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H Murofushi

Publications and source records attributed to H Murofushi.

At least 73 records · Page 4Linked to original sources
PubMed1987

Identification of the 190 kD microtubule-associated protein in cultured fibroblasts and its association with interphase and mitotic microtubules.

We previously investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of the adrenal medulla and adrenal cortex and found that they contain a new kind of MAP with a molecular weight of 190,000 (190 kD MAP) as a major species (Kotani, S., H. Murofushi, S. Maekawa, C. Sato, and H. Sakai. Eur. J. Biochem. 156, 23-29, 1986). We now have used an affinity purified anti-(190 kD MAP) antibody and show by indirect immunofluorescent microscopy the association of this MAP with microtubules in situ in TIG-3 cells (human embryonic lung fibroblasts). The 190 kD MAP was present along the interphase and mitotic microtubules, and there was no marked difference between the staining pattern with anti-tubulin and that with anti-(190 kD MAP) antibodies, evidence that the localization of 190 kD MAP is not restricted to the subset of microtubules. We also isolated MAPs from TIG-3 cells and identified their 190 kD MAP as a major heat-stable component. Several other unidentified polypeptides were recovered in the MAP fraction specifically.

Fibroblasts↗
PubMed1986

Purification and characterization of high Ca2+-requiring neutral proteases from porcine and bovine brains.

Porcine and bovine brain high Ca2+-requiring neutral proteases were purified to homogeneity by the same isolation procedures, and their properties were compared. A high degree of similarity existed between the two proteases. The purification procedures included ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on phenyl-Sepharose CL-4B, second DEAE-cellulose chromatography, second phenyl-Sepharose CL-4B chromatography, and gel filtration on Ultrogel AcA 34. Both purified enzymes were composed of Mr 75,000 and 29,000 subunits, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzymes required 250 microM Ca2+ for half-maximal activity and 700 microM Ca2+ for maximal activity. Sr2+ and Ba2+, but not Mg2+ or Mn2+, also activated both enzymes but not as effectively as Ca2+. Both enzymes displayed maximum activity at pH 7.5-8.0. Leupeptin, antipain, and trans-epoxysuccinyl-L-leucylagmatine inhibited both enzymes. Neurofilament triplet proteins and microtubule-associated proteins were extensively hydrolyzed by both proteases, but tubulin and actin were not hydrolyzed. The amino acid compositions of the two proteases were very similar. Antisera against bovine brain protease cross-reacted with porcine brain protease when examined by immunoelectrotransfer blot techniques.

Amino Acids↗
PubMed1986

Characterization of microtubule-associated proteins isolated from bovine adrenal gland.

We investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of both the adrenal medulla and the cortex. The major constituents of the adrenal MAPs isolated by the taxol-dependent procedure [Vallee, R. B. (1982) J. Cell. Biol. 92, 435-442] were several polypeptides in the high-molecular-mass region (high-Mr MAPs) and a 190000-Mr polypeptide (190-kDa MAP). In the cortex MAP fraction, the most prominent component was 190-kDa MAP, while the medulla MAP fraction was rich in high-Mr MAPs. Twice-cycled microtubule proteins prepared without taxol from the same sources also contained high-Mr MAPs and 190-kDa MAP. High-Mr MAPs contained protein species identical to MAP1 and MAP2 of mammalian brain as judged from electrophoretic mobility, heat-stability and immunoreactivity. 190-kDa MAP was classified as MAP subspecies distinct from high-Mr MAPs by several criteria. The MAP fractions had the ability to polymerize purified tubulin into microtubules, and the major MAP species (high-Mr MAPs and 190-kDa MAP) were found to cosediment with reconstituted microtubules. Tau factor, one of the major MAPs in the mammalian brain, appeared to be a minor species in the adrenal gland.

Adrenal Cortex↗
PubMed1986

Regulation of sperm flagellar movement by protein phosphorylation and dephosphorylation.

