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S Kakiuchi

Publications and source records attributed to S Kakiuchi.

At least 19 recordsLinked to original sources
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

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

[Pharmacokinetics of cefoperazone in liver diseases].

To obtain useful informations for determining the optimal dosage of drugs in patients with impaired liver function, pharmacokinetics of cefoperazone (CPZ) was studied in healthy adults (normal control group) and 35 patients with liver disease (liver disease group). CPZ is a new third generation cephalosporin which is mainly excreted into the bile and has serum half-life of about 2 hours. After a rapid intravenous injection of CPZ, peripheral blood and urine samples were obtained at the time according to the protocol of this study. Serum and urine concentrations of CPZ were determined by the bioassay method using Micrococcus luteus ATCC 9341 as the test strain. From the concentrations of CPZ in serum and urine, pharmacokinetic parameters were calculated by means of a "two-compartment open model". After an administration of CPZ, the serum concentration declined more slowly in the liver disease group as compared to the normal control group. At 1 hour after intravenous injection of CPZ, the difference of the serum level was already observed between these 2 groups. The half-life of elimination (T 1/2) was 2 to 4 times longer in the liver disease group. The elimination rate constant (K10) and total clearance (Clt) of CPZ were much lower in the liver disease group than in the control group except for hepatocellular carcinoma. On the other hand, urinary excretion rate (Ur) was lower in the normal control group than in the liver disease group. There was a close correlation between disappearance rate of indocyanine green (KICG) and parameters such as T1/2, K10, Clt and Cler with coefficients of -0.642, 0.723, 0.690 and 0.682, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
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↗
PubMed1984

Immunocytochemical demonstration of caldesmon and actin in thyroid glands of rats.

The distribution of caldesmon (a calmodulin-binding, F-actin interacting protein; Sobue et al. 1982) and actin was studied in the rat thyroid gland by means of light-microscopic immunocytochemistry, and the fine-structural distribution of actin filaments was examined by use of heavy meromyosin (HMM). Caldesmon and actin were demonstrated in the apical cytoplasm of almost all the follicle epithelial cells in normal as well as TSH-treated animals. Immunoreactivities for both caldesmon and actin showed almost the same pattern in localization. The smooth muscle cells of the blood vessels were also positive for caldesmon and actin. By electron microscopy, numerous actin filaments decorated by HMM and running perpendicularly or randomly to the apical surface were recognized in the apical cytoplasm of the follicle epithelial cell. These results suggest that caldesmon and actin, in conjugation with calmodulin, play a role in the regulation of cellular activity such as exocytosis and endocytosis in the apical portion of the follicle epithelial cell.

Actins↗
PubMed1984

Occurrence of caldesmon (a calmodulin-binding protein) in cultured cells: comparison of normal and transformed cells.

Caldesmon is a calmodulin-binding and F-actin-binding protein originally purified from chicken gizzard smooth muscle. This protein binds to F-actin filaments in a Ca2+- and calmodulin-dependent "flip-flop" fashion, thereby regulating the function of actin filaments. Here we report that various lines of cultured cells contain a Mr 77,000 protein that specifically reacts with the affinity-purified caldesmon antibody raised against chicken gizzard caldesmon . Among the fibroblast proteins that had been pulse-labeled with [35S]methionine, the Mr 77,000 protein was the only protein band detected on the NaDodSO4 gel that reacted with the anticaldesmon . The subcellular distribution of the Mr 77,000 protein was investigated by the indirect immunofluorescence technique using the anticaldesmon . In all fibroblast cell lines examined, the immunofluorescence localized along the cellular stress fibers and in leading edges of the cell. In Rous sarcoma virus-transformed cells (S7-1), however, the distribution of the fluorescence changed to a diffuse and blurred appearance. These staining patterns of anticaldesmon obtained with the normal and transformed cells coincided with those of antiactin in the corresponding states, strongly suggesting the functional linkage between the Mr 77,000 protein and actin filaments. We propose to refer to this Mr 77,000 protein as caldesmon 77. The cellular level of caldesmon 77 in transformed S7-1 cells decreased to about one-third of that in their normal counterparts (cell line no. 7). Essentially the same result was obtained with normal rat kidney cells infected with the temperature-sensitive transformation mutant Schmidt-Ruppin strain of Rous sarcoma virus (68 N2 clone). The cellular level of caldesmon 77 observed at a permissive temperature (35 degrees C) was about one-third of that at a nonpermissive temperature (38.5 degrees C). These changes of caldesmon 77 in transformed cells may correlate with the loss of Ca2+ regulation in the transformed state.

