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K Ohgi

Publications and source records attributed to K Ohgi.

At least 91 records · Page 5Linked to original sources
PubMed1986

Roles of lysine1 and lysine7 residues of bovine pancreatic ribonuclease in the enzymatic activity.

In order to investigate the roles of Lys1 and Lys7 of RNase A in the enzymatic activity, four S-peptide derivatives were prepared and their abilities to activate S-protein were measured. They are 1-norleucine-S-peptide, 7-norleucine S-peptide, 1,7-di-norleucine-S-peptide, and tri-N-acetyl S-peptide. From the analyses of the relative activity and kinetic parameters of RNase S' derivatives with UpU, UpU greater than p, and UpUpU greater than p, it was concluded that Lys7 of RNase A is a binding site for 3'-phosphate of UpU greater than p and the modification or substitution of Lys1 affects the binding of trinucleotide substrate.

Animals↗
PubMed1986

Distribution of two urinary ribonuclease-like enzymes in human organs and body fluids.

In order to determine the distribution of two human urinary RNase (RNase Us and RNase UL)-like enzymes in human tissues and body fluids, enzyme immunoassay systems were established using rabbit anti-RNase sera. The sensitivity of the assay systems was of similar order to that of radioimmunoassay systems previously reported. In the enzyme immunoassay, the cross reactivities of anti-RNase UL serum towards RNase Us, bovine kidney RNase K2, bovine RNase A, and bovine seminal RNase Vs were less than 1%. The cross reactivity of anti-RNase Us-serum towards RNase UL was less than 0.5% and cross reactivities were minimal for RNase A, RNase K2, and RNase Vs. The RNase levels in human organs and body fluids were measured by enzyme immunoassay. In milk, semen and saliva, only RNase UL-like enzyme was found. Both RNase Us- and RNase UL-like enzymes were found in kidney, stomach, and pancreas and the RNase Us/RNase UL ratios were 0.49, 1.35, and 0.34, respectively. In lung, liver, spleen, and leukocytes, most of the RNase activity was accounted for by RNase Us-like enzyme. The activity of RNase Us-like enzyme was especially high in lung, spleen, and leukocytes. The crude extracts of several tissues and body fluids were separated by phosphocellulose column chromatography and the contents of the two urinary RNase-like enzymes were determined by enzyme immunoassay. In stomach, kidney, pancreas, and serum, both enzymes were present in multiple forms. In spleen and lung, both the major RNase (RNase Us) and minor RNase (RNase UL) existed in two forms.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Site of alkylation of the major ribonuclease from Aspergillus saitoi with iodoacetate.

A base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C]iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-Ser-Tyr... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-Asp-Tyr-Ala. .... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M. The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.

Alkylation↗
PubMed1985

Peritoneoscopy as an aid in intravenous injection of indocyanine green (ICG).

Peritoneoscopy as an aid in intravenous injection of indocyanine green (ICG) was clinically evaluated. Hepatic parenchyma was stained after intravenous injection of ICG, while interstitial connective tissue, fatty deposition and hepatoma tissue were not. Regenerative hepatic cell mass including dark reddish patchy marking (Shimada's code No. 7) and semispherical areas of regeneration or nodules (Shimada's code No. 8) was well stained and clearly contrasted. There were some cases of chronic active hepatitis, in which liver surface showed spotty staining at sites expected to become regenerative nodules in the future, in contrast to being judged as "no abnormal findings" peritoneoscopically. On the other hand, periportal reddish marking (Shimada's code No. 4) representing piecemeal or bridging hepatic cell necrosis was not stained.

Carcinoma, Hepatocellular↗
PubMed1985

Purification and properties of bovine kidney ribonucleases.

