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Biomedical subjects

K Morihara

Publications and source records attributed to K Morihara.

At least 37 records · Page 2Linked to original sources
PubMed1990

Complete nucleotide sequence of the structural gene for alkaline proteinase from Pseudomonas aeruginosa IFO 3455.

The DNA-encoding alkaline proteinase (AP) of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, the gene-incorporated bacteria expressed high levels of both AP activity and AP antigens. The amino acid sequence deduced from the nucleotide sequence revealed that the mature AP consists of 467 amino acids with a relative molecular weight of 49,507. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified AP reported previously. The amino acid sequence analysis revealed that both the N-terminal side sequence of the purified AP and several internal lysyl peptide fragments were identical to the deduced amino acid sequences. The percent homology of amino acid sequences between AP and Serratia protease was about 55%. The zinc ligands and an active site of the AP were predicted by comparing the structure of the enzyme with of Serratia protease, thermolysin, Bacillus subtilis neutral protease, and Pseudomonas elastase.

Amino Acid Sequence↗
PubMed1989

Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase.

The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences. The pattern of identity of amino acid sequences was quite evident in the regions that include structurally and functionally important residues of Bacillus subtilis thermolysin.

Amino Acid Sequence↗
PubMed1988

Cloning and characterization of elastase structural gene from Pseudomonas aeruginosa IFO 3455.

An 8.3 Kb DNA fragment was cloned from Pseudomonas aeruginosa IFO 3455. This fragment-containing Escherichia clone, pEL2, produced a high level of elastase activity. A smaller EcoRI-KpnI fragment was subcloned into pUC118 and E. coli HB101 was transformed with the plasmid. A deletion mutant clone was also constructed in the same bacteria. These deletion mutants were tested for elastase activity and it became clear that the full length of the elastase gene was 1.0-1.3 Kb. DNA sequencing analysis revealed that this DNA fragment contains the DNA sequence coding N-terminal amino acid sequence of the elastase protein.

Amino Acid Sequence↗
PubMed1988

Enzymatic synthesis of delta sleep-inducing peptide.

The delta sleep-inducing peptide was assembled enzymatically from three tripeptide fragments. All the peptide bonds were prepared by either papain- or alpha-chymotrypsin-mediated synthesis. Secondary hydrolysis was suppressed by introducing N alpha-protected amino acid or peptide esters as carboxyl components and using an alkaline pH. The protected nonapeptide was oxidized with ferric chloride to deprotect the C-terminal phenylhydrazide and then hydrogenated. The homogeneous peptide was obtained by reversed phase high-performance liquid chromatography. Comparison of enzymatic and chemical preparations showed no obvious differences.

Amino Acid Sequence↗
PubMed1986

Influence of temperature on the enzymic semisynthesis of human insulin by coupling and transpeptidation methods.

The influence of temperature of enzymic semisynthesis of human insulin ester was determined by using coupling and transpeptidation methods with trypsin and Achromobacter lyticus proteinase I as catalysts. The optimal reaction conditions were studied at the selected temperatures of 25, 12 and 4 degrees C. The results showed that the synthesis rates by both methods with trypsin increased as the temperature increased, but the final product yield correspondingly decreased. Therefore the reaction with trypsin should be done below 12 degrees C, preferably at 4 degrees C. This agrees well with the stability of trypsin at these temperatures. When the catalyst was Achromobacter lyticus proteinase I, no such complex temperature effects were observed, and the findings indicated that the reactions should be conducted below 37 degrees C for enzyme stability.

Catalysis↗
PubMed1986

Comparison of the subsite specificity of the mammalian neutral endopeptidase 24.11 (enkephalinase) to the bacterial neutral endopeptidase thermolysin.

