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M Iwama

Publications and source records attributed to M Iwama.

At least 55 records · Page 3Linked to original sources
PubMed1995

Base non-specific acid ribonuclease from Irpex lacteus, primary structure and phylogenetic relationships in RNase T2 family enzyme.

Two base non-specific acid RNases (RNase Irp1 and RNase Irp2) were purified from a commercial enzyme, "Driselase" (Irpex lacteus) in a homogenous state on SDS-PAGE by several steps of chromatographic separations. RNAse Irp2 was a simple polypeptide with 235 amino acid residues and RNase Irp1 was a glycopeptide with 248 amino acid residues. The amino acid sequences of both RNases were identified by Edman degradation of the peptides derived from these RNAses. RNase Irp1 was composed of the RNase Irp2 and extra C-terminal 13 residues of peptide. The phylogenetic relation of these RNases with the other fungal RNases already known was discussed. The sequence of RNase Irp2 was very highly homologous (67.5%) with that of RNase Le2 from Lentinus edodes.

Amino Acid Sequence↗
PubMed1994

pH profile of kinetic constants of RNase Rh from Rhizopus niveus and its mutant enzymes towards UpU, and possible mechanisms of RNase Rh.

In order to elucidate the mechanism of action of Rhizopus niveus RNase Rh, we investigated the pH profiles of the kinetic parameters of RNase RNAP Rh, a derivative of RNase Rh, and its mutant enzymes, i.e., RNase RNAP Rh H104F, RNase RNAP Rh E105Q, and RNase RNAP Rh D51N. Based on comparisons of their profiles we concluded that protonation of His104 is indispensable for the enzymatic activity and Glu105 accelerates the enzymatic activity, especially at acid pH centered at pH 3.5. Based on these data and the previous data on the chemical modification and enzymatic properties of other mutant enzymes, we propose the following as a possible mechanisms of RNase Rh action. (i) His109 participates in enzymatic action as a general base catalyst which removes the hydrogen of the 2'-OH of the ribose moiety. (ii) His46 participates in the reaction as a general acid catalyst which interacts with the 5'-oxygen atom of the scissile phosphodiester bond and becomes a proton donor to the departing nucleoside or nucleotide. (iii) His104 interacts with phosphate anion and its protonation is favorable for the enzymatic activity. (iv) Since the protonated form of Glu105 is more favorable for activity, we postulate two possible roles for Glu105: (a) its stabilizes the intermediate, and (b) it interacts with the oxygen atom of P = O and polarizes the phosphorus atom.

Dinucleoside Phosphates↗
PubMed1994

Purification and primary structure of a new guanylic acid specific ribonuclease from Pleurotus ostreatus.

A guanine nucleotide-specific RNase (RNase Po1) was isolated from caps of the fruit bodies of Pleurotus ostreatus. RNase Po1 is most active towards RNA at pH 8.0. The effect of heating on the molar ellipticity at 210 nm of RNase Po1 showed that RNase Po1 is more stable than RNase T1. The primary structure of RNase Po1 was determined to be < ETGVRSCNCAGRSFTGTDVTNAIRSARAGGSGNYPHVYNNFEGFSFSCTPTFFEFPVFRGSVYSGGSPG ADRVIYD- QSGRFCACLTHTGAPSTNGFVECRF. It consisted of 101 amino acid residues, with a molecular weight of 10,760. RNase Po1 has relatively higher sequence homology with RNase T1 family RNase. It contains 6 half cystine residues. The locations of four of them are superimposable on those of RNase U1 and RNase U2. The amino acid residues forming the active site of RNase T1 were well conserved in this RNase. Therefore, RNase Po1 is a unique member of the RNase T1 family in respect of the location of one disulfide bridge, and its stability.

Amino Acid Sequence↗
PubMed1994

Effects of vitamin E deficiency on hepatic microsomal cytochrome P450 and phase II enzymes in male and female rats.

