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

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.