Flagellar motility of Triton models of sea urchin spermatozoa was reactivated by cyclic AMP-dependent protein kinase and a protein factor, termed motility activator, both of which were prepared from the detergent-extract of sea urchin spermatozoa. It was shown that phosphorylation of the motility activator by the protein kinase is necessary for the reactivation of flagellar motility [Ishiguro et al, J. Cell Biol. 92:777-782, 1982; Murofushi et al, in "Biological Functions of Microtubules and Related Structures," Academic Press, 1982]. Reactivating factor was also detected in a KCl-extract of the axoneme fraction devoid of the detergent-extractable materials. The activity of this factor was also cyclic AMP- and protein kinase-dependent. Furthermore, when freshly prepared Triton models were treated with phosphoprotein phosphatase prepared from bovine cardiac muscle, the flagellar motility was drastically suppressed. This inhibition of the motility was partially recovered by the addition of cyclic AMP and protein kinase to the phosphatase-treated models.

Adenosine Triphosphatases↗
PubMed1986

Purification and characterization of a 190-kD microtubule-associated protein from bovine adrenal cortex.

A heat-stable microtubule-associated protein (MAP) with molecular weight of 190,000, termed 190-kD MAP, was purified from bovine adrenal cortex. This MAP showed the same level of ability to promote tubulin polymerization as did MAP2 and tau from mammalian brains. Relatively high amounts of 190-kD MAP could bind to microtubules reconstituted in the presence of taxol. At maximum 1 mol of 190-kD MAP could bind to 2.3 mol of tubulin. 190-kD MAP was phosphorylated by a cAMP-dependent protein kinase prepared from sea urchin spermatozoa and by protein kinase(s) present in the microtubule protein fraction prepared from mammalian brains. The maximal numbers of incorporated phosphate were approximately 0.2 and approximately 0.4 mol per mole of 190-kD MAP, respectively. These values were lower than that of MAP2, which could be heavily phosphorylated by the endogenous protein kinase(s) up to 5 mol per mole of MAP2 under the same assay condition. 190-kD MAP had no effects on the low-shear viscosity of actin and did not induce an increase in turbidity of the actin solution. It was also revealed that 190-kD MAP does not cosediment with actin filaments. These data clearly show that, distinct from MAP2 and tau, this MAP does not interact with actin. Electron microscopic observation of the rotary-shadowed images of 190-kD MAP showed the molecular shape to be a long, thin, flexible rod with a contour length of approximately 100 nm. Quick-freeze, deep-etch replicas of the microtubules reconstituted from 190-kD MAP and brain tubulin revealed many cross-bridges connecting microtubules with each other.

Adrenal Cortex↗
PubMed1985

Role of microtubules and axolinin in membrane excitation of the squid giant axon.

The squid giant axon possesses numerous microtubules and a large pool of axolinin, both of which are preferentially localized at the peripheral region of the axoplasm just underlying the axolemma. A series of perfusion experiments presents evidence that microtubules localized in the clad are directly associated with the membrane excitability. The internal perfusons which cause microtubules to depolymerize reduce the excitability and those supporting microtubule assembly increase the peak sodium current. Direct evidence that microtubules are essential for membrane excitation is obtained by the restoration of diminished membrane excitability. The restoration system consists of squid brain or axoplasmic tubulin, axolinin, ATP, Mg2+, and cAMP or of porcine brain tubulin, tubulin-tyrosine ligase, axolinin, GTP, ATP, Mg2+, and cAMP. The former restoration system causes the diminished excitability to recover in such a way that the Na conductance, resting potential, and Na reversal potentials are much improved. One hypothesis is that tyrosinated tubulin is polymerized into microtubules underneath the axolemma. Axolinin binds to the axolemma at one end of the rod-like molecule and cross-links the assembled microtubules with the other end, thereby reconstructing cytoskeletons just underneath the axolemma which are necessary for membrane excitation.

Alkaloids↗
PubMed1985

Phosphoprotein phosphatase inhibits flagellar movement of Triton models of sea urchin spermatozoa.

