Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.
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PMID:The contractile basis of amoeboid movement. V. The control of gelation, solation, and contraction in extracts from Dictyostelium discoideum. 2 Apr 47

1. The procedure of Barrett [(1973) Biochem. J.131, 809-822] for isolating cathepsins B and D from human liver was modified for use with rat liver and skeletal muscle. The purified enzymes appeared to be similar to those reported in other species. 2. Sephadex G-75 chromatography of concentrated muscle extract resolved two peaks of cathepsin B inhibitory activity, corresponding to molecular weights of 12500 and 62000. 3. The degradation of purified myofibrillar proteins by cathepsins B and D was clearly demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After incubation with enzyme, the polypeptide bands representing the substrates decreased in intensity and lower molecular weight products appeared. 4. Cathepsins B and D, purified from either rat liver or skeletal muscle, were shown to degrade myosin, purified from either rabbit or rat muscle. Soluble denatured myosin was degraded more extensively than insoluble native myosin. Degradation by cathepsin B was inhibited by lack of reducing agent, or by myoglobin, iodoacetic acid and leupeptin, but not by pepstatin. The same potential modifiers were applied to cathepsin D, and only pepstatin produced inhibition. 5. Rat liver cathepsin B had a pH optimum of 5.2 on native rabbit myosin. The pH optimum of cathepsin D was 4.0, with a shoulder of activity about 1pH unit above the optimum. 6. Rat liver cathepsins B and D were demonstrated to degrade rabbit F-actin at pH5.0, and were inhibited by leupeptin and pepstain, respectively. 7. The degradation of myosin and actin by cathepsin D was more extensive than that by cathepsin B.
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PMID:Degradation of myofibrillar proteins by cathepsins B and D. 2 66

1. Phenylglyoxal reacts rapidly with isolated myosin heads (subfragment 1) and induces two successive and distinguishable effects on their enzymic properties: first, a twofold activation of the Ca2+ and Mg2+-dependent ATPases with no effect onthe K+-ATPase followed by inhibition of the K+, Ca2+ and actin-activated Mg2+-ATPases. A specific protein-reagent reagent complex is formed during the second phase of the modification reaction (Ki approximately 5 x 10(-3) M). 2. ADP and ATP with or without cations provide efficient protection only against the loss of ATPase activities, suggesting that the second inhibitory process is occurring at or close to the active site. 3. On the basis of [14C]phenylglyoxal-labelling experiments and the composition of modified subfragment-1 derivatives, it is demonstrated that the sequential modification of two reactive arginyl residues is responsible for the observed activation-inhibition phenomena. Blocking of the first reactive residue produces a shift in the pH/activity curves related to the Ca2+ and Mg2+-dependent ATPases with an apparent activation effect. Modification of the second guanidino group does not destroy the affinity of the protein for the nucleotide substrates but does alter the nucleotide binding site as reflected in the inability of Mg2+. ATP to dissociate the modified subfragment-1--actin complex. It is concluded that electrostatic interactions between this positively charged group and the negatively charged ATP and ADP molecules may be critical for the hydrolytic efficiency of myosin heads. 4. After dissociation and separation of the polypeptide constituents of the protein in acetic acid medium, both labelled sites are found to reside in the heavy chain.
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PMID:Involvement of an arginyl residue in the catalytic activity of myosin heads. 4 10

The contracted pellets derived from a high-speed supernate of Dictyostelium discoideum (S3) were investigated to determine the functional activity associated with this specific subset of the cellular motile apparatus. A partially purified model system of gelation and contraction (S6) was prepared from the contracted pellets, and the presence of calcium- and pH-sensitive gelation and contraction in this model demonstrated that a functional cytoskeletal-contratile complex remained at least partially associated with the actin and myosin during contraction. Semi-quantitative assays of gelation and solation in the myosin-free preparation S6 included measurements of turbidity, relative viscosity, and strain birefringence. The extent of gelation was optimal at pH 6.8 and a free calcium ion concentration of approximately 3.0 x 10(-8) M. Solation was favored when the free calcium ion concentration was greater than 7.6 x 10(-7) M or when the pH was increased or decreased from pH 6.8. Gelation was reversibly inhibited by increasing the free calcium ion concentration to approxomately 4.6 x 10(-6) M at pH 6.8. The solation-gelation process of this model has been interpreted to involve the reversible cross-linking of actin filaments. The addition of purified D. discoideum myosin to S6 served to reconstitute calcium- and pH-regulated contraction. The results from this study indicate that contraction is coupled functionally to the local breakdown (solation) of the gel. Therefore, solation has been identified as a structural requirement for extensive shortening during contraction. We have called this concept the solation-contraction coupling hypothesis. Fractionation of a preparation derived from the contracted pellets yielded a fraction consisting of actin and a 95,000-dalton polypeptide that exhibited calcium-sensitive gelation at 28 degrees C and a fraction composed of actin and 30,000- and 18,000-dalton polypeptides that demonstrated calcium-sensitive genlation at 0 degrees C.
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PMID:The contractile basis of ameboid movement. VI. The solation-contraction coupling hypothesis. 4 49

