R. E. Basford

Glutathione-dependent peroxidative metabolism in the alveolar macrophage

Phagocytosis by rabbit alveolar macrophages (AM) is accompanied by increases in O(2) consumption, glucose oxidation, and H(2)O(2) formation. Two aspects of the interrelations between these metabolic features of phagocytosis have been studied.First, the following evidence indicates that glutathione, glutathione reductase, and peroxidase serve as a cytoplasmic shuttle between H(2)O(2) and NADPH-dependent glucose oxidation: (a) AM contain 5.9 mmumoles of reduced glutathione per 10(6) cells and exhibit glutathione peroxidase and NADPH-specific glutathione reductase activity; (b) oxidized glutathione potentiates NADP stimulation of glucose oxidation; (c) an artificial H(2)O(2)-generating system stimulates glucose oxidation; (d) the cell penetrating thiol inhibitor, N-ethylmaleimide diminishes glucose oxidation. This effect largely depends on inhibition of the glutathione system rather than on inhibition of either H(2)O(2) formation or enzymes directly subserving glucose oxidation.Second, three potential H(2)O(2)-generating oxidases have been sought. No cyanide-insensitive NADH or NADPH oxidase activity could be detected. D-amino acid oxidase activity was 0.48 +/-0.07 U/10(6) cells with D-alanine as substrate.

BRAIN MITOCHONDRIA‐VI THE COMPOSITION OF BOVINE BRAIN MITOCHONDRIA 1

MITOCHONDRIA isolated from bovine brain by the method of STAHL, SMITH, NAPOLITANO and BASFORD (1963) were shown to be relatively free of contamination by non-mitochondria1 particulate material. The mitochondria1 preparation was shown to carry out citric acid cycle oxidations with concomitant oxidative phosphorylation and respiratory control comparable to mitochondria1 preparations from other tissues. Electron micrographs indicated that mitochondrial structure was also comparable to that of mitochondrial preparations from other tissues (STAHL et al., 1963). At least two significant differences in the enzymic capacity of brain mitochondria compared with mitochondria of other tissues have been demonstrated. SACKTOR, PACKER and ESTABROOK (1959) and STAHL et al. (1963) showed that the rate of cc-glycerophosphate oxidation is higher in brain mitochondria than in mitochondria isolated from heart or liver. BEATTIE, SLOAN and BASFORD (1963) showed that bovine brain mitochondria prepared by the method of STAHL et al. (1963) also contain the major portion of the two ATP-requiring enzymes of glycolysis : hexokinase and phosphofructokinase. As a further extension of the comparison of mitochondria of brain with those of other tissues, it was the purpose of the present work to investigate some aspects of the chemical composition of these mitochondria with particular emphasis on electron carriers. EXPERIMENTAL Materials. The following materials were obtained commercially: AMP, ADP, ATP, NAD+, NADH, FMN, FAD, ethylenediaminetetra-acetic acid (EDTA), tris(hydroxymethy1)aminomethane(tris), a-glycerophosphate, desiccated firefly tails, lactate dehydrogenase, Type I, and adenylate kinase from Sigma Chemical Company, St. Louis, Missouri; diphenylthiocarbazone(dithizone) from Fisher Chemical Company, Pittsburgh, Pennsylvania; malate, phosphoenolpyruvate, and pyruvate kinase from California Foundation for Biochemical Research, Los Angeles, California; succinate from Matheson, Coleman, and Bell, Cincinnati, Ohio; Difco Trypsin (1 :250) from Difco Laboratories, Detroit, Michigan; silicic acid (100 mesh, powder) from Mallinckrodt Chemical Works, St. Louis, Missouri; Silica gel G (for thin-layer chromatography) from Brinkmann Instruments, Westbury, Long Island, New York; sodium borohydride from Callery Chemical Company, Pittsburgh, Pennsylvania; Dowex A-1 (50-100 mesh) from J. T. Baker Chemical Company, Phillipsburg, New Jersey; p-(I-octy1)-phenoxy polyethoxy ethanol (Triton X-100) from Ruger Chemical Company, Irvington-On-Hudson, New York; Carbowax from Union Carbide Chemicals Company, Charleston,

The effect of bilirubin on the energy metabolism of brain mitochondria.

Abstract— Unconjugated bilirubin caused uncoupling of oxidative phosphorylation in brain mitochondria prepared from mature or weanling rats. Human serum albumin in an amount sufficient to bind 100 per cent of the added bilirubin was able to protect the mitochondria from the inhibitory effects of bilirubin. Bilirubin inhibited both the endogenous and DNP‐activated ATPase activities but had no effect upon the Mg2+‐stimulated ATPase; albumin prevented the inhibition.

