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H Murofushi

Publications and source records attributed to H Murofushi.

At least 55 records · Page 3Linked to original sources
PubMed1990

Interaction mechanism between microtubule-associated proteins and microtubules. A proton nuclear magnetic resonance analysis on the binding of synthetic peptide to tubulin.

An amino acid sequence essential for microtubule-associated proteins (MAPs) to bind to microtubules is presented [Aizawa et al. (1989) J. Biol. Chem. 264, 5885-5890]. A synthetic peptide of 23 amino acid residues which corresponded to the sequence [tubulin binding peptide (TBP)] was active in binding to tubulin and inducing its assembly. The TBP-tubulin interaction mechanism was analyzed by proton nuclear magnetic resonance spectroscopy as a simplified model for MAP-microtubule interactions. Intraresidue transferred nuclear Overhauser effects (TRNOEs) of TBP in TBP-tubulin mixtures were analyzed, and strong binding of two Val and two Lys residues of TBP to tubulin was detected. Among the sharply peaked signals from tubulin aromatic residues, those due to Tyr ring protons broadened upon mixing with TBP, suggesting the involvement of Tyr residue(s) in the binding with TBP. Irradiation of the tubulin Tyr protons resulted in an intermolecular TRNOE at TBP methyl proton resonances. Evidently, hydrophobic interactions between Val and Tyr residues are important for the binding of TBP to tubulin. Hydrophobic interactions have not been taken into account previously in the widely accepted electrostatic model for the binding of MAPs to microtubules.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of a ubiquitously distributed microtubule-associated protein with Mr 190,000.

A heat-stable microtubule-associated protein (MAP) with apparent molecular weight of 190,000 is a major non-neural MAP which distributes ubiquitously among bovine tissues (termed here MAP-U). Previously we reported that microtubule-binding chymotryptic fragments of MAP-U and tau contain a common assembly-promoting (AP) sequence of 22 amino acid residues (Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1989) J. Biol. Chem. 264, 5885-5890). We isolated cDNA clones for MAP-U containing the whole coding sequence. Northern blot analysis revealed that a major species of MAP-U mRNA is 5 kilobases in length and is expressed ubiquitously among bovine tissues. Nucleotide sequence analysis revealed the complete amino acid sequence of MAP-U which consists of 1,072 amino acid residues. Analysis of the deduced amino acid sequence of MAP-U indicated that this molecule is clearly divided into two domains in terms of electrostatic charge distribution: an amino-terminal acidic domain (residues 1-640) and a carboxyl-terminal basic domain (residues 641-1072). The amino-terminal domain of MAP-U shows no significant sequence homology with other known protein sequences including neural MAPs, tau, and MAP-2. The amino-terminal domain of MAP-U contains unique 18 1/2 repeats of 14-amino acid motif which have not been observed in other MAPs. The carboxyl-terminal domain of MAP-U is further divided into three regions: a Pro-rich region (residues 641-880), an AP sequence region (residues 881-1003), and a short hydrophobic tail (residues 1004-1072). The Pro-rich region is mainly composed of five species of amino acid residues, Pro, Ala, Lys, Ser, and Thr. The AP sequence region contains four tandem repeats of AP sequences, and thus, this region is considered to play a leading role in the interaction of MAP-U with microtubules.

Amino Acid Sequence↗
PubMed1990

The mitotic apparatus-associated 51-kDa protein from sea urchin eggs is a GTP-binding protein and is immunologically related to yeast polypeptide elongation factor 1 alpha.

