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The rates of NADH-supported superoxide/hydrogen peroxide production by membrane-bound bovine heart respiratory complex I, soluble pig heart dihydrolipoamide dehydrogenase (DLDH), and by accompanying operation of these enzymes in rat heart mitochondrial matrix were measured as a function of the pool of pyridine nucleotides and its redox state. Each of the activities showed nontrivial dependence on nucleotide pool concentration. The NAD+/NADH ratios required for their half maximal capacities were determined. About half of the total NADH-supported H2O2 production by permeabilized mitochondria in the absence of stimulating ammonium could be accounted for by DLDH activity. The significance of the mitochondrial NADH-dependent hydrogen peroxide production under physiologically relevant conditions is discussed. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Genetic defects in pyruvate dehydrogenase complex (PDC) cause lactic acidosis, neurological deficits, and often early death. Most mutations of PDC are localized in the alpha subunit of the pyruvate dehydrogenase (E1) component. We have kinetically characterized a patient's missense mutation alphaH44R in E1alpha by creating and purifying three recombinant human E1s (alphaH44R, alphaH44Q, and alphaH44A). Substitutions at histidine-15 resulted in decreased V(max) values (6% alphaH44R; 30% alphaH44Q; 90% alphaH44A) while increasing K(m) values for thiamine pyrophosphate (TPP) compared to wild-type (alphaH44R, 3-fold; alphaH44Q, 7-fold; alphaH44A, 10-fold). This suggests that the volume of the residue at site 15 is important for TPP binding and substitution by a residue with a longer side chain disrupts the active site more than the TPP binding site. The rates of phosphorylation and dephosphorylation of alphaH44R E1 by E1-kinase and phospho-E1 phosphatase, respectively, were similar to that of the wild-type E1 protein. These results provide a biochemical basis for altered E1 function in the alphaH44R E1 patient.

2-Amino-3-carboxy-1,4-naphthoquinone (ACNQ) is a novel growth stimulator for bifidobacteria. The role of ACNQ as a mediator of the electron transfer from NAD(P)H to dioxygen (O(2)) and hydrogen peroxide (H(2)O(2)), proposed in our previous paper, was examined using the cell-free extract and whole cells of Bifidobacterium longum. Continuous monitoring of ACNQ, O(2) and H(2)O(2) by several amperometric techniques has revealed that ACNQ works as a good electron acceptor of NAD(P)H diaphorase and that the reduced form of ACNQ is easily autoxidized and also acts as a better electron donor of NAD(P)H peroxidase than NAD(P)H. The generation of H(2)O(2) by B. longum under aerobic conditions is effectively suppressed in the presence of ACNQ. These ACNQ-mediated reactions would play roles as NAD(P)(+)-regeneration processes. The accumulation of ACNQ in the cytosol has been also suggested. These characteristics of ACNQ seem to be responsible for the growth stimulation of bifidobacteria. Vitamin K(3), which has an extremely low growth-stimulating activity and was used as a reference compound, exhibits much lower activity as an electron transfer mediator. The difference in the activity is discussed in terms of the redox potential and partition property of the quinones.

Lipoamide dehydrogenase was purified around 22-fold relative to the crude extracts of Streptomyces seoulensis with an overall yield of 9.5%. The enzyme was composed of two identical subunits with a molecular mass of 54 kDa and contained 1 mol of FAD per mol of subunit. The absorption spectra of the enzyme revealed the absorption maxima of flavoprotein at 272, 349, and 457 nm. Catalytically active two-electron reduced lipoamide dehydrogenase was produced by anaerobic reduction with one equivalent of NADH. Addition of excess amount of NADH led to the four-electron reduced lipoamide dehydrogenase. The reaction of the enzyme in the reduction reaction of lipoamide or lipoic acid could be explained by a ping-pong mechanism like many other lipoamide dehydrogenases reported earlier. The enzyme also catalysed the reduction of various quinone compounds with NADH as electron donor via a ping-pong mechanism. The enzyme can catalyse a single electron transfer in case of quinone-reducing process, evidenced by the production of 1,4-naphthosemiquinone radical anion. The quinone-reducing activity of the enzyme was dramatically inhibited by NAD + , indicating the involvement of four-electron reduced form. The structural gene for the enzyme was cloned using a DNA fragment PCR-amplified with the primers designed from N-terminal and internal amino acid sequences. The deduced amino acid sequence shared striking similarity with those of lipoamide dehydrogenases from prokaryotes and eukaryotes. The gene was named lpd . All tested Streptomyces contained one homologue of the lpd gene, which is consistent with the fact that most organisms contain only one lipoamide dehydrogenase.

