Ca2+-dependent activation of oxoglutarate dehydrogenase by vasopressin in isolated hepatocytes.

Vasopressin stimulated gluconeogenesis from proline in hepatocytes from starved rats; this was attributed to an activation of oxoglutarate dehydrogenase (EC 1.2.4.2) [Staddon & McGivan (1984) Biochem. J. 217, 477-483]. The role of Ca2+ in the activation mechanism was investigated. (1) In the absence of extracellular Ca2+, vasopressin caused a stimulation of gluconeogenesis and a decrease in cell oxoglutarate content that were markedly transient when compared with the effects in the presence of Ca2+. (2) Ca2+ added to cells stimulated for 2 min by vasopressin in the absence of extracellular Ca2+ sustained the initial effects of vasopressin. Ca2+ added 15 min after vasopressin, a time at which both the rate of gluconeogenesis and the cell oxoglutarate content were close to the control values, caused a stimulation of gluconeogenesis and a decrease in cell oxoglutarate content. (3) Under conditions of cell-Ca2+ depletion, vasopressin had no effect on gluconeogenesis or cell oxoglutarate content. (4) Ionophore A23187 stimulated gluconeogenesis and caused a decrease in cell oxoglutarate content, but the phorbol ester 4 beta-phorbol 12-myristate 13-acetate had no effects. (5) These data suggest that the initial activation of oxoglutarate dehydrogenase by vasopressin is dependent on an intracellular Ca2+ pool and independent of extracellular Ca2+. For activation of a greater duration, a requirement for extracellular Ca2+ occurs. The activation of oxoglutarate dehydrogenase by A23187 is consistent with a mechanism involving Ca2+, but the lack of effect of 4 beta-phorbol 12-myristate 13-acetate indicates that protein kinase C is not involved in the mechanism of activation by vasopressin.     

Kinetic characterization of the pyruvate and oxoglutarate dehydrogenase complexes from human heart

The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruvate, CoA and NAD, respectively. Acetyl-CoA produces a competitive inhibition of PDC (Ki = 35 microM) with respect to CoA, whereas NADH produces the same type of inhibition with respect to NAD (Ki = 36 microM). The oxoglutarate dehydrogenase complex (OGDC) from human heart has active sites with two different affinities for 2-oxoglutarate ([S]0.5 of 30 and 120 microM). ADP (1 mM) decreases the [S]0.5 values by a half. The inhibition of OGDC (Ki = 81 microM) by succinyl-CoA is of a competitive type with respect to CoA (Km = 2.5 microM), whereas that of NADH (Ki = 25 microM) is of a mixed type with respect to NAD (Km = 170 microM).     

The metabolic response of the kidney to acute sodium lactate alkalosis

In vivo studies were performed in the dog to verify if sodium lactate had an important effect on the metabolism of glutamine by the kidney. The animals were infused with 0.6 M sodium lactate to induce acute metabolic alkalosis with plasma bicarbonate of 29.7 mM. During these experiments, it was demonstrated that the renal uptake of glutamine increased by 46%, while the renal production of ammonia was unchanged. The renal production of alanine rose from 6.0 to 16.8 mumol/min. Plasma concentration of lactate increased from 1.3 to 19.2 mM, while that of pyruvate increased from 0.075 to 0.454 mM. In the renal tissue, alpha-ketoglutarate, malate, oxaloacetate, lactate, pyruvate, citrate, and alanine increased significantly. Similar changes were found in the liver and skeletal muscle. The observed changes are best described by transamination of pyruvate and glutamate under the influence of alanine aminotransferase (GPT). It can be calculated that this reaction was responsible for 76% of the production of ammonia from glutamine, the latter being necessary to provide glutamate for the synthesis of alanine. Dogs infused with 0.3 M sodium bicarbonate instead of sodium lactate with the same degree of acute metabolic alkalosis, showed a depression of 40% in the renal uptake of glutamine with a 38% decrease in renal ammoniagenesis and a 20% fall in the production of alanine. The present studies demonstrate that the production of ammonia from glutamine is not necessarily related to changes in acid-base balance, but may be associated with biochemical alterations related to the synthesis of alanine by the kidney.     

