Multiple forms of molybdate-stabilized glucocorticoid-receptor complexes from HeLa cell cytosol

Summary The investigation on hydrodynamic parameters of molybdate-stabilized glucocorticoid-receptor complexes from HeLa cell cytosol permitted resolution of four distinct forms. The first one could be detected in concentrated cytosols at low salt concentrations, and had the following properties: sedimentation coefficient = 9 S; R _s = 9.3 nm; M _r = 357,800; f/f _o = 1.83; axial ratio (prolate ellipsoid) = 16. When these cytosol extracts were diluted, a second form could be detected with sedimentation coefficient = 8.3 S; R _s = 9.05 nm; M _r = 320,700;f/f _o = 1.84; axial ratio = 16. Under high salt conditions, glucocorticoid-receptor complexes in concentrated cytosol had the following properties: sedimentation coefficient = 6.4 S; R _s, = 6.7 nm; M _r = 183,100;f/f _o = 1.64; axial ratio = 12. When either these cytosol extracts were diluted, or glucocorticoid-receptor complexes were subjected to repeated analysis, a fourth form was detected with sedimentation coefficient = 3.76 S; R _s = 5.67; M _r = 91,000; f/f _o = 1.75; axial ratio = 14. Besides salt concentration and dilution, the time elapsed between sample dilution and analysis appeared to affect the hydrodynamic properties of glucocorticoid-receptor complexes. On the basis of our findings, it has been concluded that the most likely structure of molybdate-stabilized glucocorticoid-receptor complexes of HeLa cell cytosol can be represented by association of monomers in homodimers, and homotetramers. A homotrimer form could not be deduced from our findings, and the 320,700 glucocorticoid-receptor complex we observed has been suggested to represent an unresolved mixture of trimers and tetramers.     

DNA methylase from HeLa cell nuclei.

A DNA methylase has been purified 270-fold from HeLa cell nuclei by chromatography on DEAE-cellulose, phosphocellulose, and hydroxyapatite. The enzyme transfers methyl groups from S-adenosyl-L-methionine to cytosine residues in DNA. The sole product of the reaction has been identified as 5-methylcytosine. The enzyme is able to methylate homologous (HeLa) DNA, although to a lesser extent than heterologous DNA. This may be due to incomplete methylation of HeLa DNA synthesized in vivo. The HeLa enzyme can methylate single-stranded DNA, and does so to an extent three times greater than that of the corresponding double-stranded DNA. In single-stranded M. luteus DNA, at least 2.4% of the cytosine residues can be methylated in vitro by the enzyme. The enzyme also can methylate poly (dG-dC-dG-dC) and poly (dG, dC). Bilateral nearest neighbors to the 5-methylcytosine have been determined with M. luteus DNA in vitro and HeLa DNA in vivo. The 5' neighbor can be either G or C while the 3' neighbor is always G and this sequence is, thus, p(G/C)pmCpG.     

Polyanion-induced release of polyribosomes from HeLa cell nuclei.

Intact detergent-washed HeLa nuclei contain a population of polyribisomes that were released by exposure to polyanions such as RNA or poly(U). The released material appeared by electron microscopic examination to be particles averaging about 200 to 300 angstroms in diameter. Sedimentation velocity analysis of the released particles indicated that the particles had S20,w values of 75 and 110. The particles stimulated amino acid incorporation in an ascites S-30 or S-100 extract at 2.5 mM Mg2+. Studies with a variety of antibiotics indicated that these polyribosomes were capable of elongating but not initiating protein synthesis. Although these polyribosomes may be of cytoplasmic origin, they appear unique in that agents thought to disperse chromatin are required for their release from the nucleus.     

A DNA-activated protein kinase from HeLa cell nuclei.

A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.     

Analysis of multiple forms of nuclear factor I in human and murine cell lines.

Nuclear factor I (NFI) is a group of related site-specific DNA-binding proteins that function in adenovirus DNA replication and cellular RNA metabolism. We have measured both the levels and forms of NFI that interact with a well-characterized 26-base-pair NFI-binding site. Five different NFI-DNA complexes were seen in HeLa nuclear extracts by using a gel mobility shift (GMS) assay. In addition, at least six forms of NFI were shown to cross-link directly to DNA by using a UV cross-linking assay. The distinct GMS complexes detected were composed of different subspecies of NFI polypeptides as assayed by UV cross-linking. Different murine cell lines possessed varying levels and forms of NFI binding activity, as judged by nitrocellulose filter binding and GMS assays. The growth state of NIH 3T3 cells affected both the types of NFI-DNA complexes seen in a GMS assay and the forms of the protein detected by UV cross-linking.     

5'-Hydroxyl polyribonucleotide kinase from HeLa cell nuclei. Purification and properties.

An enzyme, 5'-hydroxyl polyribonucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of RNA in the presence of ATP, has been isolated from extracts of HeLa cell nuclei. The kinase requires a divalent cation (Mg2+ or Mn2+) for activity, has an alkaline pH optimum, and is sensitive to the sulfhydryl antagonist N-ethylmaleimide. 5'-hydroxyl terminated polydeoxyribonucleotides are phosphorylated much less efficiently than the 5'-hydroxyl terminated polyribonucleotides, and the kinase preparation is inactive on ribonucleoside 3'-monophosphates. Enzyme activity is inhibited by ADP and by pyrophosphate. The sedimentation coefficient of the kinase is estimated to be 5.6 S from glycerol gradient centrifugation.