- Email: [email protected]
Effect of age on the crystalline rat liver lactate dehydrogenase
Arch. Gerontol. Geriatr., 5 (1986) 57-64
57
Elsevier AGG 00137
Effect of age on the crystalline rat liver lactate dehydrogenase M. Prabhakaram and S.N. Singh * Biochemistry Laboratory, Department of Zoology, Banaras Hindu University, Varanasi-221 005, India (Received 1 September 1985; revised version received 5 December 1985; accepted 9 December 1985)
Summary Lactate dehydrogenase (LDH) was purified and crystallised from the liver of immature (4 weeks), young (22 weeks) and old (116 weeks) female rats and the kinetic, physical and chemical properties of the purified enzyme were studied, Even though slight differences existed in effect of pH, temperature, storage and ratio of A2s0/26o, significant differences were not observed in kinetic properties, behaviour on polyacrylamide gels. ion-exchange chromatography column, and -SH groups among the three age groups. These data indicate that, unlike muscle LDH of old rats which had shown altered properties (earlier report from our laboratory), liver LDH is an unaltered, like LDH of heart. The possible reason for the unaltered nature of the liver LDH may be: (a) the tissue origin; (b) the 8ene responsible for its synthesis is expressing well even in old age; or (c) non-influence of post-translational modifications on the enzyme molecule.
liver LDH; aging; properties
Introduction
The different levels of enzymes in aging animals were studied in detail (Kanungo, 1980; Wilson et al., 1982). However, a limited number of enzymes was purified so far, from aging rats, to study the effect of age (Reznick and Gershon, 1977; Gafni, 1981; Dreyfus et al., 1983; Rothstein, 1983; Prabhakaram and Singh, 1984, 1985; Singh and Prabhakaram, 1984). It leads to the conclusion that post-translational rather than sequential changes may be responsible for the altered nature of enzymes. In this regard, to find out the cause of altered/unaltered nature of enzymes, we have selected lactate dehydrogenase as a model system because of its immense role in giycolysis. The biological importance of lactate dehydrogenase (L-lactate: NAD ÷ oxidoreductase, EC 1.1.1.27, LDH) is studied in detail for the last three decades. This ubiquitous enzyme occurs generally in vertebrate cells as five separable isozymic forms (Markert and Ursprung, 1962), with a predominant M-type in * To whom reprint requests should be addressed. 01674943/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
58
anaerobic tissue (e.g. skeletal muscle) and H-type in aerobic tissue (e.g. heart muscle). However, it is not well understood why liver has adapted M-type instead of H-type. Earlier we have reported that M-LDH, but not H-LDH is an altered one (Prabhakaram and Singh, 1984, 1985). The present report deals about the age-related properties of crystalline liver LDH of aging rats with a view to investigate if this enzyme exists as an altered/unaltered form during old age.
Materials and Methods
Animals Female albino Wistar strain rats, which were maintained under standard laboratory conditions, were used for the present studies. Immature (4 weeks), young (22 weeks) and old (116 weeks) rats were sacrificed and liver was excised immediately, chilled in ice-cold saline, following by processing for the purification of the lactate dehydrogenase.
Chemicals All the biochemicals were obtained from Sigma Chemical Co., USA. DEAE-Sephadex A-50 and Sephadex G-200 were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Triple distilled water was used through out the experimentation.
Analytical methods All steps were carded out at 0 + 2°C, unless otherwise mentioned. The purification procedure was that of Fritz et al. (1970) with certain modifications. To avoid hemoglobin contamination, batch step and two more cyclings of the purified enzyme on a DEAE A-50 ion-exchange chromatography column were included. The enzyme activity was measured by the spectrophotometric method of Kornberg (1955) and expressed in International Units. The protein content was measured according to the method of Lowry et al. (1951). Polyacrylamide gel electrophoresis (PAGE) was carried out following the method of Davis (1964), using 8~ acrylamide gels instead of 7.5~. Specific enzyme staining was done with lithium lactate as substrate (Dietz and Lubrano, 1967). General protein staining was carried out with 0.05~ Coomassie Brilliant Blue (Weber and Osborn, 1969). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was performed using the method of Laemmli (1970). Scanning of the gels was carried out at 500 nm in a Beckman Analytrol. The number of -SH groups was estimated with Ellman's reagent (DTNB: 5',5'-dithiobis-2-nitrobenzoic acid; Eilman, 1959). The purified enzyme was crystallised by the slow additions of ammonium sulphate (35-40~ final concentration) and crystals were photographed under the phase contrast microscope. The crystalline enzyme of all the three ages was passed through Sephadex G-200, after extensive dialysis against 0.05 M potassium phosphate buffer, pH 7.4. The enzyme thus
59
obtained was analysed in a U V range from 220-320 nm in a Unicam SP-500 spectrophotometer. The purified enzyme was used to study its kinetic, physical and chemical properties. These include K., (for pyruvate), K i (with oxalate and oxamate in presence of pyruvate), effect of pH, temperature, urea and sodium sulphite.
