Search PubMed⌕ Search

Biomedical subjects

I Ohkubo

Publications and source records attributed to I Ohkubo.

88 records · Page 5Linked to original sources
PubMed1985

Efficient purification of human plasma beta 2-microglobulin from the haemodialysate of a patient with chronic renal failure by use of high-performance liquid chromatography.

Human plasma beta 2-microglobulin was isolated in a good yield (more than 80%) from the haemodialysate (blood ultrafiltrate) of a patient with chronic renal failure. The isolation procedure consisted of Sephadex G-100 gel filtration and two steps of high-performance liquid chromatography: reversed-phase high-performance liquid chromatography and gel permeation chromatography. The purified beta 2-microglobulin was homogeneous by sodium dodecyl sulphate polyacrylamide gel electrophoresis and two-dimensional electrophoresis.

Blood Proteins↗
PubMed1984

Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen.

A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human alpha 2-thiol proteinase inhibitor that was isolated from fresh plasma. Eighteen positive clones were isolated from one million phage, and each was plaque purified. The cDNA insert of one of these phage was sequenced and shown to code for alpha 2-thiol proteinase inhibitor as identified by a partial amino acid sequence of the light chain of alpha 2-thiol proteinase inhibitor. This cDNA insert contained 1529 base pairs coding for the complete alpha 2-thiol proteinase inhibitor. It included 45 base pairs of 5' noncoding sequence, 1281 base pairs that code for pre alpha 2-thiol proteinase inhibitor, a stop codon, 160 base pairs of 3' noncoding sequence, and 40 base pairs of poly(A) tail. The noncoding sequence on the 3' end contained a potential recognition site (AATAAA) for processing and polyadenylation of precursor messenger RNA. The amino acid sequence of alpha 2-thiol proteinase inhibitor deduced from the cDNA showed a striking similarity (overall homology at 74%) to that of bovine low molecular weight (LMW) kininogen, including two internally repeated sequences and a nonapeptide sequence of bradykinin. These data clearly indicated that alpha 2-thiol proteinase inhibitor and LMW kininogen are identical. This was further supported by immunological cross-reactivity between alpha 2-thiol proteinase inhibitor and LMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1983

Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X.

The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/KM value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least five residues long spanning S4 to S1' and has favorable interactions over seven subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa beta or Xa beta or thrombin. Factor IXa beta failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved four hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3'. Factor Xa beta poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa beta substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of either factors IXa beta or Xa beta toward synthetic substrates. Both factor IXa beta and Xa beta cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. However the rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids, and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa beta and Xa beta.

Amino Acid Sequence↗
PubMed1982

Comparative studies on nucleoside diphosphatase of rat ascites hepatoma and rat liver: activity level, purification and properties.

1. The activities of nucleoside diphosphatase in various rat ascites cells of hepatoma, and fetal and neonatal rat liver were much lower than that of normal adult rat liver. 2. The enzyme was purified from ascites hepatoma (AH-66 cell lines) to an apparently homogeneous state and the enzymatic properties were studied in comparison with the enzyme from rat liver microsomes. 3. The hepatoma enzyme had less stability based on the results of heat-inactivation experiments. 4. However, the other properties of hepatoma enzyme; Km value, molecular weight, optimal pH, isoelectric point, substrate specificity and antigenicity, were similar to those of rat liver enzyme.

Acid Anhydride Hydrolases↗
PubMed1980

Purification of nucleosidediphosphatase of rat liver by metal-chelate affinity chromatography.

A procedure is presented for the purification of nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver by affinity chromatography using metal conjugated to epoxy-activated Sepharose 6B. The enzyme is eluted from the conjugate by a solution of L-histidine. The enzyme, when bound to metal-chelate gel, is active in a suspended form, suggesting that the catalytic site is different from the site that binds to the metal-chelate gels. Substrate specificity and Km value of the enzyme obtained are similar to those of the enzyme obtained from the same sources by a conventional procedure, indicating that the metal-chelate affinity chromatography does not bring about any substantial change in the catalytic properties.

Acid Anhydride Hydrolases↗
PubMed1980

Purification and characterization of nucleoside diphosphatase from rat-liver microsomes. Evidence for metalloenzyme and glycoprotein.

Nucleoside diphosphatase was purified from rat liver microsomes more than 3000-fold with a 16% yield using a procedure including concanavalin-A--Sepharose and phenyl-Sepharose column chromatography. The purified enzyme had a specific activity of 2500 units/mg protein and appeared homogeneous by gel electrophoresis. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation, and a Stokes' radius of 4.8 nm was estimated by the gel filtration technique. Its molecular weight is 130,000, but only one single band of Mr 65,000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 9% carbohydrates. The enzyme had a pH optimum of 7.5, an isoelectric point of 4.85 and a Km of 2.5 mM for UDP. On the basis of direct measurement of metal content in the native enzyme, the rat liver nucleoside diphosphatase was found to be a metalloenzyme containing 0.9 mol zinc and 0.1 mol manganese/mol 65,000-Mr subunit. Metal-free nucleoside diphosphatase has been prepared. The activity of the metal-free enzyme was restored by the addition of several divalent cations, zinc being the most effective.

Acid Anhydride Hydrolases↗
PubMed1980

[A study on purification and some characterizations of nucleoside diphosphatase from rat liver microsomes (author's transl)].

