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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Solubilization of proteins from total membranes (a mixture of cytoplasmic and outer membranes) of Rickettsia prowazekii, a typical gram-negative bacterium, was studied using three different detergents. It was shown that isolated outer membranes and sarkosyl-insoluble material contain major polypeptides of 134, 31, 29.5 and 25 kDa as well as minor polypeptides of 78, 60, 42, and 17 kDa, while the total membranes--the same plus a great number of additional minor proteins. The material solubilized by octyl glucoside in the presence of MgCl2 contains exclusively major proteins (134, 31, 29.5, and 25 kDa). No differential solubilization takes place upon membrane treatment with octyl glucoside in the absence of Mg2+ or with Triton X-100. Rickettsial proteins are insensitive to trypsin in both whole cells and total membranes, unless the latter are presolubilized with octyl glucoside. Proteinase K degrades all of the total membrane proteins but only the 134 kDa polypeptide of whole cells. Upon immunoblotting predominantly the major outer membrane proteins (134, 31, and 20.5 kDa) and, to a lesser extent, the minor proteins (60, 42, and 17 kDa) interact with human convalescent serum.
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PMID:[Selective solubilization and biochemical analysis of R. prowazekii outer membrane proteins]. 139 Dec 24

Proteinase-inhibiting components of the coelomic fluid of the earthworm Lumbricus terrestris were examined. Inhibition of proteinases of serine, aspartate and thiol families was tested. Very strong inhibition was observed only in the case of trypsin. Additional data suggest that the inhibition is related to proteins of molar mass of 42 kDa and 20 kDa, respectively. These two proteins are present in the coelomic fluid in several forms which differ in their isoelectric points.
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PMID:New trypsin inhibitors are present in the coelomic fluid of the earthworm Lumbricus terrestris. 141 1

A synthetic peptide corresponding to the first 28 amino acids of the Alzheimer disease amyloid beta/A4 peptide (3.2 kDa) aggregated to a high molecular weight (15 kDa) on SDS/urea polyacrylamide gels. Proteinase K, V8 protease, trypsin, and endopeptidase Lys-C readily degraded the aggregate. By contrast, when digested by endopeptidase Arg-C, a new polypeptide aggregate of higher molecular weight (16 kDa) was observed on denaturing gels without degraded smaller products. The new aggregate was comprised of three peptides: an intact beta/A4(1-28) and partially degraded peptides beta/A4(1-5) plus beta/A4(6-28). The results were confirmed by treatment of beta/A4 with other arginine-specific proteases: the gamma subunit of nerve growth factor and clostripain. The results indicate that arginine-specific proteases, including a growth factor processing enzyme, can nick aggregated beta/A4(1-28) amyloid and alter the configuration to produce a more complex aggregated form. If similar highly specific proteolytic mechanisms occur in the Alzheimer disease brain, the processing may promote the formation of high molecular weight aggregates that contribute to the development of relatively insoluble senile plaque core protein.
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PMID:Arginine specific endopeptidases modify the aggregation properties of a synthetic peptide derived from Alzheimer beta/A4 amyloid. 151 20

Grapevine fanleaf nepovirus (GFLV) has a bipartite plus-sense RNA genome. Its structural and functional proteins originate from polyprotein maturation by at least one virus-encoded proteinase. Here we describe the cloning of the 24-kDa proteinase cistron located between the virus-linked protein (VPg) and the RNA-dependent RNA polymerase cistron in GFLV RNA1 (nucleotides 3966 to 4622). Proteinase expressed from this clone is able to cleave GFLV polyprotein P2 in order to produce the coat protein and a 66-kDa protein which is further processed to the 38-kDa presumed movement protein. The GFLV 24-kDa proteinase sequence contains sequence similarities with other nepovirus and comovirus proteinases, particularly at the level of the conserved domains corresponding to the hypothetical catalytic triad and to the substrate-binding pocket (amino acids 192 to 200). Site-directed mutagenesis of residues His43, Glu87, and Leu197 abolished proteinase activity. Inactivation of the enzyme is also observed if the catalytic residue Cys179 was substituted by isoleucine, but replacement by a serine at the same position produced a mutant with an activity identical to that of native proteinase. All our data show that GFLV cysteine proteinase presents structure similarities to the proteinases of cowpea mosaic virus and potyviruses but is most closely related to trypsin.
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PMID:Effects of site-directed mutagenesis on the presumed catalytic triad and substrate-binding pocket of grapevine fanleaf nepovirus 24-kDa proteinase. 151 63

The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.
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PMID:The contribution of the extracellular matrix to gravisensing in characean cells. 152 45

Proteinase specificity of a proteinaceous inhibitor of subtilisin (SSI; Streptomyces subtilisin inhibitor) can be altered so as to strongly inhibit trypsin simply by replacing P1 methionine with lysine (with or without concomitant change of the P4 residue) through site-directed mutagenesis. Now the crystal structure of one such engineered SSI (P1 methionine converted to lysine and P4 methionine converted to glycine) complexed with bovine trypsin has been solved at 2.6 A resolution and refined to a crystallographic R factor of 0.173. Comparing this structure with the previously established structure of the native SSI complexed with subtilisin BPN', it was found that (i) P1 lysine of the mutant SSI is accommodated in the S1 pocket of trypsin as usual, and (ii) upon complex formation, considerable conformation change occurs to the reactive site loop of the mutant SSI. Thus, in this case, flexibility of the reactive site loop seems important for successfully changing the proteinase specificity through mere replacement of the P1 residue.
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PMID:Crystal structure of an engineered subtilisin inhibitor complexed with bovine trypsin. 158 73

