P.K. Jensen

Antimycin-insensitive oxidation of succinate and reduced nicotinamide-adenine dinucleotide in electron-transport particles I. pH dependency and hydrogen peroxide formation

Suspensions of electron-transport particles from beef heart supplied with 2 μg antimycin per mg particle protein retain a distinct capacity to oxidize succinate and reduced nicotinamide-adenine dinucleotide. The oxidation rates increase with pH until they reach a maximum where they become practically identical with the rates of the overall (uninhibited) oxidation at the same pH. The pH dependency is influenced by the cation concentration in the reaction medium. An increase in K+ or Na+ concentration shifts the pH optimum to more acid values. The oxidation leads to formation of H2O2, but the amount of this reaction product is less than required to account for the amount of substrate oxidized and O2 consumed.

Isolation of lipid particles from baker's yeast

The presence of lipid particles in yeast cells has been demonstrated by light as well as by electron microscopy [ I], but the more precise nature of the lipid is unknown. The present letter describes the isolation and subsequent chemical analysis of lipid particles from baker’s yeast (Saccharomyces cerevisiue). The results show that triacylglycerols and sterol esters are the major components of the lipid and account for 90-95% of the total particle content.

Membrane-bound lipid particles from beef heart chemical composition and structure

Abstract Lipid particles have been isolated from bovine heart muscle. Electron microscopy indicates that the particles are identical with osmiophilic particles situated between the myofibrils and intimately associated with mitochondria. The particles appear in the electron micrograph as a structureless core surrounded by tripartite structures typical of the “unit membrane”. The chemical composition of the particles is similar to that of lipid found in depot fat; triglyceride esterified with saturated and monoenoic acids. Only small amounts of protein and phospholipid are present. Data on the extractability of the lipid from the particles by different organic solvents and in suspension media of varying pH, together with data obtained after separation of “core” and “surface” material, suggest that the particles consist of a core of triglyceride surrounded by a layer of phospholipid and protein possibly forming the tripartite structures seen in the electron micrograph.

Antimycin-insensitive oxidation of succinate and reduced nicotinamide-adenine dinucleotide in electron-transport particles. II. Steroid effects.

Abstract Antimycin-insensitive oxidation in electron-transport particles at physiological pH is inhibited by deoxycorticosterone with succinate as substrate. When the substrate is reduced nicotinamide-adenine dinucleotide and the steroid concentration is low, oxidation is stimulated, but high steroid concentrations are inhibitory. Amytal has similar effects. The divergence of the effect of steroid on the oxidation of reduced nicotinamide-adenine dinucleotide is a consequence of a steroid-induced shift in acid direction of the pH curve for antimycin-insensitive oxidation, where the magnitude of the shift depends on steroid concentration. The effect of steroid appears to be associated with the formation of H 2 O 2 during antimycin-insensitive oxidation.

Triglyceride lipase activity in subcellular fractions from beef heart

Lipolytic activity in the heart has so far been studied mainly with respect to lipoprotein lipase and the role of this enzyme in transfer of fatty acids from plasma triglycerides to the muscle cells. Interest in transfer of fatty acids from intracellular stores of triglyceride to mitochondria was stimulated in the present laboratory, when membrane bound lipid particles were isolated from beef heart homogenates [1 ]. The triglyceride content of these particles was 90-95%, 3-5% was protein and the electron microscopic appearance of the isolated particles was similar to that of particles in situ in beef heart muscle cells. Here as well as in other heart muscle preparations they are seen surrounded by mitochondria [2, 3], which they probably furnish with fatty acids in connection with triglyceride hydrolysis [cf. 4]. On this background it was decided to study the intracellular localization of triglyceride lipase (TGL) activity in beef heart muscle cells, presently the only material from which lipid particles of this kind have been isolated. This letter reports the distribution of neutral to alkaline TGL activity among subcellular fractions prepared from beef heart cytoplasmic extracts (i.e. homogenates freed of nuclei and cell debris [5] ). One half of the recovered TGL activity was in the soluble fraction, the other half was evenly distributed between the microsomal and mitochondrial fractions. However, contamination of the mitochondrial fraction with microsomal protein most likely accounts for all TGL activity in the mitochondrial fraction. As judged by the effect of serum, NaC1 and heparin, lipoprotein

