Coggins P J, Zwiers H
Department of Medical Physiology, University of Calgary, Alberta, Canada.
J Neurochem. 1990 Jan;54(1):274-7. doi: 10.1111/j.1471-4159.1990.tb13311.x.
Protein kinase C phosphorylates the neurone-specific protein B-50 at a single Ser41 residue, which is also the point for a major proteolytic cleavage in vitro, and probably in vivo, that produces a B-50 phosphorylation-inhibiting N-terminal fragment and a large C-terminal metabolite B-60 (B-50(41-226]. The intact purified protein will bind to calmodulin in the absence of calcium, but the interaction has an absolute requirement for dephospho-B-50. In an attempt to unify two aspects of B-50 biochemistry, we have examined the interaction of B-50 binding to calmodulin and B-50 proteolysis. HPLC- and affinity-purified B-50 bound to calmodulin, but purified B-60 did not. To ensure that this effect was not due to the phosphorylation state of pure, isolated B-60, the metabolite was generated in vitro using a Triton extract of synaptosomal plasma membranes, which contains the as yet uncharacterized B-50 protease. B-60 derived from dephospho-B-50 also failed to bind calmodulin. The results demonstrate a direct connection between B-50 binding to calmodulin and B-50 proteolysis. The position of the proposed calmodulin-binding domain within intact B-50 is discussed in light of the failure of calmodulin to bind B-60.
蛋白激酶C在单个丝氨酸41残基处使神经元特异性蛋白B - 50磷酸化，该残基也是体外（可能在体内也是）主要蛋白水解切割的位点，此切割产生一个抑制B - 50磷酸化的N端片段和一个大的C端代谢产物B - 60（B - 50(41 - 226]）。完整纯化的蛋白在无钙情况下能与钙调蛋白结合，但这种相互作用对去磷酸化的B - 50有绝对需求。为了统一B - 50生物化学的两个方面，我们研究了B - 50与钙调蛋白结合及B - 50蛋白水解之间的相互作用。经高效液相色谱和亲和纯化的B - 50能与钙调蛋白结合，但纯化的B - 60不能。为确保这种效应不是由于纯的、分离的B - 60的磷酸化状态导致的，使用含有尚未鉴定的B - 50蛋白酶的突触体细胞膜的 Triton提取物在体外生成该代谢产物。源自去磷酸化B - 50的B - 60也未能与钙调蛋白结合。结果表明B - 50与钙调蛋白结合和B - 50蛋白水解之间存在直接联系。鉴于钙调蛋白不能结合B - 60，讨论了完整B - 50中拟议的钙调蛋白结合结构域的位置。
