Abstract
We examined the distribution of acid NADPase activity in rat hepatocytes by cytochemistry and subcellular fractionation. Cytochemical studies revealed strong NADPase activity in many lysosomes and Golgi saccules situated between the medial and trans aspect of the stack. Light, spotty deposits of reaction product also were observed frequently within elements of the Golgi-associated endoplasmic reticulum lysosome (GERL) system. Biochemical studies with liver homogenates indicated that NADPase activity was distributed across various subcellular fractions in a pattern resembling typical nonspecific acid phosphatase activity, as monitored using beta-glycerophosphate as substrate. Thus, by differential centrifugation, most cellular NADPase activity was found in the large granule (ML) fraction (61%). The microsomal (P) fraction showed about 17% whereas the nuclear (N) and cytosol (S) fractions each accounted for about 11% of the remaining NADPase activity, respectively. Separation of the ML fraction into endosomes and secondary lysosomes revealed most of the acid NADPase activity associated with lysosomes. An alternate one-step method for isolation of fractions indicated that 3% of the recovered acid NADPase activity, but 55% of the Golgi marker enzyme galactosyl transferase, was associated with a purified intact Golgi fraction. Cytochemical studies with ML and intact Golgi fractions indicated that bona fide reaction product was localized to lysosomes and to the same saccules that were reactive in vivo. We propose that saccules making up the trans half of the Golgi stack may represent a site for accumulation of a biogenetic precursor of the lysosomal acid NADPase in hepatocytes.