BIOCHIMICA ET BIOPHYSICA ACTA 531 BBA 75442 THE ISOLATION OF NUCLEAR MEMBRANE FROM A LARGE-SCALE PREPARATION OF BOVINE LIVER NUCLEI RONALD BEREZNEY, LINDA K. FUNK AND F. L. CRANE Department of Biological Sciences, Purdue University Lafayette, Ind. 47 907 (U.S.A .) (Received February i3th , 197 o) SUMMARY I. A procedure for the large-scale preparation of bovine liver nuclei is described. 2. Nuclear membrane was prepared by treatment of nuclei with deoxyribo- nuclease followed by extraction with a 0. 5 M MgC1, solution. 3. An evaluation of mitochondrial and microsomal contamination involving chemical, enzymatic and ultrastructural analysis indicated only slight contamination in both nuclear and nuclear membrane fractions. 4. The total contaminating protein averaged 2.65 % for nuclei and lO.6 % for nuclear membrane. 5. Electron microscopy showed that the membrane fraction contained both outer and inner nuclear membranes. 6. The nuclear membrane fraction contained 74.6 % protein, 8. 9 % RNA, 0.92 % DNA, and 13.7 % phospholipid. Recovery studies showed an average of lO.3 % of the nuclear protein, 23.9 % of the nuclear RNA, 0.27 % of the nuclear DNA, and 47.1% of the nuclear phospholipid recovered in the membrane fraction. INTRODUCTION Although the nuclear membrane or envelope has been well studied structur- ally 1-5, a great deal of unanswered questions of fundamental importance remain concerning its functioning. Such aspects as the chemical composition and enzymatic activity of nuclear membrane, the role of nuclear membrane in nucleo-cytoplasmic exchanges, the biogenetic relationship between endoplasmic reticulum and nuclear membrane, the relationship between the outer and inner nuclear membranes, and the functioning of nuclear pores are all but impossible to investigate without direct methods of analysis. Until recently, lack of procedures for nuclear membrane isolation have prevented its biochemical and physicochemical analysis 6-s. For the past 2 years we have been concerned with isolating nuclear membranes in amounts useful for comprehensive biochemical analysis. In this paper a procedure is described for nuclei isolation from bovine liver which combines high purity with the important advantage of being on a large scale. From this large-scale nuclear preparation, nuclear membranes were isolated. Rigorous criteria involving electron microscopic and enzymatic analysis were used in evaluating contamination in the nuclei and nuclear membrane fractions. Because of the large amount of membrane Biochim. Biopkys. Acta, 203 (197 o) 531-546