Temperature dependence of mitochondrial respiratory activities

Arrhenius plots of succinate oxidase activity in intact beef heart mitochondria show a clear transition from a low to a high activation energy at 27°C. This temperature is significantly higher than that observed for ATPase (17°C). Arrhenius plots of succinate-cytochromec reductase and cytochromec oxidase also show anomalous curves; while the latter has a breakpoint (at 26°C) only when assayed manometrically, the former has a break at only 20°C.The succinoxidase activity of lipid-deficient mitochondria depends upon addition of exogenous phospholipids. Unsaturated phospholipids are more active than saturated phospholipids but the latter become very effective in restoration of succinoxidase at increasing temperatures. It is suggested that a liquid-crystalline state of the phospholipids is required for correct binding to the lipid-depleted membrane and for restoration of respiratory activity. The is no clear correlation between the above mentioned effects in lipid deficient mitochondria and the transitions in the Arrhenius plots of intact mitochondria.     

Temperature dependence of mitochondrial oligomycin-sensitive proton transport ATPase

The temperature dependence of the oligomycin-sensitive ATPase (complex V) kinetic parameters has been investigated in enzyme preparations of different phospholipid composition. In submitochondrial particles, isolated complex V, and complex V reconstituted in dimirystoyl lecithin vesicles, the Arrhenius plots show discontinuities in the range 18–28°C, while no discontinuity is detected with dioleoyl lecithin recombinant. Van't Hoff plots ofK _m also show breaks in the same temperature interval, with the exception of the dioleoylenzyme vesicles, whereK _m is unchanged. Thermodynamic analysis of the ATPase reaction shows that DMPC-complex V has rather larger values of activation enthalpy and activation entropy below the transition temperature (24°C) than those of the other preparations, while all enzyme preparations show similar free energies of activation (14.3–18.5 kcal/mol). The results indicate that temperature and lipid composition influence to a different extent both kinetic and thermodynamic parameters of ATP hydrolysis catalyzed by the mitochondrial ATPase.     

Temperature dependence of the enzyme-substrate recognition mechanism

We determined the crystal structure of the liganded form of alpha-aminotransferase from a hyperthermophile, Pyrococcus horikoshii. This hyperthermophilic enzyme did not show domain movement upon binding of an acidic substrate, glutamate, except for a small movement of the alpha-helix from Glu16 to Ala25. The omega-carboxyl group of the acidic substrate was recognized by Tyr70* without its side-chain movement, but not by positively charged Arg or Lys. Compared with the homologous enzymes from Thermus thermophilus HB8 and Escherichia coli, it was suggested that the more thermophilic the enzyme is, the smaller the domain movement is. This rule seems to be applicable to many other enzymes already reported.     

Pituitary thyrotropin-releasing hormone receptors: local anesthetic effects on binding and responses

Pharmacological agents are widely used to probe the mechanism of action of TRH. A number of these drugs behave as local anesthetics at high concentrations. The effect of local anesthetics on the binding of [3H]Me-TRH to specific receptors was studied using the GH4C1 line of rat pituitary tumor cells. [3H]Me-TRH binding was inhibited by classical local anesthetics with the order of potency (IC50 values): dibucaine (0.37 mM) greater than tetracaine (1.2 mM) greater than lidocaine (3.3 mM) greater than procaine and benzocaine (greater than 10 mM). IC50 values for other drugs with local anesthetic properties that inhibited [3H]Me-TRH were: 100 microM trifluoperazine, 100 microM imipramine, 170 microM chlorpromazine, 300 microM verapamil, and 700 microM propranolol. Inhibition by tetracaine and verapamil increased as the pH was raised from 6 to 8.5, indicating that the free base form of the amine drugs was the inhibitory species, and the local anesthetic effect was greater at 37 C than at 24 C or 0 C. [3H]Me-TRH binding to receptors in isolated membranes was inhibited to the same extent as binding to receptors on intact cells. Local anesthetics were 3- to 20-fold less potent at inhibiting [3H]Me-TRH to digitonin-solubilized receptors than binding to intact cells. In contrast, the potency of chlordiazepoxide, a putative TRH antagonist, to inhibit [3H]Me-TRH binding was equal using cells and solubilized receptors (IC50 = 10 microM). Local anesthetics inhibited TRH-stimulated PRL release and also inhibited basal PRL secretion and secretion stimulated by two nonhormonal secretagogues, (Bu)2cAMP and a phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of local anesthetic inhibition of neuronal sodium currents. pH and hydrophobicity dependence.