Nucleoside diphosphatase from rat liver microsomes was purified about 3,000-fold to a homogenous state using a procedure including Concanavalin A-Sepharose and phenyl-Sepharose column chromatographies. The molecular weight was estimated as 130,000 +/- 2,000 for the native enzyme. Sodium dodecyl sulfate gel electrophoresis indicated the presence of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 9% carbohydrate consisting of mannose (4.2%). On the amino acid analysis, the enzyme contained 27.1% acidic, 10.7% basic and 34.6% non-polar amino acids. The isoelectric point of 4.85 with the enzyme would, at least partly, be ascribed to the higher content of glutamic acid. While the activity of the purified enzyme was inhibited by EDTA and o-phenanthroline, the inhibited enzyme activity was restored by the addition of Mg2+, Ca2+, Mn2+, Co2+ or Zn2+. The enzyme was found to be a metalloenzyme containing approximately 2 atoms or less of Zn2+ per molecule of the native enzyme.

Acid Anhydride Hydrolases↗
PubMed1978

Glucose-6-phosphatase activity in liver and blood platelets of two patients with glycogen storage disease type I.

Glucose-6-phosphatase (G-6-Pase) activity in liver and blood platelets of two patients with glycogen storage disease (GSD) type I is described. Both patients had a reduced activity of G-6-Pase in liver. The km value for glucose 6-phosphate (G-6-P) of residual activity in liver of both patients was similar to that of control liver. We could not demonstrate any reduced activity of platelet G-6-Pase in the patients. Platelet G-6-Pase with our assay method seems to represent a nonspecific phosphatase activity. Our observation suggests that it is necessary to examine platelet G-6-Pase of many other patients with GSD type I to confirm that G-6-Pase deficiency can be diagnosed by enzyme assay performed on blood platelets.

Alanine↗
PubMed1978

Affinity purification and some properties of nucleoside diphosphatase from rat liver cytosol.

Nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver cytosol was purified up to 336--fold by the procedure including affinity chromatographies of concanavalin A- and alanine-Sepharose. The final purified enzyme showed a single protein band upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a tetramer with molecular weight of 120 000 which consists of subunit with molecular weight of 30 000. The enzyme was found to be a glycoprotein on the basis of its chromatographic behaviour with concanavalin A-Sepharose and positive staining with periodate-Schiff reaction in polyacrylamide gels. Furthermore, the two molecular forms with isoelectric points of 4.7 and 5.0 were demonstrated by electrofocusing, though they did not show any significant difference with respect to their catalytic properties.

Acid Anhydride Hydrolases↗
PubMed1977

Solubilization and partial characterization of UDP-N-acetylgalactosamine: globoside alpha-N-acetylgalactosaminyltransferase from dog spleen microsomes.

UDP-N-acetylgalactosamine:globoside alpha-N-acetylgalactosaminyltransferase (EC 2.4.1.-) synthesizing Forssman hapten was solubilized from dog spleen microsomes by a combination of Triton X-100 treatment and sonication. The solubilized enzyme was partially purified by calcium phosphate gel, ammonium sulfate fractionation and then DEAE-cellulose column chromatography. The enzymatic activity of the purified preparation was stimulated by exogenously added phosphatidylserine, as found in the particulate enzyme. When the properties of the purified enzyme were examined in the presence of exogenous phosphatidylserine, the enzyme had an absolute requirement for Mn2+; this was not substituted by Ca2+ or Mg2+. Apparent Km values for UDP-N-acetylgalactosamine and globoside were 1-10(-5) and 5-10(-4) M, respectively. It had a pH optimum of 6.55 regardless of the presence or absence of exogenous lipids. Since the partially purified enzyme was completely free of uridine diphosphatase which was found in the particulate preparaton, the effect of UDP on the transferase activity could be studied. Thus, UDP inhibited 85% of the activity at a concentration of 1.5 mM. p-Cholormercuribenzoate inhibited over 90% of the activity at 2 mM, indicating the transferase to be SH-enzyme.

Animals↗
PubMedDate not supplied

Peptidases play an important role in cataractogenesis: an immunohistochemical study on lenses derived from Shumiya cataract rats.

The role of proteolytic enzymes in Shumiya cataract rats in alterations to lens proteins during cataract formation was studied immunohistochemically using antibodies against exopeptidases, such as lysosomal dipeptidyl peptidase II (DPP II), cytosolic dipeptidyl peptidase III, and soluble and membrane-bound alanyl aminopeptidases, and against cytosolic endopeptidases such as mu- and m-calpains, and 20S proteasome. AlphaB-crystallin was detected as a proteolytic marker in the lenses. A constant immunoreactivity against all the antibodies employed was observed in the lens epithelium independent of the strain and age of the rats. A weak immunoreactivity against exo- and endopeptidases and an intense reactivity against alphaB-crystallin were observed in the lens fibres of control rats at all ages. The immunoreactivity of these peptidases in lens fibres increased with age in cataract rats, but that of alphaB-crystallin decreased. No reactivity against exo- and endopeptidases was seen in the perinuclear region of lenses of control rats at all ages or in Shumiya cataract rats at 8 and 10 weeks of age, but an intense reactivity against these peptidases was observed in the lens perinuclear region of lenses in cataract rats at 12 and 14 weeks of age. AlphaB-crystallin immunoreactivity was observed with ordered striations in the lens perinuclear region of all control rats whereas the striations in this area of cataract rat lens were disorganized. Membrane-bound alanyl aminopeptidase was detected feebly in the lens epithelium and fibres of both types of rat at all weeks of age. These findings indicate that exo- and endopeptidases, except for membrane-bound alanyl aminopeptidase, are expressed intensively and are age-dependent. Conversely, the amount of alphaB-crystallin decreased with age in lens fibres of cataract rats. Calpains (mu- and m-), 20S proteasome, dipeptidyl peptidases II and III and soluble alanyl aminopeptidase are thought to induce lens opacification kinetically during cataract formation in Shumiya cataract rats through the intracellular turnover of lens proteins.

Age Factors↗