Age related changes in the activity of the glyoxalase system

m hams at aplj ELSEVIER SCIENCE IRELAND

Mechamsms of Ageing and Development 73 (1994) 39-45

Age related changes in the activity of the glyoxalase system Roopa Sharma-Luthra, R K Kale* Radtatton Btology Laboratory School of Ltfe Sctences Ja~aharlal Nel~lu Untver~ltl New Delht 110067 lndta

(Received 14 May 1993, revision recewed 3 October 1993, accepted 29 October 1993)

Abstract Male Swiss albmo mice of progresswe age (1-25 months old) were collected regularly from the Ammal Facility of the umverslty at monthly intervals and were sacrificed by cervical dislocation The specific actlvlUes of glyoxalase I (GI) and glyoxalase II (GII) were determmed tmmedtately m liver, spleen and kidney Our results indicate that the activity of GI increased with an increase m the age of mice up to 12-14 months depending on the type of the tissue, thereafter it decreased progressively m the old animals The increase in the actwlty of GI may be suggestwe of the rapid rate of cell division in these organs needed for the growth and development of the antmal, the progressive decline In the activity of GI In old age may be related to the decreased proliferative capacity of these organs This dechne may increase the oxidative damage and in turn enhance the process of ageing The actwlty of GII decreased with increase in age up to 12-13 months The mode and the magmtude of the activity of GII in old age depends on the type of tissue The pattern of the GI/GII ratio in all the three tissues was similar up to 13-14 months of age Key words Glyoxalase I, Glyoxalase II, Age

1. Introduction Development a n d agemg constitute a complex m u l t l f a c t o n a l process [i,2] Ageing affects prohferatwe homeostasis [3] and the prohferatwe capacity of cells Advanced age Is associated with reductzon in cell prollferauve responses [4] Several studies have suggested the posslblhty of involvement of the glyoxalase * Corresponding author 004%6374/94/$07 00 © 1994 Elsevier Sc2ence Ireland Ltd All rights reserved SSDI 0047-6374(93)0141 I-Z

40

R Sharma-Luthra R K Kale / Mech Ageing Dev 73 (1994)39-45

system in the regulation of cell dlvlston m animals as well as m plants [5-10] Since this system is considered a metabolic Indicator of cell proliferation [11], modulation of its activity may occur during the development and ageing The glyoxalase system consists of GI, GII and reduced glutathlone (GSH) This system catalyses the conversion of methylglyoxal to D-lactic acid It also converts c~-oxoaldehydes to (R)-2-hydroxy acids [12] GI (EC 4 4 i 5) catalyses the formation of (S)-D-lactoylglutathxone (MeCH(OH)CO-SG) from hemlthxoacetal formed nonenzymatlcaily from methylglyoxal(MeCO-CHO) and reduced glutathione (GSH) (Reaction 1) Glyoxalase II (EC 3 1 2 6) catalyses the hydrolysis of (S)-D-lactoylglutathlone to D-lactic acid (MeCH(OH)COOH) (Reaction 2) and regenerate GSH consumed in Reaction 1 [13,14] MeCOCHO + GSH ,~ MeCOCH(OH)-SG -

MeCH(OH)CO-SG

(i)

MeCH(OH)CO-SG + H20 ~ MeCH(OH)COOH + GSH Methylglyoxal, o~-oxoaldehydes and (S)-D-lactoylglutathlone are toxic compounds [13] Methylglyoxal Induces the generation of active oxygen species [15] and the same might be true for (S)-D-lactoylglutathlone and ct-oxoaldehydes Accumulation of these compounds may cause the generation of free radicals It is well established that the ageing results from deleterious damage to tissues by free radicals produced during metabolism The importance of the correlation between ageing and the alterations m the activities of regulatory enzymes, co-factors and co-enzymes that are substrates or activators for diverse enzymes have been suggested [16] The glyoxalase system is considered vital for biological functions as it is involved in the regulation of cell division, vesicle mobilization, detoxlcatlon, diabetes mellltus and cancer [13] Moreover, GSH is a substrate for diverse enzymes Since the glyoxalase system is involved m cell growth and its substrates are toxic and generate free radicals, and GSH ~s its cofactor, this system may have some important role in ageing and development The Influence of age on the glyoxalase system has not been studied fully Human erythrocytes fractlonated by age have been shown to display changes in GI activity [17] The present study deals with influence of age on the activity of GI and GII in liver, spleen and kidney of mice 2. Materials and methods 2 1 Ammals Male, Swiss albino mice, 1-25 months old, were regularly obtained from the Animal Facility of the Jawaharlal Nehru Umverslty at monthly intervals Standard food (Hindustan Lever Ltd ) and water was provided ad hbitum 2 2 Preparatton jor the enzyme assay Animals were sacrificed by cervical dislocation Liver, spleen and kidney were removed immediately and washed with chilled sodium phosphate buffer (0 2 M, pH 7) A 10% homogenate (w/v) of each organ was made in chilled buffer using Potter Elvehjem homogenizer The homogenate was centrifuged at 30 000 × g for 45 mxn

