Localization of phosphorylase kinase subunits at the sarcoplasmic reticulum of rabbit skeletal muscle by monoclonal and polyclonal antibodies.
Molecular structures related to phosphorylase kinase have been localized by light and electron microscopy in tissue sections of rabbit skeletal muscle employing polyclonal antibodies directed against the holoenzyme as well as monoclonal antibodies specific for its alpha-, beta- or gamma-subunits. In frozen sections of prefixed muscle fibres both known major regions of glycogen deposition, the intermyofibrillar space and the perinuclear area, are stained predominantly. In sections of unfixed muscle in which cytosolic phosphorylase kinase was removed by extensive washes prior to immunostaining the immunolabel is mainly associated with the sarcoplasmic reticulum (SR). This membrane location is further confirmed by immunoblot analysis of proteins solubilized from isolated SR with Triton X-114. Employing monoclonal antibodies two membrane proteins are identified as the alpha- and beta-subunits of phosphorylase kinase by Western blots. Immunoprecipitates reveal also the gamma-subunit; the delta-subunit, i.e., calmodulin, is enriched with the solubilized enzyme. It proves that a SR membrane associated form of holophosphorylase kinase exists in muscle. Functionally, this kinase might be involved in phosphorylation of phosphatidylinositol present on the SR Ca2+ transport ATPase and thereby might play a role in regulation of Ca2+ transport.
Thieleczek
R
R
Abteilung für Biochemie Supramolekularer Systeme, Ruhr-Universität, Bochum/Bundesrepublik, Deutschland.
Behle
G
G
Messer
A
A
Varsanyi
M
M
Heilmeyer
L M
LM
Drenckhahn
D
D
D000818:Animals; D000911:Antibodies, Monoclonal; D002499:Centrifugation, Density Gradient; D005455:Fluorescent Antibody Technique; D007118:Immunoassay; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008861:Microsomes / Q000201:enzymology; D009132:Muscles / Q000201:enzymology* / Q000648:ultrastructure; D010764:Phosphorylase Kinase / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D010764:Phosphorylase Kinase