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Biomedical subjects

K Horikoshi

Publications and source records attributed to K Horikoshi.

At least 163 records · Page 9Linked to original sources
PubMed1990

The nucleotide sequence of an alpha-amylase gene from an alkalopsychrotrophic Micrococcus sp.

An alpha-amylase gene from Micrococcus sp. 207 was cloned into Escherichia coli JM101 using the vector pHSG399. The constructed recombinant plasmid pYK63 contained a 4.8 kb chromosomal DNA fragment derived from strain 207 DNA. The cloned amylase isolated from E. coli JM101 (pYK63) produced mainly maltotetraose from starch, and exhibited temperature and pH activity profiles closely similar to those of the enzyme from the original strain. Nucleotide sequence analysis of the cloned DNA fragment revealed one open reading frame containing the gene which consisted of 3312 bp (1104 amino acids). When compared with several other alpha-amylases, three consensus sequences were identified in the region of the active site. About 300 amino acid residues were present both upstream and downstream of the active site region.

Amino Acid Sequence↗
PubMed1990

Purification and properties of an oligo-1,6-D-glucosidase from an alkalophilic Bacillus species.

An isomaltose-hydrolyzing alpha-D-glucosidase from the alkalophilic Bacillus designated strain F5 was purified to an electrophoretically homogeneous state. The molecular weight of the purified glucosidase was 60,000 by SDS-poly(acrylamide) gel electrophoresis, and 63,000 by Sephacryl S-200 gel-filtration chromatography. The enzyme was most active for isomaltose at pH 6.0-6.5 and 45 degrees, and stable up to 50 degrees at pH 7.0 and in the range of pH 6.0-9.0 at 50 degrees by 10-min incubation. The apparent Vmax and Km values for isomaltose were 34.5 mumol.min-1.mg-1 of protein, and 3.33 mM. Panose and isomaltotriose are the best substrates for this enzyme. The restricted substrate specificity indicated the assignment of the enzyme to be an oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase; EC 3.2.1.10), but it was suggested that it could be a new type of oligo-1,6-glucosidase on the basis of its action on a series of (1----4)-alpha-malto-oligosaccharides.

Bacillus↗
PubMed1990

[Altered alpha atrial natriuretic peptide (ANP) concentrations in plasma, atria and hypothalamus in experimentally induced hyper- and hypothyroid rats].

In order to assess a possible involvement of thyroid hormone in atrial natriuretic peptide (ANP), experimentally induced hyper- and hypothyroid rats were employed, and the immunoreactive rat ANP (IR-ANP) concentrations in plasma, atria and brain regions including the hypothalamus were measured by a specific radioimmunoassay. Plasma IR-ANP concentration in hypothyroid rats was 14.5 +/- 2.9 (mean +/- SD) fmol/ml, significantly lower than that in control rats (p less than 0.05 vs control of 24.9 +/- 9.7 fmol/ml). Plasma IR-ANP concentration in hyperthyroid rats was 66.4 +/- 9.7 fmol/min, significantly higher than that in the controls (p less than 0.01). Atrial IR-ANP concentration in hyperthyroid rats was significantly lower than that in the controls (79.9 +/- 11.1 nmol/g vs 133.5 +/- 21.2 nmol/g (control), p less than 0.05), though no significant change was observed in atrial IR-ANP concentration in hypothyroid rats. While hypothalamic ANP concentration in hypothyroid rats was significantly lower than that in the controls (17.5 +/- 3.5 pmol/g vs 31.9 +/- 1.9 pmol/g (control), p less than 0.05), there was no significant change of that in the hyperthyroid rats. On reverse phase high performance liquid chromatography, the major peak in plasma and hypothalamus extract was thought to be identical to synthetic alpha-rat ANP (1-28). These results may suggest that in the hyperthyroid state an excessive amount of ANP is released from atria into the blood, and that in the central nervous system thyroid hormone involve ANP metabolism being different from the atrium.