R Sharma-Luthra, R K Kale/Mech Agemg Dev 73 (1994,) 39-45

41

at 4°C The specific activities of the enzymes (GI and GII) were determined in the supernatant 2 3 Enzyme assay The specific activities of GI and GII were asssayed spectrophotometrlcally using Racker's method as described by Sharma and Kale [14] The GI activity is given as units/rag protein One unit of the enzyme is defined as the amount of enzyme catalyslng the formation of 1 #mol of (S)-D-lactoyiglutathlone/mln under assay conditions The activity of GII is given as unlts/mg protein One unit of the enzyme is defined as the amount of enzyme required to catalyse the hydrolysis of 1 /zmol of (S)-Dlactoylglutathlone/min under assay condition 2 4 Protein determmatton Protein concentration was determined by the method of Bradford [18] using bovine serum albumin as standard 2 5 Chemicals Methylglyoxal, GSH and (S)-D-lactoylglutathlone were from Sigma All other chemicals were of analytical grade 3. Results and discussion

The age related changes in the activity of GI are shown in Figs 1-3 The results indicate that the activity of GI increased with increase in age for about 12-14

LIVER 107 106 >.,

>

1 05

u

o

104

u a

103 I

I

4

8

I

I

12 16 Age ( m o n t h )

I

I

20

2/4

Fig 1 Specific activity of O l m the liver of Swiss albino mice of &fferent age groups

42

R Sharma-Luthra R K Kale/Mech Ageing Dev 73 (1994)39-45

SPLEEN

0'170 t 0.165

i 0.160 o

Q. uO

0.155

4

I

8

I

1

.I

12 16 20 Age (months)

24

Fig 2 Specific actlvlt,~ of Ol m the spleen of Swiss albino mice of dlflerent age groups

months, depending on the organ, and thereafter it decreased wxth age The growthrelated Increase m G! activity correlates with increased D N A synthesis [6,8] Therefore, the increase m the acUvlty of GI in all three tissues is probably suggestive of cell division needed for growth and development of the ammal The decline m the activity of GI in old age may be due to a reduction/cessation m the prohferatlve activity The prohferatlve capacity decreases w~th old age [4] Dechne m the actlwty of GI may lead to the accumulatxon of methylglyoxal and cause inactivation of glycolytlc enzymes [19] adversely affecting the major metabolic pathway, protein cross hnkmg [20], depletion in sulphydryl compounds and generation of free radicals [21], which may enhance the ageing process The decrease m the actlwty of GI among three organs was found to be in the following order spleen > liver > kidney Our observations suggest that the actw~ty of GI undergoes changes with the age of mice The activity of GI! did not show very rapid change throughout the study period However, overall, a decrease m the activity of G I I was observed until about 13 months of age in all three organs we studied and thereafter the mode and magnitude of G I I activity m old age depends on the type of tissue (Table 1) The activities of GI and GII are inversely related [13] In the present study, we also have observed

R Sharma-Luthra, R K Kale/Mech Agemg Dev 73 (1994) 39-45

43

KIDNEY

07

IIIIII

06 >

-t~= 0 5 <

u

~_-OZ, Q.

m0.3

0.2 I

1

/4

8

I

I

I

12 16 20 Age ( m o n t h s )

