Fabiani R, Ronquist G
Department of Clinical Chemistry, University Hospital, University of Uppsala, Sweden.
Clin Chim Acta. 1993 Jul 16;216(1-2):175-82. doi: 10.1016/0009-8981(93)90150-3.
Examination of prostasomes, isolated from human seminal plasma, showed that there was very little remaining paranitrophenylphosphatase activity when assayed in the presence of 10 mmol/l of tartrate and 2 mmol/l of levamisole. Under these conditions it was possible to study the prostasome membrane-bound 5'-nucleotidase activity, which was unaffected by these two inhibitors. The activity was considered to be located at the external surface of the prostasome membrane and a 50-60% increase in activity was obtained by the addition of 0.05% Triton X-100. The prostasome membrane-linked 5'-nucleotidase readily hydrolysed 5'-AMP. Two other 5'-nucleoside monophosphates, 5'-IMP and 5'-GMP, were also hydrolysed, but more slowly; 2'- or 3'-AMP were practically not attacked. The prostasome membrane-linked 5'-nucleotidase obeyed Michaelis-Menten kinetics. Apparent Km for 5'-AMP was 11.2 +/- 2.1 mumol/l and Vmax 64.7 +/- 11.4 nmol/mg protein/min. These figures were somewhat changed in presence of 0.05% Triton X-100, the Km value being reduced by 30% and the Vmax value increased by 60%. Adenosine 5' (alpha, beta methylene) diphosphate (100 mumol/l), Ni2+ (10 mmol/l) and concanavalin A (20 micrograms/ml) were all potent inhibitors of the prostasome membrane-linked 5'-nucleotidase.
对从人精浆中分离出的前列腺小体进行检测，结果显示，在10 mmol/l酒石酸盐和2 mmol/l左旋咪唑存在的情况下进行测定时，对硝基苯磷酸酶的残留活性非常低。在这些条件下，可以研究前列腺小体膜结合的5'-核苷酸酶活性，该活性不受这两种抑制剂的影响。该活性被认为位于前列腺小体膜的外表面，添加0.05% Triton X-100后活性增加了50 - 60%。前列腺小体膜连接的5'-核苷酸酶能轻易水解5'-AMP。另外两种5'-核苷单磷酸，5'-IMP和5'-GMP也能被水解，但速度较慢；2'-或3'-AMP实际上不被作用。前列腺小体膜连接的5'-核苷酸酶符合米氏动力学。5'-AMP的表观Km为11.2 +/- 2.1 μmol/l，Vmax为64.7 +/- 11.4 nmol/mg蛋白质/分钟。在0.05% Triton X-100存在的情况下，这些数值有所变化，Km值降低了30%，Vmax值增加了60%。5'（α，β亚甲基）二磷酸腺苷（100 μmol/l）、Ni2+（10 mmol/l）和伴刀豆球蛋白A（20 μg/ml）都是前列腺小体膜连接的5'-核苷酸酶的有效抑制剂。
