Metabolic pathways of the intermediate metabolism of maize root tips were identified and quantified after labeling to isotopic and metabolic steady state using glucose labeled on carbon-1, -2, or -6 with 14C or 13C. The specific radioactivity of amino acids and the 13C-specific enrichment of specific carbons of free glucose, sucrose, alanine and glutamate were measured and used to calculate metabolic fluxes. The non-triose pathways, including synthesis of polysaccharides, accumulation of free hexoses, and to a lesser extent starch synthesis, were found to consume 75% of the glucose entering the root tips. The cycle of synthesis and hydrolysis of sucrose was found to consume about 70% of the ATP produced by respiration. The comparison of the specific radioactivities of amino acids and phospholipid glycerol phosphate after labeling with [1-(14)C] or [6-(14)C]glucose revealed the operation of the pentose phosphate pathway. The transfer of label from [2-(14)C]glucose to carbon-1 of starch glucosyl units confirmed the operation of this pathway and indicated that it is located in plastids. It was found to consume 32% of the hexose phosphates entering the triose pathways. The remaining 68% were consumed by glycolysis. The determination of the specific enrichment of carbohydrate carbons -1 and -6 after labeling with [1-(13)C]glucose indicated that both the conversion of triose phosphates back to hexose phosphates and the transaldolase exchange contributed to this randomization. Of the triose phosphates produced by glycolysis and the pentose phosphate pathway, about 60% were found to be recycled to hexose phosphates, and 28% were directed to the tricarboxylic acid cycle. Of this 28%, two-thirds were found to be directed through the pyruvate kinase branch and one-third through the phosphoenolpyruvate branch. The latter essentially has an anaplerotic function since little malate was found to be converted to pyruvate (malic enzyme reaction).

A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division.

Substrate (futile) cycling involving carbohydrate turnover has been widely reported in plant tissues, although its extent, mechanisms, and functions are not well known. In this study, two complementary approaches, short and steady-state labeling experiments, were used to analyze glucose metabolism in maize (Zea mays) root tips. Unidirectional rates of synthesis for storage compounds (starch, Suc, and cell wall polysaccharides) were determined by short labeling experiments using [U-14 C]glucose and compared with net synthesis fluxes to determine the rate of glucose production from these storage compounds. Steady-state labeling with [1-13 C]glucose and [U-13 C]glucose showed that the redistribution of label between carbon C-1 and C-6 in glucose is close to that in cytosolic hexose-P. These results indicate a high resynthesis flux of glucose from hexose-P that is not accounted for by glucose recycling from storage compounds, thus suggesting the occurrence of a direct glucose-P-to-glucose conversion. An enzyme assay confirmed the presence of substantial glucose-6-phosphatase activity in maize root tips. This new glucose-P-to-glucose cycle was shown to consume around 40% of the ATP generated in the cell, whereas Suc cycling consumes at most 3% to 6% of the ATP produced. The rate of glucose-P cycling differs by a factor of 3 between a maize W22 line and the hybrid maize cv Dea, and is significantly decreased by a carbohydrate starvation pretreatment.The development of nonphotosynthetic tissues is closely related to Suc import. Suc is degraded by Suc synthase (SuSy) or invertase to provide UDP-Glc or hexoses for the biosynthesis of structural or storage compounds and for ATP production (Fig. 1). In 1988, Hargreaves and ap Rees used pulse-chase experiments to show the presence of a cycle of synthesis and degradation of Suc in pea roots. This cycle, consuming ATP without any apparent physiological function, was described as a futile cycle, according to Fell's definition (Fell, 1997), and, according to more recent terminology, which refers to a possible role for these processes, it can be described as a substrate cycle (the Suc cycle). Since then, the Suc cycle has been found in many other tissues: chenopodium cells, potato (Solanum tuberosum) tubers, ripening banana (Musa cavendishii), maize (Zea mays) root tips, tomato (Lycopersicon esculentum) cells, and tomato fruit (Hatzfeld and Stitt, 1990;Hill and ap Rees, 1994; DieuaideNoubhani et al., 1995 DieuaideNoubhani et al., , 1997N'tchobo et al., 1999;Rontein et al., 2002). One can estimate that, for the synthesis and degradation of one molecule of Suc, 1.5 to two molecules of ATP are consumed (Fig. 1). Depending on the tissue, this cycle could consume between 5% and 70% of the ATP produced by the cell (Hatzfeld and Stitt, 1990;Hill and ap Rees, 1994;Dieuaide-Noubhani et al., 1995;Fernie et al., 2002;Rontein et al., 2002).Two other substrate cycles related to central carbohydrate metabolism have been described in plants, hexose-P 4 triose-P cycling and the starch synthesi...

Recently, we purified five 3-hydroxyacyl-CoA dehydrogenases from isolated rat liver peroxisomal fractions. The enzymes were designated I-V according to their order of elution from the first column used in the purification procedure. Determination of the substrate (L-or o-hydroxyacyl-CoA) stereospecificity and (de)hydratase measurements with the different 3-hydroxyacyl-CoA stereoisomers of straight-chain fatty acids and the bile acid intermediate trihydroxycoprostanic acid, immunoblotting analysis with antibodies raised against the different enzymes and peptide sequencing, all performed on enzymes I-V and molecular cloning of enzyme I11 revealed the following picture.Rat liver peroxisomes contain two multifunctional P-oxidation proteins : (a) multifunctional protein 1 (the classical multifunctional protein ; MFP-1) displaying 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase and A', A'-enoyl-CoA isomerase activity (enzyme IV) and (b) multifunctional protein 2 (MFP-2) displaying 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activity (enzyme LII).Because of their substrate stereospecificity and because of the stereochemical configuration of the naturally occurring P-oxidation intermediates, MFP-1 and MFP-2 appear to be involved in the P-oxidation of fatty acids and bile acids intermediates, respectively.The deduced amino acid sequence of the cloned MFP-2 cDNA is highly similar to that of the recently described porcine endometrial estradiol 17P-dehydrogenase [Leenders, F., Adamski, J., Husen, B., Thole, MFP-2 is partially cleaved, most probably in vivo, in a estradiol 17P-dehydrogenasel~-3-hydroxyacyl-CoA dehydrogenase that forms a dimeric complex (enzyme I) and a hydratase. The physiological significance of enzyme I in bile acid synthesis (and steroid metabolism) remains to be determined. MFP-1 (enzyme IV) is artefactually cleaved during purification giving rise to 3-hydroxyacyl-CoA dehydrogenase V. 3-Hydroxyacyl-CoA dehydrogenase I1 is a mitochondrial contaminant similar to porcine and murine mitochondrial 3-hydroxyacyl-CoA dehydrogenase.