Mechanisms of Ageing and Development, 6 (1977) 241-257
241
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
R E C E N T D E V E L O P M E N T S IN T H E A G E - R E L A T E D A L T E R A T I O N O F ENZYMES: A REVIEW
MORTON ROTHSTEIN Division of Cell and Molecular Biology, State University of New York at Buffalo, Buffalo, N. Y. 14214 (U.S.A.)
(Received December 15, 1976)
SUMMARY Recent developments dealing with the age-related alteration of enzymes have been examined. Since the last review of this field, three enzymes have been purified to homogeneity from young and old nematodes (enolase, phosphoglycerate kinase, triosephosphate isomerase) and one from the liver of young and old rats (superoxide dismutase). In all cases except for triosephosphate isomerase, the enzymes from old animals show a reduced catalytic ability compared to those from young animals. In addition, new reports of increases in the amount of altered enzymes in late-passage cells in tissue culture have appeared, though contrary evidence has also been published. The data from these and other papers are compared and discussed. Possible explanations for the alteration of enzymes include error theory, substitution of individual amino acids and conformational change without sequence change. Though final conclusions cannot be made, the evidence favors the latter idea to explain the presence of altered enzymes in old animals.
INTRODUCTION A review dealing with age-related alterations of enzymes was published recently [ 1]. However, since the original preparation of the manuscript, enough new information has become available to warrant an updating of the subject. In the interval, the first example of a pure altered enzyme from mammalian species has been isolated from the liver of old rats; new information has become available dealing with altered enzymes in latepassage cells in tissue culture; several reports of altered enzymes in cells which carry out no protein synthesis (e.g., red blood cells) have been published and three more enzymes from young and old free-living nematodes (Turbatrix aceti) have been purified and described. Detailed study of one of these altered enzymes (enolase) has provided support for the idea that the alteration is a result of conformational modifications without changes in amino acid sequence. Other data have been obtained which support this
242 concept. However, unequivocal evidence which eliminates the possibility of minor changes in sequence or post-synthetic (covalent) modification of proteins as a cause of altered enzymes has not been reported. This paper reviews recent work in the field of altered enzymes and interprets the data in the context of new and previous results. lntact mammalian species Previous reports showing the presence of altered enzymes in mammalian species were limited to work with aldolase in crude mouse liver [2] and mouse muscle homogenates [3] and a brief abstract dealing with pure aldolase in mouse liver [4]. Recently, Reiss and Gershon [5] have shown that pure superoxide dismutase from livers of old rats (27 months old) has only 40% of the specific activity of the pure enzyme isolated from young animals (6 months old). A Wistar strain of rats was used, described as "WF". Immunotitration confirmed the fact that the "old" enzyme* has a lower catalytic ability, as more antiserum per unit of activity is required than for the "young" enzyme. In accord with most of the other reports dealing with the agerelated alteration of enzymes, "old" superoxide dismutase shows a greater sensitivity to heat, but other properties (Ki, molecular weight, charge, etc.) show no differences. The enzyme consists of 3 isozymes. A recent note, employing a complex procedure for analysis of an 18-residue peptide from aldolase [6] showed no significant difference in activity per nanomole of sequence in 24 month old rats (Fischer 344) and young animals (2 months of age). It is unfortunate that the latter were selected before maturity instead of at 6-12 months. However, the comparison would be valid if the specific activity of the enzyme in more mature animals is the same as that in the young animals used. This circumstance is a reasonable expectation as Fritz and White [7] showed that although there is an increase in the specific acitivity of phosphoglycerate kinase in rat muscle during development, it is due to an increase in the amount of enzyme. The specific activity of the molecules (based upon immunotitrations) does not change. Thus, according to the above experiments [6], there is no accumulation of "inactive" aldolase in the Fischer 344 rats at 24 months of age. Grinna and Barber [8], in a follow-up of their original paper, indicate that the agerelated loss of specific activity reported for glucose-6-phosphate in microsomal fractions from liver and kidney of male Sprague-Dawley rats is due to the presence of less enzyme molecules in the membrane of the old animals (24 vs. 6 months of age). Wulf and Cutler [9] examined the enzyme activity and thermal stability of glucose6-phosphate dehydrogenase (G6PD) in 5 different tissues of C57BL/6J female mice at four ages. Oddly, the relative enzymatic activity increased with age. Since the determinations were carried out in crude homogenates, it is not known if the observed changes in activity are due to changes in the amount of enzyme, amount of protein, or to the *Henceforth, "young" and "old" refer to preparations isolated from young and old organisms, respectively.
