Studies on lysine:N6-hydroxylation by cell-free systems of Aerobacter aerogenes 62-1.

Electron microscopic examination has revealed the vesicular nature of the membrane component, of the cell-free system of Aerobacter aerogenes 62-1, which catalyses lysine: N6-hydroxylation. Regardless of the orientation of the vesicles, N-hydroxylation process is still stimulated by pyruvate. Both pyruvate oxidation and lysine: N6-hydroxylation were inhibited by protonophores and Gramicidin S.

Purification and properties of Escherichia coli 4'-phosphopantothenoylcysteine decarboxylase: presence of covalently bound pyruvate.

4'-Phosphopantothenoylcysteine decarboxylase was purified 900-fold from Escherichia coli B with an overall yield of 6%. The enzyme migrates as a single band with a molecular weight of 35,000 +/- 3000 in 10% polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has an apparent molecular weight of 146,000 +/- 9000 as determined by a gel exclusion column. At pH 7.6 and 25 degrees C, Km = 0.9 mM and Vmax = 600 nmol/(min X mg of protein). The pH optimum for Vmax is between 7.5 and 7.7. Hydroxylamine, phenylhydrazine, potassium cyanide, and sodium borohydride as well as pyridoxal phosphate and pyridoxal inactivated the enzyme. The enzyme contains covalently bound pyruvate as suggested by the isolation of [3H]lactate and pyruvate from [3H]NaBH4-reduced enzyme and native enzyme, respectively. One mole of [3H]lactate was isolated per 39,000 g of [3H]NaBH4-reduced and completely inactivated enzyme, and 1 mol of pyruvate was isolated per 31,000 +/- 4000 g of native enzyme. Mild base treatment released lactate and pyruvate from the reduced and the native enzymes, respectively, suggesting the pyruvate is attached to the enzyme by an ester bond. These findings are in accord with similar results obtained with the horse liver enzyme (R. Scandurra, personal communication). The presence of covalently bound pyruvate in the bacterial and mammalian enzymes suggests that pyruvate plays a major role in the mechanism of action.

Secretion of pyruvate. An antioxidant defense of mammalian cells.

Cells in culture are exposed to marked oxidative stress, H2O2 being one of the predominant agents. Pyruvate and other alpha-ketoacids reacted rapidly, stoichiometrically, and nonenzymatically with H2O2, and they protected cells from its cytolytic effects. All five human and murine cell types studied, both malignant and nonmalignant, released pyruvate at an initial rate of 35-60 microM/h/2.5 X 10(6) cells when placed in 1 ml pyruvate-free medium. After 6-12 h a plateau of 60-150 microM pyruvate was attained, corresponding to concentrations reported for normal human serum and plasma. The rate of pyruvate accumulation was almost doubled in the presence of exogenous catalase, suggesting that released pyruvate functions as an antioxidant. The rate of pyruvate accumulation was dependent on cell number. Succinate, fumarate, citrate, oxaloacetate, alpha-ketoglutarate, and malate were not secreted in significant amounts from P815 cells; export was specific for pyruvate and lactate among the metabolites tested. Extracellular pyruvate was in equilibrium with intracellular stores. Thus, cells conditioned the extracellular medium with pyruvate at the expense of intracellular pyruvate, until homeostatic levels were attained in both compartments. We propose that cells plated at low density in the absence of exogenous pyruvate fail to thrive for two reasons: prolonged depletion of intracellular pyruvate and prolonged vulnerability to oxidant stress.

Cold adaptation in guinea pig at level of isolated brown adipocyte.

Isolated brown adipocytes were prepared from guinea pigs acclimated to 28 degrees C or exposed to 4-8 degrees C for periods of up to 3 wk. Cells from warm-adapted animals retained respiratory control when stimulated with norepinephrine. Cells from guinea pigs exposed to cold for 4-21 days showed a much greater respiratory response to norepinephrine due to enhanced uncoupling rather than enhanced substrate supply. After 7 days of cold acclimation, norepinephrine-stimulated respiration became uncontrolled and was limited only by the maximal respiratory capacity of the mitochondria. Three weeks of cold acclimation were accompanied by a doubling of total cell number, a doubling of the mitochondrial protein per adipocyte, and a sixfold increase in the norepinephrine-stimulated respiration per in situ mitochondrion with no change in respiratory chain capacity. The induction of norepinephrine-stimulated respiration correlated with the appearance of high-affinity purine nucleotide binding sites on the mitochondria, diagnostic of the uncoupling protein. If the results are extrapolated to the whole animal, they indicate that brown adipose tissue makes little contribution to thermogenesis in the warm-adapted guinea pig but may account for most or all the increment seen on cold adaptation.

