The effects of phosphate and electron transport on the carbonyl cyanide m-chlorophenylhydrazone-induced ATPase of rat-liver mitochondria

1. The effects of phosphate and electron transport on the ATPase induced in ratliver mitochondria by the uncoupler carbonyl cyanide m-chlorophenylhydrazone have been measured at different uncoupler concentrations and compared with those of ATP, oligomycin and aurovertin.

2. The inhibitory action of respiratory-chain inhibitors on the ATPase activity, which is independent of the actual inhibitor used, is greatly delayed or prevented by the presence of uncoupler, and, in the case of rotenone, can be reversed completely by the subsequent addition of succinate (in the absence of uncoupler). These results can be explained on the basis of the proposal previously made by others that coupled electron transfer causes a structural change in the ATPase complex that results in a decreased affinity of the ATPase inhibitor for the mitochondrial ATPase.

3. Inorganic phosphate specifically stimulates the ATPase activity at high uncoupler concentrations (> 0.2 μM), but has no effect at low concentrations. The stimulation is prevented or abolished by sufficiently high concentrations of aurovertin.

4. Aurovertin prevents the inhibition of the uncoupler-induced ATPase by high uncoupler concentrations.

5. It is proposed that the steady-state concentration of endogenous P_i may be an important regulator of the turnover of the ATPase in intact mitochondria and that the inhibition of ATPase activity by high concentrations of uncoupler is at least partially mediated via changes in the concentration of endogenous P_i.     

Coupling mechanisms in anionic substrate transport across the inner membrane of rat-liver mitochondria

The translocation of P_i, malate, -oxoglutarate, and citrate across the inner membrane of rat-liver mitochondria has been studied. Investigation on the effect of pH on anionic substrate translocation across the mitochondrial membrane shows that their distribution across the inner membrane can be governed by transmembrane pH difference. However, evidence is presented that the translocation of P_i, but not that of malate, -oxoglutarate, or citrate can bedirectly coupled to an OH^– counterflux (H₂PO ₄ ^– –OH^– exchange-diffusion). and malate-tricarboxylate exchange-diffusion reactions is directly demonstrated. The study of the effect of uncouplers on the efflux from mitochondria of substrate anions, in the absence of counteranion, and on the anion exchange-diffusions shows that uncouplers act in at least two ways: they promote the efflux of P_i from mitochondria and inhibitdirectly the exchange-diffusion reactions. The kinetics of this inhibition are described. These results are discussed in the light of previous work on the effect of uncouplers on the distribution of substrate anions across the inner membrane of isolated mitochondria. Coupling mechanisms in substrate anion translocation and aspects of the energetics of anion translocation are discussed.     

The orientation of phosphate-dependent glutaminase on the inner membrane of rat renal mitochondria

Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p-chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment.     

Conditions for activity of glutaminase in kidney mitochondria

1. Rat kidney mitochondria oxidize glutamate very slowly. Addition of glutamine stimulates this respiration two- to three-fold. Addition of glutamate also stimulates respiration in the presence of glutamine. 2. By measuring mitochondrial swelling in iso-osmotic solutions of glutamine or of ammonium glutamate it was shown that glutamine penetrates the mitochondrial membrane rapidly whereas ammonium glutamate penetrates very slowly. 3. Experiments in which reduction of NAD(P)⁺ was measured in preparations of intact and broken mitochondria indicated that glutamate dehydrogenase shows the phenomenon of `latency'. On the addition of glutamine rapid reduction of nicotinamide nucleotides in intact mitochondria was obtained. 4. During the action of glutaminase there is an accumulation of glutamate inside the mitochondria. 5. When the mitochondria were suspended in a medium containing glutamine, P_i and rotenone the rate of production of ammonia was stimulated by the addition of a substrate, e.g. succinate. Addition of an uncoupler or antimycin A abolished this stimulation. 6. The effects of succinate and uncoupler were especially pronounced in the presence of glutamate, which is an inhibitor of glutaminase activity by competition with P_i. 7. Determination of the enzyme activity in media at different pH values showed that the optimum pH for glutaminase activity in the preparation of broken mitochondria was 8, whereas for intact mitochondria it was dependent on the energy state. In the presence of succinate as an energy source it was pH 8.5, but in the presence of uncoupler or antimycin A it was 9. This displacement of the pH optimum to a higher value was especially pronounced in the presence of both glutamate and uncoupler. 8. If nigericin was present in potassium chloride medium the pH optimum for enzyme activity in intact non-respiring mitochondria was nearly the same as in the preparation of broken mitochondria; however, its presence in K⁺-free medium displaced the pH optimum for glutaminase activity to a very high value. 9. It is postulated that because of low permeability of the kidney mitochondrial membrane to glutamate the latter accumulates inside the mitochondria, and that this leads to the inhibition of the enzyme by competition with P_i and also by lowering the pH of the intramitochondrial space. With succinate as substrate for respiration there is an outward translocation of H⁺ ions, which together with accumulation of P_i increases glutaminase activity. Translocation of K⁺ ions inward increases the enzyme activity, perhaps by increasing the pH of the internal spaces and causing an accumulation of P_i. 10. The importance of the location of the enzyme in the mitochondria in relation to its biological function and conditions for activity is discussed.     

