R.L. Lester

Studies on the electron-transport system XXVII. The respiratory activity of acetone-extracted beef-heart mitochondria role of coenzyme Q and other lipids☆

Abstract The effects of acetone extraction on the electron-transport activities of beef-heart mitochondria have been investigated. Two standard preparations have been developed. Preparation I, made by extraction with acetone containing 4% water is devoid of CoQ, and requires the specific addition of CoQ to restore succinate oxidation with various electron acceptors. The restored succinoxidase activity is completely sensitive to antimycin A and KCN. Preparation II, made by extraction with acetone containing 10% water under specified conditions, has lost not only its complement of CoQ but also three-quarters of its lipid complement, and the combined addition of coenzyme Q plus unidentified mitochondrial lipids is required for restoration of succinoxidase activity. The physical state of the lipid which is added is a crucial factor in its maximum effectiveness. Particles extracted with acetone lose the capacity to oxidize DPNH by cytochrome c or oxygen, and this deficiency may be a consequence of the inability of the preparation to catalyze the oxidation of DPNH by CoQ. Comparable results were also obtained with rat-liver and beef-liver mitochondria.

Studies on the mechanism of oxidative phosphorylation III. Phosphorylating particle types from beef heart

Crude mitochondrial suspensions, prepared on a large scale from beef heart, have been fractionated by centrifugation in a buffered medium. Two principal fractions, heavy and light, have been isolated, and some of their properties with respect to the oxidation of citric acid cycle substrates and the accompanying phosphorylation have been studied. The heavy fraction, which resembles intact mitochondria much more closely than the light, catalyzes the oxidation of these substrates very efficiently with P/O ratios quite close to the assumed theoretical values. These preparations can be conveniently stored in the deep-freeze for long periods of time without considerable loss of activity.

Studies on the electron transport system XX. Chemical and physical properties of the coenzyme Q family of compounds

Abstract The chemical and physical properties of four members of a closely related group of compounds are described. These compounds, which function as oxidation-reduction coenzymes in terminal electron transport, have been isolated from beef heart mitochondria, Azotobacter vinelandii and Torula utilis . These compounds are shown to be similar in that each contains the same quinonoid chromophore, probably dialkoxylated. These compounds have different molecular weights, and this difference is at least in part explained by the composition of the mono-unsaturated isoprenoid side chain(s); that is, these compounds differ in the number of such isoprenoid units which occur in the side chain. The formulae of these four compounds which best fit the available data are C 58 H 88 O 4 , C 53 H 80 O 4 , C 48 H 72 O 4 and C 43 H 64 O 4 .

Studies on the electron transport system. XV. Coenzyme Q (Q275) and the succinoxidase activity of the electron transport particle.

Coenzyme Q can be extracted from the electron transport particle and other particles with isooctane. Such extracted particles lose the capacity to oxidize succinate, and this capacity can be restored by addition of coenzyme Q and other lipid supplements. The coenzyme role of Q275 is highly specific. A large number of quinones including vitamin K1 and tocopherol quinone were unable to replace coenzyme Q. No evidence could be found that coenzyme Q is a component of the DPNH chain. On the other hand, cytochrome c is also released in an insoluble form ETP by isooctane, and the addition of cytochrome c fully restores succinoxidase activity of the isooctane-extracted particle. A requirement for both cytochrome c and coenzyme Q in oxidation of succinate can be demonstrated by treating ETP with deoxycholate and isooctane.

Studies on the electron transport system XXVIII. The mode of reduction of tetrazolium salts by beef heart mitochondria; Role of coenzyme Q andother lipids

The pathways involved in the reduction of various tetrazolium salts catalyzed by beef-heart mitochondria were investigated. It was found that the reduction of 2,2′,5′-tetraphenyl-3,3′-(4,4′-biphenylene)ditetrazolium chloride, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride, and 2,5-diphenyl-3-α-naphthyletrazolium chloride with succinate as substrate were dependent on CoQ and were largely sensitive to antimycin. These tetrazoles were not reduced by the reduced forms of the isolated cytochromes. Hence these interact somewhere at or beyond the antimycin site and before cytochromes c1 and c. 2,2′-di-p-nitrophenyl-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′- biphenylene)ditetrazolium chloride reduction by succinate is not CoQ-dependent and appears to occur at a site responsible for CoQ reduction. Reduction of tetrazoles by reduced diphosphopyridine nucleotide would appear to take place at the flavoprotein level. Evidence indicating that different requirements exist for the reduction of K3 and CoQ is also presented. Data on the lipid requirements for tetrazole reduction in lipid-depleted mitochondrial preparations are given. A simple assay procedure is described for the enzymic reduction of tetrazolium salts.