Animals↗
PubMed1990

Characterization of an alkaline protease from Bacillus sp. no. AH-101.

The Bacillus sp. no. AH-101 alkaline protease showed higher hydrolysing activity against insoluble fibrous natural proteins such as elastin and keratin in comparison with subtilisins and Proteinase K. The optimum pH of the enzyme toward elastin and keratin was pH 10.5 and pH 11.0-12.0 respectively. The specific activity toward elastin and keratin was 10,600 units/mg protein and 3970 units/mg protein, respectively. The enzymatic activity was not inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Carbobenzoxy-glycyl-glycyl-L-phenylalanyl chloromethyl ketone completely inhibited the caseinolytic activity, but 36% elastolytic activity remained. No inhibitory effect on caseinolytic and elastolytic activity was shown by tosyl-L-phenylalanyl-chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, carbobenzoxy-L-phenylalanyl chloromethyl ketone, and elastatinal. The amino acid composition and amino terminal sequence of the enzyme were determined. The no. AH-101 alkaline protease was compared with subtilisin BPN', subtilisin Carlsberg, no. 221, and Ya-B alkaline proteases. Extensive sequence homology existed among these enzymes.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167.

The nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH2-terminal amino acid sequence analysis of the G6-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102,598. Identity of the NH2-terminal amino acid sequences among each component of the multiform G6-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G6-amylase gene showed no homology with those of other bacterial alpha-amylases although the consensus amino acid sequences of the active center were well conserved.

Amino Acid Sequence↗
PubMed1990

DNA sequences required for the alkalophily of Bacillus sp. strain C-125 are located close together on its chromosomal DNA.

Two alkali-sensitive mutants of alkalophilic Bacillus sp. strain C-125 were obtained. Mutant 38154 showed defective regulation of internal pH. Plasmids pALK1 and pALK2, containing DNA fragments different from those of the parent strain, were able to recover alkalophily in mutants 18224 and 38154, respectively. DNA analysis suggested that the two fragments overlapped on the chromosomal DNA of strain C-125.

Bacillus↗
PubMed1990

A novel antifungal antibiotic, FR-900848. I. Production, isolation, physico-chemical and biological properties.

Streptoverticillium fervens produced a new antibiotic, FR-900848, which has a high specific activity against filamentous fungi. Purified by solvent extraction and column chromatography, the compound appears as colorless crystals. Its structure is C32H43N3O6, which consists of 5'-amino-5'-deoxy-5,6-dihydrouridine with an unsaturated fatty acid having unprecedented four serial and one isolated cyclopropane rings.

Animals↗
PubMed1990

Comparison of CD composition produced by chimeric CGTases.

Using 12 chimeric cyclomaltodextrin glucanotransferases (CGTases) constructed from two genes, the CGTase gene from the alkalophilic Bacillus sp. strain No. 38-2 (CGTase 38-2 gene) and the CGTase gene from the alkalophilic Bacillus sp. strain No. 17-1 (CGTase 17-1 gene), we compared the effect of those chimeric enzymes on cyclodextrin (CD) production, especially on the composition of the CDs produced. It was found that the N-terminal and the C-terminal segments were important for CD production. Chimeric enzymes that contained the N-terminal and the C-terminal segments derived from CGTase 38-2 produced large amounts of CD, and especially a higher proportion of alpha-CD than those of other chimeric enzymes.

Bacillus↗
PubMed1990

Extracellular production of human tumor necrosis factor-alpha by Escherichia coli using a chemically-synthesized gene.

A DNA fragment of approximately 490 base pairs encoding human TNF was chemically synthesized and expressed within Escherichia coli cells. Furthermore, extracellular production of human TNF and several N-terminal deletion mutants of TNF was attempted using the excretion vector pEAP8. The TNF mutant with two N-terminal amino acids deleted (N delta 2-TNF) was efficiently excreted into the culture medium by E. coli carrying the plasmid pEXTNF3. In this clone, the signal peptide was correctly processed during the excretion. The E. coli-excreted N delta 2-TNF had higher antitumor activity than wild-type TNF or N delta 2-TNF produced intracellularly by E. coli.