We investigated the biochemical characteristics of the 51-kDa protein that is a major mitotic apparatus-associated basic protein of sea urchin eggs (Toriyama, M., Ohta, K., Endo, S., and Sakai, H. (1988) Cell Motil. Cytoskeleton 9, 117-128). The amino acid composition of the 51-kDa protein was apparently different from those of tubulin, actin, histones, and myelin basic protein; yet it was similar to those of polypeptide elongation factors 1 alpha (EF-1 alpha). In addition, antibody to EF-1 alpha from yeast cross-reacted with the 51-kDa protein. [3H] GTP binding activity was detected in the phosphocellulose-purified fraction (PC fraction) which predominantly contained the 51-kDa protein and was shown to be specific to GTP, GDP, guanylyl imidodiphosphate, and ITP. Photo-affinity labeling using [alpha-32P]8-azidoguanosine triphosphate (8-azido-GTP) demonstrated that a 51-kDa polypeptide in the PC fraction specifically bound 8-azido-GTP. This GTP-binding polypeptide was bound to a GTP affinity column, could be eluted by the addition of GTP, and was immunoreactive with anti-51-kDa protein antibodies. When the PC fraction was applied to a gel filtration chromatography column, GTP binding activity was completely coeluted with the 51-kDa protein. Furthermore, the PC fraction and the gel filtration-purified fraction had EF-1 alpha activity: [14C]Phe-tRNA transferring activity to ribosomes in the presence of poly(U) and ribosome-dependent GTPase activity. The results indicate that the mitotic apparatus-associated 51-kDa protein is a GTP-binding protein and suggest that it is structurally and functionally related to yeast EF-1 alpha.

Affinity Labels↗
PubMed1989

A common amino acid sequence in 190-kDa microtubule-associated protein and tau for the promotion of microtubule assembly.

Previously we reported that chymotryptic fragments of bovine adrenal 190-kDa microtubule-associated proteins (27-kDa fragment) and bovine brain tau (14-kDa fragment) contained microtubule-binding domain (Aizawa, H., Murofushi, H., Kotani, Hisanaga, S., Hirokawa, N., and Sakai, H. (1987) J. Biol. Chem. 262, 3782-3787; Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1988) J. Biol. Chem. 263, 7703-7707). In order to study the structure of microtubule-binding domain of the two microtubule-associated proteins, we analyzed the amino acid sequence of the 27-kDa fragment and compared the sequence with that of the 14-kDa fragment. This revealed that 190-kDa microtubule-associated protein and tau contained at least one common sequence of 20 amino acid residues in their microtubule-binding domains. A synthetic polypeptide corresponding to the common sequence (Lys-Asn-Val-Arg-Ser-Lys-Val-Gly-Ser-Thr-Glu-Asn-Ile-Lys- His-Gln-Pro-Gly-Gly-Gly-Arg-Ala-Lys) was bound to microtubules competitively with the 190-kDa MAP. The apparent dissociation constant (KD) for the binding of the polypeptide to microtubules was estimated to be 1.8 x 10(-4) M, and the maximum binding reached 1.2 mol of the synthetic polypeptide/mol of tubulin dimer. This synthetic polypeptide increased the rate and extent of tubulin polymerization and decreased the critical concentration of tubulin for polymerization. The polypeptide-induced tubulin polymers were morphologically normal microtubules and were disassembled by cold treatment. The common sequence (termed assembly-promoting sequence) was thus identified as the active site of 190-kDa microtubule-associated protein and tau for the promotion of microtubule assembly. The reconstitution system of microtubules with this synthetic polypeptide with assembly-promoting sequence may be useful to elucidate detailed molecular mechanism of the promotion of microtubule assembly by microtubule-associated proteins.

Adrenal Glands↗
PubMed1989

Microtubule-associated protein-2 stimulates DNA synthesis catalyzed by the nuclear matrix.

Microtubule-associated protein-2 (MAP-2) isolated from porcine brains stimulated DNA synthesis catalyzed by the nuclear matrix isolated from Physarum polycephalum in the presence of activated DNA as exogenous templates. The degree of the stimulation depended on the amount of the nuclear matrix, but not on that of the template. MAP-2 also stimulated DNA polymerase alpha activity solubilized from nuclei, but not DNA polymerase beta activity. These results suggest that MAP-2 stimulates DNA synthesis by interacting with the putative DNA replication machinery including DNA polymerase alpha bound to the matrix. Similar stimulation occurred in the nuclear matrix isolated from HeLa and rat ascites hepatoma cells, which strongly suggests that MAP-2 is involved in the control of DNA replication in eukaryotic cells.

Animals↗
PubMed1989

Binding of kinesin to stress fibers in fibroblasts under condition of microtubule depolymerization.

