Purification and characterization of a lysophosphatidic acid-specific phosphatase.
Purification and characterization of a lysophosphatidic acid-specific phosphatase.
复制标题
溶血磷脂酸特异性磷酸酶的纯化和表征。
作者：
M. Hiroyama;T. Takenawa
从牛脑细胞质中纯化了溶血磷脂酸（LPA）特异性磷酸酶。通过（NH4）2SO4分离和Q-Sepharose、DEAE-5PW、Superdex 200和肝素- sepharose等层析步骤进行纯化。在还原条件下，最终酶制剂在SDS/PAGE上显示分子量为44 kDa的单条带。该酶的活性完全依赖于Triton X-100、CHAPS、胆酸和辛基- β -葡萄糖苷等洗涤剂的存在。活性与Mg2+无关；其他阳离子是抑制性的。该酶能水解LPA，但不能水解心磷脂、四油基双磷脂酸、1-磷酸神经酰胺和1-磷酸鞘氨醇，磷脂酸有少量水解。纯化后的酶水解1-油基LPA的速率为1。用LPA作为Triton X-100混合胶束测定时，每毫克蛋白质为1微mol/min。LPA的Km值为38微米。NaF和n -乙基马来酰亚胺明显抑制其活性，而心得安的抑制作用较弱。LPA特异性磷酸酶可能在LPA消除中起重要作用。
Lysophosphatidic acid (LPA)-specific phosphatase was purified 3300-fold from bovine brain cytosol. The purification was achieved by (NH4)2SO4 fractionation and several chromatography steps, such as Q-Sepharose, DEAE-5PW, Superdex 200 and heparin-Sepharose. The final enzyme preparation showed a single band of molecular mass 44 kDa on SDS/PAGE under reducing conditions. The enzyme activity was completely dependent on the presence of detergents such as Triton X-100, CHAPS, cholate and octyl-beta-glucoside. The activity was independent of Mg2+; other cations were inhibitory. The enzyme hydrolysed LPA specifically but not cardiolipin, tetraoleoyl-bisphosphatidic acid, ceramide 1-phosphate or sphingosine 1-phosphate, although phosphatidic acid was hydrolysed slightly. The purified enzyme hydrolysed 1-oleoyl LPA at a rate of 1. 1 micromol/min per mg of protein when assayed with LPA as Triton X-100 mixed micelles. The Km value for LPA was 38 microM. NaF and N-ethylmaleimide markedly inhibited the activity, but propranolol had a less potent inhibitory effect. The LPA-specific phosphatase might have an important role in LPA elimination.