RESUMEN
Respiration-dependent responses of a pH probe (fluorescein isothiocyanate, FITC), covalently bound to the membrane proteins of mitochondria and submitochondrial particles (SMP) have been studied. A spectral shift indicating FITC deprotonation was observed when respiration was activated in coupled mitochondria. Such a response was increased by valinomycin and reduced by uncoupler. Some FITC deprotonation was detected in the presence of excess of an uncoupler, but the response was smaller and insensitive to valinomycin. FITC deprotonation was also observed in submitochondrial particles after succinate addition. In this case it was not affected by uncoupler. Increase in the buffer concentration was found to (i) decrease the FITC response and (ii) increase the rate of uncoupled respiration in both mitochondria and submitochondrial particles. The results are consistent with the assumption that respiration initiates appearance of local H+ activity gradients on the inner side of the internal mitochondrial membrane during the steady-state H+ pumping. We suggest that the formation of this gradient is due to kinetic barrier to proton transfer from the bulk phase to the respiratory proton pump vicinity.
Asunto(s)
Fluoresceína-5-Isotiocianato/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Protones , Acetona/análogos & derivados , Acetona/farmacología , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Bovinos , Respiración de la Célula/efectos de los fármacos , Corazón , Concentración de Iones de Hidrógeno , Hígado , Permeabilidad , Potasio/farmacología , Bombas de Protones/metabolismo , Ratas , Partículas Submitocóndricas/metabolismo , Ácido Succínico/farmacología , Desacopladores/farmacología , Valinomicina/farmacologíaRESUMEN
Preparations of coupled mitochondria were labeled with fluorescein isothiocyanate (FITC). Comparison of characteristics of FITC bound to mitochondria versus azolectin liposomes indicates that a significant part of the probe is bound to mitochondrial proteins including proteins of the matrix side of the inner membrane. The experiments with the probe indicate that mitochondrial proteins resemble typical polyelectrolytes with constant of dissociation close to 10(-7).H+ and K+ compete for binding to mitochondrial proteins at neutral pH. The slow deprotonation of matrix proteins is observed during equilibration of matrix protons with medium protons. This process is not restricted to the transmembrane proton transfer. The data are explained by the theory of immobilized buffer. Under certain conditions the extent of dissociation of the matrix immobilized buffer correlates with the rate of mitochondrial respiration in state 3P according to chance.
Asunto(s)
Mitocondrias Hepáticas/metabolismo , Consumo de Oxígeno , Animales , Unión Competitiva , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Fluoresceína-5-Isotiocianato/metabolismo , Concentración de Iones de Hidrógeno , Ionóforos/farmacología , Liposomas/metabolismo , Proteínas de la Membrana/metabolismo , Nigericina/farmacología , Fosfatidilcolinas , Fosfolípidos/metabolismo , Unión Proteica , Bombas de Protones/fisiología , Ratas , Espectrofotometría , Desacopladores/farmacologíaRESUMEN
The addition of oligomycin in the presence of Ca2+ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca(2+)-induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5-20 microM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca(2+)-induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca2+ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Calcio/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Oligomicinas/farmacología , Consumo de Oxígeno/efectos de los fármacos , Adenilato Quinasa/metabolismo , Animales , Atractilósido/análogos & derivados , Atractilósido/farmacología , Técnicas In Vitro , Mitocondrias Hepáticas/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , RatasRESUMEN
A concerted function of purine nucleotide (PN) binding and fatty acid (FA) release from the uncoupling protein (UP) resulting in the maximum coupling (potential) of brown adipose tissue (BAT) mitochondria was demonstrated. The uncoupling effect of FA was studied (at 4 mM MgCl2): 17 nmol oleate per mg protein caused a slight uncoupling with 8.9 mM ATP but with ATP below 3.6 mM almost total uncoupling was achieved. This shows that the PN-controlled gate can be stabilized in the closed conformation (with 8.9 mM ATP), also when FA is bound to UP. The sensitivity of the FA effect to ATP proves that oleate directly interacts with UP. The closed conformation of the H+ channel of UP is then abolished by oleate when a lower free ATP concentration is maintained outside.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Carnitina/metabolismo , Proteínas Portadoras , Ácidos Grasos no Esterificados/fisiología , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Ácidos Oléicos/farmacología , Desacopladores/farmacología , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Cricetinae , Guanosina Difosfato/farmacología , Homeostasis , Canales Iónicos , Cinética , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales , Ácido Oléico , Consumo de Oxígeno/efectos de los fármacos , Proteína Desacopladora 1RESUMEN
Experiments in which we investigated the possible oxidative utilization of lipoid substrates by brain and liver mitochondria were carried out with rats aged 5 and 90 days, kept under completely standardized conditions. Brain mitochondria were isolated on a Ficoll gradient after Clark and Nicklas (1970). Respiratory activity (or the respiratory control index-R.C.) was determined in the manner described in an earlier paper (Dobesová and Mourek 1980). Na succinate or Na malate was used as the testing substrate; palmityl carnitine, acetyl carnitine and acetoacetate were used as lipoid substrates. Oxygen consumption was measured with a Clark's oxygen electrode and respiration was expressed in nAt oxygen per min per mg protein, which was measured by the method of Lowry et al. (1951). When using succinate or malate, in agreement with our previous results we did not find any development changes in the respiratory activity of the brain mitochondria. The oxidation of acetoacetate by the brain mitochondria of 5-day-old rats was about five times greater, and of acetyl carnitine over two times greater, compared with the CNS mitochondria of adult rats. The oxidative utilization of lipoid substrates by the liver mitochondria of 5-day-old rats was significantly greater than their utilization by CNS mitochondria (in the case of palmityl carnitine three times greater, for example) and was always significantly greater than in the liver mitochondria of adult rats. We demonstrated that mitochondria isolated from the brain of 5-day-old rats are equipped with an enzymatic apparatus which allows them to utilize lipoid substrates on a significantly greater scale than in adulthood.
Asunto(s)
Acetoacetatos/metabolismo , Encéfalo/metabolismo , Carnitina/análogos & derivados , Mitocondrias Hepáticas/metabolismo , Mitocondrias/metabolismo , Palmitoilcarnitina/metabolismo , Acetilcarnitina/metabolismo , Animales , Animales Lactantes/metabolismo , Técnicas In Vitro , Malatos/metabolismo , Oxidación-Reducción , Ratas , Ratas Endogámicas , Succinatos/metabolismoRESUMEN
The present study reveals that the previously described effect of ATP-synthetase inhibition concomitant with inhibition of respiratory chain functioning may be observed at different absolute values of delta psi on the mitochondrial membrane. This fact points out that the membrane potential is not a unique regulator in coupling of ATP-synthetase and respiratory chain activities. We found, using the double-inhibitor titration technique, that ATP-synthetase inhibition induces proportional inhibition of respiratory chain enzymes and vice versa respiratory chain inhibition induces proportional inhibition of ATP-synthetase. This effect is shown to exist only when osmolarity is close to 150-300 (mosM) (in the physiological range). The coupling effectivity (ADP/O) of mitochondria under these conditions is maximal. Under conditions of high osmolarity (400-600 mosM) the respiratory chain and ATP-synthetase behave as if they were coupled by bulk phase delta -mu H+, from the kinetic point of view.