Open AccessA Method for the Rapid Isolation of Nuclear Membranes from Rat Liver
Author(s) -
Kay Robert R.,
Fraser Derek,
Johnston Irving R.
Publication year - 1972
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1972.tb02081.x
Subject(s) - nuclear membrane , membrane , nuclear dna , dna , biochemistry , cell nucleus , nuclear pore , inner membrane , biology , microbiology and biotechnology , chemistry , cytoplasm , gene , mitochondrial dna
1 A rapid, mild method for isolating nuclear membranes from isolated rat liver nuclei is described. The method employs a double digestion of nuclei with a low level of DNAase I at approximately 0.1 mM Mg 2+ and slightly alkaline pH, to release the membranes. 2 Electron microscopy shows excellent preservation of the nuclear membrane morphology. Large membrane fragments consisting of a double membrane with ribosomes adhering to the outer layer are produced. The layers are connected at nuclear pores, which have distinct annular subunits and occasional central granules. Contamination by other subcellular structures is minimal. 3 The recoveries of nuclear protein, DNA, RNA and phospholipid in the membrane are approximately 8%, 1–3%, 8% and 55%, respectively. The DNA associated with the membrane does not appear to result from the reassociation of released DNA. The enzymic content of the membrane is similar to that of the microsomes. It is concluded that the nuclear membrane is the sole nuclear location of glucose‐6‐phosphatase. 4 About 1% of the total nuclear DNA polymerase was recovered associated with the nuclear membrane. By three criteria this enzyme was identical with pure authentic nuclear DNA polymerase. This finding is discussed against the background of the suggestions that the nuclear membrane is involved in DNA replication.