Inactivation of mitochondrial ATPase by ultraviolet light

The present work describes experiments that show that far-ultraviolet irradiation induce the inhibition of ATPase activity in both membrane-bound and soluble F1. It was also found that ultraviolet light promotes the release of tightly bound adenine nucleotides from F1-ATPase. Experiments carried out with submitochondrial particles indicate that succinate partially protects against these effects of ultraviolet light. Titration of sulfhydryl groups in both irradiated submitochondrial particles and soluble F1-ATPase indicates that a conformational change induced by photochemical modifications of amino acid residues appears involved in the inactivation of the enzyme. Finally, experiments are described which show that the tyrosine residue located in the active site of F1-ATPase is modified by ultraviolet irradiation.     

Pig heart mitochondrial ATPase : properties of purified and membrane-bound enzyme. Effects of flavonoids.

Soluble ATPase (F1) has been purified from pig heart mitochondria. The purified enzyme had a high specific activity and was homogeneous as checked by ultracentrifugation and electrofocusing. It could be dissociated into subunits by cold-treatment or sodium dodecyl sulfate denaturation. The molecular weights of the two major and three minor subunits could be estimated by sodium dodecyl sulfate gel electrophoresis. The native enzyme had an isoelectric point of 5.2 while the cold-denatured enzyme showed three main bands focusing at pH 5.0, 5.2, and 5.4. Kinetic properties (Vm and Km (atp) have been compared for the soluble and membrane bound ATPase in presence of various anions. Inhibitory effects of Quercetin and other flavonoids have been tested in order to get an insight on the interaction between ATPase and its natural inhibitor.     

Rabbit heart mitochondrial hexokinase: Solubilization and general properties

In rabbit heart, results show that two isoenzymes of hexokinase (HK) are present. The enzymatic activity associated with mitochondria consists of only one isoenzyme; according to its electrophoretic mobility and its apparent K_m for glucose (0.065 mm), it has been identified as type I isoenzyme. The bound HK I exhibits a lower apparent K_m for ATPMg than the solubilized enzyme, whereas the apparent K_m for glucose is the same for bound and solubilized HK. Detailed studies have been performed to investigate the interactions which take place between the enzyme and the mitochondrial membrane. Neutral salts efficiently solubilize the bound enzyme. Digitonin induces only a partial release of the enzyme bound to mitochondria; this result could be explained by the existence of contacts between the outer and the inner mitochondrial membranes [C. R. Hackenbrock (1968)Proc. Natl. Acad. Sci. USA61, 598–605]. Furthermore, low concentrations (0.1 mm) of glucose 6-phosphate (G6P) or ATP^4? specifically solubilize hexokinase. The solubilizing effect of G6P and ATP^4?, which are potent inhibitors of the enzyme, can be prevented by incubation of mitochondria with P_i or Mg²⁺. In addition, enzyme solubilization by G6P can be reversed by Mg²⁺ only when the proteolytic treatment of the heart homogenate is omitted during the course of the isolation of mitochondria. These results concerning the interaction of rabbit heart hexokinase with the outer mitochondrial membrane agree with the schematic model proposed by Wilson [(1982) Biophys. J.37, 18–19] for the brain enzyme. This model involves the existence of two kinds of interactions between HK and mitochondria; a very specific one with the hexokinase-binding protein of the outer mitochondrial membrane, which is suppressed by glucose 6-phosphate, and a less specific, cation-mediated one.     

Mg2+-induced ADP-dependent inhibition of the ATPase activity of beef heart mitochondrial coupling factor F1.

Incubation of F₁ in the presence of Mg²⁺ results in a pronounced lag in its ATPase activity measured with the ATP-regenerating system. A decrease of the initial rate of ATPase induced by Mg²⁺ is also observed when free nucleotides were separated from the enzyme by Sephadex gel filtration. No inhibition is observed when F₁ treated to remove tightly bound nucleotides was preincubated in the presence of Mg²⁺. Mg²⁺-induced inhibition of ATPase activity of nucleotide-depleted F₁ can be restored by an addition of low concentrations of ADP. In all cases the inhibited ATPase can be activated by the ADP-removing system /phosphoenol pyruvate + pyruvate kinase/. It is concluded that i/ Mg²⁺-induced inhibition of the ATPase activity of F₁ is due to the formation of an inactive F₁. ADP complex; and ii/ unusual inhibition of oligomycin-sensitive ATPase by ADP /Fitin et al., Biochem. Biophys. Res. Communs. 1979, $\text{86}$, 434/ is directed to F₁ component of the complete mitochondrial ATPase system.     

