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Biomedical subjects

H Usui

Publications and source records attributed to H Usui.

At least 163 records · Page 9Linked to original sources
PubMed1985

Comparison of tyrosine protein kinases in membrane fractions from mouse liver and Ehrlich ascites tumor.

Tubulin was phosphorylated mainly at tyrosine residues by membranes from mouse liver and Ehrlich ascites tumor with ATP in the presence of MnCl2, ZnCl2, NaVO3, and Nonidet P-40 in an epidermal growth factor (EGF)- and insulin-independent manner. The tyrosine tubulin kinase activity in the tumor membranes is comparable to the activity in liver membranes. Two tyrosine tubulin kinases, namely I (Mr = 64,000) and II (Mr = 46,000), were purified 5-17-fold and were free of the EGF receptor and the insulin receptor. Phosphorylation of endogenous proteins produced major alkali-resistant phosphoproteins of 56 and 53 kDa in kinase I preparations and of 46 and 37 kDa in kinase II preparations. These kinases phosphorylated tubulin stoichiometrically at tyrosine and had a preference of alpha-subunit to beta-subunit of tubulin. Apparent Km values of kinases I and II for tubulin were about 4 and 8 microM and for ATP were about 2 and 4 microM, respectively. Thiol reagents inhibited their reactions. N alpha-p-Tosyl-L-lysine chloromethyl ketone stimulated the reactions at 1 mM but inhibited them at 5 mM. Although kinases I and II also phosphorylated angiotensin II, tyrosine-glutamate copolymers, and heavy chains of anti-pp60src IgG, they differed from each other in preferences for the substrates. More than 95% of the enzyme activities was not immunoprecipitated with the anti-pp60src IgG which cross-reacts with pp60c-src. In comparison with the corresponding liver enzymes, tumor enzymes were more stable to heat and incorporated more phosphate into tubulin, but showed lower activity toward angiotensin II and anti-pp60src IgG.

Adenosine Triphosphate↗
PubMed1985

Comparison of tyrosine phosphorylation of proteins by membrane fractions from mouse liver, Ehrlich ascites tumor and MH134 hepatoma.

When membrane fractions from mouse liver, Ehrlich ascites tumor and MH134 hepatoma were incubated with [gamma-32P]ATP at 0 degree C in the presence of MnCl2, ZnCl2 and NaVO3, proteins were phosphorylated on tyrosines to a larger extent in liver membranes than in tumor membranes. Separation of labelled proteins by SDS-gel electrophoresis showed phosphorylated alkali-resistant bands of 170, 140, 130, 80, 56, 53 and 46 kDa proteins in Ehrlich ascites tumor membranes; liver membranes exhibited more strongly phosphorylated bands of 170, 56, 53 and 46 kDa proteins. Epidermal growth factor stimulated the tyrosine phosphorylation of only a 170 kDa protein, which was more significant in liver membranes. Liver membranes exhibited slightly higher levels of tyrosine protein kinase activity compared to tumor membranes.

Animals↗
PubMed1985

Differences in contractile responses to electrical stimulation and alpha-adrenergic binding sites in isolated cerebral arteries of humans, cows, dogs, and monkeys.

The neurogenic responses of cerebral arteries of humans and the monkey were compared with those of the dog. alpha-Adrenoceptors in these cerebral arteries were also characterized by radioligand-binding studies. In human posterior communicating arteries, the contractile response to electrical transmural stimulation was attenuated by treatment with phentolamine in 41% of preparations tested, but not affected by aspirin. The responses in the monkey and canine arteries were not attenuated by treatment with phentolamine, but were reduced by aspirin in 60 and 73% of the cases, respectively. Reactivity and affinity to exogenous noradrenaline were comparable in human and monkey arteries, but greater than those of canine arteries. Radioligand-binding assays revealed binding sites for [3H]prazosin in the human and monkey cerebral arteries; the KD and Bmax values were not significantly different between the two species. Such binding sites were not detected in canine arteries. Binding sites for [3H]yohimbine were present in the arteries of the three species. Thus, the neurogenic contractile response of human cerebral arteries may be at least in part adrenergic in nature, while that of monkey and canine arteries may be due to neurogenically occurring arachidonate. alpha-Adrenoceptors in the monkey arteries are similar to those in humans.

Animals↗
PubMed1985

Metabolism of branched-chain amino acids in rats with acute hepatic failure: a tracer study using 15N-leucine.