A male child with metabolic acidosis was diagnosed as having dihydrolipoamide dehydrogenase (E3) deficiency. E3 activity of the proband's cultured fibroblasts and blood lymphocytes was 3-9% of normal, while in the parent's lymphocytes it was about 60% of normal. The proband's pyruvate dehydrogenase complex (PDC) and the alpha-ketoglutarate dehydrogenase complex activities from cultured skin fibroblasts were 12% and 6% of normal, respectively. PDC activity in the parents cultured fibroblasts was 25-31% of normal. Western and Northern blot analyses showed similar quantities of E3 protein and mRNA in cultured fibroblasts from the proband and his parents. DNA sequencing of cloned full-length E3 cDNAs, from the proband and the parents, showed two mutations on different alleles of proband were inherited from the parents. One mutation is a three nucleotide (AGG) deletion, from the mother, resulting in deletion of Gly101 in the FAD binding domain. The other mutation is a nucleotide substitution (G to A), from the father, leading to substitution of Lys for Glu340 in the central domain. The same deletion mutation was found in E3 cDNA from a chorionic villus sample and cultured fibroblasts obtained from the mother's subsequent offspring. This finding illustrates the possibility of successful prenatal diagnosis of E3 deficiency utilizing mutations characterized prior to initiation of pregnancy.

A dihydrolipoamide dehydrogenase (LPD; dihydrolipoamide:NAD oxidoreductase, EC 1.8.1.4.) activity has been detected in the cyanobacterium Synechocystis PCC 6803. The enzyme was isolated from the membraneous fraction after detergent solubilization and shown to be homogenous on the basis of SDS-PAGE and N-terminal sequencing. The isolated enzyme had a specific activity of 75 U (mg protein)(-1) and was shown to be a homodimer with an apparent molecular mass of 104 kDa for the dimer and 55 kDa for the subunits. The enzyme contains 1.75 mol noncovalently bound FAD (mol enzyme)(-1) suggesting that each subunit contains 1 mol FAD and that the FAD is fairly tightly associated with the enzyme. N-terminal sequencing gave a contiguous amino acid sequence of 17 residues and showed that the N-terminus of the LPD from Synechocystis PCC 6803 has significant homologies to other LPDs sequenced so far. Immunoblot experiments indicated that the enzyme is mainly present in the membrane fraction, and immunocytochemical investigations gave evidence that the LPD in Synechocystis PCC 6803 is located in the periplasma space between the cytoplasma membrane and the peptidoglycan layer. This is the first report on an extracellular, membrane-bound LPD in a cyanobacterium.

Norepinephrine was oxidized by the Mn(3+)-pyrophosphate complex to the corresponding o-quinone at pH 6.5. Cyclized norepinephrine ortho-quinone showed an absorption maximum at 289 and 483 nm. No oxygen consumption was observed during oxidation of norepinephrine to o-quinone by Mn3+ and subsequent cyclization. The reduction of cyclized norepinephrine ortho-quinone to the corresponding hydroquinone was catalyzed by DT-diaphorase. However, the hydroquinone formed proved to be unstable in the presence of oxygen, since reduction of cyclized norepinephrine o-quinone by DT-diaphorase was accompanied by continuous oxidation of NADH and oxygen consumption. Addition of the chelator DETAPAC or SOD to the incubation mixture during reduction of cyclized norepinephrine ortho-quinone by DT-diaphorase strongly inhibited NADPH oxidation and oxygen consumption, suggesting that manganese and superoxide radicals were involved in hydroquinone autoxidation. Elimination of the effects of superoxide radicals, manganese and H2O2 on autoxidation of hydroquinone by addition of SOD, catalase and DETAPAC to the incubation mixture resulted in a 79% inhibition of NADH oxidation, suggesting that 21% of the autoxidation is oxygen-dependent. However, the effect of these additions on oxygen consumption was even more pronounced (93% inhibition).