243 presence of altered enzyme. Small increases were found in heat-labile G6PD in the tissues of old mice (770-780 days of age) compared to those from young animals (160-180 days of age). Unfortunately, the heat lability curves for G6PD showed different slopes for each tissue studied. Since the enzyme should be the same in each case, this result is anomalous and makes it difficult to know how accurately the heat lability experiments really reflect the percentage of altered enzymes. In this reviewer's opinion, experiments of the above nature would be far more valuable if they were performed on pure enzymes. Recently, Yagil [ 10] reported evidence for the lack of altered G6PD in the livers of old C57B1 mice. The enzyme was examined by electrophoresis on acrylamide gels, electroimmunodiffusion and temperature stability. The latter detected no altered enzyme. The first two procedures would not detect "altered" G6PD as changes in charge have never been observed in any of the altered enzymes so far studied (see Discussion). Another recent paper reports no change in the properties of fructose-l,6biphosphatase from liver of young and adult Fischer rats [1 I]. The young rats utilized were less than 3 months old and the adult animals were barely 1 year old. These experiments, therefore, do not add to our information regarding aged animals.
Nematode enzymes Nematodes continue to play an important role as a source of "altered" enzymes. Bolla and Brot [12] reported that in T. aceti, the specific activity of elongation factor I (EF-1) declines with age. In old organisms, the factor shifts mostly to lower molecular weight forms. Immunotitration indicated the presence of inactive or partly active molecules. However, the antiserum was prepared to the factor obtained from young organisms. This material consists of heavier molecular species. Thus, the antiserum might not react so effectively with the "old" (smaller) EF-1 molecules. In fact, from the figures shown, it appears that even the young preparation of EF-1 is not inhibited completely by the antiserum. The above authors also reported that after an initial rise, DNA and RNA polymerase show an age-related loss of specific activity. However, the results are based upon mg of protein in crude homogenates and therefore do not distinguish between a reduced amount of enzyme and a reduced effectiveness of the enzyme molecules. Addition of ct.tocopherol to the medium was found to extend the lifespan of T. aceti, as had been reported earlier by Epstein and Gershon [13]. However, Bolla and Brot [12] showed that the relationship of enzyme activity to the age of the organisms was affected by the presence of the antioxidant. For example, DNA polymerase activity was increased and EF-I activity peaked at a later age. The first example of a pure enzyme to be compared from young and old organisms was isocitrate lyase from T. acen" [14]. The enzyme was large (mol. wt. 480,000), consisting of 4 subunits and 5 isozymes. In spite of these complexities, it is clear that the "old" enzyme has a much lower specific activity than the "young" enzyme. Moreover, at least 2 of the "old" isozymes are less stable to heat than their young counterparts [ 15]. From the heat stability curves, it is apparent that the "old" isocitrate lyase consists
244 of normally active + partly active molecules. It was also established that there were no "cuts" in the protein nor were there any charge differences. Such characteristics as Kra and Ki were unaltered. In this laboratory, three additional enzymes from young and old T. aceti have been purified and characterized. Sharma et al. [16] purified enolase from young and old T. acetL The enzyme is relatively small, having a molecular weight of 82,000 and consisting of 2 subunits of equal size. There are no isozymes detectable by gel electrophoresis (2 pH values) or by isoelectric focusing. The subunits are probably identical, an idea further supported by the fact that neither subunit possesses a free N-terminal amino group (Sharma and Rothstein, unpublished results). Typically, vertebrate enolase from other sources possesses an N-terminal acetylalanine [17]. "Young" and "old" enolase from T. aceti yielded identical patterns after gel electrophoresis and isoelectric focusing. Km and Ki (phosphate) were essentially unchanged though the "old" enzyme did show a