The localization of kinesin in EBTr (bovine embryonic trachea fibroblast) cells was studied by indirect immunofluorescence microscopy using an affinity-purified antibody against bovine adrenal kinesin. It has already been shown that in interphase cells a part of kinesin is located on microtubules and the rest diffusely distributed throughout the cytoplasm [Murofushi et al., 1988]. When microtubules were depolymerized with cold or colchicine treatment, antikinesin antibody-stained fibrous components distinct from microtubules. These fibrous structures were considered to be stress fibers because they were stained with rhodamine-phalloidin and because the fibrous staining with antikinesin antibody was completely lost by treating the cells with cytochalasin D along with colchicine. When cold-treated cells in which a major part of kinesin had been localized on stress fibers were incubated at 37 degrees C, kinesin reappeared on reconstituted microtubules. These observations strongly suggest that kinesin has affinity not only to microtubules but also to stress fibers in culture cells.

Animals↗
PubMed1989

The molecular structure of adrenal medulla kinesin.

The molecular structure of bovine adrenal kinesin was studied by electron microscopy using the low-angle rotary shadowing technique. Adrenal kinesin exhibited either a folded or an extended configuration; the ratio of the two is dependent on the salt concentration. Almost all adrenal kinesin molecules were folded in a low-ionic solution, and the ratio of extended molecules increased to 40-50% in a solution containing 1 M ammonium acetate. Kinesin in the extended configuration displayed a rod-shaped structure with a mean length of about 80 nm. The morphologies of the ends were different; one end was composed of two globular particles, similar to the two-headed structure of myosin, while the other end had a more ill-defined structure, appearing either as a globular particle, an aggregate of two to four small granules, or a frayed, fan-like structure. The folded kinesin molecule possessed a hinge region in the middle of the rod, at about 32 nm from the neck of the two heads. In our preparations, the majority of adrenal kinesin molecules were folded at physiological salt concentrations. Adrenal kinesin bound to microtubules in the presence of adenylyl imidodiphosphate (AMP-PNP) also displayed a folded morphology.

Acetates↗
PubMed1989

Immunohistochemical localization of microtubule-associated proteins in the nervous system of the small intestine of guinea pig.

Layers containing Auerbach's and Meissner's plexuses were dissected from the small intestine of guinea pig and immunostained with affinity-purified antibodies against brain-specific microtubule-associated proteins (MAPs): MAP1, MAP2 and tau and a MAP with a molecular weight of 190,000 dalton purified from bovine adrenal cortex (190-kDa MAP). MAP1 antibody stained the network of nerve fibers and the cell bodies of enteric neurons in both Auerbach's and Meissner's plexuses. Staining with anti-tau antibody gave the same results. Antibody against MAP2 stained neuronal cell bodies and short thin processes extending from them. Interganglionic strands composed mainly of long processes were unstained. Anti-190-kDa MAP antibody stained both the neuronal cell bodies and bundles of nerve fibers. However, the staining was less intense than that with anti-MAP1 and tau antibodies. Differentiation in the structure of the cytoskeleton probably exists in the neuronal processes of the enteric neurons as is shown in the dendrites and axons in some neurons of the central nervous system. Thus, enteric neurons possess axon-like processes containing MAP1, tau and probably lower amounts of 190-kDa MAP. Cell bodies and dendrite-like structures of these neurons contain MAP2 in addition to MAP1, tau and 190-kDa MAP.

Animals↗
PubMed1988

Purification and characterization of kinesin from bovine adrenal medulla.

Kinesin was purified from bovine adrenal medulla. The sedimentation coefficient was 8.8 S. Sedimentation equilibrium ultracentrifugation studies showed the molecular weight of kinesin to be 300,000. The calculated axial ratio was 1:16. The Stokes radius was estimated to be 8.9 nm by gel filtration. Circular dichroism showed the alpha-helix content to be about 50%. Purified kinesin preparation contained a major polypeptide with a molecular weight of 120,000 and minor ones with molecular weights of 71,000, 68,000, and 65,000. Bovine adrenal kinesin had an ATPase activity which was stimulated severalfold by microtubules to a specific activity of about 0.1 mumol/min.mg. Kinesin molecules adsorbed to a glass slide promoted the movement of microtubules on the glass surface at a rate of about 0.5 micron/s. Immunostaining of EBTr (bovine embryonic trachea fibroblast) cells and bovine adrenal chromaffin cells in interphase with an affinity-purified antibody against the major polypeptide of kinesin showed that some kinesin was located on microtubules and the rest distributed throughout the cytoplasm in a diffuse manner. EBTr cells in mitotic phase gave a staining pattern showing that kinesin was present throughout the cytoplasm with higher concentration in the region of mitotic apparatus.

