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 addition to its kinase activity, the myosin light chain kinase (MLCK) of smooth muscle has an actin binding activity through which it can regulate the actin-myosin interaction of smooth muscle (Kohama, K., Okagaki, T., Hayakawa, K., Lin, Y., Ishikawa, R., Shimmen, T., and Inoue, A. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211). In this study, we have analyzed the actin binding activity of MLCK and related it to its amino acid sequence by producing native and recombinant fragments of MLCK. Parent MLCK exhibited both calcium ion (Ca2+) and calmodulin (Ca2+/CaM)-sensitive and Ca2+/CaM-insensitive binding to actin filaments. The native fragment, which consists of the Met1-Lys114 sequence (Kanoh, S., Ito, M., Niwa, E., Kawano, Y., and Hartshorne, D. J. (1993) Biochemistry 32, 8902-8907), and the recombinant NN fragment, which contains this 1-114 sequence, showed only Ca2+/CaM-sensitive binding. An inhibitory effect of the NN fragment on the actin-myosin interaction was observed by assaying in vitro motility and by measuring the actin-activated ATPase activity of myosin. The recombinant NN/41 fragment, which is constructed without the Met1-Pro41 sequence of the NN fragment, lost both the actin binding activity and the inhibitory effect. We confirmed the importance of the 1-41 sequence by using a few synthetic peptides to compete against the NN fragment in binding to actin filaments. The experiments using recombinant fragments and synthetic peptides also revealed that the site for CaM-binding is the Pro26-Pro41 sequence. The site for the Ca2+/CaM-insensitive binding, which is shown to be localized between the Ca2+/CaM-sensitive site and the central kinase domain of MLCK, exerted no regulatory effects on the actin-myosin interaction.
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PMID:The structure and function of the actin-binding domain of myosin light chain kinase of smooth muscle. 940 19

Chronic asthma is characterized by hypertrophy and hyperplasia of airway smooth muscle cells (SMC) that limit airflow by a geometric effect. Whether contractility of airway SMC is altered is not clear. Cultured cells were used as a model of hyperplasia. Phenotypic changes seen indicated conversion to a synthetic, weakly contractile type. At confluence, although limited reversal of protein changes was seen, no restoration in contractility occurred. Phenotypic modulation of postconfluent cultured airway SMC under prolonged serum deprivation (arrested cells) is reported here. Two phenotypically distinct groups of cells were identified in primary airway SMC cultures: 1) elongated spindle-shaped cells, which expressed large amounts of smooth muscle contractile and regulatory proteins, and 2) flat and stellate cells, which expressed very little. The first group showed a surprising shortening capacity and a velocity that was even greater than that of the freshly isolated cells, whereas the second group became spherical and noncontractile. Even more surprising was that the myosin heavy chain (MHC) isoform (SM-B) generally said to be associated with the higher shortening velocity disappeared from the cell, while the content of the key rate-limiting regulating enzyme, myosin light chain kinase (MLCK), increased 30-fold. We conclude that a functional, contractile phenotype of airway SMC can be obtained by prolonged serum deprivation. We speculate that the increased contractility could be the result of increased phosphorylation of the 20-kDa myosin light chain resulting from increased content of smooth muscle MLCK rather than any increase in endogenous MHC ATPase activity. This model may be useful for study of SMC differentiation and contraction.
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PMID:Serum deprivation induces a unique hypercontractile phenotype of cultured smooth muscle cells. 961 7