The interaction of the potential-sensitive molecular probe merocyanine 540 with phosphorylating beef heart submitochondrial particles under equilibrium and time-resolved conditions

The interaction of the potential-sensitive extrinsic molecular probe merocyanine 540 ( M540 ) with phosphorylating submitochondrial particles has been investigated under equilibrium and time-resolved conditions. The addition of ATP to a M540 -membrane suspension produces oligomycin and CCCP-sensitive spectral changes with absolute maxima near 490, 530, and 565 nm; a 1- to 2-nm red shift of the dye absorption spectrum is also evident in the longer-wavelength region of the spectrum. In fixed-wavelength work, the energy-dependent optical signals were increased by the addition of nigericin and NH4Cl, and could be subsequently restored to the control level by valinomycin or KSCN, respectively. These observations suggest that M540 is specifically sensitive to the membrane-potential portion of the electrochemical gradient presumably present in the submitochondrial particle system in the presence of substrate. Binding analyses based on the Langmuir adsorption isotherm and the direct linear method indicate that the M540 dissociation constant is decreased by the presence of ATP with little or no change in the maximum number of binding sites. The M540 dissociation constant was markedly decreased when 0.1 M NaCl was present in the medium, suggesting that the association of this probe with the membrane may be subject to considerable surface charge repulsion. Results from the binding analyses indicate that the origin of the energy-dependent spectral changes may be an enhanced association of M540 with the submitochondrial particle membrane resulting from the transfer of dye from the aqueous phase to membrane-binding sites. The time course of the NADH-, ATP-, or succinate-induced signal developed slowly, on a time scale of tens of seconds, and follows a second-order rate law, suggesting that the rate-limiting step in the development of the ATP-induced M540 signal may be the transfer of dye from the aqueous phase to membrane-binding sites. The enhanced passive binding of M540 to the submitochondrial particle membrane in the presence of NaCl reduces the concentration of free dye apparently available to redistribute to the membrane when substrate is present, with a concomitant reduction in the observed pseudo-first-order and the second-order rate constants. If the effective free dye concentration is estimated from binding data and used in the plot from which the latter rate constant is obtained, the value of this constant compares favorably with the obtained in the absence of the electrolyte.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mitochondrial ATPase inactivation by interaction with its substrate

Purified F₁-ATPase is slowly inactivated by interaction, in a preincubation medium, with its substrate MgATP. Interaction with Mg²⁺ before addition of ATP to the preincubation medium is essential to induce the inactivation. This inactivation is different from other Mg²⁺-induced inhibitions previously described. Free ATP concentration is implicated in the inactivation process and a linear relationship can be established between this concentration and the number of turnovers which are necessary for total inactivation. ITP, 2′-dATP, and GTP can also induce inactivation. Although ITP and GTP are hydrolyzed at a lower rate than ATP and 2′-dATP, they induce inactivation after a smaller number of turnovers than the latter. This process closely follows a kinetics of the type described for suicide enzymes. A reaction scheme is suggested and discussed.     

Thiols related to mitochondrial ATPase and transports: unmasking upon conformational changes supported by the comparative effects of ethacrynate and dihydroethacrynate.