Phosphoprotein phosphatase prepared from bovine cardiac muscle was used to study the roles of axonemal phosphoproteins in the flagellar motility of sea urchin spermatozoa. When isolated axonemes were incubated with cyclic AMP-dependent protein kinase, gamma-[32P]ATP and cyclic AMP, more than 15 polypeptides were phosphorylated. Most were dephosphorylated by treatment with phosphoprotein phosphatase. When Triton models of sea urchin spermatozoa were treated with phosphoprotein phosphatase followed by an addition of ATP, the flagellar motility of the models was drastically reduced in comparison with that of the untreated models. The motility of the phosphatase-treated Triton models was partially restored by an addition of cyclic AMP and cyclic AMP-dependent protein kinase. These data give strong support to the idea that the motility of eukaryotic flagella is controlled by a protein phosphorylation-dephosphorylation system.

Adenosine Triphosphatases↗
PubMed1984

Axonal microtubules necessary for generation of sodium current in squid giant axons: I. Pharmacological study on sodium current and restoration of sodium current by microtubule proteins and 260K protein.

Effects of the reagents suppressing or supporting axoplasmic microtubule assembly were studied on the Na ionic current of squid giant axons by perfusing the axon internally with the solution containing the reagent. Among the reagents suppressing the assembly, colchicine, vinblastine, podophyllotoxin, sulfhydryl reagents such as DTNB and NEM, and chaotropic anions such as iodide and bromide, were examined. These reagents reduced maximum Na conductance and shifted the voltage dependence of steady-state Na activation in a depolarizing direction along the voltage axis. They also made the voltage dependence less steep, but did not affect sodium inactivation appreciably. Effects on Na ionic current of reagents which support microtubule assembly (Taxol, DMSO, D2O and temperature) were opposite the effects of those agents suppressing assembly. At the same time, we demonstrated that after Na currents were partially reduced, they could be restored by internally perfusing the axon with a solution containing microtubule proteins, 260K proteins and cAMP under conditions favorable for microtubule assembly. For full restoration, it was found that the following conditions were necessary: (1) The microenvironment within the axon is suitable for microtubule assembly. (2) Tubulins incorporated into microtubules are fully tyrosinated at their C-termini. (3) A peripheral protein having a molecular weight of 260,000 daltons (260K protein) is indispensable. These results suggest that axoplasmic microtubules and 260K proteins in the structure underlying the axolemma play a role in generating Na currents in squid giant axons.

Alkaloids↗
PubMed1984

33K protein--an inhibitory factor of tubulin polymerization in porcine brain.

A factor (33K protein) that modulates tubulin polymerization in vitro has been purified to homogeneity from porcine brain by ammonium sulfate fractionation and Whatman DE52, Toyo-pearl HW65C and Bio-Gel A 0.5 m column chromatographies. The purified fraction was free of nucleic acids and sugars. The activity of the purified 33K protein is pronase E sensitive but apparently heat- and trypsin-resistant though it undergoes tryptic digestion. The 33K protein inhibits polymerization of brain microtubule proteins in a dose-dependent manner and partially depolymerizes preformed microtubules. It also inhibits polymerization of purified starfish tubulin and microtubule elongation involving fragellar outer doublet microtubules and purified porcine brain tubulin. This suggests that the target of the 33K protein is tubulin rather than microtubule-associated proteins. The 33K protein causes incomplete depolymerization of microtubules and a new steady state is quickly attained which is apparently independent of microtubule mass concentration. Divalent cations such as calcium and magnesium do not modulate the inhibitory activity of the 33K protein.

Animals↗
PubMed1984

An 18K protein from ascites hepatoma cell depolymerizes actin filaments rapidly.

