Characterization and partial purification of protein fatty acyltransferase activity from rat liver.
The acylation of proteins through the addition of palmitate to cysteine residues is a common posttranslational modification for a variety of proteins, but the enzymology of this reversible modification has resisted elucidation. We developed a strategy to purify protein fatty acyltransferase (PAT) activity from rat livers that took advantage of recent knowledge on the cellular location and inhibition of PAT activity. We determined that three different thiolases have PAT activity in the presence of imidazole and therefore started the purification with a plasma membrane fraction to minimize the contamination with these enzymes. After detergent extraction of the plasma membrane fraction, the PAT activity was enriched about 90-fold by sequential chromatography including affinity chromatography to a cerulenin-based inhibitor of palmitoylation. The partially purified PAT activity (1) was lost with treatments to degrade or denature proteins, (2) could acylate tubulin, Galpha(i) and RGS16 and (3) showed a preference for palmitate and to a lesser degree other long-chain fatty acids. This purification procedure is a significant advance over previous efforts at PAT purification and a starting point for a proteomic approach for identification of mammalian PAT.
Hiol
Abel
A
Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Caron
Joan M
JM
Smith
Charles D
CD
Jones
Teresa L Z
TL
D000215:Acylation; D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D051381:Rats; D013379:Substrate Specificity
D000217:Acyltransferases; C043421:glycoprotein N-palmitoyltransferase