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)

Actin, myosin, and a high molecular weight actin-binding protein were extracted from rabbit alveolar macrophages with low ionic strength sucrose solutions containing ATP, EDTA, and dithiothreitol, pH 7.0. Addition of KCl, 75 to 100 mM, to sucrose extracts of macrophages stirred at 25 degrees caused actin to polymerize and bind to a protein of high molecualr weight. The complex precipitated and sedimented at low centrifugal forces. Macrophage actin was dissociated from the binding protein with 0.6 M KCl, and purified by repetitive depolymerization and polymerization. Purified macrophage actin migrated as a polypeptide of molecular weight 45,000 on polyacrylamide gels with dodecyl sulfate, formed extended filaments in 0.1 M KCl, bound rabbit skeletal muscle myosin in the absence of Mg-2+ATP and activated its Mg-2+ATPase activity. Macrophage myosin was bound to actin remaining in the macrophage extracts after removal of the actin precipitated with the high molecular weight protein by KCl. The myosin-actin complex and other proteins were collected by ultracentrifugation. Macrophage myosin was purified from this complex or from a 20 to 50% saturated ammonium sulfate fraction of macrophage extracts by gel filtration on agarose columns in 0.6 M Kl and 0.6 M Kl solutions. Purified macrophage myosin had high specific K-+- and EDTA- and K-+- and Ca-2+ATPase activities and low specific Mg-2+ATPase activity. It had subunits of 200,000, 20,000, and 15,000 molecular weight, and formed bipolar filaments in 0.1 M KCl, both in the presence and absence of divalent cations. The high molecular weight protein that precipitated with actin in the sucrose extracts of macrophages was purified by gel filtration in 0.6 M Kl-0.6 M KCl solutions. It was designated a macrophage actin-binding protein, because of its association with actin at physiological pH and ionic strength. On polyacrylamide gels in dodecyl sulfate, the purified high molecular weight protein contained one band which co-migrated with the lighter polypeptide (molecular weight 220,000) of the doublet comprising purified rabbit erythrocyte spectrin. The macrophage protein, like rabbit erythrocyte spectrin, was soluble in 2 mM EDTA and 80% ethanol as well as in 0.6 M KCl solutions, and precipitated in 2 mM CaCl2 or 0.075 to 0.1 M KCl solutions. The macrophage actin-binding protein and rabbit erythrocyte spectrin eluted from agarose columns with a KAV of 0.24 and in the excluded volumes. The protein did not form filaments in 0.1 M KCl and had no detectable ATPase activity under the conditions tested.
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PMID:Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages. 12 34

Actin, myosin, and a high molecular weight actin-binding protein were purified from chronic myelogenous leukemia (CML) leukocytes. CML leukocyte actin resembled skeletal muscle and other cytoplasmic actins by its subunit molecular weight, by its ability to polymerize in the presence of salts, and to activate the Mg2+-ATPase activity of rabbit skeletal muscle myosin. CML leukocyte myosin was similar to other vertebrate cytoplasmic myosins in having heavy chains and two light subunits. However, its apparent heavy-chain molecular weight and Stokes radius suggested that it was variably degraded during purification. Purified CML leukocyte myosin had average specific EDTA- AND Ca2+-activated ATPase activities of 125 and 151 nmol Pi released/mg protein per min, respectively and low specific Mg2+-ATPase activity. The Mg2+-ATPase activity of CML myosin was increased 200-fold by rabbit skeletal muscle F-actin, but the specific activity relative to that of actin-activated rabbit skeletal muscle myosin was low. CML leukocyte myosin, like other vertebrate cytoplasmic myosins, formed filaments in 0.1 M KCl solutions. Reduced and denatured CML leukocyte-actin-binding protein had a single high molecular weight subunit like a recently described actin-binding protein of rabbit pulmonary macrophages which promotes the polymerization and gelation of actin. Cytoplasmic extracts of CML leukocytes prepared with ice-cold 0.34-M sucrose solutions containing Mg2+-ATP, dithiothreitol, and EDTA at pH 7.0 underwent rapid gelation when warmed to 25 degrees C. Initially, the gel could be liquified by cooling to ice-bath temperature. With time, warmed cytoplasmic extract gels shrunk ("contracted") into aggregates. The following findings indicated that CML leukocyte actin-binding protein promoted the temperature-dependent gelation of actin in the cytoplasmic extracts and that CML leukocyte myosin was involved in the contraction of the actin gels: (a) Cytoplasmic extract gels initially contained actin as their major polypeptide component and consistent of tangled thin filaments; (b) Contracted aggregates of cytoplasmic extract gels contained by large quantities of myosin as well as actin; (c) Purified actin-binding protein underwent a temperature-dependent, reversible aggregation and caused low concentrations of purified muscle or CML leukocyte actins to gel in sucrose solutions; (d) The gels formed from purified actin plus purified actin-binding protein slowly contracted in the presence but not in the absence of purified CML leukocyte myosin; (e) Rabbit antiserum against purified CML leukocyte actin-binding protein but not against purified CML leukocyte myosin inhibited the gelation of warmed CML leukocyte extracts. Antiserum against CML leukocyte myosin had no effect on the gelation of CML leukocyte extracts but partially curtailed the contraction of the CML leukocyte extract gels and of gels formed from purified CML leukocyte actin-binding protein plus rabbit skeletal muscle actin.
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PMID:Interactions of actin, myosin, and an actin-binding protein of chronic myelogenous leukemia leukocytes. 13 21

