Comp. Biochem Physiol.. Vol. 62B, pp. 557 to 561.
0305-0491/79.'0401-0557502.00/0
© Pergamon Press Ltd 1979. Printed in Great Britain
A C I D G L Y C O H Y D R O L A S E IN C H I N E S E H A M S T E R (CRICETULUS GRISEUS) WITH S P O N T A N E O U S D I A B E T E S - - V S U B C E L L U L A R D I S T R I B U T I O N IN THE K I D N E Y A. Y. CHANG and C. S. PERRY Diabetes and Atherosclerosis Research, Upjohn Company, Kalamazoo, MI 49001, U.S.A.
(Received 4 July 1978) Abstract--l. Seven renal glycohydrolases were measured in four subcellular fractions prepared from highly inbred aglycosuric (A V-line) and glycosuric (XA-line) Chinese hamsters. 2. ~-D-galactosidase and fl-D-galactosidase were highest in the nuclear (N) and supernatant (S) fractions; both fractions showed reduced activities in the XA animals. 3. :t-D-mannosidase was chiefly a particulate enzyme and its decrease in x A animals was evident in N, lysosomal-mitochondrial (LM) and mitochondrial-microsomal (MM) fractions. 4. No significant difference in N-acetyl-fl-D-glucosaminidase was found in any of these subcellular fractions between AVand XA animals. 5. Although total Ct-L-fucosidase and fl-D-fucosidase levels were similar in AV and XA kidneys, a difference was observed in the S fraction. 6. fl-D-glucuronidase was virtually absent in N and LM fractions and the S fraction of A V kidneys showed higher activity than the XAs. and allowed food and water ad libitum. The animals were bled through the orbital sinus and killed by decapitation early in the morning. The kidneys were excised and rinsed in cold saline.
INTRODUCTION
Variations in the levels of renal (Chang, 1978ab; C h a n g & Greenberg, 1978) and plasma (Chang & Perry, 1978) glycohydrolases have been found in the U p j o h n colony of Chinese hamsters (Cricetulus griseus) with spontaneous diabetes; significant correlations between renal levels of 0t-D-galactosidase (EC 3.2.1.22), fl-o-galactosidase (EC 3.2.1.23) and N-acetylfl-o-glucosaminidase (EC 3.2.1.30) and blood sugar levels have also been established in these animals (Chang & Greenberg, 1978). In an early study on the subcellular distribution of fl-D-glucosidase (EC 3.2.1.21) and fl-o-galactosidase in the kidneys of diabetic, ketotic and nondiabetic Chinese hamsters, a bimodal distribution of fl-o-galactosidase was observed, whereas fl-o-glucosidase resided mainly in the soluble fraction (Chang, 1978c). In view of these findings, the present study was carried out in order to extend our knowledge of diabetes-related differences of other renal glycohydrolases and of their subcellular locations in the Chinese hamster. F o r this purpose, kidney homogenates were separated into nuclear (N), lysosomal-mitochondrial (LM), mitoc h o n d r i a l - m i c r o s o m a l (MM) and supernatant (S) fractions, according to Price & Dance (1967), with modifications. The activities of =t-o-galactosidase, fl-Dgalactosidase, =t-D-mannosidase (EC 3.2.3.24), N-acetyl-fl-D-glucosaminidase, ~t-L-fucosidase (EC 3.2.1.51), fl-D-fucosidase (EC 3.2.1.38) and fl-D-glucuronidase (EC 3.2.1.31) were measured in each fraction.
