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Biomedical subjects

K Sobue

Publications and source records attributed to K Sobue.

At least 109 records · Page 6Linked to original sources
PubMed1987

Cytosynalin: a Mr 35,000 cytoskeleton-interacting and calmodulin-binding protein.

A Mr 35,000 calmodulin-binding protein, which also possesses the ability to interact with a number of cytoskeletal elements, was purified from bovine synaptosomal membranes, using hydroxylapatite and phosphocellulose column chromatography. We call this protein "cytosynalin," which is derived from a combination of the words "cytoskeleton" and "synapsis." Indirect immunofluorescent staining of cytosynalin revealed that it is distributed throughout neural cells including in dendrites, varicosities, and growth cones. In 3T3 cells, cytosynalin is located on the inner surface of the plasma membrane. The staining pattern of cytosynalin observed with anti-cytosynalin antibodies was similar to that observed with antibodies against calspectin (nonerythroid spectrin or fodrin). Cytosynalin binds to calspectin and to tubulin and actin filaments, as determined by binding assays, turbidity measurements, low-shear viscometry, and electron microscopy. Furthermore, after heating at 90 degrees C for 5 min, cytosynalin retains its binding activity toward calmodulin and cytoskeletal elements. These results lead to the conclusion that cytosynalin is colocalized with calspectin at the inner surface of the plasma membrane, probably in association with other cytoskeletal elements.

Amino Acids↗
PubMed1987

Purification of an 80 kDa Ca2+-dependent actin-modulating protein, which severs actin filaments, from bovine adrenal medulla.

A protein with a molecular weight of 80 kDa, which binds Ca2+-dependently to actin, was purified chromatographically from bovine adrenal medulla by using Sephacryl S-300, DEAE-Sepharose, actin-DNase I Sepharose, and Sephacryl S-200. This protein was retained on an actin-DNase I affinity column only in the presence of Ca2+, and could be eluted from this column by EGTA. The 80 kDa protein is a monomer and binds to G-actin in a Ca2+-dependent manner at an equimolar ratio. It caused fragmentation of actin filaments at more than 4 X 10(-7) M free Ca2+ concentration, as determined by low-shear viscometry and electron microscopy. Saturating amounts of tropomyosin showed a slight protective effect on the fragmentation of actin filaments by the 80 kDa protein. Considering the mode of action on actin filaments, the 80 kDa protein reported here seems to be a gelsolin-like protein. Gel electrophoresis of this protein revealed changes in mobility depending upon the concentration of Ca2+. This result also indicates that the 80 kDa protein itself is a Ca2+-binding protein.

Actins↗
PubMed1987

Purification of caldesmon and myosin light chain (MLC) kinase from arterial smooth muscle: comparisons with gizzard caldesmon and MLC kinase.

We have developed a simple and conventional purification method for caldesmon and MLC kinase from bovine arterial smooth muscle, and compared the arterial and gizzard proteins. Arterial caldesmon shares the alternative binding to calmodulin or F-actin in a Ca2+-dependent manner and the antigenic determinants with the gizzard protein. Both caldesmons have the same association constant with F-actin (1.3-1.7 X 10(7) M-1) and the same maximum binding (1 caldesmon per 12-14 actins). However, the molecular weight of arterial caldesmon (dimer of a 148 kDa polypeptides) was slightly different from that of gizzard caldesmon (heterodimer of 150/147 kDa polypeptides). The molecular weight of arterial MLC kinase (160 kDa) was much larger than that of the gizzard enzyme (135 kDa). The enzyme activities of both MLC kinases were comparable (Km = 9.5 microM, Vmax = 12.5 mumol/min X mg). The association constant of the arterial enzyme to F-actin (5.1 X 10(6) M-1) was much larger than that of the gizzard enzyme (9.0 X 10(5) M-1) but the maximum binding was the same (1 enzyme per 12-13 actins). Immunocytochemical examinations showed that caldesmon and MLC kinase in cultured arterial cells have a restricted localization along the stress fibers, suggesting functional linkages between both proteins and actin filaments in vivo.

Animals↗
PubMed1987

Isolation and characterization of a calmodulin binding fragment of chicken gizzard caldesmon.

