Plastid glycolytic enzymes

Plastid glycolytic enzymes. The cotyledon chloroplasts capability was tested to produce pyruvate from 3-PGA. For this purpose the presence of phos-phoglyceromutase, enolase and pyruvate kinase has been examined in the stroma fraction and - for comparison - in a membrane-free 100,000 x g supernatant which contained also cytoplasmic constituents. These results are expressed in nmol/mg protein/min (values for 100,000 x g fraction in brackets) phosphoglyceromutase 12.1 (102) enolase 38 (78) pyruvate kinase 11.9 (61). 

Intact plastids were isolated from early, early to mid and mid to late cotyledon embryos (1.5, 2.5 and 3.5 mg embryo fresh weight) of B. napus cv. Topas (according to [1]). Isolated plastids were incubated with " C-labelled metabolites and the incorporation of C into fatty acids determined [1]. The activities of enzymes were measured using standard assays [1] optimized for plastid extracts. The proportions of glycolytic enzyme activities attributable to the plastid were determined by adding increasing concentrations of cytosol (supernatant) back to a plastid pellet. The plastidial activity of each enzyme was then calculated from the relationship between its total activity and that of a cytosolic and plastidial marker enzyme essentially as previously described [3]. Short term assays for the rate at which isolated plastids take up Relabelled metabolites were performed using silicone oil filtration [4]. 

Figure 3.1 Metabolic origin of plastid carbon source and enzyme cofactors required for de novo fatty acid synthesis, (a) Glycolytic enzymes (b) pyruvate kinasec (EC 2.7.1.40) (c) pyruvate kinasep (EC 2.7.1.40) (d) phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) (e) L-malate dehydrogenase (EC 1.1.1.37) (f) malic enzyme (EC 1.1.1.40) (g) pyruvate dehydrogenase (EC 1.2.4.1, EC 2.3.1.12, EC 1.8.1.4).

It is unclear whether the rate of FAS within the seed plastid is enzyme limited, or metabolite limited, or both. Unlike the chloroplast, where transport mechanisms and stromal metabolite concentrations are known, there is almost a complete dearth of similar information for developing seeds. This is an obstacle to understanding the regulation of FAS in seeds. It would be helpful to know the steady state levels of ATP, ADP, NADH, NADPH, CoA, acetyl- and malonyl-CoA, ACP, acyl-ACPs and glycolytic intermediates in the plastid. As the seed during its maturation phase is a one-way metabolic system, there may not be a fine control, as exemplified by carbon... 

During the development of oilseed rape embryos starch accumulates transiently during the early stages of oil accumulation (P. da Silva and A.M. Smith, pers. comm.). Both starch and fatty acids are synthesized in the plastids and these processes are dependent upon the import of cytosolic metabolites in non-photosynthetic cells. Plastids have been isolated from several nonphotosynthetic tissues and characterized with respect to their enzyme capacities and utilization of exogenous substrates to support starch and fatty acid synthesis in vivo. These studies have revealed that the glycolytic pathway is present although the activities of some of the enzymes in the lower half of the pathway can be low. Pyruvate dehydrogenase is also present in these plastids. 

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