Two RNases (RNases K1 and K2) were purified from bovine kidney by means of column chromatography on phospho-cellulose, Sephadex G-50, CM-cellulose, heparin-Sepharose, nd agarose-APUP. They were named RNase K1 and RNase K2 in order of elution from the heparin-Sepharose column. The purity of RNase K1 thus obtained was about 90% by SDS-disc electrophoresis. RNase K2 was purified to homogeneity by SDS- and pH 4.3 disc electrophoresis. The yield of RNase K2 was 3.4 mg from 11 kg of kidneys. The antigenic properties of the two bovine renal RNases were studied by Ouchterlony's double diffusion analysis. RNase K1 and RNase A were serologically indistinguishable. RNase K2 did not cross-react immunologically with RNase K1 or RNase A. The molecular weights of these RNases determined by gel-filtration on Sephadex G-50 were 13,400 and 14,600 for RNase K1 and RNase K2, respectively. The pH optima for RNase K1 and RNase K2 were 8.5 and 6.5, respectively. Both RNase K1 and RNase K2 were as acid stable as RNase A. RNase K2 was less heat-stable than RNase K1 and RNase A. Although both renal RNases were pyrimidine nucleotide-specific enzymes, RNase K1 and RNase A were more preferential or cytidylic acid than RNase K2. The chemical composition of RNase K2 was determined. RNase K2, like human urinary RNase Us, contained one tryptophan residue. The N-terminal sequences of RNase K2 and RNase Us were determined by Edman degradation. Rnase K2 had a homologous sequence of about 10 amino acid residues with the sequence of RNase Us, a typical non-secretory RNase, within the N-terminal 30 residues.

Amino Acids↗
PubMed1985

The subsite structures of guanine-specific ribonucleases and a guanine-preferential ribonuclease. Cleavage of oligoinosinic acids and poly I.

In order to estimate the size of the active site of guanylic acid specific RNases (RNase T1 from Aspergillus oryzae and RNase St from Streptomyces erythreus) and guanine-preferential RNase (RNase Ms from A. saitoi), the depolymerization reaction of oligoinosinic acid, (Ip)nI greater than p, having various chain lengths was studied. The kinetic parameters for depolymerization of oligoinosinic acids, (pKm, log V and log V/Km) by the three RNases increased with increase of the chain length of the substrates, and became almost constant at n = 2 or more. Thus, the size of the active site of RNase T1, RNase St, and RNase Ms was estimated to be three nucleotides in length.

Aspergillus↗
PubMed1984

Some evidence suggesting the existence of P2 and B3 sites in the active site of bovine pancreatic ribonuclease A.

In order to clarify the subsite structure of ribonuclease A (RNase A), the interactions of pdTp, pAp, dTpdAp, and pdTpdAp with RNase A were investigated by means of kinetic studies and 31P NMR spectroscopy. The pH profile of the 31P NMR spectrum of RNase A-pdTp complex indicated the interaction of the 3'- and 5'-phosphates with RNase A. The signal of 3'-phosphate of pdTp in the presence of RNase A gave a characteristic titration curve indicating the participation of more than 2 ionic groups in the P1 subsite. A similar 31P NMR titration was observed in the case of 5'-phosphate of pAp in the presence of RNase A. The results indicated that pAp interacted with RNase A at the P1, B2, and P2 sites. dTpdAp and pdTpdAp inhibited RNase A action more markedly than dTpdA, indicating the contribution of 3'- and 5'-terminal phosphate groups attached to dTpdA to the affinity of RNase A. The 31P NMR spectra of RNase A-dTpdAp and pdTpdAp complexes excluded the possible interaction of the monoester type phosphate of the inhibitors with the P1 site of RNase A, thus indicating the binding of the 3'-side phosphates with the P2 subsite of RNase A.

Animals↗
PubMed1984

Kinetic studies on the cleavage of oligouridylic acids and poly U by bovine pancreatic ribonuclease A.

Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU greater than p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25 degrees C. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n greater than or equal to 2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1)+(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.

Animals↗
PubMed1983

Radioimmunoassays for two types of human urinary ribonucleases: differential determination of ribonucleases in human serum.