Effects of endurance training and high-fat diet intake on the branched-chain 2-oxo acid dehydrogenase complex in skeletal muscle were examined in rats. The basal activities of the enzyme complex (approximately 4% in active form of the total enzyme) in the muscle of rats under the fed conditions were not different between trained and untrained rats. The basal activity in the muscle was elevated by 24 h starvation in both groups of rats, but the level of the elevation was significantly greater in the trained rats than in the untrained rats. On the other hand, high-fat diet intake did not alter the basal activity of the enzyme complex in the muscle or the profile of activation of the enzyme complex by muscle contractions elicited by the electrical stimulation, suggesting that the fat content in the diet does not affect the enzyme activity in the muscle. Neither training nor diet affected the total enzyme activity or the amount of enzyme protein. Activation by leucine administration of the enzyme complex in the muscle was greater in the trained rats than in the untrained rats, suggesting that the activity state of the enzyme complex is more responsive to regulation by the 2-oxo acid derived from leucine in the muscles of endurance-trained rats.

Plasma membranes from most mammalian cells display significant transplasma membrane oxidoreductase (PMO) activity. The enzymes use an extracellular, impermeant electron acceptor as substrate and intracellular reduced pyridine nucleotide as electron donor. The plasma membrane from a neuroblastoma cell line, NB41A3, has been biotinylated and purified by immunoprecipitation with avidin and antiavidin-antibodies. The protein recovery of an immunopurified membrane preparation was0.15% of the protein content in the cell extract. The preparation displays an increase in the specific activity of PMO's of 15- to 20-fold compared to the activity in whole cells. With this approach the presence of a NADH-diaphorase within the cell plasma membrane can be demonstrated. This activity accounts for about one third of the total cellular diaphorase activity. The PMO activity cannot be attributed to an increased permeabilization of the plasma membrane induced upon biotinylation nor to intracellular activity from lysed cells. Activation of basal metabolism (glycolysis) stimulates PMO activity up to approx. 54%, presumably through a raise of the intracellular NADH store. PMO also promotes cell growth at low substrate concentrations (0.1-1 microM). Native gel electrophoresis of iminobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several PMO activities at the plasma membrane.

The particulate fraction from sonicates of P. tularensis contained a very active DPNH oxidase system, but was almost inactive with TPNH. The TPNH-DPN transhydrogenase of the cell was located in the soluble fraction, while most of the DPNH-3-acetylpyridine DPN transhydrogenase activity was in the particulate fraction (or acetone powder residue). Almost no TPNH-3-acetylpyridine DPN transhydrogenase activity was present. TPNH-DPN transhydrogenase activity could be determined by coupling with the particulate DPNH-3-acetylpyridine DPN transhydrogenase. About 2 3 of the DPNH diaphorase activity was found in the soluble fraction. The DPNH/TPNH diaphorase ratio was about 0.7 for whole sonicate, and about 0.5 for the soluble fraction. Very little TPNH diaphorase activity was observed in the particulate fraction, even after treatment with acetone, butanol, or deoxycholate, or after further sonic oscillation.

A comparison of the rates of reaction of lipoyl dehydrogenase with dihydrolipoamide, reduced diphosphopyridine nucleotide and oxidized acetyl pyridine diphosphopyridine nucleotide have shown the involvement of the enzyme flavin in the transhydrogenase reaction catalyzed by this enzyme. The previously reported involvement of oxidized diphosphopyridine nucleotide in the catalytic oxidation of reduced diphosphopyridine nucleotide with lipoic acids as acceptors has been investigated by use of Neurospora DPNase, which by hydrolysing oxidized diphosphopyridine nucleotide has permitted a study of the effect of oxidized diphosphopyridine nucleotide on the oxidation-reduction state of the enzyme. It has been found that under certain conditions (which result in inhibition of enzyme activity) two molecules of reduced diphosphopyridine nucleotide will react to produce a fully reduced form of the enzyme, in which both the reactive disulphide and the flavin are fully reduced. Evidence is presented that the active form of the enzyme is a half-reduced form in which both flavin and the disulphide are only partially reduced. A reaction mechanism based on these findings is proposed.

