Redox state of free nicotinamide-adenine nucleotides in the cytoplasm and mitochondria of alveolar macrophages.

Cytoplasmic free NAD(+)/NADH ratios have been calculated from lactate to pyruvate ratios, and mitochondrial NAD(+)/NADH ratios, have been calculated from beta-hydroxybutyrate to acetoacetate ratios in isolated rabbit alveolar macrophages. In freshly harvested cells, assuming a pH of 7 for the two compartments, cytoplasmic NAD(+)/NADH averaged 709 +/-293 (SD), and mitochondrial NAD(+)/NADH averaged 33.2 +/-30.2, values which are significantly different. 30 min of air incubation in a relatively poorly buffered medium showed a significant reduction in calculated mitochondrial NAD(+)/NADH to 10.1 +/-4.8. 30 min of exposure of cells to a hypoxic environment (equivalent to a nonventilated, perfused alveolus) caused significant reductions of NAD(+)/NADH in both compartments. Re-exposure of hypoxic cells to air produced a change toward normal in cytoplasmic NAD(+)/NADH but did not reverse mitochondrial abnormality. Uncertainties concerning the value of cytoplasmic and mitochondrial pH under control conditions and during experimental pertubations, limit absolute interpretation of NAD(+)/NADH ratios calculated from redox pairs, but the data suggest the following: (a) separate cytoplasmic and mitochondrial compartments for NAD(+) and NADH exist in the alveolar macrophage; (b) brief periods of exposure to moderate hypoxia of the degree seen in clinical lung disease produce decreases in both cytoplasmic and mitochondrial NAD(+)/NADH; (c) the mitochondrial changes are less easily reversed than the cytoplasmic changes; (d) measurements of NAD(+)/NADH provide an early sensitive indication of biochemical abnormality; and (e) careful control of extracellular pH is required in studies involving experimental modifications of alveolar macrophage function.

Energy metabolism in human erythrocytes. I. Effects of sodium fluoride.

Exposure of red cells to fluoride produces a variety of metabolic alterations, most of which are based upon the secondary effects of enolase inhibition, which reduces pyruvate synthesis and interferes with the regeneration of diphosphopyridine nucleotide (NAD). Adenosine triphosphate (ATP) is consumed in the hexokinase and phosphofructokinase reactions but is not regenerated since the deficiency of NAD limits glyceraldehyde phosphate dehydrogenase. ATP depletion in the presence of fluoride and calcium induces a massive loss of cations and water. Of the other known sites of ATP utilization, membrane-bound ATPase is inhibited by fluoride, but the incorporation of fatty acids into membrane phospholipids is unaffected until ATP is depleted. The addition of methylene blue to fluoride-treated red cells regenerates NAD, permitting triose oxidation and the generation of 3-phosphoglycerate and 2,3-diphosphoglycerate. Enolase inhibition is then partially overcome by mass action, and sufficient glycolysis proceeds to maintain the concentration of ATP. This in turn prevents the massive cation and water loss, and permits membrane phospholipid renewal to proceed. Membrane ATPase activity is not restored by the oxidant so that normal cation leakage remains unopposed by cation pumping in red cells exposed to the combination of fluoride and methylene blue.

Inhibition of mixed-function oxidation in perfused rat liver by fluoroacetate treatment.

The effect of fluoroacetate, an inhibitor of the citric acid cycle, on the mixed-function oxidation of p-nitroanisole in isolated perfused livers from fed rats was studied. The citric acid cycle was inhibited by injection of 5 mg/kg sodium fluoroacetate into rats 3 hr prior to liver perfusion experiments. Inhibition of the citric acid cycle was marked by accumulation of citrate (5-fold) and decreases in rates of glycolysis and glycogenolysis by 50-90%. Fluoroacetate treatment inhibited mixed function oxidation in the perfused liver by about 50% without affecting p-nitroanisole O-demethylation by isolated microsomes. Fluorocitrate, at concentrations up to 50 microM, did not inhibit microsomal p-nitroanisole O-demethylation in vitro. These data support the hypothesis that mixed-function oxidation in intact hepatocytes is dependent upon reducing equivalents generated via the citric acid cycle.

Metabolic integrity of the isolated perfused lobule of human placenta.

Tissue levels of lactate, pyruvate and the adenine nucleotides were measured in samples of human placenta obtained (1) immediately on delivery, (2) after perfusion on the maternal side for one hour, and (3) after a corresponding period of warm ischaemia. Metabolic activity in the isolated perfused placental lobule was assessed in terms of these tissue metabolites and by measuring protein synthetic rate by means of determining the incorporation of a labelled amino acid. Perfusion was found to lower significantly the lactate level whereas ATP was maintained at comparable levels with those in the placenta just after delivery. Perfused placental ATP levels are lower than in vivo levels seen in other tissues with high metabolic rates such as rat liver, kidney and also in guinea-pig placenta. Protein synthetic rate was found to be lower than that observed in other fetal tissues.