We developed radioimmunoassays for two types of human urinary ribonucleases. The assays are sensitive, reproducible and specific. One human urinary ribonuclease (RNase UL) showed strong immunological identity with human pancreatic ribonuclease, and the other ribonuclease (RNase Us) showed strong immuno cross-reactivity with human liver ribonuclease. There was no immuno cross-reactivity between these two urinary ribonucleases. Serum immunoreactive RNase UL was eluted as four peaks on phosphocellulose chromatography, whereas immunoreactive RNase US was eluted as a single peak. Serum content of immunoreactive RNase UL was 354 +/- 105 ng/ml (mean +/- SD) in normal individuals, and that of immunoreactive RNase Us was 15.9 +/- 5.7 ng/ml (mean +/- SD). No correlation was demonstrated between these two ribonuclease contents in serum.

Adult↗
PubMed1983

Studies on salivary gland ribonucleases. III. Purification and properties of three ribonucleases from bovine parotid gland.

Three alkaline ribonucleases [EC 3.1.4.22] were purified 4,500- to 7,850-fold from bovine parotid gland by repeated CM-Sephadex C-25 chromatography and Sephadex G-50 gel filtration, with a total recovery of about 33%. They were designated as RNase BP1, BP2, and BP3, based on their order of elution from a CM-Sephadex C-25 column. The molecular weights of these enzymes were estimated by gel electrophoresis to be 18,500, 18,000, and 14,000, respectively. These enzymes are very similar to RNase A in that they are inhibited by heparin, show preferential hydrolysis of C5'-O-P linkages adjacent to a cytosine nucleotide rather than a uracil nucleotide, and in their antigenic properties. Spermine was found to stimulate the activities of these enzymes; the degree of stimulation was in the order of RNase BP3 greater than BP2 greater than BP1. The stimulation by spermine is mainly due to the increased cleavage of C5'-O-P linkages adjacent to cytosine nucleotides. From the results of amino acid analysis, glycosidase digestion and amino-terminal sequencing, it was suggested that the differences in molecular weights of RNase BP1, BP2, and BP3 are due to the differences in carbohydrate contents. In addition, it was suggested that RNase BP3 is identical with RNase A.

Animals↗
PubMed1983

Characterization of two forms of base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi.

Two forms of RNases (RNase ML and RNase MM) from Aspergillus saitoi which are base non-specific and adenylic acid preferential were separated from each other by DEAE-cellulose column chromatography. They are indistinguishable with respect to enzymatic properties such as base preferability, pH optimum, kinetic constants measured with 2',3'-cUMP and 2',3'-cCMP as substrates, and effects of ionic strength, physical properties such as heat stability, isoelectric point and circular dichroism spectra, amino acid composition and immunological property. They only differ in carbohydrate content. The apparent molecular weight determined by SDS-disc electrophoresis was 36,000 for RNase ML and 32,000 for RNase MM. Both RNases were reduced and carboxymethylated, and then digested with trypsin, separately. Glycopeptides were isolated from the both digests by gelfiltration and paper chromatography. The amino acid compositions of glycopeptides obtained from RNase ML (ML TS-IIC) and that obtained from RNase MM (MM TS-IIIC) were the same. The amino acid sequences of both glycopeptides determined by Edman degradation and carboxypeptidase digestion were also the same. The results indicated that RNase ML and RNase MM were the same protein having different sizes of carbohydrate chains at one site on the molecule.

Amino Acid Sequence↗
PubMed1982

Ribosome structure. Localization of 7-methylguanosine in the small subunits of Escherichia coli and chloroplast ribosomes by immunoelectron microscopy.

The minor nucleoside 7-methylguanosine occurs in Escherichia coli 16 S ribosomal RNA at a single site. High pressure liquid chromatographic analysis shows that a single residue of 7-methylguanosine is also present in chloroplast 16 S ribosomal RNA, presumably at an analogous position in the sequence. Antibodies to 7-methylguanosine were induced in rabbits and shown to be highly specific for the intact methylated base. These antibodies were reacted with 30 S ribosomal subunits from E. coli and from the chloroplasts of Alaskan peas. These two types of ribosome have been shown to be topographically similar (Trempe, M. R., and Glitz, D. G. (1981) J. Biol. Chem. 256, 11873-11879). Electron microscopy of the subunit-antibody complexes showed similar subunit-IgG monomers and antibody-linked subunit dimers. In greater than 95% of the complexes observed for each type of ribosome, antibody contact was consistent with a single binding site, which places 7-methylguanosine near the junction of the upper one-third and lower two-thirds of the subunit and maximally distant from the platform. The analogous localization in both E. coli and chloroplast 30 S ribosomal subunits lends support to their proposed common evolutionary origin.

