The significance of the attachment of rat kidney glutaminase to the inner mitochondrial membrane.

The inner mitochondrial membrane of rat kidney mitochondria was altered by 0.03% Triton X-100 treatment in such a way as to render it permeable to NAD and CoA molecules without release of phosphate-dependent glutaminase. A break of linearity in the Arrhenius plot of the enzyme activity was characteristic for a conformational change of a membrane-bound enzyme. The activity of phosphate-dependent glutaminase immobilized in the inner mitochondrial membrane, as studied in 0.03% Triton X-100-treated mitochondria, and solubilized, as in the supernatant of sonicated mitochondria, was hyperbolic with respect to glutamine concentration. Under optimal conditions (pH 8.6 and 100 mM phosphate) the Vmax and Km were 216 +/- 12 nmol/mg per min and 2.7 +/- 0.4 mM, respectively, for Triton X-100-treated mitochondria, and 121 +/- 8 nmol/mg per min and 15.9 +/- 1.8 mM for sonicated mitochondria. Under near physiological conditions (pH 7.8 and 20 mM phosphate), distinct differences in phosphate-dependent glutaminase kinetics were observed. The Vmax as 29.8 +/- 0.4 and 2.6 /- 0.3 nmol/mg per min and the apparent Km 1.55 +/- 0.06 and 24.5 +/- 6.6 mM for Triton X-100 and sonicated mitochondria, respectively. The sigmoidal activation by phosphate at pH 7.8 was significantly shifted to the left in Triton X-100-treated as compared to sonicated mitochondria. As opposed to the data obtained in sonicated mitochondria, the kinetics of phosphate-dependent glutaminase in 0.03% Triton X-100-treated mitochondria agreed quite well with those obtained in intact, rotenone-inhibited and metabolically active mitochondria. These results suggest that an attachment of phosphate-dependent glutaminase to the inner membrane of kidney mitochondria has a profound effect on its kinetics, particularly under near physiological conditions.

Glutamate metabolism in rat kidney mitochondria.

1. Kidney cortex mitochondria did not swell in ammonium or potassium salts of glutamate even in the presence of valinomycin or 2,4-dinitrophenol. Aminooxyacetate diminished the reduction of nicotinamide nucleotides in intact mitochondria in the presence of glutamate. 2. Transamination with oxaloacetate appeared to be the main pathway of glutamate metabolism in isolated rat kidney cortex mitochondria under various metabolic conditions. Ammonia formation was negligible. The gamma-aminobutyrate pathway was found to be of almost no importance.

Effect of extracellular pH and inhibitors on the gluconeogenesis and ammoniagenesis relationship in rat kidney cortex slices.

1. Gluconeogenesis from glutamine, fumarate, pyruvate, glutamine plus fumarate, and glutamine plus pyruvate, was generally higher at pH 7.1 than at pH 7.4 and 7.7, whereas ammoniagenesis did not depend on the pH of the medium. 2. The intermediates of the Krebs cycle decreased ammonia formation from glutamine, raising at the same time gluconeogenesis. 3. Arsenite, malonate, maleate, hydrazine and 2,4-dinitrophenol inhibited gluconeogenesis, and enhanced simultaneously ammonia formation irrespective of the pH of the medium.

Metabolism of glutamine in rat kidney mitochondria in the presence of aminooxyacetate.

1. Aminooxyacetate has no effect on respiration of rat kidney cortex mitochondria in the presence of glutamine but it inhibits respiration in the presence of glutamate to the values of endogenous respiration. 2. Rat kidney mitochondria in state 3 produce aspartate from glutamine in the amount corresponding to almost 50% of the glutamate formed intramitochondrially. 3. Aminooxyacetate affects neither formation of ammonia nor of aspartate from glutamine but it inhibits aspartate synthesis in the mitochondria treated with Triton X-100.

Prenatal lead exposure and the pregnancy outcome. A case-control study in southern Poland.

In an attempt to test null hypothesis (Ho): that prenatal lead exposure does not increase the risk of prematurity and the delivery of SGA infants, a case-control study was performed in four hospitals of Southern Poland (Kraków, Rabka, Limanowa, Zakopane). Lead content was determined in maternal and cord blood as well as in head and pubic hair by the GF AAS (Perkin Elmer). A significant interregional variation of lead content in maternal blood was observed. Lead concentration in maternal and cord blood was significantly higher in the group of mothers of SGA newborns when compared to the controls. This was not the case with respect to the mothers of preterm infants. Also, the comparison of lead concentration in head and pubic hair revealed no statistically significant case-control differences. For a combined population of cases and controls, a significant gradient of lead concentration between maternal and cord blood was demonstrated. The correlation between lead content in different body compartments was observed. Conclusions. Different blood lead levels observed in mothers from four hospitals suggest different exposure. Higher lead concentration in maternal blood was associated with an increased risk of the delivery of SGA infant.

