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S Tsuiki

Publications and source records attributed to S Tsuiki.

At least 37 records · Page 2Linked to original sources
PubMed1990

Immunological discrimination of intralysosomal, cytosolic, and two membrane sialidases present in rat tissues.

Cytosolic sialidase was purified from rat skeletal muscle, and the purified enzyme migrated as a single band of Mr 43,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal antibody raised against the enzyme inhibited and immunoprecipitated rat liver cytosolic sialidase as well as the muscle enzyme but failed to cross-react with the intralysosomal sialidase of rat liver and membrane sialidases I (synaptosomal) and II (lysosomal) of rat brain. The antibody against brain membrane sialidase I (anti-I) and that against sialidase II (anti-II), which could be useful to discriminate the two enzymes, did not cross-react with the intralysosomal and cytosolic sialidases of liver. Although more than 90% of liver plasma membrane sialidase was immunoprecipitated with anti-I, only 60% of liver lysosomal membrane sialidase was immunoprecipitated with anti-II, the remainder being immunoprecipitated with anti-I. In confirmation of these data, liver lysosomal membrane exhibited two peaks of ganglioside sialidase corresponding to the membrane sialidases I and II on Aminohexyl-Sepharose chromatography while only one peak of ganglioside sialidase corresponding to sialidase I was observed for liver plasma membrane. These results indicate that the four types of rat sialidase are proteins distinct from one another and that the three kinds of antisera described above are useful for discriminating these sialidases qualitatively and probably quantitatively.

Animals↗
PubMed1990

Particulate-associated protein phosphatases of rat hepatomas as compared with the enzymes of rat liver.

In the course of investigating the neoplastic alterations of protein phosphatases, the particulate fractions of rat liver and AH-13, a strain of rat ascites hepatoma, were chromatographed on DEAE-cellulose and assayed for protein phosphatase using glycogen synthase D and phosphorylase a as substrates. The synthase phosphatase activity of rapidly growing AH-13 was due almost entirely to a divalent cation-inhibited protein phosphatase, tentatively designated phosphatase N, the level of which was elevated remarkably in the hepatoma as compared with liver. Other hepatomas including primary hepatoma induced with 3'-methyl-4-dimethylaminoazobenzene also exhibited high levels of this phosphatase. Phosphatase N exhibited Mr = 49,000 (gel filtration) and has been partially purified with little alteration in properties. Partially purified phosphatase N was inhibited by divalent cations, rabbit skeletal muscle polypeptide inhibitor-2 and heparin, and released the catalytic subunit of type-1 protein phosphatase upon tryptic digestion. It is therefore apparent that phosphatase N is a type-1 protein phosphatase. There is some evidence to suggest that the high levels of phosphatase N in neoplastic cells are due primarily to enhanced synthesis of its non-catalytic (regulatory) subunit.

Adenosine Triphosphate↗
PubMed1990

Neoplastic alteration of a membrane-associated sialidase of rat liver.

Rat liver particulate fraction contains two types of membrane-associated and gangliosides-hydrolyzing sialidase, which have been shown to be identical to two membrane-associated sialidases of rat brain (I and II) chromatographically, immunologically and in substrate specificity. Chromatography on AH-Sepharose 4B of the membrane sialidases of rat primary hepatoma induced by 3'-methyl-4-dimethylaminoazobenzene (MeDAB) further revealed that hepatocarcinogenesis induces a marked decrease in sialidase II but no decrease in sialidase I. Using antisera against sialidases I and II of rat brain, immunoprecipitation studies of the solubilized particulate fractions of rat liver and MeDAB-hepatoma gave results similar to those obtained chromatographically. Using the same immunological technique, sialidase II but not sialidase I was found to be decreased in AH109 A hepatoma and in regenerating and fetal liver.

Animals↗
PubMed1990

Tumor-promoting phorbol ester induces alterations of sialidase and sialyltransferase activities of JB6 cells.

Sialidase and sialyltransferase activities were studied in JB6 mouse epidermal cells before and after exposure to phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), which irreversibly induces anchorage-independent growth and tumorigenicity. JB6 cells exhibited sialidase activities toward 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (4MU-NeuAc) and gangliosides at pH 4.5 in the particulate fraction but apparently not in the cytosol at pH 4.5 or 6.0. In JB6 cells exposed to TPA and in the anchorage-independent transformants, the sialidase activity toward 4MU-NeuAc was decreased and the activity toward gangliosides was increased compared with those in untreated JB6 cells. Immunological analysis with antisera against membrane-associated sialidases I and II revealed that plasma membrane-associated sialidase I was increased and lysosomal membrane-associated sialidase II was decreased under these conditions. TPA treatment also affected the sialyltransferase activities of JB6 cells: and elevation of the transfer activities toward asialo-orosomucoid and asialo-porcine submaxillary mucin but a reduction of GM3 and GD3 synthase activities were observed on exposure to TPA and in cells transformed by TPA to retain anchorage-independency. These results suggest that an increase in sialic acid bound to glycoproteins and a decrease in that bound to glycolipids may occur in JB6 cells exposed to TPA and in the anchorage-independent transformants.