Antigen-Antibody Complex↗
PubMed1982

Primary structure of a minor ribonuclease from Aspergillus saitoi.

1. RNase Ms, a base non-specific RNase from Aspergillus saitoi was reduced and carboxymethylated (RCM-RNase Ms). RCM-RNase Ms was hydrolyzed with trypsin, and the trypsin digests were then treated with chymotrypsin. Trypsin digests were also treated with Staphylococcus protease and with chymotrypsin, separately. 2. By the analyses of the amino acid sequences of the peptides formed, the alignment of these peptides in RCM-RNase Ms was determined. 3. From the digest of heat-denatured RNase Ms with Bacillus subtilis protease, two peptides containing disulfide bridges were isolated. From the analysis of these two peptides, the locations of the bridges were determined. 4. The amino acid sequence of RNase Ms was compared with those of RNase T1 (Asp. oryzae, guanine specific), RNase U1 (Ustilago sphaerogena, guanine specific) and RNase U2 (Ustilago sphaerogena, purine specific). There are very similar sequences between these for RNases irrespective of their differences in base specificity. These were, in RNase Ms, tripeptide sequence containing His39 (Tyr-Pro-His), the tetrapeptide containing Glu57 (Glu-Tyr-Pro-Ile), the hexapeptide containing Arg76 (Asp-Arg-Val-Ile-Phe-Asp) and the hexapeptide containing His 91 (Ile-Thr-His-Thr-Gly-Ala). The other sequences common for all four RNases are Tyr67, Phe100, and Cys103 in RNase Ms. Since among these peptides His39, Glu57, His91, and Arg76 in RNase Ms corresponded to His40, Glu58, His92, and Arg77 in RNase T1 which are known to be involved in the active site of RNase T1, the possible role of these amino acids in the active site of RNase Ms is discussed. 5. The sequence similarity of RNase Ms to that of RNase T1 was about 60% and to those of RNase U1 and RNase U2 was about 30%. 6. The details of the experimental evidence used to elucidate the amino acid sequence of RNase Ms are described in the supplemental miniprint.

Amino Acid Sequence↗
PubMed1981

Alkylation of a ribonuclease from Streptomyces erythreus with iodoacetate and iodoacetamide.

1. RNase St was inactivated by iodoacetate. The inactivation was most rapid at pH 5.0-7.0. Competitive inhibitors protected RNase St from inactivation by iodoacetate. The protective effect of 2'-GMP was most effective among nucleotides tested. 2. RNase St was inactivated with the concomitant incorporation of one molar equivalent of carboxymethyl group. The carboxymethyl group incorporated into RNase St was liberated by treatment with 0.2 N NaOH or 1 M hydroxylamine. Thus the incorporation of a carboxymethyl group into a carboxyl group was demonstrated. 3. 14C-labeled CM-RNase St was digested successively with alkaline protease and aminopeptidase M. The 14C-labeled amino acid was identified as the carboxymethyl ester of glutamic acid by means of column chromatography. 4. By digestion of reduced carboxymethylated CM-RNase St with trypsin, a peptide containing a 14C-carboxymethyl group was isolated by Dowex AG-50W colum chromatography. alpha-Chymotryptic digestion of the radioactive tryptic peptide, Glu48-Lys65, produced a tetrapeptide containing a 14C-carboxymethyl group, that is, Tyr59-His60-Glu61-Tyr62. Therefore, it was concluded that Glu61 in RNase St was the site of carboxymethylation. 5. When RNase St was inactivated by iodoacetamide at pH 8.0, about 2 histidine residues were modified. The molar ratio of the products of carboxyamidomethylation were 52.3%, 21.7%, 21.0%, and 4.8% for 3-CAM-His, 1,3-di-CAM-His, 1-CAM-His, and di-CM-Lys, respectively. 6. CD spectra of CM-RNase St and CAM-RNase St were practically the same as that of the native RNase St indicating the maintenance of the native conformation during modification. 7. The binding constants of CM-RNase St and CAM-RNase St with 2'-GMP were about 1/150 and 1/38 of that of the native enzyme, respectively.

