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)

An inhibitor of neutral subtilopeptidase [EC 3.4.24.4] was purified from porcine serum by salting out with (NH4)2SO4, chromatography on anion exchange sephadex, gel filtration with Sepharose 6B, and isoelectric focusing. The preparation was homogeneous by electrophoretic and ultracentrifugal criteria, and was shown to be a glycoprotein with a molecular weight of 740,000. It inhibited the caseinolytic activities of thermolysin, subtilisin, trypsin [EC 3.4.21.4], and alpha-chymotrypsin [EC 3.4.21.1] as well as that of neutral subtilopeptidase by an equimolar binding to those proteolytic enzymes. SDS-polyacrylamide gel electrophoresis after reduction with beta-mercaptoethanol indicated that the inhibitor was made up of four subunit monomers having a molecular weight of 190,000. From comparisons of its physiocochemical and inhibitory properties with those of well-investigated plasma proteins, the inhibitor was identified as alpha2-macroglobulin. On treatment of the inhibitor with neutral subtilopeptidase, a protein with a molecular weight of 95,000 appeared after treatment with SDS and beta-mercaptoethanol, suggesting that a peptide bond susceptible to the enzyme exists near the mid-point of the subunit chains.
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PMID:A neutral subtilopeptidase inhibitor from porcine serum some evidence for alpha2-macroglobulin. 5 60

Cells of Acinetobacter calcoaceticus contain a constitutive periplasmic metalloproteinase showing similar properties as the periplasmic metalloproteinase of Escherichia coli. The periplasmic proteinase of A. calcoaceticus was purified, starting from periplasm, by ammonium sulfate precipitation, hydrophobic interaction chromatography and chromatofocusing up to the homogeneity of the enzyme in SDS-electrophoresis with a yield of 6.7% and a purification factor of 417. The enzyme has a molecular mass of 108,000 (gel filtration) or 112,000 (native electrophoresis), and consists of four identical subunits with a molecular mass of 27,000 (SDS-electrophoresis). The purified enzyme degrades preferentially polypeptides such as glucagon and insulin. Larger proteins are accepted as substrates to a considerably lower extent. All tested synthetic substrates with trypsin, chymotrypsin, elastase and thermolysin specificity were not cleaved. Therefore, the described enzyme was designated "insulin-cleaving proteinase" (ICP).
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PMID:Purification of a periplasmic insulin-cleaving proteinase from Acinetobacter calcoaceticus. 151 May 71

Trypsin inhibitory activity from the hemolymph of Limulus polyphemus was found to co-purify with coagulogen (the clottable protein in blood coagulation) after acidification, ammonium sulfate precipitation, and gel filtration. Limulus trypsin inhibitor (LTI) was separated from coagulogen by ion-exchange chromatography on carboxymethyl-Sephadex. LTI is an inhibitor of trypsin (Ki = 3.3 nM) on both high and low molecular weight substrates. It also inhibits chymotrypsin but has little or no effect on thrombin, thermolysin, pepsin, or papain, nor does LTI inhibit the proteolytic cascade produced in endotoxin-stimulated Limulus amoebocyte lysate coagulation. Electrophoresis under nonreducing conditions on denaturing polyacrylamide gel yields a doublet migrating with an estimated Mr of 20,000. Under reducing conditions, a single broad band migrates with an estimated Mr of 15,000. The native structure is a monomer of moderate asymmetry with a molecular weight of 16,300 and a so20,w = 1.5(5), as determined by analytical ultracentrifugation. The amino acid composition of LTI yields a calculated molecular weight of 15,680 and a calculated partial specific volume of 0.71(7) ml/g. LTI does not contain methionine, tryptophan, or detectable levels of reducing carbohydrate. The NH2-terminal sequence (V-S-P-P-F-I-K-Q-T-K-F-S-T-X-F-L-G-X-S-S) consists primarily of hydrophobic amino acid residues. Comparison of the amino acid composition and amino-terminal sequence of LTI with those of other known protease inhibitors reveals no significant similarity to other trypsin inhibitors. The novel physical characteristics suggest that LTI represents a new type of protease inhibitor.
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PMID:A novel trypsin inhibitor from the hemolymph of the horseshoe crab Limulus polyphemus. 198 74

The relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine plasma fibronectin as well as human plasma fibronectin was adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5 M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0 M, indicating that both fibronectins have very high surface hydrophobicities. On digestion with thermolysin, porcine plasma fibronectin yielded five fragments (140-150, 43, 25, 17, and 14 kDa) similar to those reported for human fibronectin, although porcine fibronectin was more resistant to the digestion than human fibronectin. The three heparin-binding fragments were found to have a wide range of surface hydrophobicities, the 140-150 kDa fragment having the lowest, the 25 kDa fragment a higher, and the 14 kDa fragment the highest among all the fragments. The 43 kDa collagen-binding and 17 kDa fragments had surface hydrophobicities as high as that of fibronectin. It is noteworthy that the 43 kDa collagen-binding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbohydrates.
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PMID:Hydrophobic properties of porcine fibronectin and its functional domains. 201 77

Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205-316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu302 residue into fragments 205-302 and 303-316. Upon incubation for 2-5 days of fragment 205-302 with a 5-fold excess of peptide 303-316, prepared by solid phase synthesis, with V8-protease in 0.1 M ammonium acetate, pH 6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to approximately 90% (based on fragment 205-302). The same procedure was used to prepare also the thermolysin fragments 205-315 and 205-311 by enzymatic coupling of fragment 205-302 to peptide 303-315 or 303-311, these last prepared by proteolytic digestion of the synthetic peptide 303-316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205-316 of thermolysin at the level of its helical segment encompassing residues 301-312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.
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PMID:Semisynthesis of carboxy-terminal fragments of thermolysin. 211 42

An extracellular Zn-endopeptidase was purified to homogeneity from the culture filtrates of Streptococcus faecalis (human oral strain 0G1-10) by a procedure that comprised concentration in an Amicon Hollow Fiber System, ammonium sulfate precipitation, gel permeation chromatography, hydrophobic interaction chromatography (batch operation on phenyl-sepharose Cl-4B), followed by fast protein liquid chromatography (FPLC) on a phenyl-Superose HR 5/5 column, and finally FPLC on a Superose 12 HR 10/30 column. The enzyme is a 31.5-kDa strongly hydrophobic protein with an isoelectric point of 4.6 and a broad pH optimum of 6 to 8. The substrate specificity of the enzyme is similar to that of the mammalian membrane endopeptidase-24.11 and Streptococcus thermophilus thermolysin (EC 3.4.24.4) in hydrolyzing preferentially the Phe24-Phe25 bond in insulin B-chain, followed by cleavage of the His5-Leu6 bond. The enzyme was especially active on Azocoll and gelatin; soluble and insoluble collagens were hydrolyzed at a lower rate. S. faecalis sex pheromone-related peptides and several mammalian bioactive peptides were cleaved at sites involving pronounced hydrophobicity. The enzyme did not hydrolyze small synthetic peptide derivatives (phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg and 2-furylacryloyl-L-Leu-Gly-L-Ala) that are typically attacked by "true" bacterial collagenases. Chemical modification indicated the importance of histidyl, carboxyl, and tyrosyl groups in enzyme activity, suggesting that this enzyme may thus be classified as a metalloprotease II (EC 3.4.24.4). The enzyme is strongly inhibited by a 720-kDa factor present in rat inflammatory exudate. The pronounced ability of the enzyme to attack collagenous materials and certain bioactive peptides suggests its participation in inflammatory processes involving the presence of S. faecalis.
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PMID:Purification and substrate specificity of a strongly hydrophobic extracellular metalloendopeptidase ("gelatinase") from Streptococcus faecalis (strain 0G1-10). 253 44

