Identity of aliphatic L- -hydroxyacid oxidase and glycolate oxidase from rat livers

l-α-Hydroxyacid oxidase and glycolate oxidase have been partially purified from rat livers and found to be identical, judging by substrate specificities, K_m values for certain substrates and coenzyme (FMN), activation energy, inhibition rates by various reagents and pH optimum. K_m values are as follows; glycolate, 2.4 × 10^?4m; l-α-hydroxyisocaproate, 1.26 × 10^?3; glyoxylate, 1.41 × 10^?4m; and FMN, 1.13 × 10^?6m. K_m values for glycolate and FMN are one-tenth and one-twentieth the literature values for hepatic glycolate oxidase. Sucrose density gradient centrifugation establishes that this enzyme is located in hepatic peroxisomes.     

A durohydroquinone oxidation site in the mitochondrial transport chain

Although duroquinone had little effect upon NADH oxidation in neutral lipid depleted mitochondria, durohydroquinone was oxidized by ETP at a rate sensitive to antimycin A. Fractionation of mitochondria into purified enzyme systems showed durohydroquinone: cytochromec reductase to be concentrated in NADH: cytochromec reductase, absent in succinate:cytochromec reductase, and decreased in reduced coenzyme Q:cytochromec reductase. Durohydroquinone oxidation could be restored by recombining reduced coenzyme Q:cytochromec reductase with NADH:coenzyme Q reductase. Pentane extraction had no effect upon either durohydroquinone or reduced coenzyme Q₁₀ oxidation, indicating lack of a quinone requirement between cytochromesb andc. Both chloroquine diphosphate and acetone (96%) treatment irreversibly inhibited NADH but not succinate oxidation. Neither reagents had any effect upon durohydroquinone oxidation but both inhibited reduced coenzyme Q₁₀ oxidation 50%, indicating a site of action between Q₁₀ and duroquinone sites. Loss of chloroquine sensitive reduced coenzyme Q₁₀ oxidation after acetone extraction suggests two sites for Q₁₀ before cytochromeb.     

Catalytic properties of glutathione reductase from liver at norm and toxic hepatitis

Catalytic properties of glutathione reductase (GR; EC 1.6.4.2) have been investigated using homogenous preparations of this enzyme purified from livers of control rats and rats with toxic hepatitis. Some properties of this enzyme remained unchanged under conditions of toxic hepatitis; these included eletrophoretic mobility (R_f = 0.23 ± 0.01), molecular mass (104.5 ± 5.2 kDa), pH optimum (7.4 ± 0.37), as well as close pK values of functional groups. However, enzyme isolated from toxic liver was characterized by lower affinity for substrate and coenzyme as well as by appearance of substrate inhibition. In addition there are some differences in regulation of GR activity by metabolites of tricarboxylic acid cycle.     

Essentiality of coenzyme Q for the oxidation of -glycerophosphate by pig brain mitochondria

Serial extraction of lyophilized pig brain mitochondria with cold pentane resulted in complete loss of α-glycerophosphate oxidase activity. On titration with coenzyme Q₁₀ the activity was fully recovered. On comparing the decline of α-glycerophosphate, NADH, and succinoxidase activities during serial extraction with pentane, α-glycerophosphate oxidation was always the first to be lost. Extraction of coenzyme Q₁₀ from lyophilized brain mitochondria with pentane does not affect the activities of α-glycerophosphate or NADH dehydrogenase, but succinate dehydrogenase is partially inactivated. Reversible inactivation of the α-glycerophosphate oxidase system on depletion of the coenzyme Q content is taken as evidence that coenzyme Q is an obligatory component of this system. In accord with the conclusion that coenzyme Q is probably the physiological oxidant of α-glycerophosphate dehydrogenase, in antimycin-treated brain mitochondria α-glycerophosphate causes full activation of endogenous succinate dehydrogenase, in analogy to the previously observed activation by NAD-linked substrates in liver and heart mitochondria and by NADH in submitochondrial particles.     

