Reagents for reversible coupling of proteins to the active centres of trypsin-like serine proteinases.
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ABSTRACT:
SUBMITTER: Kalindjian SB
PROVIDER: S-EPMC1148556 | biostudies-other | 1987 Dec
REPOSITORIES: biostudies-other
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Project description:1. A comparison of the diagonal ;maps' of chymotrypsin A and ;tosylphenylalanyl chloromethyl ketone'-inhibited chymotrypsin A showed that His-57 is alkylated specifically by this substrate analogue. 2. From peptic digests of chymotrypsinogen A and B, trypsin and elastase it was demonstrated by the diagonal electrophoretic technique that a common di-histidine cystine-bridged structure is present in all four enzymes. 3. The sequences of these peptides were determined and show that the positions of the two histidine residues relative to the disulphide bond are a common feature. Thus His-40 of chymotrypsin A is only two residues removed from CyS-42, and His-57 is adjacent to the other half of this bridge, CyS-58. 4. Considerable variation in sequence occurs about His-40, where the aromatic residues 39 and 41 of the chymotrypsins and trypsin are replaced by alanine and threonine in elastase. There is a remarkable similarity in sequence following CyS-42 and preceding CyS-58 in all four enzymes.
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Project description:The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.
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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).
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Project description:The synthesis of unsymmetrical axle [2]rotaxanes through a recently developed Ni-catalyzed C(sp3)-C(sp3) cross-coupling of redox-active esters (formed directly from carboxylic acids) and organozinc reagents (derived from alkyl bromides) is reported. The method also furnishes, as a minor product, the symmetrical axle [2]rotaxanes resulting from the homo-coupling of the organozinc half-thread. The rotaxanes are formed in up to 56% yield with the ratio of unsymmetrical rotaxane increasing with the cavity size of the macrocycle. In the absence of the redox-active ester neither rotaxane is formed, even though the homo-coupling rotaxane product does not incorporate the redox-active ester building block. A Ni(iii) intermediate is consistent with these observations, providing support for the previously postulated mechanism of the Ni-catalyzed cross-coupling reaction.
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Project description:BackgroundAnopheles aquasalis is a dipteran of the family Culicidae that is widely distributed in the coastal regions of South and Central America. This species acts as a vector of Plasmodium vivax, an important etiological agent of malaria, which represents a serious public health problem. In mosquitoes, trypsin-like serine proteases are important in blood meal digestion, immune responses and reproductive functions. The study of peptidases expressed in the mosquito midgut is essential to understanding the mechanisms of parasite-host interaction and the physiological process of nutrient digestion.MethodsOur study aimed to identify and characterize the proteolytic activities in the midgut of sugar-fed An. aquasalis females using zymographic analyses (substrate-SDS-PAGE), in-solution assays and mass spectrometry.ResultsHere, we used a zymographic analysis to further biochemically characterize the proteolytic profile of the midgut of sugar-feeding An. aquasalis females. The trypsin peptidases migrated between ~17 and ~76 kDa and displayed higher proteolytic activities between pH 7.5 and 10 and at temperatures between 37 °C and 50 °C. Four putative trypsin-like serine peptidases were identified using mass spectrometry and data mining. The molecular masses of these peptidases were similar to those observed using zymography, which suggested that these peptidases could be responsible for some of the observed proteolytic bands.ConclusionsTaken together, our results contribute to the gene annotation of the unknown genome of this species, to the tissue location of these peptidases, and to the functional prediction of these crucial enzymes, which all impact further studies of this species.
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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.
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