Shoshan-Barmatz V, Orr I, Weil S, Meyer H, Varsanyi M, Heilmeyer L M
Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Biochim Biophys Acta. 1996 Aug 14;1283(1):89-100. doi: 10.1016/0005-2736(96)00079-x.
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.
在本研究中，我们研究了分子量在150,000 - 170,000范围内的磷酸化150 kDa和160 kDa蛋白与其他SR蛋白之间的关系。在SDS - PAGE上，鉴定它们的激酶，以及这些蛋白与雷诺丁受体（RyR）之间的纯化和结构相互作用。基于以下几点，磷酸化的150 kDa蛋白被鉴定为肌质网腔蛋白：（a）它与三种不同的针对肌质网腔蛋白的单克隆抗体的交叉反应性；（b）其在SDS - PAGE上的迁移率，在用内切糖苷酶H消化后发生改变；（c）它通过α - 甲基甘露糖苷从扁豆凝集素柱上洗脱；（d）它对胰蛋白酶的抗性；（e）它结合Ca2+以及用全染剂染成蓝色的能力。基于以下几点，磷酸化的160 kDa蛋白被鉴定为富含组氨酸的Ca2+结合蛋白（HCP）：（a）其Ca(2 +)结合特性以及用全染剂染成蓝色；（b）被cAMP依赖性激酶的催化亚基磷酸化；（c）在Ca2+存在下其在SDS - PAGE上迁移率增加；（d）其热稳定性；（e）其部分氨基酸序列。内源性激酶基于以下几点被鉴定为酪蛋白激酶II（CK II）：肝素、5,6 - 二氯苯并咪唑核糖核苷、聚天冬氨肽和血红素对内源性160/150 kDa蛋白磷酸化的抑制作用，以及它使用[γ - 32P]GTP作为磷酸供体的能力。使用特异性多克隆抗CK II抗体证明了CK II与肌质网（SR）膜的结合。CK II的腔定位是基于以下推测：只有在用低渗介质处理后，CK II才会被1 M NaCl从SR中提取出来，并且只有在NP - 40存在下与SR孵育后，带电抑制剂肝素和聚天冬氨肽才会抑制CK II的活性。160 kDa和150 kDa蛋白在精胺 - 琼脂糖柱上纯化，并被CK II磷酸化。与SR中150/160 kDa蛋白的内源性磷酸化一样，CK II对纯化蛋白的磷酸化被La3 +（Cl50 = 4 microM）和血红素抑制。结果表明位于腔内的肌质网腔蛋白和HCP被CK II磷酸化。