Presence of a flavoprotein in O2-evolving Photosystem II preparations from the cyanobacterium Anacystis nidulans

A highly active O₂-evolving Photosystem II complex which was greatly depleted of phycobiliproteins was isolated from the cyanobacterium Anacystis nidulans. This complex contained the flavoprotein with l-amino acid oxidase activity which we have previously shown to be present in thylakoid preparations of this cyanobacterium (Pistorius, E.K. and Voss, H. (1982) Eur. J. Biochem. 126, 203–209). One of the most prominent polypeptides in this O₂-evolving Photosystem II complex had a molecular weight of 49 kDa. This polypeptide co-chromatographed on SDS-polyacrylamide gels with the purified l-amino acid oxidase which consists of two subunits of 49 kDa. The antagonistic effect of CaCl₂ on the two examined reactions could also be demonstrated with this O₂-evolving Photosystem II complex: CaCl₂ stimulated photosynthetic O₂ evolution, but inhibited the l-amino acid oxidase activity. Both reactions were inhibited by o-phenanthroline. These results further support a functional relationship between the flavoprotein with l-amino acid oxidase activity and Photosystem II activities in A. nidulans. However, we only found 1 mol FAD per 350–650 mol chlorophyll, although 1 gatom Mn per 5–10 mol chlorophyll was present. When we assume a photosynthetic unit of about 40 chlorophylls, then in most preparations the FAD values were more than a factor of 10 too low. Results which we obtained with the purified l-amino acid oxidase showed that the FAD values were in most enzyme samples lower than the theoretically expected value of 2 mol FAD per mol enzyme. Moreover, in some cases the absorption spectrum of the enzyme showed substantial deviations from the spectrum of oxidized FAD. These experiments indicated that the flavin in the enzyme could partly exist in a form which was different from ‘authentic oxidized FAD’. We do not yet know the chemical nature of this ‘modified flavin’.     

Comparison of growth stimulation of HeLa cells,HL-60 cells,and mouse fibroblasts by coenzyme Q10

Summary The addition of coenzyme Q₁₀ to culture media stimulates the serum-free growth of HeLa, HL-60 cells, and mouse fibroblasts (Balb/3T3). With HeLa cells, the stimulation by coenzyme Q₁₀ is additive to the stimulation by ferricyanide, an impermeable electron acceptor for the transplasma membrane electron transport. This combined response to coenzyme Q₁₀ and ferricyanide is enhanced with insulin. -Tocopherylquinone can also stimulate the growth of HeLa cells, but vitamin K₁ is inactive. Specificity of quinone effects is indicated. Serum-free growth of Balb/3T3 and SV 40 transformed BaIb/3T3 (SV/T2) cells is also stimulated by coenzyme Qio with stimulation similar to HeLa cells. However, Balb/3T3 cells are not stimulated by ferricyanide, which does not increase the response to coenzyme Q₁₀. The transformed cells (SV/T2) respond better to ferricyanide alone, but the effects of coenzyme Qio and ferricyanide are not additive. Serum-free growth of HL-60 cells is stimulated dramatically by coenzyme Q₁₀. The extent of growth stimulation on HL-60 cells is almost six-fold that of HeLa or Balb/3T3 cells. The stimulation of NADH-ferricyanide reductase (a transmembrane redox enzyme) by coenzyme Q₁₀ with HL-60 cells is similar to their growth pattern in response to coenzyme Q₁₀. Unlike HL-60, HeLa and Balb/3T3 cells show little stimulation of ferricyanide reduction by coenzyme Q₁₀. The stimulatory effect on both ferricyanide reduction and cell growth by the short side-chain coenzyme Q₂ is much less than that of the long side-chain coenzyme Q₁₀. Ferricyanide reduction by HeLa cells is inhibited by coenzyme Q analogs such as 2,3-dimethoxy-5-chloro-6-naphthyl-mercapto-coenzyme Q and 2-methoxy-3-ethoxyl-5-methyl-6-hexadecyl-mercapto-coenzyme Q. However, these inhibitions are reversed by coenzyme Q₁₀. The growth inhibition of HL-60 cells by other coenzyme Q analogs, such as capsiacin can also be reversed by coenzyme Q₁₀. These data indicate that plasma membrane-based NADH oxidation or modification of the membrane quinone redox balance may be a basis for the growth stimulation.     

