Characterization of an acyltransferase acting on p21N-ras protein in a cell-free system.
We have identified a protein-acyltransferase activity in membranes from mouse fibroblasts which transfers palmitate from palmitoyl-CoA to p21N-ras. Specificity of acylation has been confirmed by linkage analysis using hydroxylamine and by peptide mapping of in vivo and in vitro acylated p21N-ras. The acylation was temperature- and time-dependent, and prevented by prior boiling of membranes, consistent with an enzymatic process. The activity was detected in membranes, but not cytosol, and co-fractionated on Percoll gradients with Golgi markers. Cytosolic p21N-ras from mouse fibroblasts, which is C-terminally modified at its CAAX sequence, was a better substrate for the enzyme that recombinant bacterially expressed, unmodified p21N-ras. The activity could be solubilised in non-ionic detergents, making it amenable to purification.
Gutierrez
L
L
National Institute for Medical Research, Mill Hill, London, U.K.
Magee
A I
AI
D000215:Acylation; D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D002855:Chromatography, Thin Layer; D005227:Fatty Acids / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D010449:Peptide Mapping; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D005227:Fatty Acids; D006898:Hydroxylamines; D019811:Hydroxylamine; D000217:Acyltransferases; D016283:Proto-Oncogene Proteins p21(ras)