Hsu H H, Munoz P A, Barr J, Oppliger I, Morris D C, Vaananen H K, Tarkenton N, Anderson H C
J Biol Chem. 1985 Feb 10;260(3):1826-31.
Alkaline phosphatase of matrix vesicles isolated from fetal bovine epiphyseal cartilage was purified to apparent homogeneity using monoclonal antibody affinity chromatography. The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Tween 20 whereas the major portion of nonspecific protein was removed by this single step. Of various agents tested, 0.6 M 2-amino-2-methyl-1-propanol, pH 10.2, was the most effective in eluting 80-100% of the enzyme initially applied. Both Tween 20 and 2-amino-2-methyl-1-propanol associated with the eluted enzyme were effectively removed by the sequential application of DEAE-cellulose and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 81,000. This molecular weight was nearer reported values for rat liver (Ohkubo, A., Langerman, N., and Kaplan, M. M. (1974) J. Biol Chem. 249, 7174-7180) and porcine kidney (Cathala, G., Brunel, C., Chapplet-Tordo, D., and Lazdunski, M. (1975) J. Biol. Chem. 250, 6040-6045) alkaline phosphatase, than to previously reported values for chicken (Cyboron, G. W., and Wuthier, R. E. (1981) J. Biol. Chem. 256, 7262-7268) and fetal calf (Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1980) Calcif. Tissue Int. 30, 217-225) cartilage matrix vesicle alkaline phosphatase. The purified alkaline phosphatase was activated by micromolar Mg2+. The amino acid composition of cartilage alkaline phosphatase was found to be similar to that previously described for porcine kidney (Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141, 273-282). Double immunoprecipitation data indicated that monoclonal antibody against cartilage alkaline phosphatase cross-reacted with fetal bovine liver or kidney enzyme but failed to react with calf intestinal or rat cartilage enzyme. Thus these observations suggest that alkaline phosphatase of matrix vesicles from calcifying epiphyseal cartilage is a liver-kidney-bone isozyme.
利用单克隆抗体亲和层析法，从胎牛骺软骨分离出的基质小泡碱性磷酸酶被纯化至表观均一。在2%吐温20存在的情况下，基质小泡丁醇提取物中的酶能特异性结合到固定化的抗体-琼脂糖上，而大部分非特异性蛋白可通过这一步骤被去除。在测试的各种试剂中，0.6M 2-氨基-2-甲基-1-丙醇（pH 10.2）在洗脱最初上样的80%-100%的酶方面最为有效。通过依次应用DEAE-纤维素和琼脂糖CL-6B层析，可有效去除与洗脱酶相关的吐温20和2-氨基-2-甲基-1-丙醇。用十二烷基硫酸钠和巯基乙醇处理后的酶制剂进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（使用银染）显示存在一条主要条带，对应分子量为81,000。该分子量更接近大鼠肝脏（大久保，A.，兰格曼，N.，和卡普兰，M.M.（1974）《生物化学杂志》249，7174 - 7180）和猪肾脏（卡塔拉，G.，布鲁内尔，C.，沙普莱特-托尔多，D.，和拉兹敦斯基，M.（1975）《生物化学杂志》250，6040 - 6045）碱性磷酸酶的报道值，而与先前报道的鸡（西博龙，G.W.，和武蒂尔，R.E.（1981）《生物化学杂志》256，7262 - 7268）和胎牛（福尔图纳，R.，安德森，H.C.，卡蒂，R.P.，和萨伊德拉，S.W.（1980）《钙化组织国际》30，217 - 225）软骨基质小泡碱性磷酸酶的值不同。纯化的碱性磷酸酶被微摩尔浓度的Mg2+激活。发现软骨碱性磷酸酶的氨基酸组成与先前描述的猪肾脏碱性磷酸酶（瓦克斯穆特，E.D.，和日和田，K.（1974）《生物化学杂志》141，273 - 282）相似。双免疫沉淀数据表明，抗软骨碱性磷酸酶的单克隆抗体与胎牛肝脏或肾脏酶发生交叉反应，但不与小牛肠或大鼠软骨酶反应。因此，这些观察结果表明，来自钙化骺软骨的基质小泡碱性磷酸酶是一种肝-肾-骨同工酶。
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