Matecki A, Stopa M, Was A, Pawelczyk T
Department of Clinical Biochemistry, Medical University of Gdansk, Poland.
Int J Biochem Cell Biol. 1997 May;29(5):815-28. doi: 10.1016/s1357-2725(97)00014-9.
In an attempt to obtain sufficient quantities of pure phospholipase C delta 1 (PLC delta 1) necessary for structural and kinetic studies, human fibroblast PLC delta 1 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6xHis) fusion protein and purified to homogeneity. From 11 of E. coli culture 21 mg of pure PLC delta 1 was obtained by a two-step purification procedure, which includes Ni(2+)-NAT agarose and Mono S cation exchange chromatography. Catalytic properties of recombinant PLC delta 1 with respect to activation by spermine and calcium ions and inhibition by sphingomyelin were similar to or identical to PLC delta 1 purified from rat liver. Calcium activation of PLC delta 1 was dependent on the presence of spermine. Half-maximal activity was attained at 250 and 170 nM of free Ca2+ in the presence and absence of spermine, respectively. Sphingomyelin and lysosphingomyelin were mixed type inhibitors with respect to PIP2. Ceramide inhibits PLC delta 1 very weakly. GM1, which is a ceramide bound glucosidically to the oligosaccharide moiety, was a strong non-competitive inhibitor of PLC delta 1. In the absence of spermine, sphingosine and phytosphingosine weakly activated PLC delta 1. The results indicate that the effect of sphingomyelin and its metabolites on PLC delta 1 activity depends on the presence of spermine. It is postulated that, among other factors, in vivo, activity of PLC delta 1 may depend on the turnover of sphingomyelin.
为了获得结构和动力学研究所需的足够量的纯磷脂酶Cδ1（PLCδ1），将人成纤维细胞PLCδ1克隆到pPROEX-1载体中，作为（6xHis）融合蛋白在大肠杆菌细胞中表达并纯化至同质。通过两步纯化程序，从1升大肠杆菌培养物中获得了21毫克纯PLCδ1，该程序包括Ni(2+)-NAT琼脂糖和Mono S阳离子交换色谱。重组PLCδ1在精胺和钙离子激活以及鞘磷脂抑制方面的催化特性与从大鼠肝脏纯化的PLCδ1相似或相同。PLCδ1的钙激活依赖于精胺的存在。在有和没有精胺的情况下，分别在250和170 nM游离Ca2+时达到半数最大活性。鞘磷脂和溶血鞘磷脂是关于PIP2的混合型抑制剂。神经酰胺对PLCδ1的抑制作用非常弱。GM1是一种与寡糖部分糖苷键结合的神经酰胺，是PLCδ1的强非竞争性抑制剂。在没有精胺的情况下，鞘氨醇和植物鞘氨醇对PLCδ1有微弱的激活作用。结果表明，鞘磷脂及其代谢产物对PLCδ1活性的影响取决于精胺的存在。据推测，在体内，除其他因素外，PLCδ1的活性可能取决于鞘磷脂的周转。
