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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Binding of 125I-labeled human chorionic gonadotropin to Pseudomonas maltophilia is dependent on time, temperature, and pH and the binding to this procaryotic species is hormone-specific and saturable. The equilibrium dissociation constant is 2.3 X 10(-9) M. There are no cooperative interactions between binding sites (Hill coefficient, 1.05). The number of sites is estimaated as 240 fmol/100 mug of protein. NaCl and KCl, at concentrations from 1 to 10 mM, have no effect on binding. Divalent cations (Mg2+ and Ca2+) and 1 mM EDTA inhibit hormone binding. Binding is destroyed by heat or by treatment with Pronase of alpha-chymotrypsin and is increased by phospholipase C. Binding of the labeled gonadotropin is not observed with other gram-negative organisms--e.g., Escherichia coli, Pseudomonas testosteroni, Pseudomonas aeruginosa, Enterobacter aerogenes, or Enterobacter cloacae.
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PMID:Specific gonadotropin binding to Pseudomonas maltophilia. 26 83

The presence of proteinase inhibitor has been proved in the hemolymph of a number of insect species from seven different insect orders. The amount of proteinase inhibitor in the hemolymph significantly increases after injection of inactivated bacteria into the hemocoelom of Galleria mellonella-larvae. Moreover the larvae show an increased resistance against normally lethal concentrations of trypsin, chymotrypsin, pronase P and extracellular proteinase produced by Pseudomonas aeruginosa. It is discussed that the proteinase inhibitor is one of the factors acting in the antibacterial defense system in the hemolymph of Galleria mellonella-larvae.
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PMID:[Occurrence and function of a proteinase inhibitor in the hemolymph of insects]. 81 Oct 27

Alginate is believed to be a major virulence factor in the pathogenicity of Pseudomonas aeruginosa in the lungs of patients suffering from cystic fibrosis. Guanosine diphospho-D-mannose dehydrogenase (GDPmannose dehydrogenase, EC 1.1.1.132) is a key enzyme in the alginate biosynthetic pathway which catalyzes the oxidation of guanosine diphospho-D-mannose (GDP-D-mannose) to GDP-D-mannuronic acid. In this paper, we report the structural analysis of GMD by limited proteolysis using three different proteases, trypsin, submaxillary Arg-C protease, and chymotrypsin. Treatment of GMD with these proteases indicated that the amino-terminal part of this enzyme may fold into a structural domain with an apparent molecular mass of 25-26 kDa. Multiple proteolytic cleavage sites existed at the carboxyl-terminal end of this domain, indicating that this segment may represent an exposed region of the protein. Initial proteolysis also generated a carboxyl-terminal fragment with an apparent molecular mass of 16-17 kDa which was further digested into smaller fragments by trypsin and chymotrypsin. The proteolytic cleavage sites were localized by partial amino-terminal sequencing of the peptide fragments. Arg-295 was identified as the initial cleavage site for trypsin and Tyr-278 for chymotrypsin. Catalytic activity of GMD was totally abolished by the initial cleavage. However, binding of the substrate, GDP-D-mannose, increased stability toward proteolysis and inhibited the loss of enzyme activity. GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.
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PMID:Characterization of guanosine diphospho-D-mannose dehydrogenase from Pseudomonas aeruginosa. Structural analysis by limited proteolysis. 137 Apr 73

