Over the next year it is planned to attempt to purify the acid-resistant nucleational factor in cartilage micropuncture fluid (Cfl). A new technique of band centrifugation and of microcolumn chromatography whereby cartilage fluid proteoglycans can be separated on Sepharose 2B into aggregate and subunit forms has already been developed. The site on these chromatographic systems where alkaline phosphatase and the nucleational factor appear have been determined and run on SDS capillary gels. The nucleational factor will be studied by precipitation in an in vitro model system of calcium and phosphate from a synthetic lymph. The calcium will be measured by our new helium glow photometric method for calcium. The purified proteoglycan aggregates will be dissociated and the nature of the link glycoprotein studied to see if it could be an alkaline phosphatase or a calcium-binding nucleating factor. Further studies for the presence of membranes or membrane fragments of matrix vesicles will be done in the Cfl and their relationships to nucleational factor determined. Most of this past year has been spent on the different technique of band centrifugation and refinements in our technique of analytical ultracentrifugation to implement the goals which were started last year and are now essentially the same. When a nucleational factor is isolated either from the proteoglycan aggregates or other fractions, these will be studied for their biochemical properties, and presence on cartilage collagen or in matrix vesicles sought. BIBLIOGRAPHIC REFERENCES: Howell, D.S.: Hypertrophic Osteoarthropathy. In Arthritis and Allied Conditions, Chapter 62, 9th. Edition, J.L. Hollander and D.J. McCarty, Jr., eds. Lea and Febiger, publishers, Philadelphia, Pa., 1978, in press. Howell, D.S.: Pathogenesis of Osteoarthritis. In Arthritis and Allied Conditions, Chapter 72, 9th Edition, J.L. Hollander and D.J. McCarty, Jr., eds. Lea and Febiger, publishers, Philadelphia, Pa., 1978, in press.