The effects of a vitamin E (VE) deficient diet given for 120 days on total cytochrome P450 (P450) contents and UDP-glucuronyl transferase (UDPGT) and sulfotransferase (ST) activities were investigated in rat liver of both sexes. The VE deficient regimen lowered the serum VE levels, increasing the hepatic malondialdehyde concentration regardless of animal sex, which shows an elevation of the tissue peroxi-disability. Hepatic total P450 contents were significantly decreased in male rats given the VE deficient diet, whereas the effects on female rats were less obvious. Concerning phase II enzymes, VE deficiency had no effect on the activities of UDPGT and ST in rats of both sexes. The data suggest that overall capacity of detoxification of xenobiotics may be impaired by VE deficiency to a much greater extent in male than in female rat liver.

Animals↗
PubMed1993

Isolation and characterization of a major allergenic component (gp55) of Aspergillus fumigatus.

IgE class antibodies specific for antigens in a water-soluble extract of Aspergillus fumigatus (strain NHL-5759) were analyzed by immunoblotting with sera from patients with allergic bronchopulmonary aspergillosis. All the sera tested were reactive with a major 50 to 60 kd protein in the extract. This allergen, designated gp55, was purified by gel filtration and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen was found to be present in the water-soluble extract in the form of a complex composed of approximately eight molecules of gp55. The carbohydrate and phosphate content of the purified antigen were 23.1% and 0.46%, respectively. The molar ratio of mannose to galactose residues was 2.76:1, and the protein was glycosylated predominantly with N-linked oligosaccharides. The serologic activity of the gp55 antigen was abolished by treatment with nonspecific protease (Pronase) but not by treatment with sodium metaperiodate or endoglycosidases. Thus the major antigenic site of the glycoprotein is located within its peptide moiety. The antigen itself displayed no chymotryptic or tryptic activity. The amino acid sequence of the 20 N-terminal residues of the antigen (ATPHEPVFFSWDAGAVTSFP) is different from that of any other protein previously reported.

Amino Acid Sequence↗
PubMed1993

Role of Asp51 and Glu105 in the enzymatic activity of a ribonuclease from Rhizopus niveus.

The active site of a base non-specific RNase from Rhizopus niveus (RNase Rh), consists of three histidine residues and one carboxyl group [Ohgi, K. et al. (1992) J. Biochem. 111, 132-138]. In order to identify this acidic amino acid residue, we chose Asp51 and Glu105 as candidates based on a comparison of the primary structures of four fungal RNases and self-incompatibility factors of Nicotiana alata which belong to the RNase T2 family. We substituted these amino acid residues with other amino acids by site-directed mutagenesis, and determined the enzymatic properties of the mutated enzymes. The enzymatic activities of E105Q, E105D, and E105A mutant enzymes were decreased markedly, but those of D51N, D51E, and D51A were decreased only slightly when RNA was used as a substrate. Therefore we concluded that Glu105 is related to the catalytic function. Kinetic constants for the enzymatic activity of E105Q and E105D toward ApU suggest that the proper size and negative charge of side chain groups are important for the catalysis of RNase Rh. However, the enzymatic activity of D51N toward ApU, but not toward UpU, decreased markedly. Therefore, we suggest that Asp51 is one of the amino acid residues forming the base recognition site. The substitution of Asp51 by Asn causes the enzyme to be more guanine nucleotide-preferential.

Amino Acid Sequence↗
PubMed1993

Characterization of poly C preferential ribonuclease from chicken liver.

