一种通过分离的基质小泡研究钙化的简单且明确的方法。ATP和小泡磷酸酶的作用。

A simple and defined method to study calcification by isolated matrix vesicles. Effect of ATP and vesicle phosphatase.

作者信息

Hsu H H, Anderson H C

出版信息

Biochim Biophys Acta. 1977 Nov 7;500(1):162-72. doi: 10.1016/0304-4165(77)90056-3.

PMID:200279

Abstract

A simplified and defined system was developed to study in vitro calcium phosphate deposition by isolated matrix vesicles from rabbit growth plate cartilage, and to examine the relationship between vesicle phosphatase and calcium deposition. Samples of suspended vesicles containing 25 microgram of protein, were incubated for 2 h in a 45Ca-labelled solution with 2.2 mM Ca2+, 1.6 mM PO 3/4-and 1 mM ATP at pH 7.6. Calcium deposition was related to the amount of PO4 hydrolysed by matrix vesicle phosphatases from ATP and other phosphate esters. Ca2+ or Mg2+ was found to stimulate matrix vesicle ATPase, but the hydrolysis of phosphoenolpyruvate, glucose 1-phosphate, beta-glycerol phosphate and AMP was independent of either cation. All of the above substrates supported calcium deposition. 1 mM ATP was more effective than 5 mM in supporting calcium deposition, indicating inhibition of mineralization at higher ATP concentrations. Our results suggest that, in addition to concentrating calcium, vesicles provide phosphate from ATP for mineral formation and at the same time remove the inhibitory effect of ATP upon mineral deposition.

摘要

开发了一种简化的特定系统，用于研究从兔生长板软骨中分离出的基质小泡的体外磷酸钙沉积，并研究小泡磷酸酶与钙沉积之间的关系。将含有25微克蛋白质的悬浮小泡样品，在pH值为7.6的含2.2 mM Ca2+、1.6 mM PO43−和1 mM ATP的45Ca标记溶液中孵育2小时。钙沉积与基质小泡磷酸酶从ATP和其他磷酸酯水解的PO4量有关。发现Ca2+或Mg2+可刺激基质小泡ATP酶，但磷酸烯醇丙酮酸、葡萄糖1-磷酸、β-甘油磷酸和AMP的水解与这两种阳离子无关。上述所有底物均支持钙沉积。1 mM ATP在支持钙沉积方面比5 mM更有效，表明在较高ATP浓度下矿化受到抑制。我们的结果表明，除了浓缩钙外，小泡还从ATP中提供磷酸盐用于矿物质形成，同时消除ATP对矿物质沉积的抑制作用。