Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.27 (thermolysin)
1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We found a novel metalloproteinase, which has high activity at low temperatures and in the presence of organic solvents, in the culture supernatant of a marine bacterium, Vibrio sp. T1800. The metalloproteinase, named vimelysin, was purified from the culture supernatant by three column chromatographies. About 150 mg of purified vimelysin was obtained from 3.3 liters of the culture supernatant with a high yield of 57%. The purified vimelysin showed a single protein band on SDS-PAGE with molecular weight of 38,000. The isoelectric point of vimelysin was 4.3 by isoelectric focusing. The optimum pH of vimelysin was pH 8.0 or pH 6.5 using casein or furylacryloyl-glycyl-leucine amide (FAGLA) as substrates, respectively. The optimum temperature of vimelysin was 50 degrees C when casein was used as a substrate, but it was 15 degrees C when FAGLA was used as a substrate. Interestingly, vimelysin activity was completely retained after 48 h of incubation at 25 degrees C in the presence of 50% ethanol. Moreover, vimelysin showed 40% activity of the control even in the presence of 10% ethanol, while thermolysin showed only 5% activity under the same conditions.
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PMID:A novel alcohol resistant metalloproteinase, vimelysin, from vibrio sp. T1800: purification and characterization. 890 Nov 4

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl] (FA)-dipeptide amides and N-carbobenzoxyl-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced by high concentrations (1-5 M) of neutral salts. The activation is due to an increase in the molecular activity, k(cat), while the Michaelis constant, K(m), is not affected by the addition of NaCl. In the present study, the effect of NaCl on the thermolysin-catalyzed hydrolysis of FA-glycyl-L-leucine amide (FAGLA) has been examined by changing the pH and temperature, and by adding alcohols to the reaction mixture. The enzyme activity, expressed by k(cat)/K(m), is pH-dependent, being controlled by two functional residues with pK(a) values of 5.4 and 7.8 in the absence of NaCl. The acidic pK(a) is shifted from 5.4 to 6.7 by the addition of 4 M NaCl, while the basic one is not changed. The degree of activation at a given concentration of NaCl is pH dependent in a bell-shaped manner with the optimum pH around 7. Although the activity increases in both the presence and absence of NaCl with increasing temperature from 5 to 35 degrees C, the degree of activation decreases. Alcohols inhibit thermolysin, and the degree of activation decreases with increasing alcohol concentration. The degree of activation tends to increase with increasing dielectric constant of the medium, although it varies considerably depending on the species of alcohol. Electrostatic interactions on the surface and at the active site of thermolysin are suggested to play a significant role in the remarkable activation by salts.
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PMID:Effects of pH, temperature, and alcohols on the remarkable activation of thermolysin by salts. 937 14

An anticancer peptide from soy protein was purified and isolated. Defatted soy protein was hydrolyzed with thermoase and hydrophobic peptides were extracted with ethanol. The peptide extract was fractionated by XAD-2 hydrophobic, gel filtration chromatography, and different C18 HPLCs. Anticancer activity of each fraction was assayed by measuring in vitro cytotoxicity on P388D1, a mouse monocyte macrophage cell line. IC50 value of a peptide fraction from Sephadex G-25 chromatography was 0.16 mg/ml. This peptide fraction at 1 mg/ml significantly affected cell cycle progression by arresting P388D1 at G2/M phases. Finally purified peptide from analytical C18 HPLC was nonapeptide of which molecular weight was 1157 Da and the sequence was X-Met-Leu-Pro-Ser-Tye-Ser-Pro-Tyr.
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PMID:Anticancer activity of hydrophobic peptides from soy proteins. 1121 78

