Oxidation of mitochondrial pyridine nucleotides by aglycone derivatives of adriamycin.

Adriamycin (AdM) and related anthracyclines are potent antineoplastic agents, the clinical utility of which is limited by severe cardiotoxicity. Aglycone derivatives of AdM have recently been reported to trigger the release of Ca2+ from isolated, preloaded rat heart mitochondria and to modify mitochondrial sulfhydryl (-SH) groups. Both mitochondrial Ca2+ retention and -SH status are sensitive to mitochondrial NAD(P)+/NAD(P)H ratios. This investigation examined the effects of AdM and its aglycone derivatives on the pyridine nucleotide redox status of isolated, intact heart mitochondria with the following results. (i) AdM aglycones induced the slow, Ca2(+)-independent oxidation of mitochondrial NAD(P)H. Oxidation was proportional to aglycone concentration between 5 and 60 microM. (ii) In terms of potency, 7-deoxy AdM aglycone greater than or equal to 7-hydroxy AdM aglycone much greater than AdM. (iii) Inhibitor data suggested that NAD(P)H oxidation reflects the rotenone-insensitive reduction of AdM aglycone and subsequent electron transfer to O2 generating superoxide. (iv) NAD(P)H oxidation mediated by AdM aglycone could be distinguished from the Ca2(+)-dependent NAD(P)H oxidation associated with mitochondrial Ca2+ release. This communication is the first to describe redox interactions of AdM with intact mitochondria.     

Inhibition of mitochondrial oxidative phosphorylation by adriamycin

The antitumour antibiotic, adriamycin, inhibited oxidative phosphorylation in freshly prepared mitochondria from the heart, liver and kidney of the rat. It abolished respiratory control and stimulated ATPase activity. Succinate oxidation by heart mitochondria was extremely sensitive to the drug when hexokinase was present in the reaction medium. The sensitive site has been identified to lie in the region between the succinate dehydrogenase flavoprotein and ubiquinone of the respiratory chain.     

Mechanism of lignin inhibition of enzymatic biomass deconstruction

Background

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process.

Results

By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose binding of TrCel7A (Y466, Y492, and Y493).

Conclusions

Lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.

     

Mechanism of inhibition of the prothrombinase complex by a covalent antithrombin-heparin complex

Factor-Xa assembly into the prothrombinase complex decreases its availability for inhibition by antithrombin + unfractionated heparin (AT + UFH). We have developed a novel covalent antithrombin-heparin complex (ATH), with enhanced anticoagulant actions compared with AT + UFH. The present study was performed to extend understanding of the anticoagulant mechanisms of ATH by determining its inhibition of Xa within the critical prothrombinase. Discontinuous inhibition assays were performed to determine final k(2) values for inhibition of Xa. Fluorescent microscopy was conducted to evaluate inhibitor-prothrombinase interactions. The k(2) for inhibition of prothrombinase versus free Xa by AT + UFH was lower, whereas for ATH were much higher. Relative to intact prothrombinase, rates for Xa inhibition by AT + UFH in complexes devoid of prothrombin/vesicles/factor-Va were higher. For ATH, exclusion of prothrombin decreased k(2), removal of vesicles increased k(2) and exclusion of factor-Va gave no effect. While UFH may displace Xa from prothrombinase, Xa is detained within prothrombinase during ATH reactions. We confirm prothrombinase hinders inhibitory action of AT + UFH, whereas ATH is less affected with prothrombin being a key component in the complex responsible for the opposing effects. Overall, the results suggest that covalent linkage between AT-heparin assists access and neutralization of complexed Xa, with concomitant inhibition of prothrombinase function compared with conventional non-conjugated heparin.     

Mechanism of inhibition of rat liver bilirubin UDP-glucuronosyltransferase by triphenylalkyl derivatives

A series of potent and competitive inhibitors of UDP-glucuronosyltransferase derived from 7,7,7-triphenylheptanoic acid has been synthesized in order to probe the active site of the isozyme involved in the glucuronidation of the endogenous toxic compound, bilirubin IXα. Like triphenylalkylcarboxylic acids, triphenyl alcohols were found to be very effective competitive inhibitors of the reaction (K_i 12 to 180 μM). Superimposition of the best inhibitors with bilirubin by computer modeling showed a marked spatial similarity, which accounts for the observed competitive-type inhibition. The bulky triphenylmethyl moiety of the inhibitor superimposed well on the part of the bilirubin molecule containing three of the four pyrrole rings. In agreement, substitution of the triphenylmethyl moiety by planar structures such as fluorenyl or indenyl rings completely suppressed the inhibition. In addition, the weak inhibition exerted by the shortest carboxylic acids could be related to the higher acidity of these molecules. The inhibition potency depended on the acidity of the molecules; the more acidic, the less inhibitory, suggesting that the presence of a negative charge on the inhibitor molecule prevents bilirubin glucuronidation. Based on these results, a reaction mechanism for bilirubin glucuronidation is postulated. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 19–27, 1998     

