Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proteolysis of rhodopsin in disc membranes of right-side out orientation by thermolysin, papain and St. aureus V8 protease allowed to identify two highly exposed regions of polypeptide chain located on the cytoplasmic membrane surface: carboxyl terminal sequence 321-348 and the fragment 236-241. Incubation with chymotrypsin reveals the third site on the cytoplasmic surface, 146-147, accessible to proteolytic enzymes. Frozen-thawed membranes comprise a mixture of vesicles with normal and inverted orientation. Both thermolytic and chymotryptic digests of rhodopsin in these membranes contain the polypeptide which represents the amino terminal sequence lacking the first 30 amino acid residues. Thus at least 30 amino acids from the N-terminus must protrude into the intradiscal space. One additional site was located on the intradiscal surface: papain digests rhodopsin in the inverted membranes at the position 186-187. Localization of the proteolytic cleavage sites allowed to propose a model for rhodopsin topography in disc membrane: the polypeptide chain traverses the bilayer thickness seven times; each of seven transmembrane segments containing approximately 40 amino acid residues includes a sequence of approximately 30 hydrophobic amino acids; which are probably in close contact with hydrocarbon matrix of the membrane. Hydrophobic sequences are terminated with fragments containing clusters of hydrophilic amino acids, possibly interacting with lipid polar head groups and orienting each segment in the bilayer.
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PMID:[Study of the molecular organization of visual rhodopsin in photoreceptor membranes by limited proteolysis]. 667 81

Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to restore the adhesive properties of pronase-treated cells, showing the protein nature of the molecules involved in adhesion to fibronectin. A peculiar feature of these proteins was their resistance to cleavage by trypsin. After prolonged trypsin treatment (1 mg/ml for 20 min at 37 degrees C), cells adhered and spread on fibronectin-coated dishes, even when protein synthesis was inhibited by 4 microM cycloheximide. Under these conditions only three glycoproteins (gp) of molecular weight 130,000, 120,000, and 80,000 were left on the cell surface. These were precipitated by a rabbit antiserum against BHK cells that also inhibited adhesion of trypsin-treated cells. gp120 and gp80 were left at the cell surface after mild pronase digestion (0.2 mg/ml for 20 min at 37 degrees C), under conditions not affecting adhesion. These data suggest that these glycoproteins may be involved in fibronectin-mediated cell adhesion in some yet unknown way.
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PMID:Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins. 674 66

Two classes of beta-sheet to beta-sheet packing can be distinguished in globular proteins. Both classes have beta sheets with the usual right-handed twist packed face to face. In orthogonal beta-sheet packings, the strand directions of the different beta sheets are 90 degrees to each other. Twisted beta sheets in this orientation have anticomplementary surfaces: one pair of diagonally opposite corners in the beta sheets is very close, and the other pairs of corners splay apart. At the close corners, the beta sheets are usually covalently connected: a strand that is part of one beta sheet turns through a right-handed bend to become part of the second beta sheet. The bend may occur at a beta bulge, or over a stretch of residues with a characteristic conformation, forming what we call a beta bend. Contacts between the beta sheets occur along the diagonal joining the close corners. They improve about one-fourth of the beta-sheet residues, and two-thirds of them are Val, Ile, or Leu. Elsewhere, the space between the beta sheets is filled by side chains from other parts of the protein, often alpha helices placed at the splayed corners. Examples of orthogonal beta-sheet packing are found in alcohol dehydrogenase, the acid proteases, the trypsin family, papain, staphylococcal nuclease, and thermolysin. In aligned beta-sheet packings, the angle between the strand directions of the packed beta sheets is approximately -30 degrees. In this orientation, the twisted beta-sheet surfaces are complementary. The principles governing this class of beta-sheet packings have been described previously. Here we discuss the difference and similarities of the aligned and orthogonal packing classes.
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PMID:Orthogonal packing of beta-pleated sheets in proteins. 675 82

1. The effect of various proteolytic enzymes was assayed on the adenylate cyclase activity in purified brain membrane preparations from the insect Ceratitis capitata. Trypsin, chymotrypsin, papain, thermolysin, elastase, subtilisin and prot. XIV were examined. 2. Trypsin treatment, at 37 degrees C, decreased the adenylate cyclase activity even in the presence of GppNHp that protects the activity from the thermal inactivation. 3. Residual basal, GppNHp- and F(-)-stimulated activities were similar when membrane preparations were preincubated either in the presence or in the absence of GppNHp and F-. 4. All proteolytic activities assayed on the brain membrane preparations, excepting papain, exerted an inhibition of adenylate cyclase in basal conditions. 5. The inhibition was stronger in the presence of F- than in the presence of other regulators. 6. Papain showed also a notable inhibition of adenylate cyclase in the presence of F-. 7. Phospholipase A2 treatment decreased both basal and stimulated activity; however, F(-)-sensitive activity was less affected than basal and GppNHp-sensitive activity. F(-)-stimulated activity was less affected by phospholipase A2 than either basal or GppNHp-stimulated activities. 8. Phospholipids are, then, essential for the highest basal activity, although the relationship between catalytic and nucleotide-regulatory components was unaffected by this treatment.
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PMID:Effect of proteolytic and lipolytic enzymes on the adenylate cyclase activity from brain membranes of Ceratitis capitata. 675 15