A TPN-specific dehydrogenase which reversibly oxidizes L -gulonate to D -glucuronate has been purified 33-fold from pig kidney. The purified enzyme is free of aldonalactonase and DPN- L -gulonate dehydrogenase. Although L -galactonate is also a substrate for this enzyme, the name TPN- L -gulonate dehydrogenase is suggested for this enzyme. The purified enzyme does not act on the γ -lactones of the substrates. Uridine diphospho- D -glucuronate is n is not reduced by the enzyme. The role of the dehydrogenase in the conversion of D -glucuronate to L -ascorbate and L -xylulose as well as the metabolic source of free D -glucuronate for ascorbic acid biosynthesis are discussed.

1. 1. Lipoyl dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) and two asparagusate dehydrogenases from asparagus mitochondria were purified by a series of steps, freezing and thawing, sodium dodecylsulfate extraction, and chromatography on Sephadex G-200 and DEAE-cellulose. 2. 2. Lipoyl dehydrogenae was highly specific for α-lipoic acid, which could not be replaced at all by asparagusic acid. Each of the asparagusate dehydrogenases was capable of reducing both asparagusic and α-lipoic acids by using NADH as hydrogen donor. 3. 3. Reduction of α-lipoic acid with NADH by lipoyl dehydrogenase was activated by NAD+, but that of asparagusic acid by asparagusate dehydrogenase was inactivated by NAD+. 4. 4. Lipoyl dehydrogenase and two asparagusate dehydrogenases differed in electrophoretic mobility on polyacrylamide gels.

A new method permitting the simultaneous evaluation of the redox states of α-lipoamide dehydrogenase and electron-transfer flavoprotein in intact rat liver mitochondria by two-channel fluorimetry is described. It is shown that correction for the partial overlap of emission spectra can readily be introduced after a calibration procedure is performed. This method was applied to the investigation into regulation of palmitoylcarnitine oxidation. It was found that in the presence of rotenone, malonate and a redox buffer for the mitochondrial NAD-system, the β-oxidation flux was sensitive to variations in redox state of respiratory chain electron carriers at low states of NAD reduction. Therefore, the concept of β-oxidation control caused solely by the NAD redox state can no longer be sustained.

Previous electron spin resonance studies have demonstrated that the decay of ascorbyl plus semiquinone radicals, produced in an aqueous mixture of ascorbate and 2,6-dimethoxy-p-quinone, is accelerated by ascites cells. This effect was concluded to involve a sulfhydryl-containing NAD(P)H-enzyme, and work on cultured cell lines showed that on neoplastic transformation the activity against the radicals was increased. We show here that at least three disulfide-oxidoreductases are able to quench the radicals in a similar way to that of viable cells. Glutathione reductase (EC 1.6.4.2) in the presence of NADPH and oxidised glutathione, and dihydrolipoamide dehydrogenase (EC 1.8.1.4) with NADH and lipoamide, are found to accelerate the radical decay by reducing the quinone or semiquinone. DT-diaphorase (EC 1.6.99.2) in the presence of NAD(P)H can also achieve this by reducing the quinone directly. Lipoamide dehydrogenase and glutathione reductase are also capable of reducing nitroxide spin labels, a finding considered of relevance to the reported reduction of such spin labels by neuroblastoma cells.

Pig heart lipoamide dehydrogenase (EC 1.6.4.3) contains ten half-cystines (as cysteic acid) per mole of enzyme bound FAD. Two of these are linked in an intrachain cystine which acts in concert with the flavin during catalysis. A peptic peptide containing this active center disulfide has been isolated and shown to have the sequence: Glu-Thr-Leu-Gly-Gly-Thr-Cys-Leu-Asn-Val-Gly-Cys-Ile-Pro-Ser (Lys, Ala, Leu). The enzyme also contains seven titratable thiols when either 5,5′-dithiobis (2-nitrobenzoic acid) or iodoacetate is used as titrant. Tryptic peptides containing alkylated thiols have been isolated and characterized by amino acid composition and by their positions in two-dimensional chromatography-electrophoresis. On the basis of map position and composition, the peptides containing thiols can be distinguished from one another. The results are compared with recent data of Brown and Perham (Brown, J. P. and Perham, R. N. (1974) Biochem. J. 138, 505–512) on the compositions and partial sequences of tryptic chymotryptic peptides containing half-cystines. The combined data associate nine of the ten half-cystines with unique compositions.

Glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) was purified to homogeneity from porcine erythrocytes by use of affinity chromatography on 2′,5′-ADP-Sepharose 4-B. Analytical ultracentrifugation experiments were analysed to give the following physical parameters for the enzyme: s 20,w = 5.7 S, D 20,w = 50 μ m 2 /s, and M w = 103 000 (protein concentration, 0.5 mg/ml). The frictional ratio was 1.37 and the Stokes radius was 4.3 nm. The enzyme molecule is a dimer composed of subunits of equal size each containing a FAD molecule. The amino acid compositions and circular dichroism spectra of the porcine and human enzymes indicated extensive structural similarities. The isoelectric point was at pH 6.85 (at 4°C). The absorption spectrum of the oxidized enzyme had maxima at 377 and 462 nm. In vivo the enzyme appears to be partially reduced. At a physiological concentration of reduced glutathione the apparent Michaelis constants for glutathione disulfide and NADPH were higher than in the absence of reduced glutathione. At 0.15 M ionic strenth the catalytic activity obtained with NADPH as reductant was optimal at pH 7 and more than 200 times higher than that obtained with NADH. S -sulfoglutathione and some mixed disulfides of glutathione were poor substrates with the exception of the mixed disulfide of coenzyme A and reduced glutathione. The purified enzyme displayed low transhydrogenase activity with oxidized pyridine nucleotide analogs and diaphorase activity with 2,6,-dichlorophenolindophenol as acceptor substrattes; both NADPH and NADH served as donors.

The pyruvate dehydrogenase (Pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor acetylating), EC 1.2.4.1) complex from Salmonella typhimurium was purified, characterized and compared to the enzyme complex from Escherichia coli. No difference could be found in the molecular weights of the native enzyme complexes or in the single polypeptide chains of the enzymes of the two organisms. Values of 100 000, 87 000 and 56 000 were obtained for the polypeptide chains of the pyruvate dehydrogenase, the dihydrolipoamide transacetylase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) and the dihydrolipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) components, respectively. Complete cross-reactivity was found with antibodies directed against the pyruvate dehydrogenase complex from E. coli and electron micrographs of both enzyme complexes reveal identical structures. A high Michaelis constant for pyruvate with a Km = 6 . 10(-4) M and a somewhat weaker cooperativity as compared to the enzyme from E. coli reflect some minor differences, while the binding of the cofactor thiamine diphosphate (Km = 1 . 10(-6) M) is identical for both enzyme complexes. Reassociation to a fully active complex molecule works with equal facility between the pyruvate dehydrogenase component and a dihydrolipoamide transacetylase: dihydrolipoamide dehydrogenase subcomplex from either organism in all possible combinations.

The pyruvate dehydrogenase complex was purified from Ascaris muscle both with and without MgCl2 treatment at the first stage of purification. The specific activity of complex purified with MgCl2 treatment was about 2-fold as high as that purified without it. In addition to three component enzymes, two unknown polypeptides of 46 and 41 kDa were found in the complex purified by the two procedures. The quantity of unknown polypeptide of 41 kDa was increased in the complex purified with MgCl2 treatment as compared with that without it. Antibodies against the three component enzymes were prepared. All the antibodies precipitated the two unknown polypeptides in addition to the three component enzymes in immunoprecipitation experiments. Antibody against the alpha-subunit of pyruvate dehydrogenase reacted with the 41 kDa polypeptide as well as the alpha-subunit in the immunoblotting method. The unknown polypeptide of 46 kDa did not react with any antibody. These results suggest that the unknown 41 kDa polypeptide is a derivative of the alpha-subunit and that the unknown 46 kDa polypeptide is not a proteolytic-degradative product of component enzymes but is a component of the Ascaris pyruvate dehydrogenase complex. When the Ascaris complex was incubated with [2-14C]pyruvate in the absence of CoASH, only lipoate acetyltransferase was acetylated. In rat heart pyruvate dehydrogenase complex, lipoate acetyltransferase and another protein (referred to as component x or protein x) were acetylated. These results indicate that the unknown polypeptide of 46 kDa is a new component.