Reversible inhibition of the mitochondrial ubiquinol-cytochrome c oxidoreductase complex (complex III) by ethoxyformic anhydride

The mitochondrial ubiquinol-cytochrome c oxidoreductase (complex III) is inhibited by ethoxyformic anhydride (EFA). The inhibition is readily reversed by hydroxylamine, suggesting the involvement of essential histidyl or possibly tyrosyl residues. The spectrum of ethoxyformylated complex III in the UV region showed a peak at 238 nm, indicative of N-(ethoxyformyl)histidine. Addition of hydroxylamine caused a large decrease of the 238-nm peak, which amounted to 16 mol of (ethoxyformyl)histidine/mol of cytochrome c1. Hydroxylamine addition to ethoxyformylated complex III also caused a small change at about 280 nm, which could be due to reversal of 1.6 O-ethoxyformylated tyrosyl residues/mol of cytochrome c1. Among many inhibitors of the cytochrome bc1 region of the respiratory chain, EFA is the only reagent known to cause reversible inhibition by covalent modification of amino acid residues. The inhibition site of EFA was determined to be between cytochromes b-562 and c1. However, unlike antimycin, which also inhibits in the same region, EFA did not promote the reduction of cytochrome b-566 in particles treated with substrates. In addition, it was found that EFA inhibits proton translocation in the cytochrome bc1 region and is a more effective electron transport inhibitor when added to reduced particles as compared to oxidized particles. These results together with the strong possibility that the EFA target is a histidyl or possibly a tyrosyl residue have been discussed in relation to the mechanism of proton translocation by complex III.     

The mechanism of the inhibition of the mitochondrial pyruvate transportater by alpha-cyanocinnamate derivatives.

Pyruvate transport into rat liver mitochondria is inhibited by a variety of thiol reagents. alpha-Cyanocinnamate and its derivates, potent and reversible inhibitors of pyruvate transport, react reversibly with mercaptoethanol and cysteine to form addition products. It is concluded that these inhibitors react with an essential thiol group on the pyruvate carrier.     

Mechanism of inhibition of human leukocyte elastase by two cephalosporin derivatives.

The cephalosporin derivatives L 658758 [1-[[3-(acetoxymethyl)-7 alpha-methoxy-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-en-2-yl]carbonyl]proline S,S-dioxide] and L 659286 [1-[[7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo- 1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-aza-(6R)-bicyclo[4.2.0]-o ct-2-en-2-yl]carbonyl]pyrrolidine S,S-dioxide] are mechanism based inhibitors of human leukocyte elastase (HLE). The mechanism involves initial formation of a Michaelis complex followed by acylation of the active site serine. The group on the 3'-methylene is liberated during the course of these reactions, followed by partitioning of an intermediate between hydrolysis to regenerate active enzyme and further modification to produce a stable HLE-inhibitor complex. The partition ratio of 2.0 obtained for the reaction with L 658758 approaches that of an optimal inhibitor. These compounds are functionally irreversible inhibitors as the recovery of activity after inactivation is slow. The half-lives at 37 degrees C of the L 658758 and L 659286 derived HLE-I complexes were 9 and 6.5 h, respectively. The complexes produced by both inhibitors are similar chemically since the thermodynamic parameters for activation to regenerate active enzyme are essentially identical. The free energy of activation for this process is dominated primarily by the enthalpy term. The stability of the final complexes likely arises from Michael addition on the active site histidine to the 3'-methylene.     

Recovery of adriamycin induced mitochondrial dysfunction in liver by selenium

Adriamycin (ADR) is a chemotherapeutic drug. Its toxicities may associate with mitochondriopathy. Selenium (Se) is a trace element for essential intracellular antioxidant enzymes. However, there is lack of data related to the effect of selenium on the liver tissue of ADR-induced mitochondrial dysfunction. The study was to investigate whether Se could restore mitochondrial dysfunction of liver-exposed ADR. Rats were divided into four groups as a control, ADR, Se, co-treated ADR with Se groups. The biochemical measurements of the liver were made in mitochondrial and cytosol. ATP level and mitochondria membrane potential (MMP) were measured. Total oxidant (TOS), total antioxidant (TAS) status were determined and oxidative stress index (OSI) was calculated by using TOS and TAS. ADR increased TOS in mitochondria and also oxidative stress in mitochondria. ADR sligtly decreased MMP, and ATP level. Partial recovery of MMP by Se was able to elevate the ATP production in cotreatment of ADR with Se. TOS in mitochondria and cytosol was diminished, as well as OSI. We concluded that selenium could potentially be used against oxidative stress induced by ADR in liver, resulting from the restoration of MMP and ATP production and prevention of mitochondrial damage in vivo.     

