Isolation of nuclear envelopes with polyanions

Abstract

Optimal conditions for the isolation of nuclear envelopes by the action of heparin on nuclei were established and a morphologic and biochemical study of the isolated envelopes is presented. An almost 100% yield of pure nuclear envelopes can be obtained by a single sedimentation step after incubation of nuclei with heparin for 40 min at 4°C. The nuclear membrane pellet obtained in this way contains whole envelopes with a preserved perinuclear space and with ribosomes present on the outer leaflet. A single band with an apparent buoyant density of 1.18 is obtained by sucrose density gradient analysis. The chemical composition of the pellet is similar to that of the purified membranes and corresponds to 62% proteins, 34% phospholipids, 3% RNA, and 0.5% DNA. The presence of low concentrations of sodium phosphate (2-10 mM) is critical for a complete solubilization of the chromatin. A less rapid and complete solubilization is obtained with the potassium salt. Low concentrations of Mg++ (1-3 mM) counteract chromatin solubilization by heparin mainly at the level of chromatin-nuclear membrane association. The presence of EDTA in the medium leads to isolated nuclear envelopes on which neither ribosomes nor nuclear pores are visible, indicating that pore structure is dependent on the presence of Ca++ or Mg++. A comparison with other polyanions indicates a decisive advantage of heparin. However, pure nuclear envelopes can also be obtained by the action of dextran sulfate (mol wt 500,000) on nuclei incubated for 5 min at 37°C, in the presence of phosphate ions.