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)

The complete amino acid sequence of the cardiac stimulatory and haemolytic protein tenebrosin-C, from the Australian sea anemone Actinia tenebrosa, has been determined by Edman degradation of the intact molecule and fragments produced by treatment of the polypeptide chain with cyanogen bromide and enzymatic cleavage with endoproteinase Asp-N, thermolysin and trypsin. The molecule is a single-chain polypeptide consisting of 179 amino acid residues with a calculated molecular mass of 19,797 Da. Tenebrosin-C shows a high degree of amino acid sequence similarity (63%) with Stoichactis helianthus cytolysin III [Blumenthal, K. M. and Kem, W. R. (1983) J. Biol. Chem. 258, 5574-5581] and is identical to a partial sequence (90 residues) reported for equinatoxin, a cardiostimulatory and haemolytic protein isolated from the European sea anemone Actinia equina [Ferlan, I. and Jackson, K. (1983) Toxicon Suppl. 3, 141-144]. No amino acid sequence similarity was detected between tenebrosin-C and other protein sequences stored in available databases. The predicted secondary structure of tenebrosin-C suggests that it is a compact, highly structured molecule.
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PMID:Complete amino acid sequence of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone Actinia tenebrosa. 197 96

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

1. Bolesatine is a toxic protein (LD50 oral 3.3 mg/kg in mice) isolated from the mushroom Boletus satanas Lenz, which inhibits protein synthesis in vitro. It induces gastroenteritis in human. 2. 14C-Bolesatine, given orally to rats (30 micrograms/kg), is distributed in the gastrointestinal, tract, kidney, liver and, to a lesser extent, in the thymus, spleen and lung. Bolesatine is eliminated in faeces and urine (80% in 24h). 3. The material excreted in urine is not proteolysed, and no protease (trypsin, chymotrypsin, pronase, proteinase K, Staphylococcus aureus (strain V8) protease and pepsin) is found to hydrolyse bolesatine in either its native or denatured form. However, thermolysin hydrolysed denatured bolesatine to a protein having a Mr of about 55 kD. 4. Bolesatine is found in all the following rat liver and kidney subcellular fractions: cytoplasm, mitochondria, ribosomes, microsomes and nuclei.
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PMID:Disposition of the toxic protein, bolesatine, in rats: its resistance to proteolytic enzymes. 200 68

Purified Escherichia coli Shiga-like toxin II variant (SLT-IIv) was characterized with regard to selected physical, chemical, and biological properties. N-terminal amino acid sequencing confirmed the identities of 33,000-, 27,500-, and 7,500-molecular-weight (MW) bands seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified SLT-IIv as the A subunit, A1 fragment, and B subunit, respectively. The arginine-serine bond between amino acids 247 and 248 in the A subunit was determined to be the site for proteolytic cleavage into A1 and A2 fragments. As with other SLTs, gel filtration chromatography of SLT-IIv gave estimates of the MW of holotoxin that were variable and less than predicted for a 1-A-subunit-5-B-subunit configuration. The MWs were estimated to be 40,000 and 43,000 by Sephacryl S-100 and Sephadex G-100 and less than 2,000 by Bio-Sil Sec-250 gel filtration chromatography. The isoelectric point of SLT-IIv holotoxin was 9.0. Cytotoxicity of SLT-IIv was destroyed by heating at 65 degrees C for 30 min and by incubation with 2-mercaptoethanol and dithiothreitol, but it increased 30-fold by incubation with trypsin, chymotrypsin, or pepsin and 2-fold by incubation with thermolysin. SLT-IIv cytotoxic activity was stable at neutral and alkaline pH values but was lost at pHs 3, 4, and 5. SLT-IIv was stable in fluid from the anterior and posterior small intestines of pigs but was not enterotoxic in pig intestinal loops. The smallest doses of SLT-IIv that inhibited protein synthesis in porcine endothelial cells and Vero cells were 0.1 ng and 0.1 fg, respectively.
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PMID:Physicochemical and biological properties of purified Escherichia coli Shiga-like toxin II variant. 200 12

