One-step isolation and characterization of nuclear membranes

Abstract

Purified fractions of nuclei from rat liver and isolated calf thymocytes are treated with 0.5 M MgCl 2 or with an alkaline buffer, pH 8.5. Such procedures dissociate nuclear membranes from the chromatin. The membranes are simply floated in a gradient. The recovery in the membrane band obtained by the Mg 2+ technique is 55 to 60 %. Freeze-etching studies of the membrane pellets provide a good way to check their purity, pores being very conspicuous. A 5'-nucleotidase and an ATPase are detected on the nuclear membranes of thymocytes, as well as a still undescribed 3'-nucleotidase. However, no marker enzymes for the nuclear membrane have been found. The topology of sugar residues on the nuclear membranes is studied by means of lectins. Concanavalin A binds in large amounts to nuclear envelopes, both on the inner and outer leaflets, but not to pores.