Amino Acid Sequence↗
PubMed1989

Amino acid sequence of Mirabilis antiviral protein, total synthesis of its gene and expression in Escherichia coli.

We have determined the complete amino acid sequence of Mirabilis antiviral protein (MAP). MAP is composed of 250 amino acids having a combined molecular weight of 27,833 and contains 23 lysine residues and 7 arginine residues. The amino acid sequence of MAP has 24% homology with the Ricin D-A chain. To carry out systematic structure-function studies of MAP, we have accomplished the total synthesis of its gene. We designed a synthetic MAP gene containing 12 unique restriction sites that were on the average 65 base pairs apart. Thirty synthetic oligonucleotides were enzymatically joined to form DNA duplexes. These were strategically synthesized to have EcoRI and HindIII cohesive ends and were cloned in pUC19. Nine blocks of the synthetic fragments were assembled in pUC19 to form the MAP gene consisting of 759 base pairs. The correctness of the connecting reactions was confirmed by step-wise sequencing of each assembled fragment as well as the total gene. When expressed under control of the tac promoter in Escherichia coli, the synthetic gene gave a protein similar to the native MAP. This was confirmed by an enzyme-linked immunosorbent assay and Western blotting analysis.

Amino Acid Sequence↗
PubMed1989

The complete nucleotide sequence of the glutamine synthetase gene (glnA) of Bacillus subtilis.

The glutamine synthetase (GS) gene from Bacillus subtilis PCI 219 was cloned in Escherichia coli using the vector pBR329. A plasmid, pSGS2, was isolated from a glnA+ transformant and the cloned GS gene was found to be located in a 3.6 kb DNA fragment. The nucleotide sequence of a 1.8 kb segment encoding the GS was determined. This segment showed an open reading frame which would encode a polypeptide of 444 amino acids. The amino acid sequence of this GS gene product has higher homology with that of the Clostridium acetobutylicum GS than that of the E. coli GS.

Amino Acid Sequence↗
PubMed1989

The nucleotide sequence of the lipo-penicillinase gene of alkalophilic Bacillus sp. strain 170.

The lipo-penicillinase (LIPEN) gene from an alkalophilic Bacillus sp. strain 170 was cloned in Escherichia coli using the vector pHSG399. A plasmid, pFAP121, was isolated from an ampicillin resistant transformant and the cloned LIPEN gene was found to be in a 2.2 kb DNA fragment. The nucleotide sequence of a 1.9 kb segment encoding the LIPEN was determined. This segment showed an open reading frame which would encode a polypeptide of 310 amino acids. The amino acid sequence of this LIPEN gene product has strong homology with those of the Bacillus cereus beta-lactamase III and Bacillus licheniformis penicillinase.

Amino Acid Sequence↗
PubMed1989

Construction of a secretion vector production of peptide hormones in Escherichia coli (extracellular production of calcitonin as fused protein).

A new secretion vector, pEAP84 which contained a unique restriction site (BglII) at the 3' end of the penicillinase gene to produce a fused protein, and the Ex-kil region to make the outer membrane permeable, was constructed from pEAP82. A recombinant plasmid p84h06, which contained a synthetic gene for human calcitonin with a cyanogen bromide cleavage site at the junction site of the fused protein, was constructed and introduced into Escherichia coli. The hybrid protein produced in E. coli carrying p84h06 was secreted into the culture medium. The amino acid composition of this product was consistent with that deduced from the DNA sequence. Mature calcitonin was obtained following cyanogen bromide cleavage of the fused protein.

Amino Acid Sequence↗
PubMed1989

The third cellulase of alkalophilic Bacillus sp. strain N-4: evolutionary relationships within the cel gene family.

