Increased matrix vesicle protein in rachitic rat epiphyseal growth plates

Abstract

Extracellular, membrane-bound vesicles are widely regarded to be the initial site of calcification in a variety of tissues under normal and pathological conditions. Alkaline phosphatase is believed to play a vital role in this process by hydrolysing ester phosphates or mineral inhibitors, e.g. inorganic phosphates. In the present study, matrix vesicles from normal and rachitic rat growth plates were compared with regard to specific activity of alkaline phosphatase, total vesicle protein and ultrastructural distribution of alkaline phosphatase activity. Matrix vesicles were released from normal or rachitic growth plates by collagenase digestion and isolated by differential centrifugation. Enzyme cytochemical localization involving a cerium capture method was performed on vesicles collected by vacuum filtration on Millipore filters. SDS gels and Western blots on fractions of both normal and rachitic matrix vesicles showed major proteins to be almost identical and confirmed the presence of alkaline phosphatase in both. Total matrix vesicle protein ((mg total matrix vesicle protein/rat) × 10 2) per rat was significantly greater for the rachitic animals (9.0 ± 2.0 vs. 4.0 ± 1.0), P < 0.0001. Alkaline phosphatase specific activity (units alkaline phosphatase/mg vesicle protein) in the rachitic and normal matrix vesicles was 25.29 ± 9.36 and 18.78 ± 3.37, respectively (0.05 < P < 0.1). Electron dense cerium phosphate deposits were localized to the outer membrane surface of matrix vesicles derived from both types of rats. This data, the first to quantify the relationship between rickets, matrix vesicle protein and alkaline phosphatase specific activity, suggests that matrix vesicles from rachitic and normal rats have biochemical and morphological similarity. Since the quantity of vesicle protein released from rachitic growth plates is 2–3 times greater per animal than that released from normals, the induction of rickets represents an approach by which matrix vesicle yield can be significantly increased.