Hironori Hanada

Bidirectional Signaling between Sarcoglycans and the Integrin Adhesion System in Cultured L6 Myocytes

The rat L6 skeletal muscle cell line was used to study expression of the dystrophin-containing glycoprotein complex and its interaction with the integrin system involved in the cell-matrix adhesion reaction. A complex of dystrophin and its associated proteins was fully expressed in L6 myotubes, from which anti-dystrophin or anti-α-sarcoglycan co-precipitated integrin α5β1 and other focal adhesion-associated proteins vinculin, talin, paxillin, and focal adhesion kinase. Immunostaining and confocal microscopy revealed that dystrophin, α-sarcoglycan, integrin α5β1, and vinculin exhibited overlapping distribution in the sarcolemma, especially at focal adhesion-like, spotty structures. Adhesion of cells to fibronectin- or collagen type I-coated dishes resulted in induction of tyrosine phosphorylation of α- and γ-sarcoglycans but not β-sarcoglycan. The same proteins were also tyrosine-phosphorylated when L6 cells in suspension were exposed to Arg-Gly-Asp-Ser peptide. All of these tyrosine phosphorylations were inhibited by herbimycin A. On the other hand, treatment of L6 myotubes with α- and γ-sarcoglycan antisense oligodeoxynucleotides resulted in complete disappearance of α- and γ-sarcoglycans and in significant reduction of levels of the associated focal adhesion proteins, which caused about 50% reduction of cell adhesion. These results indicate the existence of bidirectional communication between the dystrophin-containing complex and the integrin adhesion system in cultured L6 myocytes.

Kinetic studies of chromaffin granule H+-ATPase and effects of bafilomycin A1

Vacuolar type H+-ATPase purified from bovine chromaffin granules did not show simple Michaelis-Menten type kinetics, and had apparent Km values of 5 microM, 30 microM and 300 microM. These three Km values suggested the presence of catalytic cooperativity during steady-state hydrolysis. The single turnover rate was 10(-3)-fold the maximal velocity of the enzyme and similar to the rate estimated from the velocity of steady-state hydrolysis with the smallest Km value (5 microM). The H(+)-ATPase was inhibited by the stoichiometric binding of bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase. This inhibitor not only lowered the rate of ATP hydrolysis at the single catalytic site, but also affected the catalytic cooperativity of the enzyme.

Molecular cloning of cDNA encoding the 16 KDa subunit of vacuolar H+-ATPase from mouse cerebellum

cDNA for the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from mouse cerebellum and sequenced. The deduced polypeptide (155 amino acid residues; molecular weight, 15,808) was highly hydrophobic and homologous to the subunits of bovine adrenal medulla, Torpedo marmorata electric lobe, Drosophila and yeast. Glu-139 (supposed to be essential for proton transport) was also conserved as the potential dicyclohexylcarbodiimide binding site. The subunit had four transmembrane segments: Segment II and IV were highly homologous and Glu-139 was located in Segment IV. The roles of the non-conserved regions are discussed.

Vacuolar type H+-ATPase genes : presence of four genes including pseudogenes for the 16-kDa proteolipid subunit in the human genome

Genes for the human vacuolar type H(+)-ATPase proteolipid (16-kDa) subunit were cloned and their nucleotide sequences were determined. Comparison of the deduced sequences indicated that at least four genes including pseudogenes are present in the human genome. One of them corresponded to that for the 16-kDa subunit expressed in HeLa cells. The coding sequence was separated by two introns. The second intron was located in the DNA segment giving a loop between the second and third transmembrane helices, supporting the idea that the 16-kDa subunit was evolved by gene duplication. The primary sequence determined from the second clone had a termination codon behind the third transmembrane helix. Possible translation products from the other two clones had no putative acidic residues essential for proton transport function of the 16-kDa subunit. Thus, it is interesting to know whether these genes are transcribed, since they may have unique cellular functions.

α1-Syntrophin has distinct binding sites for actin and calmodulin

Overlay and co‐sedimentation assays using recombinant α1‐syntrophin proteins revealed that two regions of α1‐syntrophin, i.e. aa 274–315 and 449–505, contain high‐affinity binding sites for F‐actin (K d 0.16–0.45 μM), although only a single high‐affinity site (K d 0.35 μM) was detected in the recombinant full‐length syntrophin. We also found that actomyosin fractions prepared from both cardiac and skeletal muscle contain proteins recognized by anti‐syntrophin antibody. These data suggest a novel role for syntrophin as an actin binding protein, which may be important for the function of the dystrophin‐glycoprotein complex or for other cell functions. We also found that α1‐syntrophin binds calmodulin at two distinct sites with high (K d 15 nM) and low (K d 0.3 μM) affinity.

Uni-site catalysis by Escherichia coli F1-ATPase with different numbers of bound nucleotides

We prepared two types of E. coli F1 by slightly different gel filtration procedures of the purified F1: F1(II) contained about 2 mol, and F1(V) about 5 mol of bound adenine nucleotides per mol of the enzyme. Thus F1(II) had more than 2, possibly 3, vacant catalytic sites, while F1(V) had less than one vacant catalytic site. The rate of ATP hydrolysis in uni‐site catalysis (in the presence of inorganic phosphate) was about 3‐fold higher with F1(II) than with F1(V), suggesting that ADP and inorganic phosphate bound at the catalytic sites of F1(V) changed the kinetics of uni‐site catalysis significantly.

Ca2+-ATPase distributes differently in cardiac sarcolemma than dihydropyridine receptor α1 subunit and Na+/Ca2+ exchanger

We have investigated the distribution of the sarcolemmal Ca2+ transporters in hamster and dog ventricular myocytes by immunocytochemical and membrane fractionation techniques. The data suggest that the DHP receptor α1 subunit and the Na+/Ca2+ exchanger are present in surface sarcolemma as well as T‐tubule membranes located at the cardiac dyads. Compared with these Ca2+ transporters, the sarcolemmal Ca2+‐ATPase is much less abundant in the latter fraction. Thus the sarcolemmal Ca2+‐ATPase seems to be located predominantly in surface sarcolemma.