Two-dimensional gel analysis of nuclear proteins during muscle differentiation in vitro: I. Changes in nuclear protein content

Abstract

Improved methods are described for the isolation of nuclei from skeletal muscle-cell cultures by lysis with Triton X-100 and centrifugation through 65% sucrose-5 mM MgCl 2. Evidence is presented that levels of contamination by cytoplasmic and fibrillar proteins are acceptably low. Nuclear proteins were separated into saline-soluble and non-histone protein (NHP) fractions and analysed on two-dimensional gels stained with Coomassie Blue. 2D-gels of NHP and cytoplasmic proteins were compared and few proteins common to both fractions were found. Of three major cytoplasmic proteins, actin, tubulin and tropomyosin, only actin could be positively identified as a minor protein in NHP and tropomyosin was undetectable. An overall increase in nuclear NHP content (relative to DNA) occurred during the early period of cell differentiation in vitro (24–66 h of culture) and a corresponding increase in nuclear protein to DNA ratio was observed. There were differential changes between individual NHP, some major proteins showing little change, while others showed large increases. These changes may be related to increases in nuclear protein uptake which are reported to occur during differentiation and hormone-induced gene transcription in other cell types.