The long-range of this research is to elucidate the mechanism of endochondral calcification, a process essential for normal bone formation, skeletal development and fracture healing. While other factors are involved, matrix vesicles (MV) are primarily implicated in initiating calcification. Thus the goal of this research is to elucidate MV calcification. MV, when isolated from growth plate cartilage and incubated in a synthetic cartilage lymph, induces mineral formation by acquiring large amounts of Ca2+ and Pi. MV contain high levels of mineralization. The three major goals of this project are: 1) to characterize key MV proteins, 2) to characterize the nucleational complex, and 3) to reconstitute functional MV. Fist, ion porters essential for the entrance of Ca2+ and Pi into the vesicle lumen during MV mineralization will be characterized. The Ca2+ porter, annexin V, also possesses collagen-binding activities. Since interaction with type II and X collagens activates Ca2+ entrance into MV, the hypothesis that collagen binding activates the annexin Ca2+ channel will be tested. Little is known of the Pi-porter in MV; using rat kidney Pi-transporter cDNA as a probe, the chondrocyte Pi-porter will be identified and cloned to aid in characterizing its activity in MV Pi-transport. Entrance of Ca2+ into MV during MV mineralization activates phospholipases that selectively break down phosphatidylserine and sphingomyelin. Because these lipids impede outgrowth of mineral from the vesicle lumen, MV phospholipases will be isolated and characterized. Also, an acid-labile nucleational complex and Ca2+/Pi-binding proteins have been found to be critical for induction of MV mineral formation. Accordingly, this nucleational complex (electrolytes, lipids and proteins), and the Ca2+/Pi-bindings proteins in the vesicle lumen, will be isolated and characterized. Finally, with these essential components identified, the nucleational complex, and functional MV, will be reconstituted by incorporating these key proteins, mineral ions and lipids into synthetic unilamellar vesicles. To summarize: 1) the functions of annexin V and regulation of its Ca2+ channel activity will continue to be explored; 2) the MV Na+-dependent Pi-transporter, 3) the MV core proteins involved in storing Ca2+ and Pi, 4) phospholipases responsible for breakdown of the MV membrane during calcification, and 5) the nucleational complex will be isolated and characterized. Finally, with this information, 6) the nucleational complex, and 7) complete functional MV, will be reconstituted using lipids, electrolytes, proteins and enzymes shown to be key to MV function.