The plasmalemma and hyaline ectoplasm together constitute the sensory and motor organ of macrophages. The purpose of this study was to isolate this cell fraction in order to analyze it biochemically and functionally. Brief sonification of warmed rabbit lung macrophages caused release of heterodisperse hyaline blebs and filopodia, which were easily collected by differential centrifugation. Viewed in the electron microscope, these structures consisted of membrane-bounded sacs principally containing actin filaments. Some contained secondary lysosomes. They were enriched threefold over whole cell homogenates in specific adenylate cyclase activity and in trichloroacetic-acid-precipitable (125)I when derived from cells labeled with 125(I) by means of a lactoperoxidase-catalyzed reaction. These markers were found to have identical isopycnic densitites when macrophage homogenates were subjected to sedimentation in a focusing sucrose density gradient system, and these markers had densities distinct from those of other cytoplasmic organelles. These markers were therefore assumed to be associated with macrophage plasma membranes. The specific beta- glucuronidase activity of the bleb fraction was similar to that of homogenates, but the blebs had considerably lower specific succinic dehydrogenase activity and RNA content, and DNA was undetectable. Electrophoresis of blebs solubilized in sodium dodecyl sulfate on polyacrylamide gels revealed polypeptides co-migrating with macrophage actin-binding protein, myosin, and actin; blebs also had EDTA-activated adenosine triphosphatase activity characteristic of myosin. The concentrations of actin-binding protein and myosin were higher in blebs than in cells or cytoplasmic extracts, whereas actin concentrations were similar (relative to extracts) or only slightly greater (than in cells). Blebs and intact cells had high lactate dehydrogenase activities in the presence but not the absence of Triton X-100. Blebs and cells oxidased 1-[(14)C]glucose, and the rate of glucose oxidation was increased substantially in the presence of latex beads. We conclude that intact sacs of plasmalemma encasing contractile proteins and cytoplasmic enzymes can be isolated from macrophages. They are enriched in myosin and actin-binding protein, indicating that the contractile apparatus is regulated in the cell periphery. These structures have the capacity to respond to environmental signals. We suggest the name "podosomes" for them because of their resemblance to macrophage pseudopodia. We propose that podosome formation results from rapid dissolution of the cortical gel when the membrane is in an actively extended configuration.
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PMID:Peripheral hyaline blebs (podosomes) of macrophages. 92 88

Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of beta-glucoronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasma-lemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and approximately 20 percent less actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change. We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle exclusion that characterize phagocytosis.
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PMID:Evidence for contractile protein translocation in macrophage spreading, phagocytosis, and phagolysosome formation. 92 89

Actin and myosin of rabbit pulmonary macrophages are influenced by two other proteins. A protein cofactor is required for the actin activation of macrophage myosin Mg2 ATPase activity, and a high molecular weight actin-binding protein aggregates actin filaments (Stossel T.P., and J.H. Hartwig. 1975. J. Biol. Chem. 250:5706-5711)9 When warmed in 0.34 M sucrose solution containing Mg2-ATP and dithiothreitol, these four proteins interact cooperatively. Acin-binding protein in the presence of actin causes the actin to form a gel, which liquifies when cooled. The myosin contracts the gel into an aggregate, and the rate of aggregation is accelerated by the cofactor. Therefore, we believe that these four proteins also effec the temperature-dependent gelation and aggregation of crude sucrose extracts pulmonary macrophages containing Mg2-ATP and dithiothreitol. The gelled extracts are composed of tangled filaments. Relative to homogenates of resting macrophages, the distribution of actin-binding protein in homogenates of phagocytizing macrophages is altered such that 2-6 times more actin-binding protein is soluble. Sucrose extracts of phagocytizing macrophages gel more rapidly than extracts of resting macrophages. Phagocytosis by pulmonary macrophages involves the formation of peripheral pseudopods containing filaments. The findings suggest that the actin-binding protein initiates a cooperative interaction of contractile proteins to generate cytoplasmic gelation, and that phagocytosis influences the behavior of the actin-binding protein.
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PMID:Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis. 103 11

