The transport and utilization of acetyl coenzyme A by rat liver Golgi vesicles. O-acetylated sialic acids are a major product.

When intact rat liver Golgi vesicles were incubated with [acetyl-3H]acetyl coenzyme A, radioactivity was incorporated into the vesicles in a manner dependent upon temperature, time, protein, and acetyl-CoA concentration. The vesicles concentrated the label 121-fold relative to the medium within 20 min, suggesting an active transport mechanism operating in intact vesicles, and incorporated more than 50% of this label into acid-insoluble materials. This was supported by the finding that incorporation was markedly reduced by Triton X-100 at levels above its critical micellar concentration. While the intravesicular low molecular weight fraction was predominantly free acetate, acetate ions themselves were not permeant to the vesicles. Double-label experiments suggested that the transport process involved the entire acetyl-CoA molecule. This was further supported by the fact that coenzyme ASH, palmitoyl-CoA and butyryl-CoA were markedly inhibitory. Incorporation was optimal at 22 degrees C at pH 7.0, and was moderately stimulated by ATP. However, compounds known to abolish proton gradients or to inhibit the Golgi proton pump had no effect. The apparent Km for the utilization process was 0.61 microM with a Vmax of 21.3 pmol/mg of protein/min. Oligomycin and 4,4'-diisothiocyanostilbene-2,2'disulfonic acid were inhibitory, whereas CMP-NeuAc, UDP-GlcNAc, adenosine 3'-phosphate, 5'-phosphosulfate, atractylosides, tunicamycin, 2'5'-ADP, and 3',5'-ADP were not, showing that this transport process is distinct from other nucleotide transporters previously described in rat liver Golgi. 75-85% of the radioactivity incorporated was shown to be in O-acetylated sialic acids, by neuraminidase release, purification, and high pressure liquid chromatography. The majority of the neuraminidase-resistant radioactivity was released by alkaline hydroxylamine as [3H]acetylhydroxamate, but a significant fraction was resistant to this treatment. The nature of the non-sialic acid radioactivity remains unknown. The existence of this transport mechanism provides yet another level at which the O-acetylation of sialic acids could be regulated.