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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In vertebrate smooth muscle actomyosin and myofibrils a myosin light chain of molecular weight about 20,000 becomes phosphorylated at the same Ca2+ concentration as required to stimulate the actin-activated ATPase activity of myosin. Further, the degree of phosphorylation in the preparations as well as in various reconstituted actomyosins is proportional to their measured Ca2+ sensitivity. The phosphorylation process is very rapid and is essentially completed before the rise in ATPase activity. The enzyme responsible for the observed myosin phosphoylation is a specific myosin light chain kinase which is routinely co-purified with myosin. This kinase is normally present in actomyosin and its removal together with tropomyosin leads to a complete loss of the actin-activated ATPase activity. It is suggested that the Ca-dependent phosphorylation of the light chain via the light chain kinase represents the initial step in the activation of myosin that leads to contraction. Relaxation is probably effected by an as yet uncharacterised light chain phosphatase.
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PMID:Ca-linked phosphorylation of a light chain of vertebrate smooth-muscle myosin. 13 9

A fraction has been obtained from baby hamster kidney (BHK-21) cells that will stimulate the actin-moderated ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of both BHK-21 myosin and gizzard smooth muscle myosin. This activation is associated with the specific phosphorylation of the myosin 20,000-dalton light chain. The BHK-21 myosin light chain kinase preparation contains a major protein of approximately 105,000 molecular weight as determined by sodium dodecyl sulfate gel electrophoresis. Both the actin activation and phosphorylation events require the presence of Ca2+ and the so-called modulator or calcium-dependent regulator protein that has been isolated from smooth muscle, brain, and other tissues. On the basis of these results we propose that this kinase system constitutes a Ca2+-dependent regulatory mechanism for myosin-actin interactions in nonmuscle mammalian cells.
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PMID:Calcium-sensitive regulation of actin-myosin interactions in baby hamster kidney (BHK-21) cells. 15 71

Myosin was purified from rabbit alveolar macrophages in a form that could not be activated by actin. This myosin could be phosphorylated by an endogenous myosin light chain kinase, up to 2 mol of phosphate being incorporated/mol of myosin. The site phosphorylated was located on the 20,000-dalton myosin light chain. Phosphorylation of macrophage myosin was found to be necessary for actin activation of myosin ATPase activity. Moreover, the actin-activated ATPase activity was found to vary directly with the extent of myosin phosphorylation, maximal phosphorylation (2 mol of Pi/mol of myosin) resulting in an actin-activated MgATPase activity of approximately 200 nmol of Pi/mg of myosin/min at 37 degrees C. These results establish that phosphyoyration of the 20,000-dalton light chain of myosin is sufficient to regulate the actin-activated ATPase activity of macrophage myosin.
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PMID:Macrophage myosin. Regulation of actin-activated ATPase, activity by phosphorylation of the 20,000-dalton light chain. 15 17

Myosin isolated under phosphorylation conditions, showed an additional band of phosphorylated light chain. In the case of cardiac myosin, LC2 is the phosphorylated light chain whereas in skeletal myosin, it is the 18,000 dalton component known as DTNB light chain. There are no differences in K+-EDTA and Ca2+ activated myosin ATPase of cardiac and skeletal of control and phosphorylated myosins. Our experiments showed that the rat heart and skeletal muscle myosins isolated under phosphorylating conditions exhibited high phosphate content which is associated with higher actin activated Mg2+ ATPase activity of myosin as compared to control. Control myosin phosphorylated using myosin light chain kinase and Ca2+ also showed high actin activated myosin ATPase activity. Beef heart myosin isolated in the presence of phosphate buffer, also exhibited a higher level of phosphate followed by an increase in actin activation as compared to myosin isolated in the absence of phosphate buffer. All these experimental data suggest that there is a direct relationship between actin activation and the amount of phosphate incorporated as a result of phosphorylation.
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PMID:Phosphorylation and its effects on ATPase activity of cardiac and skeletal myosins. 16 48

We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.
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PMID:A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin. 142 Apr 39

Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration (greater than 20 micrograms/ml) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca2+, KCl, MgCl2-dependence. Maximal reaction was seen at [Ca2+] greater than 10(-6) M, 60 mM KCl and 2 mM MgCl2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca2+, KCl-dependence of ATPase activity but distinct MgCl2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCl2 and monophosphorylated myosin showed a maximum at 10 mM MgCl2. (4) The addition of tropomyosin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human platelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.
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PMID:Diphosphorylation of platelet myosin by myosin light chain kinase. 153 1

We propose a mechanism for the cytoplasmic Ca++ oscillator which is thought to power shuttle streaming in strands of the slime-mold Physarum polycephalum. The mechanism uses a phosphorylation-dephosphorylation cycle of myosin light chain kinase. This kinase is bistable if the kinase phosphorylation chain, through adenylate cyclase and cAMP, is activated by calcium. Relaxation oscillations can then occur if calcium is exchanged between the cytoplasm and internal vacuoles known to exist in physarum. As contractile activity in physarum myosin is inhibited by calcium, this model can give calcium oscillations 180 degrees out of phase with actin filament tension as observed. Oscillations of ATP concentration are correctly predicted to be in phase with the tension, provided the actomyosin cycling rate is comparable with ATPase rates for phosphorylation of the myosin light chain and its kinase.
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PMID:Model of the Ca2+ oscillator for shuttle streaming in Physarum polycephalum. 153 35