Proliferation of cultured glioma cells mediated by coenzyme Q<Subscript>10</Subscript> under conditions of serum deprivation

The effect of coenzyme Q₁₀ on glioma-cell proliferation under serum-deprived conditions has been studied. Our results have shown that the addition of coenzyme Q₁₀ into a serum-free culture medium enhances cell viability, stimulates cell growth, restores mitochondrial potential, and increases the quantity of energized mitochondria. It is found that coenzyme Q₁₀-induced glioma-cell proliferation in conditions of serum deficiency is a result of an intracellular reduced glutathione concentration with subsequent activation of protein kinase C, ERK1/2, and phosphoinositol-3-kinase.     

Studies on Vitamin B6 Metabolism in Microorganisms

Pyridoxine-P and pyridoxamine-P oxidase in the extract of Alcaligenes faecalis was purified and some properties of the enzyme were investigated. Several lines of evidence indicated that both pyridoxine-P oxidation and pyridoxamine-P oxidative deamination were catalyzed with a single enzyme. The enzyme is a flavoprotein, and the treatment of the enzyme with acid ammonium sulfate resolved the enzyme into apo- and coenzyme. Flavin mononucleotide reactivated the apoenzyme for the oxidation of both substrates. Physiological role of the pyridoxine-P and pyridoxamine-P oxidase was suggested in relation to the transformation of vitamin B₆ in microorganisms.     

The thermotropic properties of coenzyme Q10 and its lower homologues

The thermotropic properties of coenzymes Q₁₀, Q₉, Q₈, and Q₇ have been examined by differential scanning calorimetry and wide-angle X-ray diffraction. Typical scanning calorimetry cooling curves of coenzyme Q from the liquid state exhibit a single exothermic phase transition into a crystalline state at a temperature that decreases as the length of the polyisoprenoid side-chain substituent decreases. Upon subsequent heating, the molecules undergo a series of thermal events which precede the main crystalline-to-liquid endothermic phase transition. The temperature of these transitions increases with increasing chain length. The crystallization phase transition temperature depends markedly on the rate at which the sample is cooled and increases with decreasing scan rate; the temperature of the melting endotherm is not markedly affected by the scan rate. Detailed calorimetric studies of coenzyme Q₁₀ indicate that two crystalline states are formed, one at relatively high cooling rates to low temperatures and the other when preparations are cooled slowly from the liquid state to relatively high temperatures. Heating the crystalline phase formed by rapid cooling causes its transformation into the phase observed by cooling slowly. X-ray diffraction analysis confirmed the existence of these two crystal phases in coenzymes Q₉ and Q₁₀ and the transformation from the rapidly crystallized form to the more ordered form associated with slower cooling rates. At body temperature (310 K) under equilibrium conditions coenzyme Q₁₀ exists in an ordered crystalline phase; the implications of the thermotropic behavior of coenzyme Q₁₀ on mitochondrial functionin vitro andin vivo are discussed.     

Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration

COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q₂. Rescue of respiration by Q₂ is a characteristic of mutants blocked in coenzyme Q₆ synthesis. Unlike Q₆ deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q₆. The physiological significance of earlier observations that purified Coq10p contains bound Q₆ was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q₂. This suggests that in vivo binding of Q₆ by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p.     