A calmodulin binding portion was separated from chicken gizzard caldesmon by chymotryptic digestion and it was purified through two column chromatography steps on calmodulin-Sepharose and Ultrogel AcA 44. The isolated fragment has an estimated molecular weight of 35,000 (35K) and it was possibly derived from the C-terminal portion of caldesmon. The affinity of the 35K fragment for calmodulin was determined by using the characteristic calmodulin-dependent mobility shift in polyacrylamide gel electrophoresis. The 35K fragment retained the actin binding site of caldesmon. The interaction of the 35K fragment with actin was released in the presence of Ca2+ and calmodulin.

Actins↗
PubMed1987

Cyclosporin A inhibits late steps of T lymphocyte activation after transmembrane signaling.

The in vitro stimulation of murine splenic T lymphocytes with concanavalin A (Con A) produced interleukin 2 (IL2). The addition of cyclosporin A (CsA) to the culture resulted in complete inhibition of IL2 production. The Con A stimulation of T lymphocytes induced the breakdown of phosphatidylinositol into inositol trisphosphate and diacylglycerol, each of which could function as the second messengers in the subsequent signal transduction pathway. CsA did not inhibit the production of inositol (poly)phosphates. Further, CsA did not affect Ca2+-calmodulin functions; a) the redistribution of various cytoskeletal proteins as well as Con A-receptor aggregation, and b) the cytosolic Ca2+-calmodulin-dependent enzyme activities. Moreover, the activity of protein kinase C, which has been accepted to be the target of diacylglycerol, was not influenced in the presence of CsA. While the above steps of signal transduction are bypassed by synergy between calcium ionophore and phorbol ester, T lymphocyte activation which was induced by such stimuli was completely inhibited by CsA. These results indicate that CsA does not influence early steps of T lymphocyte activation as bypassed by calcium ionophore and phorbol ester, but rather inhibits later step(s) subsequent to the activation of protein kinase C and Ca2+-calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

General characteristics of the superprecipitation reaction of rat crude uterine myosin B: effects of L-methionine and/or transmethylation blockers on the reaction.

The characteristics of superprecipitation of uterine myosin B from 17 beta-estradiol-3-benzoate-treated rats and the effects of L-methionine and/or 3-deazaadenosine (3-DAA) plus homocysteine thiolactone (HCTL) on the reaction were investigated. The slope and plateau phases of the superprecipitation were dependent on ATP, calcium and magnesium. The calmodulin antagonists, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, inhibited the slope and plateau phases, but not the basal superprecipitation. SDS-polyacrylamide gel electrophoresis of the precipitate obtained in complete medium showed the presence of actin and myosin. Addition of 3 mM L-methionine had little affect on either the slope or plateau phases of the basal- or calcium-dependent superprecipitation, while 500 microM 3-DAA plus 1 mM HCTL caused only 9 to 20% decrease in the calcium-dependent superprecipitation in the presence of 0.1 mM or 1 mM ATP. These findings suggest that the calcium-dependent superprecipitation of rat uterine myosin B reflected the contractile response of uterine muscle, and that L-methionine and/or 3-DAA plus HCTL did not affect the calcium-dependent superprecipitation of uterine myosin B.

Animals↗
PubMed1986

Calspectin (fodrin or nonerythroid spectrin)-actin interaction: a possible involvement of 4.1-related protein.

The calspectin/actin complex extracted from the bovine brain membrane crosslinks F-actin, resulting in the increasing viscosity of F-actin determined by low-shear viscometry. We demonstrated the presence of a protein factor in this complex, which regulated the calspectin-F-actin interaction in a Ca2+- and calmodulin-dependent manner. Erythrocyte protein 4.1, but not synapsin I, mimics the function of this brain factor using a reconstitution system including purified calspectin, calmodulin and F-actin. In the brain complex, the Mr 120,000 and the Mr 80,000/77,000 polypeptides were detected to crossreact with anti-protein 4.1 antibody.

Actins↗
PubMed1985

Assignment of amides in the amino acid sequence of mammalian calmodulin by mass spectrometry.

The primary structure of mammalian calmodulin from various sources was analyzed by secondary ion mass spectrometry. The assignments of amide groups at residues 24, 60, 129 and 135, which had been controversial in the previous reports, were in perfect agreement with the amino acid sequence deduced from the cDNA sequence of calmodulin of other vertebrates. The result is against the post-translational amidation or deamidation process of this protein.

