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)

The enzymatic activities of native myosin light chain kinases are subject to modification by interaction with Ca2(+)-calmodulin (CaM). The interaction between myosin light chain kinase isolated from turkey gizzard (tgMLCK) and calmodulin isolated from bovine testes (CaMbt) and wheat germ (CaMwg) has been examined by means of the intrinsic tryptophan fluorescence of tgMLCK and the fluorescence of extrinsic fluorescent labels located at Cys-27 and Tyr-139 of CaMwg and Tyr-99 of CaMbt. Static and dynamic fluorescence measurements provide evidence for the involvement of the former two sites in the zone of contact with lesser involvement of the site marked by the probe at Tyr-99. Complex formation protected the primary cleavage site in CaMbt (Lys-77) from proteolysis by trypsin. These results are consistent with involvement of the N- and C-terminal lobes of CaM in stabilization of the complex with tgMLCK, but cannot rule out participation of the connecting strand in the interaction. CD measurements extending to 175 nm, obtained using synchroton radiation, indicate the following secondary structure content for tgMLCK: 17 +/- 2% alpha-helix, 22 +/- 3% antiparallel beta-sheet, 3 +/- 1% parallel beta-sheet, 24 +/- 2% beta-turns, and 34 +/- 2% random coil. Similar measurements of the CD spectra of CaMbt and of the 1:1::CaMbt:tgMLCK complex presently indicate that neither protein undergoes major secondary structure rearrangement during their interaction, although subtle changes in the CD spectrum of tgMLCK appear to be correlated with the interaction with CaM.
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PMID:The secondary structure of turkey gizzard myosin light chain kinase and the nature of its interaction with calmodulin. 208 Dec 70

Relaxation of rat aorta segments with sodium nitroprusside and endothelium-dependent vasodilators, such as acetylcholine, histamine, A23187, ATP, thrombin, and trypsin, is associated with cyclic-GMP (cGMP) accumulation in a concentration- and time-dependent fashion. With rat aorta segments, these agents also increase cyclic GMP-dependent protein-kinase activity and alter the incorporation of 32P into numerous smooth-muscle proteins. Identical patterns of protein phosphorylation were observed with both classes of relaxants on two-dimensional gel electrophoresis and autoradiography. The effects of nitroprusside were observed with or without the endothelium present. In contrast, the effects of the endothelium-dependent agents on all of these parameters (cGMP, cGMP-dependent protein kinase and protein phosphorylation) required the integrity of the endothelium. Various inhibitors of phospholipase and lypoxygenase prevented the effects of the endothelium-dependent agents, suggesting that a metabolite of arachidonic acid is the endothelium-relaxant factor and responsible for guanylate-cyclase activation. A smooth-muscle protein with decreased 32P incorporation after treatment with either class of relaxants has been identified as myosin light chain. A model is presented suggesting that the effects of endothelium-dependent vasodilators and directly acting nitrovasodilators converge at the level of guanylate-cyclase activation and cGMP accumulation, which explains the common biochemical and physiological effects on smooth muscle of these two classes of vasodilators.
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PMID:Role of cyclic-GMP in relaxations of vascular smooth muscle. 240 83

The formation and maintenance of stress fibers in cultured mesangial cells is associated with myosin light chain phosphorylation [Kreisberg et al. Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18): F227-F235, 1985], a biochemical indicator for activation of actin-myosin interactions. Agents that elevate intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) (e.g., isoproterenol) fragment stress fibers and cause myosin light chain dephosphorylation, whereas the addition of contractile agents such as arginine vasopressin (AVP) and prostaglandin E2 (PGE2) reverses these changes. Because stress fiber development in cultured cells is correlated with tight cell to substrate adhesion, we wanted to examine whether vasoactive agents have an effect on mesangial cell adhesion. Both isoproterenol and dibutyryl cAMP (DBcAMP) reduced mesangial cell adherence as measured by a trypsin assay (% detached cells: control 11 +/- 2.4%; isoproterenol plus isobutylmethylxanthine (IBMX) = 48.3 +/- 7.4%; DBcAMP = 29.3 +/- 3.7%; DBcAMP-IBMX = 73 +/- 4.4%). The areas of focal (adhesive) contacts between the cell and substratum as observed by interference-reflexion microscopy were also reduced, being replaced by areas of greater separation (% of the surface in contact with the substratum: control = 7.4 +/- 0.8%; isoproterenol-IBMX = 2.9 +/- 1.1%). Addition of PGE2 or AVP to the incubation medium containing the cAMP-elevating agents prevented the above changes. PGE2 or AVP alone increased mesangial cell adhesion (% detached cells: control 11 +/- 2.4%; PGE2 = 6.8 +/- 0.5%; AVP = 5.1 +/- 1.2%).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Vasoactive agents affect mesangial cell adhesion. 242 54