Mechanical and metabolic viability of a placental perfusion system in vitro under oxygenated and anoxic conditions.

In vitro dual circuit perfusion of the placenta with well-oxygenated medium results in the continuous and stable consumption of oxygen and glucose over a 2-h perfusion period. This is reflected in a stable production of lactate and an energy charge which is higher at the end of the perfusion period than that seen in fresh placental tissue immediately after vaginal delivery. Anoxic perfusion causes an increase in glucose consumption which is more than twofold higher than that seen in the oxygenated perfusion, resulting finally in placental uptake of glucose not only from the maternal but also from the fetal circulation. Lactate production is increased during the anoxic perfusion, while the final tissue energy charge value lies between the values observed for fresh tissue and for the oxygenated perfusion. The shift to anaerobic metabolism shown by placental tissue in anoxic conditions enables continued functioning of the tissue over the 2-h perfusion period but it appears that under anoxic conditions the tissue may incur an energy debt not observed in oxygenated perfusions.

Comparative glycolytic metabolism of sperm from normal, asthenospermic, and oligoasthenospermic men.

The glycolysis of spermatozoa from normal, asthenospermic, and oligoasthen ospermic men was studied using a respirometry technique to measure glucose utilization by the production of 14CO2 from glucose 14C (U-L). Lactate and pyruvate were measured by a spectrophotometric method using DNA as reference. Human spermatozoa preferred glucose to fructose as the glycolytic substrate when concentrations of these hexoses did not exceed normal concentrations in the blood. Spermatozoa from oligoasthenospermic men produced an average of 3.5 times more 14CO2 (345, 457 dpm/mg DNA/hour) than did spermatozoa from asthenospermic (88,837 dpm/mg DNA/hour) and normal men (96,595 dpm/mg DNA/hour). They also formed four times more lactate (9.63 mumoles/mg DNA/hour) than spermatozoa from normal men (2.33 mumoles/mg DNA/hour) and 6.4 times more pyruvate (2.90 mumoles/mg DNA/hour compared to 0.45 mumoles/mg DNA/hour). Spermatozoa from asthenospermic men formed amounts of lactate (3.01 mumoles/mg DNA/hour) and pyruvate (0.63 mumoles/mg DNA/hour) similar to those produced by spermatozoa from normal men.

Bacterial cysteine conjugate beta-lyase and the metabolism of cysteine S-conjugates: structural requirements for the cleavage of S-conjugates and the formation of reactive intermediates.

The cysteine conjugate beta-lyase mediated metabolism and the mutagenicity of the synthetic cysteine conjugates S-(2-chloroethyl)-L-cysteine (CEC), S-(2-chlorovinyl)-L-cysteine (CVC), S-(1,2,3,3,3-pentachloroprop-1-enyl)-L-cysteine (PCPC), S-(pentachlorophenyl)-L-cysteine (PCPhC), S-(chloro-1,2,2-trifluoroethyl)-L-cysteine (CTFEC), S-benzyl-L-cysteine (SBC) and S-methyl-L-cysteine (SMC) were investigated in Salmonella typhimurium strains TA100, TA2638, TA102 and TA98 to establish structure/activity relationships. Bacterial 100,000 X g supernatants cleaved CTFEC, PCPC, CVC, PCPhC and SBC to pyruvate; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA) in all cases. Of the compounds tested, CEC, PCPC and CVC were mutagenic in the Ames-test. CTFEC, PCPhC and SBC failed to increase the number of revertants above control levels. The mutagenicity of PCPC and CVC could be inhibited by AOAA. CEC exerted a potent mutagenic effect in the Ames-test which was not affected by AOAA; CEC was not transformed to pyruvate by bacterial beta-lyase. Neither pyruvate formation nor mutagenicity were observed with SMC. These results indicate that the structure of the substituent on the sulfur atom is an important determinant for the biological activity of cysteine S-conjugates. Electronegative and/or unsaturated substituents are required for beta-lyase catalysed beta-elimination reactions. The formation of chemically unstable thiols, which may be converted to thioacylating intermediates, seems to be a prerequisite for beta-lyase dependent mutagenicity of S-conjugates.

The effects of 1,3-dinitrobenzene and mono-(2-ethylhexyl)phthalate on hormonally stimulated lactate and pyruvate production by rat Sertoli cell cultures.