Results
Pilot experiments were carried out with liver tissue of male and female rats and it was observed that sex has no effect. Since there are no apparent differences in the properties of LDHs purified from liver of immature, young or old female rats, for brevity we compare here the data obtained for young and old rats only. In order to compare the properties of young and old enzymes, liver LDH was purified under the same conditions. Table I shows the purification protocol of LDH from the livers
young 122 weeks) 2O
0.8
~0
O.e
0.4
N
'°I
o
O m Old II1G weeks)
b
f
1~o
o.e
o
c 0.6 w IL
0.4
5O 0.2
i
10
20
30
Fraction number
Fig. 1. Elution profile of lactate dehydrogenase of liver of young and old female rats on D E A E Sephadex A-50 column.
60 TABLE 1 Purification protocol of liver lactate dehydrosenase of young (22 w) and old (116 w) rats Steps
Total activity (Units)
Specificactivity (U/mg protein)
Recovery (%)
youn8
old
young
youn8
13 780
7 420
4.8
3.8
Fractionation
8400
5900
19.3
16.8
60.95
79.51
4.02
4.37
Ion-exchange column
1785
1324
270.4
287.8
12.95
17.84
56.34
74.75
Crystallisation
1560
1180
503.2
421.4
11.32
15.90
I04.83
I09.45
18000× g supernatant 70% a m m o n i u m sulphate
®
o
b
c
old
-
Fold of purification old
young
-
-
old
®
Fig. 2. Densitometric scans of crude (a) and purified (b and c) liver lactate dehydrogenase of young (A) and old (B) rats.a and b, specific enzyme stain; c, general protein stain.
61 TABLE 11 Apparent kinetic constants of purified liver lactate dehydrogenase of young (22 w) and old (116 w) rats
K"m ( x 10-3 M pyruvate) K, (ram oxalate) K i (raM oxamate)
Young
Old
0.15 0.15 0.11
0.14 0.10 0.07
of young and old female rats. Although a decline in specific activity was observed in old enzyme (16~ lower than that of the young enzyme), the overall recovery rate and purification fold were identical in both age groups of rats. Their behaviour on ion-exchange chromatography was also similar (Fig. 1). The purity of the enzyme was checked on PAGE, Fig. 2(A and B) shows the densitometric scannings of young and old enzymes. A single cathodic, predominant M-type LDH was visualised on gels with specific enzyme stain and with general protein stain. A molecular weight of 35 000 (unimer) was obtained for young and old enzymes on SDS PAGE. Table II shows the kinetic properties of LDH of young and old liver tissues. Oxalate and oxamate inhibited the enzyme activity non-competitively and competitively, respectively, in presence of pyruvate. There is no significant difference in K m or K i of LDH in the two ages of rats and the values fall in a close range (Table II). Fig. 3 (A and B) shows the effect of pH and temperature. The pH optimum was found to be between range 5.0 and 7.5 and the optimum temperature was around 65°C. Similar values were obtained for young and old enzymes without major differences. Fig. 4 (A and B) shows the effect of sodium sulphite and urea on young and old liver LDHs, respectively. Sodium sulphite and urea inhibited the
120
40
40
35
55
75
Temperoture (oC)
2.0
4.0
6.0
8.0
I)14
Fig. 3. Effect of temperature (A) and pH (B) on the activity of purified liver lactate dehydrogenase of young (O O) and old (O e ) rats.