Pyruvate inhibition of pyruvate dehydrogenase kinase is a physiological variable.

Pyruvate inhibited pyruvate dehydrogenase kinase activity in mitochondria from adipose tissue, heart, brain and kidney of fed rats. Starvation for 24 h led to increased kinase activity in mitochondria from adipose tissue and heart but not from brain or kidney and to reduction of pyruvate inhibition of the enzyme from adipose tissue, heart and brain. Insulin injection into starved animals rapidly restored pyruvate inhibition without alteration of kinase activity in adipose tissue and heart mitochondria. Induction of streptozotocin diabetes resulted in loss of pyruvate inhibition of the kinase in heart mitochondria at 48 h but not at 24 h whereas a significant increase of kinase activity was seen at 24 h. It is concluded that the mechanisms which control fluctuations of pyruvate sensitivity of the kinase are different from the mechanisms which control fluctuations of the uninhibited kinase activity.

Pyruvate participation in the low molecular weight trophic activity for central nervous system neurons in glia-conditioned media.

Conditioned media from glial cell cultures contain low molecular weight agents which can support survival of CNS neurons in the absence of recognized protein neuronotrophic factors. A similar support is provided to CNS neurons by selected basal media, and pyruvate is the critical medium constituent responsible for their trophic competence. Eagle's basal medium, which contains no pyruvate, acquires pyruvate when conditioned over astroglial cell cultures. Enzymatic degradation of the pyruvate in the astroglia-conditioned medium leads to corresponding losses in its low molecular weight trophic activity for CNS neurons. Quantitative correlations between pyruvate content and CNS trophic activity demonstrate that pyruvate is the main trophic ingredient of the glia-conditioned medium, and other low molecular weight substances, acquired during conditioning, reduce the pyruvate concentration required for its trophic effect. The "pyruvate-sparing" substances, as yet unidentified, are not the serine and Fe3+ which have pyruvate-sparing competence for peripheral, ciliary ganglionic neurons. These findings, together with previous observations, propose that prenatal neurons fail to generate or retain endogenous pyruvate at the levels for their survival-sustaining activities.

The stimulation of hepatic gluconeogenesis by acetoacetate precursors. A role for the monocarboxylate translocator.

The regulation of the gluconeogenic pathway from the 3-carbon precursors pyruvate, lactate, and alanine was investigated in the isolated perfused rat liver. Using pyruvate (less than 1 mM), lactate, or alanine as the gluconeogenic precursor, infusion of the acetoacetate precursors oleate, acetate, or beta-hydroxybutyrate stimulated the rate of glucose production and, in the case of pyruvate (less than 1 mM), the rate of pyruvate decarboxylation. alpha-Cyanocinnamate, an inhibitor of the monocarboxylate transporter, prevented the stimulation of pyruvate decarboxylation and glucose production due to acetate infusion. With lactate as the gluconeogenic precursor, acetate infusion in the presence of L-carnitine stimulated the rate of gluconeogenesis (100%) and ketogenesis (60%) without altering the tissue acetyl-CoA level usually considered a requisite for the stimulation of gluconeogenesis by fatty acids. Hence, our studies suggest that gluconeogenesis from pyruvate or other substrates which are converted to pyruvate prior to glucose synthesis may be limited or controlled by the rate of entry of pyruvate into the mitochondrial compartment on the monocarboxylate translocator.

Studies on the formation of N6-hydroxylysine in cell-free extracts of Aerobacter aerogenes 62-1.

We have investigated conditions optimal for the conversion of L-lysine to its N6-hydroxy derivative by partially purified cell-free extracts of Aerobacter aerogenes 62-1. The enzyme system was highly specific to L-lysine: the D-isomer and, the N2- or N6-derivatives of lysine, and alpha-amino acids were not hydroxylated. Most of the latter compounds had little effect onthe hydroxylation of L-lysine. However, -l-glutamic acid and L-glutamine enhanced the hydroxylation, with half-maximal activation achieved at 100 micrometers concentration of the effector. The Km values for pyruvate and L-(+)-lactate (compounds known to stimulate N-hydroxylysine formation) were found to be approx. 100 micrometers. The data show that N-hydroxylation of the amino acid precedes acylation in the biosynthesis of hydroxamic acid in A. aerogenes 62-1.