Origin of selective inhibition of mitochondrial complex I by pyridinium-type inhibitor MP-24.

Positively charged pyridiniums are unique inhibitors to probe the structural and functional properties of the ubiquinone reduction site of bovine heart mitochondrial complex I. In this study, we synthesized a series of neutral as well as pyridinium analogues of MP-24 (N-methyl-4-[2-methyl-2-(p-tert-butylbenzyl)propyl]pyridinium), a selective inhibitor of one of the two proposed binding sites of these pyridinium-type inhibitors of complex I (H. Miyoshi et al., J. Biol. Chem. 273 (1998) 17368-17374), to elucidate the origin of its selectivity. Inhibitory potencies of all neutral and pyridinium analogues with tetraphenylboron (TPB(-)), which forms an ion-pair with pyridiniums, were comparable, although the degrees of selective inhibition by pyridiniums without TPB(-) were entirely different. In contrast to MP-24, the dose-response curves of nonselective pyridiniums and all neutral analogues were not affected by incubation conditions. These results strongly suggested that the process of the inhibitor passage to the binding sites is responsible for the selective inhibition.     

Mechanism of local anesthetic effect on mitochondrial ATP synthase as deduced from photolabelling and inhibition studies with phenothiazine derivatives

The mode of interaction between mitochondrial ATP synthase and two phenothiazine derivatives, chlorpromazine (CPZ) and trifluoperazine (TFP), was studied as a model for the interaction of local anesthetic drugs with membrane proteins. Photolabelling experiments demonstrated that CPZ and TFP interact with various subunits of either the peripheral F1 moiety of the membrane-embedded F0 sector. Both drugs, however, labelled the membrane sector much more heavily. Qualitative differences in labelling were observed between CPZ and TFP, indicating non-identical sites of interaction. These diversities appeared related to the different hydrophobicities of the two drugs since: (a) TFP, which has a higher lipid/water partition coefficient, labelled the more hydrophobic subunits more markedly than CPZ; (b) reduced glutathione, a hydrophilic free radical scavenger that does not penetrate the membrane continuum, had a negligible effect on the labelling by TFP, whereas it reduced the labelling of various subunits by CPZ; (c) the labelling by [3H]TFP was poorly antagonized by cold CPZ, whereas it was almost totally prevented by fluphenazine, a phenothiazine similar to TFP in hydrophobic character. Consistently, double-inhibition experiments showed that TFP and fluphenazine are mutually exclusive inhibitors of mitochondrial ATP synthase, whereas TFP and CPZ are mutually nonexclusive. The nature of the phospholipid bilayer influenced neither the labelling nor the inhibition patterns. The complex of these data indicate that tertiary amine local anesthetics affect the activity of membrane proteins by interacting with a multiplicity of relatively aspecific hydrophobic sites located preferentially, but not exclusively, on the membrane-embedded domains. It is suggested that at least two phenothiazine derivatives of different hydrophobicities be used in photolabelling experiments, before any generalization is made, since the molecular targets of these drugs vary according to their hydrophobic character.     

S-nitrosothiol inhibition of mitochondrial complex I causes a reversible increase in mitochondrial hydrogen peroxide production

We found that reversible inactivation of mitochondrial complex I by S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in isolated rat heart mitochondria resulted in a three-fold increase in H2O2 production, when mitochondria were respiring on pyruvate and malate, (but not when respiring on succinate or in the absence of added respiratory substrate). The inactivation of complex I and the increased H2O2 production were present in mitochondria washed free of SNAP or NO, but were partially reversed by light or dithiothreitol, treatments known to reverse S-nitrosation. Specific inhibition of complex I with rotenone increased H2O2 production to a similar extent as that caused by SNAP. The results suggest that S-nitrosation of complex I can reversibly increase oxidant production by mitochondria, which is potentially important in cell signalling and/or pathology.     

Inhibitory effects on mitochondrial complex I of semisynthetic mono-tetrahydrofuran acetogenin derivatives

Modifications in the terminal alpha,beta-unsaturated gamma-methyl-gamma-lactone moiety or in the alkyl chain that links this terminal gamma-lactone with the alpha,alpha'-dihydroxylated THF system of the natural mono-tetrahydrofuranic acetogenins, annonacin and annonacinone, led to the preparation of eight semisynthetic derivatives. Their inhibitory effects on mitochondrial complex I is discussed and compared with that of the classical complex I inhibitor, rotenone.