Search PubMed⌕ Search

Biomedical subjects

E Ichishima

Publications and source records attributed to E Ichishima.

At least 55 records · Page 3Linked to original sources
PubMed1993

An active-site peptide containing the second essential carboxyl group of dextransucrase from Leuconostoc mesenteroides by chemical modifications.

The treatment of Leuconostoc mesenteroides B-512F dextransucrase with 10 mM 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and glycine ethyl ester (GEE) inactivated the enzyme almost completely within 24 min where the modification of one carboxyl group/mol of the enzyme by EDC was attained. Though 30 mM diethyl pyrocarbonate (DEP) also inactivated the enzyme, about 35% of the activity remained during a 36-min incubation. When 10 mol of imidazole residues/mol of the enzyme was modified by DEP, 50% of the activity was still retained. The addition of the substrate sucrose greatly retarded the enzyme inactivation by EDC. However, the addition of dextran slightly protected the inactivation of the glucosyl-transferring activity and accelerated the inactivation of the sucrose-cleaving activity. In the case of DEP, the addition of sucrose or dextran gave no influence on the inactivation of the enzyme. Therefore, the carboxyl group seemed to play a more important role in the substrate binding and in the catalytic activity of the dextransucrase than the imidazolium group. Differential labeling of Leuconostoc dextransucrase by EDC was conducted in the presence of a sucrose analog, sucrose monocaprate. The fluorescent probe N-(1-naphthyl)ethylenediamine (EDAN) was used as the nucleophile instead of GEE. A fluorescent labeled peptide was isolated from a trypsin digest of the EDC-EDAN modified enzyme. The amino acid sequence of the isolated peptide was Leu-Gln-Glu-Asp-Asn-Ser-Asn-Val-Val-Val-Glu-Ala.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

1,2-alpha-D-mannosidase from Penicillium citrinum: molecular and enzymic properties of two isoenzymes.

Two isoforms of acidic 1,2-alpha-D-mannosidases have been isolated from culture filtrate of Penicillium citrinum. The pI values of the two forms, designated 1,2-alpha-mannosidase Ia and Ib, were 4.6 and 4.7 respectively. Isoenzymes Ia and Ib exhibited the same molecular mass which was determined to be 53 kDa by SDS/PAGE and 54 kDa by gel-permeation chromatography. Enzymes Ia and Ib hydrolysed yeast mannan and 1,2-alpha-linked mannooligosaccharides, but did not hydrolyse p-nitrophenyl alpha-D-mannoside. The optimal pH for the hydrolysis of Man(alpha 1-->2)Man was 5.0 for both isoenzymes. Similar kinetic parameters were determined for the two forms. Activation energy was a little lower for Ia than Ib. There was little difference between the enzymes with regard to their performance at acidic or alkaline pH. The N-terminal amino acid sequences of the two enzymes were identical. Analysis of C-terminal peptides, which were prepared by tryptic digestion and anhydrotrypsin-agarose chromatography, showed that Ia and Ib had the same amino acid sequences in the C-terminal region. Tryptic digestion revealed a slight difference between the isoenzymes in the pattern of cleaved peptides on SDS/PAGE.

Amino Acid Sequence↗
PubMed1993

The carbohydrate moiety of the acid carboxypeptidase from Aspergillus saitoi.

Acid carboxypeptidase from Aspergillus saitoi is a glycoprotein that contains both N- and O-linked sugar chains. The N-glycanase released high-mannose type oligosaccharides that were separated into eight components on HPLC. One, which had a unique structure of Man11GlcNAc2, was characterized. Mild alkali treatment of the carboxypeptidase, under conditions that effect beta-elimination, yielded D-mannose. Deglycosylation of the carboxypeptidase with endo-beta-N-acetylglucosaminidase and alpha-mannosidase effected the reduction of the molecular mass from 72 kDa to 60 kDa. Partial changes of CD spectra of the native and the deglycosylated enzymes indicate that some conformational changes on the peptide of the enzyme occurred after deglycosylation. Other enzymatic properties, such as catalytic activity, pH, and thermal stability and resistivity to protease digestion, did not appear to change. Tunicamycin halted secretion of the carboxypeptidase extracellularly.

Aspergillus↗
PubMed1993

Molecular properties of aminopeptidase Ey as a zinc-metalloenzyme.

