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N-acetyl-β-glucosaminidase activity in regenerating rat liver
378
SHORT COMMUNICATIONS
BBA 23557
N.Acetyl.p.91ucosaminidase activity in regeneratin 9 rat liver During the early stage of liver regeneration, a striking increase in activities of hepatic lysosomal enzymes such as acid phosphatase, fl-glucuronidase and acid deoxyribonuclease has been described and it has been suggested that the increase of these enzyme activities is attributable to, or is directly associated with, an increase of liver cell population or cell mitosis 1, ~. N-Acetyl-fl-glucosaminidase occurs in the lysosome of the liver and nothing is known regarding the change of this enzyme activity in the regenerating l{ver. I t was of interest to know whether the changes of this enzyme activity in damaged liver after partial hepatectomy were different from those in healthy liver. Male rats of the Sprague-Dawley strain, weighing approx. 15o g, were used. Chronic hepatic damage was induced by carbon tetrachloride inhalation twice a week for 4 weeks. Partial hepatectomy was carried out by the method of HIGGINS AND ANDERSON3. Control groups of healthy and damaged rats were subjected to a sham operation. The rats were sacrificed 24 and 48 h after the hepatectomy, and the livers were perfused with cold 0.25 M sucrose solution immediately. The liver homogenates and subcellular fractions were prepared as described previously 4. Estimation of N-acetyl-fl-glucosaminidase activity was carried out according to the method of WALKER et al. 5, and the activity was expressed as a specific activity (nmoles of p-nitrophenol liberated per mg of protein per rain) and a total activity (/,moles of p-nitrophenol liberated per g of wet liver per rain). Protein was determined b y the method of LOWRY et al. 6. Changes of N-aeetyl-fl-glucosaminidase activity in the whole homogenates and in each of the subfractions of the liver are summarized in Table I. The control groups of the healthy and damaged rats with sham operation showed no significant changes of the enzyme activities in either the whole homogenates or the subcellular fractions. An increase of the enzyme activity in whole homogenates was observed both in healthy and in damaged liver 24 h after the operation. In the healthy liver, the activity decreased to the control value after 48 h. In the damaged liver, on the contrary, the activity remained at a higher level. A similar trend was seen in lysosomal fraction, although the increase was much more pronounced. No significant changes in the enzyme activity were seen after the hepatectomy in either the mitochondrial and microsomal fractions. GRISHAM7 demonstrated that the number of parenchymal cells in rat liver increased rapidly until a peak value was attained at 20 h after partial hepatectomy and thereafter decreased slowly toward a normal level. From the present study, the increase of N-acetyl-fl-glucosaminidase activity observed in lysosomal fraction of the liver is assumed as the consequence of parenchymal cell division rather than proliferation of the connective tissue cells of the liver. Difference in the activity seen in the lysosomal fraction and in the whole homogenates between healthy and damaged rat livers 48 h after the operation is considered to be caused by abnormalities of the parenchymal cell division or of the reaction of the interstitial cells in the liver of the damaged rats. In supernatant fraction of the healthy liver, the enzyme activity was increased after 24 h of the hepatectomy and showed continued higher value after 48 h. In Biochim. Biophys. Acta, 2o1 (197o) 378-38o
SHORT
COMMUNICATIONS
379
Biochim. Biophys. Acta, ZOI (1970) 378-380
380
SHORT COMMUNICATIONS
damaged liver, however, the activity showed a decrease to the control value after 48 h. This result is considered to be attributable mainly to the release of the enzyme from the liver cells, which is also suggested by the earlier finding that N-acetyl-fl-glucosaminidase activity in serum of rats with chronic hepatic damage is significantly higher than that of healthy rats 4.
Department of Medicine, Osaka University Medical School, Osaka (Japan)
T. SUEMATSU N . IWABORI
T. KOIZUMI
I M. KLOCKARS AND O. WEGELINS, Proc. Soc. Exptl. Biol. Med., 131 (1969) 218. 2 R. L. P. ADAMS, Biochem. J., 87 (1963) 532. 3 G. M. HIGGINS AND 1R. M. ANDERSON, Arch. Pathol., 22 (1936) 658. 4 T. KOlZUMI, T. SUEMATSU, N. IWABORI AND H. ABE, Biochim. Biophys. Acta, 151 (1968) 628. 5 ]?. G. WALKER, M. E. WOOLEN AND D. J. PUGH, J. Clin. Pathol., 13 (196o) 353. 6 0 . H. LOWRY, ~N. J. ROSEBROUGH, A. L. FARR AND 1~. J. I~ANDALL, J. Biol. Chem., 193 (1951) 265. 7 J. W. GRISHAM, Cancer Res., 22 (1962) 842.
