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I Mabuchi

Publications and source records attributed to I Mabuchi.

At least 73 records · Page 4Linked to original sources
PubMed1985

Barbed end-capping protein regulates polarity of actin filaments from the human erythrocyte membrane.

The directional polymerization of G actin on single-layered erythrocyte membranes has been examined in the presence or absence of a barbed end-capping protein isolated from sea urchin eggs. When in the absence of the capping protein the single-layered erythrocyte membranes were incubated with 2 microM of G actin, exceeding the critical concentrations, about half of polymerized actin filaments became orientated with arrowheads of heavy meromyosin pointing toward the membrane at 2 microM of G actin. In contrast, in the presence of the capping protein, nearly 90% of the polymerized filaments were directed with arrowheads of HMM pointing away from the membranes. Furthermore, only preincubation of the erythrocyte membranes with the capping protein is effective to a similar extent in regulating the polarity of actin filaments from the membranes. The results obtained are discussed particular as regards to the physiological roles of the barbed end-capping protein in situ.

Actins↗
PubMed1985

Alpha-actinin from sea urchin eggs: biochemical properties, interaction with actin, and distribution in the cell during fertilization and cleavage.

A protein similar to alpha-actinin has been isolated from unfertilized sea urchin eggs. This protein co-precipitated with actin from an egg extract as actin bundles. Its apparent molecular weight was estimated to be approximately 95,000 on an SDS gel: it co-migrated with skeletal-muscle alpha-actinin. This protein also co-eluted with skeletal muscle alpha-actinin from a gel filtration column giving a Stokes radius of 7.7 nm, and its amino acid composition was very similar to that of alpha-actinins. It reacted weakly but significantly with antibodies against chicken skeletal muscle alpha-actinin. We designated this protein as sea urchin egg alpha-actinin. The appearance of sea urchin egg alpha-actinin as revealed by electron microscopy using the low-angle rotary shadowing technique was also similar to that of skeletal muscle alpha-actinin. This protein was able to cross-link actin filaments side by side to form large bundles. The action of sea urchin egg alpha-actinin on the actin filaments was studied by viscometry at a low-shear rate. It gelled the F-actin solution at a molar ratio to actin of more than 1:20, at pH 6-7.5, and at Ca ion concentration less than 1 microM. The effect was abolished by the presence of tropomyosin. Distribution of this protein in the egg during fertilization and cleavage was investigated by means of microinjection of the rhodamine-labeled protein in the living eggs. This protein showed a uniform distribution in the cytoplasm in the unfertilized eggs. Upon fertilization, however, it was concentrated in the cell cortex, including the fertilization cone. At cleavage, it seemed to be concentrated in the cleavage furrow region.

Actinin↗
PubMed1984

A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin.

A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase l-Sepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein-actin complex can thus be categorized as a capping protein.

Actins↗
PubMed1984

Capping one end of an actin filament affects elongation at the other end.

The rates of elongation at the free ends of actin filaments were compared to those of intact filaments, when the one end was masked with muscle beta-actinin or cytochalasin D, using fixed actoheavy meromyosin and Limulus acrosomal actin bundles as seeds. Experimental conditions were chosen so as to prevent spontaneous filament formation as far as possible. The rate of elongation at the barbed end of fixed actoheavy meromyosin was reduced to about one-fourth when the other pointed end was capped by beta-actinin, and that at the pointed end was reduced to one-third when the barbed end was blocked by cytochalasin D. Similar effects were also observed with the packed actin bundles of horseshoe crab sperm, although the decreases in elongation were less marked: 50-60% of the control both in the presence of beta-actinin and cytochalasin D. To explain the peculiar "end effect" described above, it is proposed that possible conformational changes at one end of an actin filament caused by the binding of a capping substance are transmitted successively to the other end so as to affect the elongation there.

Actinin↗
PubMed1983

An actin-depolymerizing protein (depactin) from starfish oocytes: properties and interaction with actin.