slightly smaller value for the former (0.6 × 10-4M vs. 1.1 × 10-4M). The heat stability pattern was linear for the pure "young" enzyme but biphasic for the "old" enzyme. Both components of the latter curve showed a more rapid loss of activity at 52.5 °C than did the single component of the "young" preparation. Thus, it would appear that "old" enolase from T. aceti consists entirely of "altered" molecules. Another difference between "young" and "old" enolase was found in the respective elution patterns from columns of DE-52 [16]. The latter yields an inactive protein peak which appears just before the elution peak of enolase. This inactive material reacts with the antiserum prepared against "young" or "old" enolase. It is therefore an inactive form of the enzyme. Pure phosphoglycerate kinase (PGK) from old T. aceti appears to be typical of the altered enzymes thus far studied [18]. It has a reduced specific activity compared to the "young" enzyme (approximately 35% reduction) but other parameters such as Ki, molecular weight, charge, etc. are unchanged. Km (ATP) shows a small increase, from 0.26 mM (young) to 0.52 mM (old). The heat stability of "old" PGK is the same as that for "young" PGK, both showing an identical, slightly biphasic pattern at each of two temperatures. Analogous results were obtained with "young" and "old" nematode aldolase studied in crude homogenates [19]. In mouse liver, pure "old" aldolase is reported to be more heat sensitive than the "young" enzyme, though a linear response is obtained in each case [4]. A third enzyme, triosephosphate isomerase (TPI) has recently been purified from T. aceti [20]. This enzyme, as do the others studied, shows a continuously declining, age-related reduction in specific activity based upon the amount of protein in the crude homogenate. "Old" homogenates (24-28 days) show an activity of 14-18 U/mg protein, whereas the value for "young" preparations (7-11 days) is 29-37 U/mg. The "old" enzyme retains its apparent lower specific activity through the first two purification steps (ammonium sulfate precipitation; chromatography on Sephadex G-100). However, after treatment on DEAE-Sephadex A-50, the specific activity of the "old" enzyme rises to match that of the "young" product. Proof that both the enzymes are identical was obtained by immunotitration and temperature stability experiments.
245 The finding, in T. aceti, that "young" and "old" TPI are identical [18] means that the reduced' activity of the latter in crude homogenates is due to a reduction of approximately 50% in the number of TPI molecules present.
Altered proteins in blood components (red blood cells, platelets, leukocytes) Red blood cells would seem to be an interesting model in which to study the effect of age on enzymes. Since new enzymes are not synthesized, any alterations in their properties must necessarily be caused by post-translational modification. Indeed, Kahn et al. found that the specific activity of G6PD decreased in old red blood cells [21]. Since the decrease was based upon the ratio of enzyme activity to immunological reactivity, the data reflect a true loss of specific activity and not merely the presence of fewer enzyme molecules. At the same time, it was observed that in old cells, new forms of G6PD appeared which moved toward the anode during electrofocusing. The lower specific activity of the enzyme was related to these anodic forms. A soluble factor in the red cells appears to be responsible for the changed properties of the enzyme. Similar results were obtained with pure G6PD from human platelets [22]. Addition of extracts of leukemic granulocytes resulted in a decrease in the specific activity and increased amounts of anodic forms of the enzyme. Kahn et al. [23] concluded that the altered isoelectric focusing pattern and reduced specific activity of G6PD were a result of the presence of an increased concentration of G6PD "modifying factors". A partial purification of the "factors" from leukemic granulocytes showed it to be a heat-stable material of small molecular weight (
241
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
R E C E N T D E V E L O P M E N T S IN T H E A G E - R E L A T E D A L T E R A T I O N O F ENZYMES: A REVIEW
MORTON ROTHSTEIN Division of Cell and Molecular Biology, State University of New York at Buffalo, Buffalo, N. Y. 14214 (U.S.A.)