Amides↗
PubMed1985

Caldesmon150 regulates the tropomyosin-enhanced actin-myosin interaction in gizzard smooth muscle.

Using a reconstituted system in which myosin was preferentially phosphorylated, we examined the regulatory action of caldesmon150 on the smooth muscle actin-myosin interaction. Caldesmon150 inhibited the tropomyosin-enhanced actomyosin ATPase activity in a Ca2+-independent manner. This inhibitory effect of caldesmon150 was observed to be overcome by the addition of calmodulin in a Ca2+-dependent manner. In accordance with the observations of ATPase activity, we demonstrated evidence that the regulatory action of caldesmon150 on the actin site was mainly through control of the tropomyosin-enhanced actin-myosin interaction and calmodulin confers the Ca2+-sensitivity upon the caldesmon150 action determined by the cosedimentation method.

Actins↗
PubMed1985

Linear relationship between diphosphorylation of 20 kDa light chain of gizzard myosin and the actin-activated myosin ATPase activity.

The addition of large amounts of myosin light chain kinase to the reconstituted gizzard actomyosin shows diphosphorylation of 20 kDa myosin light chain. Accompanying diphosphorylation, the actin-activated myosin ATPase activity was also enhanced. The extent of diphosphorylation and the myosin ATPase activity were clearly demonstrated to be in a linear relationship. From the time course experiment, the conversion of monophosphorylated light chain into one which was diphosphorylated seemed to be a sequential process. Moreover, analyzing phospho-amino acid by using a two-dimensional electrophoresis technique revealed that monophosphorylated light chain contained phosphoserine and diphosphorylated one contained phosphothreonine in addition to phosphoserine.

Actins↗
PubMed1985

Ca2+ and calmodulin regulate microtubule-associated protein-actin filament interaction in a flip-flop switch.

MAP2 (microtubule-associated protein 2) and tau factor are calmodulin-binding and actin filament-interacting proteins, respectively. We have examined the effect of Ca2+ and calmodulin on MAP-induced actin gelation by the low-shear falling-ball method, the high-speed centrifugation method, and electron microscopy using negative staining. Each MAP crosslinks actin filaments to increase the apparent viscosities and finally to form gels. Calmodulin inhibited MAP2- and tau factor-induced actin gelation (MAP2- and tau factor-actin interaction) only in the presence of Ca2+, but not in its absence. There were no differences in actin filament crosslinking activity of respective MAPs with or without Ca2+. MAP2 was not coprecipitated with F-actin only in the presence of Ca2+ and calmodulin determined by the high-speed centrifugation method. But MAP2 was found to bind to F-actin under any other conditions examined. In contrast, the tau factor-actin filament interaction could only be detected by the low-shear viscosity, but not by the high-speed centrifugation method. MAP2 and tau factor aggregated to form actin bundles as shown by electron microscopy. MAP2- or tau factor-induced bundle formation of actin filaments was inhibited only in the presence of Ca2+ and calmodulin, but not in the presence or absence of Ca2+. In conclusion, the interaction of MAP2- and tau factor-actin filaments is regulated by Ca2+ and calmodulin in a flip-flop switch.

Actins↗
PubMed1985

Crosslinking of actin filaments is caused by caldesmon aggregates, but not by its dimers.

A recent report by Bretscher [(1984) J. Biol. Chem. 259, 12873-12880] showed that caldesmon prepared by his method crosslinks actin filaments to form thick bundles. This is in contrast to the results of previous work that caldesmon binds to F-actin but does not cause any gelation [(1981) Proc. Natl. Acad. Sci. USA 78, 5652-5655]. The present work clearly showed that caldesmon purified according to Bretscher does not cause any gelation of F-actin. However, caldesmon aggregates formed by concentration or by freeze-thawing gelated F-actin to form bundles.

Actins↗
PubMed1985

Purification and characterization of caldesmon77: a calmodulin-binding protein that interacts with actin filaments from bovine adrenal medulla.

