Biomedical subjects

E Ichishima

Publications and source records attributed to E Ichishima.

At least 19 recordsLinked to original sources

PubMed1998

Action of serine carboxypeptidase from paecilomyces carneus on oligopeptides containing carboxy-terminally amidated peptides

Paecilomyces carneus carboxypeptidase sequentially liberated amino acids from the carboxy-terminus of neurotensin, angiotensin I, bradykinin, and delta sleep-inducing peptide, indicating that the sequential hydrolysis of peptides was limited by the occurrence of intermediates with the structure of -Gly-X (X = L-amino acid), -Pro-X, -X-Gly, and -X-Pro. The enzyme had carboxyamidase and/or amidase activities for the carboxy-terminally amidated peptides. The enzyme essentially acted as a carboxyamidase for the long carboxy-terminally amidated peptides; an amidase became dominant for the substrates in the presence of bulky amino acids such as Arg, Met, Leu, and Phe in the penultimate (P1) and P2 positions, corresponding with the S1 and S2 sites of the enzyme, and the P3 position of carboxy-terminally amidated peptides played a significant role in the action as a carboxyamidase or a amidase.

Journal Article↗

PubMed1997

Five crucial carboxyl residues of 1,2-alpha-mannosidase from Aspergillus saitoi (A. phoenicis), a food microorganism, are identified by site-directed mutagenesis.

An acidic 1,2-alpha-mannosidase from fungus, Aspergillus saitoi (now designated Aspergillus phoenicis), is highly specific for 1,2-alpha-mannosidic linkage in the high-mannose type oligosaccharide at pH 5.0. The predicted amino acid sequence of several peptide regions, including aspartic acid and glutamic acid, bears striking similarities to 1,2-alpha-mannosidases from fungi, yeast and mouse. Active site determination of the enzyme expressed in Saccharomyces cerevisiae cells was performed by site-directed mutagenesis. Substitutions of Asp-269 to Glu and of the Glu-residues, Glu-273, Glu-411, Glu-414 and Glu-474, to Asp altered the drastic decrease of specific activities with Man alpha 1-2Man-OMe and Man9-GlcNAc2-PA as substrates and shifted the optimal pH of the mutant enzymes. From the present results, Asp-269 is probably in the ionized COO- form, whereas one of four glutamic acid residues, probably Glu-411, is the un-ionized COOH form according to the analogy of a plausible mechanism for lysozyme catalysis. It is assumed that three glutamic acid residues, Glu-273, Glu-414, and Glu-474, are probably binding sites of substrate.

Aspergillus↗

PubMed1997

Engineering of porcine pepsin. Alteration of S1 substrate specificity of pepsin to those of fungal aspartic proteinases by site-directed mutagenesis.

The S1 substrate specificity of porcine pepsin has been altered to resemble that of fungal aspartic proteinase with preference for a basic amino acid residue in P1 by site directed mutagenesis. On the basis of primary and tertiary structures of aspartic proteinases, the active site-flap mutants of porcine pepsin were constructed, which involved the replacement of Thr-77 by Asp (T77D), the insertion of Ser between Gly-78 and Ser-79 (G78(S)S79), and the double mutation (T77D/G78(S)S79). The specificities of the mutants were determined using p-nitrophenylalanine-based substrates containing a Phe or Lys residue at the P1 position. The double mutant cleaved the Lys-Phe(4-NO2) bonds, while wild-type enzyme digested other bonds. In addition, the pH dependence of hydrolysis of Lys-containing substrates by the double mutant indicates that the interactions between Asp-77 of the mutant and P1 Lys contribute to the transition state stabilization. The double mutant was also able to activate bovine trypsinogen to trypsin by the selective cleavage of the Lys6-Ile7 bond of trypsinogen. Results of this study suggest that the structure of the active site flap contributes to the S1 substrate specificity for basic amino acid residues in aspartic proteinases.

Amino Acid Sequence↗

PubMed1997

Miltpain, new cysteine proteinase from the milt of chum salmon, Oncorhynchus keta.

A new cysteine proteinase, salmon miltpain, was isolated and purified from the milt of chum salmon (Oncorhynchus keta). Native molecular mass was estimated as 67,000 by gel filtration column chromatography (Shodex WS2003) and 22,300 by SDS-polyacrylamide gel electrophoresis. Isoelectoric point was determined to be 3.9 by isoelectric focusing. The first 15 amino acid residues in the N-terminal region were LPSFLY-AEMVGYNIL. The cysteine proteinase, which had a pH optimum of 6.0 for Z-Arg-Arg-MCA hydrolysis, required a thiol-reducing reagent for activation and was inhibited by E-64, iodacetamide, CA-074 Me, TLCK, TPCK and ZPCK. The cysteine proteinase exhibited unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg at the subsites of P2-P1 and had a K(m) of 16.3 microM and kcat of 20.3 s-1 with Z-Arg-Arg-MCA as substrate and a K(m) of 52.9 microM and kcat of 1.79 s-1 with Z-Phe-Arg-MCA. This proteinase was found to considerably hydrolyze basic proteins such as histone, salmine and clupaine but not milk casein.