A comparison has been made of the specificity of the mammalian neutral metalloendopeptidase, endopeptidase 24.11, with that of the bacterial neutral metalloendopeptidase thermolysin. A series of synthetic oligopeptides which have previously been studied as substrates for thermolysin and used in computer modeling were examined as substrates for the mammalian enzyme. It was found that P1, P2, and P'3 subsite interactions in the mammalian enzyme, although similar to those found in thermolysin, are less restrictive spatially and are considerably less dependent on hydrophobic interactions. This difference was maximally expressed with the synthetic substrate dansyl-D-alanylglycylnitrophenylalanylglycine which is a substrate for the mammalian enzyme, but not for the bacterial enzyme. A comparison of substrates in the free acid form with their corresponding amides showed that binding to the mammalian enzyme is dependent in part on an ionic interaction between the substrate carboxylate group and the enzyme. Such an ionic interaction was not observed with the bacterial enzyme.

Amino Acid Sequence↗
PubMed1986

Affinity purification of kallikrein and elastase from hog pancrease powder.

The present report describes a method that is efficient for simultaneous isolation of kallikrein and elastase from hog pancrease powder. Both enzymes were separated by successive column chromatography on CM-cellulofine and p-aminobenzamidine-Sepharose 4B. Kallikrein was further purified by column chromatography on DEAE-Sephadex and elastase was purified by repeated gel chromatography on Sephadex G-75. The kallikrein obtained was composed of two components, which were separable by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and the elastase had one component. The activity yields of kallikrein and elastase were 49 and 38%, respectively.

Amino Acids↗
PubMed1986

Specific cleavage of human type III and IV collagens by Pseudomonas aeruginosa elastase.

Purified Pseudomonas aeruginosa elastase cleaved human type III and IV collagens with the formation of specific cleavage products. Furthermore, type I collagen appeared to be slowly cleaved by both P. aeruginosa elastase and alkaline protease. These cleavage fragments from type III and IV collagens were separated from the intact collagen chains by SDS polyacrylamide gradient gel electrophoresis run under reducing conditions, and they were detected by their characteristic Coomassie blue staining pattern. The results of these studies suggest that the pathogenesis of tissue invasion and hemorrhagic tissue necrosis observed in P. aeruginosa infections may be related to the degradation of these collagen types by bacterial extracellular proteases.

Collagen↗
PubMed1986

Degradation of soluble laminin and depletion of tissue-associated basement membrane laminin by Pseudomonas aeruginosa elastase and alkaline protease.

Purified Pseudomonas aeruginosa elastase and alkaline protease rapidly cleaved soluble laminin, with each enzyme yielding different cleavage products. These cleavage fragments were separated from the intact laminin A and B polypeptide chains by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and detected by their characteristic Coomassie blue staining patterns. Pseudomonas elastase produced rapid and extensive degradation of both A and B chains, including the disulfide-rich regions. Apparently complete degradation to limit digests was obtained after 30 min with a substrate/enzyme ratio of 30:0.5. Under similar conditions, alkaline protease rapidly degraded the A chain while slowly degrading the B chain. In addition, immunoreactive laminin was released from authentic basement membranes after incubation with either enzyme as detected by an enzyme-linked immunoabsorption assay and by immunofluorescence. The results from these studies suggest a direct role for elastase and alkaline protease in both tissue invasion and hemorrhagic tissue necrosis in P. aeruginosa infections.

Animals↗
PubMed1986

New method of preparing elastase toxoid from Pseudomonas aeruginosa.

A new method for the preparation of elastase toxoid of Pseudomonas aeruginosa was developed. A chloroacetyl peptide derivative (CICH2CO-HOLeu-Ala-Gly-NH2; HOLeu, N-hydroxy-L-leucine), an active-site-directed irreversible inhibitor of Pseudomonas aeruginosa elastase, was used to prepare elastase toxoid with or without pretreatment with Formalin and L-lysine. Elastase toxoid thus obtained appeared to be ideal, possessing negligible enzyme activity while retaining full antigenicity and immunogenicity.

Animals↗
PubMed1986

Enzymatic semisynthesis of [LeuB30] insulin.

