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H Tokumitsu

Publications and source records attributed to H Tokumitsu.

At least 55 records · Page 3Linked to original sources
PubMed1992

Site-directed mutation makes rabbit calcyclin dimer.

Unlike human, rat and mouse calcyclin, purified rabbit calcyclin did not form a dimer on Tricine SDS-PAGE under non-reduced conditions. Based on the internal peptide sequence of rabbit calcylin, we isolated and sequenced a cDNA clone encoding calcyclin. The sequence of this clone (pCalC) is 629 bp long and codes 90 amino acid residues of a protein with a molecular mass of 10,153 Da. By Northern blot analysis, a major band of 0.9 kbp and a minor band of 2.6 kbp were detected in the lung. The recombinant calcyclin mutated serine at the third position to cysteine was expressed in E. coli and made dimer formation under non-reduced conditions on SDS-PAGE. Whether or not this type of mutation which prevents dimer formation of calcyclin plays a physiological role in the rabbit lung is the subject of an ongoing study.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of rabbit CAP-50, a calcyclin-associated annexin protein.

CAP-50 is a member of annexin family proteins which binds specifically to calcyclin in a Ca2+ dependent manner (Tokumitsu. H., Mizutani. A., Minami. H., Kobayashi. R., and Hidaka. H. (1992) J. Biol. Chem. 267,8919-8924). The cDNA representing the rabbit form of this protein has been cloned from rabbit lung cDNA library. Sequence analysis of two overlapping clones revealed a 81-nucleotides 5'-nontranslated region, 1512-nucleotides of open reading frame, a 672-nucleotides 3'-nontranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of portions of the deduced amino acid sequence with eight sequences of proteolytic peptides obtained from rabbit lung protein. CAP-50 cDNA encodes a 503 residue protein with a calculated M(r) of 54,043 and shows that the protein is composed of four imperfect repeats and hydrophobic N-terminal region. C-terminal region including four imperfect repeats shows 58.1% identity with human synexin (annexin VII), 48.0% identity with annexin I, 47.4% identity with annexin II, 60.1% identity with annexin IV, 54.5% identity with annexin V. Hydrophobic N-terminal region composed of 202 amino acid residues is not homologous with other annexin proteins suggesting that CAP-50 is a novel member of annexin family proteins.

Amino Acid Sequence↗
PubMed1992

Specific binding of CAP-50 to calcyclin.

CAP-50, a calcyclin-associated protein with an apparent molecular mass of 50 kDa, was purified and proved to be a novel annexin [Tokumitsu, H. et al. (1992) J. Biol. Chem. 267, 8919-8924]. We examined the binding of CAP-50 to other Ca(2+)-binding proteins which have two of four EF-hand structures, by a co-precipitation assay with phospholipid (phosphatidylserine). Among nine Ca(2+)-binding proteins (calcyclin, S-100 proteins, p11, calgizzarin, calvasculin, calmodulin and troponin C) examined, only calcyclin interacted with CAP-50. These results clearly show that the interaction of CAP-50 to calcyclin is specific, i.e. other Ca(2+)-binding proteins with the EF-hand structure could not substitute for calcyclin, thereby suggesting the possible role in specific regulation of the function of CAP-50 by Ca2+/calcyclin.

Animals↗
PubMed1992

CAP-50, a newly identified annexin, localizes in nuclei of cultured fibroblast 3Y1 cells.

A 50-kDa protein, which binds to the growth-regulated gene (2A9) product, calcyclin in a calcium-dependent manner, was purified from bovine lung. Partial amino acid sequencing of the protein revealed it to be the bovine equivalent of rabbit lung CAP-50 (calcyclin-associated protein, 50 kDa), which is a member of the annexin family and binds to calcyclin in a calcium-dependent manner. Specific polyclonal antibodies to bovine lung CAP-50 were prepared. Comparative studies between CAP-50 and synexin (annexin VII) on the immunoreactivity against anti-CAP-50 antibodies and the ability of binding to calcyclin revealed that CAP-50 was a distinct molecule from synexin. Using specific polyclonal antibodies to bovine lung CAP-50, tissue distribution and subcellular distribution of CAP-50 were investigated. In most rat tissues, except those in the central nervous systems and kidney, CAP-50 is expressed at a high or moderate level. Both studies by subcellular fractionation and by indirect immunofluorescence staining of the rat embryonic fibroblast cell line, 3Y1, revealed that CAP-50 mainly localized in nuclei. Moreover, between the cells at interphase and at mitotic phase, different distributions of CAP-50 were observed. That is, in the cells at interphase, CAP-50 seemed to localize throughout the nucleoplasm. On the other hand, in the cells during mitosis, CAP-50 was concentrated at the loop-like structure around the mitotic apparatus. CAP-50 was found in isolated 3Y1 nuclei lacking outer nuclear membranes, and approximately 50% of CAP-50 was extracted from the nuclei by chelating calcium. Thus, CAP-50, a unique annexin, localizes in nuclei.

