Comp. Biochem. Physiol. Vol. 61B, pp. 133-137 © Pergamon Press Ltd., 1978. Printed in Great Britain
0305-0491/78/0815-01331502.00/0
DIFFERENCE IN RENAL a-GALACTOSIDASE LEVELS IN MALE AND FEMALE CHINESE HAMSTERS (CRICETULUS GRISEUS) A. Y. CHANG Diabetes and Atherosclerosis Research, The Upjohn Co., Kalamazoo, MI 49001, U.S.A. (Received 6 December 1977) Abstract--1. The activity of a-galactosidase was found to be significantly higher in the kidney of female than that of male Chinese hamsters in a highly inbred colony but its activity in liver, heart and spleen remained similar between female and male animals. 2. Partially purified renal c~-galactosidase by sequential column chromatography on Sepharose 6B and DEAE-Sepharose CL-6B showed identical elution profiles, pH optima (4.5), Kms (4.4 mM) and heat-inactivation curves between enzymes of male and female animals. 3. Thus, the observed higher activity of renal a-galactosidase in the females was due to elevated enzyme concentration, not a result of enzyme polymorphism.
NaCI in the same buffer. Fractions of 3.5 ml and 1.5 ml were collected, respectively. The fractions containing A colony of Chinese hamsters (Cricetulus griseus) a-galactosidase activity from the first column were pooled with spontaneous diabetes has been successfully and directly applied to the second column without being established and line-dependent variation was found concentrated. The major a-galactosidase peak from the in tissue lactate dehydrogenase (EC 1.1.1.27) second column was concentrated in Minicon®. Heat-inactivation study was carried out by heating activity and isozyme pattern (Chang et al., 1977). In a 0.075 ml aliquots of partially purified fraction at indicated previous study on renal acid glycohydrolase level in temperature for 15 min and the tubes were cooled rapidly two highly inbred lines with different phenotypes, in ice-water. Activity was measured with heated and i.e. glycosuria and aglycosuria, it was discovered ' non-heated fractions simultaneous}y. Sources of materials that the female Chinese hamsters showed and protein determination were described previously significantly higher activity of a-galactosidase than (Chang, 1978b). the males in the same line (Chang, 1978a). Question thus arose as to whether the high activity observed RESULTS in the female animal kidney represented an elevated enzyme level or additional isozymes in the female Table 1 shows the age, body and tissue weights were responsible for the observation. Moreover, is and tissue a-galactosidase activity in six pairs of the difference also present in tissues other than the male and female Chinese hamsters closely matched kidney? The present study was carried out to answer with age (24 days and 13 months). The females in the these questions. mature group were significantly lighter and also had smaller livers, kidneys and hearts than the males; but, in relation to their body weights, these tissues MATERIALS AND METHODS were not significantly different between the males The Chinese hamsters were selected from the non- and females. The activity of a-galactosidase in liver, diabetic A V line (Chang et al., 1977) and six pairs of males spleen and heart was similar in male and female and females were matched closely with age. The animals animals but the females showed twice as much renal were killed by decapitation and the tissues were excised a-galactosidase as the male animals. Among the and homogenized in 0.5% Triton X-100, 0.45 M sucrose, tissues, a-galactosidase showed the highest activity 0.68 mM EDTA, pH 7.0 with Polytron®at a setting of 7 for three separate 10 sec runs. The volumes of the added in the kidney and the lowest in the heart; its activity medium were 2 x tissue weight for the livers, hearts and was about the same in the spleen and the liver. The kidneys of the mature animals, 4 x for the kidneys of the female and male Chinese hamsters of 24 days were weanlings and 14 x for the spleens. The homogenates were similar in size and had similar kidney weights. The spun at 12,000g for 20 min and the supernatants were mean value of renal a-galactosidase activity in the assayed for a-galactosidase using p-nitrophenyl-a-l> female weanlings appeared to be slightly higher than galactoside as substrate (Chang, 1978b). Enzyme unit was that in the males but, statistically, no significance defined as ample substrate turnover per min. was found at the 0.05 level, i.e. 0 . 1 0 > P >0.05. Sequential chromatography of the 12,000g supernatant fractions was carried out successively on Sepharose 6B Liver, heart and spleen tissues were not studied in (2.6 x 40 cm), equilibrated in 0.01 M NaH2PO4-Na2HPO4, the weanlings since no significant sex-dependent pH 6.0, 0.1% NaN3 and 5 rnM NaCl, and DEAE-Sepharose difference in these tissues was found in the mature CL-6B (0.9x 15cm) columns, equilibrated in 0.01 M animals. The elution profiles of the kidney 12,000 g superNaH2POt-Na2HPO4, pH 6.0, 0.1% NaN~ and eluted with a linear gradient made by mixing 50 ml each of 0 and 0.25 M natant on Sepharose 6B column are shown in Fig. 1, INTRODUCTION
133
[34
A. Y. CHANG Table 1. Age, body and tissue weights and tissue a-galactosidase activity of male and female Chinese hamsters
Measurements
Male a
Female a
pb
Ad.ultAnimals Age (months) Body wt.
