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Biomedical subjects

A Sugihara

Publications and source records attributed to A Sugihara.

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

Purification and some properties of a protease from Streptomyces limosus.

Streptomyces limosus was selected because it secreted a novel protease that catalyzed the synthetic reaction forming Pro-Pro-Pro from Pro-Pro. The protease was purified to an electrophoretically homogeneous state and an activity of more than about 20,000-fold that of the culture broth. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was most active in alkaline pH for the synthetic reaction producing Pro-Pro-Pro from Pro-Pro, although for the hydrolytic reaction forming proline it was most active in neutral pH. The enzyme was inhibited by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and diazoacetyl-DL-norleucine methyl ester (DAN). It can be considered that this enzyme belongs to the class of aspartic proteases. The substrate specificity indicates that this enzyme has a strong affinity for proline as a N-terminal amino acid of peptides.

Amino Acid Sequence↗
PubMed1995

[An autopsy case of immature teratoma with choriocarcinoma in the mediastinum].

The autopsy of a 28-year-old Japanese male patient, which revealed immature teratoma combined with choriocarcinoma in the mediastinum, is presented. The tumor, which was removed after chemotherapy, was localized in the superior-anterior mediastinum. Chemotherapy was performed several times after operation. However, HCG increased again, and a roentgenogram revealed metastatic shadows in both lungs. The metastatic tumors in the lungs showed only the choriocarcinoma.

Adult↗
PubMed1995

[Neuron specific enolase-producing IgD multiple myeloma with high serum amylase activity].

A 68-year-old man who was admitted to our hospital because of general fatigue and anorexia. He was diagnosed as suffering from neuron specific enolase (NSE)-producing IgD-lambda type multiple myeloma with high activity of serum amylase, based on the detection of monoclonal IgD-lambda M-protein in the serum and urine, markedly high activities of salivary-type amylase and NSE in the serum, and immunohistochemical evidence of NSE and IgD in the myeloma cells. After two courses of alpha-IFN and VMCP chemotherapy, serum IgD, amylase and NSE decreased to normal levels. These observations indicate that NSE was ectopically produced by myeloma cells.

Aged↗
PubMed1994

Cloning and nucleotide sequence of cDNA encoding a lipase from Fusarium heterosporum.

Fusarium heterosporum produces a solvent-tolerant lipase. A 1.3-kbp lipase cDNA was isolated from the cDNA library by colony hybridization with an oligonucleotide probe corresponding to the N-terminal amino acid sequence. Nucleotide sequence analysis showed an open reading frame of 999 bp, and the deduced amino acid sequence contained the N-terminal sequence determined by Edman degradation and the consensus pentapeptide (-Gly-X-Ser-X-Gly-), which is conserved in lipase, esterase, and serine protease. The mature lipase consisted of 301 amino acid residues with a molecular mass of 32.7 kDa, preceded by the putative signal peptide or preprosequence. The enzyme was homologous to lipases from Humicola lanuginosa (39% homology), Rhizomucor miehei (32%), Rhizopus delemar (32%), and Rhizopus niveus (32%), and to mono- and diacylglycerol lipase from Penicillium camembertii (38%). Comparison of this lipase with these homologous enzymes suggested that the catalytic triad was composed of Ser144, Asp198, and His256, and that the oxyanion hole was formed with Ser 82.

Amino Acid Sequence↗
PubMed1994

Positional and fatty acid specificities of Geotrichum candidum lipases.

Geotrichum candidum ATCC34614 produces a major (I) and three minor (II, III and IV) forms of lipase with similar molecular masses, but different positional and fatty acid specificities. The major and one of the minor (II) forms were confirmed to cleave both the inside and outside ester bonds of triolein indiscriminately. The non-positional specificity was also retained on 1,3-dipalmitoyl-2-oleoyl-glycerol (POP) hydrolysis. In contrast, the remaining two forms (III and IV) showed unusual positional specificity; they cleaved the inside (2-position) ester bond of triolein at nearly twice the rate of cleavage at the 1(3)-position. The preference for the inside ester bond was increased upon POP hydrolysis.

Geotrichum↗
PubMed1994

Purification and characterization of a carboxylesterase from Pseudomonas sp. KWI-56.

An intracellular carboxylesterase from Pseudomonas sp. was overproduced in E. coli, and purified to homogeneity by a combination of hydrogen bond chromatography, gel filtration, and hydrophobic interaction chromatography. Gel filtration and SDS-PAGE suggested that the purified enzyme consisted of two subunits of molecular mass of 28 kDa. Its isoelectric point was 5.9. The enzyme was thermolabile, and showed its maximum activity at 22 degrees C (pH 7.5). Methyl propionate was hydrolyzed at the highest rate among the fatty acid methyl esters tested. PMSF, DFP, PCMB, and HgCl2 inhibited the enzyme markedly, suggesting that serine and/or cysteine is in or near the active site.