Adenosine Triphosphatases↗
PubMed1988

Microtubule-binding domain of tau proteins.

Limited chymotryptic digestion of whole tau proteins produced a fragment of Mr 14,000 (CT14), which was able to bind to microtubules reconstituted from tubulin alone in the presence of taxol. This fragment was also found to persist in microtubules when microtubules consisting of tau proteins and tubulin were digested by chymotrypsin. Analysis of amino acid composition revealed that CT14 was rich in lysine and proline residues, suggesting unique structure of microtubule-binding domain of tau proteins. Amino-terminal sequence of CT14 was determined to be Ser-Ser-Pro-Gly-Ser-Pro-Gly-Thr-Pro-Gly-Ser-Arg-Ser-Arg-X-Pro-Ser-Leu-Pr o. No heterogeneity was detected in this amino-terminal sequence of 19 residues. Five species of polypeptides consisting of tau proteins were separated from each other by gel electrophoresis and subjected to chymotryptic digestion. CT14 was produced from each of the tau polypeptides by chymotryptic digestion, indicating that all tau polypeptides have a common microtubule-binding domain.

Amino Acids↗
PubMed1988

Isolation of rat liver microtubule-associated proteins. Evidence for a family of microtubule-associated proteins with molecular mass of around 200,000 which distribute widely among mammalian cells.

Heat-stable microtubule-associated proteins (MAPs) were isolated from rat liver crude extract. The most prominent species showed a molecular mass close to that of bovine adrenal 190-kDa MAP (Kotani, S., Murofushi, H., Maekawa, S., Sato, C., and Sakai, H. (1986) Eur. J. Biochem. 156, 23-29), termed rat 190-kDa MAP. Immunological studies with antiserum against the rat 190-kDa MAP showed that this MAP exists in a variety of rat cells and tissues. The characteristics of the rat 190-kDa MAP, including molecular mass, heat stability, and distribution pattern, were very similar to those of bovine adrenal 190-kDa MAP. However, one-dimensional peptide mapping revealed considerable difference, and there is little mutual immunological cross-reactivity. We also identified in mouse neuroblastoma Neuro 2a cells a MAP of around 200 kDa which is considered to be MAP4 (Parysek, L. M., Asnes, C.F., and Olmsted, J.B. (1984) J. Cell Biol. 99, 1309-1315). MAP4 was slightly immunoreactive to both anti-(rat 190-kDa MAP) antiserum and anti-(bovine 190-kDa MAP) antiserum. Taking these results together, we conclude that mammalian tissues ubiquitously contain heat-stable MAPs of 200 kDa and that these 200-kDa MAPs should be considered as species-specific homologues.

Animals↗
PubMed1988

Dual effect of antimitotic drugs on steroid secretion in mouse adrenocortical Y-1 tumor cells.

Mouse adrenocortical Y-1 tumor cells were examined in a monolayer culture and their steroid secretion was measured. The Y-1 cells constantly released a small amount of steroids in the absence of adrenocorticotropin (ACTH). When synthetic ACTH (tetracosactide acetate) was added to the medium, an increase in the steroid secretion of approximately 5-fold was observed. The Y-1 cells also showed a typical cytoplasmic retraction in response to ACTH. Incubation of the cells with an antimitotic drug, colchicine, prior to ACTH-stimulation resulted in a 30-50% reduction in ACTH-induced steroid secretion. Under the conditions used in these experiments, viable numbers of cell and of total amount of protein per dish were not measurably changed, indicating that the condition was not lethal. Another antimitotic drug, colcemid, caused similar reactions, while lumicolchicine showed no effect. This suggests that the disruption of the microtubular system is the main cause of the inhibition. On the other hand, the ACTH-independent secretion was slightly enhanced by colchicine. The enhancement was also observed in prolonged incubation with colchicine, a condition which caused death in some of the cells.

