Alkaline phosphatase activity, characterization, and subcellular distribution during initial skeletogenesis in the prenatal rat limb.

The specific activity, tissue specificity, and subcellular distribution of alkaline phosphatase were studied in the fetal rat limb during initial cartilage calcification and bone formation. The pH optimum, Km, activation, and inhibition characteristics of the enzyme assayed for in 900 X g supernates of whole limb homogenates indicated that the activity represented a fetal bone alkaline phosphatase. Studies examining temporal changes of the enzyme in these preparations demonstrated a substantial increase in activity over each of the days during which they were studied (days 15-18). Fractions derived from the discontinuous density gradient centrifugation of the limb preparations were used to study the chronological subcellular distribution of the enzyme. Enzyme activity was found in all of the fractions with the greatest activity occurring in fractions consisting of ribosomes and small vesicles. The vesicular component was similar to the matrix vesicles dexcribed by others in calcifying tissues. The daily increase in activity measured in the curde supernate was further reflected in the distribution studies. The association of alkaline phosphatase with the vesicular structure is compatible with the theorized functions of matrix vesicles, and the substantial increase in activity between days 15 and 18 further demonstrates an intimate association of alkaline phosphatase with skeletal development.