I

2/-*

Fig 3 Specific actwlty of GI m the kidney of Swiss albino mice of different age groups

a slmdar reciprocal relatlonahlp between the actwmes of G1 and GII The relatwely lower acUwty of GII m old age, may result m the accumulation of (S)-D-lactoylglutath~one and affect the m~crotubule assembly contributing to the effects leading to senescence or cell death [13]

Table 1 Specific actwlty of GII m spleen, hver and kidney of Swiss albino mice of &fferent age groups Age (months)

Spleen

Lwer

2 4 6 8 10 12

0054 0052 0048 0048 0047 0047

4-0002 4- 0006 4- 0016 4- 0002 4- 0004 4- 0007

0336 0334 0330 0 294 0284 0274

4-004 4- 003 ± 002 4- 003 ± 001 4- 0 0 3

0177 0 176 0 175 0 170 0 169 0 170

4-003 4- 001 4- 0 0 4 4- 002 4- 001 4- 0 0 6

14 16 18 20 22 24

0048 0052 0051 0 047 0 049 0050

444. 4. 44-

0273 0 269 0268 0 265 0 265 0263

444. 444-

0 0 0 0 0 0

4± 4. 444-

0008 0007 0012 0 011 0 009 0013

Kidney

004 009 007 0 09 0 08 0 13

Each value represents an average of at least four experiments 4- S D

167 160 161 163 162 162

007 003 005 0 03 0 07 007

44

R Sharma-Luthra R K Kale/Mech Agemg Dev 73 (1994) 39-45

Table 2 Rano of GI and GII actlwt~ (GI/GII) m hver spleen and kidney ol mice of dllterent age groups Age (months)

Liver

Spleen

Kidne~

2 4 6 8 10 12 14 17 20 24

3 10 3 12 3 19 3 59 3 72 3 89 ~ 92 3 94 ~ 94 3 96

2 87 3 02 g 27 ~ 39 3 48 3 49 ~ 12 3 26 ~ 05 3 06

I 52 I 9~ 2 ]4 2 58 2 68 ~ 29 ~ 8~ ~ 82 ~ 82 "~69

Each value represents an average of at least four experiments

The ratio o f the activities o f G I a n d G I I ( G I / G I I ) was similar in all three organs until a b o u t 13 m o n t h s (Table 2) In liver the ratio was found to increase with age In the case o f the kidney a n d spleen this ratio Increased up to a b o u t 13 m o n t h s and then decreased progressively in old age The m e t a b o h s m o f methylglyoxal m these organs a p p e a r s to be u n d e r the c o n t r o l o f some c o m m o n factors, at least d u r i n g the d e v e l o p m e n t and growth phase Such a possibility was suggested m erythrocytes [22] as well as in liver and spleen [14] Alternatively, the m a g m t u d e o f age-related changes in the activity o f G I was quite h~gh c o m p a r e d with G I I Therefore the G I / G I I ratio p a t t e r n m~ght have also arisen from the changes m the actlwty o f G I [14]

4. Conclusion It can be concluded that the activity o f the glyoxalase system changes with age The activities o f G I and G I I vary d e p e n d i n g on the age and organ o f the animal The activity o f G I increased progresswely d u r i n g the growth phase o f the a m m a l and may be indicative o f the r a p i d rate o f cell division taking place during this phase o f the d e v e l o p m e n t The specific activity o f G I I was low m this period T o w a r d s old age, the activities o f the enzymes become s o m e w h a t irregular, particularly that o f G I I The deelme In the activity o f the glyoxalase system m a y increase free radical f o r m a t i o n due to the a c c u m u l a t i o n o f methylglyoxal and (S)-o-lactoylglutath~one leading to oxidative d a m a g e which m a y c o n t r i b u t e to the process o f ageing

5. Acknowledgements The a u t h o r s are thankful to University G r a n t Commlss~on ( U G C ) for financial s u p p o r t In the form o f fellowship to one o f the a u t h o r s (R S -L) The a u t h o r s are grateful to Professor A R R a o for his c r m c a l suggestions

