Isolation and biochemical characterization of nuclei from chick embryo liver.
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 MgCl(2) followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg(2+) ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl(2). 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 micromoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg(2+) activated RNA polymerase, 4.3 micromicromoles UMP-2-C(14) incorporated into RNA/microg DNA per 10 min; and Mn(2+)-(NH(4))(2)SO(4) activated RNA-polymerase, 136 micromicromoles UMP-2-C(14) incorporated into RNA/microg DNA per 45 min.
D000818:Animals; D002118:Calcium; D002458:Cell Fractionation; D002466:Cell Nucleolus / Q000032:analysis; D002467:Cell Nucleus / Q000032:analysis* / Q000201:enzymology / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D002843:Chromatin / Q000032:analysis; D004247:DNA / Q000032:analysis; D003576:Electron Transport Complex IV / Q000032:analysis; D005952:Glucose-6-Phosphatase / Q000032:analysis; D005964:Glucosyltransferases / Q000032:analysis; D005969:Glutamate Dehydrogenase / Q000032:analysis; D006651:Histocytochemistry; D008099:Liver / Q000166:cytology* / Q000196:embryology; D008274:Magnesium; D008722:Methods; D008854:Microscopy, Electron; D009243:NAD / Q000096:biosynthesis; D009249:NADP; D009708:Nucleotidases / Q000032:analysis; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis / Q000096:biosynthesis; D012316:RNA Nucleotidyltransferases / Q000032:analysis
D002843:Chromatin; D011506:Proteins; D009243:NAD; D009249:NADP; D012313:RNA; D004247:DNA; D005969:Glutamate Dehydrogenase; D003576:Electron Transport Complex IV; D005964:Glucosyltransferases; D012316:RNA Nucleotidyltransferases; D009708:Nucleotidases; D005952:Glucose-6-Phosphatase; D008274:Magnesium; D002118:Calcium