Proteoliposome System Research Articles

Effect of starvation on the activity of the mitochondrial tricarboxylate carrier

The effect of starvation on the activity of the tricarboxylate carrier has been investigated in intact rat liver mitochondria and in a reconstituted system. In both experimental conditions, the rate of citrate transport, when compared to control, is greatly reduced (35–40%) in starved rats. Similar behaviour is shown by the cytosolic lipogenic enzymes. Kinetic analysis of the carrier activity in intact mitochondria and in the proteoliposomal system has showed that during starvation only the V max of this process decreases while there is no change in the K m. No difference in the Arrhenius plot and in the lipid composition has been detected, which indicates that the reduced transport activity in fasted animals is not due to a change in the carrier lipid microenvironment. In starved rats, a reduction of the carrier activity has occurred even after the addition of increasing cardiolipin concentrations to proteoliposomes. These findings thus suggest that starvation-induced decrease of citrate carrier activity could be due to a change of the intrinsic properties of the transport protein.

Functional Reconstitution of ATP‐Dependent Transporters from the Solubilized Hepatocyte Canalicular Membrane

The hepatocyte canalicular membrane contains several primary-active ATP-dependent export carriers including one for bile salts and one for leukotriene C4 and related conjugates. The molecular identity of both transporters has not been fully elucidated. To establish a transport assay that allows the purification and identification of the proteins involved in ATP-dependent bile salt transport and in leukotriene C4 transport, we reconstituted solubilized hepatocyte canalicular membranes into phospholipid bilayers using a rapid dilution method. The proteoliposomes formed exhibited both [3H]taurocholate and [3H]leukotriene C4 uptake, which was much higher in the presence of ATP than in the presence of the non-hydrolyzable ATP-analog AdoPP[CH2]P or in the absence of nucleotides. Nucleotide requirement and osmotic sensitivity of [3H]taurocholate transport indicates true transport into the vesicle lumen. Optimized conditions for reconstitution included the addition of a high concentration of an osmolyte (glycerol) and the presence of exogenous phospholipids (0.3%) during solubilization. Highest transport rates were obtained by reconstitution into acetone/ether-precipitated Escherichia coli phospholipid supplemented with 20% cholesterol and by use of octylglucoside concentrations between 30 mM and 50 mM. Taurocholate transport was non-competitively inhibited by vanadate (Ki = 39 microM). The kinetic parameters of cyclosporin A inhibition (Ki = 2.6 microM for taurocholate and 4.3 microM for leukotriene C4 transport) as well as the affinities of taurocholate (Km = 12 microM) and leukotriene C4 (Km = 0.5 microM) in the proteoliposome system indicate that the reconstitution resulted in functionally active transport systems, which are representative of ATP-dependent transport in the intact plasma membrane.

Ca2+/H+ countertransport and electrogenicity in proteoliposomes containing erythrocyte plasma membrane Ca-ATPase and exogenous lipids.

A reconstituted proteoliposomal system was obtained with Ca-ATPase purified from human erythrocyte membrane (plasma membrane, PM ATPase), and liposomes prepared by reverse-phase evaporation. The reconstituted PM ATPase behaved as an electrogenic Ca2+/H+ exchanger and, under optimal conditions, utilization of 1 mol of ATP was accompanied by uptake of one Ca2+ by the vesicles, and ejection of one H+ from the lumen of the vesicles. Ca2+ uptake was greatly (5-fold) stimulated by the addition of calmodulin, and by collapsing the H+ gradient with the ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of calmodulin and p-trifluoromethoxyphenylhydrazone, the reconstituted system sustained transport rates of 1.00 +/- 0.12 mumol of Ca2+/mg of protein min-1 (30 degrees C), reaching asymptotic levels of 8.05 +/- 0.41 mumol of Ca2+/mg of protein (i.e. 20 mM lumenal Ca2+). The corresponding net charge transfer produced a maximal electrical gradient of 40.5 +/- 1.8 mV at steady state. Demonstration of the electrogenic behavior of the PM ATPase, obtained for the first time with these experiments, was critically dependent on the detergent used in the reconstitution procedure. The lumenal pH rise had a much greater rate-limiting effect on the pump, than the electrical potential developed by the pump.