The present study was designed to obtain further information regarding the molecular nature of the corneal receptor(s) facilitating Pseudomonas aeruginosa adhesion to cornea. Scarified adult mouse corneas in organ culture were treated for 10 or 60 min with a panel of lipase-free proteases [each at 20 micrograms ml-1 or 0.22 Units (U) ml-1, activity] including trypsin, chymotrypsin, V8 protease, elastase, subtilisin A, pronase protease and proteinase K. All of these, except proteinase K treatment (20 micrograms ml-1 for 60 min), either significantly elevated or had no effect (proteinase K 20 micrograms ml-1 for 10 min) on subsequent bacterial adhesion at 60 min following topical application of the inoculum to the scarified corneal surface. Enzyme treatment times of 10, 30 or 60 min at a higher concentration (50 micrograms ml-1) of proteinase K, significantly decreased binding at 60 min after bacterial application for each enzyme treatment time. The combined effects of proteases and lipase on bacterial binding also was examined. Eyes treated with proteinase K (20 micrograms ml-1 for 1 hr) or protease-free lipase (50,000 U ml-1 for 1 hr) alone or in combination, all reduced bacterial binding, but the effect was not additive. Trypsin or lipase alone significantly enhanced or decreased binding, respectively. In contrast, trypsin (20 micrograms ml-1 for 1 hr) followed by lipase treatment (50,000 U ml-1 for 1 hr) resulted in binding which was not significantly different than phosphate-buffered saline (PBS) control binding, indicating that the trypsin exposed receptor was lipase sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proteinase K decreases Pseudomonas aeruginosa adhesion to wounded cornea. 148 4

Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa. They possessed both enzymatic activity and residual antigenicity. Their N-terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365-404). As a result, the polypeptides contained a large portion (13-34 amino acids) of domain Ib linked to the adjacent C-terminal domain III (amino acids 405-613). The major fragment derived from subtilisin cleavage, at a final yield of 35% (S-fragment; residues 392-613; 24201 Da; pI 4.7) possessed the same level of ADP-ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37-fold higher NAD-glycohydrolase activity. Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP-ribosyltransferase activity of both exotoxin A and the S-fragment, but not the NAD-glycohydrolase activity of the S-fragment. Antibodies against the S-fragment neutralized the ADP-ribosyltransferase activity of exotoxin A. These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392-404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF-2) and show that antibodies recognize the EF-2-binding site but not the NAD(+)-binding site.
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PMID:Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa. 217 Jan 23

The primary structure of Pseudomonas cytochrome c peroxidase is presented. The intact protein was fragmented with cyanogen bromide into five fragments; partial cleavage was observed at a Met-His bond of the protein. The primary structure was established partly by automatic Edman degradations, partly by manual sequencing of peptides obtained with trypsin, thermolysin, chymotrypsin, pepsin, subtilisin and Staphylococcus aureus V8 endopeptidase. The order of the cyanogen bromide fragments was further confirmed by overlapping peptides obtained by specific cleavage of the whole protein. Pseudomonas cytochrome c peroxidase consists of 302 amino acid residues giving a calculated Mr of 33690.
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PMID:The primary structure of Pseudomonas cytochrome c peroxidase. 254 94

The occasional cleavage of the Pseudomonas cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) molecule into two well-defined fragments during the preparation of the enzyme is shown to be identical to that caused by elastase isolated from the culture solution of Pseudomonas aeruginosa. A cyanogen bromide fragmentation of proteolytically cleaved and of intact enzyme shows the cleaved peptide bond to be situated in cyanogen bromide fragment II. The amino-acid sequence of this fragment was established by sequencing peptides obtained with trypsin, thermolysin, chymotrypsin and o-iodosobenzoate. It is concluded from the sequence homology that the polypeptide chain of Pseudomonas peroxidase is wrapped around the high-potential heme in a similar manner as in high-potential cytochromes c in general. The specific proteolytic cleavage occurs at a Ser-Val (Leu-Pro) region which is assumed to be the site of attachment between enzyme and membrane. The cleavage of the Ser-Val bond renders the peroxidase molecule enzymatically inactive by impeding the conformational changes essential for the function of the native enzyme.
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PMID:Specific cleavage of Pseudomonas cytochrome-c peroxidase by elastase from Pseudomonas aeruginosa. 282 23