Poly C preferential RNase previously reported by Levy and Karpetsky [J. Biol. Chem. 255, 2153-2159 (1980)] and Miura et al. [Chem. Pharm. Bull. 32, 4053-4060 (1984)] was extensively purified from chicken liver to homogeneity as determined by SDS-PAGE (RNase CL2). The poly C preference over poly U was slightly higher than that of bovine pancreatic RNase A. However, the kinetic constants for 8 dinucleoside phosphates, CpY and UpY (Y = one of A, G, U, and C) as substrates showed that RNase CL2 was preferential for cytidylic acid, but less so than RNase A, and the influence of Y base on the rate of hydrolysis of CpY or UpY was less marked than in the case of RNase A. The primary structure of RNase CL2 was determined. The molecular weight calculated from the sequence was 13,420. Comparison of the amino acid sequence of RNase CL2 with those of other vertebrate RNases showed that RNase CL2 is a member of the RNase A family, but is not a non-secretory RNase. It retains 3 disulfide bridges of RNase A, but Cys65-Cys72 of RNase A is missing. As for the active site, the amino acid residues of the P0 and P1 sites of RNase A are completely conserved. Among the B1 site components, Thr45 (RNase A numbering) is conserved, but Phe120 and Ser123 are substituted by Leu and Thr, respectively. Among the B2 site residues, Gln69, Asn71, and Glu111 are substituted by other amino acids.

Amino Acid Sequence↗
PubMed1992

Evidence that three histidine residues of a base non-specific and adenylic acid preferential ribonuclease from Rhizopus niveus are involved in the catalytic function.

In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or His109 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the 1H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pKa values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pKa value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the 1NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.

Adenosine↗
PubMed1992

Primary structure of a base non-specific and adenylic acid preferential ribonuclease from the fruit bodies of Lentinus edodes.

The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase Le2) from the fruit bodies of Lentinus edodes was analyzed. The sequence was mostly determined by analysis of the peptides generated by V8 protease digestion and BrCN cleavage (including alpha-chymotryptic, and V8 protease digest of BrCN fragments). It consists of 239 amino acid residues. The molecular weight is 25831. The location of 10 half cystine residues were almost superimposable on those of known fungal RNases of the RNase T2 family. The sequence homologies between RNase Le2 and four known fungal RNases of the RNase T2 family, RNase T2, RNase M, RNase Trv, and RNase Rh, are 102, 103, 109, and 74, respectively. The homologous sequences are concentrated around the three histidines, which are supposed to form the active site of RNase T2 family RNases.

Agaricales↗
PubMed1992

Long-term exposure of Wistar rats to high dietary sodium chloride level. I. Changes in aortic connective tissue components.

The effect of chronic salt loading on aortic components of connective tissue was evaluated in normotensive Wistar rats maintained on a high sodium chloride (NaCl; 8%) regime for a period of 30, 90, 180 and 360 days. The blood pressure remained unchanged until 90 days and was significantly elevated thereafter. The high NaCl diet for 180 days influenced neither the serum levels of both total cholesterol and triglyceride nor the aortic contents of collagen, elastin and glycosaminoglycans (GAG) derived from the proteoglycans. The only significant change associated with a 360-day massive NaCl ingestion was the increased aortic concentration of total GAG due to an increase in chondroitin sulfate, dermatan sulfate and heparan sulfate rather than hyaluronic acid. This study suggests that an increase in the contents of sulfated GAG occurred in the late stage of hypertensive aorta of NaCl loaded rats might be responsible, at least in part, for the pathogenesis of atherosclerotic cardiovascular disease.

Animals↗
PubMed1992

Long-term exposure of Wistar rats to high dietary sodium chloride level. II. Changes in hepatic drug-metabolizing and glutathione-related enzyme systems.

The effect of excess sodium chloride (NaCl) intake on hepatic drug-metabolizing and glutathione-related enzyme systems in rats was investigated for a period of 360 days. There was no change in any parameters studied until day 180 of the experiment. However, feeding of a 8% NaCl diet for 360 days produced a decrease in cytochrome P-450 content and an increase in glutathione peroxidase activity, and resulted in a lowered capacity of hepatic tissue to activate benzo[a]pyrene to mutagens detectable in the Ames bacterial mutagenesis assay. These observations suggest that although there may be little possibility of the involvement of high NaCl diet in the carcinogenic effects of benzo[a]pyrene, long-term excess NaCl intake could reduce the potential for detoxification of many xenobiotic compounds leading to various pathological process.

Animals↗
PubMed1991

Comparative base specificity, stability, and lectin activity of two lectins from eggs of Rana catesbeiana and R. japonica and liver ribonuclease from R. catesbeiana.

