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Biomedical subjects

M Irie

Publications and source records attributed to M Irie.

At least 271 records · Page 15Linked to original sources
PubMed1982

Modification of a glucoamylase from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.

1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4)-glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with water-soluble carbodiimides was studied. 2. Gluc M1 was inactivated most effectively by 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide (CMC) at pH 4.5. 3. Inactivation of Gluc M1 with [14C]CMC proceeded with the incorporation of about 12 CMC moieties. From the results of amino acid analysis, titration of SH group with Ellman's reagent and hydroxylamine treatment at pH 7.0, it was concluded that the crucial sites of modification were carboxyl groups of Gluc M1. 4. The CD spectrum of CMC-modified Gluc M1 (residual activity, ca. 9.8%) suggested that the gross conformation of the native enzyme was retained. 5. In the presence of maltose, when Gluc M1 was incubated with [14C]CMC, ca. 10 CMC moieties were incorporated with a simultaneous decrease in enzymatic activity (30%). The Gluc M1 modified in the presence of maltose was remodified with CMC after elimination of maltose. The CMC-modified Gluc M1 was inactivated completely with the incorporation of ca. 4 CMC moieties. 6. The logarithm of the half-life of the inactivation of Gluc M1 by CMC was a linear function of log [CMC] indicating that one carboxyl group among the modified ones was crucial for inactivation of Gluc M1. 7. The protection by maltose of Gluc M1 from inactivation and the increase in K1 values for maltose of CMC-modified Gluc M1's suggested that a crucial carboxyl group(s) was located near or on subsites 2 and 3.

Aspergillus↗
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↗
PubMed1982

N-bromosuccinimide oxidation of a glucoamylase from Aspergillus saitoi.

1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4) glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with NBS was studied. 2. The tryptophan residues in Glu M1 were oxidized at various NBS/Gluc M1 ratios. The enzymatic activity decreased to about 80% of that of the native Gluc M1 with the oxidation of the first 2 tryptophan residues. The oxidation of these 2 tryptophan residues occurred within 0.2-0.5 s. On further oxidation of ca. 4-5 more tryptophan residues of Glu M1, the enzymatic activity of Gluc M1 decreased to almost zero (NBS/Gluc M1 = 20). Thus, the most essential tryptophan residue(s) is amongst these 4-5 tryptophan residues. 3. 7.5 tryptophan residues were found to be eventually oxidized with increasing concentrations of NBS up to NBS/Gluc M1 = 50. This value is comparable to the number of tryptophan residues which are located on the surface of the enzyme as judged from the solvent perturbation difference spectrum with ethylene glycol as perturbant. 4. In the presence of 10% soluble starch, about 5 tryptophan residues in Gluc M1 were oxidized at an NBS/Gluc M1 ratio of 20. The remaining activity of Glu M1 at this stage of oxidation was about 76%. On further oxidation, after removal of soluble starch, the enzymatic activity decreased to zero with the concomitant oxidation of 2 tryptophan residues. The results indicated that the essential tryptophan residue(s) is amongst these 2 tryptophans. 5. The UV difference spectrum induced by addition of maltose and maltitol to Gluc M1 showed 4 troughs at 281, 289, 297, and 303 nm. The latter 3 troughs were probably due to tryptophan residues of Gluc M1 and decreased with NBS oxidation.

Aspergillus↗
PubMed1982

Isolation of two inactive fragments of a Rhizopus sp. glucoamylase: relationship among three forms of the enzyme and the isolated fragments.

Two inactive fragments of glucoamylase [EC 3.2.1.3] from a Rhizopus sp. were isolated from the glucoamylase fraction obtained by CM-Sephadex chromatography in the previous purification of the glucoamylase (T. Takahashi et al. (1978) J. Biochem. 84, 1183-1194); the fraction contained at most 2.5% of the fragments, besides a mixture (97.5%) of three glucoamylases, designated as Gluc1 (M.W. 74,000), Gluc2 (M.W. 58,600), and Gluc3 (M.W. 61,400) in order of content. The two isolated fragments, named fragments H and L in order of size, were found to be homogeneous in polyacrylamide gel electrophoresis, isoelectric focusing, and ultracentrifugation analysis. The molecular weights of fragments H and L were 16,700 and 14,400, respectively. Fragment H had alanine as its N-terminal residue although fragment L was heterogeneous at the N-terminal amino acid. The N-terminal amino acids of Gluc1, Gluc2, and Gluc3 were found to be alanine, glutamic acid, and lysine, respectively; the three enzymes had the same C-terminal amino acid sequence of -Ser-Ala . OH. Immunodiffusion demonstrated that both fragments cross-reacted with Gluc1 and Gluc3 but not with Gluc2, although the three enzymes had common antigenicity. These results, together with the amino acid and sugar compositions of the fragments, indicate that the two fragments were derived from the N-terminal glycopeptide moiety of Gluc1 by the action of a proteolytic enzyme(s) with concomitant formation of Gluc2; Gluc3 also seems to be produced by proteolytic modification of Gluc1 with loss of its N-terminal peptide moiety.

Amino Acids↗
PubMed1981

Purification and characterization of a glucoamylase from Aspergillus saitoi.

1. A major glucoamylase [EC 3.2.1.3] of Aspergillus saitoi was purified by ultrafiltration followed by successive chromatography on DEAE-Sephadex, Ultrogel AcA 44 and SP-Sephadex. The purification achieved was 23-fold from crude extract with a yield of 21%. The purified enzyme, named Gluc M1, was proved homogeneous as judged by polyacrylamide gel electrophoresis, isoelectric focusing, ultracentrifugation, and also from the absence of the glycosidase activities detected in crude extract. 2. Gluc M1 was a glycoprotein containing 18% neutral sugar and 0.77% glucosamine, and its molecular weight was estimated to be about 90,000 by SDS-polyacrylamide gel electrophoresis and amino acid composition. The N-terminal amino acid was identified as alanine. 3. The pH optimum of Gluc M1 was 4.5 with soluble starch as a substrate. The enzyme was stable between pH 2.5 and 7.5 and retained full activity at temperatures up to 50 degrees C. The enzyme activity was inhibited by Hg2+ and, to a lesser extent, by Pb2+ and Mn2+. 4. The Km value for malto-oligomer markedly decreased with increasing chain length of substrate in glucose unit (n) and the Vmax value increased with n, thus resulting in the increase in the Vmax/Km value with n. The kinetic parameters for other substrates such as soluble starch, glycogen and isomaltose as well as the K1 values for some saccharides were also determined.

Amino Acids↗
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↗