Several actin binding proteins were isolated from ascites hepatoma cells AH7974 by DNase I affinity chromatography. Among them, a protein having a molecular weight of 18,000 was further purified by DEAE cellulose and hydroxyapatite column chromatographies and gel filtration on a Sephadex G-75 column. The 18K protein not only inhibits actin polymerization but also depolymerizes actin filaments. This conclusion was supported by viscosity and fluorescence intensity measurements and the DNase I inhibition assay. A chemical cross-linking experiment suggested that the 18K protein binds to monomeric actin and forms and 18K-actin 1:1 complex. The net depolymerization rate by the 18K protein measured by the DNase I inhibition assay was slower than the rapid reduction of the fluorescence intensity of pyrene-labeled F-actin upon addition of the 18K protein. This result suggests that the 18K protein not only binds to monomeric actin but also binds to actin filaments directly. The sedimentation assay showed that a part of the 18K protein was cosedimented with actin filaments. Electron microscopic observations demonstrated that the 18K protein decreased the amount of actin filaments and the remaining filaments appeared to be decorated and distorted by the 18K protein. The 18K protein had no Ca2+ ion sensitivity and exhibited the same effect on both this tumor actin and muscle actin.

Actins↗
PubMed1983

Bundling of microtubules in vitro by a high molecular weight protein prepared from the squid axon.

A high molecular weight protein has been partially purified from sheaths of squid giant axons. This protein fraction was capable of restoring the membrane excitability of the squid axon which had been destroyed by internal perfusion of microtubule poison, when perfused along with microtubule proteins (Matsumoto et al. (1979) J. Biochem. 86, 1155-1158). This protein, designated as 260 K protein, was purified by gel filtration and Con A-Sepharose affinity chromatography. The apparent molecular weight of the axonal protein was estimated to be 260,000 by electrophoresis in the presence of sodium dodecylsulfate. This protein was revealed to be a glycoprotein. When phosphocellulose-purified tubulin was incubated with 260 K protein at 36 degrees C in the presence of dimethylsulfoxide, turbidity of the solution was much increased. 260 K protein co-sedimented with microtubles assembled from purified tubulin. Light microscopic and electron microscopic observations revealed that the high turbidity was due to bundling of microtubules which was caused by 260 K protein. On the other hand, the effect of this protein on the turbidity increase was not so prominent when microtubules were assembled from microtubule proteins consisting of tubulin and microtubule-associated proteins. High shear and low shear viscometry and co-sedimentation experiments revealed that 260 K protein had little effect on actin polymerization under the same medium conditions as used in tubulin polymerization.

Amino Acids↗
PubMed1983

Bundling of microtubules in vitro by fodrin.

Fodrin is a spectrin-like protein present in the cortical cytoplasm of neurons and binds to F-actin to induce gelation of actin. We found that fodrin purified from porcine brains co-sedimented with microtubules which were assembled from phosphocellulose-purified tubulin prepared from porcine brains. This indicates that fodrin bound to microtubules. An unusual enhancement of turbidity at 350 nm was observed when microtubules were assembled in the presence of fodrin. Microscopic observations showed that fodrin bundled the microtubules. The interaction between fodrin and microtubules was decreased by microtubule-associated proteins (MAPs), indicating that fodrin and MAPs interacted with microtubules competitively. These data raise the possibility that microtubules are involved in the submembranous cytoskeleton of neurons with fodrin.

Animals↗
PubMed1982

Purification of an endogenous 68,000-dalton inhibitor of Ca2+-activated neutral protease from chicken skeletal muscle.

An endogenous inhibitor of Ca2+-activated neutral protease has been purified from chicken skeletal muscle. The inhibitor, which was isolated by acid treatment and four subsequent chromatographic procedures, is a protein composed of a single polypeptide having a molecular weight of 68,000. It contains large amounts of glycine, proline and glutamic acid but the contents of aromatic amino acids are low. The inhibitor appeared to act on Ca2+-activated neutral protease in a stoichiometric manner, suggesting a complex formation. The inhibitory action was not caused by sequestering of Ca2+. The inhibitor did not inhibit other well-known thiol proteases, but was slightly effective for trypsin.

