Sosa B P, Alagón A C, Martin B M, Possani L D
Biochemistry. 1986 May 20;25(10):2927-33. doi: 10.1021/bi00358a029.
A phospholipase A2 was isolated from the venom of the mexican beaded lizard (Heloderma horridum horridum) by phenyl-Sepharose chromatography followed by Sephadex G-75 gel filtration and two additional steps on ion exchange resins (DE-32 cellulose). The affinity chromatographic method (PC-Sepharose 4B) reported for the isolation of other phospholipases [Rock, Ch. O., & Snyder, F. (1975) J. Biol. Chem. 250, 2564-2566; King, T. P., Alagon, A. C., Kwan, J., Sobotka, A. K., & Lichteinstein, L. M. (1983) Mol. Immunol. 20, 297-308; King, T. P., Kochoumian, L., & Joslyn, A. (1984) Arch. Biochem. Biophys. 230, 1-12] was uneffective for the separation of this enzyme. The monomeric form of the Heloderma phospholipase has an apparent Mr of 18 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 19 060 as calculated from amino acid analysis. It also contains on the order of 7% carbohydrates per mole of enzyme. The N-terminal amino acid sequence was shown to be very different from that of phospholipases isolated from mammalian pancreas and crotalids and elapids snake venoms. The first 39 amino acid residues at the N-terminal region have 56% homology with bee venom phospholipase but differ from the bee phospholipase in that its isoelectric point is acidic (pI = 4.5), instead of basic, and it has approximately 50 amino acid residues more in the molecule. The specificity of the enzyme is mainly A2 type with possible residual B-type activity. The enzymatic activity is Ca2+-dependent. Half-cystine alignment of the Heloderma phospholipase sequence with those of other known phospholipases shows the lack of an octadecapeptide at the N-terminal region, the existence of an extra hexapeptide at positions 42-47, and an exact correspondence of Heloderma Gly-12, Gly-14, His-36, and Asp-37 with Gly-30, Gly-32, His-48, and Asp-49 from other phospholipases shown to be important for Ca2+ binding (( Dijkstra, B. W., Drenth, J., Kalk, K. H., & Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60 )).(ABSTRACT TRUNCATED AT 250 WORDS)
通过苯基 - 琼脂糖凝胶层析，随后进行葡聚糖G - 75凝胶过滤以及在离子交换树脂（DE - 32纤维素）上的另外两步操作，从墨西哥珠毒蜥（Heloderma horridum horridum）的毒液中分离出一种磷脂酶A2。报道用于分离其他磷脂酶的亲和层析方法（PC - 琼脂糖4B）[罗克，Ch. O.，& 斯奈德，F.（1975年）《生物化学杂志》250，2564 - 2566；金，T. P.，阿拉贡，A. C.，关，J.，索博特卡，A. K.，& 利希滕斯坦，L. M.（1983年）《分子免疫学》20，297 - 308；金，T. P.，科乔米安，L.，& 乔斯林，A.（1984年）《生物化学与生物物理学报》230，1 - 12]对于分离这种酶无效。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，墨西哥珠毒蜥磷脂酶的单体形式的表观分子量为18000，根据氨基酸分析计算为19060。每摩尔酶还含有约7%的碳水化合物。已表明其N端氨基酸序列与从哺乳动物胰腺以及蝰蛇科和眼镜蛇科蛇毒中分离出的磷脂酶有很大不同。N端区域的前39个氨基酸残基与蜂毒磷脂酶有56%的同源性，但与蜂磷脂酶不同的是，其等电点呈酸性（pI = 4.5），而非碱性，并且分子中大约多了50个氨基酸残基。该酶的特异性主要为A2型，可能有残余的B型活性。酶活性依赖于Ca2 +。墨西哥珠毒蜥磷脂酶序列与其他已知磷脂酶的半胱氨酸比对显示，其N端区域缺少一个十八肽，在42 - 47位存在一个额外六肽，并且墨西哥珠毒蜥的甘氨酸 - 12、甘氨酸 - 14、组氨酸 - 36和天冬氨酸 - 37与其他已表明对Ca2 +结合很重要的磷脂酶的甘氨酸 - 30、甘氨酸 - 32、组氨酸 - 48和天冬氨酸 - 49精确对应（（迪克斯塔，B. W.，德伦特，J.，卡尔克，K. H.，& 万德马伦，P. J.（1978年）《分子生物学杂志》124，53 - 60））。（摘要截断于250字）