The significance of the attachment of rat kidney glutaminase to the inner mitochondrial membrane

Chicken gizzard has been considered to be an exceptional organ of smooth musculature in which a myoglobin is present. Since the characterization of the gizzard myoglobin has to date, been very incomplete, we studied the structures and functions in detail. The main component, which constituted roughly 90% of the protein, isolated by chromatofocusing, was homogeneous by electrophoretic and ultracentrifugal analyses. The molecular weight was consistently 1.8 X 10(4) by equilibrium sedimentation and iron analysis, and the isoelectric point was 7.8. Spectroscopic properties of the oxy-, carboxy- and deoxy-derivatives were typical of myoglobin. The oxygenation equilibria were also typical of myoglobin, showing neither homotropic nor heterotropic allosteric interactions, and the temperature-dependence (delta H0) was estimated as -16.6 kcal/mol. All these characteristics of the gizzard myoglobin were identical with those of the protein from the skeletal muscles. The amino acid composition and peptide mapping results also concluded that identical myoglobin was present in the gizzard, skeletal and probably cardiac muscles.     

Protein transport machineries for precursor translocation across the inner mitochondrial membrane

The mitochondrial inner membrane has a central function for the energy metabolism of the cell. The respiratory chain generates a proton gradient across the inner mitochondrial membrane, which is used to produce ATP by the F1Fo-ATPase. To maintain the electrochemical gradient, the inner membrane represents an efficient permeability barrier for small molecules. Nevertheless, metabolites as well as polypeptide chains need to be transported across the inner membrane while the electrochemical gradient is retained. While specialized metabolite carrier proteins mediate the transport of small molecules, dedicated protein translocation machineries in the inner mitochondrial membrane (so called TIM complexes) transport precursor proteins across the inner membrane. Here we describe the organization of the TIM complexes and discuss the current models as to how they mediate the posttranslational import of proteins across and into the inner mitochondrial membrane.     

Dicarboxylate transport across the inner membrane of the chloroplast envelope.

The uptake of radioactively labeled dicarboxylates into the sorbitol-impermeable 3H2O space (the space surrounded by the inner envelope membrane) of spinach chloroplasts has been studied by means of silicone layer filtering centrifugation. 1. Malate, aspartate and a number of other dicarboxylates are rapidly transported across the envelope leading to an accumulation in the chloroplasts. This uptake proceeds mainly by a counterexchange with the dicarboxylates present there. 2. The dicarboxylate transport shows saturation characteristics allowing the determination of Km and V. 3. All dicarboxylates transported act as competitive inhibitors of the transport. 4. The activation energy of the transport as determined from the temperature dependency is evaluated to be 7 kcal/mol. 5. The rate of dicarboxylate transport is influenced by illumination, the countertransported molecules and the pH in the medium. These changes effect the transport velocity, whereas the corresponding Km values are not altered. 6. It is discussed whether there is more than one carrier involved in dicarboxylate transport in spinach chloroplasts.     