Role of AMP in regulation of the citric acid cycle in mitochondria from baker's yeast

The role of AMP in regulation of the citric acid cycle has been investigated in mitochondria prepared from mechanically disrupted bakers yeast (Saccharomyces cerevisiae). Addition of citric acid cycle intermediates and the acetyl donors: pyruvate, acetaldehyde and acetate in micromolar concentrations caused discrete and reproducible oxygen uptake with a stoicheiometric relationship to substrate amount and metabolite formation, which allowed calculations of the extent of oxidation separately for acetyl donors and auxiliary substrate. AMP increased the rate of citrate oxidation in the mitochondrial suspensions to about the same level as found for oxidation of succinate and 2-oxoglutarate. The total oxygen consumption and malate formation agreed with oxidation through the citric acid cycle also for citrate, indicating insignificant contamination with peroxisomes and enzymes of the glyoxylate pathway. In the absence of AMP, addition of acetyl donor plus auxiliary substrate (malate for pyruvate and 2-oxoglutarate for acetaldehyde and acetate) led to citrate accumulation and an oxygen consumption which indicated that the auxiliary substrate had been utilized only once for citrate formation. Malate, succinate and citrate were unable to support acetate and acetaldehyde oxidation to the same extent as found with malate supported pyruvate oxidation. AMP increased the rate of acetyl donor oxidation, reduced citrate accumulation almost to zero and gave rise to complete oxidation of acetyl donors at molar acetyl donor/auxiliary substrate ratios up to 3. Complete oxidation of acetate was obtained also with citrate as auxiliary substrate, but not with malate or succinate. Half maximal stimulation of the rate of citrate and of acetate oxidation was acetate oxidation in the absence of AMP despite the concomitant oxidation 2-oxoglutarate. The reason for this failure is not clear. The low rate of citrate (isocitrate) oxidation might create conditions unfavourable for acetate activation e.g., by allowing side reactions to compete successfully for intermediates in substrate phosphorylation at the expense of reactions leading to acetate activation. In this case the AMP effects on acetate oxidation could be explained solely as a result of activation of the NAD-dependent isocitrate dehydrogenase. The results obtained in the present investigation are in general agreement with such a “one site” suggestion, but the results are on the other hand difficult to reconcile with the role of AMP in acetate activation considering that the activating system and isocitrate dehydrogenase are both localized at or inside the inner mitochondrial membrane. A more detailed study of acetate activation under the conditions employed in the present study seems therefore required.

Steroid inhibition of reduced diphosphopyridine nucleotide-oxidase activity in electron-transport particles I. Kinetic studies

Abstract When deoxycorticosterone and several other steroid hormones are added to a suspension of electron-transport particles oxidizing DPNH, the rate of oxidation spontaneously decreases. The influence of experimental conditions on this inhibitory effect has been studied. It was found that the inhibition is independent of the level of specific activity of the oxidase and of prolonged storage at −15° of the electron-transport particles preparation. The composition of the reaction medium, however, especially with respect to buffer concentration, influences the degree of inhibition. The interaction of steroid and enzyme is reversible and the inhibition is non-competitive. The concentrations at which seven corticosteroids produce 50% inhibition have been determined. The results together with results of experiments, in which the distribution of four corticosteroids between electron-transport particles and suspension medium was estimated, indicated that the differences among the steroids with respect to their potency as inhibitors of the DPNH-oxidase activity of electron-transport particles reflect differences in their solubility in electron-transport particles more than differences in the affinity of the enzyme per se for the individual steroid.

NADH oxidation in phospholipid‐enriched cytoplasmic membrane vesicles from Escherichia coli

NADH oxidation in Escherichia coli cytoplasmic membrane vesicles enriched in anionic phospholipids by de novo synthesis of lipid in the vesicles from acyl‐CoA esters and sn‐glycerol 3‐phosphate has been studied. NADH‐oxidase but not NADH‐dehydrogenase activity was found to decrease during synthesis and accumulation of phospholipid in the vesicles. Density gradient fractionation showed that NADH‐oxidase activity was reduced to ≈30% in vesicles with a 3–6‐fold increase in anionic phospholipid, whereas vesicles with a > 10‐fold increase in phospholipid had virtually no NADH oxidase activity.