The effects of various proteases on the enzymatic or biological activity and structure of exotoxin A from Pseudomonas aeruginosa were systematically studied. The toxin was extremely resistant to treatment with various enzymes. The lethality of the toxin disappeared upon treatment with P. aeruginosa protease and elastase, thermolysin, and trypsin with a long incubation time (5h) in the presence of a high enzyme concentration (molar concentration of enzyme to toxin, 1:10 or 1:20), but was little altered by either alpha-chymotrypsin or subtilisin. The decrease of adenosine diphosphate ribosylation activity was moderate when the same treatment was used, regardless to the protease source, except in the case of papain, which was tested in the presence of reducing agents. The increase in activation of the treated toxin determined in the presence of a denaturant and a reducing agent was less than that of the intact toxin, except in the case of trypsin. The differences in disc and sodium dodecyl sulfate gel electropherograms of the toxins treated with these proteases, except for those treated with papain, suggested that the toxins had been nicked by the protease, which resulted in their degradation by sodium dodecyl sulfate treatment. Papain degraded the toxin into fragments and caused the disappearance of lethality or a marked decrease of adenosine diphosphate ribosylation activity.
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PMID:Effects of proteases on the structure and activity of Pseudomonas aeruginosa exotoxin A. 703 Sep 58

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.
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PMID:L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. 704 72

Human beta-lipotropin isolated in Hungary from frozen pituitary glands was purified by high-performance liquid chromatography in Canada. The amino acid sequence of the first 30 residues was determined. Trypsin, trypsin/papain, and trypsin/thermolysin fragments were obtained for the disputed region containing residues 9-25 of beta-lipotropin. Their amino acid composition and sequence established beyond doubt that only one human beta-lipotropin sequence is present. These results suggest the presence of only one gene coding for human pro-opiomelanocortin, the precursor of adrenocorticotropin and beta-endorphin and resolve the controversy over the sequence of human beta-lipotropin.
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PMID:The primary structure of human beta-lipotropin. Further peptide sequencing resolves the controversy and suggests the existence of only one human beta-lipotropin. 717 97

Hidden 19S IgM rheumatoid factors (RF)-i.e., RF detected in the IgM-containing fraction after separation of the serum at an acid pH-have been found in 68% of patients with seronegative juvenile rheumatoid arthritis (JRA). Inhibition studies utilizing a hemolytic assay for RF were performed to determine the specificity of hidden 19S IgM RF. Sera from 14 children with JRA were separated by gel filtration at pH 4.05. Two were seropositive for RF and 12 were seronegative; the latter had high titer hidden 19S IgM RF. The IgM-containing fractions were preincubated with monomeric human IgG, rabbit IgG, or bovine IgG, and the complement-dependent hemolytic assay ws performed. The RF in the IgM fraction from the 2 seropositive patients were inhibited most strongly by rabbit IgG, whereas hidden RF in the IgM fraction of 9 seronegative patients were inhibited markedly by human IgG (homologous IgG equal to autologous IgG), poorly by rabbit IgG, and not at all by bovine IgG. Further inhibition studies with the hidden 19S IgM RF demonstrated inhibition by the human IgG1 subclass in all patients and only minimal inhibition by the IgG3 subclass in 3 patients. Inhibition with IgG1 Fc fragments produced by papain and thermolysin digestion demonstrated inhibition by only those fragments that contained the G1m (a) antigenic area which is found in the C gamma 3 homology area of the IgG1 molecule. These data indicate that hidden 19S IgM RF possibly circulate as immune complexes bound to the IgG1 molecule and the binding chiefly occurs in th G1m (a) homology area.
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PMID:Specificity of hidden 19S IgM rheumatoid factor in patients with juvenile rheumatoid arthritis. 730 29

A novel total enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives was accomplished in low-water content systems at a preparative scale. alpha-Chymotrypsin, papain, thermolysin and bromelain adsorbed on Celite were used as catalysts. Organic solvents such as acetonitrile and ethyl acetate with small amounts of buffer added or at specific water activity were used as reaction media. Simple readily available amino acid ester derivatives were used as starting building blocks. This feature allowed the possibility of using the products in one step directly as acyl-donor ester, without any chemical or enzymatic modification, in the next enzymatic coupling. The optimal strategy for the synthesis of the enkephalin derivatives was different depending on the carboxy terminal group. The preparation of the carboxy-terminal amide derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-NH2) was achieved via 4 + 1 fragment condensation catalyzed by alpha-chymotrypsin. The carboxy-terminal ethyl ester derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-OEt) were obtained via 2 + 3 condensation catalyzed by bromelain, a quite unusual protease for peptide synthesis but more effective than papain in this coupling. Both syntheses were carried out in four enzymatic steps and one or two chemical deprotection steps routinely used in peptide synthesis. The overall yields of pentapeptide derivatives were between 40-54% of pure product.
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PMID:Enzymatic peptide synthesis in low water content systems: preparative enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives. 760 86

A rapid, cheap, and sensitive method has been developed for determining proteolytic activity of different classes of endoproteinases. The method is based on a solid-phase assay employing as substrate biotinylated gelatin adsorbed onto microtiter plates. Enzymatic activity is measured by incubating proteinase with the immobilized biotin-protein. Any remaining, undigested substrate bound to the microtiter plate is assayed with streptavidin-alkaline phosphatase. It was established that papain, pepsin, thermolysin, and trypsin all hydrolyzed the biotinylated substrate to varying degrees. Furthermore, the activity of these proteinases was blocked by their respective inhibitors. The assay presented is quick, highly reproducible, inexpensive, and useful for detecting all classes of endoproteolytic enzymes. By using different biotinylated proteins or peptides as substrates, and employing specific buffers and inhibitors, this assay may be utilized for detecting other and more specific endoproteinases.
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PMID:An assay for detecting nanogram levels of proteolytic enzymes. 766 84


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