Witcher D R, Strifler B A, Jones L R
Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis 46202-4800.
J Biol Chem. 1992 Mar 5;267(7):4963-7.
An antiserum raised against the region of the cardiac ryanodine receptor (residues 2805-2819) containing the phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) was used to identify the brain ryanodine receptor. This antiserum, which is cardiac isoform-specific, immunoprecipitated greater than 90% of the [3H]ryanodine receptor binding sites solubilized from guinea pig brain membranes. The immunoprecipitated brain receptor exhibited the characteristic cardiac-type mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The brain ryanodine receptor, like the cardiac ryanodine receptor, was a substrate for CaM kinase. Affinity-purified, site-specific antibodies completely blocked phosphorylation of both brain and cardiac receptors by CaM kinase, and two-dimensional peptide mapping identified the same major 32P-labeled peptide in receptors from both tissues. 125I-Labeled receptors also gave the same peptide maps. These results confirm that mammalian brain expresses the cardiac isoform of the ryanodine receptor. Furthermore, the unique CaM kinase phosphorylation site, which has been shown to regulate Ca2+ channel activity, is conserved.
一种针对心脏兰尼碱受体（残基2805 - 2819）中包含多功能钙/钙调蛋白依赖性蛋白激酶（CaM激酶）磷酸化位点区域制备的抗血清，被用于鉴定脑兰尼碱受体。这种抗血清具有心脏异构体特异性，能免疫沉淀从豚鼠脑膜中溶解的超过90%的[3H]兰尼碱受体结合位点。免疫沉淀的脑受体在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上呈现出特征性的心脏型迁移率。脑兰尼碱受体与心脏兰尼碱受体一样，是CaM激酶的底物。亲和纯化的位点特异性抗体完全阻断了CaM激酶对脑和心脏受体的磷酸化，二维肽图分析确定了来自两种组织的受体中相同的主要32P标记肽。125I标记的受体也给出了相同的肽图。这些结果证实哺乳动物脑表达兰尼碱受体的心脏异构体。此外，已证明能调节钙通道活性的独特CaM激酶磷酸化位点是保守的。