Association of low-density lipoproteins (LDL) with arterial chondroitin sulfate proteoglycans (CSPG) appears to contribute to their deposition in the extracellular intimal compartment and to its internalization by macrophages. CSPG and LDL interact by ionic bridges with formation of soluble and insoluble complexes. We studied the alterations on LDL structure induced by its association with arterial CSPG and other glycosaminoglycans (GAG). In soluble complexes, at low and at physiological ionic strength, arterial CSPG and sulfated GAG modify the kinetics of apoB-100 proteolysis by trypsin. However, less marked alterations in the peptide patterns were observed with proteinase V8 and almost none with thermolysin. This is indirect evidence that the presence of CSPG and GAG modified the exposure of polar regions of apoB-100 in LDL. Competitive binding experiments with agarose-bound heparin and soluble GAG also suggest that after formation of insoluble complexes with arterial CSPG and resolubilization the exposure of Lys, Arg-rich segments of apoB-100 is increased. Results from differential scanning calorimetry and differential thermal spectrophotometry showed that the CSPG and GAG-induced modifications reduced the thermal stability of the surface and core in LDL. If present in vivo, the structural alterations of polar segments of the LDL protein moiety may influence the outcome of its interaction with the arterial mesenchyma.
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PMID:Modifications of low-density lipoprotein induced by arterial proteoglycans and chondroitin-6-sulfate. 201 99

Gluten from the wheat variety Rektor was extracted with 70% aqueous ethanol. The residual protein (glutenin) was hydrolysed with trypsin. The partial hydrolysate was separated into seven fractions by gel permeation chromatography on Sephadex G 25. Four cystine-containing peptides were isolated from fraction 5 by reversed-phase high-performance liquid chromatography on ODS-Hypersil. The cystine peptides were detected by differential chromatography of the non-reduced and the reduced samples. The primary structure of the peptides was solved by the Edman degradation reaction and by partial hydrolysis with thermolysin. Three peptides derive from the alpha 2- and beta-purothionins. The structure of the fourth peptide was determined as (Formula; see text) This sequence corresponds to positions 44-48 of known sequences of the high molecular weight (HMW) subunits 9, 10, and 12. Since Rektor contains the HMW subunits 9 and 10, it could be concluded that two HMW subunits 9 or 10, or one subunit 9 and one subunit 10 were linked parallel via two disulphide bridges.
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PMID:Disulphide bonds in wheat gluten: isolation of a cystine peptide from glutenin. 203 94

Sphingosine-1-phosphate lyase is responsible for the ultimate step in sphingolipid breakdown, converting phosphorylated long chain bases into ethanolamine phosphate and a fatty aldehyde. Using tritiated dihydrosphingosine-1-phosphate, prepared enzymatically from [4,5-3H]dihydrosphingosylphosphocholine, we have reinvestigated the subcellular distribution of this enzyme in rat liver. Upon cell fractionation by differential centrifugation, the enzyme showed a microsomal distribution. Further separation of the microsomal fraction by sucrose gradient centrifugation confirmed an association with the endoplasmic reticulum. By means of constrained nonlinear regression, no evidence for a significant association with mitochondrial membranes, as reported previously (Stoffel, W., LeKim, D., and Sticht, G. (1969) Hoppe Seyler's Z. Physiol. Chem. 350, 1233-1241), nor with other cell compartments was found. The lyase activity, which appeared to be sensitive to different detergents, but not to Triton X-100, was not latent. It could be solubilized with Triton X-100, but not by high ionic strength, indicating that it is an integral membrane protein whose catalytic site is most probably exposed to the cytosol. Treatment of intact microsomal vesicles with trypsin or thermolysin inactivated the lyase activity, confirming that its catalytic site(s) or other domains essential for activity face the cytosol.
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PMID:Subcellular localization and membrane topology of sphingosine-1-phosphate lyase in rat liver. 206 24

