Degradation of myofibrillar proteins by trypsin-like serine proteinases.
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ABSTRACT:
SUBMITTER: Kay J
PROVIDER: S-EPMC1163641 | biostudies-other | 1982 Feb
REPOSITORIES: biostudies-other
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Project description:1. Hydrolysis of the myosins from smooth and from skeletal muscle by a rat trypsin-like serine proteinase and by bovine trypsin at pH 7 is compared. 2. Proteolysis of the heavy chains of both myosins by the rat enzyme proceeds at rates approx. 20 times faster than those obtained with bovine trypsin. Whereas cleavage of skeletal-muscle myosin heavy chain by both enzymes results in the generation of conventional products i.e. heavy meromyosin and light meromyosin, the heavy chain of smooth-muscle myosin is degraded into a fragment of mol. wt. 150000. This is dissimilar from heavy meromyosin and cannot be converted into heavy meromyosin. It is shown that proteolysis of the heavy chain takes place in the head region. 3. The 'regulatory' light chain (20kDa) of smooth-muscle myosin is degraded very rapidly by the rat proteinase. 4. The ability of smooth-muscle myosin to have its ATPase activity activated by actin in the presence of a crude tropomyosin fraction on introduction of Ca2+ is diminished progressively during exposure to the rat proteinase. The rate of loss of the Ca2+-activated actomyosin ATPase activity is very similar to the rate observed for proteolysis of the heavy chain and 3-4 times slower than the rate of removal of the so-called 'regulatory' light chain. 5. The significance of these findings in terms of the functional organization of the smooth muscle myosin molecule is discussed. 6. Since the degraded myosin obtained after exposure to very small amounts of the rat proteinase is no longer able to respond to Ca2+, i.e. the functional activity of the molecule has been removed, the implications of a similar type of proteolysis operating in vivo are considered for myofibrillar protein turnover in general, but particularly with regard to the initiation of myosin degradation, which is known to take place outside the lysosome (i.e. at neutral pH).
| S-EPMC1163640 | biostudies-other
Project description:In order to extend the scope for the application of acyl-enzymes as fibrinolytic agents, p-amidinophenyl ester enzyme substrates were prepared that gave stabilized acyl-enzymes capable of being coupled to other proteins. Coupling was achieved by reaction of protein thiol functions with a 2-pyridyldithio moiety within the acyl group. Acyl-enzymes derived from such substrates were stable enough to permit isolation of reversible conjugates between proteins and the active centres of plasminogen activators. Hydrolytic release of active enzyme from a conjugate of human immunoglobulin G and urokinase could be demonstrated.
| S-EPMC1148556 | biostudies-other
Project description:1. The procedure of Barrett [(1973) Biochem. J.131, 809-822] for isolating cathepsins B and D from human liver was modified for use with rat liver and skeletal muscle. The purified enzymes appeared to be similar to those reported in other species. 2. Sephadex G-75 chromatography of concentrated muscle extract resolved two peaks of cathepsin B inhibitory activity, corresponding to molecular weights of 12500 and 62000. 3. The degradation of purified myofibrillar proteins by cathepsins B and D was clearly demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After incubation with enzyme, the polypeptide bands representing the substrates decreased in intensity and lower molecular weight products appeared. 4. Cathepsins B and D, purified from either rat liver or skeletal muscle, were shown to degrade myosin, purified from either rabbit or rat muscle. Soluble denatured myosin was degraded more extensively than insoluble native myosin. Degradation by cathepsin B was inhibited by lack of reducing agent, or by myoglobin, iodoacetic acid and leupeptin, but not by pepstatin. The same potential modifiers were applied to cathepsin D, and only pepstatin produced inhibition. 5. Rat liver cathepsin B had a pH optimum of 5.2 on native rabbit myosin. The pH optimum of cathepsin D was 4.0, with a shoulder of activity about 1pH unit above the optimum. 6. Rat liver cathepsins B and D were demonstrated to degrade rabbit F-actin at pH5.0, and were inhibited by leupeptin and pepstain, respectively. 7. The degradation of myosin and actin by cathepsin D was more extensive than that by cathepsin B.
