Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria

1. 1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside.

2. 2. [³⁵S]carboxyatractyloside and [³H]bongkrekic acid bind to potato mitochondria with high affinity (K_d = 10 to 20 nM, n = 0.6–0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [³H]atractyloside binds to potato mitochondria with a much lower affinity (K_d = 0.45 μM) than carboxyatractyloside or bongkrekic acid.

3. 3. Bound [³H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [³⁵S]carboxyatractyloside by bongkrekic acid and of bound [³H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP.

4. 4. Bongkrekic acid competes with [³⁵S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [³⁵S]carboxyactratyloside binding.

5. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.

Substrate-site interactions in the membrane-bound adenine-nucleotide carrier as disclosed by ADP and ATP analogs

The binding parameters of a number of ADP or ATP analogs to the adenine nucleotide carrier in mitochondria and inside-out submitochondrial particles have been explored by means of two specific inhibitors, carboxyatractyloside and bongkrekic acid. The nucleotides tested fell into two classes depending on the shape of the binding curve. Curvilinear Scatchard plots were obtained for the binding of ADP, ATP, adenosine 5'-triphospho-gamma-1-(5-sulfonic acid)naphthylamidate [gamma-AmNS)ATP) and adenylyl (beta,gamma)-methylenediphosphate (p[CH2]ppA); on the other hand, rectilinear Scatchard plots were obtained in the case of naphthoyl-ADP (N-ADP) and 8-bromo ADP (8Br-ADP) binding. The total number of binding sites for N-ADP and 8Br-ADP could be extrapolated with good accuracy to 1.3-1.5 nmol/mg protein; this value corresponds to the number of carboxyatractyloside-binding sites in heart mitochondria (Block, M.R., Pougeois, R. and Vignais, P.V. (1980) FEBS Lett. 117, 335-340). On the other hand, because of the curvilinearity of the Scatchard plots for the binding of ADP, ATP, (gamma-AmNS)ATP and p[CH2]ppA, the total number of binding sites for these nucleotides could only be approximated to a value higher than 1 nmol/mg protein, the exact value being probably equal to that found for N-ADP and 8Br-ADP binding, i.e. 1.3-1.5 nmol/mg protein. Curvilinearity of Scatchard plots was discussed in terms of negative interactions between nucleotide-binding sites located on the same face of the adenine nucleotide carrier. A possible relationship between the features of the binding plots and the transportable nature of the nucleotide is discussed. Contrary to the enhancing effect of bongkrekic acid on [14C]ADP uptake observed essentially in nucleotide-depleted heart mitochondria (Klingenberg, M., Appel, M., Babel, W. and Aquila, H. (1983) Eur. J. Biochem. 131, 647-654), binding of bongkrekic acid to nondepleted heart mitochondria was found to partially displace previously bound [14C]ADP. These opposite effects of bongkrekic acid may be explained by assuming that bongkrekic acid is able to abolish negative cooperativity between external (cytosolic) ADP-binding sites.     

Adenine nucleotide transport in sonic submitochondrial particles. Kinetic properties and binding of specific inhibitors.