Characterization of the inhibitor sensitivity of the coenzyme A transport system in isolated rat heart mitochondria.

The effect of protein labeling agents on coenzyme A (CoA) transport into isolated rat heart mitochondria was studied. CoA transport was substantially inhibited by sulfhydryl reagents (mersalyl, pCMB) as well as by the tyrosine-selective reagent N-acetylimidazole. The effect of pCMB was reversed by DTT. Moreover, CoA uptake was completely abolished by agents selective for lysine and amino terminal residues (pyridoxal 5-phosphate, dansyl chloride). In contrast arginine-selective reagents (2, 3-butanedione, phenylglyoxal) caused considerably less inhibition of CoA uptake. Moreover, partial inhibition of transport was observed with the stilbene disulfonic acid derivatives DIDS and SITS. Finally, measurement of the effects of the labeling agents on the mitochondrial membrane potential indicated that the inhibition of CoA transport into mitochondria is not a secondary effect that arises from an alteration in the electric potential gradient across the inner mitochondrial membrane. These results provide the first information on the types of amino acid residues that may be essential to the CoA transport mechanism and provide additional support for the existence of a CoA transport protein within the mitochondrial inner membrane. Furthermore, the identification of effective inhibitors of the CoA transport system will greatly facilitate the functional reconstitution of this transporter in a proteoliposomal system following its solubilization and purification.

Functional levels of mitochondrial anion transport proteins in non-insulin-dependent diabetes mellitus

The effect of non-insulin-dependent diabetes mellitus (i.e., NIDDM; type 2 diabetes) on the levels of functional mitochondrial anion transport proteins has been determined utilizing a chemically-induced neonatal model of NIDDM. We hypothesized that moderate insulin deficiency exacerbated by the insulin resistance, which is characteristic of NIDDM, would cause changes in mitochondrial anion transporter function that were similar to those we have previously shown to occur in insulin-dependent diabetes mellitus (i.e., IDDM; type 1 diabetes) (Arch. Biochem. Biophys. 280: 181-191, 1990). Our experimental approach consisted of the extraction of the pyruvate, dicarboxylate and citrate transport proteins from the mitochondrial inner membrane with Triton X-114 using rat liver mitoplasts (prepared from diabetic and control animals) as the starting material, followed by the functional reconstitution of each transporter in a proteoliposomal system. This strategy permitted the quantification of the functional levels of these three transporters in the absence of the complications that arise when such measurements are carried out with intact mitochondria (or mitoplasts). We found that experimental NIDDM did not cause significant changes in the extractable and reconstitutable specific (and total) transport activities of the pyruvate, dicarboxylate, and citrate transporters. These results are in marked contrast to our previous findings obtained using rats with IDDM and negated our hypothesis. The present results, in combination with our earlier findings, allow us to conclude that insulin plays an important role in the regulation of mitochondrial anion transporter function.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of insulin supplementation on diabetes-induced alterations in the extractable levels of functional mitochondrial anion transport proteins

The effect of insulin supplementation on diabetes-induced alterations in the levels of functional mitochondrial anion transport proteins has been determined. The experimental approach consisted of the extraction of the pyruvate, dicarboxylate, and citrate transport proteins from the mitochondrial inner membrane with Triton X-114 using rat liver mitoplasts (prepared from control, diabetic, or insulin-supplemented diabetic animals) as the starting material, followed by the reconstitution of the function of each transporter in a proteoliposomal system. This experimental strategy permitted the quantification of the functional levels of these three transporters in the absence of the complications that arise when such measurements are carried out with intact mitochondria (or mitoplasts). We found that treatment of diabetic rats (i.e., animals that were injected with streptozotocin 3 weeks earlier) on a daily basis with insulin for 3 weeks resulted in a reversal of the diabetes-induced (a) increase in the extractable and reconstitutable total (and specific) transport activities of the pyruvate and dicarboxylate transporters and (b) decrease in the activity of the citrate transporter. These findings indicate that diabetes-induced alterations in the functional levels of mitochondrial anion transport proteins are a direct consequence of the insulin insufficiency that characterizes this disease. Furthermore, this study provides the first demonstration that insulin participates in the regulation of the functional levels of liver mitochondrial anion transport proteins.