12.90 ± 0.47
(g)
]2,95
*
0.49
NS
35,6 ± 0,6
26.8 i 1,0
0305-0491/78/0815-01331502.00/0
DIFFERENCE IN RENAL a-GALACTOSIDASE LEVELS IN MALE AND FEMALE CHINESE HAMSTERS (CRICETULUS GRISEUS) A. Y. CHANG Diabetes and Atherosclerosis Research, The Upjohn Co., Kalamazoo, MI 49001, U.S.A. (Received 6 December 1977) Abstract--1. The activity of a-galactosidase was found to be significantly higher in the kidney of female than that of male Chinese hamsters in a highly inbred colony but its activity in liver, heart and spleen remained similar between female and male animals. 2. Partially purified renal c~-galactosidase by sequential column chromatography on Sepharose 6B and DEAE-Sepharose CL-6B showed identical elution profiles, pH optima (4.5), Kms (4.4 mM) and heat-inactivation curves between enzymes of male and female animals. 3. Thus, the observed higher activity of renal a-galactosidase in the females was due to elevated enzyme concentration, not a result of enzyme polymorphism.
NaCI in the same buffer. Fractions of 3.5 ml and 1.5 ml were collected, respectively. The fractions containing A colony of Chinese hamsters (Cricetulus griseus) a-galactosidase activity from the first column were pooled with spontaneous diabetes has been successfully and directly applied to the second column without being established and line-dependent variation was found concentrated. The major a-galactosidase peak from the in tissue lactate dehydrogenase (EC 1.1.1.27) second column was concentrated in Minicon®. Heat-inactivation study was carried out by heating activity and isozyme pattern (Chang et al., 1977). In a 0.075 ml aliquots of partially purified fraction at indicated previous study on renal acid glycohydrolase level in temperature for 15 min and the tubes were cooled rapidly two highly inbred lines with different phenotypes, in ice-water. Activity was measured with heated and i.e. glycosuria and aglycosuria, it was discovered ' non-heated fractions simultaneous}y. Sources of materials that the female Chinese hamsters showed and protein determination were described previously significantly higher activity of a-galactosidase than (Chang, 1978b). the males in the same line (Chang, 1978a). Question thus arose as to whether the high activity observed RESULTS in the female animal kidney represented an elevated enzyme level or additional isozymes in the female Table 1 shows the age, body and tissue weights were responsible for the observation. Moreover, is and tissue a-galactosidase activity in six pairs of the difference also present in tissues other than the male and female Chinese hamsters closely matched kidney? The present study was carried out to answer with age (24 days and 13 months). The females in the these questions. mature group were significantly lighter and also had smaller livers, kidneys and hearts than the males; but, in relation to their body weights, these tissues MATERIALS AND METHODS were not significantly different between the males The Chinese hamsters were selected from the non- and females. The activity of a-galactosidase in liver, diabetic A V line (Chang et al., 1977) and six pairs of males spleen and heart was similar in male and female and females were matched closely with age. The animals animals but the females showed twice as much renal were killed by decapitation and the tissues were excised a-galactosidase as the male animals. Among the and homogenized in 0.5% Triton X-100, 0.45 M sucrose, tissues, a-galactosidase showed the highest activity 0.68 mM EDTA, pH 7.0 with Polytron®at a setting of 7 for three separate 10 sec runs. The volumes of the added in the kidney and the lowest in the heart; its activity medium were 2 x tissue weight for the livers, hearts and was about the same in the spleen and the liver. The kidneys of the mature animals, 4 x for the kidneys of the female and male Chinese hamsters of 24 days were weanlings and 14 x for the spleens. The homogenates were similar in size and had similar kidney weights. The spun at 12,000g for 20 min and the supernatants were mean value of renal a-galactosidase activity in the assayed for a-galactosidase using p-nitrophenyl-a-l> female weanlings appeared to be slightly higher than galactoside as substrate (Chang, 1978b). Enzyme unit was that in the males but, statistically, no significance defined as ample substrate turnover per min. was found at the 0.05 level, i.e. 0 . 1 0 > P >0.05. Sequential chromatography of the 12,000g supernatant fractions was carried out successively on Sepharose 6B Liver, heart and spleen tissues were not studied in (2.6 x 40 cm), equilibrated in 0.01 M NaH2PO4-Na2HPO4, the weanlings since no significant sex-dependent pH 6.0, 0.1% NaN3 and 5 rnM NaCl, and DEAE-Sepharose difference in these tissues was found in the mature CL-6B (0.9x 15cm) columns, equilibrated in 0.01 M animals. The elution profiles of the kidney 12,000 g superNaH2POt-Na2HPO4, pH 6.0, 0.1% NaN~ and eluted with a linear gradient made by mixing 50 ml each of 0 and 0.25 M natant on Sepharose 6B column are shown in Fig. 1, INTRODUCTION
133
[34
A. Y. CHANG Table 1. Age, body and tissue weights and tissue a-galactosidase activity of male and female Chinese hamsters
Measurements
Male a
Female a
pb
Ad.ultAnimals Age (months) Body wt.
12.90 ± 0.47
(g)
]2,95
*
0.49
NS
35,6 ± 0,6
26.8 i 1,0
