title: The identification of the phosphorylated 150/160-kDa proteins of sarcoplasmic reticulum, their kinase and their association with the ryanodine receptor.
abstract: In the present work we studied the relationship between the phosphorylated 150- and 160-kDa proteins and other SR proteins in the 150,000-170,000 range of molecular masses. on SDS-PAGE, the identification of their kinase, as well as the purification and structural interactions between these proteins and the rynodine receptor (RyR). The phosphorylated 150-kDa protein was identified as sarcalumenin based on: (a) its cross-reactivity with three different monoclonal antibodies specific for sarcalumenin. (b) its mobility in SDS-PAGE which was modified upon digestion with endoglycosidase H, (c) its elution from lentil-lectin column by alpha-methyl mannoside, (d) its resistance to trypsin, (e) its ability to bind Ca2+ and to stain blue with Stains-All. The phosphorylated 160-kDa protein was identified as the histidine-rich Ca2+ binding protein (HCP) based on: (a) its Ca(2+)-binding property and staining blue with Stains-All, (b) phosphorylation with the catalytic subunit of cAMP-dependent kinase. (c) its increased mobility in SDS-PAGE in the presence of Ca2+ (d) its heat stability and (e) its partial amino acid sequence. The endogenous kinase was identified as casein kinase II (CK II) based on the inhibition of the endogenous phosphorylation 160/150-kDa proteins by heparin, 5.6-dichlorobenzimidazole riboside, polyaspartyl peptide and hemin, and its ability to use [gamma-32P]GTP as the phosphate donor. The association of CK II with SR membranes, was demonstrated using specific polyclonal anti-CK II antibodies. The luminal location of CK II is suggested because CK II was extracted from the SR by l M NaCl only after their treatment with hypotonic medium, and CK II activity was inhibited with the charged inhibitors heparin and polyaspartyl peptide only after their incubation with the SR in the presence of NP-40. The 160- and 150-kDa proteins were purified on spermine-agarose column, and were phosphorylated by CK II. Like the endogenous phosphorylation of the 150/160-kDa proteins in SR. the phosphorylation of the purified proteins by CK II was inhibited by La3+ (Cl50 = 4 microM) and hemin. The results suggest the phosphorylation of the luminally located sarcalumenin and HCP with CK II.
authors.affiliation: Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
authors.forename: V
authors.initials: V
authors.lastname: Shoshan-Barmatz
authors.forename: I
authors.initials: I
authors.lastname: Orr
authors.forename: S
authors.initials: S
authors.lastname: Weil
authors.forename: H
authors.initials: H
authors.lastname: Meyer
authors.forename: M
authors.initials: M
authors.lastname: Varsanyi
authors.forename: L M
authors.initials: LM
authors.lastname: Heilmeyer
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D047390:Casein Kinase II; D004396:Coloring Agents; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D004396:Coloring Agents; C489419:HRC protein, Oryctolagus cuniculus; D008565:Membrane Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; D019837:Ryanodine Receptor Calcium Release Channel; C070323:sarcalumenin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D006026:Glycoside Hydrolases; D014357:Trypsin; D002118:Calcium