325
BlOchlmu'a et BlOphyslca Acta. 1077 (1991) 325-331
" 1991 ElseVier Science Publisher> BY 0167-4838/91/$03.50 ADONIS 0167483891U0171P
BBAPRO 33880
Age-related alterations of enzyme activities and subunits of hepatic glutathione S-transferases in male and female Fischer-344 rats Maria-Cristina Carrillo 1, Munetaka Nokubo 2, Kenichi Kitani and Kiyomi Sato 3 j
2,
Kimihiko Satoh 3
Jnstltuto de Fwologw Expenmental Comeet, VnwerstdlJd NlJelOnaJ De Ro.wnfJ. RosorlO (Argentma). .' Department of CllflJcal Physrology. Tokyo MC!tropohtan Institute of Geromology. Tokyo (Japan) and ~ Second Department of BIOchemIstry. Unll'er.Hty of HITO,mkl School uf MedIcine, Hlrosakl (Japan)
(Received 26 June 1990) (Revised manuscripl received 26 November 1990)
Key words: Age; Glutathiooe S·transfer.se; Enzyme activity; Enzyme subunit; (Rat liver)
Enzyme activities of glutathione S-transferases (GSTs) toward five different substrates (benzalacetone (PBO), styrene oxide (STOX), sulfobromophthalein (BSP), 1,2-dicWoro-4-nitrobenzene (DCNB) and l-chloro-2,4-dinitrobenzene (CDNB» as well as concentrations of four subunits of GST isozymes 0, 2, 3 and 4) were determined using cytosol fractions obtained from livers of young (6 months) and old (26 months) Fischer-J44 rats of both sexes. Values for enzyme activities for three substrates (DCNB, BSP and PBO) in young male rats were significantly higher than the corresponding values in female rats. In old male rats, values were generally lower than the corresponding values in young malc rats, becoming close to corresponding values in young female rats. Old female rats, however, exhibited values close to those in young female rats, except for DCNB and STOX values, which were slightly lower in old female rats. GST subunits 3 and 4, as determined by high-performance liquid chromatography after purification by affinity chromatography using S-hexyl-glutathione, were predominant in young males, whereas concentrations of subunits 1 and 2 were higher in females than in males. In male rat livers, concentrations of subunits 3 and 4 decreased considerably with age while those of subunits 1 and 2 increased, so that the subunit pattern in old male rats tended to be similar to that of young female rats. In old females, a decrease in the concentration of subunits 3 and 4 and an increase in the concentration of subunit 1 were also observed as in old male rats, while the subunit 2 concentration tended to decline. Furthermore, the elution pattern of affinity chromatography changed with age, yielding an earlier elution of most subunits in old male rats and of subunit 1 in old female rats. The results suggest that age·related changes that occur with GSTs in livers of male rats are essentially a feminization of the isozyme pattern. However, despite rather unremarkable changes in enzyme activities with age in females, considerable changes of subunit pattern (a general decrease in concentration of subunits 2, 3 and 4 and an increase in the concentration of subunit 1) were also observed in female rats, and these were much greater than could be predicted from enzyme activity changes with age in this sex.
Introduction Glutathione S-transferases (EC 2.5.1.18. RX; glutathione R-transferase) (GSTs) are a family of enzymes
Abbreviations: GST, glutathione S-transfcrase; PBO, benzalacetone; STOX. styrene oxide; BSP. sulfobromophthale"in; DeNB. 1,2-dichloro-4-mtrobenzene; eDNB. l-chloro-2,4-dinilrobenzene. Correspondence: K. Kitani. Department of Clinical Physiology. Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku. Tokyo. Japan.
present in the soluble fraction of many tissues. They are dimeric proteins having a molecular mass of approx. 50 kDa and consist of similar or dissimilar subunits [1]. Several different subunits have been identified in rats. mice and humans [2]. Within each isozyme species, these different subunits are combined to form a variety of dimeric enzymes that have different but frequently overlapping substrate specificities [2]. It has also been demonstrated that these subunits are kinetically independent [3]. This kinetic independence. however. does not exclude a functional dependence [4]. Since these enzymes represent one of the most important detoxify-
326 ing systems in the liver for numerous endogenous as well as exogenous toxicants, possible alterations in these enzymes during aging may be important for the health care of the elderly. Furthermore, this detoxifying system has been suggested to be directly related to maximal life span of many animal species [5]. A number of reports [6~16] described alterations in this system in the liver cytosol during aging; however, most studies concerned the alterations only in enzyme activities during aging. Only one group of researchers did examine alterations of isozyme cencentrations during aging [9-11]. So far, there is no information on the basis of the subunit composition for this enzyme in terms of the aging effect. Therefore, the aim of the present study was to clarify how the reported aging effects on this system are related to alterations in the subunit composition of GST. In view of the enormous differences between sexes in age effect of hepatic biotransformation reactions in the rat, we investigated in the present study both male and female rat livers. Materials and Methods Chemicals. S-Hexyl-glutathione (S-hexyl-GSH) and GST isozymes (GST 1-2 and 3-4) were prepared and purified by one of us (K. Satoh). Sulfobromophthalein sodium tetrahydrate (BSP), GSH reductase and reduced GSH were purchased from Sigma (St. Louis, MO), benzalacetone (trans-4-phenyl-3-buten-2-one, PBO) was purchased from Aldrich Chemical (Milwaukee, WI); 1,2-epoxyethylbenzene (styrene oxide; STOX) from Wako Pure Chemical (Japan); and 1,2-dichloro-4nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) were purchased from Tokyo Kasei Kogyo , M.D. and Woodhouse. K. (1986) in Liver and Agmg - 1982. Liver and Drugs (Kitani, K., cd.). pp. 395-408. Elsevier Biomedical Press, Amsterdam.