62
B
19
120
.~ so
0.001
001
GI
0o5
I
3
5
Fig. 4. Effect of sodium sulphite (A) and urea (B) on the activity of purified liver lactate dehydrogenase of young (O O) and old (0 O) rats.
enzyme activity of both young and old rats to 80~$ and 70~ (0.5 M sodium sulphite and 3 M urea), respectively. Crystalline enzyme of both the ages yielded a ratio of 1.7 and 1.9 for old and young enzymes, respectively, which is the ratio of A 28o/26o. The estimated -SH groups (residues/mole) of young and old enzymes are 90 and 85, respectively.
Discussion
The presence of age-dependent modifications in the properties of enzymes may give an insight in the altered nature of enzymes (being proteins) as a function of age. While the origin of the age-dependent phenomena has not been identified, they may result from changes in primary structure, due to biosynthetic error or somatic mutations (Hoiliday, 1975), or alternatively be the result of post-translational modifications (Dreyfus et al., 1983). In the present article we have compared the properties of lactate dehydrogenase purified from young and old liver tissues of rats. Although liver consists of predominant M-type of LDH, it has failed to show any marked differences with advancing age. Both, young and old, enzymes have been excluded as a sharp peak in the same volume on ion-exchange column. If 'old' enzyme consists of inactive molecules, they should occur in the void volume as a separate peak. However, such results were not obtained. In addition, the migration of both 'young' and 'old' enzymes is also similar on PAGE, indicating that charge differences do not exist between them. Nonetheless, the observed lower specific activity (16~) of" old enzyme as compared to young enzyme may be due to less number of active enzyme molecules. The K m for pyruvate, K~ for non-competitive inhibitor (oxalate) and competitive inhibitor (oxamate) are almost similar for young and old enzymes (where the initial activity was adjusted to the same level in both the forms). Likewise, the effect of pH, temperature, urea and sodium sulphite are
63
the same for young and old enzymes. Same number of -SH groups (residues/mole) and similar ratio of A2~0/26oconfirms that major structural differences do not exist between young and old enzymes. Studies on aldolase of the liver of mice (Gershon and Gershon, 1973a, b) and cytoplasmic superoxide dismutase of the liver, brain and heart of rats and mice (Reiss and Oershon, 1976a,b) have revealed that a n t i g e n i c i t y , K m, K i, electrophoretic mobility in polyacrylamide gels and SDS PAGE, are the same for both, young and old, individuals. Furthermore, isoelectric focussing of these enzymes does not show any difference in their electrical charges (Goren et al., 1977). Interestingly, we have observed that liver LDH resembles H-LDH in some aspects, like optimum pH, temperature and inhibition in presence of urea. Other properties are similar to that of M-LDH. Several authors have suggested that age-related modifications are due to errors in protein synthesis or to post-translational modifications (Rothstein, 1977: Dreyfus et al., 1983). However, it is not yet known whether age-related alterations have any general pattern or not. Szajnert and Schapira (1983) have reported that purified induced tyrosine aminotransferase of adult and old rat liver tissues behave identically even in presence of digestive enzymes. Purified glutamate dehydrogenase of immature, young and old rats has also failed to show any structural differences (Singh and Prabhakaram, 1984). The unaltered nature of liver LDH, as observed in the present study, can be attributed as a significant event to old animals. Since the muscle LDH shows altered nature in old age, part of its burden may be shared by liver LDH in conversion of pyruvate to lactate or vice versa. During vigorous muscular activity, the level of lactic acid increases and accumulates. This toxic lactic acid thus produced, easily escapes into blood stream and then enters the liver, where it is converted back to glucose (Cori cycle). Thus, it may be concluded that coexistence of a similar enzyme in different tissues as altered and unaltered forms is quite possible. Probably it is having a significant role in aging tissues. The reason for their altered/unaltered nature may be due to: (1) their utility by different tissues in different levels; (2) expression/repression of the gene(s) responsible for the synthesis of a specific enzyme at various stage of growth and development under the influence of a variety of modulators/regulators.
Acknowledgements The authors wish to thank Prof. M.S. Kanungo for encouragement and providing laboratory facilities. Financial support of U.G.C. (Special Assistance Programme grant to Department of Zoology) to M.P. is gratefully acknowledged.
