Schröder E, Willis A C, Ponting C P
Department of Biochemistry, University of Oxford, UK.
Biochim Biophys Acta. 1998 Apr 2;1383(2):279-91. doi: 10.1016/s0167-4838(97)00217-3.
Natural-killer-enhancing factor-B (NKEF-B) (monomeric mass = 21.82 kDa) was purified from the cytosol of porcine red blood cells and its identity was established by microsequencing. NKEF-B oligomerisation was investigated by gel filtration and small-angle X-ray scattering (SAXS). Native NKEF-B readily forms disulphide-linked dimers, but when fully reduced, the protein forms discrete oligomers containing 16 +/- 1 monomers. A total of 40% of the purified enzyme was deduced to be cysteinylated, which is consistent with the modification of one or both of two putative active site cysteine residues. In vitro, NKEF-B was found to be a specific substrate of mu- and m-calpains, the calcium-dependent cysteine proteases. The cleavage events were followed by SDS-PAGE and the cleavage sites pinpointed by N-terminally sequencing the resulting digestion fragments. This in vitro cleavage data provides support to the hypothesis that calpromotin (NKEF-B), an erythron peroxiredoxin involved in the regulation of calcium-dependent potassium transport across the plasma membrane, is cleaved by calpain in vivo.
自然杀伤增强因子B（NKEF-B）（单体质量 = 21.82 kDa）从猪红细胞胞质溶胶中纯化得到，其身份通过微量测序确定。通过凝胶过滤和小角X射线散射（SAXS）研究了NKEF-B的寡聚化。天然NKEF-B很容易形成二硫键连接的二聚体，但完全还原后，该蛋白质会形成含有16±1个单体的离散寡聚体。推断纯化酶中共有40%被半胱氨酸化，这与两个假定活性位点半胱氨酸残基中的一个或两个的修饰一致。在体外，发现NKEF-B是μ-和m-钙蛋白酶（钙依赖性半胱氨酸蛋白酶）的特异性底物。通过SDS-PAGE跟踪切割事件，并通过对所得消化片段进行N端测序来确定切割位点。该体外切割数据支持了以下假设：参与调节跨质膜钙依赖性钾转运的红细胞过氧化物酶钙促进素（NKEF-B）在体内被钙蛋白酶切割。
