Subcellular fractionation of epiphyseal cartillage isolation of matrix vesicles and profiles of enzymes,phospholipids, calcium and phosphate

Epiphyseal cartilage was fractionated into subcellular components by non-enzymatic methods, and analyzed for activity of marker enzymes, for phospholipids, and for calcium and inorganic phosphate. Alkaline phosphatase, a marker enzyme for matrix vesicles and plasma membranes, was concentrated in the 100 000 × g (microsomal) pellet and, upon subsequent frationalism, in the low-density fractions from the sucrose gradient. Mitochondrial and endoplasmic reticular enzymes were localized primarily in the 20 000 × g pellet, lysosomal enzymes predominantly in the supernate from the microsomal pellet. Two phospholipids characteristic of matrix vesicles, sphingomyelin and phosphatidylserine, were enriched in the low-density sucrose fractions; however, unlike matrix vesicles, there was no depletion in phosphatidylcholine or increase in lysophospholipids. Ca and inorganic P were concentrated in the higher-density fractions, the amounts in the lower-density fractions being some- what lower than those seen in matrix vesicles. The alkaline phosphatase-rich, low-density fractions were thus not identical to matrix vesicles isolated by collagenase digestion, but rather appear to be composed primarily of plasma membranes. Enzyme profiles indicate they were relatively free of mitochondrial, endoplasmic reticular and lysosomal contaminants. The data further indicate that significant modification of the phospholipid, electrolyte, and possibly enzyme content of chondrocyte plasma membranes, must occur during blebbing and matrix vesicle formation.