Electrophoretic analysis, using polyacrylamide gels, has been applied to purified plasma membrane preparations derived from 3T3 mouse fibroblasts, 3T3 cells transformed by SV40 and polyoma viruses, as well as to ouabain-resistant 3T3 cells. Differences have been detected at the level of the membrane peptides and the glucosamine-containing macromolecules between normal and virus-transformed cells.

Lactyl-Coa dehydrase (lactyl-CoA hydro-lyase), acyl-CoA dehydrogenase (acyl-CoA: (acceptor) oxidoreductase), lactate dehydrogenase (lactate: (acceptor) oxidoreductase), diaphorase (NADH2: (acceptor) oxidoreductase), phosphotransacetylase (acetyl-CoA: orthophosphate acetyltransferase, EC 2.3.1.8), and CoA transphorase (acetyl-CoA: proprionate CoA-transferase, EC 2.8.3.1) have been identified, partially purified, and characterized in extracts of the remen microorganism Peptostreptococcus elsdenii. On the basis of the results presented, it is suggested that the conversion of lactate to propionate occurs via the CoA esters of the lactate acrylate and propionate respectively.

Summary 1. The reactivity with tetrazolium salts has been examined for each of the enzymatic systems of oxidation of NADH and NADPH present in mammalian brain. Tetrazolium reduction has been measured using seven tetrazolium salts and purified preparations of each system in order to exclude interference from similar enzymatic activities. The effects of the concentration of substrate and tetrazolium salts, of pH and of possible inhibitory compounds have been investigated. 2. The NADH-tetrazolium reductase activity is due to the mitochondrial and microsomal systems whose relative contributions depend markedly upon the conditions of assay. PCMB in relatively low concentrations completely inhibits the activity of the latter system while leaving essentially unaffected that of the former pathway. NADH-ferricyanide reductase, lipoamide dehydrogenase (EC 1.6.4.3) and, in absence of added menadione, NAD(P)H dehydrogenases (EC 1.6.99.2), do not contribute significantly to the tetrazolium reductase activity. With added menadione, however, the largest fraction of this activity is due to the latter enzymes which can be inhibited completely by dicumarol. 3. The NADPH-tetrazolium reductase activity is essentially due to the microsomal system, since the NADPH-glutathione reductase (EC 1.6.4.2) does not contribute significantly to this activity. With regard to the NAD(P)H dehydrogenases, the same findings obtained with NADH have also been obtained with NADPH.

Bacteriol enzymes release lipoic acid from human branched chain α-ketoacid oxidase multienzyme complex and reincorporate it again. The release of lipoic acid causes formation of isobutyric coenzyme A to cease and supports evidence for the involvement of a lipoic acid-containing transcylase.

1. 1. The sensitivity of lipoamide dehydrogenase towards trace metals has been investigated. 2. 2. Incubation of the enzyme with trace metals brings about the inactivation of many of the reactions catalysed by the enzyme, while the activity with the artificial hydrogen acceptors 2,6-dichlorophenolindophenol and methylene blue is stimulated. The changes in the enzyme are irreversible. It appears that Cu2+ acts catalytically, since the enzyme is irreversibly altered by less than 1 atom Cu2+ per mole FAD, and no Cu2+ bound to the enzyme could be detected. 3. 3. Slight changes in the visible part of the spectrum are brought about by the Cu2+ treatment. These may be due to a loosened bond between the flavin and the protein. 4. 4. Upon reduction with substrate the semiquinone of the Cu2+-treated enzyme is initially produced, but is no longer stable; the enzyme has a much greater tendency to be reduced to the four-electron reduction stage. 5. 5. The implications of these results for the reaction mechanism of the enzyme are discussed.