References Davis, B.J. (1964): Disc electrophoresis. Methods and application to human serum proteins. Ann. NY Acad. 5ci., 121,404-427.
64 Dietz, A.A. and Lubrano, T. (1967): Separation and quantitation of lactic dehydrogenase iscenzymes by disc electrophoresis. Anal. Biochem., 20, 246-257. Dreyfus, J.C., Khan, A. and Schapira, F. (1983): In: Aimed proteins and aging, pp. 35-54. Editors: R.C. Adeinum and G.S. Roth. CRC Press inc., Florida. Ellman, G.L. (1959): Tissue sullhydryl groups. Arch. Biochem. Biophys., 82, 70-77. Fritz, PJ., Morrison, WJ., White, E.L. and Vesell, E.S. (1970): Comparative study of methods for quantitative measurement of lactate dehydrogenase isozymes. Anal. Biochem., 36, 443-453. Gafni, A. (1981): Location of age-related modifications in rat muscle glyceraldehyde-3-phosphate dehydrogenase. J. Biol. Chem., 256, 8875-8877. Gershon, H. and Gershon, D. (1973a): Altered enzymes in senescent organisms, mouse muscle aldolase. Mech. Ageing Dev., 2, 33--41. Gershon, H. and Gershon, D. (1973b): Inactive enzymes in aging mice: liver aldolase. Proc. Nat. Acad. Sci. USA, 70, 909-913. Goren, P., Reanick, A.Z., Reiss, U. and Gershon, D. (1977): lsoelectric properties of nematode aldolase and rat liver superoxide dismutase from young and old animals. FEBS Lett., 84, 83-86. Holliday, L. (1975): Testing the protein error theory of ageing: a reply to Baird, Samis, Massie and Zimmerman. Gerontologla, 21, 64-68. Kanungo, M.S. (1980): In: Biochemistry of Ageing, Academic Press, London. Komberg, A. (1955): In: Methods in Enzymology, VOl. !, pp. 441-443. Editors: N.O. Kaplan and S.P. Colowick. Academic Press, New York. Laemmli, U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature (London), 227, 680-685. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951): Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275. Markert, C.L. and Ursprung, H. (1962): The ontogeny of isozyme patterns of lactate dehydrogenase in the mouse. Dev. Biol., 5, 363-381. Prabhakaram, M. and Singh, S.N. (1984): Alterations in rat lactate hydrogenase: age-related comparison of M- and H-LDH. Biochem. Int. 9, 399-404. Prabhakaram, M. and Singh, S.N. (1985): Age-related properties of lactate dehydrogenase of different tissues of rats: activity and relative percentage of isozyme distribution. Life Sci. Adv., 4, 1-4. Reiss, U. and Gershon, D. (1976a): Rat liver superoxide dismutase: purification and age-related modifications. Fur. J. Biochem., 63, 617-623. Reiss, U. and Gershon, D. (1976b): Comparison of cytoplasmic superoxide dismutase in liver, heart and brain of aging rats and mice. Biochem. Biophys. Res. Commun., 73, 255-262. Rer.nick, A.Z. and Gershon, D. (1977): Age-rclated alterations in purified fructose-l,6-diphosphate aldolase from the nematode, TwbatrJx aceti. Mech. Ageing Dev., 6, 345-353. Rothstein, M. (1977): Recent developments in the age-related alterations of enzymes. Mech, Agein8 Dev., 6, 241-257. Rothstein, M. (1983): In: Altered proteins and aging, pp. 1-8. Editors: R.C. Adelman and G.S. Roth. CRC Press Inc., Florida. Singh, S.N. and Prabhakaram, M. (1984): Age-related alterations of subcellular rat liver glutamate dehydrogenase (GDH): purification of mitochondrial GDH and effect of nucleotides. J. Sci. Res. (BHU), 34, 189-195. Szajnert, M.F. and Schapira, F. (1983): properties of purified tyrosine aminotransferase from adult and senescent rat liver. Gerontology, 29, 311-319. Weber, K. and Osborn, H. (1969): The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem., 244, 4406-4412. Wilson, P.D, Watson, R. and Knook, O.L. (1982): Effects of age on rat liver enzymes. A study using isolated hepatocytes, endothelial and Kupffer cells. Gerontology, 28, 32-43.
