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)

Acanthamoeba myosin IB is a single-headed enzyme containing one heavy chain of 125,000 daltons, one light chain of 27,000 daltons, and one light chain of 14,000 daltons. The 125,000- and 27,000-dalton polypeptides are consistently found in a molar ratio of 1:1. The content of the 14,000-dalton peptide is usually only 0.1 to 0.2, and always less than 0.5, relative to the other two chains and might be a contaminant or a degradation product of one of the other chains. The specific activities of the Ca2+-ATPase, (K+, EDTA)-ATPase, and (after phosphorylation of its heavy chain by a specific kinase) actin-activated Mg2+-ATPase of Acanthamoeba myosin IB are similar to those of rabbit skeletal muscle myosin. After treatment of the enzyme with 2 M LiCl, the 125,000-dalton heavy chain of Acanthamoeba myosin Ib can be obtained, by chromatography on Sephadex G-200, essentially free of the 14,000-dalton peptide and more than 90% free of the 27,000-dalton peptide. This isolated heavy chain has the same specific ATPase activities as the original enzyme. Therefore, the heavy chain of Acanthamoeba myosin IB contains the ATPase catalytic site, the actin-binding site, and the phosphorylation site and is fully active enzymatically in the absence of light chains.
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PMID:The isolated heavy chain of an Acanthamoeba myosin contains full enzymatic activity. 15 Apr 18

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NH2-terminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-terminal 68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH2-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH+4,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Mg2+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80-kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NH2 terminus, and the second actin-binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.
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PMID:Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity. 296 46

Acanthamoeba myosins IA and IB were found to have molecular weights of 159,000 and 150,000 and Stokes radii of 6.2 and 5.9 nm, respectively. Both enzymes have frictional ratios of 1.7. Myosin IA consists of 22% alpha-helix, 32% beta-structure, and 46% unordered structure, while myosin IB is 16% alpha-helix, 46% beta-structure, and 38% unordered. Both myosins remain monomolecular under conditions in which other myosins form filaments. Beads coated with myosin IA or IB move unidirectionally on actin cables of Nitella. Movement requires ATP and phosphorylation of the myosin I heavy chain which is also required for actin-activated Mg2+-ATPase activity. Movement is inhibited by myosin I antiserum that inhibits actin-activated ATPase activity. These studies establish that these nonfilamentous, monomolecular myosins with single heavy chains of 130,000 and 125,000 daltons (IA and IB, respectively) can support actin-dependent movement analogous to that supported by filamentous myosins.
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PMID:Monomeric Acanthamoeba myosins I support movement in vitro. 316 Jun 92

Myosins IA and IB from Acanthamoeba castellanii are single-headed molecules which, upon phosphorylation of their heavy chains by a specific kinase, express actin-activated Mg2+-ATPase activity. These myosins show no tendency to self-associate under assay conditions, a property which allows unambiguous kinetic and actin-binding data to be obtained. Both myosin isoenzymes exhibit a complex dependence of actomyosin ATPase activity on F-actin concentration. A conventional hyperbolic dependence is observed at low concentrations of F-actin but at higher F-actin concentrations, inhibition and then apparent reactivation are seen to occur. From those early portions of the velocity profiles which do not deviate from simple Michaelis-Menten type kinetics, values for the Vmax (10 s-1 for myosin IA, 18 s-1 for myosin IB) and KATPase (0.25 microM for myosin IA, 0.30 microM for myosin IB) were calculated. Similar Vmax values were obtained from the reactivation segment of the kinetic data. The KATPase values are very similar to the directly measured dissociation constants (KD) of 0.10 microM for myosin IA and 0.25 microM for myosin IB. Phosphorylation of the myosin heavy chain, which elicits a greater than 20-fold activation of the actomyosin ATPase, has no effect on the binding of myosin to F-actin. This finding supports the conclusion that phosphorylation of myosins IA and IB accelerates one or more catalytic steps of the actomyosin I ATPase reaction at both low and high concentrations of F-actin.
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PMID:The interaction of F-actin with phosphorylated and unphosphorylated myosins IA and IB from Acanthamoeba castellanii. 613 3

Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy-chain phosphorylation, and (d) ADP release is fast. Thus, during steady-state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily, other than myosin-II, providing the opportunity to obtain insights about the evolution of all myosin isoforms.
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PMID:Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase. 860 84

Class I myosins have a single heavy chain comprising an N-terminal motor domain with actin-activated ATPase activity and a C-terminal globular tail with a basic region that binds to acidic phospholipids. These myosins contribute to the formation of actin-rich protrusions such as pseudopodia, but regulation of the dynamic localization to these structures is not understood. Previously, we found that Acanthamoeba myosin IC binds to acidic phospholipids in vitro through a short sequence of basic and hydrophobic amino acids, BH site, based on the charge density of the phospholipids. The tail of Dictyostelium myosin IB (DMIB) also contains a BH site. We now report that the BH site is essential for DMIB binding to the plasma membrane and describe the molecular basis of the dynamic relocalization of DMIB in live cells. Endogenous DMIB is localized uniformly on the plasma membrane of resting cells, at active protrusions and cell-cell contacts of randomly moving cells, and at the front of motile polarized cells. The BH site is required for association of DMIB with the plasma membrane at all stages where it colocalizes with phosphoinositide bisphosphate/phosphoinositide trisphosphate (PIP(2)/PIP(3)). The charge-based specificity of the BH site allows for in vivo specificity of DMIB for PIP(2)/PIP(3) similar to the PH domain-based specificity of other class I myosins. However, DMIB-head is required for relocalization of DMIB to the front of migrating cells. Motor activity is not essential, but the actin binding site in the head is important. Thus, dynamic relocalization of DMIB is determined principally by the local PIP(2)/PIP(3) concentration in the plasma membrane and cytoplasmic F-actin.
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PMID:Molecular basis of dynamic relocalization of Dictyostelium myosin IB. 2236 11