Amino Acid Sequence↗

PubMed1996

Production, purification, and properties of serine carboxypeptidase from Paecilomyces carneus.

Seventeen strains of the genus Paecilomyces were examined for their ability to produce serine carboxypeptidase. Paecilomyces carneus IFO 7012 exhibited the highest potency for serine carboxypeptidase production. A maximum yield of serine carboxypeptidase was obtained by koji culture of the strain at 22 degrees C for 7 days. The serine carboxypeptidase was purified to homogeneity from an extract of the koji culture. The molecular weight of the enzyme was estimated to be 47,000 by HPLC. The isoelectric point of the enzyme was determined to be 4.0, and the optimum pH was 4.0 toward benzyloxycarbonyl-L-glutamyl-L-tyrosine (Z-Glu-Tyr) and benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala), respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and p-chloromercurybenzoate. Relative hydrolysis rates of N-acylpeptides and kinetic studies indicated that the enzyme preferred substrates having bulky amino acids in the penultimate position from their carboxy-termini.

Amino Acid Sequence↗

PubMed1996

Characterization of the S1 subsite specificity of aspergillopepsin I by site-directed mutagenesis.

The structural determinants of S1 substrate specificity of aspergillopepsin I (API; EC 3.4.23.18), an aspartic proteinase from Aspergillus saitoi, were investigated by site-directed mutagenesis. Aspartic proteinases generally favor hydrophobic amino acids at P1 and P1'. However, API accommodates a Lys residue at P1, which leads to activation of trypsinogen. On the basis of amino acid sequence alignments of aspartic proteinases, Asp-76 and Ser-78 of API are conserved only in fungal enzymes with the ability to activate trypsinogen, and are located in the active-site flap. Site-directed mutants (D76N, D76E, D76S, D76T, S78A, and delta S78) were constructed, overexpressed in Escherichia coli cells and purified for comparative studies using natural and synthetic substrates. Substitution of Asp-76 to Ser or Thr and deletion of Ser-78, corresponding to the mammalian aspartic proteinases, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at P1. In contrast, substrates with a hydrophobic residue at P1 were effectively hydrolyzed by each mutant enzyme. These results demonstrate that Asp-76 and Ser-78 residues on the active site flap play important roles in the recognition of a basic amino acid residue at the P1 position.

Amino Acid Sequence↗

PubMed1996

HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae.

We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S. cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S. Kasahara, H. Yamada, T. Mio, Y. Shiratori, C. Miyamoto, T. Yabe, T. Nakajima, E. Ichishima, and Y. Furuichi, J. Bacteriol. 176:1488-1499, 1994). HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain. Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells. This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells. Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele. Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface. The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells. These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S. cerevisiae.

Bacterial Outer Membrane Proteins↗

PubMed1995

Molecular cloning and nucleotide sequence of the 1,2-alpha-D-mannosidase gene, msdS, from Aspergillus saitoi and expression of the gene in yeast cells.

A full-length cDNA encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) from Aspergillus saitoi was cloned. Analysis of the 1718 bp nucleotide sequence of the cDNA revealed a single open reading frame with 1539 nucleotides of 1,2-alpha-D-mannosidase gene, msdS. The predicted amino-acid sequence of 1,2-alpha-D-mannosidase consists of 513 residues with a molecular mass of 55,767 and is 70%, 26% and 35% identity with those of Penicillium citrinum 1,2-alpha-D-mannosidase, yeast alpha-mannosidase, and mouse alpha-mannosidase. The cDNA of the msdS gene has been cloned and expressed in yeast cells. To identify the activity of expression product methyl-2-O-alpha-mannopyranosyl-alpha-mannopyranoside (Man alpha 1-->2Man-OMe) was used as a substrate at pH 5.0.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and nucleotide sequence of the genomic DNA for 1,2-alpha-D-mannosidase gene, msdC from Penicillium citrinum.

A gene encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) was cloned from Penicillium citrinum genomic DNA using the polymerase chain reaction (PCR). The coding region of the gene, msdC, occupied 1737 bp and was separated into four exons by three introns. The predicted protein consisted of 511 amino acid residues with M(r) 56,569. Penicillium enzyme had a hydrophobic signal peptide at the N-terminal region as did mammalian membrane-bound alpha-mannosidases, but in this case a proteolytic cleavage occurred at Lys-35-Ser-36 to remove the signal sequence during cell growth. Parts of amino acid sequences were similar to those of mammalian Golgi alpha-mannosidase IA and IB, but the sequence around the aspartic acid residue which interacted with 1-deoxymannojirimycin (Yoshida et al. (1994) Biochem. J. 303, 97-103) was unique in Penicillium enzyme.