The rate of release of inorganic phosphate (Pi) from cycling cross-bridges in rabbit portal-anterior mesenteric vein smooth muscle was determined by following the fluorescence of the Pi-reporter, MDCC-PBP (Brune, M., J. L. Hunter, S. A. Howell, S. R. Martin, T. L. Hazlett, J. E. T. Corrie, and M. R. Webb. 1998. Biochemistry. 37:10370-10380). Cross-bridge cycling was initiated by photolytic release of ATP from caged-ATP in Triton-permeabilized smooth muscles in rigor. When the regulatory myosin light chains (MLC20) had been thiophosphorylated, the rate of Pi release was biphasic with an initial rate of 80 microM s-1 and amplitude 108 microM, decreasing to 13.7 microM s-1. These rates correspond to fast and slow turnovers of 1.8 s-1 and 0.3 s-1, assuming 84% thiophosphorylation of 52 microM myosin heads. Activation by Ca2+-dependent phosphorylation subsequent to ATP release resulted in slower Pi release, paralleling the rate of contraction that was also slower than after thiophosphorylation, and was also biphasic: 51 microM s-1 and 13.2 microM s-1. These rates suggest that the activity of myosin light chain kinase and phosphatase ("pseudo-ATPase") contributes <20% of the ATP usage during cross-bridge cycling. The extracellular "ecto-nucleotidase" activity was reduced eightfold by permeabilization, conditions in which the ecto-ADPase was 17% of the ecto-ATPase. Nevertheless, the remaining ecto-ATPase activity reduced the precision of the estimate of cross-bridge ATPase. We conclude that the transition from fast to slow ATPase rates reflects the properties and forces directly acting on cross-bridges, rather than the result of a time-dependent decrease in activation (MLC20 phosphorylation) occurring in intact smooth muscle. The mechanisms of slowing may include the effect of positive strain on cross-bridges, inhibition of the cycling rate by high affinity Mg-ADP binding, and associated state hydrolysis.
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PMID:Time-resolved measurements of phosphate release by cycling cross-bridges in portal vein smooth muscle. 982 23

Calponin is a thin filament-associated protein which has been implicated in the modulation of the contractile state of smooth muscle via its interaction with actin and inhibition of the actin-activated myosin Mg-ATPase. This inhibitory effect is alleviated by phosphorylation of calponin at Ser175 in vitro by protein kinase C. The issue of calponin phosphorylation in intact smooth muscle in response to agonists that activate protein kinase C is controversial. We have produced a monoclonal antibody that specifically recognizes calponin phosphorylated at Ser175 and used it to analyze calponin phosphorylation in porcine coronary arterial smooth muscle stimulated with prostaglandin F2alpha or phorbol 12,13-dibutylate (PDB). Calponin phosphorylation increased rapidly in response to prostaglandin F2alpha concomitant with the increase in tension. Calponin was then dephosphorylated while force was maintained. Tension development in response to PDB was significantly slower, but again calponin phosphorylation paralleled force development. In this case, calponin dephosphorylation was very slow, consistent with prolonged activation of protein kinase C. The protein kinase inhibitors, HA1077 (1-5-(isoquinoline sulfonyl)-homopiperazine HCl) and HA1100 (1-hydroxy HA1077; 1-(hydroxy-5-isoquinoline sulfonyl-homopiperazine), inhibited tension development and calponin phosphorylation in a concentration-dependent manner with similar ED50 values in response to prostaglandin F2alpha and PDB. These results support physiological roles for calponin in force development in smooth muscle in response to agonists which trigger protein kinase C activation and in the latch state, i.e., force maintenance at low energy cost. Furthermore, the vasodilator effect of HA1077 and HA1100 is more likely due to inhibition of protein kinase C than of myosin light chain kinase.
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PMID:HA1077, a protein kinase inhibitor, inhibits calponin phosphorylation on Ser175 in porcine coronary artery. 985 93

Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was purified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrophoresis) and identified as the ROKalpha isoform. Several substrates were phosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myosin, and the 20-kDa myosin light chain were higher than other substrates. Thiophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphorylation of myosin at serine 19 increased actin-activated Mg+-ATPase activity, i.e. similar to myosin light chain kinase. Myosin phosphorylation was increased at higher ionic strengths, possibly by formation of 6 S myosin. Phosphorylation of the isolated light chain and myosin phosphatase was decreased by increasing ionic strength. Rho-kinase was stimulated 1.5-2-fold by guanosine 5'-O-3-(thio)triphosphate.RhoA, whereas limited tryptic hydrolysis caused a 5-6-fold activation, independent of RhoA. Several kinase inhibitors were screened and most effective were Y-27632, staurosporine, and H-89. Several lipids caused slight activation of Rho-kinase, but arachidonic acid (30-50 microM) induced a 5-6-fold activation, independent of RhoA. These results suggest that Rho-kinase of smooth muscle may be involved in the contractile process via phosphorylation of MYPT1 and myosin. Activation by arachidonic acid presents a possible regulatory mechanism for Rho-kinase.
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PMID:Rho-associated kinase of chicken gizzard smooth muscle. 992 Sep 27

