Portilla D, Dai G
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199, USA.
J Biol Chem. 1996 Jun 28;271(26):15451-7. doi: 10.1074/jbc.271.26.15451.
We have recently identified a cytosolic calcium-independent phospholipase A2 (PLA2) that represents the major measurable PLA2 activity in rabbit proximal tubules (Portilla, D., Shah, S. V., Lehman, P. A., and Creer, M. H.(1994) J. Clin. Invest. 93, 1609-1615). We now report the 3200-fold purification of this PLA2 to homogeneity from rabbit kidney cortex through sequential column chromatography including anion exchange, hydrophobic interaction, Mono Q, hydroxylapatite, phenyl-Sepharose, and chromatofocusing fast protein liquid chromatography from rabbit kidney cortex. The purified enzyme had a molecular mass of 28 kDa, possessed a specific activity of 1.2 micronol/mg min and a neutral pH optimum, and exhibited a preferential hydrolysis toward sn-2 fatty acid from diradylglycerophospholipids. The purified polypeptide hydrolyzed plasmenylcholine > phosphatidylcholine glycerophospholipids and selectively cleaved phospholipids containing arachidonic acid at the sn-2 position in comparison to oleic acid. Antibodies against the purified protein precipitated all of the soluble calcium-independent PLA2 activity from rabbit kidney cortex. These data altogether suggest that the 28-kDa protein in the kidney represents a novel class of calcium-independent PLA2.
我们最近鉴定出一种胞质钙非依赖性磷脂酶A2（PLA2），它是兔近端小管中主要可测量的PLA2活性物质（波蒂利亚，D.，沙阿，S.V.，莱曼，P.A.，和克里尔，M.H.（1994年）《临床研究杂志》93卷，1609 - 1615页）。我们现在报告，通过包括阴离子交换、疏水相互作用、单Q、羟基磷灰石、苯基 - 琼脂糖和色谱聚焦快速蛋白质液相色谱等连续柱色谱法，从兔肾皮质中对这种PLA2进行了3200倍的纯化，使其达到同质。纯化后的酶分子量为28 kDa，比活性为1.2微摩尔/毫克·分钟，最适pH为中性，并且对二酰基甘油磷脂的sn - 2脂肪酸表现出优先水解作用。与油酸相比，纯化后的多肽对缩醛磷脂>磷脂酰胆碱甘油磷脂的水解作用更强，并且能选择性地在sn - 2位置切割含有花生四烯酸的磷脂。针对纯化蛋白的抗体沉淀了兔肾皮质中所有的可溶性钙非依赖性PLA2活性。这些数据共同表明，肾脏中的28 kDa蛋白代表了一类新型的钙非依赖性PLA2。