Amino Acid Sequence↗
PubMed1992

A calcyclin-associated protein is a newly identified member of the Ca2+/phospholipid-binding proteins, annexin family.

A calcyclin-associated protein with an apparent molecular weight of 50,000 (CAP-50) was purified from rabbit lung. The procedure included ammonium sulfate precipitation, anion and cation ion-exchange, and calcyclin affinity chromatographies. Interestingly, partial amino acid sequences of lysyl-endpeptidase-digested fragments indicated that CAP-50 was a member of the Ca2+/phospholipid-binding proteins, the annexin family. The sequence of a proteolytic peptide with Staphylococcus aureus V8 protease on NH2-terminal region is not homologous with any other annexin family proteins. Phospholipid binding studies showed that CAP-50 bound to phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid-containing vesicles, in a Ca(2+)-dependent manner. In the presence of Ca2+/calcyclin, CAP-50 formed a complex with calcyclin and bound to the PS-containing vesicles. The apparent Kd value of calcyclin for CAP-50 was calculated to be 1.61 x 10(-6) M. Zero-length cross-linking studies indicated that 1 mol of CAP-50 bound to an equimolar unit of calcyclin. CAP-50 inhibited the phospholipase A2 activity, dose-dependently (IC50 = 0.2 microM), however, calcyclin did not alter the inhibitory effect. With the 125I-calcyclin gel overlay method, calcyclin bound tightly to CAP-50 in a Ca(2+)-dependent manner after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that rabbit lung CAP-50 is a newly identified member of the annexin family. Ca2+/calcyclin apparently regulates the function of CAP-50 on cytosolic face of the plasma membrane.

Amino Acid Sequence↗
PubMed1992

Acidic calmodulin binding protein, ACAMP-81, is MARCKS protein interacting with synapsin I.

ACAMP-81 is an acidic calmodulin binding protein with molecular mass of 81 kDa. We report partial amino acid analysis of ACAMP-81 and its interaction with synapsin I. 123 amino acids of ACAMP-81 were determined and the sequence was completely identical with that of MARCKS protein which was thought to be a substrate for calcium/phospholipid dependent protein kinase (PKC). We found ACAMP-81 bound to synapsin I with 125I-labeled ACAMP-81 overlay method. ACAMP-81 bound to the cysteine specific cleaved 51 kDa fragment derived from middle/tail region of synapsin I.

Amino Acid Sequence↗
PubMed1992

Neutron-capture therapy of murine ascites tumor with gadolinium-containing microcapsules.

Gadolinium-containing microcapsules were evaluated as an agent for gadolinium neutron-capture therapy. Mice were inoculated intraperitoneally with 10(7) Ehrlich ascites tumor cells and gadolinium microcapsules and exposed to thermal neutrons for 12 min (approximately 1.86 x 10(12) neutrons cm-2). Significantly more mice given gadolinium microcapsules than those given placebo microcapsules or control survived for 60 days and considerably longer (P < 0.0001), indicating that gadolinium neutron-capture reactions effectively suppressed the growth of ascites tumor cells in mice. The results suggest that these microcapsules are an effective gadolinium carrier for neutron-capture therapy.

Animals↗
PubMed1991

Phosphorylation of bovine brain 81-kDa acidic calmodulin binding protein (ACAMP-81) in vitro.

We found a novel 81-kDa acidic protein (ACAMP-81) in the bovine brain membrane fraction, which bound to calmodulin in a Ca(2+)-dependent manner. The present study reveals physicochemical properties and phosphorylation of this protein with various protein kinases in vitro. The Stokes radius and sedimentation coefficient were calculated to be 52 A and 2.05 S, respectively, suggesting that the structure of ACAMP-81 is highly elongated. Purified Ca2+/phospholipid-dependent protein kinase (protein kinase C), cAMP-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) catalyzed the incorporation of 1.46, 0.72, and 0.44 mol of phosphate/mol of ACAMP-81, respectively. The amino acid residues of ACAMP-81 phosphorylated by either protein kinase C or cAMP-dependent protein kinase were almost exclusively on serine. Sequential phosphorylation of ACAMP-81 by cAMP-dependent protein kinase and protein kinase C resulted in the additional incorporation of 1.15 mol of [32P]phosphate into ACAMP-81. Comparison of phosphopeptide maps of ACAMP-81 phosphorylated by each kinase revealed that there are two classes of phosphorylatable polypeptide, one is phosphorylatable by both protein kinases which contained two polypeptides and the others are specific sites for protein kinase C.

Amino Acids↗
PubMed1991

A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding proteins.