Studies on reduced and oxidized coenzyme Q (ubiquinones). II. The determination of oxidation-reduction levels of coenzyme Q in mitochondria,microsomes and plasma by high-performance liquid chromatography

Reduced and oxidized coenzyme Q₁₀ (Q₁₀H₂ and Q₁₀) in guinea-pig liver mitochondria were rapidly extracted and determined by high-performance liquid chromatography (HPLC). The percentages of Q₁₀H₂ as compared to the total (sum of Q₁₀ and Q₁₀H₂) were increased by the addition of respiratory substrates such as succinate, malate and β-hydroxybutyrate (State 4). The levels of Q₁₀H₂ in State 4 were increased more extensively with electron-transport inhibitors such as KCN, NaN₃ and antimycin A. These results indicate that the method for determining Q₁₀H₂ and Q₁₀ by HPLC is quite useful for investigation of the physiological function of coenzyme Q in mitochondria and other organelles. The reduced and oxidized coenzyme Q levels of rat liver mitochondria, which contain both coenzyme Q₉ and coenzyme Q₁₀, were measured simultaneously. The results suggest that coenzymes Q₉ and Q₁₀ play a similar role as an electron carriers. The liver microsomes of guinea-pig contained approx. 133 nmol total coenzyme Q₁₀ per g protein. The Q₁₀H₂ levels of microsomes were increased from 46.5 to 67.5 and 64.8% with NADH and NADPH, respectively. The plasma levels of total coenzyme Q were 0.92 μg/ml for man, 0.35 μg/ml for guinea-pig and 0.27 μg/ml for rat. The reduced coenzyme Q were also present in those plasma samples. The levels of reduced coenzyme Q were 51.1, 48.9 and 65.3%, respectively.     

On the Partly Reduced Coenzyme Q Isolated from “Rhodotorula lactose” IFO 1058 and Its Relation to the Taxonomic Position

From the intact cells of “Rhodotorula lactosa” R1 (IFO 1058), a new coenzyme Q, which has a different mobility on paper chromatograms from other five naturally occurring homologs of the coenzyme Q series, was isolated and purified as a crystalline state. The chemical analyses such as UV and IR absorption spectrophotometries, and NMR and mass spectrometries revealed that the material, mp 28.7~28.9°C, was identified as a Co Q₁₀ derivative with the reduced C₅ unit in the isoprenoid side chain terminal remote from the quinone nucleus, Co Q₁₀ (H–10). The strain R 1 with such a unique coenzyme Q system is, concerning its taxonomic position, discussed in connection with other criteria.     

Coordinated, coenzyme Q reversible, 2,5-dibromothymoquionine inhibition of electron transport and ATPase in Escherichia coli.

2,6-dibromothymoquinone (DBMIB) and other coenzyme Q analogs partially inhibit electron transport and the membrane-bound Mg⁺⁺ stimulated ATPase of $\text{E. coli}$ membranes. The inhibitions by DBMIB are fully reversed by coenzyme Q₆, and other analogs show partial reversal by coenzyme Q₆. Electron transport reactions inhibited are NADH and lactate oxidase, NADH menadione reductase, lactate phenazinemethosulfate reductase and duroquinol oxidase. The concentrations of DBMIB required are similar for electron transport and ATPase inhibition and inhibitions are all increased by uncouplers. Electron transport and ATPase are not inhibited in a DBMIB insensitive mutant. Soluble ATPase extracted from the membranes does not show DBMIB inhibition under either high or low Mg⁺⁺ conditions. Lipophilic chelators show additional inhibition over DBMIB. It appears that coenzyme Q functions at three sites in $\text{E. coli}$ electron transport where ATPase activity is controlled. Coenzyme Q deficient mutants also show decreased electron transport and ATPase activity which is restored by coenzyme Q.     

A novel caffeine dehydrogenase in Pseudomonas sp. strain CBB1 oxidizes caffeine to trimethyluric acid

A unique heterotrimeric caffeine dehydrogenase was purified from Pseudomonas sp. strain CBB1. This enzyme oxidized caffeine to trimethyluric acid stoichiometrically and hydrolytically, without producing hydrogen peroxide. The enzyme was not NAD(P)⁺ dependent; coenzyme Q₀ was the preferred electron acceptor. The enzyme was specific for caffeine and theobromine and showed no activity with xanthine.     