(Received December 15, 1976)
SUMMARY Recent developments dealing with the age-related alteration of enzymes have been examined. Since the last review of this field, three enzymes have been purified to homogeneity from young and old nematodes (enolase, phosphoglycerate kinase, triosephosphate isomerase) and one from the liver of young and old rats (superoxide dismutase). In all cases except for triosephosphate isomerase, the enzymes from old animals show a reduced catalytic ability compared to those from young animals. In addition, new reports of increases in the amount of altered enzymes in late-passage cells in tissue culture have appeared, though contrary evidence has also been published. The data from these and other papers are compared and discussed. Possible explanations for the alteration of enzymes include error theory, substitution of individual amino acids and conformational change without sequence change. Though final conclusions cannot be made, the evidence favors the latter idea to explain the presence of altered enzymes in old animals.
INTRODUCTION A review dealing with age-related alterations of enzymes was published recently [ 1]. However, since the original preparation of the manuscript, enough new information has become available to warrant an updating of the subject. In the interval, the first example of a pure altered enzyme from mammalian species has been isolated from the liver of old rats; new information has become available dealing with altered enzymes in latepassage cells in tissue culture; several reports of altered enzymes in cells which carry out no protein synthesis (e.g., red blood cells) have been published and three more enzymes from young and old free-living nematodes (Turbatrix aceti) have been purified and described. Detailed study of one of these altered enzymes (enolase) has provided support for the idea that the alteration is a result of conformational modifications without changes in amino acid sequence. Other data have been obtained which support this
242 concept. However, unequivocal evidence which eliminates the possibility of minor changes in sequence or post-synthetic (covalent) modification of proteins as a cause of altered enzymes has not been reported. This paper reviews recent work in the field of altered enzymes and interprets the data in the context of new and previous results. lntact mammalian species Previous reports showing the presence of altered enzymes in mammalian species were limited to work with aldolase in crude mouse liver [2] and mouse muscle homogenates [3] and a brief abstract dealing with pure aldolase in mouse liver [4]. Recently, Reiss and Gershon [5] have shown that pure superoxide dismutase from livers of old rats (27 months old) has only 40% of the specific activity of the pure enzyme isolated from young animals (6 months old). A Wistar strain of rats was used, described as "WF". Immunotitration confirmed the fact that the "old" enzyme* has a lower catalytic ability, as more antiserum per unit of activity is required than for the "young" enzyme. In accord with most of the other reports dealing with the agerelated alteration of enzymes, "old" superoxide dismutase shows a greater sensitivity to heat, but other properties (Ki, molecular weight, charge, etc.) show no differences. The enzyme consists of 3 isozymes. A recent note, employing a complex procedure for analysis of an 18-residue peptide from aldolase [6] showed no significant difference in activity per nanomole of sequence in 24 month old rats (Fischer 344) and young animals (2 months of age). It is unfortunate that the latter were selected before maturity instead of at 6-12 months. However, the comparison would be valid if the specific activity of the enzyme in more mature animals is the same as that in the young animals used. This circumstance is a reasonable expectation as Fritz and White [7] showed that although there is an increase in the specific acitivity of phosphoglycerate kinase in rat muscle during development, it is due to an increase in the amount of enzyme. The specific activity of the molecules (based upon immunotitrations) does not change. Thus, according to the above experiments [6], there is no accumulation of "inactive" aldolase in the Fischer 344 rats at 24 months of age. Grinna and Barber [8], in a follow-up of their original paper, indicate that the agerelated loss of specific activity reported for glucose-6-phosphate in microsomal fractions from liver and kidney of male Sprague-Dawley rats is due to the presence of less enzyme molecules in the membrane of the old animals (24 vs. 6 months of age). Wulf and Cutler [9] examined the enzyme activity and thermal stability of glucose6-phosphate dehydrogenase (G6PD) in 5 different tissues of C57BL/6J female mice at four ages. Oddly, the relative enzymatic activity increased with age. Since the determinations were carried out in crude homogenates, it is not known if the observed changes in activity are due to changes in the amount of enzyme, amount of protein, or to the *Henceforth, "young" and "old" refer to preparations isolated from young and old organisms, respectively.
