Plasma membrane isolated from astrocytes in primary cultures. Its acceptor oxidoreductase properties.
Rat astrocytes in primary cultures were employed to isolate the plasma membrane. The method for the isolation of plasma membrane was based on the capacity of the cytoskeleton to adhere to the substratum entrapping intracellular organelles during freezing-thawing cycle performed on the cell. By washing the 'surface adherent framework', the untrapped plasma membrane were recovered and density equilibrium centrifugation resulted in the isolated membrane. The isolated plasma membrane was characterized on the basis of a variety of marker enzymes positive to the plasma membrane such as (Na+ + K+)-ATPase or 5'-nucleotidase as well as the lack of conventional markers of other endomembranes. Ultrastructurally the membranes, as isolated here, were mainly vesicular in nature. The isolated plasma membrane was devoid of the dehydrogenase responsible for NADH-cytochrome c reductase activity. However, NADH-ferricyanide reductase activity and the dehydrogenase system catalyzing the transfer of reducing equivalents from NADH or NADPH to dichloroindophenol seems plasma membrane redox system. The identical specific activity employing dichloroindophenol as an electron acceptor with NADH or NADPH as donor indicate a DT-diaphorase (EC 1.6.99.2) like activity in the astrocytes plasma membrane.