57
Elsevier AGG 00137
Effect of age on the crystalline rat liver lactate dehydrogenase M. Prabhakaram and S.N. Singh * Biochemistry Laboratory, Department of Zoology, Banaras Hindu University, Varanasi-221 005, India (Received 1 September 1985; revised version received 5 December 1985; accepted 9 December 1985)
Summary Lactate dehydrogenase (LDH) was purified and crystallised from the liver of immature (4 weeks), young (22 weeks) and old (116 weeks) female rats and the kinetic, physical and chemical properties of the purified enzyme were studied, Even though slight differences existed in effect of pH, temperature, storage and ratio of A2s0/26o, significant differences were not observed in kinetic properties, behaviour on polyacrylamide gels. ion-exchange chromatography column, and -SH groups among the three age groups. These data indicate that, unlike muscle LDH of old rats which had shown altered properties (earlier report from our laboratory), liver LDH is an unaltered, like LDH of heart. The possible reason for the unaltered nature of the liver LDH may be: (a) the tissue origin; (b) the 8ene responsible for its synthesis is expressing well even in old age; or (c) non-influence of post-translational modifications on the enzyme molecule.
liver LDH; aging; properties
Introduction
The different levels of enzymes in aging animals were studied in detail (Kanungo, 1980; Wilson et al., 1982). However, a limited number of enzymes was purified so far, from aging rats, to study the effect of age (Reznick and Gershon, 1977; Gafni, 1981; Dreyfus et al., 1983; Rothstein, 1983; Prabhakaram and Singh, 1984, 1985; Singh and Prabhakaram, 1984). It leads to the conclusion that post-translational rather than sequential changes may be responsible for the altered nature of enzymes. In this regard, to find out the cause of altered/unaltered nature of enzymes, we have selected lactate dehydrogenase as a model system because of its immense role in giycolysis. The biological importance of lactate dehydrogenase (L-lactate: NAD ÷ oxidoreductase, EC 1.1.1.27, LDH) is studied in detail for the last three decades. This ubiquitous enzyme occurs generally in vertebrate cells as five separable isozymic forms (Markert and Ursprung, 1962), with a predominant M-type in * To whom reprint requests should be addressed. 01674943/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
58
anaerobic tissue (e.g. skeletal muscle) and H-type in aerobic tissue (e.g. heart muscle). However, it is not well understood why liver has adapted M-type instead of H-type. Earlier we have reported that M-LDH, but not H-LDH is an altered one (Prabhakaram and Singh, 1984, 1985). The present report deals about the age-related properties of crystalline liver LDH of aging rats with a view to investigate if this enzyme exists as an altered/unaltered form during old age.
Materials and Methods
Animals Female albino Wistar strain rats, which were maintained under standard laboratory conditions, were used for the present studies. Immature (4 weeks), young (22 weeks) and old (116 weeks) rats were sacrificed and liver was excised immediately, chilled in ice-cold saline, following by processing for the purification of the lactate dehydrogenase.
Chemicals All the biochemicals were obtained from Sigma Chemical Co., USA. DEAE-Sephadex A-50 and Sephadex G-200 were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Triple distilled water was used through out the experimentation.
Analytical methods All steps were carded out at 0 + 2°C, unless otherwise mentioned. The purification procedure was that of Fritz et al. (1970) with certain modifications. To avoid hemoglobin contamination, batch step and two more cyclings of the purified enzyme on a DEAE A-50 ion-exchange chromatography column were included. The enzyme activity was measured by the spectrophotometric method of Kornberg (1955) and expressed in International Units. The protein content was measured according to the method of Lowry et al. (1951). Polyacrylamide gel electrophoresis (PAGE) was carried out following the method of Davis (1964), using 8~ acrylamide gels instead of 7.5~. Specific enzyme staining was done with lithium lactate as substrate (Dietz and Lubrano, 1967). General protein staining was carried out with 0.05~ Coomassie Brilliant Blue (Weber and Osborn, 1969). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was performed using the method of Laemmli (1970). Scanning of the gels was carried out at 500 nm in a Beckman Analytrol. The number of -SH groups was estimated with Ellman's reagent (DTNB: 5',5'-dithiobis-2-nitrobenzoic acid; Eilman, 1959). The purified enzyme was crystallised by the slow additions of ammonium sulphate (35-40~ final concentration) and crystals were photographed under the phase contrast microscope. The crystalline enzyme of all the three ages was passed through Sephadex G-200, after extensive dialysis against 0.05 M potassium phosphate buffer, pH 7.4. The enzyme thus
59
obtained was analysed in a U V range from 220-320 nm in a Unicam SP-500 spectrophotometer. The purified enzyme was used to study its kinetic, physical and chemical properties. These include K., (for pyruvate), K i (with oxalate and oxamate in presence of pyruvate), effect of pH, temperature, urea and sodium sulphite.