Caldesmon150, a protein composed of the Mr 150,000/147,000 doublet, alternately binds to calmodulin and actin filaments in a Ca2+-dependent "flip-flop" fashion. In all fibroblast cell lines examined, we also found a Mr 77,000 protein that crossreacts with anti-caldesmon150 antibody by using an immunoprecipitation technique [Owada, M.K., Hakura, A., Iida, K., Yahara, I., Sobue, K. & Kakiuchi, S. (1984) Proc. Natl. Acad. Sci. USA 81, 3133-3137]. In this report, we examine the tissue distribution of caldesmon by the method of immunoblotting, using caldesmon-specific antibody. Both caldesmon150 and caldesmon77 show widespread distribution in the tissues examined. Caldesmon77 is more widely distributed than caldesmon150, and we have purified caldesmon77 from bovine adrenal medulla. Its molecular weight estimated by NaDodSO4/polyacrylamide gel electrophoresis was 77,000, and a tetramer of this polypeptide may constitute the native molecule (Mr, 300,000). Caldesmon77 possesses a number of features in common with caldesmon150, including flip-flop binding to calmodulin and actin filaments depending on the concentration of Ca2+ and crossreactivity with caldesmon150-specific antibody. Analysis of caldesmon77-F actin interaction by sedimentation and electrophoresis revealed that 0.5 mg of caldesmon77 bound to 1 mg of F actin. This indicated that the molar ratio between caldesmon77 (tetramer) and actin monomer was calculated to be 1:12-14. In addition, caldesmon77 regulated the actin-myosin interaction in Ca2+-sensitive actomyosin obtained from adrenal medulla. These results suggest that caldesmon77 might be a ubiquitous actin-linked regulator of nonmuscle contractile processes, including those in adrenal medulla.

Actins↗
PubMed1985

Phosphorylation of myosin light chain and the actin-activated ATPase activity of adrenal medullary myosin.

Myosin light chain kinase was partially purified from bovine adrenal medulla. A polypeptide of Mr 165,000 dalton was identified as kinase by using anti-gizzard myosin light chain kinase IgG on immunoreplica. Phosphorylation of medullary myosin was Ca2+- and calmodulin-dependent. The phosphorylated myosin was showed to enhance the actin-activated Mg2+-ATPase activity. In contrast, the myosin ATPase activity was dramatically decreased by dephosphorylation of myosin.

Actins↗
PubMed1985

Calcium-dependent interaction of actin filaments with actin binding protein in the presence of calmodulin and caldesmon.

Caldesmon, calmodulin-, and actin-binding protein of chicken gizzard did not affect the process of polymerization of actin induced by 0.1 M KCl. Caldesmon binds to F-actin, thus inhibiting the gelation action of actin binding protein (ABP; filamin). Low shear viscosity and flow birefringence measurements revealed that in a system of calmodulin, caldesmon, ABP, and F-actin, gelation occurs in the presence of micromolar Ca2+ concentrations, but not in the absence of Ca2+. Electron microscopic observations showed the Ca2+-dependent formation of actin bundles in this system. These results were interpreted by the flip-flop mechanism: in the presence of Ca2+, a calmodulin-caldesmon complex is released from actin filaments on which ABP exerts its gelating action. On the other hand, in the absence of Ca2+, caldesmon remains bound to actin filaments, thus preventing the action of ABP.

Actins↗
PubMed1985

Correlation between multiple phosphorylation of gizzard myosin light chains and actin-activated myosin ATPase activity.

With large amounts of gizzard Mr 135,000 calmodulin-binding protein (myosin light chain kinase), the phosphate incorporation into myosin light chains was determined to be 2 mol/mol of myosin light chain. The actin-activated ATPase activity was dramatically enhanced when myosin light chains were phosphorylated by more than 1 mol of phosphate incorporated/mol of myosin light chain.

Actins↗
PubMed1984

Immunocytochemical demonstration of caldesmon (a calmodulin-binding, F-actin-interacting protein) in smooth muscle fibers and absorptive epithelial cells in the small intestine of the rat.

The distribution of caldesmon (a calmodulin-binding, F-actin-interacting protein) (Sobue et al. 1982) and of actin was studied in the rat's small intestine by means of light-microscopic immunocytochemistry. Positive immunostaining for caldesmon was seen in smooth muscle cells of the intestinal wall, and of blood vessels, and in the apical portion of the absorptive epithelial cells. The immunoreactivity in goblet cells was difficult to recognize. The positive reaction to immunostaining for actin showed almost the same pattern as that for caldesmon. These results suggest that this calmodulin-binding protein may play an important role in the control of actin-myosin interaction in smooth muscle cells and in non-muscle cells.

Actins↗