The hydrophobic component to the binding affinities of one acyclic phosphinate (4) and three macrocyclic phosphonamidate inhibitors (1-3) to the zinc peptidase thermolysin was probed by varying the solvent composition. Increasing the percentage of ethanol in the buffer solution over the range 0-9% increases the inhibition constants, K(i), by up to an order of magnitude. This approach represents an experimental method for distinguishing solvation from conformational or other effects on protein-ligand binding. The size of the "antihydrophobic effect" is correlated with the amount of hydrophobic surface area sequestered from solvent on association of the inhibitor and enzyme, although it is attenuated from that calculated from the surface tension of ethanol-water mixtures. The results are consistent with the Lum-Chandler-Weeks explanation for the size dependence of the hydrophobic effect.
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PMID:Antihydrophobic solvent effects: an experimental probe for the hydrophobic contribution to enzyme-inhibitor binding. 1194 21

Basal keratinocytes of human epidermis strongly express the cell surface glycoprotein beta(1)-integrin, and putatively harbour epidermal stem cells. Selective sorting and culturing of keratinocyte stem cells forms the basis for studies on the role of these cells as targets for therapeutic intervention and gene therapy. Here we have studied variables which affect cell surface labelling for beta(1)-integrin, flow sorting and subsequent culturing of beta(1)-integrin-positive and beta(1)-integrin-negative keratinocytes. Keratinocytes were derived from small human skin punch biopsies (3 or 4 mm in diameter), and we tested a number of variables such as choice of proteolytic enzyme for cell isolation, cell concentration, fixation, storage of fixed cell suspensions and labelling conditions. In contrast to thermolysin treatment for cell isolation, trypsin treatment left most cell surface beta(1)-integrin molecules intact. Ethanol and paraformaldehyde fixation interfered with beta(1)-integrin detection, and unfixed cells gave the best results. Optimisation of all the individual steps resulted in a labelling protocol for reproducible staining and sorting of the cells. Sorted cells were seeded in 96-well plates (300 cells/well) and colonies were obtained in more than 50% of the wells with beta(1)-integrin-positive keratinocytes. In plates with beta(1)-integrin-negative cells, only 10% of the wells contained keratinocyte colonies. Flow sorted keratinocytes obtained by trypsin formed numerous colonies in cell culture experiments. In cell suspensions obtained with thermolysin, only sparse colonies were formed. We conclude that our methodology permits the use of small human tissue samples for cell labelling and sorting, while preserving the clonogenic potential.
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PMID:Immunofluorescent surface labelling, flow sorting and culturing of putative epidermal stem cells derived from small skin punch biopsies. 1216 32

Alcohols inhibit the thermolysin-catalyzed hydrolysis of N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH(2) and decrease the NaCl-induced activation of thermolysin in a concentration-dependent manner [K. Inouye et al. (1997) J. Biochem. 122, 358-364]. In this study, the inhibitory effects of alcohols on thermolysin activity were examined in detail using 10 different alcohols and a fluorescent substrate, (7-methoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2). The inhibition by all alcohols examined is completely reversible, and thermolysin activity is recovered by dilution. The inhibitor constants (K(i)) are in the range of 35-430 mM, and the order of the inhibitory effect is 1-pentanol, 1-propanol, 2-butanol, 2-methyl-1-propanol > 1-butanol > 2-propanol > ethanol, tert-amyl alcohol >> tert-butyl alcohol >> methanol. Linear and secondary alcohols whose mains chains consist of more than 3 carbons inhibit thermolysin effectively. Thermolysin activity is decreased by decreasing the dielectric constant, D, of the reaction medium containing the alcohol, and the decrease depending on the D value was almost the same manner for all alcohols except methanol, tert-butyl alcohol, and tert-amyl alcohol. Alcohols may inhibit thermolysin activity both by binding to the active site, most possibly to the S1' subsite, of thermolysin and by altering the electrostatic and hydrophobic environment around the thermolysin molecule.
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PMID:Inhibitory effects of alcohols on thermolysin activity as examined using a fluorescent substrate. 1247 97

We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.
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PMID:[Modified proteinases in peptide synthesis in organic media]. 1460 9