The differential cleavage of surface proteins of Borrelia burgdorferi IRS strains by several proteases was examined. Proteinase K, trypsin, chymotrypsin and thermolysin all cleaved the outer surface protein B (OspB) to undetectable levels by Coomassie Brilliant Blue staining, whereas some residual protein was detected by immunoblotting with polyclonal and monoclonal antibodies. Not even antigenic fragments were detectable by immunoblotting with 1A8 monoclonal antibody reactive with OspB. Less effective or ineffective was the cleavage of OspB by V8 protease and proteinase A, respectively. The outer surface protein A was cleaved only by proteinase K. The effect of trypsin on borreliae viability and adhesion to cultured cells was also studied. The trypsin treatment of borreliae did not impair the viability of organisms which continued to synthesize the cleaved OspB. The attachment of B. burgdorferi to HEp-2 cells was reduced by 41% after treatment with trypsin, whereas preincubation of borreliae with monoclonal antibody 1A8 and guinea pig immune serum reduced the adhesion of borreliae to the cells by 32% and 87%, respectively.
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PMID:Differential cleavage of surface proteins of Borrelia burgdorferi by proteases. 160 94

Metal-binding proteins (ceruloplasmin, transferrin, ferritin, and lactoferrin), proteinase inhibitors (alpha 1-antitrypsin, alpha 2-macroglobulin and inter-alpha-trypsin inhibitors), and albumin were assayed in synovial fluid obtained from 20 patients with rheumatoid arthritis (RA) and 15 with osteoarthritis (OA). The levels of proteinase inhibitors and metal-binding proteins, except transferrin, were significantly increased in synovial fluid from RA patients as compared with synovial fluid from OA patients. Metal-binding proteins significantly correlated with rheumatoid factor and immune complexes in synovial fluid from RA patients. Proteinase inhibitor levels also significantly correlated with C-reactive protein, and complement components. These results suggest that the raised level of metal-binding proteins and proteinase inhibitors in synovial fluid from RA patients reflect inflammatory activity, and hence may play an important role in the pathogenesis of inflammatory joint diseases.
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PMID:Correlation of metal-binding proteins and proteinase inhibitors with immunological parameters in rheumatoid synovial fluids. 170 87

Alpha 2-Macroglobulin (alpha 2M) is a plasma proteinase inhibitor that binds up to 2 mole of proteinase per mole of inhibitor. Proteinase binding or reaction with small primary amines causes a major conformational change in alpha 2M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7H11D6 is exposed. The association of alpha 2M-proteinase or alpha 2M-methylamine with alpha 2M cellular receptors is prevented by 7H11D6. In this investigation, the binding of 7H11D6 to alpha 2M was studied by electron microscopy. 7H11D6 bound to alpha 2M-methylamine and alpha 2M-trypsin but not to native alpha 2M. The structure of alpha 2M after conformational change resembled the letter "H." 7H11D6 epitopes were identified near the apices of the four arms in the alpha 2M "H" structure. 7H11D6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to alpha 2M-trypsin but not to native alpha 2M). alpha 2M conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete alpha 2M conformational change is not necessary for 7H11D6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary alpha 2M-trypsin (1 mole trypsin per mole alpha 2M instead of 2). Structures resembling the letter "H" were most common; however, each field showed some atypical molecules with arms that were compacted instead of thin and elongated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Immunoelectron microscopy studies with a monoclonal antibody directed against a receptor recognition site epitope in human alpha 2-macroglobulin. 172 27

Porcine heart muscle pyruvate dehydrogenase (PDH, EC 1.2.4.1) with subunit composition alpha 2 beta 2 catalyzes the initial decarboxylation step of an oxidative decarboxylation sequence of pyruvate. Highly purified PDH, was further activated several-fold by limited digestion with trypsin, Staphylococcus aureus V8 proteinase (V8) or papain. The activation with these proteinases required about 10 min to attain a maximal level, lasted 1/2-2 h and thereafter decreased gradually. Addition of an inhibitor of each proteinase resulted in an immediate cessation of any further changes in the enzymatic activity. The optimal pH of the proteinase-activated PDH was not affected. Proteinases increased the maximum velocity and the apparent Km values for pyruvate, but the Hill coefficients for pyruvate were unchanged. Proteinase-activated PDH was capable of associating two other component enzymes to produce large unit resembling the native complex. The Coomassie brilliant blue stained gels after SDS-PAGE showed that the PDH alpha subunit (41 kDa) was cleaved by trypsin or V8 into two major fragments (31 and 10 kDa), whereas PDH beta was unaffected. By amino-terminal sequence analyses of these fragments the trypsin cleavage sites were identified as Arg-273 and Arg-282 and the V8 cleavage sites were Glu-277 and Glu-280.
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PMID:Proteinase-catalyzed activation of porcine heart muscle pyruvate dehydrogenase and identification of its cleavage site. 173 46


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