The identification of the phosphorylated 150/160-kDa proteins of sarcoplasmic reticulum, their kinase and their association with the ryanodine receptor

Varda Shoshan-Barmatz, Irit Orr, Simy Weil, Helmut Meyer, Magdolna Varsanyi, Ludwig M.G. Heilmeyer

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Abstract

In the present work we studied the relationship between he 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 ryanodine 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 α-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 Ca2+-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 [γ-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 1 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 phospholation of the 150/160-kDa proteins in SR, the phosphorylation of the purified proteins by CK II was inhibited by La3+ (CI50 = 4 μM) and hemin. The results suggest the phosphorylation of the luminally located sarcalumenin and HCP with CK II.

Original languageEnglish
Pages (from-to)89-100
Number of pages12
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1283
Issue number1
DOIs
StatePublished - 14 Aug 1996

Keywords

  • Casein kinase II
  • Protein phosphorylation
  • Ryanodine receptor
  • Sarcalumenin
  • Sarcoplasmic reticulum

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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