Results
Pilot experiments were carried out with liver tissue of male and female rats and it was observed that sex has no effect. Since there are no apparent differences in the properties of LDHs purified from liver of immature, young or old female rats, for brevity we compare here the data obtained for young and old rats only. In order to compare the properties of young and old enzymes, liver LDH was purified under the same conditions. Table I shows the purification protocol of LDH from the livers
young 122 weeks) 2O
0.8
~0
O.e
0.4
N
'°I
o
O m Old II1G weeks)
b
f
1~o
o.e
o
c 0.6 w IL
0.4
5O 0.2
i
10
20
30
Fraction number
Fig. 1. Elution profile of lactate dehydrogenase of liver of young and old female rats on D E A E Sephadex A-50 column.
60 TABLE 1 Purification protocol of liver lactate dehydrosenase of young (22 w) and old (116 w) rats Steps
Total activity (Units)
Specificactivity (U/mg protein)
Recovery (%)
youn8
old
young
youn8
13 780
7 420
4.8
3.8
Fractionation
8400
5900
19.3
16.8
60.95
79.51
4.02
4.37
Ion-exchange column
1785
1324
270.4
287.8
12.95
17.84
56.34
74.75
Crystallisation
1560
1180
503.2
421.4
11.32
15.90
I04.83
I09.45
18000× g supernatant 70% a m m o n i u m sulphate
®
o
b
c
old
-
Fold of purification old
young
-
-
old
®
Fig. 2. Densitometric scans of crude (a) and purified (b and c) liver lactate dehydrogenase of young (A) and old (B) rats.a and b, specific enzyme stain; c, general protein stain.
61 TABLE 11 Apparent kinetic constants of purified liver lactate dehydrogenase of young (22 w) and old (116 w) rats
K"m ( x 10-3 M pyruvate) K, (ram oxalate) K i (raM oxamate)
Young
Old
0.15 0.15 0.11
0.14 0.10 0.07
of young and old female rats. Although a decline in specific activity was observed in old enzyme (16~ lower than that of the young enzyme), the overall recovery rate and purification fold were identical in both age groups of rats. Their behaviour on ion-exchange chromatography was also similar (Fig. 1). The purity of the enzyme was checked on PAGE, Fig. 2(A and B) shows the densitometric scannings of young and old enzymes. A single cathodic, predominant M-type LDH was visualised on gels with specific enzyme stain and with general protein stain. A molecular weight of 35 000 (unimer) was obtained for young and old enzymes on SDS PAGE. Table II shows the kinetic properties of LDH of young and old liver tissues. Oxalate and oxamate inhibited the enzyme activity non-competitively and competitively, respectively, in presence of pyruvate. There is no significant difference in K m or K i of LDH in the two ages of rats and the values fall in a close range (Table II). Fig. 3 (A and B) shows the effect of pH and temperature. The pH optimum was found to be between range 5.0 and 7.5 and the optimum temperature was around 65°C. Similar values were obtained for young and old enzymes without major differences. Fig. 4 (A and B) shows the effect of sodium sulphite and urea on young and old liver LDHs, respectively. Sodium sulphite and urea inhibited the
120
40
40
35
55
75
Temperoture (oC)
2.0
4.0
6.0
8.0
I)14
Fig. 3. Effect of temperature (A) and pH (B) on the activity of purified liver lactate dehydrogenase of young (O O) and old (O e ) rats.