Animals↗
PubMed1990

Effects of OK-432 activation on the sialidase activities of rat peritoneal macrophages.

Intraperitoneal treatment of rat peritoneal macrophages with OK-432 results in more than 9-fold increase in the activity of ganglioside sialidase, which seems to coincide with the appearance of a cell surface antigen, asialo-GM1. The results of subsequent studies suggest that the ganglioside sialidase is located in the plasma membrane, where the enzyme may be responsible for the formation of asialo-GM1 from GM1. In the macrophages activated with OK-432, sialidase activity toward 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc) is also increased. It appears that the 4MU-NeuAc sialidase is intralysosomal and is increased together with other acid hydrolases present in the lysosomes.

Animals↗
PubMed1990

Characterization of protein phosphatases associated with the particulate fraction from rat liver.

Protein phosphatases associated with the particulate fraction from rat liver were studied by chromatographing the fraction on a DEAE-cellulose column and assaying the eluate with phosphorylase alpha and glycogen synthase D as substrates. Phosphorylase phosphatase activity emerged as two peaks, termed P-1 and P-2 in order of elution, both of which were inhibited by Mn2+ and Mg2+. P-1 and P-2 were Mr = 50,000 and 32,000 proteins, respectively, and when treated with trypsin, P-1 converted to a form indistinguishable from P-2, to which protein phosphatase inhibitor-2 was a potent inhibitor. Thus P-2 appears to be the catalytic subunit of type-1 protein phosphatase even though it has been degrated proteolytically as evidenced by its relatively low Mr. The elution profile of glycogen synthase phosphatase activity was entirely different. The activity obtained with 5 mM Mn2+ resolved into three peaks, the second-migrating M-2 being the largest. M-2 is an Mr = 70,000 protein; but an attempt to purify it has been unsuccessful giving a product of Mr = 40,000 and closely similar to the type-1 catalytic subunit in properties including inhibition by inhibitor-2. These results suggest that phosphatases P-1 and M-2 have a common catalytic subunit (type-1), which is bound to different "regulatory" subunits. M-2 distributes in glycogen particles and microsomes evenly while P-1 is almost exclusively in microsomes.

Animals↗
PubMed1990

[Three patients with typical sandblaster's silicosis proven by mineralogical analysis].

Three family members who had worked as sandblasters in their own sandblasting factory showed innumerable small nodularities in both lung fields of their chest radiograms. One of those showed conglomerate shadows in the upper lung fields. Those shadows seemed to be consistent with those of silicosis. One of the patients was examined by transbronchial lung biopsy (TBLB) and showed typical silicotic nodules. Mineralogical studies were done on the abrasive particles and the deposited particles in the lung tissue specimen obtained via TBLB and bronchoalveolar lavage fluid sample (BALF) using polarized microscopy, X-ray diffraction and analytical electron microscopy. The particles which had accumulated on the floor of the factory mineralogically consisted of mostly (over 90%) silica quartz containing small amount of chlorite, and the deposited particles in the lung tissue and those in the BALF showed similar composition.

Adult↗
PubMed1989

Expression of rat protein phosphatase 2C (IA) in Escherichia coli.

A cDNA containing the entire coding sequence of rat type 2C (IA) protein phosphatase was expressed in Escherichia coli. An extract of bacterial cells harboring the recombinant plasmid contained a major (Mr = 41,000 - 43,000) and a minor (Mr = 30,000) protein band; both of these reacted with an anti-type 2C protein phosphatase serum. The size of the major protein band agrees well with that of the 2C phosphatase conceptualized from the cognate cDNA. A Mg2+-dependent protein phosphatase activity was detected in extracts containing the recombinant protein, but not in host cell extracts. Based on these results, it is concluded that the isolated cDNA clone encodes a functional type 2C protein phosphatase.

Animals↗
PubMed1989

Molecular cloning of rat type 2C (IA) protein phosphatase mRNA.

A full-length cDNA encoding rat type 2C (IA) protein phosphatase was isolated from a kidney cDNA library. The cDNA was identified by screening the library with oligonucleotides based on a partial amino acid sequence determined from purified rat liver phosphatase. This clone is 2.35 kilobase pairs long and has a single extended translation reading frame that predicts a 382-amino acid protein of 42,416 daltons. The deduced amino acid sequence contains segments corresponding to three peptides from rat liver type 2C protein phosphatase and two peptides from rabbit skeletal muscle type 2C phosphatase. Rat kidney type 2C protein phosphatase is distantly related to yeast adenylate cyclase but is not related to the catalytic subunits of two other protein phosphatases (types 1 and 2A).

