ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF NUCLEI FROM CHICK EMBRYO LIVER

Abstract

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.