Ultrastructural demonstration of p-nitrophenyl phosphatase (p-NPPase) activity in the epiphyseal growth plate.

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Abstract
A cytochemical procedure for p-nitrophenylphosphatase (p-NPPase) activity was applied to epiphyseal growth-plate cartilage in order to study maturational changes in some of the phosphatases present in this tissue. In the plasma membrane and matrix vesicles, two types of p-NPPases were observed. One was insensitive to aldehyde fixation, was located on the outside of the membrane, and diminished in potency as maturation progressed. The other was sensitive to fixation, occurred on the inside of the membranes, and was most intense in the hypertrophic zone. Matrix vesicles rich in p-NPPase activity contained crystals of mineral while those which had no activity did not calcify. In fixed samples, p-NPPase was also detectable in mitochondria, Golgi complex and lysosomes, while in unfixed samples the only non-plasma membrane location observed was in the endoplasmic reticulum. A variety of inhibitors with known specificities (ouabain, vanadate, NaF, duramycin, levamisole) had no effect on activity. K-dependency was not demonstrable. Consequently, it is not possible to assign the p-NPPase activity to a specific phosphatase. However, the p-NPPase activity is distinct from Na+,K+-ATPase, Ca-ATPase, K+,H+-ATPase and alkaline phosphatase. The results of this study provide further evidence for the plasma-membrane origin of matrix vesicles. Further, the phosphatase which hydrolyzes p-NPPase observed is associated with matrix-vesicle calcification.