1. Aminopeptidase Ey from hen's egg yolk contains 1.0 g atom of zinc/mol of a subunit having molecular weight of 150 kDa. The inactive, Zn(2+)-free apoenzyme was reactivated by Co2+, Mn2+, Ca2+, Cd2+, Cu2+ and Ni2+ in addition to Zn2+, whereas Mg2+ and Fe2+ were ineffective. 2. The enzymatical properties of reconstituted enzymes, except for Zn(2+)-reconstituted enzyme, differed from native enzyme. The values for the activation energy were calculated by aminopeptidase Ey and Co(2+)-reconstituted enzyme. 3. The isoelectric point of the enzyme was about 2.8 as determined by isoelectric focusing. An asialo form of the enzyme, obtained by treatment with Arthrobacter sialidase, had an isoelectric point of 4.4. 4. The amino terminal sequence of aminopeptidase Ey was determined to be acyl-Xaa-Xaa-Pro-Glu-Ala-Ala-Ser-Leu-Pro-Gly. There was no identity with any known sequences of aminopeptidase.

Amino Acid Sequence↗
PubMed1993

Specificity and molecular properties of penicillolysin, a metalloproteinase from Penicillium citrinum.

The specificity and mode of action of penicillolysin, a metalloproteinase from Penicillium citrinum, were investigated with several bioactive-oligopeptides. The enzyme showed a high affinity toward the Pro-X (X = Gln, Lys, Leu or Arg) bonds of substance P, dynorphin A (1-13), neurotensin and chicken brain pentapeptide, and the R-R bonds in dynorphin A and neurotensin. Preferential cleavage of bonds by the enzyme with hydrophobic amino acid residues at the P1 position were observed on the peptides used. The specificity of penicillolysin differs from that of other metalloproteinases. The M(r) and pI were determined as 18,000 and 9.6, respectively. The first 50 amino acids in the N-terminal region were TKETCSNASRKSALEKALSNTVKLANAAATAARSGSASKFSEYEKTTSSS. CD spectra on the hollo- and apo-enzymes of penicillolysin were studied.

Amino Acid Sequence↗
PubMed1993

A heat-labile serine proteinase from Penicillium citrinum.

A serine proteinase from Penicillium citrinum was purified. The M(r) and isoelectric point were determined as about 26,000 and 9.5, respectively. Activity was retained up to above 40 degrees at pH 7 for 30 min, but the enzyme was completely inactivated at 50 degrees. The first amino acids in the N-terminal region were ANVVQSNVPSWGLARISSKRPGTTSYTYDSTAGEGVVFYGVDTG. The specificity differs from that of other serine proteinases. Kinetic studies on fluorogenic substrates were determined.

Amino Acid Sequence↗
PubMed1993

Substrate specificity of aminopeptidase Ey from hen's (Gallus domesticus) egg yolk.

1. Aminopeptidase Ey, purified from the egg yolk of the hen (Gallus gallus domesticus), was studied for its specificity against oligopeptides at pH 7.5. The enzyme has a broad specificity for amino acid residues at P1 position. 2. The enzyme hydrolyzed N-terminal Xaa-Pro bonds in chicken brain peptide (Leu-Pro-Leu-Arg-PheNH2), substance P fragment 1-4 (Arg-Pro-Lys-Pro) and bradykinin fragment 1-5 (Arg-Pro-Pro-Gly-Phe), but did not hydrolyze substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2) or bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). 3. The enzyme released proline from Pro-Phe-Gly-Lys, while it was unable to release proline from melanocyte, stimulating the hormone release-inhibiting factor (Pro-Leu-GlyNH2) and schistoFMRF-amide (Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-PheNH2).

Amino Acid Sequence↗
PubMed1992

Modification of subsite Lys residue induced a large increase in maltosidase activity of Taka-amylase A.

A cross-linked modification of Taka-amylase A (TAA) by o-phthalaldehyde gave two enzymes, M1- and M2-TAA, which had optimum pH lower than that of native TAA by 0.5 to 1.0 pH units. The modified enzymes showed higher maltosidase activity, and produced glucose even at the initial period of hydrolysis, in contrast to the native TAA. The modification caused more than a 500-fold decrease in the k0 value of native TAA for alpha-amylase activity, but a definite increase in k0 value of 109. 1 min-1 (native TAA) to 460.0 min-1 (M1-TAA) and 147.1 min-1 (M2-TAA) for maltotriose was evidence for improvement of maltosidase activity of modified enzymes.