| S-EPMC1183729 | biostudies-other
Project description:BackgroundDermatomyositis and polymyositis are the best known idiopathic inflammatory myopathies (IIMs). Classic histopathologic findings include the infiltration of inflammatory cells into muscle tissues. Neutrophil serine proteinases (NSPs) are granule-associated enzymes and play roles in inflammatory cell migration by increasing the permeability of vascular endothelial cells. In this study, we aimed to find the roles of NSPs in pathogenesis of IIMs.MethodsRNA and DNA were isolated to measure the relative expression of NSPs and their methylation levels. The expression of NSPs in serum and muscle tissues was tested by enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence, respectively. Serum from patients was used to culture the human dermal microvascular endothelial cells (HDMECs), and then we observed the influence of serum on expression of VE-cadherin, endothelial cell tube formation, and transendothelial migration of peripheral blood mononuclear cells (PBMCs).ResultsWe found that the expression of NSPs was increased in PBMCs, serum, and muscle tissues of IIM patients; these NSPs were hypomethylated in the PBMCs of patients. Serum NSPs were positively correlated with clinical indicators of IIM patients, including lactic dehydrogenase, erythrocyte sedimentation rate, C-reactive protein, immunoglobulin G, immunoglobulin M, and immunoglobulin A. Patients with anti-Jo-1, with anti-Ro-52, or without interstitial lung disease had lower levels of proteinase 3. Serum NSPs degraded the VE-cadherin of HDMECs, and serum NSP application increased the permeability of HDMECs.ConclusionsOur studies indicate, for the first time, that NSPs play an important role in muscle inflammatory cell infiltration by increasing the permeability of vascular endothelial cells in IIM patients.
| S-EPMC6034343 | biostudies-literature
Project description:BackgroundWe have recently reported the expression of murine Implantation Serine Proteinase genes in pre-implantation embryos (ISP1) and uterus (ISP1 and ISP2). These proteinases belong to the S1 proteinase family and are similar to mast cell tryptases, which function as multimers.ResultsHere, we report the purification and initial characterization of ISP1 and 2 with respect to their physico-chemical properties and physiological function. In addition to being co-expressed in uterus, we show that ISP1 and ISP2 are also co-expressed in the pre-implantation embryo. Together, they form a heterodimer with an approximate molecular weight of 63 kD. This complex is the active form of the enzyme, which we have further characterized as being trypsin-like, based on substrate and inhibitor specificities. In addition to having a role in embryo hatching and outgrowth, we demonstrate that ISP enzyme is localized to the site of embryo invasion during implantation and that its activity is important for successful implantation in vivo.ConclusionOn the basis of similarities in structural, chemical, and functional properties, we suggest that this ISP enzyme complex represents the classical hatching enzyme, strypsin. Our results demonstrate a critical role for ISP in embryo hatching and implantation.
| S-EPMC1713233 | biostudies-literature
Project description:The inhibition of six serine proteinases by a tumour-associated trypsin inhibitor (TATI) was studied using synthetic peptide substrates. Physiological concentrations of TATI inhibited the amidolytic activities of trypsin, plasmin, urokinase and tissue plasminogen activator (tPA). Chymotrypsin, kallikrein and thrombin were also inhibited, but by much higher concentrations of TATI. The ability of TATI to inhibit trypsin, plasmin, urokinase and tPA suggests that it has a role in proteolytic processes in vivo involving these enzymes.
| S-EPMC1135171 | biostudies-other
Project description:Periodontitis is a chronic destructive infection of the tooth-supportive tissues, which is caused by pathogenic bacteria such as Actinobacillus actinomycetemcomitans. A severe form of periodontitis is found in Papillon-Lefèvre syndrome (PLS), an inheritable disease caused by loss-of-function mutations in the cathepsin C gene. Recently, we demonstrated that these patients lack the activity of the polymorphonuclear leukocyte (PMN)-derived serine proteinases elastase, cathepsin G, and proteinase 3. In the present study we identified possible pathways along which serine proteinases may be involved in the defense against A. actinomycetemcomitans. Serine proteinases are capable to convert the PMN-derived hCAP-18 into LL-37, an antimicrobial peptide with activity against A. actinomycetemcomitans. We found that the PMNs of PLS patients released lower levels of LL-37. Furthermore, because of their deficiency in serine proteases, the PMNs of PLS patients were incapable of neutralizing the leukotoxin produced by this pathogen, which resulted in increased cell damage. Finally, the capacity of PMNs from PLS patients to kill A. actinomycetemcomitans in an anaerobic environment, such as that found in the periodontal pocket, seemed to be reduced. Our report demonstrates a mechanism that suggests a direct link between an inheritable defect in PMN functioning and difficulty in coping with a periodontitis-associated pathogen.
