NH2
+
Again, anticipating recent work. Dr Kinoshita showed that the lens contains only a few percent of glutathione in the oxidized form. With more sophisticated techniques, it has been found that only trace amounts of GSSG are found in lens epithelial cell preparations (Spector et al.. 1987). What then is the function of glutathione.; This is a question that has concerned Dr Kinoshita throughout his career. The common belief is that glutathione protects protein thiol groups from oxidation. However, as Dr Kinoshita’s early work demonstrates, the overall mechanism is not simple. Working with calf lens homogenates, it was shown that while GSH thiol groups were rapidly oxidized by 100 % 0, in the presence of 10 ,u~ cupric ion, the protein thiols were hardly affected (Merola and Kinoshita, 1957). Indeed, removing GSH had little effect on protein thiol oxidation. Thus, it was clear that the GSH thiols were much more reactive than the protein thiols. After perturbation of the native protein structure, it could be demonstrated that a significant fraction of the protein thiol was rapidly oxidized. In later work reported from Dr Kinoshita’s laboratory, it was found that the human lens proteins were easily oxidized (Kinoshita and Merola, 1973). However, recent work has now shown that in protein isolated from young human lenses,the thiols are buried and unreactive, but with aging and development of cataract, the protein thiols become exposed, and with cataract formation, oxidation is observed (Garner and Spector, 1980). Unpublished work from this laboratory has shown that the loss of glyceraldehyde- 3-phosphate dehydrogenase (GPD) activity caused by H,O, oxidation was not effectively restored by GSH concentrations of 34 mM. In contrast, 200 /AM thioredoxin restored GPD concentration activity to the normal range. Thus, there is now considerable evidence to suggestthat the GSH protection of protein does not involve the direct reduction of oxidized thiol. Indeed, work reported from this laboratory and from M. Lou suggests that GSH forms mixed disulfide with protein thiols, a situation which contributes to protein perturbation (Lou, McKellar and Chang, 1985; Spector, Wang and Huang. 1986). It now seemsthat the primary function of GSH involves the detoxification of potent oxidants which are capable of oxidizing protein. Thus. for example, the major lens enzyme metabolizing H,O., - requires GSH.
H2N-C
f Scheme 3.
2GSH + H,O, -
t:bH
GSSG+ 2H,O. ,wr’,~xi
Some
Aspects
of Dr Kinoshita’s Contributions Lens Protein Chemistry ABRAHAM
Biochemistry
to
SPECTOR
and Molecular Biology Laboratqry, Department of Ophtha/mo/ogy, College and Surgeons, Columbia University, New York, NY 70032, U.S.A.
of Physicians
A review of some of Dr Kinoshita’s contributions to our understanding of lens protein and glutathione biochemistry is presented. Particular emphasis is placed on Dr Kinoshita’s work involved with the relationship of carbohydrate metabolism and the maintenance of reduced glutathione. the question of the biological function of glutathione in the lens, the effect of oxidative stress provided by diamide and azoester on glutathione, membrane pump function and protein and also ascorbate and H,O, effects on Na+, K+-ATPase. The importance of oxidative stress was recognized early by Dr Kinoshita and he has continued to make significant contributions in this area as illustrated by his work with Dr Zigler on posterior subcapsular cataracts and with Drs Garland and Zigler on mixed function oxidation. It is concluded that Dr Kinoshita’s overall contributions in the areas mentioned above have been broad and of considerable importance. Key words: glutathione: lensprotein; oxidative stress: cataract. discussion omits aspects of his work. The choice is based, in part, upon the interests of the writer as well asthe assumption that it was Dr Kinoshita’s interest in glutathione that significantly influenced his decision to accept Dr David Cogan’sinvitation to join the Howe Laboratory of Harvard Medical School, for it was already known in the early 1950s that the lens had a high glutathione concentration. At the time Dr Kinoshita was doing his graduate work, there was considerable interest in transpeptidation and transamidation reactions. Working with kidney extracts, Dr Kinoshita isolated and purified an enzyme from kidney which was shown to catalyze the reaction (Kinoshita and Ball, 1952 1:
Dr Kinoshita’s work in lens carbohydrate chemistry, the development of the osmotic hypothesis to explain sugar-induced cataract, the demonstration of the pivotal role of aldose reductase in that process, and finally, the development of aldosereductase inhibitors which were shown initially to delay and, with more effective drugs, prevent sugar cataracts, is considered his most important contribution to eye research. But Dr Kinoshita’s contributions extend far beyond his seminal work in carbohydrate chemistry and diabetic cataract. He has made fundamental contributions to almost every aspect of lens biochemistry. In reviewing his contributions to peptide and protein chemistry, it quickly became apparent that in the short time available, it was not possibleto review adequately this large body of work and show its impact on the present viewpoint. Thus, from his contributions to ,8- and ycrystallin structure, a-crystallin aggregation, aspects of the molecular biology of the crystallins, membrane chemistry, the effect of cataract development on protein structure and the impact of oxidative stress upon lens protein and glutathione (GSH), only the last subject is considered, and even in this case, the
i F-” (332
1
yGlu-Cys-Gly
+ Arg -+ Glu-Arg
+ Cys-Gly.