This study of the slow component of axonal transport was aimed at two problems: the specific identification of polypeptides transported into the axon from the cell body, and the identification of structural polypeptides of the axoplasm. The axonal transport paradigm was used to obtain radioactively labeled axonal polypeptides in the rat ventral motor neuron and the cat spinal ganglion sensory neuron. Comparison of the slow component polypeptides from these two sources using sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis revealed that they are identical. In both cases five polypeptides account for more than 75% of the total radioactivity present in the slow component. Two of these polypeptides have been tentatively identified as tubulin, the microtubule protein, on the basis of their molecular weights. The three remaining polypeptides with molecular weights of 212,000, 160,000, and 68,000 daltons are constitutive, and as such appear to be associated with a single structure which has been tentatively identified as the 10-nm neurofilament. The 212,000-dalton polypeptide was found to comigrate in SDS gels with the heavy chain of chick muscle myosin. The demonstration on SDS gels that the slow component is composed of a small number of polypeptides which have identical molecular weights in neurons from different mammalian species suggests that these polypeptides comprise fundamental structures of vertebrate neurons.
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PMID:The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons. 4 55

A new high-molecular-weight protein, named filamin, was isolated from chicken gizzard. In chicken gizzard, filamin is present in an amount approximately 30-40% of that of myosin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly purified filamin revealed a single polypeptide of about 250,000 daltons. Rabbit antibody directed against purified chicken gizzard filamin did not crossreact with myosin purified from the same source. By the use of microcomplement fixation and indirect immunofluorescent staining with antibodies to chicken gizzard filamin, an antigenically similar or identical protein was found to be widely distributed both in other organs of the chicken and in cultured cells of other species, but not in chicken skeletal muscle. In cultured cells, filamin was found largely to be arranged as a filamentous array very similar to that found for myosin. These data imply that filamin is a widely occurring and chemically conserved component of filaments is smooth muscle and non-muscle cells.
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PMID:Filamin, a new high-molecular-weight protein found in smooth muscle and non-muscle cells. 5 35

Antibodies prepared against actomyosins can be shown to behave similarly, if not identically to more recently prepared antibodies against highly purified myosins. Details of the purification of the antigens, and of the production of antibodies to chick myosins from smooth gizzard muscle and from striated pectoral muscle are given. The antibody specificity appears to be directed against the heavy chains of the myosin molecules, since these antibodies specifically inhibit the myosin ATPase reaction, and since in situ staining of myosin polypeptide chains on an SDS gel using the antibodies in indirect fluorescence shows staining only in the heavy band region. Use of the antibodies in immunofluorescence microscopy suggest that the antibodies are tissue, but not species, specific. Example of their use in staining tissue sections are shown.
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PMID:Production of specific antibodies to contractile proteins, and their use in immunofluorescence microscopy. I. Antibodies to smooth and striated chicken muscle myosins. 5 8

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

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.
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PMID:Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin. 12 55

A myosin-like protein was identified in isolated rabbit liver cells. It was extracted with high-ionic-strength buffer containing ATP, and purified by gel filtration in the presence of iodide. The myosin polypeptide was indistinguishable in size from the heavy chain of muscle myosin as determined by electrophoresis on polyacrylamide gels and gel filtration in the presence of sodium dodecyl sulfate. The hepatic myosin had an amino acid composition similar to that of muscle myosin, but lacked 3-methylhistidine. The Mg2+ -ATPase of the myosin was not activated by muscle actin. At low ionic strength, in the presence of Mg2+, the protein aggregated to form bipolar filaments 0.3 mum in length. A protein which resembled muscle actin in size and amino acid composition was extracted along with the myosin. Based on scans of stained sodium dodecyl sulfate polyacrylamide gels, the myosin content was estimated as 0.3% to 0.4% of the cell protein. The actin-like component was present in approximately ten-fold excess by weight. This ratio suggests that the organization and function of myosin in the hepatocyte is very different from that in the muscle cell.
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PMID:The identification of myosin in rabbit hepatocytes. 13 48


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