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Oleate stimulates glucose production and concomitantly decreases lactate and pyruvate production by rat hepatocyte suspensions incubated with dihydroxyacetone as substrate. The actions of oleate could be blocked by D-(+)dodecanoylcarnitine, which inhibits transport of the fatty acid into the mitochondria and the subsequent oxidation. beta-Hydroxybutyrate, but not acetoacetate, also stimulated glucose synthesis and inhibited lactate and pyruvate production. Furthermore, both beta-hydroxybutyrate and oleate stimulated oxygen consumption to the same extent. This suggests that oleate stimulates glucose production by the provision of energy subsequent to mitochondrial beta-oxidation of the fatty acids. The content of ATP itself did not appear to be responsible for the effects of oleate. Crossover analysis of the gluconeogenic intermediates implicated a site of oleate action between fructose 1,6-bisphosphate and fructose 6-phosphate, suggesting phosphofructokinase and/or fructose-bisphosphatase as possible regulatory sites. Coupled with the finding that intracellular citrate accumulates upon addition of oleate or beta-hydroxybutyrate, but not acetoacetate, the results suggest that citrate inhibition of phosphofructokinase accounts for the redirection of carbon flow from lactate and pyruvate formation and towards that of glucose.

Rates of phosphatidate synthesis from dihydroxyacetone phosphate via acyl dihydroxyacetone phosphate or glycerol phosphate are compared in homogenates of 13 tissues, most of which are deficient in glycerol phosphate dehydrogenase (EC 1.1.1.8). In all tissues examined, dihydroxyacetone phosphate entered phosphatidate more rapidly via acyl dihydroxyacetone phosphate than via glycerol phosphate. Tissues with a relatively low rate of phosphatidate synthesis via glycerol phosphate, showed no compensating increase in the rate of synthesis via acyl dihydroxyacetone phosphate. The rates at which tissue homogenates synthesize phosphatidate from dihydroxyacetone phosphate via glycerol phosphate increase as glycerol phosphate dehydrongenase increase. Both glycerol phosphate dehydrogenase and glycerol phosphate: acyl CoA acyltransferase (EC 2.3.1.15) are more active than dihydroxyacetone phosphate : acyl CoA acyltransferase (EC 2.3.1.42). Thus, all the tissue homogenates possessed an apparently greater capability to synthesize phosphatidate via glycerol phosphate than via acyl dihydroxyacetone phosphate, but did not express this potential. This result is discussed in relation to in vivo substrate limitations.

1. The formation of metabolites in the stroma compartment of isolated chloroplasts during carbon fixation, and their export to the medium, have been investigated using improved techniques. 2. Rapid separation of photosynthesising chloroplasts from the medium, accompanied by simultaneous quenching of metabolism was achieved by using silicone oil layer filtering centrifugation under illumination. Metabolites were separated by microscale ion-exchange chromatography. Quantitative determination of each metabolite was based on labelling with 32P. 3. It was found that fixed carbon was exported from the chloroplasts only as triose phosphate and phosphoglycerate, and to a minor extent, as pentose monophosphate. The main compounds accumulating in the stroma were hexose and heptose monophosphates and phosphoglycerate. A marked decrease in the concentration of inorganic phosphate in the stroma during the first 5 min of illumination was accompanied by a complementary increase in organic phosphate so that the total amount of phosphate within the chloroplasts remained constant. 4. The concentration difference for phosphoglycerate between the stroma and the medium was much higher than for triose phosphate or inorganic phosphate, although all three compounds are transported across the inner membrane of the chloroplast envelope by the same carrier. It was concluded that the efflux of phosphoglycerate was restricted.

Dihydroxy [3-14C]acetone phosphate was prepared enzymatically from [1-14C]glucose and use as a substrate in a partially purified quinolinate synthetase system prepared from Escherichia coli mutants. Carbon-by-carbon degradation of the resulting [14C]quinolinate showed that 96% of the 14C was located in carbon-4, indicating that carbon-3 of dihydroxyacetone phosphate condenses with carbon-3 of aspartate in quinolinate synthesis in E. coli.

The uptake of phosphate and phosphorylated compounds into the chloroplast stroma has been studied by silicone layer filtering centrifugation. 1. Inorganic phosphate, 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde phosphate are transported across the envelope leading to an accumulation in the chloroplast stroma. This uptake proceeds by a counter exchange with phosphate and phosphorylated compounds present there. 2. The transport shows saturation characteristics allowing the determination of Km and V. 3. The phosphorylated compounds transported act as competitive inhibitors of the transport. From measurements of the Km and Ki the specificity of the transport is described. 4. The transport of inorganic phosphate and 3-phosphoglycerate is inhibited by p-chloromercuriphenyl sulfonate, pyridoxal 5'-phosphate and trinitrobenzene sulfonate. 5. The activation energy of phosphate transport as determined from the temperature dependence is evaluated to be 16 kcal (0--12 degrees C). 6. It is concluded that inorganic phosphate, 3-phosphoglycerate, dihydroxy-acetone phosphate and glyceraldehyde phosphate are transported by the same carrier, which has been nominated phosphate translocator. 7. Simultaneous measurements of the proton concentration in the medium and the transport into the chloroplasts show that the transfer of 3-phosphoglycerate involves a transfer of a proton into the same direction. 8. Measurements of the pH dependence of the transport indicate that all substances including 3-phosphoglycerate are transported by the phosphate translocator as divalent anions. 9. The physiological function of the phosphate translocator is discussed.

A p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing alpha-glucosidase of a thermophile, Bacillus thermoglucosidius KP 1006, was purified to an electrophoretically-homogeneous state. Its molecular weight was estimated as 60 000 by gel electrophoresis. The molecular activity (ko) and the Km value at 60 degrees C and pH 6.8 for p-nitrophenyl-alpha-D-glucopyranoside were 233 s-1 and 0.24 mM, respectively. The enzyme cleft the non-reducing terminal alpha-1,6-glucosidic bonds of isomaltose, panose, isomaltotriose, isomaltotetraose, and isomaltopentaose. The ko values were 72.4, 194, 208, 233 and 167 s-1, and the Km values were 3.3, 9.5, 11, 13 and 21 mM, respectively. Each isomaltosaccharide was hydrolyzed to glucose by the cleavage of single glucose units from its nonreducing end. The present study suggests that the enzyme is an oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase, EC 3.2.1.10) and an exo-glucosidase.