[Frontiers in Bioscience 2, a26-30, September 15, 1997]|
SPERMINE STIMULATES THE PHOSPHORYLATION OF THE NUCLEAR MATRIX PROTEINS CATALYZED BY NUCLEAR KINASE II
Rati Verma and Kuang Yu Chen
Department of Chemistry and The Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08855-0939
Received 8/19/97 Accepted 8/25/97
3. MATERIALS AND METHODS
Nucleoplasm was prepared by extraction of isolated nuclei as previously described (13), and dialyzed against buffer B (20 mM Tris-HCI, pH 8.0, 0.1 mM EDTA, 10 mM beta-mercaptoethanol, 10% glycerol and 0.25 mM PMSF) containing 0.3 M NaCl. It was then concentrated by an Amicon stirred cell equipped with a PM-10 membrane. The concentrated nucleoplasm was chromatographed twice through phosphocellulose column (1.5 x 20 cm) as previously described (13, 14). Active fractions were pooled and 400 micrograms of Pentax BSA (Fraction V) was added. They were then concentrated and stored at 40 C in buffer B containing 0.65 M NaCl. Under such conditions the enzyme activity could be preserved up to six months.
Nuclei and various subnuclear fractions (hnRNP and the HMG and matrix proteins) were prepared as previously described (14). They were briefly heated (650 C for 5 min) to inactivate the endogenous kinase activities before being used as kinase substrates. The phosphorylation mixture generally contained 20 microliters of substrate, 5 to 10 units of NII kinase, 50 micromolar ATP (2.5 x 106 cpm/nmol), 5 mM MgCl2, 1 mM DTT, 30 mM NaCl in 50 mM Tris-HCl (pH 7.4). The reaction mixture was incubated at 300 C for 15 min and then processed for SDS-PAGE and autoradiographic analysis.
Tissue culture, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and immunoblotting were carried out as described (14). Protein concentration was determined by the method of Bradford (15).