Adenylyl Cyclases↗
PubMed1988

Molecular cloning of cDNA for the catalytic subunit of rat liver type 2A protein phosphatase, and detection of high levels of expression of the gene in normal and cancer cells.

A cloned cDNA encoding a catalytic subunit of type 2A protein phosphatase from a rat liver cDNA library was obtained by use of a synthetic oligonucleotide corresponding to the tryptic peptide sequence of the purified enzyme. There was only a single amino acid difference between the deduced amino acid sequence of the clone obtained and those of the catalytic subunits, 2A alpha, of the rabbit skeletal muscle, porcine kidney and human liver enzymes, suggesting that this clone was a rat 2A alpha cDNA. On Northern blot analysis using a cDNA fragment as a probe, three mRNA species were detected in rat liver: a major mRNA of 2.0 kb and a minor one of 2.7 kb under high stringency conditions, and also a 1.1 kb mRNA under low stringency conditions. The 2A alpha gene was found to be highly expressed in various tissues of rat, especially the brain. High levels of expression of the gene were also detected in mouse NIH3T3 cells and their transformants, and in human cancer cell lines as well as a human immortalized cell line.

Amino Acid Sequence↗
PubMed1988

Tyrosine protein kinase in preneoplastic and neoplastic rat liver.

When rats are subjected to chemical hepatocarcinogenesis according to the protocol of D. Solt and E. Farber ((1976) Nature (London) 263, 701-703), the liver exhibits elevated levels of tyrosine protein kinase activity as early as 3 weeks after the injection of diethylnitrosoamine. A more striking elevation in tyrosine protein kinase activity is noted in rat hepatomas induced by administration of chemical carcinogens, in particular that of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). Tyrosine protein kinase solubilized from the particulate fraction of 3'-Me-DAB-induced hepatoma has a molecular weight identical to that of p60v-src, cross-reacts with p60v-src immunologically, phosphorylates the heavy chain of anti-p60v-src IgG, and probably belongs to a family of p60c-src. The tyrosine protein kinase from the particulate fraction of normal rat liver is indistinguishable from the hepatoma kinase in these properties; thus it apparently differs only in the level of activity. Whether the liver and hepatoma kinases differ merely quantitatively or whether they differ even qualitatively, however, remains to be elucidated.

Affinity Labels↗
PubMed1988

Membrane-associated sialidase of rat liver and its decrease in hepatomas.

Using the particulate fraction of tissue homogenate, plasma membrane-associated sialidase was assayed at pH 4.5 with bovine brain mixed gangliosides as the substrate. The activity was lower in rat hepatoma induced by 3'-methyl-4-dimethylaminoazobenzene (MeDAB) and transplantable AH-109A rat hepatoma than in normal rat liver. The enzyme was almost quantitatively solubilized from liver particulate fraction by using 0.5% (w/v) sodium deoxycholate plus 0.2% (w/v) Triton X-100. When chromatographed on DEAE-cellulose, the solubilized activity emerged as a single peak. The enzyme thus obtained was maximally active at pH 4.5, and readily hydrolyzed mixed gangliosides but was less active toward 4-methylumbelliferyl-alpha-N-acetylneuraminic acid, 3'-sialyllactose and fetuin. The corresponding enzyme from MeDAB-induced hepatoma was indistinguishable from the liver enzyme in terms of ease of solubilization, pH-activity relationship, chromatographic behavior and substrate preference. It therefore appears that the plasma membrane-associated sialidase of hepatomas differs from that of liver only in the tissue level of activity.

Animals↗
PubMed1988

Comparative study of the levels of sialyltransferases responsible for the formation of sugar chains in glycoproteins and gangliosides in rat liver and hepatomas.

Sialyltransferases responsible for the formation of sugar chains in glycoproteins were studied in rat hepatoma in comparison with rat liver. Hepatoma induced by feeding Wistar rats with 3'-methyl-4-dimethylaminoazobenzene (MeDAB) was more active than Wistar liver in sialylating asialo-orosomucoid, and this was due to an increased activity of Gal(beta 1----4)GlcNAc (alpha 2----6) sialyltransferase, the major sialyltransferase in these tissues. Gal(beta 1----3,4)GlcNAc (alpha 2----3) sialyltransferase and the sialyltransferase acting on asialo-bovine submaxillary mucin were, however, decreased in the hepatoma. A similar pattern of sialyltransferase alterations was observed in regenerating liver and other tumors such as AH-109A hepatoma and Sato lung cancer, both of which had been inoculated into Donryu rats. In contrast to these sialyltransferases, the activities of the sialyltransferases responsible for the formation of gangliosides were markedly different even between Wistar and Donryu livers. When compared with Wistar liver, MeDAB-induced hepatoma was higher in lactosylceramide- and lower in GM3-sialyltransferase activity, but these two activities were both lower in AH-109A compared with Donryu liver.