Monoclonal antibodies raised against chicken gizzard smooth muscle myosin light chain kinase were used for immunological and structural studies of this enzyme. Epitope mapping of trypsin-digested chicken gizzard enzyme showed that MM-1, 2, 3, 4, 5, 6, and 7 bind to 65 kDa (trypsin-digested) and 60 kDa (chymotrypsin-digested) fragments which contain the catalytic domain of the kinase. Kinetic analysis demonstrated that MM-7 inhibited kinase activity competitively with respect to ATP and noncompetitively with respect to myosin light chain, thereby indicating that MM-7 binds at or near the ATP binding site of the enzyme. Immunoblot analysis revealed that all these antibodies (MM-1 to 12) reacted with the enzyme (130 kDa) from intestinal and vascular smooth muscles, whereas 5 (MM-1, 3, 4, 6, and 9) or 3 (MM-1, 3, and 4) of 12 antibodies did not cross-react with chicken cardiac muscle or with blood platelet myosin light chain kinase (130 kDa), respectively. None of these antibodies showed cross-reactivity against skeletal muscle myosin light chain kinase. As for mammalian species, MM-11 and 12 reacted with myosin light chain kinase of vascular smooth muscle (140 kDa) and MM-11 cross-reacted with the enzyme (140 kDa) from cardiac muscle of rat and rabbit. These data suggest the existence of at least 4 subspecies of myosin light chain kinase in chicken tissues and the heterogeneity of tissue- and species-specific isozyme forms.
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PMID:Monoclonal antibody assessment of tissue- and species-specific myosin light chain kinase isozymes. 247 31

Following our study in Balb/c 3T3 cells and other cultured fibroblasts of the changes in myosin light chain phosphorylation associated with alterations in cell shape, attachment, and receptor patching, we have now determined the corresponding changes in cytoskeletal myosin distribution, and in the cellular calcium concentration, since this might, in part, mediate such responses. Immunofluorescence microscopy showed that myosin assembly into ordered forms such as actomyosin bundles and myosin sheath almost always correlated with previously shown high phosphorylation levels of myosin regulatory light chain, whereas diffuse distributions usually correlated with low or undetectable levels. An exception was observed in treatment to alter cellular cAMP levels when, in a biphasic response, assembly was correlated inversely with the phosphorylation states shown previously. Fluorescent indicators for intracellular calcium concentration, [Ca++]i, showed that myosin disassembly by trypsin or EGTA acting externally on the cells was preceded by a transient increase in [Ca++]i. For EGTA this was associated with transient recruitment of myosin into dorsal sheath structure as well as the transient enhancement of phosphorylation shown earlier. Blockage of EGTA-induced disassembly could be achieved by azide, which also caused an immediate increase in [Ca++]i and inhibited its subsequent decline. Trypsin-induced dephosphorylation did not appear to involve an eventual reduction of [Ca++]i. Therefore, in many but not all of the systems studied, correlated changes were observed in myosin assembly, [Ca++]i, and the myosin phosphorylation levels shown earlier.
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PMID:Myosin regulation and calcium transients in fibroblast shape change, attachment, and patching. 254 42

