Lysosomal enzymes associated with vitreous hyalocyte granules: I. Intracellular distribution patterns of enzymes

Abstract

Utilizing a combination of centrifugal and enzymatic digestion techniques, a method was developed for the isolation of the subcellular granules of calf vitreous hyalocytes. The granules of varying size were separated according to their sedimentation properties into “nuclear”, “granular” and “mitochondrial-microsomal” fractions. The association of lysosomal enzyme activity with the granules was first indicated by the histochemical demonstration of acid phosphatase activity, followed by in vitro colorimetric determinations for the hydrolytic enzymes acid phosphatase and β-glucuronidase, and for alkaline phosphatase. Enzymatic activities were present only after the cells were mechanically disrupted, and the various fractions had distinctive differences in their enzyme distribution. A structure-linked latency of enzyme activity was demonstrated in the individual fractions, maximum activity being dependent on fragmentation of the granular membrane. Confirmation of the composition and morphology of the separated constituents at each step of the procedures was obtained by light and electron microscopic studies.