Control of the citric acid cycle by glyoxylate. The mechanism of inhibition of oxoglutarate dehydrogenase, isocitrate dehydrogenase and aconitate hydratase

1. The effects of glyoxylate on partially purified preparations of aconitate hydratase, isocitrate dehydrogenase and oxoglutarate dehydrogenase were compared with those of oxalomalate and hydroxyoxoglutarate (obtained by condensation of glyoxylate with oxaloacetate and pyruvate respectively). 2. Glyoxylate (1mm) did not affect aconitate hydratase and isocitrate dehydrogenase, whereas oxalomalate (1mm) inhibited the enzyme activities completely. 3. Glyoxylate (0.025mm) inhibited oxoglutarate dehydrogenase irreversibly, whereas the same concentrations of oxalomalate and hydroxyoxoglutarate were ineffective. This inhibitory effect was prevented if oxoglutarate, pyruvate or oxaloacetate was mixed with the enzyme before the glyoxylate. 4. Incubation of oxoglutarate dehydrogenase with radioactive glyoxylate produced radioactive carbon dioxide; radioactivity was also recovered in the portion of the enzyme identified with thiamin pyrophosphate. 5. The behaviour of glyoxylate in producing multiple inhibitions of the citric acid cycle, either by direct interaction with oxoglutarate dehydrogenase, or by means of its condensation compounds which inhibit aconitate hydratase and isocitrate dehydrogenase, is discussed.     

Phosphate-dependent glutaminase from rat kidney. Cause of increased activity in response to acidosis and identity with glutaminase from other tissues.

Immune serum was prepared against phosphate-dependent glutaminase purified from rat kidney and was used to investigate the cause of increased renal glutaminase activity in acidotic rats. Crude kidney homogenates from acidotic rats exhibited a fourfold greater specific activity for phosphate-dependent glutaminase. The glutaminase was solubilized initially by lyophilization of borate treated mitochondria with a 40–60% recovery and with maintenance of threefold difference in specific activity. Both preparations showed the same equivalence point in a quantitative precipitin experiment. To confirm these results, phosphate-dependent glutaminase was also solubilized by treatment of mitochondria isolated from normal and acidotic rat kidney cortex with 1% Triton X-100. The two preparations exhibited a fivefold difference in specific activity and again showed the same equivalence point in a quantitative precipitin experiment. These results indicate that the cause of increased phosphate-dependent glutaminase activity during acidosis is due to the presence of an increased amount of this enzyme. The antiserum prepared against the kidney phosphate-dependent glutaminase did not crossreact with glutaminase solubilized from rat liver mitochondria. But, rat brain mitochondria do contain a phosphate-dependent glutaminase that is immunologically identical to the enzyme from rat kidney.     

31P NMR studies on kidney intracellular pH in acute renal acidosis

• 1.1. Phosphorus nuclear magnetic resonance (³¹P NMR) provides a non-destructive method for the quantitative observation of the most abundant phosphate-containing metabolites in isolated perfused kidney and in kidneys inside an anaesthetised animal.
• 2.2. In this paper, the NMR method is introduced and the intracellular pH changes following acute renal acidosis are measured (from the resonance position of P_i) for the isolated perfused rat kidney.
• 3.3. The results are discussed in relation to the biochemical data obtained by freeze extraction.

     

Characteristics of kinetics of the enzymatic oxoglutarate dehydrogenase reaction in a system with 2,6-dichlorophenolindophenol

The 2-oxoglutarate: 2,6-dichlorophenolindophenol (DCPIP)--oxidoreductase reaction catalyzed by the oxoglutarate dehydrogenase complex from bovine adrenal glands corresponds to the kinetic mechanism of a "ping-pong" type. There are signs of positive cooperativity of the oxoglutarate dehydrogenase interaction with the substrate and negative cooperativity of that with the electron acceptor. The half-maximal rate of the model reaction is provided by 0.01 mM concentrations of 2-oxoglutarate and DCPIP. The exceeding of the DCPIP optimum concentration (0.1 mM) results in the enzyme inhibition.     

Distinct effects of glucagon and vasopressin on proline metabolism in isolated hepatocytes. The role of oxoglutarate dehydrogenase.

The hormonal regulation of gluconeogenesis and ureogenesis in isolated rat hepatocytes with 5 mM-proline as precursor was studied, with the following results. (1) The formation of glucose and urea in a 30 min interval were stimulated more by vasopressin than by glucagon, and the effects of the two hormones in combination were additive. (2) The rates of gluconeogenesis during the 30 min were constant under control, glucagon-stimulated and glucagon-plus-vasopressin-stimulated conditions. The stimulated rate in the presence of vasopressin diminished with time; glucagon in combination with vasopressin prevented this diminution, resulting in an additive effect. (3) Coincident with these changes in gluconeogenesis, vasopressin caused a decrease in cell oxoglutarate concentration, which, in contrast with the decrease caused by glucagon, was greater, but not sustained unless glucagon was also present. Changes in cell glutamate concentration similar to those observed for oxoglutarate occurred. (4) The data suggest that activation of oxoglutarate dehydrogenase (EC 1.2.4.2) by glucagon and vasopressin by different mechanisms may explain the relative effects of the hormones alone and in combination on gluconeogenesis from proline.     