Amino Acids↗
PubMed1992

Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action.

A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto). Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al. and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA. The cleavage sites in the oxidized B-chain of insulin by the proteinase were CySO3H7-Gly8, Val12-Glu13, Try16-Leu17, and Phe25-Tyr26. The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or alpha 2-macroglubulin.

Amino Acid Sequence↗
PubMed1991

Affinity labeling of a subsite of Taka-amylase A by the fluorescent reagent o-phthalaldehyde.

A cross-linked modification of Lys residue located at the subsite of the enzyme active site of Taka-amylase A was attained by the use of the fluorescent reagent of o-phthalaldehyde (OPA). The fluorescence and uv absorption at 337 nm derived from the isoindole ring, which was produced by cross-linking through the epsilon-amino group of Lys and the thiol group of the Cys residue, provided the evidence for the OPA-mediated inactivation of Taka-amylase A. Kinetic analysis showed that 1 mol of OPA per mole of enzyme was incorporated, which corresponded closely with the value obtained by the uv absorption. Because the OPA inactivation was retarded by the substrate analog of alpha-cyclodextrin, OPA modification was classified as a type of affinity labeling reaction. A remarkable increase in the pI value from 4.0 to 5.6 upon the modification led to clear separation of the modified enzyme from the native Taka-amylase A by a DEAE-Sephacel column and led to the charge isomer pattern on gel electrophoresis performed according to the method of Hedrick and Smith. Moreover, the affinity gel electrophoresis showed that the modified enzyme completely lost the affinity for the substrate soluble starch, which indicated that the subsite modification occurred.

Affinity Labels↗
PubMed1990

Use of water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for the fluorescent determination of uronic acids and carboxylic acids.

Reaction between glucuronic acid and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was monitored by the o-phthalaldehyde (OPA) method, which was developed for the fluorescent assay of compounds containing an amino group. About 1 nmol of glucuronic acid was detected by this method. This EDC-OPA method was effective in detecting not only acidic sugar but also carboxylic acid. Although the sensitivity of the EDC-OPA method was somewhat lower than that of amino acid determination by OPA, a very simple and convenient assay was attained for compounds containing a carboxyl group.

Carbohydrates↗
PubMed1986

Specificities of extracellular and ribosomal serine proteinases from Bacillus natto, a food microorganism.

The specificities of extracellular and ribosomal serine proteinase from Bacillus natto, a food microorganism, were investigated. Both proteins have highly restricted and characteristic specificities. With the extracellular serine proteinase, initial cleavage site was observed at Leu15-Tyr16, secondary site at Ser9-His10 and additional cleavage sites at Gln4-His5 and His5-Leu6 in the oxidized insulin B-chain. Hydrolysis of proangiotensin with the extracellular serine proteinase was observed primarily at Phe8-His9 and secondary at Tyr4-Ile5. The extracellular serine proteinase has a Km of 0.08 mM and kcat of 3 S-1 for angiotensin hydrolysis. With the ribosomal proteinase, initial cleavage site of the oxidized insulin B-chain was observed at Leu15-Tyr16 and additional cleavage site at Phe24-Phe25. Hydrolysis of proangiotensin was observed at Tyr4-Ile5 bond with the ribosomal proteinase.

Angiotensinogen↗
PubMed1984

Multiple forms of protyrosinase from Aspergillus oryzae and their mode of activation at pH 3.0.

This paper describes the isolation of three molecular forms (I-III) of protyrosinase and catalytically active tyrosinase ( monophenol ,dihydroxyphenylalanine: oxygen oxidoreductase, E.C. 1.14.18.1) from fresh mycelia of Aspergillus oryzae BIR 128 by (NH4)2SO4 fractionation, successive chromatographies and disc-gel electrophoresis. Experimental evidence is presented that purified protyrosinase is contaminated with firmly attached proteinases, and that this contamination may account for both the multiple molecular forms of protyrosinase /tyrosinase and the activation of the proenzyme at acidic pH (2.5-3.0). The activation process of protyrosinase seems to be the result of a cleavage of the polypeptide chain by aspartic proteinase from Aspergillus (EC 3.4.23.6) in the purified protyrosinase preparation. Protyrosinase and tyrosinase have different conformation, different stabilities and some different properties.

Aspergillus↗