Base Sequence↗
PubMed1993

Cloning and sequence analysis of an esterase gene from Pseudomonas sp. KWI-56.

The gene encoding an esterase from Pseudomonas sp. KWI-56 was cloned and sequenced. The nucleotide sequence contained an open reading frame encoding a polypeptide comprising 262 amino acids, whose molecular weight agreed well with the value obtained by SDS-PAGE. Comparison of the amino acid sequence with those of the other homologous enzymes suggested that Ser-92 and His-24l might be included in the catalytic triad.

Amino Acid Sequence↗
PubMed1993

Characterization of Geotrichum candidum lipase III with some preference for the inside ester bond of triglyceride.

A new form of Geotrichum candidum lipase with a unique positional specificity was found to exist in the culture broth as a minor component together with the well-documented major form. Unlike the major form, which cleaves both the inside and outside ester bonds of triglyceride indiscriminately, the newly isolated form showed some preference for the inside (2-position) ester bond. The new enzyme was also characterized by its own fatty acid specificity, i.e., an outstandingly high activity towards triolein and methyl oleate among the simple triglycerides and fatty acid methyl esters tested. Moreover, the enzyme possessed a specific activity three times as high as the major form. Notable difference in circular dichroism spectra were observed between the two forms, indicating distinct conformational differences. Edman degradation revealed that the N-terminal sequence of the new form differed from that of the major form, thus demonstrating the existence of a novel lipase gene on the chromosome.

Amino Acid Sequence↗
PubMed1993

Cloning and sequencing of two chromosomal lipase genes from Geotrichum candidum.

Two chromosomal lipase genes (lip1 and lip2 encoding lipases I and II, respectively) were cloned from Geotrichum candidum by colony hybridization using lipase cDNAs as probes, and their nucleotide sequences were determined. Both genes contained 5'- and 3'-flanking regions in addition to their coding regions. The coding regions contained no intron, and there was 86% homology between their nucleotide sequences. However, no homology was observed between the flanking regions. The primer extension analysis showed that four transcription initiation sites were localized around 100-bp upstream from the ATG start codon of the lip2 gene. The putative promoter sequences of eukaryotic genes, CCAAT-----TATAAA, were found in the 5'-flanking regions of two lipase genes. These sequences might participate in the lipase induction by long-chain fatty acid. There were two inverted repeat sequences and a pyrimidine-rich sequence in the 5'-flanking region of lip2, but not in the 5'-flanking region of lip1. This specific structure of lip2 might reflect the higher transcriptional efficiency.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia.

A thermostable lipase from Pseudomonas cepacia has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification included treatment of the culture supernatant with acrinol, hydrophobic interaction chromatography, and gel filtration. The enzyme was a monomeric protein with M(r) of 36,500 and pI of 5.1. The optimal pH at 50 degrees C and optimal temperature at pH 6.5 were 5.5-6.5 and 55-60 degrees C, respectively, when olive oil was used as the substrate. Simple triglycerides of short and middle chain fatty acids (C < or = 12) were the preferred substrates over those of long chain fatty acids. The enzyme cleaved all the ester bonds of triolein, with some preference for the 1,3-ester bonds. The enzyme retained all its activity even after incubation at 75 degrees C (pH 6.5) for 30 min. Further, the activity was not impaired during 21 h storage at pH 6.5 in 40% water-miscible solvents including methanol, ethanol, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, and dioxane. The addition of dimethylsulfoxide or acetone to the assay mixture in the range of 0-35% stimulated the enzyme, whereas benzene or n-hexane had an inhibitory effect. These properties together with the N-terminal amino acid sequence confirmed that the enzyme differs from the known Pseudomonas sp. lipases.

Amino Acid Sequence↗
PubMed1991

Cloning, nucleotide sequencing, and expression in Escherichia coli of a lipase and its activator genes from Pseudomonas sp. KWI-56.

A lipase gene (lip) and its activator gene (act) on a 2.9 kb BglII-EcoRI fragment from Pseudomonas sp. KWI-56 were cloned in Escherichia coli using pUC19 as a vector plasmid. From the sequencing results, the open reading frames of the lip and the act were found to contain 1092 and 1032 nucleotides, respectively. The act existed downstream of the lip with the same orientation. When the lip was expressed in E. coli using the lac promoter on the pUC plasmid vector, the lipase activity of E. coli carrying both the lip and the act was 200-fold greater than that carrying only the lip. This result suggested the act was important in the expression of the lip in E. coli.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of a novel thermostable lipase from Bacillus sp.