Alkylation↗
PubMed1981

Purification and properties of human urine ribonucleases.

1. Two RNases (RNase UL and RNase US) were purified from the urine of human adults by means of column chromatographies on SP-Sephadex C-50, phospho-cellulose and CM-cellulose and gel-filtration on Sephadex G-75 in homogeneous states obtained by SDS-disc electrophoresis. 2. Molecular weights of these RNases determined by gel-filtration were 38,000 and 13,000 for RNase UL and RNase US, respectively. 3. Optimal pH's of urine RNases were 8.0 and 6.75 for RNase UL and RNase US, respectively. 4. Chemical composition of urine RNases was determined. RNase UL contains about 20.7% of neutral sugar and 7.8% of hexosamine. RNase US contains a very small amount of carbohydrate moiety. 5. Base specificity of urine RNases studied with 2',3'-cyclic nucleotides and dinucleoside phosphates as substrates indicated that both RNases were pyrimidine specific and cytosine preferential enzyme, as is bovine pancreatic RNase A. Although base specificity of RNase UL was qualitatively similar to RNase A, that of RNase US was slightly different. That is, RNase US did not hydrolyze UpU and hydrolyzed UpC and 2',3'-cyclic UMP very slowly. 6. Antigenic properties of human urine RNases were studied by Ouchterlony's double diffusion analysis. RNase UL, RNase US, and RNase A were serologically distinguishable.

Adult↗
PubMed1980

Carboxamidomethylation of a ribonuclease from Aspergillus saitoi.

1) The inactivation of a RNase from Aspergillus saitoi (RNase Ms) was studied to obtain information on its active site. 2) Inactivation of RNase Ms by iodoacetamide was greater at an alkaline pH, and was protected more by 2',(3')-AMP than by 2',(3')-GMP. 3) Analysis of the hydrolysis products with 6 N HCl and alkaline treatment of carboxamidomethylated RNase Ms showed that the sites of reaction were one carboxyl group and one histidine residue. 4) Since the incorporation of a carboxamidomethyl group into carboxylic acid was not protected by 2',(3')-AMP, it was concluded that the formation of N1-carboxamidomethylhistidine was responsible for the loss of enzymatic activity of RNase Ms.

Adenosine Monophosphate↗
PubMed1979

Modification of an arginine residue of a base-nonspecific ribonuclease from Aspergillus saitoi.

1. A base-nonspecific ribonuclease from Aspergillus saitoi [RNase Ms, EC 3.1.4.23; molecular weight, 12,500] was modified with phenylglyoxal (PG) and 1,2-cyclohexanedione (CHD) in order to determine whether a single arginine residue was involved in the active site of the enzyme. 2. RNase Ms was inactivated by both PG and CHD with concomitant loss of one arginine residue. A competitive inhibitor of RNase Ms, 2',(3')-AMP, protected the enzyme from inactivation by PG. These findings strongly suggest that one arginine residue is involved in the active site of RNase Ms. 3. Difference CD spectra were measured at pH 5.5 for the binding of 2'-AMP and adenosine to native RNase Ms and the CHD- and PG-modified enzyme derivatives to determine the association constants. The arginine modification brought about a marked decrease in the binding affinity of 2'-AMP for the enzyme, but only a slight decrease for adenosine, suggesting that the arginine residue had interacted with the phosphate groups of the substrate.

Arginine↗