Molluscan myosins are regulated molecules that control muscle contraction by the selective binding of calcium. The essential and the regulatory light chains are regulatory subunits. Scallop myosin is the favorite material for studying the interactions of the light chains with the myosin heavy chain since the regulatory light chains can be reversibly removed from it and its essential light chains can be exchanged. Mutational and structural studies show that the essential light chain binds calcium provided that the Ca-binding loop is stabilized by specific interactions with the regulatory light chain and the heavy chain. The regulatory light chains are inhibitory subunits. Regulation requires the presence of both myosin heads and an intact headrod junction. Heavy meromyosin is regulated and shows cooperative features of activation while subfragment-1 is non-cooperative. The myosin heavy chains of the functionally different phasic striated and the smooth catch muscle myosins are products of a single gene, the isoforms arise from alternative splicing. The differences between residues of the isoforms are clustered at surface loop-1 of the heavy chain and account for the different ATPase activity of the two muscle types. Catch muscles contain two regulatory light chain isoforms, one phosphorylatable by gizzard myosin light chain kinase. Phosphorylation of the light chain does not alter ATPase activity. We could not find evidence that light chain phosphorylation is responsible for the catch state.
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PMID:Regulation by molluscan myosins. 1009 69

Cellular Ca2+ transients and Ca2+-binding proteins regulate physiological phenomena as diverse as muscle contraction, neurosecretion, and cell division. When Ca2+ is rapidly mixed with slow Ca2+ chelators, EGTA, or Mg2+/EDTA, artificial Ca2+ transients (ACTs) of varying duration (0.1-50 ms half-widths (hws)) and amplitude can be generated. We have exposed several Ca2+ indicators, Ca2+-binding proteins, and a Ca2+-dependent enzyme to ACTs of various durations and observed their transient binding of Ca2+, complex formation, and/or activation. A 0.1 ms hw ACT transiently occupied approximately 70% of the N-terminal regulatory sites of troponin C consistent with their rapid Ca2+ on-rate (8.7 +/- 2.0 x 10(7) M-1 s-1). A 1.1 ms hw ACT produced approximately 90% transient binding of the N-terminal of calmodulin (CaM) to the RS-20 peptide, but little binding of CaM's C-terminal to RS-20. A 0.6 ms hw ACT was sufficient for the N-terminal of CaM to transiently bind approximately 60% of myosin light chain kinase (MLCK), while a 1.8 ms hw ACT produced approximately 22% transient activation of the sarcoplasmic reticulum (SR) Ca2+/ATPase. In both cases, the ACT had fallen back to baseline approximately 10-30 ms before maximal binding of CaM to MLCK or SR Ca2+/ATPase activation occurred and binding and enzyme activation persisted long after the Ca transient had subsided. The use of ACTs has allowed us to visualize how the Ca2+-exchange rates of Ca2+-binding proteins dictate their Ca2+-induced conformational changes, Ca2+-induced protein/peptide and protein/protein interactions, and enzyme activation and inactivation, in response to Ca2+ transients of various amplitude and duration. By characterizing the response of these proteins to ACTs, we can predict with greater certainty how they would respond to natural Ca2+ transients to regulate cellular phenomena.
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PMID:Characterizing the response of calcium signal transducers to generated calcium transients. 1019 40

