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S Tsuiki

Publications and source records attributed to S Tsuiki.

At least 55 records · Page 3Linked to original sources
PubMed1986

Identification and characterization of Mg2+-dependent phosphotyrosyl protein phosphatase from rat liver cytosol.

Although highly purified preparations of Mg2+-dependent phosphoseryl protein phosphatase (also designated phosphatase IA or phosphatase 2C) dephosphorylated phosphotyrosyl histone, the activity has been resolved from phosphatase IA by polyacrylamide gel electrophoresis at pH 9.5. This novel phosphotyrosyl-specific protein phosphatase absolutely requires Mg2+ or Mn2+ for activity, is inhibited by Zn2+, vanadate and fluoride, and has an optimal pH of 9.0 and Mr = 50,000. Certain properties of this phosphatase so closely resemble those of phosphatase IA that the two enzymes tend to be copurified through various separation procedures.

Animals↗
PubMed1986

Evidence for sialidase hydrolyzing gangliosides GM2 and GM1 in rat liver plasma membrane.

Rat liver plasma membrane removed sialic acid from mixed bovine brain gangliosides more efficiently than from sialyllactose and orosomucoid with an optimal pH of 4.5. When individual gangliosides, each labeled with [14C]sialic acid or [3H]sphingosine, were tested, not only GD1a and GM3 but also GM2 and GM1, both of which had been considered to resist mammalian sialidases, were desialylated. The products of GM2 and GM1 hydrolysis were identified as asialo-GM2 and asialo-GM1, respectively, by thin-layer chromatography.

Animals↗
PubMed1986

Activation of a D-form of rabbit muscle glycogen synthase by Ca2+-activated protease.

Glycogen synthase isolated from rabbit skeletal muscle as a D-form (synthase D2) is activated by a rat liver cytosolic protein differing from any of the known protein phosphatases (D2 activase). Although reversible by phosphorylation by cyclic AMP-dependent protein kinase, the activation is a result of limited proteolysis by D2 activase, which has been identified as the Ca2+-activated protease.

Animals↗
PubMed1986

Cytosolic sialidases of rat tissues with special reference to skeletal muscle enzyme.

Using 4-methylumbelliferyl-N-acetylneuraminic acid as substrate, the cytosolic fractions of various rat tissues were assayed for sialidase activity. The activity was about 16 times greater in skeletal muscle than in liver, and the enzymes from the two sources were identical in chromatographic behavior, pH optimum, and substrate specificity. Apparently the same enzyme was found to be distributed in brain, heart, stomach, intestine, and testis, but not in lung and spleen. Sialidase was also present in kidney cytosol at a high level, but the enzyme resembled liver lysosomal sialidase rather than liver cytosolic sialidase in substrate specificity.

Animals↗
PubMed1985

Purification and characterization of cytosolic sialidase from rat liver.

Sialidase has been purified from rat liver cytosol 83,000-fold by sequential chromatography on DEAE-cellulose, CM-cellulose, Blue-Sepharose, Sephadex G-200, and heparin-Sepharose. When subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the purified cytosolic sialidase moved as a single protein band with Mr = 43,000, a value similar to that obtained by sucrose density gradient centrifugation. The purified enzyme was active toward all of the sialooligosaccharides, sialoglycoproteins, and gangliosides tested except for submaxillary mucins and GM1 and GM2 gangliosides. Those substrates possessing alpha 2----3 sialyl linkage were hydrolyzed much faster than those with alpha 2----6 or alpha 2----8 linkage. The optimum pH was 6.5 for sialyllactose and 6.0 for orosomucoid and mixed brain gangliosides. The activity toward sialyllactose was lost progressively with the progress of purification but restored by addition of proteins such as bovine serum albumin. In contrast, neither reduction by purification nor restoration by albumin was observed for the activity toward orosomucoid. When mixed gangliosides were the substrate, bile acids were required for activity and this requirement became almost absolute after the enzyme had been purified extensively. Intracellular distribution study showed that about 15% of the neutral sialidase activity was in the microsomes. The enzyme could be released by 0.5 M NaCl; the released enzyme was indistinguishable from the cytosolic sialidase in properties.

Albumins↗
PubMed1985

Casein and histone kinases of a rat ascites hepatoma as compared with those of rat liver.

Seryl/threonyl-protein kinases in cytosolic and particulate fractions from rat liver and AH-13, a rat ascites hepatoma, have been studied by chromatographing these fractions on DEAE-cellulose and assaying the eluates with casein, phosvitin, histone and protamine as substrates. Liver cytosolic fraction contains a group of well-characterized seryl/threonyl-protein kinases, namely, casein kinases I and II and histone kinases I and II. Liver particulate fraction, on the other hand, is almost totally devoid of casein kinase I and histone kinase I but contains an additional peak of casein kinase tentatively designated casein kinase III. In AH-13, cytosolic casein kinase I is markedly increased and particulate-associated casein kinases II and III are moderately increased as compared with liver. Moreover, it was found that in AH-13, the histone kinase I level is high in the particulate fraction but markedly decreased in the cytosolic fraction. It is suggested that particulate-associated histone kinase I may be of cytosolic origin.

