Say J C, Ciuffi K, Furriel R P, Ciancaglini P, Leone F A
Departamento de Química - Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brasil.
Biochim Biophys Acta. 1991 Jul 8;1074(2):256-62. doi: 10.1016/0304-4165(91)90161-9.
A soluble form of an alkaline phosphatase obtained from rat osseous plates was purified 204-fold with a yield of 24.3%. The purified enzyme showed a single protein band of Mr 80,000 on SDS-PAGE and an apparent molecular weight of 163,000 by gel filtration on Sephacryl S-300 suggesting a dimeric structure for the soluble enzyme. The specific activity of the enzyme at pH 9.4 in the presence of 2 mM MgCl2 was 19,027 U/mg and the hydrolysis of p-nitrophenyl phosphate (K0.5 = 92 microM) showed positive cooperativity (n = 1.5). The purified enzyme showed a broad substrate specificity, however, ATP, bis(p-nitrophenyl) phosphate and pyrophosphate were among the less hydrolyzed substrates assayed. Surprisingly the enzyme was not stimulated by cobalt and manganese ions, in contrast with a 20-25% stimulation observed for magnesium and calcium ions. Zinc ions exerted a strong inhibition on p-nitrophenylphosphatase activity of the enzyme. This paper provides a simple experimental procedure for the isolation of a soluble form of alkaline phosphatase which is induced by demineralized bone matrix during endochondral ossification.
从大鼠骨板中获得的一种可溶性碱性磷酸酶被纯化了204倍，产率为24.3%。纯化后的酶在SDS-PAGE上显示出一条分子量为80,000的单一蛋白条带，通过在Sephacryl S-300上的凝胶过滤测得其表观分子量为163,000，这表明该可溶性酶具有二聚体结构。在2 mM MgCl2存在下，该酶在pH 9.4时的比活性为19,027 U/mg，对磷酸对硝基苯酯的水解作用（K0.5 = 92 microM）显示出正协同性（n = 1.5）。纯化后的酶表现出广泛的底物特异性，然而，在所检测的底物中，ATP、双（对硝基苯基）磷酸酯和焦磷酸是水解较少的底物。令人惊讶的是，该酶不受钴离子和锰离子的刺激，而镁离子和钙离子可产生20 - 25%的刺激作用。锌离子对该酶的对硝基苯磷酸酶活性有强烈抑制作用。本文提供了一种简单的实验方法，用于分离在软骨内骨化过程中由脱矿骨基质诱导产生的可溶性碱性磷酸酶。