62
B
19
120
.~ so
0.001
001
GI
0o5
I
3
5
Fig. 4. Effect of sodium sulphite (A) and urea (B) on the activity of purified liver lactate dehydrogenase of young (O O) and old (0 O) rats.
enzyme activity of both young and old rats to 80~$ and 70~ (0.5 M sodium sulphite and 3 M urea), respectively. Crystalline enzyme of both the ages yielded a ratio of 1.7 and 1.9 for old and young enzymes, respectively, which is the ratio of A 28o/26o. The estimated -SH groups (residues/mole) of young and old enzymes are 90 and 85, respectively.
Discussion
The presence of age-dependent modifications in the properties of enzymes may give an insight in the altered nature of enzymes (being proteins) as a function of age. While the origin of the age-dependent phenomena has not been identified, they may result from changes in primary structure, due to biosynthetic error or somatic mutations (Hoiliday, 1975), or alternatively be the result of post-translational modifications (Dreyfus et al., 1983). In the present article we have compared the properties of lactate dehydrogenase purified from young and old liver tissues of rats. Although liver consists of predominant M-type of LDH, it has failed to show any marked differences with advancing age. Both, young and old, enzymes have been excluded as a sharp peak in the same volume on ion-exchange column. If 'old' enzyme consists of inactive molecules, they should occur in the void volume as a separate peak. However, such results were not obtained. In addition, the migration of both 'young' and 'old' enzymes is also similar on PAGE, indicating that charge differences do not exist between them. Nonetheless, the observed lower specific activity (16~) of" old enzyme as compared to young enzyme may be due to less number of active enzyme molecules. The K m for pyruvate, K~ for non-competitive inhibitor (oxalate) and competitive inhibitor (oxamate) are almost similar for young and old enzymes (where the initial activity was adjusted to the same level in both the forms). Likewise, the effect of pH, temperature, urea and sodium sulphite are
63
the same for young and old enzymes. Same number of -SH groups (residues/mole) and similar ratio of A2~0/26oconfirms that major structural differences do not exist between young and old enzymes. Studies on aldolase of the liver of mice (Gershon and Gershon, 1973a, b) and cytoplasmic superoxide dismutase of the liver, brain and heart of rats and mice (Reiss and Oershon, 1976a,b) have revealed that a n t i g e n i c i t y , K m, K i, electrophoretic mobility in polyacrylamide gels and SDS PAGE, are the same for both, young and old, individuals. Furthermore, isoelectric focussing of these enzymes does not show any difference in their electrical charges (Goren et al., 1977). Interestingly, we have observed that liver LDH resembles H-LDH in some aspects, like optimum pH, temperature and inhibition in presence of urea. Other properties are similar to that of M-LDH. Several authors have suggested that age-related modifications are due to errors in protein synthesis or to post-translational modifications (Rothstein, 1977: Dreyfus et al., 1983). However, it is not yet known whether age-related alterations have any general pattern or not. Szajnert and Schapira (1983) have reported that purified induced tyrosine aminotransferase of adult and old rat liver tissues behave identically even in presence of digestive enzymes. Purified glutamate dehydrogenase of immature, young and old rats has also failed to show any structural differences (Singh and Prabhakaram, 1984). The unaltered nature of liver LDH, as observed in the present study, can be attributed as a significant event to old animals. Since the muscle LDH shows altered nature in old age, part of its burden may be shared by liver LDH in conversion of pyruvate to lactate or vice versa. During vigorous muscular activity, the level of lactic acid increases and accumulates. This toxic lactic acid thus produced, easily escapes into blood stream and then enters the liver, where it is converted back to glucose (Cori cycle). Thus, it may be concluded that coexistence of a similar enzyme in different tissues as altered and unaltered forms is quite possible. Probably it is having a significant role in aging tissues. The reason for their altered/unaltered nature may be due to: (1) their utility by different tissues in different levels; (2) expression/repression of the gene(s) responsible for the synthesis of a specific enzyme at various stage of growth and development under the influence of a variety of modulators/regulators.
Acknowledgements The authors wish to thank Prof. M.S. Kanungo for encouragement and providing laboratory facilities. Financial support of U.G.C. (Special Assistance Programme grant to Department of Zoology) to M.P. is gratefully acknowledged.