This study assesses the importance of local anesthetic charge and hydrophobicity in determining the rates of binding to and dissociation from neuronal Na channels. Five amide-linked local anesthetics, paired either by similar pKa or hydrophobicity, were chosen for study: lidocaine, two tertiary amine lidocaine homologs, a neutral lidocaine homolog, and bupivacaine. Voltage-clamped nodes of Ranvier from the sciatic nerve of Bufo marinus were exposed to anesthetic externally, and use-dependent ("phasic") block of Na current was observed. Kinetic analysis of binding (blocking) rates was performed using a three parameter, piecewise-exponential binding model. Changes in extracellular pH (pHo) were used to assess the role of drug protonation in determining the rate of onset of, and recovery from, phasic block. For those drugs with pKa's in the range of pHo tested (6.2-10.4), the forward binding rate during a depolarizing pulse increased at higher pH, consistent with an increase in either intracellular or intramembrane concentration of drug. The rate for unbinding during depolarization was independent of pHo. The dissociation rate between pulses also increased at higher pHo. The pHo dependence of the dissociation rate was not consistent with a model in which the cation is trapped relentlessly within a closed channel. Quantitative estimates of dissociation rates show that the cationic form of lidocaine dissociates at a rate of 0.1 s-1 (at 13 degrees C); for neutral lidocaine, the dissociation rate is 7.0 s-1. Furthermore, the apparent pKa of bound local anesthetic was found to be close to the pKa in aqueous solution, but different than the pKa for "free" local anesthetic accessible to the depolarized channel.     

Possible mechanism of long-term anti-arrhythmia and local anesthetic effect of quaternary ammonium compounds

In experiments with dialized neurons of L. stagnalis mollusc the recovery of Na-current (INa) after its depression by local anesthetic trimecaine and its quaternary derivative N-ethyltrimecaine (G-88) was studied. A full recovery of INa within tens of seconds after washing off trimecaine but not G-88 was observed. The half-time for vanishing of INa use-dependent depression by G-88 was 17 minutes, and there was no substantial vanishing of tonic INa block even after an hour of G-88 washing off. A hypothesis is advanced that the long recovery time of INa is one of the mechanisms providing long pharmacological action of quaternary antiarrhythmic and local anesthetic ammonium compounds.     

Temperature dependence of the atractyloside-induced mitochondrial Ca2+ release

1. Mitochondrial Ca2+, accumulated by succinate oxidation was released by addition of 50 microM atractyloside. Beside this Ca2+ efflux, a large oxidation of pyridine nucleotides and sustained membrane depolarization occurs. An absolute requirement for acetate to support Ca2+ release is demonstrated. 2. Membrane de-energization, NAD(P)H oxidation, and Ca2+ efflux as induced by atractyloside were temperature-dependent, since it occurs when mitochondria are incubated at 22 degrees C and was abolished at 4 degrees C. 3. Taking into account this latter, the effects of atractyloside on mitochondrial Ca2+ release appears not to be a simple result of the binding of the inhibitor to adenine nucleotide translocase. 4. It is proposed that the mechanism involved in atractyloside-driven membrane permeability to Ca2+ must be related with the transference of the conformational change of the carrier, to another membrane structure responsible for the maintenance permeability to ions.     

Ionic mechanism of action of a spin-labeled local anesthetic on squid axon membranes.

