Addition of oleoyl-CoA (1µM), or other acyl-CoA thioesters with a chain length of C16 or greater, to oilseed rape plastids (Brassica napus L.) inhibited the rate of D-glucose 6-phosphate (Glc6P) uptake by 70% after 2min. The IC50 value for oleoyl-CoA inhibition of the transporter was approx. 0.2–0.3µM. Inhibition was alleviated by the addition of acyl-CoA binding protein (ACBP) or BSA at slightly higher concentrations. Oleic acid (5–25µM), Tween 40 (10µM), Triton-X 100 (10µM) and palmitoylcarnitine (5µM) had no effect on Glc6P uptake. The uptake of [1-14C]Glc6P occurred in exchange for Pi, 3-phosphoglycerate or Glc6P at a typical rate of 30nmol Glc6P/min per unit of glyceraldehyde-3-phosphate dehydrogenase (NADP+). The Km(app) of the Glc6P transporter for Glc6P was 100µM. Neither CoA (0.3mM) nor ATP (3mM) inhibited Glc6P uptake, but the transporter was inhibited by 72% when ATP and CoA were added together. This inhibition was attributable to the synthesis of acyl-CoA thioesters, predominantly oleoyl-CoA and palmitoyl-CoA, by long-chain fatty acid-CoA ligase (EC 22.214.171.124) from endogenous fatty acids in the plastid preparations. Acyl-CoA thioesters did not inhibit the uptake of [2-14C]pyruvate or D-[1-14C]glucose into plastids. In vivo quantities of oleoyl-CoA and other long-chain acyl-CoA thioesters were lower than those for ACBP in early cotyledonary embryos, 0.7±0.2pmol/embryo and 2.2±0.2pmol/embryo respectively, but in late cotyledonary embryos quantities of long-chain acyl-CoA thioesters were greater than ACBP, 3±0.4pmol/embryo and 1.9±0.2pmol/embryo respectively.
- acyl-CoA binding protein
- glucose 6-phosphate
- The Biochemical Society, London © 2000