References Davis, B.J. (1964): Disc electrophoresis. Methods and application to human serum proteins. Ann. NY Acad. 5ci., 121,404-427.
64 Dietz, A.A. and Lubrano, T. (1967): Separation and quantitation of lactic dehydrogenase iscenzymes by disc electrophoresis. Anal. Biochem., 20, 246-257. Dreyfus, J.C., Khan, A. and Schapira, F. (1983): In: Aimed proteins and aging, pp. 35-54. Editors: R.C. Adeinum and G.S. Roth. CRC Press inc., Florida. Ellman, G.L. (1959): Tissue sullhydryl groups. Arch. Biochem. Biophys., 82, 70-77. Fritz, PJ., Morrison, WJ., White, E.L. and Vesell, E.S. (1970): Comparative study of methods for quantitative measurement of lactate dehydrogenase isozymes. Anal. Biochem., 36, 443-453. Gafni, A. (1981): Location of age-related modifications in rat muscle glyceraldehyde-3-phosphate dehydrogenase. J. Biol. Chem., 256, 8875-8877. Gershon, H. and Gershon, D. (1973a): Altered enzymes in senescent organisms, mouse muscle aldolase. Mech. Ageing Dev., 2, 33--41. Gershon, H. and Gershon, D. (1973b): Inactive enzymes in aging mice: liver aldolase. Proc. Nat. Acad. Sci. USA, 70, 909-913. Goren, P., Reanick, A.Z., Reiss, U. and Gershon, D. (1977): lsoelectric properties of nematode aldolase and rat liver superoxide dismutase from young and old animals. FEBS Lett., 84, 83-86. Holliday, L. (1975): Testing the protein error theory of ageing: a reply to Baird, Samis, Massie and Zimmerman. Gerontologla, 21, 64-68. Kanungo, M.S. (1980): In: Biochemistry of Ageing, Academic Press, London. Komberg, A. (1955): In: Methods in Enzymology, VOl. !, pp. 441-443. Editors: N.O. Kaplan and S.P. Colowick. Academic Press, New York. Laemmli, U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature (London), 227, 680-685. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951): Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275. Markert, C.L. and Ursprung, H. (1962): The ontogeny of isozyme patterns of lactate dehydrogenase in the mouse. Dev. Biol., 5, 363-381. Prabhakaram, M. and Singh, S.N. (1984): Alterations in rat lactate hydrogenase: age-related comparison of M- and H-LDH. Biochem. Int. 9, 399-404. Prabhakaram, M. and Singh, S.N. (1985): Age-related properties of lactate dehydrogenase of different tissues of rats: activity and relative percentage of isozyme distribution. Life Sci. Adv., 4, 1-4. Reiss, U. and Gershon, D. (1976a): Rat liver superoxide dismutase: purification and age-related modifications. Fur. J. Biochem., 63, 617-623. Reiss, U. and Gershon, D. (1976b): Comparison of cytoplasmic superoxide dismutase in liver, heart and brain of aging rats and mice. Biochem. Biophys. Res. Commun., 73, 255-262. Rer.nick, A.Z. and Gershon, D. (1977): Age-rclated alterations in purified fructose-l,6-diphosphate aldolase from the nematode, TwbatrJx aceti. Mech. Ageing Dev., 6, 345-353. Rothstein, M. (1977): Recent developments in the age-related alterations of enzymes. Mech, Agein8 Dev., 6, 241-257. Rothstein, M. (1983): In: Altered proteins and aging, pp. 1-8. Editors: R.C. Adelman and G.S. Roth. CRC Press Inc., Florida. Singh, S.N. and Prabhakaram, M. (1984): Age-related alterations of subcellular rat liver glutamate dehydrogenase (GDH): purification of mitochondrial GDH and effect of nucleotides. J. Sci. Res. (BHU), 34, 189-195. Szajnert, M.F. and Schapira, F. (1983): properties of purified tyrosine aminotransferase from adult and senescent rat liver. Gerontology, 29, 311-319. Weber, K. and Osborn, H. (1969): The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem., 244, 4406-4412. Wilson, P.D, Watson, R. and Knook, O.L. (1982): Effects of age on rat liver enzymes. A study using isolated hepatocytes, endothelial and Kupffer cells. Gerontology, 28, 32-43.