Antagonism of [3H]fatty acid incorporation into vimentin by sodium pyruvate: pitfalls of protein acylation.
In the course of studying possible fatty acid acylation of vimentin by cultured bovine lens epithelial cells, several potential pitfalls of protein-fatty acid acylation were recognized. Even exhaustive delipidation of vimentin with organic solvents failed to remove all noncovalently associated [3H]palmitate and [3H]myristate. Hydroxylamine treatment of vimentin, separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), failed to remove either palmitate or myristate derived radiolabel. Hydroxylamine treatment did remove palmitate label from a group of lower molecular weight proteins. The myristate radiolabel associated with vimentin recovered after SDS-PAGE and subjected to acid hydrolysis was shown due to incorporated [3H]amino acids, mainly glutamic acid, generated from the fatty acid. Adding excess sodium pyruvate to labeling media has been used by others to reduce the metabolic conversion of fatty acids to amino acids; however, no direct evidence in support of this antagonism was presented. We observed that inclusion of sodium pyruvate at between 5 and 20 mM in the labeling medium produced a dramatic decrease in incorporation of myristic acid radiolabel into vimentin. However, inclusion of even 20 mM pyruvate did not completely antagonize the metabolic conversion of fatty acid label to amino acids. Furthermore, the sodium pyruvate antagonism could be totally obscured if the exposure of X-ray film by fluorography was even slightly prolonged. The results illustrate the danger in assuming that solvent extraction totally delipidates proteins and that adding sodium pyruvate to labeling media prevents the transfer of fatty acid label to amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)
Cenedella
R J
RJ
Department of Biochemistry, Kirksville College of Osteopathic Medicine, Missouri 63501.
Mitchell
J
J
D000595:Amino Acid Sequence; D000818:Animals; D016477:Artifacts; D002417:Cattle; D002478:Cells, Cultured; D003459:Crystallins / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D007908:Lens, Crystalline / Q000378:metabolism; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011773:Pyruvates / Q000494:pharmacology*; D014746:Vimentin / Q000378:metabolism*
D003459:Crystallins; D005227:Fatty Acids; D009227:Myristic Acids; D010169:Palmitic Acids; D011773:Pyruvates; D014746:Vimentin; D019814:Myristic Acid; D019308:Palmitic Acid