Experimental conditions for the preparation of [LeuB30] insulin by coupling of des-AlaB30 insulin with Leu-OBu(t) were determined using Achromobacter protease I and trypsin as catalysts. Successful coupling required a large excess of the amine component (0.8 M), a high concentration of organic cosolvent (35-50%) and neutral pH of the reaction mixture. The coupling yield of Achromobacter protease I after 24 h at 37 degrees C was almost the same or a little higher than that at 25 degrees C. With trypsin, the coupling yield at 37 degrees C after 24 h was considerably lower than at 25 degrees C. This was partly ascribed to the difference in concentration of organic cosolvent at 37 degrees C and 25 degrees C; 35% and 50%, respectively, or possibly of enzyme stability at these temperatures. The maximum product yield was about 90% with both enzymes under optimal conditions. A preparative scale experiment was performed with Achromobacter protease I; the yield of [LeuB30] insulin was 51% using porcine insulin as the starting material. This semisynthetic insulin was identified by HPLC and amino acid analysis. No difference was observed in CD spectra between [LeuB30] insulin and human insulin.

Alcaligenes↗
PubMed1985

Is the site of action of grayanotoxin the sodium channel gating of squid axon?

An attempt was made to elucidate the site of action of grayanotoxin (GTX) in the nerve membrane by using various endopeptidases. The experiment was conducted on squid axons isolated from Doryteuthis bleekeli with both voltage clamp and internal perfusion methods. Intracellular application of various endopeptidases for more than 30 min eliminated the gating action from both Na current and K current systems. When GTX (100 microM) was subsequently applied to the internal medium, the membranes could depolarize to various extents. This finding strongly suggests that the site of action of GTX is not confined to the channel gating but is present in a part of the Na channel having both voltage sensor and ion filter functions. With the application of trypsin, St. fradiae trypsin, pronase, BPN', and St. fradiae protease (group B), GTX-induced depolarization was much smaller than that with the application of alpha-chymotrypsin, N-protease, and thermolysin (group A). The difference in the sensitivity to GTX between group A and group B became remarkable as the time for application of the enzymes was prolonged. Since all enzymes belonging to group B retain trypsin-like activity and are more effective in removing the sensitivity to GTX, it is suggested that the molecular moiety around the binding site of GTX is rich in basic amino acids or the essential part for opening the Na channel should be protected by basic amino acids.

Animals↗
PubMed1984

Purification of human plasma alpha 1-proteinase inhibitor and its inactivation by Pseudomonas aeruginosa elastase.

Human alpha 1-proteinase inhibitor was purified according to a modification of the method of Kurecki et al. (Anal. Biochem. 99, 415 (1979) ), with Affi-Gel Blue treatment before Zn-affinity column chromatography. The inhibitor was inactivated in the presence of Pseudomonas aeruginosa elastase (1/2,000 molar ratio) for 2 h at pH 7.5 and 25 degrees C. The inactivated inhibitor was purified by column chromatography on Sephadex G-75 and DE-52. Little or no difference was observed between the native and inactive inhibitors in immunological response, amino acid composition or far-ultraviolet CD spectrum. On the other hand, a considerable difference was observed in the near-ultraviolet CD spectrum. Two amino-terminal sequences were found in the inactive inhibitor in almost the same ratio; one was the same as that of the intact inhibitor and the other was Met-Ser-Ile-Pro-. The two components were separated by high-performance liquid chromatography using 0.1% trifluoroacetic acid containing 30-70% CH3CN (gradient) as the eluent. Amino acid analysis and N- and C-terminal amino acid sequence analyses indicated that one fraction corresponded to the sequence of 1-357 of the alpha 1-proteinase inhibitor and the other to 358-394. We concluded that P. aeruginosa elastase can inactivate human alpha 1-proteinase inhibitor by splitting the peptide bond of Pro357-Met358, leading to local change near the active site but little change in the structure as a whole. The split carboxy-terminal fragment binds tightly to the rest of the inhibitor.

Amino Acids↗