Vimelysin is a unique metalloproteinase from Vibrio sp. T1800 exhibiting high activity at low temperature and high stability in organic solvents such as ethanol. A 1,821 bp open reading frame of the vimelysin gene encoded 607 amino acid residues consisting of an N-terminal pro-region, a mature enzyme, and a C-terminal pro-region. The mature enzyme region showed 80%, 57% and 35% sequence identity with the mature forms of vibriolysin from V. vulnificus, pseudolysin from Pseudomonas aeruginosa, and thermolysin from Bacillus thermoproteolyticus, respectively. The catalytic residues and zinc-binding motifs of metalloproteinases are well conserved in vimelysin. The vimelysin gene was expressed in E. coli JM109 cells and the recombinant enzyme was purified as a 38-kDa mature form from cell-free extracts. The purified recombinant enzyme is indistinguishable from the enzyme purified directly from Vibrio. To obtain mutants exhibiting higher stability in organic solvents, random mutations were introduced by error-prone PCR and 600 transformants were screened. The N123D mutant exhibits two times higher stability in organic solvents than the wild-type enzyme. A plausible mechanism for the stability of the N123D mutant in organic solvents was discussed based on homology models of vimelysin and the N123D mutant.
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PMID:Molecular cloning of the gene encoding Vibrio metalloproteinase vimelysin and isolation of a mutant with high stability in organic solvents. 1642 99

The synthesis of decanoate beta-cyclodextrin esters (beta-CDd) and hexanoate beta-cyclodextrin esters (beta-CDh) was biocatalyzed by thermolysin from native beta-cyclodextrin (beta-CD) and vinyl hexanoate or vinyl decanoate used as acyl donors. The products were chemically characterized by infrared, NMR, and mass spectrometry. Both beta-CDd and beta-CDh esters were identified as a mixture of beta-CD preferentially substituted on the C2 position by the corresponding acyl chain. The degree of substitution varied from 2 to 7 for beta-CDd and from 4 to 8 for beta-CDh. The ability of beta-CD esters to self-organize into nanoparticles was tested using a nanoprecipitation technique in various solvents. The mean size diameter and polydispersity measured by quasi-elastic light scattering were dramatically affected by the nature of solvent (acetone, ethanol, or tetrahydrofuran) used in the nanoprecipitation technique. When directly observed using cryo-transmission electron microscopy, beta-CDh appeared as uniformly dense nanospheres, whereas beta-CDd exhibited a multilamellar onion-like organization. A structural model was rationalized for the beta-CDd nanoparticles.
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PMID:Nanoparticles of beta-cyclodextrin esters obtained by self-assembling of biotransesterified beta-cyclodextrins. 1647 24

Three colipases were purified from pancreas of two birds (ostrich and turkey) and one mammal (dromedary). After acidic and/or heat treatment and precipitation by sulfate ammonium and then ethanol, cofactors were purified by Sephadex G-50 gel filtration followed by ion-exchange chromatography first on Mono S and then on Mono Q. One molecular form was obtained from each species with a molecular mass of approximately 10 kDa. Cofactors were not glycosylated. The N-terminal sequences of the three purified cofactors showed high sequence homology. A 90 amino acid sequence of the ostrich cofactor was established based on peptide sequences from four different digests of the denaturated protein using trypsin, chymotrypsin, thermolysin, or staphylococcal protease. This sequence exhibited a high degree of homology with chicken and mammal cofactors. Bile salt-inhibited pancreatic lipases from five species were activated to variable extents by colipases from bird and mammal origins. The bird pancreatic lipase-colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. Our comparative study showed that mammal colipase presents a lower activation level toward bird lipases than the bird counterpart. Three-dimensional modeling of ostrich colipase suggested a structural explanation of this fact.
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PMID:Biochemical and structural comparative study between bird and mammal pancreatic colipases. 1695 80


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