ABSTRACT
Rat liver nuclear protein kinases NI and NII have been purified to homogeneity by an improved method. This method includes a casein-phosvitin-Sepharose column step, which separates the enzymes from the other chromosomal non-histone proteins, and a gel filtration at high ionic strength in the presence of a high concentration of protease inhibitors to separate the two enzymes from each other. NI has an apparent molecular mass of approximately 50 kDa and is composed of a single subunit. NII has an apparent molecular mass of 133 kDa and is composed of two subunits of identical molecular mass. The V and the Km of the two enzymes were determined for several substrates. Both enzymes phosphorylate chromosomal non-histone proteins with partly different specificities as shown by two-dimensional electrophoreses. When incubated in the absence of protease inhibitors, the enzymes were degraded into discrete polypeptides. Autophosphorylation of a polypeptide derived from NII was observed after incubation of the enzyme with ATP. This phosphorylation stimulated the enzyme activity. Several chromosomal proteins coeluted with NII from the casein-phosvitin-Sepharose column. They remained associated with the enzyme in sucrose gradients, during gel filtration performed at physiological ionic strength, and are dissociated at high ionic strength. These proteins were highly phosphorylated when the protein-NII complex was incubated with ATP.
Subject(s)
Cell Nucleus/enzymology , Liver/enzymology , Protein Kinases/isolation & purification , Amino Acids/analysis , Animals , Binding Sites , Centrifugation, Density Gradient , Chromatography/methods , Chromosomal Proteins, Non-Histone/isolation & purification , Electrophoresis, Polyacrylamide Gel , Kinetics , Male , Molecular Weight , Phosphorylation , Protein Kinases/metabolism , Rats , Rats, Inbred Strains , Substrate SpecificitySubject(s)
Cell Nucleus/metabolism , Histones/metabolism , Liver/metabolism , Acetyl Coenzyme A/metabolism , Acetylation , Animals , Kinetics , Male , Rats , Rats, Inbred StrainsABSTRACT
The iron stimulation of ferritin synthesis is a translational phenomenon. Iron acts through a modification of the microsomal apparatus as shown previously. In the present study we have shown that the factor(s) involved in this stimulation are present in the KC1 extracts from polysomes of iron-treated rats.