243 presence of altered enzyme. Small increases were found in heat-labile G6PD in the tissues of old mice (770-780 days of age) compared to those from young animals (160-180 days of age). Unfortunately, the heat lability curves for G6PD showed different slopes for each tissue studied. Since the enzyme should be the same in each case, this result is anomalous and makes it difficult to know how accurately the heat lability experiments really reflect the percentage of altered enzymes. In this reviewer's opinion, experiments of the above nature would be far more valuable if they were performed on pure enzymes. Recently, Yagil [ 10] reported evidence for the lack of altered G6PD in the livers of old C57B1 mice. The enzyme was examined by electrophoresis on acrylamide gels, electroimmunodiffusion and temperature stability. The latter detected no altered enzyme. The first two procedures would not detect "altered" G6PD as changes in charge have never been observed in any of the altered enzymes so far studied (see Discussion). Another recent paper reports no change in the properties of fructose-l,6biphosphatase from liver of young and adult Fischer rats [1 I]. The young rats utilized were less than 3 months old and the adult animals were barely 1 year old. These experiments, therefore, do not add to our information regarding aged animals.
Nematode enzymes Nematodes continue to play an important role as a source of "altered" enzymes. Bolla and Brot [12] reported that in T. aceti, the specific activity of elongation factor I (EF-1) declines with age. In old organisms, the factor shifts mostly to lower molecular weight forms. Immunotitration indicated the presence of inactive or partly active molecules. However, the antiserum was prepared to the factor obtained from young organisms. This material consists of heavier molecular species. Thus, the antiserum might not react so effectively with the "old" (smaller) EF-1 molecules. In fact, from the figures shown, it appears that even the young preparation of EF-1 is not inhibited completely by the antiserum. The above authors also reported that after an initial rise, DNA and RNA polymerase show an age-related loss of specific activity. However, the results are based upon mg of protein in crude homogenates and therefore do not distinguish between a reduced amount of enzyme and a reduced effectiveness of the enzyme molecules. Addition of ct.tocopherol to the medium was found to extend the lifespan of T. aceti, as had been reported earlier by Epstein and Gershon [13]. However, Bolla and Brot [12] showed that the relationship of enzyme activity to the age of the organisms was affected by the presence of the antioxidant. For example, DNA polymerase activity was increased and EF-I activity peaked at a later age. The first example of a pure enzyme to be compared from young and old organisms was isocitrate lyase from T. acen" [14]. The enzyme was large (mol. wt. 480,000), consisting of 4 subunits and 5 isozymes. In spite of these complexities, it is clear that the "old" enzyme has a much lower specific activity than the "young" enzyme. Moreover, at least 2 of the "old" isozymes are less stable to heat than their young counterparts [ 15]. From the heat stability curves, it is apparent that the "old" isocitrate lyase consists
244 of normally active + partly active molecules. It was also established that there were no "cuts" in the protein nor were there any charge differences. Such characteristics as Kra and Ki were unaltered. In this laboratory, three additional enzymes from young and old T. aceti have been purified and characterized. Sharma et al. [16] purified enolase from young and old T. acetL The enzyme is relatively small, having a molecular weight of 82,000 and consisting of 2 subunits of equal size. There are no isozymes detectable by gel electrophoresis (2 pH values) or by isoelectric focusing. The subunits are probably identical, an idea further supported by the fact that neither subunit possesses a free N-terminal amino group (Sharma and Rothstein, unpublished results). Typically, vertebrate enolase from other sources possesses an N-terminal acetylalanine [17]. "Young" and "old" enolase from T. aceti yielded identical