Studies on the degradation of HeLa non-histone proteins.
The hydrolysis of HeLa non-histone nuclear proteins over 24 h has been monitored in dilute alkali at 4, 15 and 25 degrees C using the standard ninhydrin estimation, dansylation and various electrophoresis techniques. Under conditions (up to 0.2 N NaOH, 4 degrees C) that do not release a significant quantity of ninhydrin-positive material or new N-terminal end group considerable breakdown was observed by two-dimensional electrophoresis analysis. The number of stained spots decreased from approx. 140 to 25--30. No internal protease activity could be found. Labelling studies (14C-labelled amino acids) showed that much of the hydrolysed material was extracted from the gel during normal staining and destaining procedures. Peptides could be extracted from alkali-hydrolysed non-histone protein with acid/ethanol and could be further separated by thin-layer chromatography on silica gel G. Short-term labelling of HeLa cells (14C-labelled amino acids for up to 60 min) revealed that these peptides probably have a high rate of turnover. [14C]Glucosamine studies also indicated the presence of considerable carbohydrate material in the low molecular weight products of this alkaline hydrolysis. Various standard proteins and histones were unaffected by hydrolysis in up to 0.2 N NaOH (4 degrees C, 24 h) as judged by gel electrophoresis. Seven different phosphate-splitting enzymes and an esterase had no effect on the non-histone protein electrophoresis patterns but a preparation of phospholipase C which had no protease activity towards eight standard proteins did produce considerable breakdown in HeLa non-histone proteins similar to that produced by 0.2 N NaOH at 4 degrees C.
D000143:Acids; D002241:Carbohydrates / Q000032:analysis; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000378:metabolism*; D000431:Ethanol; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D009555:Ninhydrin; D010738:Type C Phospholipases / Q000494:pharmacology
D000143:Acids; D002241:Carbohydrates; D002868:Chromosomal Proteins, Non-Histone; D000431:Ethanol; D010738:Type C Phospholipases; D009555:Ninhydrin