The actin-based motor protein myosin II plays a critical role in many cellular processes in both muscle and non-muscle cells. Targeted disruption of the Dictyostelium regulatory light chain (RLC) caused defects in cytokinesis and multicellular morphogenesis. In contrast, a myosin heavy chain mutant lacking the RLC binding site, and therefore bound RLC, showed normal cytokinesis and development. One interpretation of these apparently contradictory results is that the phenotypic defects in the RLC null mutant results from mislocalization of myosin caused by aggregation of RLC null myosin. To distinguish this from the alternative explanation that the RLC can directly influence myosin activity, we expressed three RLC point mutations (E12T, G18K and N94A) in a Dictyostelium RLC null mutant. The position of these mutations corresponds to the position of mutations that have been shown to result in familial hypertrophic cardiomyopathy in humans. Analysis of purified Dictyostelium myosin showed that while these mutations did not affect binding of the RLC to the MHC, its phosphorylation by myosin light chain kinase or regulation of its activity by phosphorylation, they resulted in decreased myosin function. All three mutants showed impaired cytokinesis in suspension, and one produced defective fruiting bodies with short stalks and decreased spore formation. The abnormal myosin localization seen in the RLC null mutant was restored to wild-type localization by expression of all three RLC mutants. Although two of the mutant myosins had wild-type actin-activated ATPase, they produced in vitro motility rates half that of wild type. N94A myosin showed a fivefold decrease in actin-ATPase and a similar decrease in the rate at which it moved actin in vitro. These results indicate that the RLC can play a direct role in determining the force transmission and kinetic properties of myosin.
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PMID:Regulatory light chain mutations affect myosin motor function and kinetics. 1021 54

The catalytic domain of myosin light chain kinase (MLCK) not only exerts kinase activity to phosphorylate the 20 kDa light chain but also inhibits the actin-myosin interaction. The site of action of this novel role of the domain has been suggested to be myosin [Okagaki et al. (1999) J. Biochem. 125, 619-626]. In this study, we have analyzed the amino acid sequences of MLCK and myosin that are involved in the inhibition. The ATP-binding peptide of Gly526-Lys548 of chicken gizzard MLCK exerted the inhibitory effect on the movement of actin filaments on a myosin-coated glass surface. However, the peptide that neighbors the sequence failed to inhibit the movement. The inhibition of the ATP-binding peptide was confirmed by measuring ATPase activities of the myosin. The inhibition by parent MLCK of the movement was relieved by the 20 kDa light chain, but not by the 17 kDa myosin light chain. The peptide of the 20 kDa light chain sequence of Ser1-Glu29 also relieved the inhibition. Thus, the interaction of the ATP-binding sequence with the 20 kDa light chain sequence should cause the inhibition of the actin-myosin interaction. Concerning the regulation of the inhibition, calmodulin relieved the inhibitory effect of MLCK on the movement of actin filaments. The calmodulin-binding peptide (Ala796 Ser815) prevented the relief, suggesting the involvement of this sequence. Thus, the mode of regulation by Ca2+ and calmodulin of the novel role of the catalytic domain is similar, but not identical, to the mode of regulation of the kinase activity of the domain.
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PMID:Inhibitory effect of the catalytic domain of myosin light chain kinase on actin-myosin interaction: insight into the mode of inhibition. 1034 7

The basally located actin cytoskeleton has been demonstrated previously to regulate Cl- secretion from intestinal epithelia via its effects on the Na+-K+-2Cl- cotransporter (NKCC1). In nontransporting epithelia, inhibition of myosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1. The aim of this study was to investigate the role of myosin in the regulation of secretagogue-stimulated Cl- secretion in intestinal epithelia. The human intestinal epithelial cell line T84 was used for these studies. Prevention of myosin light chain phosphorylation with the MLCK inhibitor ML-9 or ML-7 and inhibition of myosin ATPase with butanedione monoxime (BDM) attenuated cAMP but not Ca2+-mediated Cl- secretion. Both ML-9 and BDM diminished cAMP activation of NKCC1. Neither apical Cl- channel activity, basolateral K+ channel activity, nor Na+-K+-ATPase were affected by these agents. Cytochalasin D prevented such attenuation. cAMP-induced rearrangement of basal actin microfilaments was prevented by both ML-9 and BDM. The phosphorylation of mosin light chain and subsequent contraction of basal actin-myosin bundles are crucial to the cAMP-driven activation of NKCC1 and subsequent apical Cl- efflux.
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PMID:Myosin regulation of NKCC1: effects on cAMP-mediated Cl- secretion in intestinal epithelia. 1048 31


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