A testosterone-binding protein (Mr = 50,500) has been isolated from the Gram-negative bacterium Pseudomonas testosteroni. The protein was partially purified by a combination of ion exchange chromatography and chromatofocusing. Final purification was achieved by electroelution of the 50 kDa protein from SDS-polyacrylamide gels. Following renaturation from a diluted solution of guanidine-HCl, specific binding of [3H]testosterone to the purified protein was observed. The native protein has a pI of 6.8. It appears to contain 428 amino acids, 39% of which are hydrophobic. There is only one cysteine residue. Both chymotrypsin and V8 protease were used to produce peptide maps of the protein for use in future identification. The first 10 amino acids situated at the N-terminal of the protein were Ser-Pro-Phe-Asp-Leu-Arg-Pro-Leu-Ser-Gly. Testosterone binding to the protein was saturable at approximately 3.8 nmol/mg protein; the binding constant was approximately 25 nM. Unlabelled testosterone, androstenedione, 5 alpha-dihydrotestosterone and 5 beta-dihydrotestosterone were able to compete for [3H]testosterone bound to the protein; 17 beta-estradiol also competed for [3H]testosterone but to a lesser degree. Neither progesterone nor desoxycorticosterone competed for the testosterone-binding site. Binding of testosterone to the protein was stable at pH's ranging from 5.5 to 9.0 and at various temperatures ranging from 4 to 30 degrees C. The protein was unable to metabolize testosterone in either the presence or absence of the cofactor NAD.
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PMID:Isolation and characterization of a 50 kDa testosterone-binding protein from Pseudomonas testosteroni. 291 97

Human alveolar macrophages (AM) were obtained from eight normal volunteers using fiberoptic bronchoscopic lavage to explore potential interrelationships among leukocytes in pulmonary defense against infection. AM placed in monolayer tissue cultures released material into culture supernatants with the capacity to enhance the bactericidal capacity of human neutrophils. Neutrophils preexposed to supernatants killed Pseudomonas aeruginosa from 70 to 90% more efficiently than control cells (P less than 0.02). AM culture supernatants contained this material by 4 h of incubation, and in vitro stimulation of AM cultures with heat-killed P. aeruginosa further increased its production. Gel filtration of AM culture supernatants with a G-50 Sephadex column allowed isolation of a 6,000-D neutrophil-activating factor (NAF) that was resistant to heat (56 degrees C, 30 min). The isoelectric point of NAF, as determined by chromatofocusing, was approximately 7.6. Enzyme digestion of NAF specimens, prepared sequentially by gel filtration and chromatofocusing, demonstrated 50-70% loss of activity after incubations with trypsin, chymotrypsin, and neuraminidase. NAF was only minimally chemotactic and eluted from Sephadex G-50 with particles of a different molecular size than those of AM-derived chemotactic factors (i.e., approximately 10,000 D and less than 500 D). Preincubation of neutrophils with NAF resulted in greater release of superoxide anion upon their subsequent stimulation by either bacterial phagocytosis or by phorbol myristate acetate, as compared with control neutrophils stimulated in a like manner. These studies indicate that human AM secrete a heat-stable, low molecular weight basic protein, with the capacity to enhance oxidative microbicidal activity of neutrophils.
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PMID:Isolation and partial characterization of a human alveolar macrophage-derived neutrophil-activating factor. 298 53

The effect of various proteases (trypsin, chymotrypsin, subtilisin, protease 401, and thermolysin) on the mitochondrial isoenzyme (m-AST) and cytoplasmic isoenzyme (c-AST) of human and swine aspartate aminotransferase (AST;EC 2.6.1.1) was evaluated. All procedures including the reaction with proteases and the subsequent determination of the AST activity were carried out in an automatic analyzer. The mammalian c-AST was efficiently inactivated by chymotrypsin, subtilisin and protease 401 while m-AST activity decreased very slowly with these proteases. Thermolysin and trypsin showed much less effect on c-AST activity. Especially, chymotrypsin at concentrations of 0.5-1.0 g/L inactivated human c-AST almost completely but showed no detectable inactivating effect on m-AST. Thus chymotrypsin appears to be the most suitable protease for the differential determination of AST isoenzymes in human serum. Further studies on the effects of proteases with AST from other species showed that Escherichia coli AST resembled mammalian m-AST while Pseudomonas AST resembled c-AST.
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PMID:Determination of human aspartate aminotransferase isoenzymes by their differential sensitivity to proteases. 306 46


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