R Sharma-Luthra, R K Kale/Mech Agemg Dev 73 (1994) 39-45

45

6. References 1 L Hayfhck. Theories of biological ageing E,:p Gerontol. 20 (1985) 145-159 2 C E Finch. Mechamsm of senescence some thoughts m April 1990 E,:p Gerontol 27 (1992) 7-16 3 E J Ma~oro. Ageing and prohferatwe homeostasis modulation by food restncuon m rodents Lab Amm Set 42(2)) (1992) 132-137 4 D C Powers. J E Morley and J M Flood Age related changes in LFA-I expression, cell adhesion and PHA reduced prohferatlon by lymphocytes from senescence acclerated mouse (SAM)-pl8 and SAM-R/I substrams Cell lmmunol 141 (1992) 444-456 5 N M Alexander and J C Bouyer. Glyoxalase activity m sham and partially hepatectomtzed rats Cancer Res 310 (1971) 1875-1878 6 P S u t r a v e a n d A R Rao. Studlesmglyoxalaselmregneratmgmousespleen LtJeStl Adv 1(1982) 141-145 7 A Dlxlt. L C Garg. P Sutrave and A R Rao. Glyoxalase 1 m regenerating mouse liver ecxposed to carcinogens Btochem lnt. 7 (1983) 207-213 8 O Ramaswamy. S Pal. S Guha-Mukherjee and S K Sopory. Presence of glyoxalase I m pea Biothem lnt. 7 (1983) 307-318 9 S Bagga. R Das and S K Sopory. Inhibition of cell prohferatlon and glyoxalase 1 activity by calmoduhn mhlbltors m Brasstta oleratea J Plant Phv3wl 129 (1987) 149-153 10 U Seth1. A Basu and S Guha-Mukherjee. Control of cell prohferatlon and dlfferentmtion by regulating polyamme biosynthesis in cultures of Brasstta and its corelatlon with glyoxalase I actw~ty Plant Set. 56 (1988) 167-175 11 N I Hooper. M J T~sdale and P J Thornalley. Modification of glyoxalase system m human HL60 promyelocytlc leukemia cells during differentiation to neutnphfls in vitro Btothtm Btoph~s At ta 966 (1988) 362-369 12 J D Clelland and P J Thornalley. S-2-Hydroxyacyleglutathlone derivatives enzyme preparaUon purification and charactensatlon J Chem Sot Perkm Trans 1 (1991) 3009-3015 13 P J Thornalley. Rewew the glyoxalase system new developments towards functional charactenzatmn of metabohc pathway fundamental to bmloglcal hfe Bto~hem J 269 (1990) 1-11 14 R Sharma and R K Kale. Effect of radmtlon on glyoxalase 1 and glyoxalase I1 act)vines m spleen and I,ver of mice Int J Radtat Bwl 63(2)(1993)233-238 15 M P Kalapos. L Braun. A Llttauer and H de Groot Methylglyoxal toxicity and reactive oxygen . Abstract No KI4 In 645th Meeting of the Btotherm~al Sotwtt The Biochemical Society London p 35 16 E R Stadtman. Protein modification m ageing J Gerontol 43 (1988) 112-120 17 A C McLellan and P J Thornalley. Glyoxalase act~wty m human RBC tractJonated b.~ age Meth Ageing Dev 48 (1989) 63-71 18 M M Bradford. Rap~d and sensmve method of quantJtatlon of m~crogram quantmes of protien utdlsmg the principle of protlen-dye binding Anal Bw~hem 72 (1976) 248-254 19 G Leoncml M Maresca and A Bonslgnore. The effect of methylglyoxal on glycohtlc enzymes FEBS Lett 117 (1980) 17-18 20 E Schauenstem, H Esterbauer and H Zollner, ot-Dicarbonyls In 41deh~de~m Btoloettal S~tem~ Plon Ltd, London, 1977, 112-157 21 S Baskaran and K A Balasubramanmn, Effect ofmethylglyoxal on protein thlol and amino groups ,n isolated rat erythrocytes and colonocytes and actwlty of various brush border enzymes lmhan J Btothem Btophls 27(1990)13-17 22 D J Cre~ghton M MJghonm T Pourmotabbed and M K Guha Optimization of the efficiency of glyoxalase pathway Btothemtstrt 27 (1988) 7376-7384