Amino Acid Sequence↗

PubMed1995

Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis.

Carboxypeptidase from Aspergillus saitoi removes acidic, neutral and basic amino acids as well as proline from the C-terminal position at pH 2-5. cpdS, a cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. Analysis of the 1816-nucleotide sequence revealed a single open reading frame coding for 523 amino acids. When A. saitoi carboxypeptidase cDNA was expressed in yeast cells, carboxypeptidase activity was detected in the cell extract and was immunostained with a 72 kDa protein with polyclonal anti-(A. saitoi carboxypeptidase) serum. The recombinant enzyme treated with glycopeptidase F migrated with an apparent molecular mass of 60 kDa on SDS/PAGE, which was the same as that of the de-N-glycosylated carboxypeptidase from A. saitoi. Site-directed mutagenesis of the cpdS indicated that Ser-153, Asp-357 and His-436 residues were essential for the enzymic catalysis. It can be concluded that A. saitoi carboxypeptidase has a catalytic triad comprising Asp-His-Ser and is a member of serine carboxypeptidase family (EC 3.4.16.1).

Amino Acid Sequence↗

PubMed1995

The structure of subtilisin ALP I from alkalophilic Bacillus sp. NKS-21.

The gene for an alkaline serine protease from alkalophilic Bacillus sp. NKS-21 (subtilisin ALP I) was cloned, and its nucleotide sequence was determined. The gene (aprQ) contained an open reading frame of 1125 bp, encoding a primary product of 374 amino acids. The mature protease, composed of 272 amino acids, was preceded by a putative signal sequence of 37 amino acids and a pro-sequence of 65 amino acids. The mature protease conserved the catalytic triad, Asp, His, and Ser, as subtilisin BPN' or other subtilisins, and the subtilisin ALP I might belong to the subtilisin super family. The primary structure of subtilisin ALP I was compared and discussed with those of 13 subtilisins, 5 subtilisins from alkalophilic Bacillus, and 8 from neutrophiles. Low homology was shown between subtilisin ALP I and subtilisins from alkalophiles or subtilisins from neutrophiles. Forty-five amino acid residues of the mature protein of subtilisin ALP I were entirely independent of other subtilisins. According to the homology of ALP I with other subtilisins, subtilisin ALP I might be in the middle point between alkaline subtilisins and neutral ones.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and nucleotide sequence of the 90k serine protease gene, hspK, from Bacillus subtilis (natto) No. 16.

We previously reported purification and characterization of a 90k serine protease with pI 3.9 from Bacillus subtilis (natto) No. 16 [Kato et al. 1992 Biosci Biotechnol Biochem 56:1166]. The enzyme showed different and unique substrate specificity towards the oxidized B-chain of insulin from those of well-known bacterial serine proteases from Bacillus subtilisins. The structural gene, hspK, for the 90k serine protease was cloned and sequenced. The cloned DNA fragment contained a single open reading frame of 4302 bp coding a protein of 1433 amino acid residues. The deduced amino acid sequence of the 90k-protease indicated the presence of a typical signal sequence of the first 30 amino acids region and that there was a pro-sequence of 164 amino acid residues after the signal sequence. The mature region of the 90k-protease started from position 195 of amino acid residue, and the following peptide consisted of 1239 amino acid residues with a molecular weight of 133k. It might be a precursor protein of the 90k-protease, and the C-terminal region of 43k might be degraded to a mature protein from the precursor protein. The catalytic triad was thought to consist of Asp33, His81, and Ser259 from comparison of the amino acid sequence of the 90k-protease with those of the other bacterial serine proteases. The high-molecular-weight serine protease, the 90k-protease, may be an ancient form of bacterial serine proteases.

Amino Acid Sequence↗

PubMed1995

A new alkaline serine protease from alkalophilic Bacillus sp.: cloning, sequencing, and characterization of an intracellular protease.

To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence. The molecular weight was 34,624. The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out. The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants. The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.

Amino Acid Sequence↗

PubMed1995

Molecular and enzymatic properties of an aspartic proteinase from Rhizopus hangchow.

An aspartic proteinase, rhizopuspepsin (EC 3.4.23.21), from Rhizopus hangchow was purified. The M(r) and isoelectric point were determined as ca 37,000 and 4.5, respectively. The first 19 amino acids in the N-terminal region were SGSGVVPMTDYEYDIEYYG. The contents of the alpha-helix, beta-structure and random coil were calculated to be ca 7.5, 88.9 and 2.7%, respectively. The enzyme can activate trypsinogen at pH 3.0. The activity was completely inactivated by pepstatin A. The specificity and mode of action of the enzyme were investigated with oxidized insulin B-chain at pH 3. The enzyme hydrolysed primarily two peptide bonds, the Leu15-Tyr16 bond and the Tyr16-Leu17 bond, while additional cleavage of the bonds, Ala14-Leu15 and Phe24-Phe25 was also noted.