Animals↗
PubMed1984

Rat-liver lysosomal sialidase. Solubilization, substrate specificity and comparison with the cytosolic sialidase.

Purified liver lysosomes, prepared from rats previously injected with Triton WR-1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity. The intralysosomal sialidase was solubilized from the mitochondrial-lysosomal fraction of rat liver and partially purified by Sephadex G-200, or Sephadex G-200 followed by CM-cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60 000 +/- 5000 by gel filtration; it hydrolyzed a variety of sialooligosaccharides , those containing (alpha 2----3)sialyl linkages being better substrates than those with (alpha 2----6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and transferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin. In contrast to the intralysosomal sialidase, the sialidase partially purified from rat liver cytosol by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose and CM-cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maximally active at pH 5.8 and had a relative molecular mass of approximately 60 000. It also hydrolyzed gangliosides but not submaxillary mucin.

Animals↗
PubMed1984

Cytosolic protein phosphatases of rat ascites hepatoma AH-13 as compared with those of rat liver: isolation and characterization of a novel protein phosphatase.

To investigate the alterations of phosphoseryl/phosphothreonyl-protein phosphatases in neoplastic tissues, the cytosols of rat liver and AH-13, a strain of rat ascites hepatoma, were chromatographed on DEAE-cellulose and the fractions obtained were assayed for protein phosphatase with glycogen synthase D and phosphorylase alpha as phosphoprotein substrates. While the glycogen synthase phosphatase and phosphorylase phosphatase activities of liver cytosol were largely due to phosphatases IA and II, respectively, as previously reported, these phosphatases were absent or present in only small amounts in AH-13 cytosol, whose glycogen synthase phosphatase and phosphorylase phosphatase activities were due almost wholly to a novel protein phosphatase that appeared to be absent in liver. This phosphatase, termed phosphatase H, was purified further by aminohexyl-Sepharose-4B and Sephadex G-200 chromatography without altering its glycogen synthase D/phosphorylase alpha activity ratio. Purified phosphatase H required Mg2+ or Mn2+ for activity and had a molecular weight of about 330,000. It displayed a substrate specificity broader than that of either phosphatase IA or II.

Animals↗
PubMed1984

Sialidase of rat hepatomas: qualitative and quantitative comparison with rat liver sialidase.

The subcellular distribution of sialidase in rat hepatoma induced by 3'-methyl-4-dimethylaminoazobenzene was studied by using sialyllactose as a substrate in the pH range of 4.0-7.0. As found in rat liver, the activity was recovered largely in the mitochondrial/lysosomal fraction with an optimal pH of 4.5 and in the cytosolic fraction with an optimal pH of 6.0, although hepatoma lysosomal (acidic) sialidase was also distributed in the microsomal fraction. The lysosomal and cytosolic sialidases of the hepatoma were indistinguishable from the corresponding enzymes of liver in chromatographic behavior, kinetics and substrate specificity. The levels of lysosomal and cytosolic sialidase activities in liver and hepatomas were then studied in the pellet and supernatant fractions, respectively, obtained by centrifuging the postnuclear supernatant at 105,000g for 1 hr. All the hepatomas tested, one primary and three transplanted, showed higher lysosomal sialidase and lower cytosolic sialidase activities as compared with liver. Quantitative changes similar to those seen in hepatomas were observed in regenerating liver after partial hepatectomy.

Animals↗
PubMed1983

Hypertriacylglycerolemia and adipose tissue lipoprotein lipase activity in the Nagase analbuminemic rat.

In Nagase analbuminemic rats, serum triacylglycerol levels were significantly elevated. This abnormality was accompanied by decreased adipose tissue fat stores, and both were more marked in female than in male rats. Parametrial adipose tissue lipoprotein lipase activity was determined in normally fed female rats. When expressed per mg protein, the activity in analbuminemic rats was only 35% of that in control rats. The activity in analbuminemic rats, however, could be increased as in control rats by refeeding starved animals with a fat-free and carbohydrate-rich diet, and the peak values recorded were the same with the two groups. Treatment of animals with streptozotocin lowered adipose tissue lipoprotein lipase activity in both groups to similar levels. These results suggest that hypertriacylglycerolemia associated with analbuminemia may be caused, at least in part, by altered hormonal control of adipose tissue lipoprotein lipase activity.

Adipose Tissue↗
PubMed1983

Increase in serum sialyltransferase in tumor-bearing rats: the origin and nature of the increased enzyme.