A nucleophilic group in the active site of aldehyde dehydrogenase, which covalently binds the aldehyde moiety during the enzyme-catalyzed oxidation of aldehydes to acids, was acylated with the chromophoric aldehyde trans-4-(N,N-dimethylamino)cinnamaldehyde (DACA). Acyl-enzyme trapped by precipitation with perchloric acid was digested with trypsin, and the peptide associated with the chromophoric group was isolated and shown to be Gln-Ala-Phe-Gln-Ile-Gly-Ser-Pro-Trp-Arg. After redigestion with thermolysin, the chromophore was associated with the C-terminal hexaresidue part. If the chromophore is attached to this peptide, serine would be expected to bind the aldehyde and lead to the required acylated derivative. Differential labeling experiments were performed in which all free thiol groups on the acylated enzyme were blocked by carboxymethylation. The acyl chromophore was then removed by controlled hydrolysis and the protein reacted with [14C]iodoacetamide. No 14C-labeled tryptic peptides were isolated, suggesting that the sulfur of a cysteine cannot be the acylated residue in the precipitated acyl-enzyme.
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PMID:Evidence for reactivity of serine-74 with trans-4-(N,N-dimethylamino)cinnamaldehyde during oxidation by the cytoplasmic aldehyde dehydrogenase from sheep liver. 210 32

The overall folding of neutral protease from Bacillus subtilis has been predicted by computer-aided modelling, taking as a basis the known three-dimensional structure of thermolysin. As expected from the 50% similarity of sequence between the two proteins, the structure of B. subtilis protease is similar to that of thermolysin, including the two-domain topology and location of elements of regular secondary structure (helices and strands), whereas specific differences were predicted in loop regions. A protruding and loose loop predicted in B. subtilis has been detected also experimentally by a limited proteolysis approach. Incubation of B. subtilis protease at pH 9.0 for 24 h at room temperature with trypsin at 20:1 ratio (by mass) leads to a specific and almost quantitative fission of the Arg214-Asn215 peptide bond located in a highly exposed, and thus probably flexible, loop of the protease. On the other hand, thermolysin was completely resistant to tryptic hydrolysis when reacted under identical conditions. The 'nicked' B. subtilis protease can be isolated by gel filtration chromatography at neutral pH, whereas the two constituting fragments 1-214 and 215-300 are separated under protein-denaturing conditions. Overall, these results indicate that the limited proteolysis approach can pinpoint a peculiar difference in surface structure between the two similar protein molecules of B. subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins.
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PMID:Structural features of neutral protease from Bacillus subtilis deduced from model-building and limited proteolysis experiments. 211 Aug 95

The primary structures of ascidian trypsin inhibitors (iso-inhibitors I and II) were reported in the preceding paper (Kumazaki, T. et al. (1990) J. Biochem. 107, 409-413). Both of them have eight half-cystines in a molecule composed of 55 amino acid residues with a sequence showing no extensive homology to other known protease inhibitors. To locate the four disulfide bridges in the molecule, native inhibitor I was digested with thermolysin to yield cystine-containing peptides. The peptides were separated from each other by reversed-phase HPLC. A core peptide still containing six closely located half-cystines (e.g. -Cys-Arg-Cys and -Cys-Cys-) was further digested with Streptomyces griseus trypsin for cleavage of the Arg-Cys bond. On the other hand, the Cys-Cys bond was split by applying manual Edman degradation to the core peptide. Amino acid composition analyses of the resulting cystine peptides allowed us to define the whole disulfide bridge structure in the parent molecule. The topological relation between the disulfide loops and the reactive site suggested that the ascidian trypsin inhibitor may be classified as a member of the Kazal-type inhibitor family.
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PMID:Disulfide bridge structure of ascidian trypsin inhibitor I: similarity to Kazal-type inhibitors. 211 16


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