| S-EPMC1594863 | biostudies-literature
Project description:Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our current knowledge of their components is limited-prophenoloxidase-activating proteinases (PAPs) are among the few hemolymph proteinases (HPs) with known functions. To identify components of proteinase systems in the hemolymph of Manduca sexta, we amplified cDNAs from larval fat body or hemocytes using degenerate primers coding for two conserved regions in S1 family serine proteinases. PCR yielded fragments encoding seven known (HP1-HP4, PAP-1, PAP-2 and PAP-3) and 18 unknown (HP5-HP22) serine proteinases. We screened cDNA libraries and isolated clones for 17 of the newly discovered HPs (HP5-HP22 except for HP11) and prepared antibodies to 14 recombinant proteins (HP6, HP8-HP10, HP12, HP14-HP19, HP21 and HP22). Fourteen of the HPs contain regulatory clip domain(s) at their amino-terminus--HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, HP22 and PAP-1 have one, whereas HP12, HP15, PAP-2 and PAP-3 have two clip domains. Multiple sequence alignment of catalytic domains in these and other arthropod serine proteinases provided useful clues for future functional analysis. Northern blot and reverse transcription PCR (RT-PCR) analyses showed increases in HP2, HP7, HP9, HP10, HP12-HP22 mRNA levels at 24h after a bacterial challenge, and immunoblot analysis confirmed elevated concentrations of HP12, HP14-HP19, HP21 and HP22 proteins in plasma in response to injected bacteria. Hemocytes express HP13 and HP18; fat body produces HP12, HP20-HP22; both tissues synthesize the other HPs. These results collectively indicate the existence of a complex serine proteinase network in M. sexta hemolymph, predicted to mediate rapid defense responses upon wounding and/or microbial infection.
| S-EPMC2042105 | biostudies-literature
Project description:Seven arginylfluoroalkanes ('arginine fluoroalkyl ketones') were synthesized by using a modified Dakin-West procedure. The structure of benzoyl-Arg-CF2CF3 was analysed by 19F-n.m.r. spectroscopy and m.s. and the compound was shown to exist primarily as a hydrate or cyclic carbinolamine. Arginylfluoroalkanes are good inhibitors of blood-coagulation serine proteinases and were found to be slow-binding inhibitors for bovine trypsin with Ki values of 0.2-56 microM. Benzoyl-Arg-CF2CF3 was the best inhibitor for bovine thrombin and human Factor XIa, and inhibited thrombin and Factor XIa competitively with Ki values of 13 microM and 62 microM respectively. The best inhibitor for pig pancreatic kallikrein was p-toluoyl-Arg-CF3, with a Ki value of 35 microM. Benzoyl-Arg-CF3 and benzoyl-Arg-CF2CF3 inhibited human plasma kallikrein competitively, with Ki values of 50 microM. None of the seven arginylfluoroalkanes was a good inhibitor of human factor Xa or of Factor XIIa. The arginylfluoroalkanes were tested in the prothrombin time (PT) and activated partial thromboplastin time (APTT) coagulant assays. Two fluoroketones, benzoyl-Arg-CF2CF3 and 1-naphthoyl-Arg-CF3, had significant anticoagulant activity. Benzoyl-Arg-CF2CF3 was found to prolong the PT 1.8-fold at 120 microM and to prolong the APTT 2.4-fold at 90 microM, whereas 1-naphthoyl-Arg-CF3 only prolonged the APTT 1.7-fold at 100 microM.
| S-EPMC1136917 | biostudies-other
Project description:Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.
| S-EPMC8124345 | biostudies-literature