María del Mar Sola

Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney

The evolution of pyruvate carboxylase has been studied in rat liver and kidney during perinatal development. The pyruvate carboxylase activity, amount of enzyme and mRNA levels have been assayed from 2 days before delivery to weaning. In liver, there is a peak of activity and amount of enzyme 24 h before delivery and 2 peaks, at 12 h and 6 days, after parturition. The transcription of the enzyme gene followed a similar pattern, with mRNA peaks preceding those of activity and amount of enzyme. However, in kidney, pyruvate carboxylase activity, amount and mRNA remain low until weaning. These results confirm the limited role of renal gluconeogenesis during the perinatal development. Since all carboxylases contain biotin as prosthetic group, the biotinylation of pyruvate carboxylase during the perinatal period was investigated by western-blot using streptavidin-biotin peroxidase. In the mitochondrial samples from liver and kidney, all the pyruvate carboxylase detected was fully biotinylated, indicating an early development of the holocarboxylase synthetase activity in the perinatal period. This Western-blot technique also allowed us the detection of other biotin-enzymes based on their molecular weight. In liver, during the perinatal development propionyl-coA and 3-methyl-crotonyl-coA carboxylases followed a pattern of induction similar to pyruvate carboxylase. In kidney, the expression of mitochondrial carboxylases was lower compared to liver and propionyl-coA carboxylase was not detected during the studied period

Regulation of Hexose-Phosphate Cycle Determines Glucose and Fructose Accumulation in Cherimoya (Annona cherimola Mill.) during Ripening

Summary Cherimoya ( Annona cherimola Mill. cv. Fino de Jete ) is a climacteric fruit. Starch stores are converted mainly to glucose and fructose in equimolar concentrations during ripening. There is little accumulation of sucrose, which is consistent with the induction of invertase 2 days after harvesting. Activities of enzymes involved in the hexose-phosphate cycle have been determined during ripening. ATP-dependent phosphofructokinase shows a slight increase in maximum velocity independent of the presence of fructose 2,6-bisphosphate. Pyrophosphate-dependent phosphofructokinase and fructose 1,6-bisphosphatase, on the other hand, are activated and inhibited by fructose 2,6-bisphosphate, respectively, at the onset of ripening. Activities of these enzymes, as well as concentrations of fructose 2,6-bisphosphate, fructose 1,6bisphosphate and fructose 6-phosphate, suggest that glycolysis and gluconeogenesis are differentially activated and hexose-phosphate cycling is important in the regulation of carbon flux from starch during ripening.

Postharvest Changes of Sugar Concentrations in Chilled-Injured Cherimoya (Annona cherimola Mill.)

Summary Starch is the main form of carbon stores in cherimoya ( Annona cherimola Mill. cv. Fino de Jete ), constituting 10% to 12% of the fresh weight of mature-green cherimoya. During ripening starch was completely hydrolyzed when fruits were stored at non-chilling temperatures (22 °C or 12 °C), being mainly converted to glucose and fructose in equimolar concentrations, with transient accumulation of sucrose. Storage at chilling temperatures (4 °C or 1 °C) produced a decrease in the starch hydrolysis rate and consequently in the concentration of monosaccharides. When chilled-injured cherimoya were rewarmed to 22 °C after 11 days of storage in the chilling conditions, an activation of starch hydrolysis took place. In these rewarmed fruit, accumulation of glucose and fructose was smaller than in non chilling fruit. The final monosaccharide concentrations were dependent on the severity of the chilling-injury.