Fulminant hepatic failure (FHF) was produced in rats with intraperitoneal injection of D-galactosamine. Control rats received only physiological saline. 15N-leucine (200 mg/kg of body weight) was injected into the rats via the tail vein. Arterial blood was drawn before and 5, 15, 30 and 60 min after the injection of 15N-leucine. 15N-amino acids were determined quantitatively by gas chromatography and mass spectrometry. The plasma 15N-leucine level decreased logarithmically in the same manner in both groups. This result suggests that leucine is mainly metabolized in extrahepatic tissues. The incorporation of 15N into plasma isoleucine and valine was not significantly different between the groups. Plasma alanine and glutamine concentrations increased in controls and decreased in FHF rates after the injection. The incorporation of 15N into plasma alanine in rats with FHF was significantly later than in controls. This result may suggest that undergoing hyperammonemia causes to form more glutamine from glutamate in extrahepatic sites as the same manner as for chronic hepatic failure. Additionally, insulin levels increased temporarily after the injection of leucine in both groups. This increase may play a role in the decrease in plasma isoleucine and valine concentrations after injection of leucine.

Alanine↗
PubMed1984

Molecular cloning and the complete nucleotide sequence of cDNA to mRNA for S-100 protein of rat brain.

The complete nucleotide sequence of mRNA for beta-subunit of rat brain S-100 protein was determined from recombinant cDNA clones. The sequence was composed of 1488 bp which included the 276 bp of the complete coding region, the 120 bp of the 5'-noncoding region and the 1092 bp of the 3'-noncoding region containing two polyadenylation signals. In addition, the poly(A) tail was also found. The amino acid sequence deduced from the nucleotide sequence was homologous to the amino acid sequence of bovine S-100 beta subunit except 4 residues showing species differences. From the viewpoint of evolutionary implications, the homology between the nucleotide sequence of S-100 and those of rat intestinal Ca-binding protein (ICaBP) and calmodulin (CaM) was examined. A dot-blot hybridization of poly(A) RNA from the developing rat brains using a labeled cDNA showed a rapid increase in S-100 mRNA at 10-20 postnatal days. The presence of S-100 mRNA in C-6 glioma cells is also described.

Age Factors↗
PubMed1984

Isolation and immunohistochemical localization of a cerebellar protein.

Our previous report described that a protein called spot 35 is found in the cerebellar cytosol of adult rats by two-dimensional gel electrophoresis. In this paper we isolated this protein from the soluble fraction of bovine and rat cerebella and then prepared an antiserum against the bovine protein. This protein shows pI around 5.3 and Mr around 27 kdalton. Determination of the amino acid composition of this protein shows high glutamic acid, aspartic acid and leucine contents. Using the antiserum we examined the immunohistochemical localization of this protein by the peroxidase-antiperoxidase method. Purkinje cells, their dendrites and axons were immunohistochemically stained in the cerebella of adult rats, rabbits and humans. Other cells, such as granule cells and glial cells, and myelin did not react to the antiserum.

Amino Acids↗
PubMed1984

Effects of polyamine hydrochlorides on dephosphorylation of phosphorylated H2B histone integrated into nucleosome core particles.

The rate of dephosphorylation of isolated P-H2B histone by pig heart phosphoprotein phosphatase (Mr = 224,000) and its catalytic subunit (Mr = 31,000) was suppressed more than 95% at low ionic strength when the substrate was integrated into nucleosome core particles. The suppressed rate was increased 13-43-fold by polyamine hydrochlorides and Mg(CH3COO)2 at the optimal ionic strength of 0.2-0.38 M. Phosphatase activity toward integrated P-H2B histone was distributed in both rat liver cytosol and nuclear extracts. The phosphatase activities in these fractions showed similar specific activities and were also increased 10-32-fold by 10 mM spermine.4HCl.

Animals↗
PubMed1984

Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor.

A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.

Amino Acid Sequence↗
PubMed1984

Synthesis of 8,2'-ethano-cyclo-2'-deoxyadenosines.

The radical- or photo-cyclization of protected 2'-iodoethyl or 2'-phenylthioethyl derivatives of 2'-deoxyadenosine afforded the 8,2'-ethano-cyclo-2'-deoxyadenosine, a new carbon-bridged adenine cyclonucleoside.

Deoxyadenosines↗
PubMed1983

The effect of heart and skeletal muscle troponin complexes and calmodulin on the Ca2+-dependent reactions of phosphorylase kinase isoenzymes.

The dephosphorylated form of phosphorylase kinase was purified 700-fold from rabbit heart extract. The purified enzyme had a pH 6.8/pH 8.2 activity ratio of 0.04-0.08 and was completely dependent on Ca2+ with an apparent Ka value for Ca2+ of 2.59 microM at pH 6.8. At free Ca2+ concentrations between 0.057 microM and 400 microM, 1.5 microM rabbit heart troponin complex had no significant effect on the reaction. However, 1.5 microM rabbit skeletal muscle troponin complex stimulated the reaction 1.5-2-fold with a concomitant decrease in the Ka value for Ca2+ to 1.40 microM. No differences in the effects of these troponin complexes were observed when heart-type and skeletal muscle-type phosphorylase b isoenzymes from either rabbit or pig were used as substrate. Similar effects of heart and skeletal muscle troponin complexes were observed on the Ca2+-dependent reaction of the dephosphorylated form of phosphorylase kinase partially purified from rabbit skeletal muscle. A saturating concentration (1.36 microM) of bovine brain calmodulin stimulated 2-5-fold the Ca2+-dependent reaction of skeletal muscle phosphorylase kinase, but not the reaction of heart phosphorylase kinase. Heart troponin complex (12 microM) suppressed 80-100% the stimulatory effect of skeletal muscle troponin complex on the reactions of phosphorylase kinase isoenzymes, but had no significant effect on the stimulation by calmodulin of skeletal muscle phosphorylase kinase reaction.

