R M Kawamoto

University of Miami Miller School of Medicine, Miami, FL, United States

Are you R M Kawamoto?

Claim your profile

Publications (10)36.27 Total impact

  • R M Kawamoto, J P Brunschwig, A H Caswell
    [show abstract] [hide abstract]
    ABSTRACT: Polyclonal antibodies have been developed against the junctional feet or spanning protein from skeletal muscle triads. These probes in combination with immunogold labels have been used to localize the spanning protein by electron microscope of isolated vesicles from terminal cisternae/triads. The spanning protein antibodies specifically bind to the electron dense junctional feet. In vesicles permeabilized by hypotonic treatment or by saponin, some gold particles may be seen on the luminal side of the vesicle. Trypsin treatment of vesicles causes complete loss of the 300 K spanning protein from SDS gels while dot blots show that some but not all the antigenic activity is lost. This treatment is associated with the loss of the electron dense projections from the membrane surface and is coincident with the loss of immunogold staining when antibody is added to the intact vesicles. On the other hand, in experiments in which the luminal portions of the isolated vesicles have been made accessible to the polyclonal antibodies by sectioning lightly fixed vesicles before immunogold tagging, extensive gold labelling was found to occur in trypsin treated vesicles which have lost detectable projections from the cytoplasmic side of the membrane. These data support the view that the spanning protein projects from the sarcoplasmic reticulum towards the transverse tubules but further suggest that spanning protein extends into and probably through the sarcoplasmic reticulum membrane in accord with the proposition that it is a Ca2+ channel.
    Journal of Muscle Research and Cell Motility 09/1988; 9(4):334-43. · 1.36 Impact Factor
  • Methods in Enzymology 02/1988; 157:68-84. · 2.00 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: A monoclonal antibody has been developed against the putative junctional protein or spanning protein (SP) from skeletal muscle triads. By immuno-affinity chromatography, we have purified this protein. The native protein has a molecular mass of 630-800 kD, as determined by gel filtration and rate zonal centrifugation. Within the limits of the methods used, the basic unit of the SP appears to be a dimer. In electron micrographs, it is shown to exhibit a circular profile with a diameter of approximately 100 A. In thin section analysis, the protein is frequently observed as parallel tracks of electron-dense particles bordering a translucent core. We suggest that the basic unit of the junctional structure is a dimer of 300-kD subunits and that four such entities constitute the intact SP. The purified protein has been used to develop polyclonal antibodies. By immunoelectron microscopy using immunogold probes, the SP has been localized to the junctional gap of the triad. By attaching the SP to an affinity resin, three proteins have been identified as forming associations with the SP. The Mrs of the proteins are 150, 62, and 38 kD; the 62-kD protein is calsequestrin.
    The Journal of Cell Biology 11/1986; 103(4):1405-14. · 10.82 Impact Factor
  • R M Kawamoto, A H Caswell
    [show abstract] [hide abstract]
    ABSTRACT: Glyceraldehydephosphate dehydrogenase purified from rabbit skeletal muscle is auto-phosphorylated with MgATP. Half-maximal phosphorylation is achieved around 0.3 mM. The phosphorylation is Ca2+ independent. The phosphoenzyme complex is labile in alkaline conditions and stable in moderately acid media. The complex is readily hydrolyzed by 0.1 M neutral hydroxylamine, indicating the complex formed is a high-energy acyl phosphate. The phosphorylation is reduced by nicotinamide adenine dinucleotides, reduced form (NADH), glyceraldehyde 3-phosphate, and nicotinamide adenine dinucleotide (NAD+). The enzyme is also dephosphorylated by these metabolites although to a lesser extent by NAD+. Calsequestrin isolated from rabbit skeletal muscle inhibits the phosphorylation of the enzyme. The phosphoenzyme behaves as a kinase catalyzing the phosphorylation of proteins of Mr 80 000 and 72 000 found in the skeletal muscle terminal cisternae/triad preparation. This reaction is enhanced by NADH. The phosphate found in the protein substrate has been shown to be the same phosphate initially involved in the phosphorylation of glyceraldehydephosphate dehydrogenase.
    Biochemistry 03/1986; 25(3):657-61. · 3.38 Impact Factor
  • R M Kawamoto, R J Baskin
    [show abstract] [hide abstract]
    ABSTRACT: Purified sarcoplasmic reticulum vesicles from normal and dystrophic chicken skeletal muscle have been isolated by a procedure employing pressure disruption of a microsomal suspension. The dystrophic sarcoplasmic reticulum (SR) exhibited reduced Ca++ transport and phosphoenzyme formation, but the Ca++-ATPase activity was normal. Normal and dystrophic SR showed similar lipid profiles, except for a significant increase in free fatty acids in the dystrophic SR. Investigations involving the interaction of oleic acid with normal SR showed fatty acids can induce conditions similar to those found in dystrophic SR.
    Muscle & Nerve 01/1986; 9(3):248-56. · 2.31 Impact Factor
  • N R Brandt, R M Kawamoto, A H Caswell
    [show abstract] [hide abstract]