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. I. Effects of fructose-2,6-bisphosphate and divalent cations

1. The native rat-kidney cortex Fructose-1,6-BPase is differentially regulated by Mg2+ and Mn2+. 2. Mg2+ binding to the enzyme is hyperbolic and large concentrations of the cation are non-inhibitory. 3. Mn2+ produces a 10-fold rise in Vmax higher than Mg2+. [Mn2+]0.5 is much larger than [Mg2+]0.5. At elevated [Mn2+] inhibition is observed. 4. Mg2+ and Mn2+ produce antagonistic effects on the inhibition of the enzyme by high substrate. 5. Fru-2,6-P2 inhibits the enzyme by rising the S0.5 and favouring a sigmoidal kinetics. 6. The inhibition by Fru-2,6-P2 is released by Mg2+ and more powerfully by Mn2+ increasing the I0.5.

cAMP-Independent Synergistic Effects of Insulin and Dexamethasone on Fructose 2,6-Bisphosphate Metabolism in H4IIE Cells

Hormonal regulation of fructose 2,6-bisphosphate (Fru-2,6-P2) content was studied in H4IIE cells. These cells were found to be very sensitive to physiological concentrations of insulin. Addition of either insulin or dexamethasone alone increased Fru-2,6-P2 in a time- and dose-dependent manner, and the maximal effect of the hormones was seen at 1 h. Neither hormone had any measurable effect on cAMP levels. The effect of addition of both insulin and dexamethasone on Fru-2,6-P2 was synergistic. Insulin, but not dexamethasone, rapidly increased 6-phosphofructo-2-kinase (6PF-2-K) activity by causing dephosphorylation of the enzyme as judged by a decrease in the Km for fructose-6-phosphate. Addition of both hormones also resulted in a synergistic 10-fold increase in enzyme protein as measured by kinase activity and phosphoenzyme formation. Dexamethasone increased liver 6PF-2-K/Fru-2,6-P2 mRNA abundance by 10- to 12-fold as measured by a ribonuclease protection assay, and insulin increased it by only 4-fold. Effects were observed as early as 1 h after hormone addition, but addition of both hormones together showed no synergy. We conclude that the synergistic effects of insulin and dexamethasone on Fru-2,6-P2 content are mediated by a combination of stimulation of expression of the bifunctional enzyme gene by both hormones and insulin-induced modulation of the activation state of the bifunctional enzyme, both of which are mediated by cAMP-independent mechanisms.

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. II: Effects of adenine nucleotides

1. The native rat-kidney cortex Fructose-1,6-bisphosphatase is differentially regulated by adenine nucleotides in the presence of divalent cations. 2. Binding of AMP and ADP to the enzyme is co-operative. The inhibition by both nucleotides show an uncompetitive mechanism AMP being the most efficient inhibitor. 3. Mg2+ decreases the inhibition produced by AMP and ADP by enhancing their I0.5 and completely annulates the inhibitory effect of ATP. 4. In the presence of Mn2+ ADP behaves as an inhibitor but no inhibition is evident with AMP, suggesting the existence of different allosteric sites for each nucleotide.

Kinetic characterization of phosphofructokinase isolated from rat kidney cortex

1. Phosphofructokinase from rat kidney cortex has been purified by affinity chromatography to a final specific activity of 15 units per mg of protein, measured at 25 degrees C and pH 8. 2. This lower spec. act., compared with that of the enzyme from other sources, shows the enzyme in proximal tubules to be less active, which would account for the main gluconeogenic role of these nephron sections. 3. The binding of fructose-6-phosphate to the enzyme is co-operative. ATP increases the Hill coefficient and produces a marked allosteric inhibition on the activity. 4. Fructose-2,6-bis-phosphate is a potent activator of the enzyme from this source. It reduces the Hill coefficient of the enzyme and the inhibition constant of ATP. A marked difference between this and the liver enzyme is that the activation is not co-operative.

Stimulation of mitochondrial pyruvate transport in rat renal cortex by phenylephrine

Phenylephrine effect on liver and kidney cortex mitochondrial pyruvate concentration was investigated. While in liver the alpha 1-adrenergic agent produced a decrease in pyruvate content, a significant increase was observed in kidney, even in the presence of 0.5 mM alpha-cyano-4-hydroxy-cinnamate. These changes were not observed when pyruvate was formed by intramitochondrial transamination of alanine, suggesting a role for the pyruvate transport across mitochondrial membranes in the regulation of mitochondrial pyruvate metabolism in kidney cortex. This was corroborated measuring the phenylephrine effect on pyruvate carboxylation.