Comparison between the effects on various rat liver mitochondrial functions of ethacrynate, a thiol reagent inhibitor of oxidative phosphorylations [3, 4] and those of dihydroethacrynate its saturated derivative which is not a thiol reagent, has been performed. Both, ethacrynate and dihydroethacrynate increase oxygen consumption by mitochondria in state 4 (succinate as substrate) in a concentration dependent way (from 1 to 5 X 10(-4) M EA or DHEA). This activation is followed, only with ethacrynate, by an inhibition appearing sooner with higher concentrations. After preincubation or mitochondria with ethacrynate (1 to 5 X 10(-4) M), the stimulation of respiration by (ADP + Pi) is completely inhibited whereas it is only weakly affected by dihydroethacrynate at the same concentrations. Ethacrynate and dihydroethacrynate provoke variations of intramitochondrial Mg2+ and K+ levels which need energy from the respiratory chain. These are affected by Pi or (Pi + ADP) in a different way with ethacrynate and with dihydroethacrynate. After preincubation with mitochondria, ethacrynate and to a smaller extent dihydroethacrynate, inhibit partially ADP translocation; ADP increases the inhibitory effect of EA on translocation and not that of dihydroethacrynate. Ethacrynate increases the oligomycin sensitive ATPase activity and dihydroethacrynate still more. After a ten minutes preincubation with mitochondria, ethacrynate and dihydroethacrynate hardly affect the 2.4 DNP stimulated ATPase activity. Preincubation with succinate or ADP strongly increases the ethacrynate inhibition whereas it decreases dihydroethacrynate inhibition. Ethacrynate and dihydroethacrynate do not affect the efflux of Pi produced by ATP hydrolysis but ethacrynate enforces the inhibitory effect of mersalyl (Mg2+ containing medium). After ten minutes of preincubation with mitochondria, ethacrynate binds 25 nmoles of -SH/mg protein (DTNB titration) and dihydroethacrynate has no effect. These results show an effect of ethacrynate on two types of thiols linked with energy conservation mechanisms and ADP translocation. These thiols could be unmasked or made accessible by conformational modifications of the inner membrane upon energization or addition of ADP.     

Computer modeling of muscle phosphofructokinase kinetics

The kinetics of the phosphofructokinase reaction were studied by computer modeling. A general random order, two-state allosteric model, of which the Monod--Wyman--Changeux model is a limiting case, was found to most accurately reproduce the experimental observations of Pettigrew & Frieden (1979 a,b). A simplified model with Hill coefficients was found to fit almost as well. In these models substrates bind preferentially to and stabilize the enzyme in the R state, and ATPH3-, the inhibitory species, binds preferentially to and stabilizes the enzyme in the T state. Enzymatic activity is regulated by conversion from the R to the T state, which is effected by protonation, especially of the uncomplexed enzyme, but the experimental data are inadequate for accurate estimation of the pKa of the enzyme. Random order binding of substrates is an important cause of sigmoidal kinetics. Additional experiments that would aid in the discrimination among rival models are described.     

The distribution of ATPase activities in purified transverse tubular membranes

Vesiculated fragments of transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes were purified from heterogeneous microsomal membrane fractions of chicken breast muscle by a modification of an iterative calcium-oxalate loading technique. The distribution of ATPase activities were determined for the TT and SR and were compared to enriched fractions of sarcolemma (SL) membranes. The TT membranes were characterized by high rates of magnesium-stimulated ATPase (Mg-ATPase) and 5′-nucleotidase activities but were virtually devoid of calcium-stimulated, magnesium-dependent ATPase (Ca,Mg-ATPase) activity. Moderate levels of a latent sodium and potassium-stimulated ATPase (Na,K-ATPase) were observed for TT membranes when unmasked with valinomycin and monensin. In contrast to the behavior of TT membranes, highly purified SR membranes displayed an active Ca,Mg-ATPase but negligible Na,K-ATPase, Mg-ATPase, and 5′-nucleotidase activities. High levels of Na,K-ATPase and 5′-nucleotidase activities were observed for SL membranes; however, the SL displayed no appreciable Ca,Mg-ATPase and Mg-ATPase activities. The lack of significant Mg-ATPase activity in the SR and SL fractions suggested that the Mg-ATPase was uniquely associated with the TT membranes. The TT Mg-ATPase was further characterized by its pH and temperature dependences, and its sensitivity to pharmacologic agents. The Mg-ATPase of the TT was insensitive to inhibition by sodium azide and oligomycin in concentrations shown to exert maximum inhibition on the F₁ ATPase of submitochondrial particles. The Mg-ATPase was also resistant to the effects of ouabain and orthovanadate in concentrations which abolished the Na,K-ATPase and Ca,Mg-ATPase activities of the SL and SR, respectively. The Mg-ATPase displayed temperature and pH optima (25 °C, pH 7.3) which were distinguishable from the Ca,Mg-ATPase (45 °, pH 7.0) of highly purified SR fractions but which were very similar to the temperature and pH dependencies of the mixed microsomal fractions (MMF) from which the TT membranes were derived. Similarities in the pH and temperature dependencies of the TT and MMF Mg-ATPases plus the absence of appreciable Mg-ATPase activity in highly purified SR membranes suggests that the “basic” Mg-ATPase often seen in crude SR fractions may originate from TT membrane contamination. The resistance of the TT Mg-ATPase to inhibition by the pharmacologic agents tested plus its unique temperature and pH dependences indicate that this ATPase is distinguishable from other ATPases and may, therefore, be of value as a specific biochemical marker for TT membranes.     