The relaxant effects of amiloride and its analogues, benzamil, 5-(N,N-diethyl)-amiloride (DEAM) and 5-(N-ethyl-N-isopropyl)-amiloride (EIAM), were investigated using smooth muscle of guinea-pig taenia caeci and chicken gizzard. High K+-induced contractions of intact taenia and gizzard were inhibited by these compounds (1-100 microM) with the order of potency; benzamil greater than or equal to EIAM greater than DEAM greater than amiloride. Contractions of permealized taenia and gizzard were also inhibited by these compounds at concentrations 8-35 times higher than those needed to inhibit the contractions of intact tissues. These compounds inhibited 20 K myosin light chain (MLC) phosphorylation at the concentrations needed to inhibit the contraction in the permealized muscles. Calmodulin (CaM) activity, as monitored by erythrocyte membrane (Ca2+ + Mg2+)-ATPase and phosphodiesterase activities, was inhibited by DEAM and EIAM at similar concentrations as those to inhibit the MLC phosphorylation. Benzamil also inhibited CaM activity at concentrations 4-8 times higher than those required to inhibit MLC phosphorylation. However, amiloride failed to inhibit CaM activity. Among these compounds, amiloride and benzamil inhibited Ca2+/CaM-independent MLC phosphorylation due to trypsin-treated MLC kinase. Taenia tissue gradually accumulated these compounds and the tissue/medium ratio exceeded 3.5-17 after a 3-hr incubation period. These results indicate that amiloride and its analogues inhibit smooth muscle contraction mainly by the direct inhibition of MLC phosphorylation. The inhibitory effect of amiloride may be attributable to the inhibition of MLC kinase, whereas the inhibitory effect of DEAM and EIAM may largely be attributable to the inhibition of CaM. Benzamil may inhibit contraction by the inhibition of both MLC kinase and CaM. Differences in the drug-sensitivity between intact and permealized tissues may be attributable to the difference in drug accumulation by the cell.
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PMID:Direct inhibition of contractile apparatus by analogues of amiloride in the smooth muscle of guinea-pig taenia caecum and chicken gizzard. 293 May 91

Three forms of 20-kDa myosin light chain (MLC), unphosphorylated, monophosphorylated, and diphosphorylated MLC (designated 20K, 20K-P, and 20K-PP) were demonstrated in thrombin-stimulated human platelets by two different gel electrophoretic methods: in the presence of glycerol urea or in two dimensions (isoelectric and sodium dodecyl sulfate). The diphosphorylation of platelet 20-kDa MLC increased, dose dependently, up to 0.4 U/ml thrombin and reached 25% of platelet 20-kDa MLC. After mono- or diphosphorylated 20-kDa MLC from thrombin-stimulated platelets was digested with trypsin, the analysis using two-dimensional peptide mapping demonstrated that two different sites were phosphorylated by MLC kinase and protein kinase C, as noted in the case of 12-O-tetradecanoylphorbol-13-acetate-stimulated platelets (M. Naka, et al. (1983) Nature (London) 306, 490-492). The more rapid monophosphorylation was catalyzed preferentially by MLC kinase while the slower and additional phosphorylation was catalyzed mainly by protein kinase C. These results suggest the importance of distinguishing multiple site phosphorylation of 20-kDa MLC in thrombin-activated human platelets.
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PMID:Two phosphorylated forms of myosin in thrombin-stimulated platelets. 335 50