Caldesmon is an actin-binding protein present in smooth muscle cells that also inhibits actin-activated myosin ATPase activity. To assess the possible role of caldesmon in the regulation of smooth contraction, we investigated the effects of synthetic peptides on force directly recorded from single hyperpermeable smooth muscle cells of ferret aorta and portal vein. GS17C, a peptide that contains the residues from Gly651 to Ser667 of the caldesmon sequence plus an added cysteine at the C terminus, binds calmodulin in a Ca(2+)-dependent manner and also binds to F-actin but does not inhibit actomyosin ATPase activity (Zhan, Q., Wong, S.S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814). In cells in which Ca2+ was clamped at pCa 7.0, GS17C induced a dose-dependent contraction (EC50 = 0.92 microM) in aorta cells, whereas it evoked little or no contraction in portal vein cells. The GS17C-induced contraction in aorta cells was inhibited at higher Ca2+ concentrations (above pCa 6.6) and by pretreatment with calmodulin. Another peptide, C16AA, which contains the residues from Ala594 to Ala609 and does not bind actin or calmodulin, did not induce contraction. Our results strongly suggest that GS17C induces contraction by the displacement of the inhibitory region of endogenous caldesmon and, furthermore, that caldesmon present in these smooth muscle cells regulates contraction by providing a basal resting inhibition of vascular tone.
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PMID:Regulation of vascular smooth muscle tone by caldesmon. 138 78

Rat seminal vesicles and the lateral prostate secrete a glycoprotein designated as SVS II in an androgen-dependent manner. SVS II, which has a M(r) of 49,000 and a pI of 10.5, is an actin-binding protein. G- and F-actins cosediment with SVS II at a ratio of 2:1 (actin:SVS II). SVS II affects the kinetics of actin polymerization in the same way as do barbed end capping proteins. Interaction with actin is specific for the skeletal and cardiac muscle isoforms and there is no corresponding interaction with cytoplasmic actins. The binding site is close to the C-terminus of actin. Monospecific polyclonal antibodies directed against the N-terminus of actin cross-react with SVS II, but there is no cross-reaction by a monoclonal antibody directed against a C-terminal epitope on actin. Recent sequence analysis of SVS II shows a sequence of about 14 residues that is repeated 13 times between residues 86 and 298. The consensus sequence based on these repeats is homologous to residues 10 to 25 of actin; this may account for the immunological cross-reactivity. Like actin, SVS II binds and inhibits the activity of DNase I, but SVS II has no effect on the ATPase activity of myosin subfragment 1. Thus, SVS II is an actin-binding protein which retains some properties of actin itself.
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PMID:SVS II--an androgen-dependent actin-binding glycoprotein in rat semen. 151 6

Calponin, an actin-binding protein, inhibited the acto-heavy meromyosin (HMM) MgATPase and lowered the binding of HMM to actin. The amount of calponin bound to actin or tropomyosin-actin was the same when the ATPase was inhibited 80-90%. While the KATPase was diminished only less than 2-fold in the presence of calponin, the Vmax was decreased 6-fold and 2-fold with actin and tropomyosin-actin, respectively. A comparison of the kinetic constants for the ATP hydrolysis obtained in the presence of actin-calponin and tropomyosin-actin-calponin revealed that the tropomyosin augmented the Vmax 5-fold from the inhibited level, but there was no effect on the KATPase.
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PMID:The mechanism for the inhibition of actin-activated ATPase of smooth muscle heavy meromyosin by calponin. 182 52

We have purified an actin-binding protein from the plasmodia of a lower eukaryote, Physarum polycephalum, with an apparent molecular mass of 210,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein bound to actin filaments with a stoichiometry of 1:7-8 in a Ca(2+)-calmodulin-dependent manner. Antibody raised against caldesmon from smooth muscle cross-reacted with the 210-kDa protein. In vitro motility assay revealed that the 210-kDa protein increased the sliding velocity of actin filaments on Physarum myosin. The 210-kDa protein more than doubled the actin-activated ATPase activity of Physarum myosin under comparative conditions of in vitro motility assay. Further increases in the concentration of the 210-kDa protein decreased its stimulatory effects. Ca(2+)-calmodulin prevented the stimulatory effects of the 210-kDa protein. Unexpectedly, smooth muscle caldesmon also increased the sliding velocity of actin filaments on smooth muscle myosin at lower concentrations. The well-known inhibitory effect of smooth muscle caldesmon on the actin-myosin interaction was observed with this motility assay when the concentration of the caldesmon was increased further. The stimulatory and inhibitory effects were confirmed by measurements of actin-activated ATPase activity of smooth muscle myosin. From estimations of the intracellular concentrations of the 210-kDa protein and smooth muscle caldesmon in vivo, it appears that effects of the former and the latter on actin-myosin interactions in vivo are stimulatory and inhibitory, respectively.
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PMID:Stimulation of the interaction between actin and myosin by Physarum caldesmon-like protein and smooth muscle caldesmon. 183 62

Calponin, a 35 kDa actin-binding protein, was shown to be a normal component of 'native' thin filaments prepared from sheep aorta. Actin, tropomyosin, caldesmon and calponin were present in molar ratios 14:2:1:0.9. Calponin was isolated from thin filaments in yield 0.5 mg/100 mg thin filament protein. Calponin inhibited actomyosin ATPase up to 85%, half maximal at 0.2 calponin/actin. Inhibition did not depend on tropomyosin, Ca2+ or Ca2+ calmodulin. Caldesmon inhibited actomyosin with a 10-fold greater potency than calponin in the presence of tropomyosin and inhibition could be reversed by Ca2+ calmodulin under certain conditions. Calponin had no effect on caldesmon inhibition or the reversal of inhibition.
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PMID:Properties of calponin isolated from sheep aorta thin filaments. 183 99


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