The uptake of oxalate by rat liver and kidney mitochondria.

Oxalate, a metabolic end product, forms calcium oxalate deposits in the tissues under a variety of pathological conditions. In order to determine whether oxalate is able to penetrate the mitochondrial matrix, the uptake of oxalate by rat liver and kidney cortical mitochondria was characterized. Mitochondria did not swell in an iso-osmotic medium of ammonium oxalate unless a small amount of phosphate was provided. This phosphate-induced swelling was prevented by N-ethylmaleimide. The uptake of [14C]oxalate by liver and kidney mitochondria followed first order kinetics and was inhibited by mersalyl an inhibitor of the phosphate and dicarboxylate carriers. Accumulation of [14C]oxalate at equilibrium was significantly higher by mitochondria energized with succinate than by rotenone-inhibited mitochondria due to higher matrix pH as determined by the [14C]5,5'-dimethyloxazolidine-2, 4-dione distribution ratio. The velocity of oxalate accumulation by mitochondria was temperature dependent. The activation energy was 81.5 and 86.5 J/mol for liver and kidney mitochondria, respectively. In both types of mitochondria, the rate of oxalate uptake was hyperbolic with respect to the concentration of oxalate. The apparent Km was 28.8 +/- 0.6 and 13.4 +/- 1.2 mM and the Vmax 87.1 +/- 1.1 and 66.1 +/- 3.1 nmol X mg-1 X min-1 at 12 degrees C for liver and kidney mitochondria, respectively. Phenylsuccinate exhibited mixed inhibition of the rate of oxalate uptake. Oxalate exhibited also a mixed inhibition of the uptake and oxidation of malate by mitochondria. The data obtained provide evidence that oxalate is transported across the mitochondrial membrane by a phosphate-linked, carrier-mediated system similar to or identical to the dicarboxylate transporter.

Effects of maleate on the content of CoA and its derivatives in rat kidney mitochondria.

Effects of maleate on the content of CoA derivatives in isolated mitochondria and in the tissues of maleate-intoxicated rats have been studied. The addition of maleate to kidney mitochondria incubated with 2-oxo-glutarate decreased CoA-SH and acid-soluble acyl-CoA concentrations while acid insoluble acyl-CoA content remained unchanged. As a result, a substantial loss (depletion) of the total CoA occurred. Similar changes in CoA content were found in vivo in the kidneys of maleate-treated rats. Neither in the isolated liver mitochondria nor in the liver of intoxicated animals have such changes been observed before. Acetoacetate, the substrate for CoA transferase, added to kidney mitochondria before maleate, abolished its inhibitory effect on oxidation of 2-oxoglutarate and prevented the decrease of CoA content. The data are in accord with the previous findings indicating that maleate can bind and sequester CoA in the form of a stable and metabolically inert compound.

The purine nucleotide cycle and ammonia formation from glutamine by rat kidney slices.

To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant.

Regulation of rat kidney mitochondrial metabolism in acute acidosis.

Proximal tubular ammoniagenesis is amplified under conditions of acute and chronic metabolic acidosis. Current hypotheses postulate that alterations in intracellular pH (pHi) or in the pH gradient across the inner mitochondrial membrane (delta pHm) influence mitochondrial glutamine metabolism. Enhanced glutamine transport across the inner mitochondrial membrane might constitute a key regulatory factor in acidosis. To examine changes in delta pHm, a technique was used to determine pHi and intramitochondrial pH (pHm) simultaneously. Regulation of the enzyme alpha ketoglutarate dehydrogenase (alpha KGDH) was assessed by evaluating enzyme activity at varied levels of medium pH, Ca++, and adenosine diphosphate (ADP). The results indicate that pHi decreased with an acid external pH. A fall in pHi correlated to increase activity of alpha KGDH associated with increased affinity for the substrate, alpha KG. Increments in either buffer Ca++ or ADP concentration increased enzyme affinity for alpha KG at pH 7.6 but not at pH 6.8. These results, compatible with previous reports, indicate that pH, Ca++, and ADP are effectors of the enzyme alpha KGDH. Alterations in pH across the inner mitochondrial membrane might augment flux through alpha KG by accelerating glutamine metabolism. Increased alpha KG oxidation over the range of 10 to 500 mumol/L Ca++ concentration is compatable with data for Ca++ regulation reported for the solubilized enzyme. These studies provide evidence that the above factors, through enhancing alpha KGDH activity, participate in regulation of ammoniagenesis during states of acidosis.