Animals↗
PubMed1988

Analysis of glucose-6-phosphate translocase and hexose-6-phosphate phosphohydrolase, the two obligatory components of microsomal glucose-6-phosphatase system, in rat liver.

A membrane filter procedure developed by Igarashi et al. (1984) for the measurement of glucose 6-phosphate uptake by the microsomes has been demonstrated to be a good method for assaying glucose-6-phosphate translocase, an obligatory component of the microsomal glucose-6-phosphatase system. When glucose-6-phosphate translocase was assayed in developing and diabetic rat livers independently of hexose-6-phosphate phosphohydrolase, another obligatory component of the glucose-6-phosphatase system, the two activities were found to undergo alterations, whose profiles, however, were quite distinct from each other. The profile of the microsomal glucose-6-phosphatase activity resembles the profile of the phosphohydrolase activity rather than that of the translocase activity, suggesting that the phosphohydrolase may be rate-limiting at least under these conditions. AH-109A, a strain of transplantable rat ascites hepatoma, was found to lack both glucose-6-phosphate translocase and hexose-6-phosphate phosphohydrolase activities.

Aging↗
PubMed1987

ATP-Mn2+ stimulates the generation of a putative mediator of insulin action.

Substantial evidence suggests that insulin receptor-associated protein kinase may play a pivotal role in the expression of the intracellular effects of insulin. This study was undertaken to determine whether insulin receptor kinase contributes to the generation of putative insulin mediators. The effect of ATP and divalent cation addition on the production of insulin mediators from liver plasma membranes was investigated. ATP (1 mM) added to liver plasma membranes in the absence of divalent cations enhanced insulin-stimulated release/generation of mediator slightly (approximately 3-fold). ATP in the presence of Mn2+ further increased release/generation of mediator markedly (approximately 100-fold). In contrast, ATP in the presence of Mg2+ had no stimulatory effect. Mn2+ and Mg2+ alone were ineffective. Addition of EDTA completely diminished the stimulatory effects of insulin, ATP, and Mn2+. The stimulation was ATP-specific since other nucleotides and nonhydrolyzable analogues of ATP had no or very weak activity. ATP-Mn2+ stimulated insulin-dependent mediator release/generation in a dose-dependent manner. These results suggest that insulin mediator release/generation is markedly stimulated by an ATP-Mn2+-dependent phosphorylation reaction, similar to insulin-stimulated receptor tyrosine kinase phosphorylation.

Adenosine Triphosphate↗
PubMed1987

Activation of rat liver and rabbit skeletal muscle glycogen synthases by rat liver cytosolic protein phosphatases.

To gain more insight into the nature of the substrate specificity of protein phosphatases, four forms of glycogen synthase D were used as substrates for previously characterized protein phosphatases, IA, IB, and II, from rat liver cytosol. The phosphatase activity was measured as the conversion of glycogen synthase D to synthase I. While glycogen synthase isolated from rat liver as the D-form was activated mainly by phosphatase IA, rabbit skeletal muscle glycogen synthase previously phosphorylated in vitro by cyclic AMP-dependent protein kinase or phosphorylase kinase was activated efficiently by phosphatases IA, IB, and II. Glycogen synthase isolated from rabbit skeletal muscle as the D-form, however, was a poor substrate for all three phosphatases. These results suggest that the phosphorylation state as well as the primary structure of synthase D markedly affects the rate of its activation by individual protein phosphatases. A protein phosphatase released from rat liver particulate glycogen, on the other hand, activated all forms of synthase D used here readily and at about the same rate.

Animals↗
PubMed1987

[Liver phosphotyrosine protein phosphatase and tyrosine protein kinase in chemical hepatocarcinogenesis].

We found and characterized three forms of phosphotyrosine protein phosphatase, subsequently designated PTPP-1, -2 and -3, respectively, in rat liver. Chemical hepatocarcinogenesis according to Solt and Farber was accompanied by a slight increase in liver phosphotyrosine protein phosphatase activity and a remarkable increase in liver tyrosine protein kinase activity. A maximum 8-fold increase in tyrosine kinase activity was observed in hepatomas induced with 3'-methyl-4-dimethylaminoazobenzene (MeDAB). Tyrosine protein kinase that increased with the progress of chemical hepatocarcinogenesis was solubilized from the particulate fraction of MeDAB-induced hepatoma. The enzyme was shown to require Mg2+ for its activity, to immunoprecipitate with anti-pp-60src-IgG and to phosphorylate the IgG. Rat liver also contains another tyrosine protein kinase which requires Mn2+ and does not immunoprecipitate with anti-pp60src; the level of this enzyme appears to diminish with the progress of chemical hepatocarcinogenesis.

Animals↗