Physico-chemical properties and interaction with actin of an actin-depolymerizing protein from mature starfish oocytes were studied. This protein, which is called depactin, exists in a monomeric form under physiological conditions. Its molecular weight is approximately 20,000 for the native protein and approximately 17,000 for denatured protein. The Glu + Asp/Lys + Arg molar ratio of this protein is 1.55. The apparent pl of the denatured depactin is approximately 6. The extent of actin polymerization is reduced by the presence of depactin; however, the rate of polymerization seems to be accelerated as measured spectrophotometrically at 238nm. This effect is interpreted to indicate that depactin cut the newly formed filaments into small fragments, thereby increasing the number of the filament ends to which monomers are added. The apparent critical concentration of actin for polymerization, as determined by viscometry or flow birefringence measurement, is increased by the presence of depactin in a concentration-dependent manner. Raising the pH of the solution does not reverse the action of depactin. The molar ratio of actin and depactin, which interact with each other, is estimated to be 1:1 by means of a cross-linking experiment using a water-soluble carbodiimide. Depactin binds to a DNase I-Sepharose column via actin and is selectively eluted with 0.6 M KCl or 0.6 M Kl. The association constant between actin and depactin is estimated, using the column, to be 2-3 X 10(6) M-1. The content of depactin in the high-speed supernatant of the oocyte extract is determined to be 1%; this can act upon approximately 63% of the actin in the supernatant.

Actin Depolymerizing Factors↗
PubMed1983

Electron microscopic determination of the actin filament end at which cytochalasin B blocks monomer addition using the acrosomal actin bundle from horseshoe crab sperm.

G-actin freed from exogenous ATP was added to the pieces of isolated acrosomal actin bundles from horseshoe crab sperm to form filaments as reported earlier (Tilney, L.G., Bonder, E.M., & DeRosier, D.J. (1981) J. Cell Biol. 90, 485-494). The growth of a filament was far more rapid at one end (the preferred end) than the other end. These ends were shown to correspond to the barbed and pointed ends, respectively, by decoration of the filaments with myosin subfragment 1. Cytochalasin B inhibited the monomer addition at the preferred end. This technique is useful in determining the ends to which actin filament end-binding proteins from nonmuscle cells bind, which are considered to regulate the actin polymerization in the cells.

Acrosome↗
PubMed1983

[Cytokinesis].

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Animals↗
PubMed1982

Effects of phalloidin microinjection and localization of fluorescein-labeled phalloidin in living sand dollar eggs.

Effects of microinjection of phalloidin on fertilization and cleavage of sand dollar (Clypeaster japonicus and Scaphechinus mirabilis) eggs were studied. The drug, previously injected into unfertilized eggs, showed no effect on the elevation of the fertilization membrane upon insemination up to an intracellular concentration of 50 microM. However, the movement of the egg pronucleus to the sperm pronucleus was inhibited and the fusion of pronuclei did not occur. The subsequent development no longer took place. When phalloidin was injected into fertilized eggs, the thickness of the cortical layer increased and the microvilli became conspicuous. Both nuclear division and cleavage were inhibited at the intracellular concentration of more than 20 microM, though the latter seemed to be more sensitive to phalloidin than the former. Fluorescein-labeled phalloidin (FL-phalloidin) was injected into eggs in order to investigate F-actin localization by fluorescence microscopy. In both unfertilized and fertilized eggs, FL-phalloidin was localized in the cortical layer within 1 min after injection. It was also localized in the cortical layer as radially oriented rod-like structures when injected into fertilized eggs before the disappearance of the nuclear membrane. No distinct fluorescence was detected in the mitotic apparatus or in the cleavage furrow. FL-phalloidin redistributed gradually into egg cytoplasm. In unfertilized eggs, fluorescent rods were found especially in the egg pronucleus 30 min after injection.

Actins↗
PubMed1982

Evidence that myosin does not contribute to force production in chromosome movement.