Further work demonstrated the general reaction catalyzed by the enzyme is in Scheme 1. While this enzyme was only present in the kidney and pancreas and did not appear to use glutamine as a substrate, it anticipated an important class of enzymes discovered by Waelsch, the trans-
ii F- R’ (332
+R’-
I
CH2
+R
(332
I WJAH‘OH
W
I PH‘COOH
R = Cys- Gly, Gly R’ = aminoacid
Scheme1. ‘)0144835/90/060689+06
$03.00/O
0 1990 AcademicPressLimited
690
A. SPECTOR
““ii”
R-NH2
+
T
C=O
e
Y=O NH I R
+
NH3
Scheme 2.
glutaminases (Waelsch. 1962 1. These enzymes are activated by Ca”+ and catalyze the general reaction shown in Scheme 2, where the amino acid utilized by the enzyme encompassesa broad spectrum of compounds including lysine sometimespresent in peptide linkage and the amide represents glutamine, in this example, in peptide linkage. Recently, work from Lorand’s and Bloemendal’s laboratories (Lorand et al., 1981; Berbers et al., 1983) has suggested that this enzyme may be activated by increasing Ca”+ concentrations found in cataract formation and specifically induce cross-linking between P-crystallin peptides shown in Scheme 3. It is interesting to note that the addition of amines will inhibit the reaction. But, it was with GSH, itself that Dr Kinoshita was fascinated. Thus, in the late 195Os, he published a number of papers concerned with GSH that developed into a general interest in oxidation of the lens. He was, at that time, evaluating the relative contributions to glucose metabolism of the glycolytic pathway, Krebs cycle, and the hexose monophosphate shunt. In the course of this work, Dr Kinoshita recognized that the unusually active shunt activity in the lens would result in the production of a significant amount of NADPH which could be utilized to reduce oxidized glutathione (GSSG).Anticipating current concepts, he was able to show that the presence of oxidized glutathione stimulated hexosemonophosphateactivity almost twofold (Kinoshita and Masarat, 19 57). Recent work has shown that H,O, causes rapid oxidation of GSH (Spector et al., 198 7) and has confirmed the stimulation of shunt activity (Giblin, McCready and Reddy, 1982). With characteristic care, Dr Kinoshita demonstrated that there was sufficient GSH reductase activity to reduce the oxidized glutathione. Indeed examining a number of tissues an interesting correlation between glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the shunt pathway, and GSHreductase activities was found. Where the activity of the G6Pd was high, GSHreductase activity was also high and where one enzyme had a low activity so did the other. Thus, the activities of the two enzymes appeared to be linked.
-Lys-NH2
+
Again, anticipating recent work. Dr Kinoshita showed that the lens contains only a few percent of glutathione in the oxidized form. With more sophisticated techniques, it has been found that only trace amounts of GSSG are found in lens epithelial cell preparations (Spector et al.. 1987). What then is the function of glutathione.; This is a question that has concerned Dr Kinoshita throughout his career. The common belief is that glutathione protects protein thiol groups from oxidation. However, as Dr Kinoshita’s early work demonstrates, the overall mechanism is not simple. Working with calf lens homogenates, it was shown that while GSH thiol groups were rapidly oxidized by 100 % 0, in the presence of 10 ,u~ cupric ion, the protein thiols were hardly affected (Merola and Kinoshita, 1957). Indeed, removing GSH had little effect on protein thiol oxidation. Thus, it was clear that the GSH thiols were much more reactive than the protein thiols. After perturbation of the native protein structure, it could be demonstrated that a significant fraction of the protein thiol was rapidly oxidized. In later work reported from Dr Kinoshita’s laboratory, it was found that the human lens proteins were easily oxidized (Kinoshita and Merola, 1973). However, recent work has now shown that in protein isolated from young human lenses,the thiols are buried and unreactive, but with aging and development of cataract, the protein thiols become exposed, and with cataract formation, oxidation is observed (Garner and Spector, 1980). Unpublished work from this laboratory has shown that the loss of glyceraldehyde- 3-phosphate dehydrogenase (GPD) activity caused by H,O, oxidation was not effectively restored by GSH concentrations of 34 mM. In contrast, 200 /AM thioredoxin restored GPD concentration activity to the normal range. Thus, there is now considerable evidence to suggestthat the GSH protection of protein does not involve the direct reduction of oxidized thiol. Indeed, work reported from this laboratory and from M. Lou suggests that GSH forms mixed disulfide with protein thiols, a situation which contributes to protein perturbation (Lou, McKellar and Chang, 1985; Spector, Wang and Huang. 1986). It now seemsthat the primary function of GSH involves the detoxification of potent oxidants which are capable of oxidizing protein. Thus. for example, the major lens enzyme metabolizing H,O., - requires GSH.
H2N-C
f Scheme 3.
2GSH + H,O, -
t:bH
GSSG+ 2H,O. ,wr’,~xi
