title: NAP-2 is part of multi-protein complexes in HeLa cells.
abstract: We previously reported that a complex of nuclear proteins from HeLa cells, among them histone H1 and casein kinase 2 co-eluted from immobilized nucleosome assembly protein 2 (NAP-2)-Sepharose. Here, using HeLa cell nuclear extracts, we found NAP-2 migrates in a blue-native polyacrylamide gel with an apparent molecular weight of 300 kDa. HeLa cell NAP-2, labeled in vivo with radioactive orthophosphate, co-precipitated with at least two phosphoproteins, with an apparent mass of 100 and 175 kDa, respectively, as determined by SDS-PAGE. NAP-2 from total HeLa cell extract co-purified with other proteins through two sequential chromatographic steps: first, a positively charged resin, Q-Sepharose, was used, which purified NAP-2 more easily with other proteins that eluted as a single peak at 0.5 M NaCl. This fraction possessed both relaxing and supercoiling activities, and it was able to assemble regularly spaced nucleosomes onto naked DNA in an ATP-dependent manner. Second, a negatively charged resin (heparin) was used, which retained small amounts of NAP-2 (a very acidic polypeptide) and topoisomerase I. This fraction, although able to supercoil relaxed DNA, did so to a lesser extent than the Q-Sepharose fraction. The data suggest that NAP-2 is in complex(es) with other proteins, which are distinct from histones.
authors.affiliation: McGill Cancer Center, McGill University, Montreal, Quebec, Canada H3G 1Y6.
authors.forename: Pedro
authors.initials: P
authors.lastname: Rodriguez
authors.forename: Marcia T
authors.initials: MT
authors.lastname: Ruiz
authors.forename: Gerald B
authors.initials: GB
authors.lastname: Price
authors.forename: Maria
authors.initials: M
authors.lastname: Zannis-Hadjopoulos
mesh_terms: D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D006367:HeLa Cells; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding
chemical_list: D046912:Multiprotein Complexes; C104356:NAP1L4 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D006493:Heparin