    ABSTRACT: The binding of nitrendipine to transverse (T) tubules isolated from skeletal muscle triads is inhibited by dithiothreitol (KI approximately 0.05 mM) and glutathione (KI approximately 3 mM). The t 1/2's of inhibition (18.3 and 11.5 min, respectively) suggest that these hydrophylic reagents act upon the exposed surface of the vesicles. Dithiothreitol shifts the apparent KD for nitrendipine from 8.5 nM to 30 nM without altering the Bmax extrapolated by Scatchard analysis. That T-tubules isolated by disruption of triad junctions are constrained to have the protoplasmic (P) face uniformly exposed was experimentally confirmed. These studies show that a sulfhydryl residue on the P-face of the T-tubule influences the affinity of the receptor for dihydropyridines.
    Biochemical and Biophysical Research Communications 03/1985; 127(1):205-12. · 2.41 Impact Factor
  • N R Brandt, R M Kawamoto, A H Caswell
    [show abstract] [hide abstract]
    ABSTRACT: Receptors for the dihydropyridine class of Ca2+ channel antagonists are present on transverse tubules isolated by mechanical disruption of skeletal muscle triads. These observations account for the previously reported presence of nitrendipine binding sites in heavy sarcoplasmic reticulum. Nitrendipine receptors were not found in the terminal cisternae after disruption of the triad junctions. The number of sites in junctional T-tubules (27 pmol/mg) is only half that reported for transverse tubules isolated as free vesicles (pmol/mg). The presence of nitrendipine receptors in that region of the transverse tubules held in close apposition to the SR cisternae is consistent with the architectural requirements for the Ca2+ induced Ca2+ release phenomenon to be the mechanism of excitation-contraction coupling.
    Journal of receptor research 02/1985; 5(2-3):155-70.
  • R J Baskin, R Kawamoto
    [show abstract] [hide abstract]
    ABSTRACT: Vesicles isolated from the transverse tubules and sarcoplasmic reticulum of normal and dystrophic chicken skeletal muscle were analyzed for enzymatic activity and examined following freeze-fracture. A stereological procedure was used to determine particle density distributions on the resulting membrane fracture faces. The particle densities measured in this investigation were compared with those of an earlier study on intact muscle. Isolated sarcoplasmic reticulum vesicles showed a characteristically high P-face (cytoplasmic leaflet) particle density (5108 +/- 169 particles/micron2) and a low E-face (luminal leaflet) particle density (505 +/- 57 particles/micron2). Transverse tubule fractions showed a high E-face particle density (2346 +/- 179 particles/mu2) as well as a substantial P-face particle density (1019 +/- 129 particles/micron2). The high transverse tubule E-face particle density represents a characteristic morphological feature in the same way that the very high P-face particle density is characteristic of sarcoplasmic reticulum membranes. The major morphological alteration in dystrophic membranes was a shift in the E-face particle density distribution of isolated transverse tubules to a lower average particle density. (The E-face particle density of sarcoplasmic reticulum fractions showed no differences.)
    Biochimica et Biophysica Acta 05/1984; 771(2):109-18. · 4.66 Impact Factor
  • R M Kawamoto, R J Baskin
    [show abstract] [hide abstract]
    ABSTRACT: Two new lines of chickens with near identical genotypes (greater than 90% isogeneity), one demonstrating avian dystrophy, were used for isolation of sarcoplasmic reticulum vesicles. Vesicles from line 433 (dystrophic) displayed reduced Ca2+-ATPase activity, phosphoenzyme formation and steady-state calcium transport capabilities in comparison with vesicles from line 03 (normal). Lipid analyses show that dystrophic vesicles have greater amounts of cholesterol and lesser amounts of phosphatidylcholine. The results support the use of isogenic chickens in further studies of avian dystrophy. However, the results also suggest that current sarcoplasmic reticulum vesicle purification procedures dependent on differential calcium accumulation may not fully achieve the intended purpose.
    Biochimica et Biophysica Acta 09/1983; 732(3):620-6. · 4.66 Impact Factor
  • S Hanna, R Kawamoto, M McNamee, R J Baskin
    [show abstract] [hide abstract]
    ABSTRACT: We have isolated sarcoplasmic reticulum from normal and dystrophic chicken muscle, using an improved isolation procedure. Dystrophic sarcoplasmic reticulum has a reduced level of calcium-sensitive ATPase activity, phosphoenzyme formation, and steady-state calcium transport. Anion-stimulated calcium transport by dystrophic sarcoplasmic reticulum is also reduced when measured under the proper conditions, and dystrophic sarcoplasmic reticulum shows no alteration in calcium efflux rate. Active calcium phosphate loading of the normal and dystrophic sarcoplasmic reticulum preparations indicates that a reduced percentage jof the dystrophic vesicles are capable of active calcium transport. The loaded dystrophic sarcoplasmic reticulum vesicles exhibit the same relative reductions in enzymatic activity as the starting sarcoplasmic reticulum preparations. However, the enzyme activities of normal and dystrophic sarcoplasmic reticulum are similar in the presence of detergent and exogenous phospholipid. On the basis of these results, we suggest that the lipid microenvironment of the dystrophic enzyme is altered.
    Biochimica et Biophysica Acta 05/1981; 643(1):41-54. · 4.66 Impact Factor

Publication Stats

114 Citations
255 Views
36.27 Total Impact Points

Institutions

  • 1985–1988
    • University of Miami Miller School of Medicine
      • Division of Clinical Pharmacology
      Miami, FL, United States
  • 1981–1984
    • University of California, Davis
      Davis, California, United States