Lactate and pyruvate production by rat Sertoli cell cultures was measured after treatment with two toxicants (1,3-dinitrobenzene, DNB; mono-(2-ethylhexyl)phthalate, MEHP) in the presence or absence of follicle-stimulating hormone (FSH) or dibutyryl-cAMP (dbcAMP). 1,3-DNB together with FSH or dbcAMP produced increases in media lactate and pyruvate concentrations significantly greater than those induced by individual treatments. MEHP in conjunction with either hormone also produced greater increases in lactate concentrations compared with separate additions. However, MEHP produced a significant diminution in the hormonal stimulation of pyruvate secretion. Thus, 1,3-DNB appears to act independently of these hormones in stimulating the secretion of both metabolic products, whereas MEHP interferes with the hormonal stimulation of pyruvate (but not lactate) production.

Regulation of biosynthesis of secondary metabolites. XVII. Purification and properties of malate dehydrogenase (decarboxylating) in Streptomyces aureofaciens.

The process of isolation and purification of malate dehydrogenase (decarboxylating) (EC 1.1.1.40) from the mycelium of the actinomycete Streptomyces aureofaciens has been worked out. The enzyme was purified 35 fold. The kinetic characters of the purified enzyme are very similar to the figures for malate dehydrogenase (decarboxylating) from other sources. Km for L-malate = 2.1 X 10(-3)M, Km for NADP = 4.6 X 10(-5)M (at pH 7.4). The reaction requires metal divalent ions, Mn2+ being more effective than Mg2+. The enzyme reaches its maximal activity at pH 8.75.

Physiological characteristics of chemostatically grown Citrobacter freundii as a function of the specific growth rate and type of nutrient limitation.

Citrobacter freundii was grown aerobically in a chemostat on a mineral medium with galactose or glucose as carbon and energy sources under limitation by carbon or nitrogen source respectively. At various specific growth rates ranging from 7 to 95% mumax the culture in steady state was analysed and growth yield, specific metabolic rate of substrate utilization, intracellular concentration of pyruvate, ATP, ADP, AMP and energy charge were determined and plotted as functions of dilution rate. In all four types of experiments the physiological state of cells remained practically independent of dilution rate up to D=0.6 mumax, and at a given specific growth rate nearly independent on mumax and type of limitation. At approximately D=0.6 mumax, which is close to the maximum output dilution rate Dm, the physiological state of the cells changed: growth yields decreased and intr cellular pyruvate and adenylates concentrations increased. Consequently, in a given medium two dilution rates exist at which growth rate dx/dt is the same but the physiology of the population is quite different.

Effect of sodium arsenite on the biosynthesis of mitomycins by Streptomyces caespitosus and mode of action of mitomycin C on Bacillus subtilis NRRL B-543.

Addition of different concentrations of sodium arsenite to the fermentation medium used for the production of mitomycin antibiotics by Streptomyces caespitosus hindered the biosynthesis of mitomycins and led to the accumulation of 2-oxoglutarate, pyruvate and acetone. Mitomycin C isolated and purified using thin-layer chromatography in low concentration of about 0.1 mug/ml did not affect the RNA, DNA and protein biosynthesis of the growing Bacillus subtilis, while at 10 mug/ml mitomycin C markedly affected RNA, DNA and protein biosynthesis.

[Dynamics of biologically active enterotoxin release by E. coli]. / Dinamika vydeleniia biologicheski aktivnogo énterotoksina E. coli.

Accumulation of E. coli enterotoxin in the Finkelshtein's culture medium in growing the cells in a 30-litre reactor was studied. Accumulation of active highly molecular enterotoxin occurred in the course of a 6-hour cultivation of E. coli, strain P-99 (O141: K85ab; K88ab: H4) in the fluid medium under aeration. Oxygen utilization, synthesis and release into the nutrient medium of pyruvic acid, and protein accumulation were observed. The preparation obtained was stable to the lyophylic drying, contained thermolabile and thermostable toxins and marked edema of mouse limbs. The data obtained were of significance for the industrial production of active enterotoxin preparations.

[Splitting of fructose diphosphate by muscle and tumor homogenates]. / Rasshcheplenie fruktozodifosfata gomogenatami myshts i opukholei.

Effect of MgCl2 and ATP on the elevation in lactate, pyruvate and methylglyoxale content was studied on incubation of fructosediphosphate with muscle and tumor homogenates. Muscle homogenate accumulated lactate, pyruvate and methyglyoxale more intensively as compared with tumor homogenate. Pyruvate was formed only in presence of MgCl2 and ATP, although it did not practically affect the elevation of lactate in muscle homogenate. The data obtained suggest that tumor homogenate does not contain the enzymatic system involved in methylglyoxale formation.