Studies on the electron transport system: XVI. Enzymic oxidoreduction reactions of coenzyme Q

Oxidoreduction reactions of coenzyme Q as catalyzed by beef heart mitochondria and derivative particles have been studied. Coenzyme Q is rapidly reduced by substrates, such as pyruvate plus malate, β-hydroxybutyrate, DPNH and succinate. It is also oxidized rapidly by molecular oxygen in the presence of mitochondrial enzymes. The reduction of coenzyme Q by the pyridinoprotein substrates is inhibited by Amytal as well as by antimycin A. The latter compound also inhibits the reduction of coenzyme Q by succinate. The oxidation of reduced coenzyme Q catalyzed by mitochondrial particles is inhibited by cyanide. The possible position of coenzyme Q relative to other members of the electron transport system of beef heart mitochondria is discussed.

Studies on the electron transport system: XVIII. Isolation of coenzyme Q (Q275) from beef heart and beef heart mitochondria

Abstract Two different methods for purification of coenzyme Q from beef heart mitochondria and from beef heart have been described. Selective extraction following saponification or direct extraction of total lipids have been used for initial extraction. The coenzyme Q in these extracts has then been purified by chromatography on Decalso or silicic acid followed by crystallization from ethanol. The coenzyme Q obtained by these two procedures have melting points which range from 49.3 to 50°, E1cm1% at 275 mμ ranges from 162 to 165. All purified preparations have the same Rf when chromatographed on silicone-treated paper and identical visible, ultraviolet and infrared spectra.

Studies on the electron transport system: XIX. The isolation of coenzyme Q from Azotobacter vinelandii and Torula utilis

Procedures are described for the isolation of 3 crystalline compounds of the coenzyme Q family. One of these compounds was isolated from the nonsaponifiable fraction of cells of Azotobacter vinelandii. Two distinct compounds were isolated from Torula utilis by direct solvent extraction with or without saponification. Chromatographic procedures followed by crystallization techniques are used for the final purification of these three compounds.

Studies on the mechanism of oxidative phosphorylation: IV. Pyridine nucleotide binding and its relation to activity in heart mitochondria

Abstract The oxidative phosphorylation activity of beef-heart particles with respect to the substrates of pyridino-protein enzymes is directly proportional to the amount of particle-bound pyridine nucleotides. The pyridine nucleotide content of the different particle types decreases in the following order: heavy particles, light particles, PETP and ETP. When particles are incubated aerobically, the pyridine nucleotides are released and rapidly destroyed, and simultaneously the capacity for pyridine nucleotide-linked oxidation declines. This loss of activity can be restored completely by addition of DPN and EDTA to the assay. After long-term aerobic incubations of mitochondria, the oxidation induced by DPN and EDTA is largely uncoupled. However, further addition of bovine serum albumin during the assay leads to restoration of a considerable proportion of the lost capacity for oxidative phosphorylation. The inactivation due to aerobic incubation can be prevented under anaerobic conditions or by addition to the incubation medium of substrates, EDTA, Mn ++ or Mg ++ . These effects, particularly in the case of protection by EDTA, are due to inhibition of the release of particle-bound pyridine nucleotides. The implications of these results with respect to the possible factors involved in binding of pyridine nucleotides are discussed.

Oxidative phosphorylation by an electron transport particle from beef heart

Abstract A stable phosphorylating electron transfer particle has been isolated from beef heart mitochondria which retains the capacity for oxidatice phosphorylation almost unimpaired. Some new aspects of the mechanism of oxidative phosphorylation have been recognized.