A thermostable lipase from Bacillus sp. has been purified to homogeneity as judged by disc-PAGE, SDS-PAGE, and isoelectric focusing. The purification included ammonium sulfate fractionation, treatment with acrinol, and sequential column chromatographies on DEAE-Sephadex A-50, Toyopearl HW-55F, and Butyl Toyopearl 650M. The purified enzyme was found to be a monomeric protein with Mr of 22,000, and pI of 5.1. The optimal pH at 30 degrees C, and optimal temperature at pH 5.6 were 5.5-7.2, and 60 degrees C, respectively, when olive oil was used as the substrate. The substrate specificity towards simple triglycerides was broad and 1- and 3-positioned ester bonds were hydrolyzed in preference to a 2-positioned ester bond. The addition of acetone to the assay mixture in the range of 0-60% (v/v) stimulated the enzyme remarkably, whereas n-hexane had an inhibitory effect.

Amino Acids↗
PubMed1990

Separation and characterization of two molecular forms of Geotrichum candidum lipase.

Southern blot analysis of the Geotrichum candidum genome with a cloned lipase cDNA as the probe indicated the existence of two genes on the chromosome of the fungus which are homologous to the cDNA. As expected, two forms of lipase (lipases I and II) were actually isolated by hydrophobic interaction chromatography after a multistep procedure including ammonium sulfate fractionation, anion exchange chromatography, and gel filtration of the culture filtrate. Lipase I, the first eluted fraction, was the predominant form, and more than 80% of the total activity was attributed to this form. Amino acid sequence analysis of the amino and carboxyl termini of these two enzyme preparations indicated that lipase I was the product of the lipase gene whose cDNA had previously been cloned and sequenced [Shimada et al. (1989) J. Biochem. 106, 383-388]. Lipase II, on the other hand, had similar amino acid composition, but different terminal sequences which were not found in the primary structure of lipase I deduced from the cDNA sequence. These results gave lines of evidence for the expression of truely different lipase genes and ruled out the possibility that the observed multiple forms are caused by proteolytic digestion. The molecular mass estimated by SDS-PAGE and the isoelectric point of lipase I were 64 kDa and 4.3, while those of lipase II were 66 kDa and 4.3, respectively. The two lipases had essentially the same specific activities, substrate specificities, pH stabilities, and optimal temperatures, but different pH optima and thermal stabilities.

Amino Acid Sequence↗
PubMed1990

cDNA cloning and characterization of Geotrichum candidum lipase II.

Geotrichum candidum produces two extracellular lipases, I and II. A lipase II cDNA clone was isolated from a cDNA library by colony hybridization using the 32P-labeled fragment of lipase I cDNA isolated previously. The nucleotide sequence of lipase II cDNA determined by the dideoxy chain terminating method includes the N- and C-terminal amino acid sequences of lipase II, and the overall amino acid composition deduced from the cDNA coincides with that deduced on amino acid analysis of this protein. The cloned lipase II cDNA codes a protein of 544 amino acids and a part of the signal sequence of 13 amino acids. The peptide chain lengths of lipases I and II are the same, their overall identity being 84%. Furthermore, four Cys residues are completely conserved, which may participate in the formation of disulfide bridges. A homology search indicated that the G. candidum lipases and Candida cyclindracea lipase are homologous enzymes and that they are members of the cholinesterase family.

Amino Acid Sequence↗
PubMed1989

cDNA molecular cloning of Geotrichum candidum lipase.

The cDNA clone of Geotrichum candidum (Geo.) lipase was isolated from the Geo. cDNA library by colony hybridization using 32P-labeled oligonucleotides corresponding to a partial amino acid sequence of this enzyme. The nucleotide sequence of the cDNA determined by the dideoxy chain terminating method included some partial amino acid sequences determined by Edman degradation, and the overall amino acid composition deduced from the cDNA coincided with that from amino acid analysis of this protein. The cloned cDNA coded a protein of 554 amino acids and a hydrophobic signal sequence of 19 amino acids. Geo. lipase contained the -Gly-X-Ser-X-Gly- sequence which is believed to form part of the interfacial lipid recognition site.

Amino Acid Sequence↗
PubMed1987

Amino acid sequence of thermostable direct hemolysin produced by Vibrio parahaemolyticus.

The complete amino acid sequence of the subunit of thermostable direct hemolysin, a dimeric protein composed of identical subunits isolated from Vibrio parahaemolyticus, was determined by sequencing BrCN-peptides, their tryptic peptides, and overlaps obtained by Achromobacter protease I digestion. The subunit consists of 165 amino acid residues with the sole disulfide bond between Cys 151 and Cys 161. It is deduced that the biologically active hemolysin is formed by noncovalent association of subunits which are not linked together by disulfide bonds. The primary structure of hemolysin elucidated in the present study is essentially the same as that deduced from the nucleotide sequence of a gene encoding the protein but differs in 9 amino acid residues, suggesting the possibility of the presence of multiple genes for the thermostable direct hemolysin in Vibrio parahaemolyticus.

Alcaligenes↗