The ionic mechanism of action of a spin-labeled local anesthetic (SLA), 2-[N-methyl-N-(2,2,6,6-tetramethylpiperidonooxyl)]-ethyl 4-ethoxylbenzoate, was studied by means of voltage clamp technique with squid giant axons in comparison with the parent compound without spin label moiety, 2-(N,N-dimethyl)ethyl 4-ethoxylbenzoate (GS-01). Like other local anesthetics, they suppressed both sodium and potassium conductance increases. However, three remarkable differences have been noted between SLA and GS-01: (1) SLA is more effective than GS-01 in suppressing the sodium and potassium conductance increases; (2) SLA induces a potassium inactivation, whereas GS-01 is lacking this ability; (3) SLA has no effect on the time to peak sodium current, whereas GS-01 prolongs it. GS-01 resembles procaine with respect to (2) and (3) above. SLA will become a useful probe for the study of the molecular mechanism of local anesthetic aciton and of ionic channel function.     

Circular dichroism and nucleotide and phosphate-induced conformational changes of mitochondrial adenosinetriphosphatase

The conformational changes induced by the binding of different effectors on F1-ATPase are investigated by using circular dichroism and are related to enzyme activity. The hydrophilic part of the terminal enzyme of oxidative phosphorylation, F1-ATPase, solubilized from the pig heart mitochondrial membrane contains both regulatory and catalytic sites which can bind nucleotides and phosphate. The circular dichroic spectra of F1-ATPase in the absence or in the presence of ADP, Mg2+, phosphate, and the substrate analogue guanosine 5'-(beta, gamma-imidotriphosphate) [GMP-P-(NH)P] were recorded and analyzed in terms of secondary structure. The most significant result is a sizable increase from 35% to 42% of the alpha-helix content when the enzyme is incubated with all the effectors. Since the kinetic study showed that GMP-P(NH)P is a competitive inhibitor of MgATP with or without preincubation of the enzyme with ADP and phosphate, it was concluded that the catalytic and regulatory sites can be simultaneously occupied by ADP and GMP-P-(NH)P. The increase of alpha-helix content is then interpreted by a conformational change that occurs only after occupation of both types of sites.     

Differential scanning calorimetry study of local anesthetic effects on F1ATPase and submitochondrial particles

The differential scanning calorimetry trace of F1ATPase, prepared from beef heart submitochondrial particles, has a single sharp endothermic transition at 80.5 +/- 1.0 degrees C and a half-height peak width of 2.0 +/- 0.2 degrees. The transition enthalpy is 19 +/- 2 cal/g of protein. Submitochondrial particles (SMP) have a similar peak at 75.1 +/- 0.5 degrees C with a half-height peak width of 1.8 +/- 0.1 degrees and an enthalpy of 5 +/- 1 cal/g of SMP protein. The SMP transition is provisionally identified as being due to membrane-bound F1ATPase. Tetracaine and dibucaine cause these transitions to shift to lower temperatures; addition of 0.3 mM dibucaine gives peaks at 71.7 and 64.9 degrees C for F1ATPase and SMP, respectively, and 1.0 mM tetracaine gives peaks at 70.0 and 60.5 degrees C for F1ATPase and SMP, respectively. These anesthetic concentrations also give appreciable inhibition of enzyme activity at 25 degrees C. We conclude that the local anesthetics induce conformational alterations in the F1ATPase-protein complex which result both in enzyme inhibition and in the lowering of the thermal denaturation transition temperature.     

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.     

Electron microscopic observations on the mitochondrial adenosinetriphosphatase in the rat spinal cord

Summary Nucleosidetriphosphatase activity of the rat spinal cord was studied with a modified Wachstein-Meisel method at ultrastructural level. A short formaldehyde perfusion fixation favoured the preservation of the mitochondrial activity.The reaction product was localized intramitochondrially in the neuropil, while the pericaryonal mitochondria were nonreactive. Similar results were obtained, when ITP was substituted for ATP. No membrane activity was observed by Mg⁺⁺ activation only, but a certain membrane affinity was noticed in the neuropil, when Na⁺ and K⁺ were included.The specificity of the ATPase reaction reported in this paper is further discussed.Abbreviations used ATP adenosinetriphosphate - ATPase adenosinetriphosphatase - IDP inosinediphosphate - ITP inosinetriphosphate - TPP thiaminepyrophosphate - UDP uridinediphosphate