Yeast mitochondrial DNA characterization after ultraviolet irradiation

Yeast mitochondrial (mtDNA) ³H-labelled was isolated from exponential phase cells after ultraviolet light irradiation. Both the size and amount of mtDNA were found to be reduced during a 40-h liquid-holding (LH) period in non-growth medium following irradiation as compared to the mtDNA recovered from nonirradiated cells under similar conditions. After the LH period, previously irradiated cells were suspended in growth medium containing [¹⁴C]adenine. Double labelled mtDNA (³H and ¹⁴C) was isolated from cells samples removed during new growth. A recovery in the amount and size of mtDNA was observed in irradiated cells during new growth. These biochemical studies agree with the observed loss and recovery of mtDNA genetic markers in UV-irradiated exponential phase yeast after a period of LH and new growth resp.     

Effect of steroid implants on sexual behavior of beef calves

The sexual behavior of 53 beef heifers and 66 beef steers was observed and recorded for 7 days following insertion of subcutaneous ear implants at 2 months of age. Thirty-five of the heifers were implanted with Synovex S (200 mg of progesterone and 20 mg of estradiol benzoate). Forty-four of the steers were implanted with Synovex H (200 mg of testosterone and 20 mg of estradiol benzoate). The remaining 18 heifers and 22 steers served as nonimplanted controls. Synovex S treatment increased the incidence of mounting (P < 0.005) and mounting receptivity (P < 0.005) of heifers, while Synovex H treatment had only slight effect, if any, on the mounting behavior (P < 0.25) and mounting receptivity (P < 0.25) of steers. Both steers and heifers mounted heifers more frequently than steers (P < 0.005). Heifers were mounted with similar frequencies by both heifers and steers (P < 0.90), while steers were mounted only by heifers (P < 0.15).     

Improvement of the anoxia-induced mitochondrial dysfunction by membrane modulation

The mitochondrial dysfunction induced by anoxia in vitro was improved with chlorpromazine, cepharanthine, bromophenacyl bromide, and mepacrine without affecting phospholipid or adenine nucleotide metabolisms. The drugs inhibited lipid peroxidation by Fe²⁺, mitochondrial disruption by Ca²⁺, and membrane perturbation by lysolecithin, and retained the activity to control H⁺ permeability across mitochondrial membranes. The drugs appeared to preserve the functions by acting to suppress the development of membrane deterioration which may have resided in the deenergization of mitochondria in the absence of oxygen.     

Inhibitory properties of endogenous subunit epsilon in the Escherichia coli F1 ATPase.

Homogeneous ? bound tightly to the purified Escherichia coli ATPase (ECF₁ from which ? had been removed and strongly inhibited its ATPase activity. ECF₁ containing ? had a lower specific activity than ECF₁ missing ?, provided that the ATPase assay was carried out at relatively high concentrations of enzyme. Antiserum specific for the ? subunit stimulated the ATPase, as did diluting the enzyme, apparently by dissociating ?. When the ATPase reaction was started by the addition of enzyme, the rate of ATP hydrolysis increased progressively during the first 3 min until a linear steady-state rate was reached. A prior incubation with ATP abolished the lag period and ADP prevented the ATP effect. ECF₁ missing ? gave a linear rate of ATP hydrolysis without a lag, unless ? was rebound to it before the assay. These results suggest that ECF₁ as purified is in an inhibited state due to the presence of the ? subunit, whose interaction with ECF₁ is governed by an equilibrium binding. ATP appears to convert ECF₁ to a form which more readily binds and releases ?.     