Reduction of ascorbate free radical by the plasma membrane of synaptic terminals from rat brain

Synaptic plasma membranes (SPMV) decrease the steady state ascorbate free radical (AFR) concentration of 1 mM ascorbate in phosphate/EDTA buffer (pH 7), due to AFR recycling by redox coupling between ascorbate and the ubiquinone content of these membranes. In the presence of NADH, but not NADPH, SPMV catalyse a rapid recycling of AFR which further lower the AFR concentration below 0.05 μM. These results correlate with the nearly 10-fold higher NADH oxidase over NADPH oxidase activity of SPMV. SPMV has NADH-dependent coenzyme Q reductase activity. In the presence of ascorbate the stimulation of the NADH oxidase activity of SPMV by coenzyme Q₁ and cytochrome c can be accounted for by the increase of the AFR concentration generated by the redox pairs ascorbate/coenzyme Q₁ and ascorbate/cytochrome c. The NADH:AFR reductase activity makes a major contribution to the NADH oxidase activity of SPMV and decreases the steady-state AFR concentration well below the micromolar concentration range.     

Inhibition by styrylpyridines of purified rat and bovine brain choline acetyltransferase

Nine styrylpyridine analogs were tested as inhibitors of choline acetyltransferase which had been highly purified from rat cerebrum and bovine caudate nuclei. In general, concentrations required to achieve 50% inhibiion (I₅₀ values) were in the micromolar range. For some analogs, I₅₀ values were similar to those obtained previously by other investigators who used less purified enzyme preparations. With certain analogs, however, the measured values of I₅₀ changed as the transferase became more purified, which may indicate the presence in the extract of other molecules which can interact with the enzyme. The methods used in purification of the enzyme suggest that the molecule which modifies the activity of CAT is probably a protein. The mode of inhibition by naphthylvinylpyridinium was found to be uncompetitive with respect to both choline and acetyl coenzyme A for both the rat and bovine transferases.     

Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.     

Effect of the side chain structure of coenzyme Q on the steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase

Steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase using coenzyme Q with two isoprenoid unit (Q₂) or with a decyl group (DQ) show an ordered sequential mechanism in which the order of substrate binding and product release is NADH-Q₂ (DQ) -Q₂H₂ (DQH₂)-NAD⁺ in contrast to the order determined using Q₁ (Q₁-NADH-NAD⁺-Q₁H₂) (Nakashima et al., J. Bioenerg. Biomembr. 34, 11–19, 2002). The effect of the side chain structure of coenzyme Q suggests that NADH binding to the enzyme results in a conformational change, in the coenzyme Q binding site, which enables the site to accept coenzyme Q with a side chain significantly larger than one isoprenoid unit. The side chains of Q₂ and DQ bound to the enzyme induce a conformational change in the binding site to stabilize the substrate binding, while the side chain of Q₁ (one isoprenoid unit) is too short to induce the conformational change.     

Chromate reduction by rabbit liver aldehyde oxidase

Chromate was reduced during the oxidation of 1-methylnicotinamide chloride by partially purified rabbit liver aldehyde oxidase. In addition to 1-methylnicotinamide, several other electron donor substrates for aldehyde oxidase were able to support the enzymatic chromate reduction. The reduction required the presence of both enzyme and the electron donor substrate. The rate of the chromate reduction was retarded by inhibitors of aldehyde oxidase but was not affected by substrates or inhibitors of xanthine oxidase. These results are consistent with the involvement of aldehyde oxidase in the reduction of chromate by rabbit liver cytosolic enzyme preparations.     

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc₁ complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q_i site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q_o site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A₃ in a fashion similar to the Q_o site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q_i and the Q_o sites of the fungal cytochrome bc₁ complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc₁ complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q_o and Q_i sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc₁ complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q_i and Q_o sites. Moreover, crystal structure of chicken cytochrome bc₁ complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q_i and Q_o sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc₁ inhibitor that acts at both of the active sites of this enzyme.