patterns after gel electrophoresis and isoelectric focusing. Km and Ki (phosphate) were essentially unchanged though the "old" enzyme did show a slightly smaller value for the former (0.6 × 10-4M vs. 1.1 × 10-4M). The heat stability pattern was linear for the pure "young" enzyme but biphasic for the "old" enzyme. Both components of the latter curve showed a more rapid loss of activity at 52.5 °C than did the single component of the "young" preparation. Thus, it would appear that "old" enolase from T. aceti consists entirely of "altered" molecules. Another difference between "young" and "old" enolase was found in the respective elution patterns from columns of DE-52 [16]. The latter yields an inactive protein peak which appears just before the elution peak of enolase. This inactive material reacts with the antiserum prepared against "young" or "old" enolase. It is therefore an inactive form of the enzyme. Pure phosphoglycerate kinase (PGK) from old T. aceti appears to be typical of the altered enzymes thus far studied [18]. It has a reduced specific activity compared to the "young" enzyme (approximately 35% reduction) but other parameters such as Ki, molecular weight, charge, etc. are unchanged. Km (ATP) shows a small increase, from 0.26 mM (young) to 0.52 mM (old). The heat stability of "old" PGK is the same as that for "young" PGK, both showing an identical, slightly biphasic pattern at each of two temperatures. Analogous results were obtained with "young" and "old" nematode aldolase studied in crude homogenates [19]. In mouse liver, pure "old" aldolase is reported to be more heat sensitive than the "young" enzyme, though a linear response is obtained in each case [4]. A third enzyme, triosephosphate isomerase (TPI) has recently been purified from T. aceti [20]. This enzyme, as do the others studied, shows a continuously declining, age-related reduction in specific activity based upon the amount of protein in the crude homogenate. "Old" homogenates (24-28 days) show an activity of 14-18 U/mg protein, whereas the value for "young" preparations (7-11 days) is 29-37 U/mg. The "old" enzyme retains its apparent lower specific activity through the first two purification steps (ammonium sulfate precipitation; chromatography on Sephadex G-100). However, after treatment on DEAE-Sephadex A-50, the specific activity of the "old" enzyme rises to match that of the "young" product. Proof that both the enzymes are identical was obtained by immunotitration and temperature stability experiments.
245 The finding, in T. aceti, that "young" and "old" TPI are identical [18] means that the reduced' activity of the latter in crude homogenates is due to a reduction of approximately 50% in the number of TPI molecules present.
Altered proteins in blood components (red blood cells, platelets, leukocytes) Red blood cells would seem to be an interesting model in which to study the effect of age on enzymes. Since new enzymes are not synthesized, any alterations in their properties must necessarily be caused by post-translational modification. Indeed, Kahn et al. found that the specific activity of G6PD decreased in old red blood cells [21]. Since the decrease was based upon the ratio of enzyme activity to immunological reactivity, the data reflect a true loss of specific activity and not merely the presence of fewer enzyme molecules. At the same time, it was observed that in old cells, new forms of G6PD appeared which moved toward the anode during electrofocusing. The lower specific activity of the enzyme was related to these anodic forms. A soluble factor in the red cells appears to be responsible for the changed properties of the enzyme. Similar results were obtained with pure G6PD from human platelets [22]. Addition of extracts of leukemic granulocytes resulted in a decrease in the specific activity and increased amounts of anodic forms of the enzyme. Kahn et al. [23] concluded that the altered isoelectric focusing pattern and reduced specific activity of G6PD were a result of the presence of an increased concentration of G6PD "modifying factors". A partial purification of the "factors" from leukemic granulocytes showed it to be a heat-stable material of small molecular weight (
