Bartolf M, Franson R C
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0164.
Biochim Biophys Acta. 1990 Feb 6;1042(2):247-54. doi: 10.1016/0005-2760(90)90016-q.
Soluble, cation-dependent, lysosomal phospholipase A2 in bovine adrenal medulla has been biochemically characterized and partially purified, and its unique pH-dependent modulation by cations has been investigated. Chromatographically distinct activities with somewhat broad pI ranges centered at 7.8, 8.1, and 8.4 have been purified 83-, 1900- and 4400-fold, respectively, from the soluble fraction of tissue homogenates. With a specific activity of 4.2 mumol phospholipid hydrolyzed per mg protein per min, the fraction of pI 8.4 is the most highly purified lysosomal phospholipase A2 reported to date; yet silver staining of isoelectric focusing gels indicates that all three species are still only minor components of the protein mixtures with which they co-purify. Lysosomal phospholipase(s) A2 has an apparent molecular weight of 30,600, as determined by gel permeation chromatography; and is probably an oligomannose-containing glycoprotein as indicated by binding to concanavalin A-Sepharose and elution by methyl alpha-D-mannopyranoside. Cation concentrations modulate hydrolysis of biomembranous phospholipid, but not neat liposomal phospholipids, in a complex manner over a broad pH range (pH 4.0-8.0). Triton X-100 stabilizes the enzyme(s) but is inhibitory when present during assay; consequently, detergent-phospholipid mixed micelles are poor substrates. Thus, experimental results are dramatically dependent on the physicochemical nature of the substrate. The role of this phospholipase(s) A2 in the membrane fusion and lysis events of catecholamine secretion, as well as its regulation by cellular proteins, can now be investigated utilizing this partially purified enzyme(s).
牛肾上腺髓质中的可溶性、阳离子依赖性溶酶体磷脂酶A2已进行了生物化学特性分析并部分纯化，同时研究了其独特的阳离子依赖性pH调节作用。已从组织匀浆的可溶部分分别纯化出具有不同色谱活性的三种酶，其pI范围较宽，中心值分别为7.8、8.1和8.4，纯化倍数分别为83倍、1900倍和4400倍。pI为8.4的组分的比活性为每分钟每毫克蛋白质水解4.2 μmol磷脂，是迄今为止报道的纯化程度最高的溶酶体磷脂酶A2；然而，等电聚焦凝胶的银染表明，这三种酶在与其共纯化的蛋白质混合物中仍然只是次要成分。通过凝胶渗透色谱法测定，溶酶体磷脂酶A2的表观分子量为30,600；如与伴刀豆球蛋白A-琼脂糖结合并经α-D-甘露吡喃糖苷洗脱所示，它可能是一种含寡甘露糖的糖蛋白。在较宽的pH范围（pH 4.0 - 8.0）内，阳离子浓度以复杂的方式调节生物膜磷脂的水解，但不调节纯脂质体磷脂的水解。Triton X-100可使该酶稳定，但在测定过程中存在时具有抑制作用；因此，去污剂-磷脂混合胶束不是良好的底物。因此，实验结果极大地依赖于底物的物理化学性质。现在可以利用这种部分纯化的酶来研究这种磷脂酶A2在儿茶酚胺分泌的膜融合和裂解事件中的作用及其受细胞蛋白的调节。
