Mitochondrial pyruvate metabolism in liver and kidney during acidosis.

Pyruvate transport and carboxylation have been determined in mitochondria from liver and kidney cortex isolated from Wistar rats with acidosis produced by three different treatments: fasting, exercise and ingestion of ammonium chloride. Fasting for 48 h or swimming for 2 h resulted in an increased rate of CO2 fixation by mitochondria from both organs incubated with pyruvate. This increase was accompanied by a rise in the rate of pyruvate transport in all cases except in mitochondria derived from the kidney of the fasted animals. Acute acidosis produced by the ingestion of ammonium chloride resulted in increases in pyruvate transport and carboxylation in kidney mitochondria, but a drop in pyruvate carboxylation was observed in mitochondria from the liver. The results are discussed in terms of the differential regulation of the mitochondria steps for gluconeogenesis from three carbon precursors in liver and kidney, taking into consideration the hormonal status of the animals and the prevailing available substrates in each condition.

Citrate inhibition of rat-kidney cortex phosphofructokinase.

The regulatory properties of citrate on the activity of phosphofructokinase (PFK) purified from rat-kidney cortex has been studied. Citrate produces increases in the K0.5 for Fru-6-P and in the Hill coefficient as well as a decrease in the Vmax of the reaction without affecting the kinetic parameters for ATP as substrate. ATP potentiates synergistically the effects of citrate as an inhibitor of the enzyme. Fru-2,6-P2 and AMP at concentrations equal to Ka were not able to completely prevent citrate inhibition of the enzyme. Physiological concentrations of ATP and citrate produce a strong inhibition of renal PFK suggesting that may participate in the control of glycolysis in vivo.

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. 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.

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.

Effects of AMP and fructose 2,6-bisphosphate on fluxes between glucose 6-phosphate and triose-phosphate in renal cortical extracts.

Gluconeogenic flux exceeds glycolytic flux at the hexose-phosphate steps when measured in extracts of kidney cortex from well-fed rats. Addition of AMP and/or fructose 2,6-bisphosphate to the assay medium partially eradicates the difference. Using principles developed by Kacser, H., and Burns, J. A. ((1973) in Rate Control of Biological Processes (Davies, D. D., ed) pp. 65-104, Cambridge University Press, London) and Heinrich R., and Rapoport T. A. ((1974) Eur. J. Biochem. 42, 97-105), flux control coefficients of enzymes participating in the pathway segments from glucose 6-phosphate to triose-phosphates and from glycerol 3-phosphate to glucose 6-phosphate were determined by additions of the respective enzyme to the system. Results show that the flux control coefficients are highly modulated by the presence of allosteric effectors, as might be expected according to the regulatory properties of phosphofructokinase and fructose-1,6-bisphosphatase purified from this origin. Measured reductions of fructose 2,6-bisphosphate and AMP levels during acidosis, starvation, or after phenylephrine treatment suggest that these changes contribute to enhanced gluconeogenesis under these conditions.

Regulation of rat-renal cortex phosphofructokinase activity by pH.

The activity of phosphofructokinase purified from rat kidney cortex has been assayed at two different pH values. At pH 7 the enzyme showed cooperativity for the binding of fructose 6-phosphate (Fru-6-P) and a strong allosteric inhibition by ATP. When the assays were done at pH 8 hyperbolic kinetics were observed for both substrates, a smaller inhibition by ATP was observed and the Vmax for ATP and for Fru-6-P was higher than at pH 7. A sequential reaction mechanism was inferred. Results are discussed in terms of the importance of a reduced hexose-phosphate cycling rate during metabolic acidosis induced by exercise.

The control of protein phosphatase-1 by targetting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1.

The major protein phosphatase that dephosphorylates smooth-muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130, 37 and 20 kDa, the most likely structure being a heterotrimer. The 37-kDa component was the catalytic subunit, while the 130-kDa and 20-kDa components formed a regulatory complex that enhanced catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppressed its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the beta isoform of protein phosphatase-1 (PP1) and the 130-kDa subunit as the PP1-binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PP1 beta with different proteins and (in conjunction with earlier analysis of the glycogen-associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits.

p34cdc2 phosphorylation sites in histone H1 are dephosphorylated by protein phosphatase 2A1.

The protein phosphatase activity in rat liver cytosol or nuclear extracts that dephosphorylates histone H1 which has been phosphorylated by p34cdc2 is inhibited completely by okadaic acid, but unaffected by inhibitor-2 or magnesium ions, demonstrating that the only enzyme in this tissue capable of dephosphorylating this substrate is a type 2A phosphatase. Fractionation of the cytosol by anion-exchange chromatography and gel filtration demonstrated that histone H1 phosphatase activity coeluted with the major species of protein phosphatase 2A, termed PP2A1 and PP2A2. PP2A1 was the most active histone H1 phosphatase, its histone phosphatase phosphorylase phosphatase activity ratio being 6-fold higher than PP2A2 and 30-fold higher than the free catalytic subunit PP2AC. It is concluded that PP2A1 is likely to be the enzyme which dephosphorylates p34cdc2-labelled histone H1 in vivo and that the A and B subunits which interact with PP2AC in this species each play a key role in facilitating dephosphorylation of this substrate. The results demonstrate that PP2A, in addition to being involved in suppressing the activation of p34cdc2 in vivo, can also function to reverse at least one of its actions.

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.

Inhibition by substrate of fructose 1,6-bisphosphatase purified from rat kidney cortex. Calculation of the kinetic constants of the enzyme.

Fructose 1,6-bisphosphatase is a typical enzyme that is severely inhibited by its own substrate. This makes it difficult to determine all the parameters involved in its kinetics. It has been shown recently that if Vm is satisfactorily estimated the remaining parameters can be determined using the Hill plot (Bounias, M. (1988) Biochem. Int. 17, 147-154). The enzyme has been purified from rat kidney cortex nearly to homogeneity, and its kinetic constants have been calculated using a rigorous algebraic method. The most interesting result is that the substrate is unable to bind to the free enzyme as an inhibitor, which indicates that the enzyme lacks an allosteric site for hexose bisphosphates.

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.