Antibody against cytoplasmic myosin, when microinjected into actively dividing cells, provides a physiological test for the role of actin and myosin in chromosome movement. Anti-Asterias egg myosin, characterized by Mabuchi and Okuno (1977, J. Cell Biol., 74:251), completely and specifically inhibits the actin activated Mg++ -ATPase of myosin in vitro and, when microinjected, inhibits cytokinesis in vivo. Here, we demonstrate that microinjected antibody has no observable effect on the rate or extent of anaphase chromosome movements. Neither central spindle elongation nor chromosomal fiber shortening is affected by doses up to eightfold higher than those require to uniformly inhibit cytokinesis in all injected cells. We calculate that such doses are sufficient to completely inhibit myosin ATPase activity in these cells. Cells injected with buffer alone, with myosin-absorbed antibody, or with nonimmune gamma-globulin, proceed normally through both mitosis and cytokinesis. Control gamma-globulin, labeled with fluorescein, diffuses to homogeneity throughout the cytoplasm in 2-4 min and remains uniformly distributed. Antibody is not excluded from the spindle region. Prometaphase chromosome movements, fertilization, pronuclear migration, and pronuclear fusion are also unaffected by microinjected antimyosin. These experiments demonstrate that antimyosin blocks the actomyosin interaction thought to be responsible for force production in cytokinesis but has no effect on mitotic or meiotic chromosome motion. They provide direct physiological evidence that myosin is not involved in force production for chromosome movement.

Actins↗
PubMed1982

Effects of muscle proteins on the interaction between actin and an actin-depolymerizing protein from starfish oocytes.

The effects of myosin, heavy meromyosin (HMM), alpha-actinin, tropomyosin, and calmodulin on the interaction between an actin-depolymerizing protein (depactin) from starfish oocytes and rabbit skeletal actin were investigated. 1. Alpha-actinin or tropomyosin did not affect the inhibitory effect of depactin on salt-induced polymerization of actin and did not induce actin polymerization when added to a mixture of actin and depactin in a buffer solution which was designed to keep actin in F-form. Myosin or HMM allowed polymerization of actin under similar conditions except that ATP was not included. Addition of ATP, inorganic pyrophosphate, adenyl-5'-yl imidodiphosphate, or ADP abolished the effect of myosin. 2. None of these proteins could keep actin in a polymerized form when depactin was added to the mixture of actin and these proteins in the presence of ATP. In the absence of ATP, however, myosin protected actin against depactin. 3. The association constant between actin and depactin was estimated from competitive binding experiments using HMM to be around 4.5 X 10(6) M-1 assuming the association constant between actin and HMM to be 3 X 10(9) M-1 (Greene, L.E. & Eisenberg, E. (1980) J. Biol. Chem. 255, 549-555). 4. Actin did not activate Mg-ATPase activity of HMM in the presence of depactin. From the above results, the mode of interaction of myosin with actin in the presence of depactin and the possible role of myosin in actin assembly in the cell are discussed.

Actin Depolymerizing Factors↗
PubMed1982

Actin modulating proteins in the sea urchin egg. I. Analysis of G-actin-binding proteins by DNase I-affinity chromatography and purification of a 17,000 molecular weight component.

Two groups of protein species which interact with G-actin were detected in unfertilized sea urchin eggs by DNase-I affinity chromatography in the presence of Ca2+. One of the protein groups, which comprised of six major proteins, was eluted by EGTA. One of these proteins was tentatively identified as calmodulin. The other protein group, comprising of four major proteins, could be dissociated from the immobilized DNase I at a higher ionic strength. One of these proteins, showing a molecular weight of 17,000 (17 K protein), was purified to homogeneity. In its action on actin, 17 K protein revealed properties quite similar to those of a protein called depactin isolated from unfertilized starfish oocytes, but different from those of profilins isolated from mammalian tissues or Acanthamoeba. 17 K protein co-migrated with depactin on an SDS-gel. It inhibited actin polymerization and quickly depolymerized F-actin. When added to G-actin before polymerization, 17 K protein suppressed the final extent of actin polymerization. This inhibition was not released by the addition of sonicated F-actin nuclei. When added to F-actin, 17 K protein rapidly reduced the viscosity and increased the G-actin concentration of the actin solution. In both cases, the final extent of actin polymerization strictly depended on the molar ratio of 17 K protein to actin, indicating a stoichiometric association between 17 K protein and actin.

Actins↗
PubMed1981

Purification from starfish eggs of a protein that depolymerizes actin.