Sympathetic secretory response to hypercapnic acidosis in swine

We studied the effect of graded acute hypercapnic acidosis (HA) on sympathetic neural activation in 15 juvenile farm swine in vivo. In seven animals with acute HA, plasma norepinephrine (NE) concentration increased progressively from 189 +/- 34 to 483 +/- 80 pg/ml (P less than 0.04) as arterial CO2 partial pressure (PaCO2) increased in steps from 40 to 80 Torr (pH 7.17 +/- 0.01). Plasma epinephrine (EPI) concentration increased from 30 +/- 15 to 125 +/- 66 pg/ml (P = NS) over the same change in PaCO2. At PaCO2 of 110 Torr, plasma NE increased 3.4-fold above maximal basal concentrations; plasma EPI was 1.8-fold greater than basal under the same conditions. With HA, systemic vascular resistance (SVR) decreased from 1,748 +/- 110 to 1,392 +/- 145 dyn.s.cm-5 (P less than 0.0002), cardiac output (CO) increased from 3.4 +/- 0.3 to 4.3 +/- 0.3 l/min (P less than 0.01), and heart rate (HR) increased from 117 +/- 11 to 154 +/- 17 beats/min (P less than 0.03). To demonstrate that catecholamine secretion was related directly to acidosis caused by an increase in PaCO2, HCO3- was infused in eight other swine to buffer extracellular acute HA (pH 7.37 +/- 0.01 at PaCO2 of 80 Torr). Buffering attenuated the increase in plasma NE, which remained within the normal range at PaCO2 of 80 Torr. The decrease in SVR and increases in CO and HR also were also attenuated by HCO3- buffering of HA. We demonstrate the effects of graded acute HA on endogenous secretion of catecholamine and on the associated hemodynamic responses in swine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses of trout gill ion transport systems to acute acidosis.

The response of rainbow trout Na+ and Cl- uptake systems to acute acidosis was tested by slow infusion of lactic acid into anaesthetized animals. Depression of blood pH by 0-4 pH unit had no effect on influx rates for either ion, and we conclude that gill ion uptake systems do not respond rapidly to blood pH changes.     

Beef liver L-glutamate dehydrogenase mechanism: Presteady state study of the catalytic reduction of 2.oxoglutarate by NADPH

The mechanism of the reduction of 2.oxoglutarate by NADPH and addition of NH₄⁺ catalyzed by L-glutamate dehydrogenase was investigated with the stopped flow technique, following dihydronicotinamide absorbance and fluorescence changes. We make conspicuous, as in the reverse reaction, the existence of prestationnary events, the characteristics of which strongly depend on the ligand addition order. If the E-NADPH-2.oxoglutarate complex is preformed, saturated with regard to each ligand, there is a burst associated with the oxidation of 0.7 mole of NADPH per mole of protomer, a first order process with a velocity constant of 300 ± 50 sec^?1. If this complex is not preformed, the burst has reduced amplitude and velocity constants, differences that suggest an isomerization step following the binding processes. Whichever the incubation order, the later evolution follows an exponential decrease towards equilibrium with a velocity constant depending on the ligand concentration up to an extrapolated value of 27 sec^?1, a step corresponding to the dissociation of the reaction products.     

Serum lactate dehydrogenase fails to predict response to treatment and survival in acute non-lymphocytic leukemia

Total serum lactate dehydrogenase (LDH) activity was measured in 514 adult patients with de novo acute non-lymphocytic leukemia (ANLL) prior to any treatment and was compared with several disease features, with response to induction treatment, and with relapse-free survival. LDH was higher in the M4 and M5 FAB cytological subtypes and was positively correlated with the white blood cell count (WBC). The proportion of remissions, of deaths during induction, and of failure, and the duration of relapse-free survival, were clearly unrelated to LDH activity, in the whole series as well as in different age groups (below 40 years, and 40 to 60 years) and in any FAB cytological subtype. Multivariate analysis showed that only WBC and sex (female better than male) were marginally related with relapse-free survival. These data provide conclusive evidence that LDH does not help in defining the prognosis of adult ANLL, either because enzyme activity fails to reflect the number and proliferation rate of leukemic cells efficiently, or because with current standard treatment these features are of borderline importance, in contrast with acute lymphocytic leukemia and malignant lymphomas.     

Regulation of the kidney xanthine dehydrogenase by glutaminase.

The steady-state concentrations of glutamine, glutamate and ammonia in the kidney cells might regulate the rate of renal xanthine dehydrogenase activity. Both glutamate and glutamine were found to be effective inhibitors of the renal xanthine dehydrogenase activity in vivo. The inhibition by glutamate depends essentially on the glutaminase inhibition.