Amino Acids↗
PubMed1982

Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of triton X-100 models of sea urchin and starfish spermatozoa.

A fraction obtained from detergent-extract of sea urchin or starfish spermatozoa using DEAE-cellulose chromatography reactivated Triton X-100 models of the spermatozoa in a cAMP-dependent manner. The DEAE fraction contained cAMP-dependent protein kinase with a high level of specific activity. Rabbit muscle inhibitor protein highly specific for cAMP-dependent protein kinases inhibited the ability of the deae fraction to induce reactivation of Triton X-100 models.l This inhibition paralleled inhibition of cAMP-dependent protein kinase activity of the DEAE fraction, suggesting participation of the enzyme in the cAMP-dependent reactivation of Triton X-100 models. However, cAMP-dependent protein kinase further purified from the DEAE fraction was incapable of reactivating these models by itself. A protein factor which was separated from the protein kinase in the course of purification of the enzyme was found to also be necessary for the reactivation. When cAMP-dependent protein kinase was pretreated with protein kinase inhibitor before addition of the protein factor, the reactivation of Triton X-100 models was no longer detected. However, after the protein factor had been incubated with cAMP and cAMP-dependent protein kinase, protein kinase inhibitor did not repress reactivation of Triton X-100 models. We propose that the reactivation needs phosphorylation of the protein factor by cAMP-dependent protein kinase.

Animals↗
PubMed1982

Interaction of tubulin with neurofilaments: formation of networks by neurofilament-dependent tubulin polymerization.

Interaction between porcine brain tubulin and neurofilaments was investigated with reference to network formation in vitro. When a mixture of tubulin and neurofilaments was incubated at 33--34 degrees C, its low-shear viscosity measured with a falling ball viscometer, was far above the sum of viscosities of the separate components and gelation was observed upon increasing the concentrations of both tubulin and neurofilaments above a certain level. Gelation that required Mg2+ and GTP was inhibited by Ca2+,Ca2+-calmodulin or ATP. Antimitotic drugs suppressed gelation at substoichiometric concentrations and cold treatment destroyed the formed gel, which indicated that gelation occurred in conjunction with tubulin polymerization. Assaying by centrifugation revealed that the amount of tubulin co-sedimented with neurofilaments was evidently larger in the presence of neurofilaments than in their absence. Furthermore, electron micrographs showed that a large number of microtubules which were shorter than usual were formed in the presence of neurofilaments. Interestingly, measurements with an Ostwald-type viscometer demonstrated that neurofilaments elevated the viscosity of the tubulin solution in a concentration-dependent fashion. In other words, neurofilaments had the ability to stimulate polymerization of tubulin. We conclude that polymerization of tubulin with neurofilaments produces three-dimensional networks in vitro.

Animals↗
PubMed1980

Isolation of calmodulin from the protozoan, Tetrahymena pyriformis, by the use of a tubulin-Sepharose 4B affinity column.

We developed a simple new method for the isolation of calmodulin, a ubiquitous Ca2+-binding protein, by using tubulin-Sepharose 4B column chromatography, and succeeded in rapid isolation of calmodulin from Tetrahymena pyriformis. The procedure was also shown to be successfully applicable to the isolation of calmodulins from starfish ovary, porcine brain, and monkey brain and, therefore, may be of general use for the rapid isolation of calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Purification and characterization of tubulin-tyrosine ligase from porcine brain.

A tubulin-tyrosine ligase was purified from porcine brains using DEAE-cellulose chromatography, Sepharose-sebacic acid hydrazide-ATP affinity chromatography and Sepharose-tubulin affinity chromatography. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 46,000. The apparent molecular weight was 37,000 on a high speed liquid chromatograph equipped with gel filtration columns. The pH optimum for the activity was around 8 and a second peak was observed at around 6.5 8.5 microM ATP or 30 microM tyrosine gave half-maximal activity. The purified enzyme catalyzed the tyrosination of the alpha subunit of tubulin in vitro.

Amino Acids↗