Fatty-acid synthesis in chloroplasts from mustard (Sinapis alba L.) cotyledons: formation of acetyl coenzyme A by intraplastid glycolytic enzymes and a pyruvate dehydrogenase complex.

Chloroplasts from the cotyledons of mustard (Sinapis alba L.) seedlings were isolated on Percoll gradients, and showed a high degree of intactness (92%) and purity as judged by electron microscopy and marker-enzyme analysis (cytoplasmic contamination lower than 0.4% on a protein basis). The chloroplasts synthesized longchain fatty acids from both precursors [1-(14)C] acetate and [2-(14)C]pyruvate; maximum incorporation rates were 96 nmol·(mg Chl)(-1)·h(-1) for acetate and 213 nmol·(mg Chl)(-1)·h(-1) for pyruvate. Acetyl-CoA-producing enzymatic activities, namely acetyl-CoA synthetase (EC 6.2.1.1.) and a pyruvate dehydrogenase complex, showed specific activities of 14.8 nmol·(mg protein)(-1)·min(-1) and 18.2 nmol·(mg protein)(-1)·min(-1), respectively. The glycolytic enzymes phosphoglyceromutase (EC 2.7.5.3) phosphopyruvate hydratase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were all found to be components of these chloroplasts, thus indicating a possible pathway for intraplastid acetyl-CoA formation.