Amino Acid Sequence↗

PubMed1995

Subtilisin Sendai from alkalophilic Bacillus sp.: molecular and enzymatic properties of the enzyme and molecular cloning and characterization of the gene, aprS.

We purified a new extracellular serine proteinase (designated subtilisin Sendai) from the culture broth of alkalophilic Bacillus sp. G-825-6, and its properties were characterized. Its optimum pH was at 10.0, when succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-4-methylcoumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) was used as a substrate. The substrate specificity of subtilisin Sendai was determined with oxidized insulin B-chain and fluorogenic peptidyl-MCA substrates. The isoelectric point of subtilisin Sendai was over 11.0. The molecular mass of the enzyme was estimated as 28,000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The circular dichroism spectrum of the enzyme was measured, and we discuss the relationship between the secondary structure of the enzyme and alkaline stability at pH 12 in comparison with that of subtilisin NAT. The structural gene (aprS) was cloned and sequenced. The deduced amino acid sequence for the mature protein (269 amino acids) was preceded by a putative signal sequence of 27 residues and a putative pro-sequence of 86 amino acids. The homology of the primary structure for 13 subtilisins was compared. The catalytic triad (Asp32, His64, and Ser221 with the numbering of subtilisin BPN') and the amino acid sequences near these amino acid residues were well conserved. As a special feature, it was observed that there was an extensive number of negatively charged amino acids in the pro-region of subtilisin Sendai and alkaline subtilisins. This was different from those of subtilisin from neutrophiles.

Amino Acid Sequence↗

PubMed1995

Aggregated form of dextransucrases from Leuconostoc mesenteroides NRRL B-512F and its constitutive mutant.

Purified dextransucrases [EC 2.4.1.5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively. DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS-PAGE were all 170 kDa. DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V0) on Sepharose 6B, while the DSM-G was eluted at 1.2 x V0 volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.

Amino Acid Sequence↗

PubMed1994

Chemical modification of Penicillium 1,2-alpha-D-mannosidase by water-soluble carbodi-imide: identification of a catalytically important aspartic acid residue.

1,2-alpha-D-Mannosidase from Penicillium citrinum was inactivated by chemical modification with 1-ethyl-3-(3-dimethylamino-propyl)carbodi-imide (EDC). Most of the activity was lost after modification in the absence of a nucleophile, glycine ethyl ester. 1-Deoxymannojirimycin (dMM), a competitive inhibitor of the enzyme, showed partial protection against the inactivation. After the modification by EDC without the presence of a nucleophile, proteolytic digests of the enzyme were analysed by reversed-phase h.p.l.c. and a unique peptide was shown to decrease when dMM was present during the modification. The peptide was absent from the digests of unmodified enzyme. The amino acid sequence of the peptide (A; Ile-Gly-Pro) was identical in part with that of the adjacent peptide (B; Ile-Gly-Pro-Asp-Ser-Trp-Gly-Trp-Asp-Pro-Lys). When cholecystokinin tetrapeptide (Trp-Met-Asp-Phe-NH2) was modified by EDC alone, the modified peptide could be separated from unmodified peptide by reversed-phase h.p.i.c., and Edman degradation was stopped before the modified aspartic acid residue. This suggested that, in the enzyme, peptide A was derived from peptide B by the modification. Consequently, Asp-4 in peptide B was assumed to be masked by dMM during the modification, and to be involved in the interaction of the enzyme with its substrate.

1-Deoxynojirimycin↗

PubMed1994

Molecular cloning and nucleotide sequence of the complementary DNA for penicillolysin gene, plnC, and 18 kDa metalloendopeptidase gene from Penicillium citrinum.

A full-length cDNA encoding the penicillolysin, an 18 kDa metalloendopeptidase from Penicillium citrinum, was cloned. Analysis of the 1284 base pair nucleotide sequence of the cDNA revealed a single open reading frame coding for 351 amino acid residues. The coding region of penicillolysin gene, plnC, occupies 1053 base pairs of the cDNA. The sequence consists of a putative 19-residue signal sequence, a 155-residue propeptide segment, and the 177-residues of penicillolysin with a molecular weight of 18,529. The deduced primary structure of penicillolysin is unique and the enzyme is a member of a new metalloendopeptidase family. Two histidine residues, His-128 and His-132, and glutamic acid residue, Glu-65 in penicillolysin were assumed to correspond to zinc ligands in the homologous thermolysin.

Amino Acid Sequence↗