NUCLEAR NONHISTONE PROTEINS IN MURINE MELANOMA CELLS .1. QUANTITATIVE ISOLATION AND FRACTIONATION

Abstract

Nuclear nonhistone proteins (NHP''s) were implicated as regulatory agents involved in controlling genetic expression. Utilizing murine melanoma cells, a method for isolating and fractionating NHP''s which greatly increases the yield of these proteins as well as the level of resolution required for detecting small differences in particular NHP''s. Mouse melanoma cells were grown in medium labeled with [3H]leucine. Following 48 h of incubation, the cells were harvested and nuclei were isolated. The NHP''s were extracted from the nuclei in a series of steps which yielded 4 major fractions: NHP1, NHP2, NHP3, NHP4. This method solubilized 80-90% of the protein from the nuclear homogenate. The NHP fractions were separated on DEAE-cellulose columns in a series of salt steps increasing in concentration from 0.05-0.50 M NaCl, followed by steps of 2 M NaCl and 4 and 7 M guanidine hydrochloride. The 40 NHP fractions eluted from these columns were further separated on polyacrylamide-SDS [sodium dodecylsulfate] gels and ranged in MW from 9000-110,000 daltons. Differences were observed in the electrophoretic pattern of each of these 40 fractions. The high resolution of these fractionation procedures greatly enhances the possibility of observing small changes in proteins which may play a role in gene regulation.