The fundamental hypothesis on which these studies is based is that non-collagenous proteins (NCPs) of mineralized connective tissues are involved in the formation of the tissues. Few of the NCPs of bone have been thoroughly studied and their functions are unknown; thus this hypothesis has not been adequately tested. A number of uncharacterized proteins are found in the calcified matrix of bone and many of them are synthesized and secreted by osteoblasts. The long term objective of this proposed research is to isolate uncharacterized proteins that are made by osteoblasts and are found in bone, and to perform experiments that would help delineate their properties and functions. The specific aims are: 1. To purify several proteins present in bone and synthesized by osteoblasts, 2. To characterize the purified bone proteins with regard to chemical composition, molecular weight, and (to some degree) primary structure, 3. To raise monoclonal and polyclonal antibodies against the bone proteins, for use in biosynthetic and immunolocalization experiments, 4. To study the biosynthesis of the bone proteins. Studies will be designed to demonstrate their synthesis by osteoblast-like or osteoblast-enriched cell cultures, 5. To determine if their biosynthesis is regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 and to delineate the nature of the regulation, 6. To determine the tissue and cellular localization of the bone proteins by immunolocalization experiments and the time of appearance in a developmental sense, and 7. To investigate possible biologic activities of purified proteins, including effects on cell attachment and spreading, on hydroxyapatite nucleation and growth and on differentiation of mesenchymal cells. These experiments will bring about further understanding of the fundamental biochemical mechanisms involved in bone formation. This information would be useful in understanding the basic causes of certain genetic and systemic diseases that affect bone. They may lead to diagnostic tests to evaluate the severity and treatment of the diseases. And this research will contribute to the understanding of the vitamin regulation of calcium and phosphate.