This paper deals with the partial purification of the pyruvate dehydrogenase complex (PDC) from chloroplasts of spinach and maize mesophyll which hitherto has been isolated only from pea chloroplasts. Starting with membrane free suspensions of lyophilized chloroplasts, and following a high-speed (140000 Xg) centrifugation of this “stromal extract”, the initial specific PDC-activities were concentrated by a factor 10 in the sediment. While most of the purification procedures described earlier resulted in almost complete loss of enzyme activities, a rate zonal sedimentation on linear glycerol gradients allowed for an additional up to 100-fold enrichment of the labile multienzyme complex, albeit with low yields. In contrast to chloroplast PDC from maize mesophyll, inactivation of the spinach complex during glycerol fractionation was due to the dissociation of its loosely bound dihydrolipoyl dehydrogenase component which collected in a lower density fraction of the gradient. Its recombination with PDC constituents of the bottom layer nearly restored initial activities. The chloroplast complex has been identified as true PDC by its substrate specificity for pyruvate, NAD+, and coenzyme A and the 1:1:1 stoichiometry of its reaction products NADH, CO2, and acetyl-CoA. The chloroplast PDC of both plant species showed the well known higher pH-and Mg-requirements than the mitochondrial complex. The observed species-specific differences in the stability of this multienzyme system suggest a connection with the aggregation state of its components. Apparently, the individual subcomplexes are able to function either together in acetyl-CoA formation or independently from each other, e.g. in the synthesis of acetolactate via hydroxy-ethyl-thiamine pyrophosphate or dihydrolipoyl dehydrogenase activities.
Acetyl-CoA and Fatty Acid Synthesis, Chloroplasts The present investigation indicates that photosynthetically active chloroplasts can synthesize acetyl-CoA either from acetate via acetyl-CoA synthetase (ACS) or from pyruvate via the pyru vate dehydrogenase complex (PDC). Both enzyme systems have been assayed in rapidly prepared extracts of chloroplasts isolated from spinach, peas and maize mesophyll. Their kinetic properties showed few species-specific differences. The differing pyruvate and acetate concentrations within the corresponding leaf tissues have been interpreted, therefore, as constituting a major factor determining the relative involvement of both acetyl-CoA synthesizing systems within the different types of chloroplasts. The idea that acetate originates from mitochondria and pyruvate from the cytosol has been supported by nonaqueous fractionation studies. Diffusion-mediated faster up take of acetate may indicate a predominant role of the ACS in spinach chloroplasts. Higher cellular pyruvate/acetate-ratios (2-5) in pea and maize leaves may enhance pyruvate uptake into chloroplasts and thus PDC-driven acetyl-CoA synthesis in pea and maize mesophyll chloroplasts. Maize mesophyll chloroplasts even show a light-driven pyruvate uptake accompanied by a stimulated acetyl-CoA and fatty acid formation. Assuming light-dependent increasing parameters in the stroma space, like Mg2+-concentrations, pH and ATP, as further control criteria in chloroplast acetyl-CoA formation, the ACS appears better adapted to the circumstances in illuminated chloroplasts because of the fact that 1. the ACS requires these cofactors altogether; 2. the PDC is stimulated by increasing pH (up to 8) and Mg-levels (up to 5 mᴍ) alone.
The enzymatic activities of the pyruvate dehydrogenase complex (PDC) and acetyl-CoA synthetase (ACS) have been compared in extracts of plastids isolated from spinach leaves and from both green and etiolated pea seedlings. A ll plastid preparations were shown to be capable of synthesizing acetyl-CoA, not only via acetyl-CoA synthetase, but also via the pyruvate dehydroge nase complex, though, with different activities. Both pathways are apparently under metabolic control. Thus, the substrate levels in photosynthetically active spinach chloroplasts appear to favor acetyl-CoA synthesis via ACS (apparent Km for acetate of 0.1 mм) , because calculated stromal pyruvate levels (0.1 m M) appear to limit its formation via the PDC (apparent Km for pyruvate of 0.2-0.3 nм) . In spinach chloroplasts, therefore, the PDC pathway seems to be predominantly involved in providing precursors for branched-chain amino acid biosynthesis (valine, leucine and isoleucine). Acetyl-CoA, synthesized via ACS, may additionally function as an inhibitor of the chloroplast PD C , because, as in mitochondria, relatively low concentrations of the end products NADH and acetyl-CoA strongly inhibit the PD C in chloroplast extracts. On the other hand, comparatively high concentrations of MgATP, a cofactor for ACS, inhibited the PDC complex. The pH optimum of about 8 and the high Mg-requirement distinguishes both enzymes from mitochondrial PDC and reflects an accomodation to stromal conditions in photosynthetically active chloroplasts.
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