Animals↗
PubMed1983

Caldesmon, a calmodulin-binding, F actin-interacting protein, is present in aorta, uterus and platelets.

Caldesmon, a protein originally found in chicken gizzard, was concluded also to be present in bovine aorta, uterus, and human platelets by demonstration of a protein with the following properties: (a) Ca2+-dependent calmodulin-binding; (b) binding to F actin in such way that the binding was broken on Ca2+-dependent binding of calmodulin; (c) cross-reactivity in immune blotting procedures with affinity-purified antibody against gizzard caldesmon; (d) similar subunit Mr-values on SDS-gel to those of gizzard caldesmon. Like gizzard caldesmon, platelet caldesmon was composed of two polypeptide bands of Mr 150 000 and 147 000, but caldesmon in aorta and uterus gave a single band of Mr 150 000. A polypeptide of Mr 165 000 that was immunologically distinct from caldesmon but, like caldesmon, bound to calmodulin and F actin in a flip-flop fashion, was also demonstrated in aorta and uterus.

Animals↗
PubMed1983

Heterogeneous pathways of Ca2+ metabolism in the triggering of the proliferative process in rat thymocytes.

Concanavalin A induces in rat thymocytes a calcium uptake at 1 h exposure and proliferative response after 24 h exposure. Phosphodiesterase activity parallels the proliferative response (thymidine uptake). Valinomycin, monensin and a small dose of ouabain also induce calcium uptake, but do not lead to thymidine uptake later. The latter treatments reduce, in some instances drastically, the concanavalin A response with respect to thymidine uptake. Trifluoperazine reduces the unstimulated thymidine uptake and the concanavalin A induced thymidine uptake. These results suggest that calcium has a decisive role in inducing proliferation but that some ways of increasing cellular Ca2+ concentration interfere in other steps with the DNA synthesis.

Animals↗
PubMed1983

Effects of phospholamban phosphorylation catalyzed by adenosine 3':5'-monophosphate- and calmodulin-dependent protein kinases on calcium transport ATPase of cardiac sarcoplasmic reticulum.

To elucidate the role of 22000-dalton protein phospholamban, a putative regulator of Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum, we examined the relationship between cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban and their effects on ATPase activity and calcium transport of cardiac sarcoplasmic reticulum. Cardiac microsomes were incubated with [gamma-32P]ATP or unlabeled ATP, catalytic subunit of cyclic AMP-dependent protein kinase and/or exogenous calmodulin, and subsequently assayed for ATPase activity and calcium uptake by cardiac sarcoplasmic reticulum. Cyclic AMP-dependent phosphorylation of phospholamban was independent of Ca2+, whereas calmodulin-dependent phosphorylation of phospholamban was dependent on Ca2+ within a range between 0.2 and 50 microM. Cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban occurred independently; when both kinases were operative, the amounts of phosphorylation were additive. Under these conditions, the phosphoproteins formed by cyclic AMP- and calmodulin-dependent protein kinases electrophoretically migrated as 11000-dalton components when sodium dodecyl sulfate-solubilized phosphoproteins were boiled prior to polyacrylamide gel electrophoresis. The ATPase activity was stimulated by either cyclic AMP- or calmodulin-dependent phosphorylation of phospholamban at Ca2+ concentrations up to 2 microM. The extents of stimulation of ATPase activity were additive when both types of phosphorylation were functional. Calcium uptake was similarly augmented by cyclic AMP- and/or calmodulin-dependent phosphorylation of phospholamban. These results indicate that Ca2+-dependent ATPase and calcium transport of cardiac sarcoplasmic reticulum are regulated by phospholamban phosphorylation catalyzed by cyclic AMP- and calmodulin-dependent protein kinases, thus suggesting a dual role of phospholamban in active calcium transport.

Biological Transport, Active↗