The role of protein kinase C (PKC) in regulating the contractile state of smooth muscle was investigated using the constitutively active catalytic fragment of PKC (PKM) with skinned (demembranated) chicken gizzard fibres. PKM attenuated a submaximal contraction in gizzard smooth muscle skinned fibres, but not in rabbit cardiac skinned fibres. PKM-mediated relaxation of submaximal contractions of smooth muscle was accompanied by a reduction in the rate of ATP hydrolysis in the fibre and by phosphorylation of the 20 kDa light chain of gizzard myosin at the PKC sites (serine-1, serine-2 and threonine-9). In addition, several other endogenous proteins were phosphorylated by PKM. However, the inhibitory effects on tension and ATPase are consistent with the biochemical effects of PKC-catalysed phosphorylation of myosin, i.e. reduction of the actin-activated MgATPase activity of myosin prephosphorylated at serine-19 by myosin light chain kinase. Pretreatment of skinned fibres with PKM and ATP gamma S in the absence of Ca2+ had no inhibitory effect on the subsequent submaximal Ca(2+)-activation of force. Consistent with this observation, PKC was not able to utilize ATP gamma S as a substrate, confirming that the observed effects were the result of PKM-catalysed protein phosphorylation. We suggest that PKC may have two distinct effects on smooth muscle contraction: translocation of PKC to the sarcolemma on stimulation results in phosphorylation of a protein(s) other than myosin and a slow, sustained contraction; in some circumstances PKC may undergo proteolysis to PKM resulting in myosin phosphorylation at PKC-specific sites, a reduction in ATPase activity and relaxation of the muscle.
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PMID:Effects of the constitutively active proteolytic fragment of protein kinase C on the contractile properties of demembranated smooth muscle fibres. 153 85

A peptide inhibitor, myosin kinase inhibitor (MKI), of myosin light chain kinase (MLCK) was tested for its effects on contractility and myosin light chain phosphorylation in Triton X-100 skinned guinea pig taenia coli. MKI is based on the amino acid sequence of the myosin light chain (residues 11-19 LC20) and is a competitive inhibitor [inhibitory constant (Ki) congruent to 10 microM] of purified MLCK with respect to myosin light chain (LC20). MKI inhibited unloaded shortening velocity (V(us)) and the calcium-sensitive ATPase activity of the skinned fibers but had no significant effect on steady-state isometric force or myosin light chain phosphorylation, as measured by IEF-polyacrylamide gel electrophoresis analysis. MKI had no significant effect on V(us) of thiophosphorylated fibers in the absence of calcium. MKI inhibited MLCK activity in protein extracts from taenia coli, as measured by radioactive phosphate incorporation into LC20. Surprisingly, MKI also inhibited the phosphatase activity of these same extracts. This peptide slowed the rate and extent of relaxation of calcium-contracted fibers and elicited a contraction in relaxed fibers. These results are consistent with the hypothesis that MKI may be a phosphatase inhibitor as well as an inhibitor of MLCK. Our data further suggest that the rate of phosphorylation-dephosphorylation turnover may be important in regulating V(us) in smooth muscle.
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PMID:Effects of myosin kinase inhibiting peptide on contractility and LC20 phosphorylation in skinned smooth muscle. 153 80

The 20,000-dalton light chain of smooth muscle myosin was exchanged with exogenous light chain in a solution containing 0.5 M NaCl and 10 mM EDTA at 40 degrees C. The light chain was almost completely exchanged within 30 min under the above conditions. The exchange was markedly inhibited either below 37 degrees C or in the presence of Mg2+ concentrations higher than 10 microM. The 20,000-dalton light chain was selectively labeled of a single thiol (Cys-108) with 5-[[2-[(iodoacetyl)amino]ethyl]amino-naphthalene-1-sulfonic acid (1,5-IAEDANS). The labeled light chain was exchanged stoichiometrically into myosin and was used as a probe to investigate the conformation of smooth muscle myosin. The resulting myosin hybrids showed enzymatic properties virtually identical with those of the control, untreated myosin; i.e., actin-activated ATPase activity was dependent on the 20,000-dalton light-chain phosphorylation catalyzed by myosin light chain kinase, and the 10S-6S conformational transition of myosin correlating with the changes in ATPase was also affected either by the light-chain phosphorylation or by the change in the ionic strength. Steady-state fluorescence antisotropy measurements were performed by varying the temperature. The Perrin-Weber plots were constructed in order to obtain information about the average rotational mobility of the probe and to estimate the rotational correlation time for the AEDANS-myosin head. The fluorescence probe on the 20,000-dalton light chain was found to be quite immobile as indicated by its limiting anisotropy (A0 = 0.33).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin. 183 59


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