BlOchlmu'a et BlOphyslca Acta. 1077 (1991) 325-331
" 1991 ElseVier Science Publisher> BY 0167-4838/91/$03.50 ADONIS 0167483891U0171P
BBAPRO 33880
Age-related alterations of enzyme activities and subunits of hepatic glutathione S-transferases in male and female Fischer-344 rats Maria-Cristina Carrillo 1, Munetaka Nokubo 2, Kenichi Kitani and Kiyomi Sato 3 j
2,
Kimihiko Satoh 3
Jnstltuto de Fwologw Expenmental Comeet, VnwerstdlJd NlJelOnaJ De Ro.wnfJ. RosorlO (Argentma). .' Department of CllflJcal Physrology. Tokyo MC!tropohtan Institute of Geromology. Tokyo (Japan) and ~ Second Department of BIOchemIstry. Unll'er.Hty of HITO,mkl School uf MedIcine, Hlrosakl (Japan)
(Received 26 June 1990) (Revised manuscripl received 26 November 1990)
Key words: Age; Glutathiooe S·transfer.se; Enzyme activity; Enzyme subunit; (Rat liver)
Enzyme activities of glutathione S-transferases (GSTs) toward five different substrates (benzalacetone (PBO), styrene oxide (STOX), sulfobromophthalein (BSP), 1,2-dicWoro-4-nitrobenzene (DCNB) and l-chloro-2,4-dinitrobenzene (CDNB» as well as concentrations of four subunits of GST isozymes 0, 2, 3 and 4) were determined using cytosol fractions obtained from livers of young (6 months) and old (26 months) Fischer-J44 rats of both sexes. Values for enzyme activities for three substrates (DCNB, BSP and PBO) in young male rats were significantly higher than the corresponding values in female rats. In old male rats, values were generally lower than the corresponding values in young malc rats, becoming close to corresponding values in young female rats. Old female rats, however, exhibited values close to those in young female rats, except for DCNB and STOX values, which were slightly lower in old female rats. GST subunits 3 and 4, as determined by high-performance liquid chromatography after purification by affinity chromatography using S-hexyl-glutathione, were predominant in young males, whereas concentrations of subunits 1 and 2 were higher in females than in males. In male rat livers, concentrations of subunits 3 and 4 decreased considerably with age while those of subunits 1 and 2 increased, so that the subunit pattern in old male rats tended to be similar to that of young female rats. In old females, a decrease in the concentration of subunits 3 and 4 and an increase in the concentration of subunit 1 were also observed as in old male rats, while the subunit 2 concentration tended to decline. Furthermore, the elution pattern of affinity chromatography changed with age, yielding an earlier elution of most subunits in old male rats and of subunit 1 in old female rats. The results suggest that age·related changes that occur with GSTs in livers of male rats are essentially a feminization of the isozyme pattern. However, despite rather unremarkable changes in enzyme activities with age in females, considerable changes of subunit pattern (a general decrease in concentration of subunits 2, 3 and 4 and an increase in the concentration of subunit 1) were also observed in female rats, and these were much greater than could be predicted from enzyme activity changes with age in this sex.
Introduction Glutathione S-transferases (EC 2.5.1.18. RX; glutathione R-transferase) (GSTs) are a family of enzymes
Abbreviations: GST, glutathione S-transfcrase; PBO, benzalacetone; STOX. styrene oxide; BSP. sulfobromophthale"in; DeNB. 1,2-dichloro-4-mtrobenzene; eDNB. l-chloro-2,4-dinilrobenzene. Correspondence: K. Kitani. Department of Clinical Physiology. Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku. Tokyo. Japan.
present in the soluble fraction of many tissues. They are dimeric proteins having a molecular mass of approx. 50 kDa and consist of similar or dissimilar subunits [1]. Several different subunits have been identified in rats. mice and humans [2]. Within each isozyme species, these different subunits are combined to form a variety of dimeric enzymes that have different but frequently overlapping substrate specificities [2]. It has also been demonstrated that these subunits are kinetically independent [3]. This kinetic independence. however. does not exclude a functional dependence [4]. Since these enzymes represent one of the most important detoxify-
326 ing systems in the liver for numerous endogenous as well as exogenous toxicants, possible alterations in these enzymes during aging may be important for the health care of the elderly. Furthermore, this detoxifying system has been suggested to be directly related to maximal life span of many animal species [5]. A number of reports [6~16] described alterations in this system in the liver cytosol during aging; however, most studies concerned the alterations only in enzyme activities during aging. Only one group of researchers did examine alterations of isozyme cencentrations during aging [9-11]. So far, there is no information on the basis of the subunit composition for this enzyme in terms of the aging effect. Therefore, the aim of the present study was to clarify how the reported aging effects on this system are related to alterations in the subunit composition of GST. In view of the enormous differences between sexes in age effect of hepatic biotransformation reactions in the rat, we investigated in the present study both male and female rat livers. Materials and Methods Chemicals. S-Hexyl-glutathione (S-hexyl-GSH) and GST isozymes (GST 1-2 and 3-4) were prepared and purified by one of us (K. Satoh). Sulfobromophthalein sodium tetrahydrate (BSP), GSH reductase and reduced GSH were purchased from Sigma (St. Louis, MO), benzalacetone (trans-4-phenyl-3-buten-2-one, PBO) was purchased from Aldrich Chemical (Milwaukee, WI); 1,2-epoxyethylbenzene (styrene oxide; STOX) from Wako Pure Chemical (Japan); and 1,2-dichloro-4nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) were purchased from Tokyo Kasei Kogyo , M.D. and Woodhouse. K. (1986) in Liver and Agmg - 1982. Liver and Drugs (Kitani, K., cd.). pp. 395-408. Elsevier Biomedical Press, Amsterdam.