Adrenal Cortex Neoplasms↗
PubMed1987

Expression of poriferasterol monoglucoside associated with differentiation of Physarum polycephalum.

A glycolipid which was expressed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephalum, has been examined. In the amoeboid stage, cells did not contain this glycolipid, but after conjugation of the haploid cells, this substance appeared and increased in its amount. From structural studies of the purified glycolipid, it has been identified as poriferasterol monoglucoside.

Cell Differentiation↗
PubMed1987

Limited chymotryptic digestion of bovine adrenal 190,000-Mr microtubule-associated protein and preparation of a 27,000-Mr fragment which stimulates microtubule assembly.

A heat stable microtubule-associated protein of Mr 190,000 (190-kDa MAP) has been purified from bovine adrenal cortex (Murofushi, H., Kotani, S., Aizawa, H., Hisanaga, S., Hirokawa, N., and Sakai, H. (1986) J. Cell Biol. 103, 1911-1919). Limited chymotryptic digestion of 190-kDa MAP produced a fragment of Mr 27,000 (27-kDa fragment), which bound to microtubules reconstituted in the presence of taxol. This fragment was purified with the aid of cosedimentation with microtubules. The purified 27-kDa fragment showed an ability to stimulate tubulin polymerization in the absence of taxol. Electron microscopic observation of microtubules reconstituted from purified 27-kDa fragment and tubulin revealed that the microtubules were in the form of thick bundles and that lateral projections which can be seen in microtubules reconstituted from intact 190-kDa MAP and tubulin were not observed. These results indicate that 27-kDa fragment includes or is a part of microtubule-binding domain of 190-kDa MAP and that this fragment is active in stimulating microtubule assembly. Amino acid analysis revealed that the 27-kDa fragment was rich in lysine, proline, and alanine, the sum of these three being about 45% of the total amino acids and that the contents of methionine, tyrosine, phenylalanine, and histidine were very low. These data suggest that the microtubule binding domain of the 190-kDa MAP comprises an unique structure.

Adrenal Cortex↗
PubMed1987

Comparison of a major heat-stable microtubule-associated protein in HeLa cells and 190-kDa microtubule-associated protein in bovine adrenal cortex.

A heat-stable microtubule-associated protein (MAP) with a molecular weight of 190,000, termed 190-kDa MAP, has been purified from bovine adrenal cortex (Murofushi, H. et al. (1986) J. Cell Biol. 103, 1911-1919). Immunoblotting experiments using an antibody against this MAP revealed that several kinds of culture cells derived from human tissues contain proteins with an apparent molecular weight of 180,000 reacting with the antibody. Indirect immunofluorescence microscopic observation of HeLa cells showed that the immunoreactive protein co-exists with microtubules, indicating that the protein is one of the HeLa MAPs. A heat-stable MAP with a molecular weight of 180,000, termed here HeLa 180-kDa MAP, was purified by the taxol-dependent procedure (Vallee, R.B. (1982) J. Cell Biol. 92, 435-442) and successive co-polymerization with brain tubulin. This protein was the most abundant MAP in HeLa cells, suggesting that the MAP is identical to the major HeLa MAP previously reported by Bulinski and Borisy (Bulinski, J.C. & Borisy, G.G. (1980) J. Biol. Chem. 255, 11570-11576) and Weatherbee et al. [1980) Biochemistry 19, 4116-4123). It was shown that, like bovine adrenal 190-kDa MAP, yet distinct from brain MAP2 and tau, purified HeLa 180-kDa MAP does not interact with actin filaments. This common characteristic of the two MAPs along with the same heat-stability strongly suggests that they are members of the same group of MAPs. The fact that HeLa 180-kDa MAP reacts with an antibody against bovine adrenal 190-kDa MAP means that they share common epitopes, in other words, common local amino acid sequences. However, the limited proteolytic patterns of the two MAPs with S. aureus V8 protease and chymotrypsin were distinct from each other, suggesting the presence of large differences in the overall primary structures between bovine adrenal 190-kDa MAP and HeLa 180-kDa MAP.

Actins↗