Preparation of subcellular fractions The procedure followed that of Price & Dance (1967) for rat kidney with modifications. The kidneys were halved, decapsulated and weighed. They were homogenized in 3 volumes of 0.45 M sucrose, 0.68 mM EDTA, pH 7.0, in a Potter-Elvehjem apparatus with nine strokes and centrifuged at 800g for 2 min. The supernatant was saved and the pellets were washed with 3 volumes of the same medium and spun again. The pellets were saved and designated as the N fraction. The supernatants, after these two centrifugations, were combined and spun at 10,000 g for 1 min. The pellets were washed once with 3 volumes of the same medium and designated as the LM fraction. The combined supernatant-wash was centrifuged in a Spinco Rotor 40 at 40,000 rev/min for 1 hr. The pellets were designated as the MM fraction and the final supernatant (S) contained mostly cytoplasmic materials. The N, LM and MM pellets were homogenized in 9 volumes of the same medium with Polytron ® at settings of 7 for 10sec thrice. These fractions were frozen and assayed for glycohydrolases within a week. All fractions were frozen and thawed only once and diluted properly to yield a linear time course at either 0, 10, 20, 30min or 0, 20, 40, 60 min of incubation. Assays All glycohydrolases were assayed using the individual p-nitrophenylglycosides as substrates at 37°C, as described previously (Chang, 1978a). et-D-mannosidase and fl-D-glucuronidase were measured at pH 5.0 and the other five enzymes at pH 4.5. Protein and blood sugar were measured as before (Chang, 1978a).
MATERIALS AND METHODS
Animals
RESULTS
The Chinese hamsters were chosen from the nondiabetic A V and the diabetic XA lines (Chang & Greenberg, 1978). The AV animals belonged to the 5--7th generations and the XA 16-17th. They were all females, 15-25 month old 557
Animals The Chinese hamsters of A V and X A lines used in this study were m a t u r e females closely matched
558
A.Y. CHANG and C. S. PERRY Table 1. Age, body and kidney weights, blood sugar, protein concentration and renal glycohydrolase activities in A V and XA line Chinese hamsters Measurements
Age
Units
AV-llne a
XA-line b
pC
months
21.0 ± 1.8
21.8 ± 0.8
Body weight
g
27.7 ± 0.8
25.6 ± 0.8
Kidney weight
g
0.267 ± 0.01
0.329 ± 0.01
0305-0491/79.'0401-0557502.00/0
© Pergamon Press Ltd 1979. Printed in Great Britain
A C I D G L Y C O H Y D R O L A S E IN C H I N E S E H A M S T E R (CRICETULUS GRISEUS) WITH S P O N T A N E O U S D I A B E T E S - - V S U B C E L L U L A R D I S T R I B U T I O N IN THE K I D N E Y A. Y. CHANG and C. S. PERRY Diabetes and Atherosclerosis Research, Upjohn Company, Kalamazoo, MI 49001, U.S.A.
(Received 4 July 1978) Abstract--l. Seven renal glycohydrolases were measured in four subcellular fractions prepared from highly inbred aglycosuric (A V-line) and glycosuric (XA-line) Chinese hamsters. 2. ~-D-galactosidase and fl-D-galactosidase were highest in the nuclear (N) and supernatant (S) fractions; both fractions showed reduced activities in the XA animals. 3. :t-D-mannosidase was chiefly a particulate enzyme and its decrease in x A animals was evident in N, lysosomal-mitochondrial (LM) and mitochondrial-microsomal (MM) fractions. 4. No significant difference in N-acetyl-fl-D-glucosaminidase was found in any of these subcellular fractions between AVand XA animals. 5. Although total Ct-L-fucosidase and fl-D-fucosidase levels were similar in AV and XA kidneys, a difference was observed in the S fraction. 6. fl-D-glucuronidase was virtually absent in N and LM fractions and the S fraction of A V kidneys showed higher activity than the XAs. and allowed food and water ad libitum. The animals were bled through the orbital sinus and killed by decapitation early in the morning. The kidneys were excised and rinsed in cold saline.