Two lectins with RNase activity obtained from eggs of Rana catesbeiana and R. japonica and RNase obtained from R. catesbeiana liver show 65-83% protein homology. The base specificity of these frog proteins was studied with 8 dinucleoside phosphates as substrates and 8 nucleotides as inhibitors. The base specificities of the B1 and B2 sites of these proteins are U greater than C and G greater than U greater than A, C, respectively. The three frog proteins are more resistant than RNase A to heat treatment, guanidine-HCl and pH-induced denaturation; i.e., they retain their native conformation up to at least 70 degrees C at pH 7.5. Differences in stability and base specificity among RNase A and the three frog proteins are discussed in relation to the primary structures. Although the two lectins agglutinate tumor cells (e.g., Ehrlich, S-180 and AH109A ascites carcinoma cells), the liver RNase has no such activity. Agglutination of AH109A cells by the two lectins is inhibited by nucleotides. Our results indicate that the agglutination sites are not identical with, but are related to, the active sites of the three frog proteins.

Amino Acid Sequence↗
PubMed1991

Purification and properties of a novel surface-active agent- and alkaline-resistant protease from Bacillus sp. Y.

In the course of a search for an alkaline stable protease for industrial use, an alkaline protease (protease BYA) was isolated from an alkalophilic Bacillus sp. Y, and its properties were characterized. Its optimum pH was pH 10.0-12.5, when casein was used as a substrate. In addition to the stability of protease BYA at pH 6.5-13.0, it was also very stable towards various surface-active agents, such as sodium dodecyl sulfate and sodium linear alkylbenzene sulfonate. Protease BYA was most active at 70 degrees C. The isoelectric point (pI) of protease BYA was about 10.1. Protease BYA was characterized as a serine protease because of its sensitivity to phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The protease seems to be related to proteases of the subtilisin family, such as subtilisin BPN', subtilisin Carlsberg, and No. 221 protease.

Amino Acid Sequence↗
PubMed1990

Net increase of platelet membrane tyrosine specific-protein kinase activity by phorbol myristate acetate.

Tyrosine protein kinase (TPK) activity in rabbit platelets after stimulation by phorbol myristate acetate (PMA) or thrombin was directly estimated by 32P incorporation from (gamma-32P)ATP (adenosine triphosphate) into synthetic peptide angiotensin II. By PMA-treatment a net increase of TPK activity was obtained, while thrombin acted on the TPK quickly but stimulation was limited within the range attained by the control after lengthy incubation. The responsive TPK to these stimulators was localized mainly in membrane but much less in cytosol. The specific activity of the particulate TPK was low in the sonicate of control ice cold platelets but increased about 6-fold when the platelets were incubated at 37 degrees C. On a brief contact of platelets with PMA at 37 degrees C the TPK was fully activated and reached a maximum value about 130% of the control. Determination of phosphotyrosine phosphatase in the stimulated platelet sonicate revealed that its participation in the above described increase of 32P-incorporation was meagre. The quick response suggested a possible role of TPK in the signal transduction through the platelet cell membrane.

Adenosine Triphosphate↗
PubMed1990

Effects of capsaicin and ethanol on hepatic drug-metabolizing enzymes in rat.

Capsaicin (CAP; 50 mg/kg body wt., p.o.) and/or ethanol (EtOH; 10% (v/v) in the drinking water) were given for a period of 30 days to male rats, and changes in hepatic drug-metabolizing enzymes were investigated. The EtOH alone tended to increase the microsomal parameters such as cytochrome P-450 content and enzyme activities of aniline hydroxylase, aminopyrine demethylase and UDP-glucuronyl transferase, while it did not affect the cytosolic enzyme activities of sulfotransferase and glutathione S-transferase. The administration of CAP without EtOH tended to decrease all of the parameters studied. In contrast, the ingestion of CAP with EtOH tended to increase even the elevated microsomal enzyme activities by EtOH alone. These data suggest that chronic alcohol ingestion could modify the potential for detoxification of xenobiotics in persons who regularly consume a large amount of hot peppers.

Animals↗