Elevated intramitochondrial adenine nucleotides and mitochondrial function

Several groups of investigators have shown that treatment of rats with glucagon produces an increase in the adenine nucleotide content of hepatic mitochondria. It has been suggested that this enlarged pool of exchangeable nucleotides may be responsible for several of glucagon's stimulatory effects on mitochondrial functions by accelerating the transport of adenine nucleotides across the inner mitochondrial membrane. This hypothesis was tested by loading rat liver mitochondria in vitro with adenine nucleotides to supranormal levels. This procedure did result in stimulation of several metabolic and bioenergetic functions including pyruvate carboxylation, uncoupler-dependent ATPase, and succinic dehydrogenase activity but not formation of citrulline. However, a sham loading that did not increase the nucleotide content of the mitochondria was essentially as effective as the loading procedure in stimulating those functions assayed. Mitochondria, loaded in vitro with supranormal levels of adenine nucleotides, were shown to have an enlarged pool of exchangeable nucleotides. This exchange was atractyloside sensitive, but the rate of exchange was only slightly increased as a consequence of enlargement of the pool. Similarly, mitochondria isolated from glucagon-treated rats showed no increase in the rate of exchange, although the exchangeable pool was increased. There was no correlation between the rate of nucleotide exchange and the rate of the uncoupler-dependent ATPase.     

Inhibition of yeast mitochondrial ATPase by concanavalin A

Concanavalin A binds to and inhibits enzyme activity of the energy transducing ATPase from yeast mitochondria. Activity loss is completely reversed by glucose or α-methyl-d-mannose. Concanavalin A reacts with the F₁ portion of the ATPase complex, suggesting that this enzyme unit may be a glycoprotein. A major concanavalin A binding site is associated with the largest subunit of the F₁ enzyme.     

Effect of mild heat treatment on the ATPase activity and proteolytic sensitivity of myosin subfragment-1

The K+-EDTA-activated ATPase activity of chymotryptic myosin subfragment-1 (S-1) decreased by 85-90% when S-1 was incubated over a 2-h period at 35 degrees C. Addition of F-actin, ATP, or ATP analogs, such as ADP or PPi, to S-1 before incubation at 35 degrees C prevented the loss of ATPase activity. The decrease in ATPase activity was also accompanied by changes in tryptic sensitivity. Instead of the normal peptide pattern--which is comprised of three heavy chain fragments (27K, 50K, and 20K)--only two fragments (27K and 20K) appeared on the sodium dodecyl sulfate-gel electrophoregram after limited tryptic digestion of thermally treated S-1. Addition of any ligand--e.g. ATP, ADP, pyrophosphate, or actin--which prevented the loss of ATPase activity during incubation at 35 degrees C also prevented the observed change in the tryptic peptide pattern of S-1. Tryptic digested S-1, whose heavy chain has been cleaved to 27K, 50K, and 20K fragments, also lost its ATPase activity upon mild heat treatment. The heat-treated trypsin-digested S-1 was subjected to a second tryptic digestion, which resulted in the disappearance of the 50K fragment, while the 50K fragment of tryptic S-1 not subjected to heat treatment was not susceptible to additional tryptic hydrolysis. The results indicate that the structural changes, that take place specifically in the 50K region of S-1 upon mild heat treatment, lead to both the loss of the ATPase activity and the changed tryptic sensitivity of S-1.     

An inhibitory high affinity binding site for ADP in the oligomycin-sensitive ATPase of beef heart submitochondrial particles.

Kinetic evidence are presented for the existence of a high affinity inhibitory site for ADP /K_i < 10^?7 M/ in the oligomycin-sensitive ATPase of beef heart submitochondrial particles. The ATPase·ADP complex is completely inactive in the ATPase reaction; it can be converted into active ATPase in a slow ATP-dependent reaction. The dependence of a first order rate constant for activation of the enzyme·ADP complex on concentration of ATP gives a K_m value equal to that for ATP in the ATPase reaction. The data obtained suggest that the membrane-bound ATPase complex contains two kinetically distinct nucleotide-binding centers, i.e. center 1 binds ATP or ADP with a formation of enzyme-substrate or enzyme-competitive inhibitor complexes: center 2 binds ADP with a formation of a complex which is able to bind ATP in center 1 and unable to hydrolyze the bound ATP. The binding of ATP or ADP in center 1 changes the reactivity of center 2 towards ADP.