A protein which is capable of depolymerizing F-actin was purified from an extract of unfertilized starfish eggs by the use of DEAE-cellulose column chromatography and hydroxylapatite column chromatography. This protein has an apparent molecular weight of 17,000. It inhibited the extent of actin polymerization as well as depolymerizing F-actin rapidly. It was shown that this protein reacts with actin at a molar ratio of 1 : 1. The properties of this protein were compared with those of profilin from mammalian tissues.

Actins↗
PubMed1980

Purification and properties of soluble actin from sea urchin eggs.

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were homogenized in a buffer containing 0.1 M KCl and 2 mM MgCl2 at pH 6.85. About 50% of the actin was recovered in the high-speed supernate of the homogenate. More than 80% of the actin in this supernate was found to be monomeric upon gel filtration chromatography through a Sephadex G-150 column or by a DNase I inhibition assay. The critical concentration for polymerization of this actin prior to further purification was 0.3-0.9 mg/ml under various conditions. Actin was purified to near homogeneity from the Sephadex G-150 pool with high yield. The purified actin had a critical concentration for polymerization of 0.02-0.03 mg/ml. The isoelectric point of the crude actin and the purified actin was the same. Indeed, we found that there is only one isoelectric focusing species of actin in the sea urchin egg, and it has an isoelectric point more basic than rabbit skeletal muscle actin. The discrepancy between the polymerizability of the crude and purified actin may be due to the presence of factors in the crude fraction which inhibit the polymerization of actin.

Actins↗
PubMed1979

Comparison of the 95,000 molecular weight protein from Limulus sperm with muscle alpha-actinin.

The 95,000 molecular weight protein (95K protein) of the false discharges of Limulus sperm, purified by means of preparative gel electrophoresis in the presence of sodium dodecyl sulfate, was compared with a 95K protein from Limulus muscle and chicken gizzard alpha-actinin. The results were as follows. 1) One-dimensional peptide mapping using four different proteases showed differences among these proteins. 2) Two-dimensional peptide mapping using trypsin showed that about 30% of the peptides in the digest of the sperm 95K protein were similar to those of chicken gizzard alpha-actinin and about 50% of the peptides were similar to those of the Limulus muscle 95K protein. 3) The sperm 95K protein contained relatively large amounts of Gly, Pro, and Ser and relatively small amounts of Glu and Leu compared to the muscle proteins. 4) Antibodies against the sperm 95K protein did not cross-react with the Limulus muscle 95K protein or chicken gizzard alpha-actinin. These results suggest that the 95K protein of sperm is different from alpha-actinin in primary structure.

Actinin↗
PubMed1977

The effect of myosin antibody on the division of starfish blastomeres.

Antiserum against starfish egg myosin was produced in rabbits. Antibody specificity to myosin was demonstrated by Ouchterlony's immunodiffusion test and by immunoelectrophoresis in the presence of sodium dodecylsulfate (SDS). The latter technique showed that the antibody binds to both heavy and light chains of egg myosin. Furthermore, the antibody reacted with starfish sperm mysosin and starfish adult muscle myosin at both the heavy and light chains. It did not react with bovine platelet mysosin or rabbit skeletal muscle myosin in Ouchterlony's test; however, a weak reaction was observed in the presence of SDS between the antibody and these myosin heavy chains. Ca- and Mg-ATPase activities of egg myosin were not affected by the antibody, but it did inhibit actin-activated ATPase activity of egg myosin. Microinjection of the antibody into blastomeres of starfish eggs at the two-cell stage was carried out. Anti-egg myosin gamma-globulin inhibited the subsequent cleavages at an amount of more than 0.3 ng when injected at interphase. The inhibition was reduced when the injection was carried out near the initiation of cleavage. At the onset of the second cleavage the antibody was not inhibitory; however, an appropriate amount inhibited the third cleavage. Although the disappearance of the nuclear membrane was observed in the presence of the antibody, the formation of the mitotic apparatus was more or less disturbed. However the formation of daughter nuclei seemed to be scarcely affected by the antibody except that the distance between the nuclei was significantly smaller than normal.

Actins↗