In rats bearing a solid form of AH-109A hepatoma, serum asialofetuin sialyltransferase activity was significantly increased. In order to identify the source of the increased serum sialyltransferase, the asialofetuin sialyltransferase activities of normal and host liver, tumor, and normal and host serum were studied by phosphocellulose column chromatography. While normal and host (day 17) livers exhibited two peaks, namely, transferases I and II, which were previously shown to be the sialylated and unsialylated species, respectively, of beta-galactoside alpha 2 leads to 6 sialyltransferase, the tumor exhibited a single peak of the enzyme, which was a sialylated species (as was transferase I) but was eluted at a position clearly distinguishable from that of either transferase I or transferase II. Under these conditions, both normal and host (day 17) sera were found to contain transferase I but not the tumor-type enzyme. The results have been interpreted as indicating that in rats with AH-109A, the tumor is not the source of the increased serum sialyltransferase. In these rats as well as in normal rats, serum sialyltransferase appears to originate mainly from the liver, whose sialyltransferase activity was also increased in rats with AH-109A.

Animals↗
PubMed1982

Purification and characterization of beta-galactoside (alpha 2 leads to 6)sialyltransferase from rat liver and hepatomas.

Asialofetuin sialyltransferase from Triton X-100 extracts of rat liver was resolved by phosphocellulose chromatography into two fractions, designated I and II in order of elution. When previously treated with Arthrobacter ureafaciens neuraminidase, fraction I eluted at about the same position as II while no alteration occurred in II. Primary rat hepatomas contained only a single asialofetuin sialyltransferase, identical to fraction I in chromatographic behavior. Transferases I and II were purified to near homogeneity. Transferase II, as well as neuraminidase-treated I, could be sialylated auto-catalytically, indicating that the lack of sialic acid in II is not due to the lack of a sialic-acid-accepting site. Both enzymes formed an (alpha 2 leads to 6)sialylgalactoside linkage with asialo-glycoproteins of the glycosylamine-type and with lactose, and were indistinguishable immunologically. Nevertheless, the transferases exhibited different molecular weights of 37000 (I) and 43000 (II). When heated at 50 degrees C, transferase I lost half its original activity within 20 min while II was scarcely inactivated. Kinetically, transferase I showed three-times higher affinity than II for CMP-N-acetylneuraminic acid and for desialylated plasma membrane. Asialofetuin sialyltransferase was also purified from primary rat hepatoma. The purified enzyme was identical to transferase I in every respect examined. We conclude that hepatomas contain transferase I but lack transferase II.

Animals↗
PubMed1981

Studies on UDP-N-acetylglucosamine : alpha-mannoside beta-N-acetylglucosaminyltransferase of rat liver and hepatomas.

When homogenates of rat liver and hepatomas were centrifuged at 78 000 X g, over 90% of liver N-acetylglucosaminyltransferase assayed with beta-galactosidase- and beta-N-acetylhexosaminidase-treated asialofetuin as acceptor was recovered in the particulate fraction, while as much as 24% of hepatoma transferase was in the supernatant fraction. The particulate transferase solubilized by 0.2% sodium deoxycholate emerged from a DEAE-cellulose column at 0.04 M NaCl (transferase A). The supernatant fractions from all the hepatomas tested contained a second N-acetylglucosaminyltransferase eluted from the column at 0.02 M NaCl (transferase B). Transferase B was absent from liver supernatant fraction. The activities of these transferases toward various acceptors and the effect of beta-N-acetylhexosaminidase on their products suggest that both transferases are UDP-N-acetylglucosamine : alpha-mannoside beta-N-acetylglucosaminyltransferase. Although ovalbumin and glycopeptide V, which was isolated from pronase digest of ovalbumin, were good acceptors, transferase A utilized ovalbumin and glycopeptide V with apparent Km values of 0.44 and 0.33 mM, respectively, whereas the corresponding values for transferase B were 4.5 and 0.050 mM.

Animals↗
PubMed1981

Purification and characterization of Mg2+-dependent glycogen synthase phosphatase (phosphoprotein phosphatase IA) from rat liver.

Phosphoprotein phosphatase IA, which represents the major glycogen synthase phosphatase activity in rat liver cytosol, has been purified to apparent homogeneity by chromatography on DEAE-cellulose, histone - Sepharose-4B and Sephadex G-100. The molecular weight of the purified enzyme was 40 000 by gel filtration and 48 000 by sodium dodecyl sulfate gel electrophoresis, Phosphatase IA is therefore a monomeric protein. When treated with 80% ethanol at room temperature, phosphatase IA underwent an inactivation which was totally prevented by 2 mM MgCl2. Catalytically, phosphatase IA has a preference for glycogen synthase D compared with phosphatases IB and II and obligatorily requires Mg2+ or Mn2+ for activity. Maximum activity was attained at 5 mM MgCl2. Since Mg2+ does not activate other phosphoprotein phosphatases in rat liver cytosol, we propose the term 'Mg2+-dependent glycogen synthase phosphatase' for phosphatase IA.

Animals↗