The third cellulase gene (celC) of Bacillus sp. strain N-4 was cloned in plasmid pBR322 and was located within a 5.5-kb HindIII fragment. The cellulase encoded by this fragment had an Mr of about 100,000 and showed optimum activity around pH 9. These properties were different from those of the enzymes encoded by the celA and celB genes of the same organism. The amino acid sequence deduced from the nucleotide sequence was found to be highly homologous to the CEL-F enzyme from Bacillus sp. strain No. 1139 [Fukumori et al., J. Gen. Microbiol. 132 (1986) 2329-2335]. An evolutionary relationship observed among the four cellulases of alkalophilic Bacillus strains and that of Bacillus subtilis endoglucanase suggested that ancestral genes for alkaline and neutral cellulases diverged early in the evolution of these enzymes.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the glutamine synthetase gene (glnA) and its upstream region from Bacillus cereus.

We have determined the complete nucleotide sequence of a 2.4 kb chromosomal EcoT22I-NspV fragment, containing the Bacillus cereus glnA gene (structural gene of glutamine synthetase). The deduced amino acid sequence indicates that the glutamine synthetase subunit consists of 444 amino acid residues (50,063 Da). Comparisons are made with reported amino acid sequences of glutamine synthetases from other bacteria. Upstrem of glnA we found an open reading frame of 129 codons (ORF129) preceded by the consensus sequence for a typical promoter. Maxicell experiments showed two polypeptide bands, with molecular weights in good agreement with that of glutamine synthetase and that of ORF129, in addition to vector-coded protein. It is possible that the product of this open reading frame upstream of glnA has a regulatory role in glutamine synthetase expression.

Amino Acid Sequence↗
PubMed1989

Construction of a chimeric series of Bacillus cyclomaltodextrin glucanotransferases and analysis of the thermal stabilities and pH optima of the enzymes.

The cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from the alkalophilic Bacillus sp. strain no. 17-1 was cloned in Escherichia coli. The cloned CGTase gene consisted of a single open reading frame which would encode a polypeptide of 713 amino acids, and the first 27 amino acid residues comprised a signal peptide. The nucleotide sequence and the amino acid sequence of this CGTase (CGTase 17-1) gene had strong homology with those of the CGTase (CGTase 38-2) gene previously cloned in our laboratory from the alkalophilic Bacillus sp. strain no. 38-2, although the enzymic properties of the CGTase 17-1 were distinct from those of the CGTase 38-2. To analyse those enzymic properties further, we constructed 12 chimeric CGTases using three restriction nuclease sites and compared the enzymic properties of the chimeric CGTases. The N-terminal part of the enzyme was important for heat stability, and the pH-activity profile was influenced by both the N- and the C-terminal parts. A third segment was less important for enzymic properties.

Amino Acid Sequence↗
PubMed1989

Two Bacillus beta-mannanases having different COOH termini are produced in Escherichia coli carrying pMAH5.

The nucleotide sequence was determined for the alkalophilic Bacillus sp. strain AM-001 beta-mannanase gene which produced two beta-mannanases (A and B) in Escherichia coli transformants. The putative beta-mannanase gene was 1,539 base pairs long and encoded a mature beta-mannanase protein of 487 amino acids and a signal peptide of 26 amino acids. The COOH-terminal amino acid of beta-mannanase A is an arginine residue located at amino acid 513 of the deduced amino acid sequence, and that of beta-mannanase B is a valine residue located at amino acid 365. Deletion derivatives having 1,098 base pairs from the ATG start codon maintained the beta-mannanase activity of the encoded polypeptide. However, clones harboring DNA fragments (1,051 base pairs) shorter than the gene which encoded beta-mannanase B (1,095 base pairs) did not exhibit the beta-mannanase activity. The simultaneous production of both beta-mannanases A and B in an E. coli transformant was demonstrated by the maxicell procedure.

Amino Acid Sequence↗