Further characterization of a nucleational agent in hypertrophic cell extraceilniar cartilage fluid

Abstract

Our studies on an (acid-resistant) nucleational factor aspirated by micropuncture techniques from the upper tibial growth plates of 42 day old rats have recently been extended. Partition of the fluid by ultracentrifugation into top fifth, next to top fifth and bottom half of the tube following high speed ultracentrifugation provided separate fractions for nucleation experiments. At Ca × Pi products of 2.4 mM 2, a mineral forming agent could be demonstrated in the bottom fraction and in the top fraction but not in the next-to-top fraction. When total calcium (Ca t) in the whole fluid was measured by a dialysis technique vs a high speed ultracentrifugation technique, Ca t by both techniques was the same. However, free calcium (Ca f) was 18% less by the dialysis method. This discrepancy could only be attributed to the presence of Ca binding — 0.32 mM — to a low density component in the top fifth of the tube and registrating therefore in the ultracentrifugation technique as Car. In other words, some of the previously reported so-called ⪡free⪢ calcium by this technique is bound to a lipid component. In the absence of the top fifth fraction there were almost identical results of Car by the two methods for free calcium measurement in serum. It was postulated that the calcium-binding factor might be matrix vesicle components such as Ca-binding phospholipid embedded in a protein-poor lipid component of vesicle membranes. Other features consistent with the function of matrix vesicles was the high activity of alkaline phosphatase and substantial activity of pyrophosphatase, AMPase and ATPase in the cartilage fluids.