Summary It has been found that a number of agents greatly affects the release by Triton-X-ioo of certain dehydrogenases from rabbit-brain mitochondria. The enzymes studied were DPN-dependent isocitrate dehydrogenase ( L s-isocitrate:DPN+ oxidoreductase (decarboxylating), EC 1.1.1.41) and TPN-dependent isocitrate dehydrogenase ( L s-isocitrate:TPN+ oxidoreductase (decarboxylating), EC 1.1.1.42), gluta-mate dehydrogenase ( L -glutamate:DPN+ (TPN+) oxidoreductase (deaminating), EC 1.4.1.3) and lipoamide dehydrogenase (DPNH:lipoamide oxidoreductase, EC 1.6.4.3). The effects of Triton X-100 on the morphology of the mitochondria were checked with the electron microscope. 1. The detergent was shown to disrupt the architecture of the mitochondria, leaving single and double membranes and amorphous material. 2. ATP, ADP, pyrophosphate, citrate and isocitrate enhanced release of the enzymes. DPN was less effective, and α-ketoglutarate, adenylic acid and adenosine were ineffective, as was EDTA. 3. K+ and Na+ (80 mM) enhanced release of both isocitrate dehydrogenases, but partially prevented release of glutamate dehydrogenase. Ca2+ and Mg2+ at 4–5 mM enhanced release of TPN-isocitrate dehydrogenase but inhibited that of DPN-isocitrate dehydrogenase and glutamate dehydrogenase. Mg2+ had little effect on the release of lipoamide dehydrogenase. 4. It is suggested that metal ions play a role in the binding of these four dehydrogenases to the mitochondria, but that the details of the attachments may be different in each case.

Diaphorase and cytochrome reductase have been extracted differentially from a pig heart-muscle preparation under various conditions. The results indicate that there is no foundation for the suggestion that diaphorase is a degraded form of cytochrome reductase.

1. 1. Two membrane fractions were isolated from homogenates of the first stellar nerve of Dosidicus gigas by a sequence of differential, discontinuous and gradient centrifugations in sucrose solutions. Membrane Fraction I has an apparent density of 1.090 g/ml and membrane Fraction II a value of 1.140 g/ml. 2. 2. At low and high magnifications in the electron microscope, both fractions appear as rounded membrane profiles similar to the plasma membrane observed in the unfractionated tissue. No other subcellular component was observed. Fraction I has a thickness of 95–110 A and that of Fraction II is 72–100 A. 3. 3. The percentage lipid composition of the two membranes was established. The following lipid classes were tentatively identified: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, cholesterol, free fatty acids and the hydrocarbon n-pentacosane. The relative distribution of the fatty acids present in the phospholipids of the two membrane fractions was different. 4. 4. The morphological appearance, the yield of the two membranes and the distribution of (Na+-K+)-dependent, ouabain-sensitive ATPase has led us tentatively to relate Fraction I with the axolemma and Fraction II with the Schwann cell plasma membrane.

In the presence of NADH, lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) catalyzes reduction of p -benzoquinone by way of a mixed mechanism involving one- and two-electron transfers. The ratio of rates of semiquinone formation and NADH oxidation is 0.3, the two-electron transfer mechanism being dominant. When the enzyme is treated with cupric ions, phenylmercuric acetate, N -ethylmaleimide and NADH + arsenite the electron transfer mechanism is shifted to a one-electron transfer type accompanied with remarkable changes in the pH-activity curve of diaphorase activity. Upon treatment of the enzyme with Cu 2+ a typical alkaline shift of pH optima for NADH-ferricyanide and NADH-benzoquinone reductase activities is observed. Changes taking place in the early phase of copper treatment can be mostly reversed by treatment with cysteine. It appears that the type of electron transfer mechanism is closely related to the extent of modification of lipoamide dehydrogenase by sulfhydryl reagents.

Summary Lipoyl dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) has been prepared from Saccharomyces cerevisiae in what appears to be a pure state. It is a flavoprotein with a molecular weight of approx. 100 000. On reduction of the enzyme with NADH the flavin moiety does not reach the fully reduced state, even with excess reducing agent. In this form the enzyme solution has a red colour and is probably present as a semi-quinone by analogy with the enzyme from pig heart. A comparison between this enzyme and those prepared from other sources is given.