Bidirectional transport of glutamine across the cell membrane in rat liver.

Hepatocytes isolated from fed rats were used to investigate glutamine transport. Glutamine transport appears as a composite process involving at least two saturable components. The Na+-dependent component probably represents the entry through the N system. The Na+-independent component was also inhibited by histidine and exhibited trans-stimulation, suggestive of a facilitated diffusion process. Kinetic parameters for both systems suggest that facilitated diffusion only plays a minor role in glutamine influx. In contrast, the Km for glutamine efflux was consistent with a physiological role of the facilitated-diffusion component in glutamine release. In Na+ medium, relatively constant distribution ratios (about 8) between intra- and extra-cellular concentrations were observed, with external glutamine ranging from 0.5 to 5 mM. The present observations suggest that glutamine influx might largely be mediated by the N system, whereas facilitated diffusion allows hepatocytes to release glutamine when intracellular concentrations are elevated. The physiological consequences of this bidirectional transfer of glutamine across the liver cell membrane is discussed.     

Control of phosphate transport across the plasma membrane of Chara corallina

This paper examines the control of phosphate uptake into Chara corallina. Influxes of inorganic phosphate (Pi) into isolated single internodal cells were measured with ³²Pi. Pretreatment of cells without Pi for up to 10 d increased Pi influx. However, during this starvation the concentrations of Pi in both the cytoplasm and the vacuole remained quite constant. When cells were pre-treated with 0.1 mM Pi, the subsequent influx of Pi was low. Under these conditions the Pi concentrations in the cytoplasm was almost the same as that of Pi-starved cells, but vacuolar Pi increased with time. Transfer of cells from medium containing 0.1 mM Pi to Pi-free medium induced an increase of Pi influx within 3 d irrespective of the concentration of Pi in the vacuole.During Pi starvation, neither the membrane potential nor the cytoplasmic pH changed. Manipulation of the cytoplasmic pH by weak acids or ammonium decreased the Pi influx slightly.Pi efflux was also measured, using cells loaded with ³²Pi. Addition of a low concentration of Pi in the rinsing medium rapidly and temporarily induced an increase in the efflux.The results show that Pi influx is controlled by factors other than simple feedback from cytoplasmic or vacuolar Pi concentrations or thermodynamic driving forces for H⁺-coupled Pi uptake. It is suggested that uptake of Pi is controlled via the concentration of Pi in the external medium through induction or repression of two types of plasma membrane Pi transporters.Key words: Chara corallina, membrane transport, phosphate influx, phosphate starvation      

Regulation of flux through glutaminase and glutamine synthetase in isolated perfused rat liver

1. Glutaminase and glutamine synthetase are simultaneously active in the intact liver, resulting in an energy consuming cycling of glutamine at a rate up to 0.2 mumol per g per min. 2. An increase in portal glutamine concentration was followed by an increased flux through glutaminase, but flux through glutamine synthetase remained unchanged. Glutaminase flux was also increased by ammonium ions or glucagon; these effects were additive. 3. Glutamine synthetase flux was increased by ammonium ions, but this activation was partly overcome by increasing portal glutamine concentrations. Glutamine synthetase flux was slightly increased by glucagon at portal glutamine concentrations of about 0.2-0.3 mM, but was strongly inhibited above 0.6 mMs. 4. During experimental metabolic acidosis there was an increased net release of glutamine by the liver, being due to opposing changes of flux through glutaminase and glutamine synthetase. Conversely, an increased glutamine uptake by the liver during metabolic alkalosis was observed due to an inhibition of glutamine synthetase and an activation of glutaminase. However, the two enzyme activities respond differently depending on whether glucagon or ammonium ions are present.     