SODIUM‐STIMULATED ADENOSINE TRIPHOSPHATASE ACTIVITY OF RAT BRAIN MITOCHONDRIA

Abstract The endogenous ATPase activity of rat brain mitochondria was stimulated 30‐50 per cent by 15‐50 mm concentrations of NaCl or Na acetate. The Na stimulation was completely abolished by small amounts of oligomycin but unaffected by ouabain. The differential effects of these inhibitors indicated that the Na‐induced ATPase activity did not result from microsomal or synaptosomal contamination of mitochrondria. Sodium salts decreased the stimulatory effects of DNP, gramicidin, or Ca, but not that of Mg on the endogenous ATPase activity. These interactions were specific for Na+ as the corresponding salts of K+ did not affect the endogenous ATPase or inhibit the DNP‐stimulated ATPase activity except at high concentrations. The Na‐induced increases in ATPase activity and respiration were more sensitive to aging of the mitochondria than were ADP/O and respiratory control ratios, or the DNP‐induced ATPase activity. These results suggest that Na+ may interact in brain mitochondria with the same high‐energy intermediate of oxidative phosphorylation proposed for DNP and Ca.

Intractable Unphysiologically Low Adenylate Energy Charge Values in Synaptosome Fractions: An Explanatory Hypothesis Based on the Fraction's Heterogeneity

Abstract: Synaptosomes prepared and incubated in a variety of ways from rat cerebra exhibited intractable, unphysiologically low adenylate energy charge values (∼0.37–0.60), low total adenine nucleotide contents (∼8–10 nmol/mg protein), and much higher adenylate kinase apparent Kcq values (∼3–8) as compared to intact brain tissue (values of ∼0.90, 25 nmol/mg, and 0.74. respectively). Synaptosomes prepared from mouse, dog, and chicken cerebra had values essentially identical to those from rat. When incubated under oxygen in a physiological salt solution containing glucose, synaptosomes metabolized more glucose to lactic acid than to CO2, and the addition of 100 μM veratridine caused a two‐ to threefold stimulation of O2 uptake, lactate accumulation, and CO2 output. It is known that synaptosome fractions contain a substantial number (at least 30–45% by volume) of cytoplasm‐containing particles devoid of mitochondria (henceforth termed “cytosolic particles”), and that ∼80% of brain hexokinase is bound to the outer mitochondrial membrane. For the cytosolic particles, lacking oxidative phosphorylation, to maintain their “in vivo” ATP turnover would require about a 19‐fold increase in the glycolytic rate, which is not possible due to limiting amounts of hexokinase, and thus these particles are postulated to be responsible for the high level of aerobic lactate accumulation and the intractable low energy charge values found in synaptosome fractions. The mitochondria‐containing particles are postulated to have a normal energy charge, a submaximal glycolytic rate, and minimal lactate production, on the basis of the capacity of veratridine to stimulate synaptosomal O2 uptake and CO2 and lactate output. Calculations based on this “two populations of particles” hypothesis indicate that for synaptosome fractions in general, (1) the cytosolic particles contain ∼35–64% of the total adenine nucleotides and maintain an energy charge of ∼0.12; (2) the cytosolic particles and mitochondria‐containing particles have adenylate kinase apparent Keq values of ∼0.21–1.66 and 0.74, respectively, revealing that the higher apparent Kcq values of the synaptosome fractions probably are not real departures from equilibrium; and (3) ∼31–45% of synaptosome fraction protein is contained in debris, which, when taken into account, yields total adenine nucleotide contents in the cytosolic particles and mitochondria‐containing particles of ∼15–24 and ∼11 −19 nmol/mg of particle protein, respectively.

Sodium ion and fatty acid oxidation in bovine brain mitochondria

Abstract In bovine brain mitochondria fatty acids are activated, in part, by high-energy intermediates of oxidative phosphorylation (Beattie and Basford, 1966) . A similar activation of fatty acids by high-energy intermediates has also been proposed for liver mitochondria (Wojtczak, 1965). This paper reports an inhibition of fatty acid oxidation in brain mitochondria by the addition of Na+ ion which has been shown by Krall (1965) to stimulate the respiratory rate of brain mitochondria in the absence of phosphate acceptor with a decrease in respiratory control. The inhibition of fatty acid oxidation by Na+ persists in the presence of oligomycin and is reversed by increasing concentrations of fatty acid suggesting that fatty acids and Na+ may interact with an identical high-energy intermediate of oxidative phosphorylation.

REPLY FROM DRS. BASFORD AND KYRIAZI

Kyriazi H. T. and Basford R. E. (1986) Intractable unphysiologically low adenylate energy charge values in synaptosome fractions: an explanatory hypothesis based on the fraction’s heterogeneity. J. Nmrochem. 47, 5 12-528. Whittaker V. P. (1968) The morphology of fractions of rat forebrain synaptosomes separated on continuous sucrose density gradients. Biochern. J. I06,412-417. Whittaker V. P. (1984) The synaptosome, in Handbook of Neurochemisfry. Vol. 7(Lajtha A,, ed), pp. 1-39. Plenum Press, New York.