We investigated the effects of a newly synthesized compound, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), a myosin light chain kinase (MLCK) inhibitor of superprecipitation of actomyosin, isometric tension development, and phosphorylation of the 20,000-Da myosin light chain (LC20) in vascular smooth muscle. Superprecipitation of actomyosin from bovine aorta was inhibited by the addition of ML-9 in a dose-dependent manner. In chemically skinned smooth muscles of the rabbit mesenteric artery, ML-9 inhibited the Ca2+-independent contraction provoked by application of trypsin-treated MLCK. In the intact rabbit mesenteric artery, increases in LC20 phosphorylation reached a maximal value of 0.49 mol of Pi/mol of LC20 within 10 sec from a resting value of 0.15 mol of Pi/mol of LC20 and then declined to near the basal level during the maintained isometric force developed in response to 50 mM KCl. Preincubation with 10-30 microM ML-9 for 30 min significantly inhibited both the maximal rate and extent of KCl-induced contraction and the phosphorylation of LC20, in a dose-dependent manner. There was a linear relationship between the initial rate of tension development and the extent of LC20 phosphorylation at 10 sec after stimulation. ML-9 nonspecifically antagonized the contraction induced by various contractile agonists, such as CaCl2, norepinephrine, serotonin, histamine, and angiotensin II. ML-9 dose dependently produced a shift to the right and down, in the dose-response curves, to all the agonists tested. These results suggest that ML-9 inhibits the actin-myosin interaction through the modulation of LC20 phosphorylation via the inhibition of MLCK activity. Thus, ML-9 may be a useful compound for investigating the physiologic role of myosin light chain phosphorylation by MLCK in living cells and tissues as well as in vitro.
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PMID:ML-9 inhibits the vascular contraction via the inhibition of myosin light chain phosphorylation. 338 76

Myofibrils from bovine longissimus muscle were obtained at 2 h postmortem and incubated in .10 to .35 M ionic strength buffers under various conditions in vitro. Increasing ionic strength or increasing the incubation time from 1 to 72 h decreased the turbidity of suspensions of myofibrils and increased myofibrillar solubilization (P less than .01 for both measures). The use of KCl or NaCl to elevate ionic strength gave essentially identical results, but lactate generally was ineffective in changing either the percentage myofibrillar solubilization or the turbidity of suspensions of myofibrils. Gel electrophoresis under denaturing conditions indicated that KCl was more effective than NaCl in causing the release of C-protein from myofibrils, and both salts were quite effective in dissociating M-protein, actin, troponin-T, tropomyosin, myosin light chain-3 and a 30,000-dalton molecular weight protein from myofilaments. Small increases in alpha-actinin also were observed, especially in samples incubated for 72 h. Substantially more myosin light chain-3, tropomyosin (or paratropomyosin) and troponin-T, and less actin and the 30,000-dalton protein, were released in samples incubated at pH 5.5 than at pH 7.0 (P less than .05). Electron micrographs indicated loss of thick filament ultrastructure after incubation for 24 h in either .1 or .3 M ionic strength, but the Z-lines were largely unaffected. In samples that had first been incubated with trypsin for 10 min, the Z-lines were virtually indistinguishable at .1 M ionic strength, and absolutely no myofibrillar structures could be discerned in samples incubated in .3 M ionic strength buffer.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ionic strength and myofibrillar protein solubilization. 362 3

The level of phosphorylation of myosin regulatory light chain in BALB/c 3T3 and certain other cultured substrate-attached fibroblasts has been shown to be altered by several agents which influence cell shape, attachment and/or surface receptors. This was investigated by metabolic labelling with [32P]orthophosphate, followed by exposure of the cells to the chosen conditions, rapid freezing to 'fix' phosphorylation levels, extraction and concentration in the presence of kinase and phosphatase inhibitors, and final analysis by two-dimensional gel electrophoresis. Gel patterns were interpreted by comparison with immunoprecipitates with antiserum to mouse nonmuscle myosin. Treatment of cells either with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or dibutyryl-cAMP suppressed light chain phosphorylation as predicted from the control mechanisms proposed previously from in vitro studies for Ca++ calmodulin and cAMP-dependent protein kinase respectively. Other effects were less easily explained: in BALB/c 3T3 cells, contrasting with previously reported behaviour of CHO cells, the cAMP-induced decline was small and transitory; and in at least one cell line (16C) the EGTA-induced decline was preceded by a strong pulse of enhanced phosphorylation. A striking and unexpected result was that azide, almost certainly acting on mitochondrial function, caused myosin light chain phosphorylation to be maintained over a long period even in the presence of EGTA which would otherwise bring about an immediate drop. The cleavage (by trypsin) or binding (by con A) of surface receptors was also shown to trigger the biochemical modulation of cellular myosin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Myosin light chain phosphorylation in fibroblast shape change, detachment and patching. 379 39


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