Animals↗
PubMed1983

Phosphoprotein phosphatases in human erythrocyte cytosol.

More than 97% of spectrin phosphatase activity in human erythrocyte hemolysate was recovered in cytosol. The cytosolic phosphatase activity was resolved into four peaks, namely phosphatases I (22%, Mr = 180,000), II (3%, Mr = 42,000), III (8%, Mr = 177,000), and IV (62%, Mr = 104,000), by aminohexyl-Sepharose column chromatography. Although these phosphoprotein phosphatases also catalyzed the dephosphorylation of phosphorylase a, glycogen synthase b, and phosphorylated H1 and H2B histones, the phosphatases differed from each other in preferences for substrates and the Mg2+ or Mn2+ requirements for their activities. The treatment with 80% ethanol converted phosphatases I, III, and IV to Mr = 31,000 forms which had essentially the same physical and catalytic properties. By contrast, the molecular weight and catalytic properties of phosphatase II, which was Mg2+- or Mn2+-dependent, were not changed by the same ethanol treatment. The major spectrin phosphatase, phosphatase IV, was purified to near homogeneity, as judged by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-gel electrophoresis revealed that the enzyme was composed of one 32,000-Da polypeptide (alpha) and one 69,000-Da polypeptide (beta). Km values of the enzyme for phosphorylated spectrin and H2B histone were 1.63 +/- 0.45 and 48.2 +/- 7.6 microM, respectively. The spectrin phosphatase activity was stimulated about 2-fold by 5-25 mM Mg2+, but was completely inhibited by the same concentration of Mn2+. Physiological concentrations of adenine nucleotides, 2,3-diphosphoglyceric acid, cyclic nucleotides, or Ca2+ and/or calmodulin had no significant effect on the reaction, but 20 mg/ml of hemoglobin inhibited the reaction by 60%. -SH-blocking agents but not iodoacetate inhibited the reaction.

Chromatography, Gel↗
PubMed1983

Resolution and reassociation of three distinct components from pig heart phosphoprotein phosphatase.

A partially purified pig heart phosphoprotein phosphatase was dissociated into three distinct components, namely alpha, beta, and gamma, by gel filtration on Sephacryl S-200 followed by chromatography on DEAE-Sephadex in the presence of 6 M urea. Although alpha itself had phosphatase activities toward P-H2B histone, P-H1 histone, phosphorylase a, and glycogen synthase b, beta and gamma had no activity toward these substrates even in the presence of 1 mM Mn2+. The beta component (Mr = 80,000) combined with alpha (Mr = 31,000) in the absence of urea to produce Form 2 (Mr = 123,000) with concomitant increase in P-H1 histone phosphatase activity and Mg2+ requirement for P-H2B histone phosphatase activity (Imazu, M., Imaoka, T., Usui, H., Kinohara, N., and Takeda, M. (1981) J. Biochem. 90, 851-862). The gamma component (Mr = 62,000) reassociated with Form 2 to produce Form 1 (Mr = 199,000) which was similar to the original phosphoprotein phosphatase in substrate specificity and Mg2+ requirement. Binding of gamma to Form 2 strongly suppressed the phosphatase activities toward phosphorylase a and glycogen synthase b with marginal effects on the other phosphatase activities and Mg2+ requirement. However, gamma alone could not associate with alpha. The gamma component was sensitive to treatment with heat (60 degrees C for 2 min) or trypsin and was resistant to treatment with DNase or RNase. The pig heart phosphoprotein phosphatase was further purified to near homogeneity, as judged by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis revealed that the purified enzyme (Mr = 171,000) was composed of three polypeptide components, namely alpha', beta', and gamma' with molecular weights of 34,000, 69,000, and 56,000, respectively. The component stoichiometry was determined to be alpha' 1 beta' 1 gamma' 1 by densitometric tracing of the Coomassie blue-stained bands on the acrylamide gel. After dissociation of alpha ' and other components by gel filtration of the purified enzyme on Sephacryl S-200 in the presence of 6 M urea, one alpha ' combined with one beta' to produce Form 2' of Mr = 106,000. Since Form 1 and the purified enzyme as well as Form 2 and Form 2' had similar catalytic properties and s20,w values, respectively, component compositions are suggested to be alpha 1 beta 1 gamma 1 for Form 1 and alpha 1 beta 1 for Form Form 2.

Animals↗