INTRODUCTION
Variations in the levels of renal (Chang, 1978ab; C h a n g & Greenberg, 1978) and plasma (Chang & Perry, 1978) glycohydrolases have been found in the U p j o h n colony of Chinese hamsters (Cricetulus griseus) with spontaneous diabetes; significant correlations between renal levels of 0t-D-galactosidase (EC 3.2.1.22), fl-o-galactosidase (EC 3.2.1.23) and N-acetylfl-o-glucosaminidase (EC 3.2.1.30) and blood sugar levels have also been established in these animals (Chang & Greenberg, 1978). In an early study on the subcellular distribution of fl-D-glucosidase (EC 3.2.1.21) and fl-o-galactosidase in the kidneys of diabetic, ketotic and nondiabetic Chinese hamsters, a bimodal distribution of fl-o-galactosidase was observed, whereas fl-o-glucosidase resided mainly in the soluble fraction (Chang, 1978c). In view of these findings, the present study was carried out in order to extend our knowledge of diabetes-related differences of other renal glycohydrolases and of their subcellular locations in the Chinese hamster. F o r this purpose, kidney homogenates were separated into nuclear (N), lysosomal-mitochondrial (LM), mitoc h o n d r i a l - m i c r o s o m a l (MM) and supernatant (S) fractions, according to Price & Dance (1967), with modifications. The activities of =t-o-galactosidase, fl-Dgalactosidase, =t-D-mannosidase (EC 3.2.3.24), N-acetyl-fl-D-glucosaminidase, ~t-L-fucosidase (EC 3.2.1.51), fl-D-fucosidase (EC 3.2.1.38) and fl-D-glucuronidase (EC 3.2.1.31) were measured in each fraction.
Preparation of subcellular fractions The procedure followed that of Price & Dance (1967) for rat kidney with modifications. The kidneys were halved, decapsulated and weighed. They were homogenized in 3 volumes of 0.45 M sucrose, 0.68 mM EDTA, pH 7.0, in a Potter-Elvehjem apparatus with nine strokes and centrifuged at 800g for 2 min. The supernatant was saved and the pellets were washed with 3 volumes of the same medium and spun again. The pellets were saved and designated as the N fraction. The supernatants, after these two centrifugations, were combined and spun at 10,000 g for 1 min. The pellets were washed once with 3 volumes of the same medium and designated as the LM fraction. The combined supernatant-wash was centrifuged in a Spinco Rotor 40 at 40,000 rev/min for 1 hr. The pellets were designated as the MM fraction and the final supernatant (S) contained mostly cytoplasmic materials. The N, LM and MM pellets were homogenized in 9 volumes of the same medium with Polytron ® at settings of 7 for 10sec thrice. These fractions were frozen and assayed for glycohydrolases within a week. All fractions were frozen and thawed only once and diluted properly to yield a linear time course at either 0, 10, 20, 30min or 0, 20, 40, 60 min of incubation. Assays All glycohydrolases were assayed using the individual p-nitrophenylglycosides as substrates at 37°C, as described previously (Chang, 1978a). et-D-mannosidase and fl-D-glucuronidase were measured at pH 5.0 and the other five enzymes at pH 4.5. Protein and blood sugar were measured as before (Chang, 1978a).
MATERIALS AND METHODS
Animals
RESULTS
The Chinese hamsters were chosen from the nondiabetic A V and the diabetic XA lines (Chang & Greenberg, 1978). The AV animals belonged to the 5--7th generations and the XA 16-17th. They were all females, 15-25 month old 557
Animals The Chinese hamsters of A V and X A lines used in this study were m a t u r e females closely matched
558
A.Y. CHANG and C. S. PERRY Table 1. Age, body and kidney weights, blood sugar, protein concentration and renal glycohydrolase activities in A V and XA line Chinese hamsters Measurements
Age
Units
AV-llne a
XA-line b
pC
months
21.0 ± 1.8
21.8 ± 0.8
Body weight
g
27.7 ± 0.8
25.6 ± 0.8
Kidney weight
g
0.267 ± 0.01
0.329 ± 0.01