Control of proteoliposomal cytochrome c oxidase: the overall reaction

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (delta psi) and pH gradient (delta pH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse delta psi and increase delta pH; the respiration rate decreases. High levels of valinomycin, however, decrease delta pH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases delta psi and collapses delta pH; the respiration rate increases. On a millivolt equivalent basis delta pH is a more effective inhibitor of activity than is delta psi. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both delta pH and delta psi components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing delta pH but in a nonlinear way to delta psi ("non-ohmically"). High levels of both delta psi and delta pH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both delta pH and delta psi components of the proton-motive force, but is more sensitive to delta pH than to delta psi at an equivalent delta mu H+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella.

A Time lag in the onset of ATP- Pi exchange catalyzed by purified ATP-synthase (CF 0-CF 1) proteoliposomes and by chloroplasts

The time course of ATP- P i exchange which is catalyzed by the isolated chloroplast ATP synthase in phospholipid vesicles was studied. The following observations were made, ( i) The onset of 32 P i incorporation into ATP lags behind ATP hydrolysis. The lag lasts for about 2 min at 37 °C and is followed by a steady-state rate which is constant for more than 30 min. Under the same experimental conditions, ATP hydrolysis shows an initial burst followed by a constant, slower rate, ( ii) The initial lag is independent of Mg-ATP concentration in the range 0.2–5 m m and of the presence of ADP. In contrast, the steady-state rate of ATP- P i exchange has an apparent K m of 0.3 m m for Mg-ATP and is stimulated by ADP. ( iii) Increasing the temperature from 30 to 45 °C decreases the lag from 6 min to zero. The steady-state rate of ATP- P i exchange is affected to a much smaller extent by the temperature in this range, ( iv) The lag is insensitive to valinomycin or tetraphenylboron, while the steady-state rate is partially inhibited. Nigericin and protonophores affect both the lag and steady-state rate, ( v) ATP-induced membrane potential formation, as followed by oxonol VI, does not correlate with the lag in its kinetics and temperature dependence. ATP-induced pH gradient formation could not be detected in the proteoliposome system, ( vi) Light-triggered ATP- P i exchange in chloroplasts shows essentially the same time course as the proteoliposome system, but the lag lasts for only about 20 s at room temperature and is unaffected by a preexisting proton gradient. These results suggest that the initial lag in ATP- P i exchange does not reflect the time required for the buildup of a protomotive force (Δ − μ H+) nor the time required to produce ADP. It is suggested, therefore, that the lag reflects an internal autocatalytic conformational change in the ATP-synthase complex which is initiated by ATP hydrolysis and which converts the enzyme from an “exclusive ATPase state” to a “reversible ATP-synthase state”. This slow transition is not directly coupled to a transmembrane pH or potential gradient.

A model proteoliposomal system for proline transport using a purified proline carrier protein from Mycobacterium phlei

The proteoliposomes prepared from purified proline carrier protein isolated from membrane vesicles of Mycobacterium phlei exhibited an uptake of proline, which was dependent upon a proton gradient generated across the lipid bilayer. Although a proton gradient was generated by the reduction of the entrapped ferricyanide by ascorbate oxidation with benzoquinone serving as a lipid soluble hydrogen carrier, transport of proline was dependent on the addition of sodium ion. The movement of sodium and proline across the artificial membrane resulted in a simultaneous collapse of the proton gradient.