O-acetylation and de-O-acetylation of sialic acids. O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues--a transmembrane reaction?
Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.
Higa
H H
HH
Department of Medicine, San Diego Veterans Administration Medical Center, California.
Butor
C
C
Diaz
S
S
Varki
A
A
D000085:Acetates / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism; D000107:Acetylation; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003065:Coenzyme A / Q000378:metabolism; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006639:Histidine; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008239:Lysine; D008247:Lysosomes / Q000378:metabolism; D008954:Models, Biological; D051381:Rats; D012794:Sialic Acids / Q000378:metabolism*
D000085:Acetates; D012794:Sialic Acids; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D006639:Histidine; D000105:Acetyl Coenzyme A; D008239:Lysine; D004047:Diethyl Pyrocarbonate; D003065:Coenzyme A