Received September 8th, 1969 Biochim. Biophys. Acta, 2Ol (197 o) 378-38o
BBA 23561
Autolytic enzyme from a staphylococcal mutant There is considerable interest at present in enzymes which lyse bacterial cell walls, especially since they are used as a tool for the investigation of cell wall structure 1. Here we report on the occurrence of an autolytic system in the walls of a staphylococcal mutant, HS 968 (ref. 2). The crude enzyme preparation digests the cell walls of another mutant, HS 116o, a coagulase-negative derivative of Staphylococcus aureus NCTC 85I i (ref. 2), and among the products of digestion the disaccharide MurNAc-GlcNAc* and the tetrasaccharide MurNAc-GlcNAc-MurNAc-GIcNAc have been isolated and characterized. The point of cleavage of the cell wall carbohydrate backbone is thus the same as that reported for lysostaphin 3 and the autolytic enzyme from Staphylococcus aureus Copenhagen 4. In addition, the crude preparation contains at 6-O-acetylase, since it converts 6-O-acetyl-MurNAc-GlcNAc to MurNAc-GlcNAc. Cell walls of HS 968 were prepared as previously described 6, the only modification was that the walls were collected from the top of the sucrose gradient rather than from the bottom. They were washed exhaustively with water and lyophilized. The walls were suspended in o.I M ammonium acetate (pH 6.8) at a concentration of IO mg/ml and incubated for 24 h at 37 °. The clear autolysate served as crude enzyme ("968 enzyme"). As substrate, we used the cell walls of HS 116o, prepared A b b r e v i a t i o n s : M u r N A c , N - a c e t y l m u r a m i c acid; GlcNAc, N - a c e t y l - D - g l u c o s a m i n e ; all oligosaccharides are fl(I-4) linked; M~,, r a t e of m i g r a t i o n on h i g h - v o l t a g e p a p e r e l e c t r o p h o r e s i s p H 6.5, relative to t h e t e t r a s a c c h a r i d e G l c N A c - M u r N A c - G l c N A c - M u r N A c . RGIeN , rate of mig r a t i o n on p a p e r c h r o m a t o g r a p h y relative to g l u c o s a m i n e .
Biochim. Biophys. Acta, 2Ol (i97 o) 380-383
SHORT COMMUNICATIONS
BBA 23557
N.Acetyl.p.91ucosaminidase activity in regeneratin 9 rat liver During the early stage of liver regeneration, a striking increase in activities of hepatic lysosomal enzymes such as acid phosphatase, fl-glucuronidase and acid deoxyribonuclease has been described and it has been suggested that the increase of these enzyme activities is attributable to, or is directly associated with, an increase of liver cell population or cell mitosis 1, ~. N-Acetyl-fl-glucosaminidase occurs in the lysosome of the liver and nothing is known regarding the change of this enzyme activity in the regenerating l{ver. I t was of interest to know whether the changes of this enzyme activity in damaged liver after partial hepatectomy were different from those in healthy liver. Male rats of the Sprague-Dawley strain, weighing approx. 15o g, were used. Chronic hepatic damage was induced by carbon tetrachloride inhalation twice a week for 4 weeks. Partial hepatectomy was carried out by the method of HIGGINS AND ANDERSON3. Control groups of healthy and damaged rats were subjected to a sham operation. The rats were sacrificed 24 and 48 h after the hepatectomy, and the livers were perfused with cold 0.25 M sucrose solution immediately. The liver homogenates and subcellular fractions were prepared as described previously 4. Estimation of N-acetyl-fl-glucosaminidase activity was carried out according to the method of WALKER et al. 5, and the activity was expressed as a specific activity (nmoles of p-nitrophenol liberated per mg of protein per rain) and a total activity (/,moles of p-nitrophenol liberated per g of wet liver per rain). Protein was determined b y the method of LOWRY et al. 6. Changes of N-aeetyl-fl-glucosaminidase activity in the whole homogenates and in each of the subfractions of the liver are summarized in Table I. The control groups of the healthy and damaged rats with sham operation showed no significant changes of the enzyme activities in either the whole homogenates or the subcellular fractions. An increase of the enzyme activity in whole homogenates was observed both in healthy and in damaged liver 24 h after the operation. In the healthy liver, the activity decreased to the control value after 48 h. In the damaged liver, on the contrary, the activity remained at a higher level. A similar trend was seen in lysosomal fraction, although the increase was much more pronounced. No significant changes in the enzyme activity were seen after the hepatectomy in either the mitochondrial and microsomal fractions. GRISHAM7 demonstrated that the number of parenchymal cells in rat liver increased rapidly until a peak value was attained at 20 h after partial hepatectomy and thereafter decreased slowly toward a normal level. From the present study, the increase of N-acetyl-fl-glucosaminidase activity observed in lysosomal fraction of the liver is assumed as the consequence of parenchymal cell division rather than proliferation of the connective tissue cells of the liver. Difference in the activity seen in the lysosomal fraction and in the whole homogenates between healthy and damaged rat livers 48 h after the operation is considered to be caused by abnormalities of the parenchymal cell division or of the reaction of the interstitial cells in the liver of the damaged rats. In supernatant fraction of the healthy liver, the enzyme activity was increased after 24 h of the hepatectomy and showed continued higher value after 48 h. In Biochim. Biophys. Acta, 2o1 (197o) 378-38o
SHORT
COMMUNICATIONS
379
Biochim. Biophys. Acta, ZOI (1970) 378-380
380
SHORT COMMUNICATIONS
damaged liver, however, the activity showed a decrease to the control value after 48 h. This result is considered to be attributable mainly to the release of the enzyme from the liver cells, which is also suggested by the earlier finding that N-acetyl-fl-glucosaminidase activity in serum of rats with chronic hepatic damage is significantly higher than that of healthy rats 4.