Mitochondrial phosphate transport and the N-ethylmaleimide binding proteins of the inner membrane

Mitochondria have been prepared from the flight muscles of mature blowflies (Sarcophaga bullata). Phosphate transport by these mitochondria, determined by rates of passive swelling in ammonium phosphate, is sensitive to inhibition by N-ethylmaleimide. 20 nmol of N-ethylmaleimide/nmol cytochrome a inhibit the swelling by 90%. When the mitochondria are inhibited by $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide}$, then solubilized in dodecyl sulfate/mercaptoethanol at 100°C and then electrophoresed on dodecyl sulfate-polyacrylamide gels, many labeled protein bands can be detected, including a large labeled peak that has the same mobility as the tracking dye, bromophenol blue. Sonic submitochondrial particles that are prepared from the $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimidelabeled}$ mitochondria, solubilized, and electrophoresed on dodecyl sulfatepolyacrylamide gels, possess only seven major labeled protein bands with no radioactive peak at the tracking dye. These labeled proteins have molecular weights of 71, 68, 64, 45, 32, 30, and approx. 10 · 10³. The nmol $\text{N-[}{}^3\text{H}\text{]-}\text{ethylmaleimide}$ bound to each of these proteins per nmol cytochrome a are 0.15, 0.19, 0.35, 0.45, 0.87, 0.10, and 0.17, respectively, when the mitochondria are inhibited with 21.5 mol $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide/mol}$ cytochrome a at 10 μM cytochrome a. Coty and Pedersen ((1975) J. Biol. Chem. 250, 3515–3521) sensitized rat liver mitochondria to $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide}$ and identified five labeled proteins. Only the labeled 32 · 10³ dalton and the 45 · 10³ dalton proteins are common to both systems     

Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of inner membrane.     

Dicarboxylate transport in the inner membrane matrix fraction obtained from rat liver mitochondria

Dicarboxylate transport was studied in the inner membrane matrix fraction (mitoplasts) and compared to that in intact rat-liver mitochondria from which the former was obtained.It is concluded that, kinetics of dicarboxylate exchange measured in mitoplasts, are very similar to those observed with mitochondria. These results would indicate that the preparation technique preserves the integrity of the inner membrane and that neither the outer membrane nor the components of the peripheral space affect these results.     

Syrinx fluid transport: modeling pressure-wave-induced flux across the spinal pial membrane

Syrinxes are fluid-filled cavities of the spinal cord that characterize syringomyelia, a disease involving neurological damage. Their formation and expansion is poorly understood, which has hindered successful treatment. Syrinx cavities are hydraulically connected with the spinal subarachnoid space (SSS) enveloping the spinal cord via the cord interstitium and the network of perivascular spaces (PVSs), which surround blood vessels penetrating the pial membrane that is adherent to the cord surface. Since the spinal canal supports pressure wave propagation, it has been hypothesized that wave-induced fluid exchange across the pial membrane may play a role in syrinx filling. To investigate this conjecture a pair of one-dimensional (1-d) analytical models were developed from classical elastic tube theory coupled with Darcy's law for either perivascular or interstitial flow. The results show that transpial flux serves as a mechanism for damping pressure waves by alleviating hoop stress in the pial membrane. The timescale ratio over which viscous and inertial forces compete was explicitly determined, which predicts that dilated PVS, SSS flow obstructions, and a stiffer and thicker pial membrane-all associated with syringomyelia-will increase transpial flux and retard wave travel. It was also revealed that the propagation of a pressure wave is aided by a less-permeable pial membrane and, in contrast, by a more-permeable spinal cord. This is the first modeling of the spinal canal to include both pressure-wave propagation along the spinal axis and a pathway for fluid to enter and leave the cord, which provides an analytical foundation from which to approach the full poroelastic problem.     

Studies on the ferrochelatase activity of mitochondria and submitochondrial particles with special reference to the regulatory function of the mitochondrial inner membrane

The question addressed in the title was examined by measuring fluorescence emission spectra and light-induced fluorescence-yield changes of chloroplasts which had been frozen to ?196 °C rapidly, as very thin samples adsorbed into substrates which were plunged directly into liquid nitrogen, or slowly by the cooling action of liquid nitrogen through the wall of the cuvette. Contrary to previous reports, we found that the rate of cooling had no influence on the shape of the emission spectrum, the extent of the variable fluorescence or the fraction of the absorbed quanta which are delivered initially to Photosystem I.