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-77)-coupled Sepharose 4B column, we purified two different Ca(2+)-binding proteins from rabbit lung extracts. The molecular weights of these proteins were estimated to be 17 kDa (calmodulin) and 10 kDa, respectively. The partial amino acid sequence of the 10-kDa protein revealed that it has two EF-hand structures. In addition, the 10-kDa protein was highly homologous (91%) to the product of growth-regulated gene, 2A9 (calcyclin). The Ca(2+)-binding property of the 10-kDa protein was observed by a change in the uv difference spectrum. Equilibrium dialysis showed that 1 mol of the 10-kDa protein bound to 2.04 +/- 0.05 mol of Ca2+ in the presence of 10(-4) M Ca2+. However, the protein failed to activate calmodulin-dependent enzymes such as Ca2+/CaM kinase II, myosin light chain kinase, and phosphodiesterase. We found that a 50-kDa cytosolic protein of the rabbit lung, intestine, and spleen bound to the 10-kDa protein, in a Ca(2+)-dependent manner. The distribution of calcyclin and calcyclin binding proteins was unique and seems to differ from that of calmodulin and calmodulin-binding proteins. Thus, calcyclin probably plays a physiological role through its binding proteins for the Ca(2+)-dependent cellular response.

Amino Acid Sequence↗
PubMed1991

Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells.

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.

Animals↗
PubMed1990

KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II.

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Purification and characterization of 81K, heat stable calmodulin-binding protein from bovine brain.

Heat stable calmodulin-binding protein has been purified from Triton X-100 soluble particulate fraction of bovine brain. Considerable purification was achieved with calmodulin coupled Sepharose 4B affinity chromatography. SDS-PAGE of the purified protein revealed the apparent homogeneity being 92% at Mr 81,000. Isoelectric focusing of purified 81K protein gave isoelectric point of 4.3. The amino acid composition was notable for high contents of acidic amino acids (15.0 mol% of glutamic acid and 8.1 mol% of aspartic acid) and 17.4 mol% of alanine. On alkaline 1 M urea gel electrophoresis, mobility of the purified 81K protein in the presence of Ca2+ and calmodulin became lower than 81K protein alone toward the anode; however, Ca2+ solely did not affect the mobility of this protein. Similarly, S-100 protein and troponin C showed the interaction with 81K protein and a decrease of mobility in the presence of Ca2+ in alkaline urea PAGE. Binding assay of 125I-labeled calmodulin revealed that 81K protein could bind to an equimolar of 125I-calmodulin as apparent dissociation constant (Kd) of 0.65 x 10(-6) M.

Animals↗
PubMed1989

Monoclonal antibody assessment of tissue- and species-specific myosin light chain kinase isozymes.

Monoclonal antibodies raised against chicken gizzard smooth muscle myosin light chain kinase were used for immunological and structural studies of this enzyme. Epitope mapping of trypsin-digested chicken gizzard enzyme showed that MM-1, 2, 3, 4, 5, 6, and 7 bind to 65 kDa (trypsin-digested) and 60 kDa (chymotrypsin-digested) fragments which contain the catalytic domain of the kinase. Kinetic analysis demonstrated that MM-7 inhibited kinase activity competitively with respect to ATP and noncompetitively with respect to myosin light chain, thereby indicating that MM-7 binds at or near the ATP binding site of the enzyme. Immunoblot analysis revealed that all these antibodies (MM-1 to 12) reacted with the enzyme (130 kDa) from intestinal and vascular smooth muscles, whereas 5 (MM-1, 3, 4, 6, and 9) or 3 (MM-1, 3, and 4) of 12 antibodies did not cross-react with chicken cardiac muscle or with blood platelet myosin light chain kinase (130 kDa), respectively. None of these antibodies showed cross-reactivity against skeletal muscle myosin light chain kinase. As for mammalian species, MM-11 and 12 reacted with myosin light chain kinase of vascular smooth muscle (140 kDa) and MM-11 cross-reacted with the enzyme (140 kDa) from cardiac muscle of rat and rabbit. These data suggest the existence of at least 4 subspecies of myosin light chain kinase in chicken tissues and the heterogeneity of tissue- and species-specific isozyme forms.

Animals↗
PubMed1989

Anti-gizzard MLCK monoclonal antibody MM13 inhibits superprecipitation and phosphorylation of bovine aortic smooth muscle actomyosin.

We prepared monoclonal antibodies directed against chicken gizzard myosin light chain kinase (MLCK) and used them to study the contractile system of aortic smooth muscle. One monoclonal antibody, MM13, dose dependently inhibited actomyosin superprecipitation of bovine aortic smooth muscle, in accord with the suppression of 20 kDa myosin light chain phosphorylation by endogenous kinase. Immunoblotting analysis demonstrated that MM13 cross-reacted with the 150,000 Mr peptide of bovine aortic actomyosin preparation. The bovine aortic MLCK was purified approximately 2,400-fold to apparent homogeneity by three steps of column chromatography. The purified enzyme has a molecular weight of 150,000 and a slower mobility than chicken gizzard MLCK (130,000 Mr), as determined by SDS-polyacrylamide gel electrophoresis. MM13 also cross-reacted with purified bovine aortic MLCK and inhibited the kinase activity, in vitro. We interpret these findings to mean that binding of the anti-gizzard MLCK monoclonal antibody directly to aortic smooth muscle MLCK (150,000 Mr) decreases the phosphorylation of the 20 kDa myosin light chain, thus suppressing the aortic smooth muscle myosin-actin interaction.

Actomyosin↗