Department of Medicine, Osaka University Medical School, Osaka (Japan)
T. SUEMATSU N . IWABORI
T. KOIZUMI
I M. KLOCKARS AND O. WEGELINS, Proc. Soc. Exptl. Biol. Med., 131 (1969) 218. 2 R. L. P. ADAMS, Biochem. J., 87 (1963) 532. 3 G. M. HIGGINS AND 1R. M. ANDERSON, Arch. Pathol., 22 (1936) 658. 4 T. KOlZUMI, T. SUEMATSU, N. IWABORI AND H. ABE, Biochim. Biophys. Acta, 151 (1968) 628. 5 ]?. G. WALKER, M. E. WOOLEN AND D. J. PUGH, J. Clin. Pathol., 13 (196o) 353. 6 0 . H. LOWRY, ~N. J. ROSEBROUGH, A. L. FARR AND 1~. J. I~ANDALL, J. Biol. Chem., 193 (1951) 265. 7 J. W. GRISHAM, Cancer Res., 22 (1962) 842.
Received September 8th, 1969 Biochim. Biophys. Acta, 2Ol (197 o) 378-38o
BBA 23561
Autolytic enzyme from a staphylococcal mutant There is considerable interest at present in enzymes which lyse bacterial cell walls, especially since they are used as a tool for the investigation of cell wall structure 1. Here we report on the occurrence of an autolytic system in the walls of a staphylococcal mutant, HS 968 (ref. 2). The crude enzyme preparation digests the cell walls of another mutant, HS 116o, a coagulase-negative derivative of Staphylococcus aureus NCTC 85I i (ref. 2), and among the products of digestion the disaccharide MurNAc-GlcNAc* and the tetrasaccharide MurNAc-GlcNAc-MurNAc-GIcNAc have been isolated and characterized. The point of cleavage of the cell wall carbohydrate backbone is thus the same as that reported for lysostaphin 3 and the autolytic enzyme from Staphylococcus aureus Copenhagen 4. In addition, the crude preparation contains at 6-O-acetylase, since it converts 6-O-acetyl-MurNAc-GlcNAc to MurNAc-GlcNAc. Cell walls of HS 968 were prepared as previously described 6, the only modification was that the walls were collected from the top of the sucrose gradient rather than from the bottom. They were washed exhaustively with water and lyophilized. The walls were suspended in o.I M ammonium acetate (pH 6.8) at a concentration of IO mg/ml and incubated for 24 h at 37 °. The clear autolysate served as crude enzyme ("968 enzyme"). As substrate, we used the cell walls of HS 116o, prepared A b b r e v i a t i o n s : M u r N A c , N - a c e t y l m u r a m i c acid; GlcNAc, N - a c e t y l - D - g l u c o s a m i n e ; all oligosaccharides are fl(I-4) linked; M~,, r a t e of m i g r a t i o n on h i g h - v o l t a g e p a p e r e l e c t r o p h o r e s i s p H 6.5, relative to t h e t e t r a s a c c h a r i d e G l c N A c - M u r N A c - G l c N A c - M u r N A c . RGIeN , rate of mig r a t i o n on p a p e r c h r o m a t o g r a p h y relative to g l u c o s a m i n e .
Biochim. Biophys. Acta, 2Ol (i97 o) 380-383