The deposition of calcium pyrophosphate and phosphate by matrix vesicles isolated from fetal bovine epiphyseal cartilage

Summary Since calcium (Ca) deposition by isolated fetal bovine matrix vesicles is selectively supported by nucleoside triphosphate, and since the Ca deposits appear to be amorphous by transmission electron microscopy 1], attempts were made to study further the nature of these Ca deposits. Calcification of isolated matrix vesicles was allowed to occur in a calcifying medium in which either inorganic phosphate (Pi) or γ-P]ATP was labeled with³²P.³²P in Ca P (pyrophosphate) deposits were analyzed by a Dowex 1×10 anion exchange chromatography. The results of the analysis indicate that the (³²P) radioactivity was mainly associated with Pi when Pi in the calcifying media was labeled with³²P. In contrast,³²P was found to be associated with inorganic pyrophosphate (PPi) when γ-³²P]ATP was used. Using a specific enzyme coupling assay for PPi, the presence of PPi in the Ca deposits was demonstrated. The amounts of Pi and PPi in the Ca deposits initiated by fetal calf matrix vesicles were found to be approximately equal. To exclude the possibility that the major part of PPi of Ca P deposit existed as adsorbed form, the deposition was performed under the conditions in which Pi was omitted from calcifying medium. The results of these experiments showed that substantial amount of PPi and Ca deposits remained the same and was not correlated to the amount of Pi in these deposits. In contrast, Pi of CaP was decreased if Pi was omitted from the calcifying medium. Thus, it appears that the major portion of PPi exists as mineral rather than adsorbed form. The moles of Ca deposited were approximately equal to the sum of moles Pi and PPi deposited. Levamisole at 10 mM inhibited 70% of ATPase (specific release of³²Pi from γ-³²P]ATP at pH 7.6) activity, 18% of Ca deposition, and 34% of Pi deposition during 3 hours of incubation. Adenosine monophosphate (AMP) or adenosine diphosphate (ADP) at 1 mM exerted earlier and greater inhibition on Ca and P (Pi and PPi) deposition than did 10 mM levamisole. Adenosine (1 mM) had little effect on Ca P deposition. Prolonged incubations which allowed enzymatic degradation of ADP and AMP substantially reduced the ADP and AMP inhibition. Levamisole was able to potentiate the AMP and ADP inhibition since it prevents further breakdown of AMP or ADP by alkaline phosphatase. Thus, we concluded that alkaline phosphate is not the major factor in thein vitro Ca P deposition by fetal bovine matrix vesicles. Instead, it appears that nucleoside trisphosphate pyrophosphohydrolase is directly involved in the early stages of deposition since both the enzyme and Ca P deposition were inhibited by AMP or ADP. The possible involvement of ATP phyophosphohydrolase in chondrocalcinosis is suggested.