A 39 kD protein has been extracted from barley flour with 0.1 M monothioglycerol at pH 5.0 and purified by (NH4)2SO4-precipitation, anion exchange and molecular sieve chromatography. It is an N-terminally blocked, non-glycosylated, single-chain protein present in at least two molecular forms of isoelectric points 5.18 and 5.22. The amino acid composition and partial sequence analysis reveal a relationship to barley endosperm Z protein which belongs to the serpin superfamily. The 39 kD protein inhibits alpha-chymotrypsin while little or no effect could be demonstrated on trypsin, subtilisin, proteinase K, S. aureus V8 protease, thermolysin or two malt thiol endoproteinases. The 39 kD protein is immunochemically related to the major protein component in beer.
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PMID:A 39 kD barley seed protein of the serpin superfamily inhibits alpha-chymotrypsin. 263 81

The positions of the inter- and intra-chain disulfide bonds of human plasma alpha 2 HS-glycoprotein were determined. alpha 2 HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)--Cys-14 (A-chain), Cys-71--Cys-82, Cys-96--Cys-114, Cys-128--Cys-131, Cys-190--Cys-201 and Cys-212--Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of alpha 2 HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the S--S bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two S--S bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second S--S loops and the end of domains A and B, and the positions of the ordered structures.
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PMID:The position of the disulfide bonds in human plasma alpha 2 HS-glycoprotein and the repeating double disulfide bonds in the domain structure. 264 41

The tripeptides Z-Pro-Leu-Gly-OEt, Z-Pro-Leu-Gly-NH2 and Z-Pro-Leu-Gly-OBzl were synthesized by thermolysin and alpha-chymotrypsin catalysis. The optimum conditions for the couplings between Z-Pro-OH and H-Leu-OEt or H-Leu-Gly-OEt catalysed by thermolysin were determined by a systematic study involving analysis of pH effect, ammonium sulfate concentration, reaction time, enzyme concentration, and relative proportion of the carboxyl and amine components. The best yield obtained for Z-Pro-Leu-OEt was 77% and for Z-Pro-Leu-Gly-OEt, 100%. Z-Pro-Leu-OEt was coupled to H-Gly-OEt, H-Gly-NH2 and H-Gly-OBzl. The best conditions to obtain Z-Pro-Leu-Gly-OEt and Z-Pro-Leu-Gly-NH2 were determined by the study of some factors that affect the reaction yield, such as organic solvent presence, substrate ratio and aqueous and organic solvent ratio. The yield obtained under optimum synthesis conditions was 55% for Z-Pro-Leu-Gly-OEt and 61% for Z-Pro-Leu-Gly-NH2. Z-Pro-Leu-Gly-OBzl was synthesized with 42% yield.
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PMID:Thermolysin and alpha-chymotrypsin mediated synthesis of tripeptides containing proline. 336 45

A common protease-resistant fragment (Mr = 27,000) was generated from purified rat hepatic actinogelin, and rat skeletal muscle and chicken gizzard alpha-actinins by limited proteolysis using thermolysin. A monoclonal antibody (A-1) which was raised against the rat hepatic actinogelin and has a cross-reactivity with rat skeletal muscle and chicken gizzard alpha-actinins was found to bind to all of the 27-kDa fragments selectively. Furthermore, one-dimensional peptide maps of the 27-kDa fragments showed a close similarity indicating the presence of some conservation in primary structure of the fragments. The 27-kDa fragments were purified to homogeneity by the same procedure using ammonium sulfate fractionation and hydrophobic chromatography. As the fragments were easily separated from other peptides during purification, they might be present as an independent structural domain. Purified 27-kDa fragments were found to bind to F-actin in a Ca2+-insensitive manner. The fragments failed to affect the low-shear viscosity of F-actin up to a molar ratio to actin monomer of 1:3.2, indicating that gelation activity of the parental molecules was lost and the fragments have only a single binding site on F-actin. Binding of the fragments to F-actin was almost completely inhibited by respective parental molecules, while binding of the whole molecules was blocked partly by their 27-kDa fragments. Thus, the interaction of the fragments with F-actin seemed to be specific, although apparent affinity was lower than the parental molecules. Considering these results, we concluded that the 27-kDa fragments are a conserved, monovalent, and Ca2+-insensitive actin-binding domain of the actinogelin and muscle alpha-actinins.
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PMID:Isolation and characterization of a conserved actin-binding domain from rat hepatic actinogelin, rat skeletal muscle, and chicken gizzard alpha-actinins. 373 25


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