Electron microscopic examination of hepatic subcellular fractions for phosphatases.

Abstract

Lead capture techniques have been investigated for demonstrating phosphatase activity, with the aid of the electron microscope, in subcellular fractions isolated from rat liver. Controls with no exposure to the enzyme substrate showed little ‘staining’ (lead phosphate deposits). Results with preparations where the enzyme incubation period had been prolonged, and the lead phosphate deposits were heavy (‘over staining’), were also reassuring: the staining did not tend to become generalized. Particularly satisfactory results were obtained when the tissue fractions were used in the form of unfixed suspensions for ‘incubation’ with the substrate, this being performed with the minimum temperature and time needed to obtain representative staining of the elements present. Thus, for 5′-nucleotidase in plasma-membrane fragments there is good staining after only 10 min at 2°C. The favoured concentration of lead ions is 2mm. When the reaction period has elapsed, glutaraldehyde is added, and the tissue material repelleted; only then should osmium tetroxide be added. Glucose-6-phosphatase staining, particularly in rough microsomes, showed a time-lag which could be abolished by prior sonication or treatment with deoxycholate. In general, the cytochemical findings tallied well with the biochemical characterization of the various fractions examined, including zonalrotor fractions containing smooth vesicles the origin of which could not be established merely by morphology.