A procedure is described for the isolation of enzymatically active nuclei from chick embryo liver. It consists of the homogenization of the pooled tissue in 0.32 M sucrose-3 mM MgCl2 followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg2+ ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl2. Based on DNA recovery, the nuclear fraction isolated by the procedure described contained an average of 62% of the nuclei in the original filtered homogenate. Light and electron microscope examinations showed that 90% of the isolated nuclei were derived from hepatocytes. They appeared intact with well preserved nucleoplasmic and nucleolar components, nuclear envelope, and pores. The isolated nuclei were quite pure, having a very low level of cytoplasmic contamination as indicated by cytoplasmic enzyme marker activities and electron microscope studies. The nuclear fraction consisted of 19.9% DNA, 6.2% RNA, 74% protein, the average RNA/DNA ratio being 0.32. Biosynthetic activities of the two nuclear enzymes NAD-pyrophosphorylase and DNA-dependent RNA polymerase were preserved. The specific activities of these enzymes were: NAD-pyrophosphorylase, 0.049 µmoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg2+ activated RNA polymerase, 4.3 µµmoles UMP-2-C14 incorporated into RNA/µg DNA per 10 min; and Mn2+-(NH4)2SO4 activated RNA-polymerase, 136 µµmoles UMP-2-C14 incorporated into RNA/µg DNA per 45 min.
Article|
August 01 1971
ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF NUCLEI FROM CHICK EMBRYO LIVER
Genevieve S. Incefy
,
Genevieve S. Incefy
From The Rockefeller University, New York 10021
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Attallah Kappas
Attallah Kappas
From The Rockefeller University, New York 10021
Search for other works by this author on:
Genevieve S. Incefy
From The Rockefeller University, New York 10021
Attallah Kappas
From The Rockefeller University, New York 10021
Received:
October 14 1970
Revision Received:
December 16 1970
Online Issn: 1540-8140
Print Issn: 0021-9525
Copyright © 1971 by The Rockefeller University Press
1971
J Cell Biol (1971) 50 (2): 385–398.
Article history
Received:
October 14 1970
Revision Received:
December 16 1970
Citation
Genevieve S. Incefy, Attallah Kappas; ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF NUCLEI FROM CHICK EMBRYO LIVER . J Cell Biol 1 August 1971; 50 (2): 385–398. doi: https://doi.org/10.1083/jcb.50.2.385
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