On the control of long-chain-fatty acid synthesis in isolated intact spinach (Spinacia oleracea) chloroplasts

Abstract

1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510–512] had higher rates of HCO3−-dependent oxygen evolution (up to 369μmol/h per mg of chlorophyll) and higher rates of [1-14C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H14CO3− in assays permitting high rats of photosynthesis. Incorporation of H14CO3− into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3–4 times in excess of that required to account for rates of [1-14C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-14C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-14C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [14C]acetyl-CoA plus [14C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO3− concentrations. Endogenous [14C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO3− concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-14C]acetate concentrations and optimal HCO3− concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.