1. A procedure for preparation of sonic submitochondrial particles competent for adenine nucleotide transport is described. ADP or ATP transport was assayed, in the presence of oligomycin, in a saline medium made of 0.125 M KCl, 1 mM EDTA, 10 mM 4-morpholinopropane sulfonic acid buffer, pH 6.5. 2. Sonic particles transport ADP and ATP by an exchange diffusion process. Externally added ADP (or ATP) is exchanged with internal ADP and ATP with a stoichiometry of one to one. The V value for ADP transport 5 degrees C was between 2 and 3 nmol/min per mg protein. 3. The transport system in sonic particles is specific for ADP and ATP. It is strongly dependent on temperature. The activation energy between 0 and 9 degrees C is approx. 35 kcal/mol. The optimum pH is 6.5, 4, Like in intact mitochondria, externally added ADP is transported into sonic particles faster at a given concentration than externally added ATP. The V value for ADP transport is 1.5-2 times higher than the V value for ATP transport. 5. The transition from the energized to the deenergized state in sonic particles results in a decrease of the pH gradient across the membrane (internal pH less than external pH) and in a 2-4 fold increase in the Km value for ATP. This latter effect is opposite that found for transport of added ATP in intact mitochondria (Souverijn, J.H.M., Huisman, L.A., Rosing J. and Kemp, Jr., A. (1973) Biochim. Biophys. Acta 305, 185-198). Energization has no effect on the V value of ATP transport in sonic particles. 6. In contrast to intact mitochondria, inhibition of ADP transport in sonic particles by bongkrekic acid does not have any lag-time and does not depend on pH. The inhibition caused by bongkrekic acid is a mixed type inhibition with a Ki value of 1.2 micronM. Atractyloside and carboxyatractyloside do not inhibit ADP transport in sonic particles, unless the particles have been preloaded with these inhibitors during the sonication. 7. Palmityl-CoA added to sonic particles inhibits efficiently ADP transport. The mixed type inhibition found with palmityl-CoA has a Ki value of 1.6 micronM. 8. [3H]Bongkrekic acid binds to sonic particles readily and with high affinity. Bongkrekic acic binding to sonic particles does not depend on pH and it has a saturation plateau, corresponding approximately to 1.3 mol of site per mol of cytochrome a. The number of [3H]atracytloside binding sites is much lower (one-fifth of the bongkrekic acid). External carboxyatractyloside does not compete with [3H]bongkrekic acid for binding to sonic particles. However, when carboxyatractyloside is present inside the particles, it inhibits the binding of [3H]bongkrekic acid.     

Isobongkrekic acid, a new inhibitor of mitochondrial ADP-ATP transport: radioactive labeling and chemical and biological properties.

An isomer of bongkrekic acid, designated as isobongkrekic acid, has been isolated from ethereal extracts of Pseudomonas cocovenenans grown on defatted coconut. Isobongkrekic acid was also obtained by alkaline treatment of bongkrekic acid. Isobongkrekic acid possesses the same ultraviolet spectrum and the same molecular weight as bongkrekic acid; it has a similar infrared spectrum but not the same nuclear magnetic resonance (NMR) spectrum. The differences in NMR data were interpreted to mean that isobongkrekic acid differs from bongkrekic acid by the configuration of the dicarboxylic end; whereas the two carboxylic groups of the dicarboxylic end have the trans configuration in bongkrekic acid, they have the cis configuration in isobongkrekic acid. Differences between bongkrekic and isobongkrekic acids are lost after catalytic hydrogenation of the molecules. Isobongkrekic acid, like bongkrekic acid, is an uncompetitive inhibitor of ADP transport in mitochondria, provided the mitochondria are preincubated in the presence of the inhibitor and a minute concentration of ADP. The inhibitory and binding efficiency of isobongkrekic acid is considerably increased below pH 7. The number of high affinity sites for [3H] isobongkrekic acid is 0.13 to 0.20 nmol/mg protein in rat liver mitochondria and about 1 nmol/mg protein in rat heart mitochondria, i.e., similar to the number of high affinity sites for [3H] bongkrekic acid. Isobongkrekic and bongkrekic acids compete for the same site, but the affinity of isobongkrekic acid for mitochondria is one-half to one-fourth that of bongkrekic acid.     

Dependence of mitochondrial oxidative phosphorylation on activity of the adenine nucleotide translocase

The coupled reactions of electron transport and ATP synthesis for the first two sites of mitochondrial oxidative phosphorylation have been previously reported to be near equilibrium in isolated respiring pigeon heart (Erecińska, M., Veech, R. L., and Wilson, D. F. (1974) Arch. Biochem. Biophys. 160, 412-421) and rat liver mitochondria (Forman, N. G., and Wilson, D. F. (1982) J. Biol. Chem. 257, 12908-12915). Measurements are presented in this paper which demonstrate that the same relationship exists for both forward and reverse electron transport in rat heart mitochondria. This conclusion implies that adenine nucleotide translocation, a partial reaction of the system, is also near equilibrium, contrasting with proposals that the translocase is rate-limiting for oxidative phosphorylation. To resolve this controversy, the respiratory rates of suspensions of isolated rat liver and rat heart mitochondria were controlled by varying either the added [ATP]/[ADP][Pi] ratios ratios or [ADP] (by varying hexokinase in a regenerating system). Titrations with carboxyatractyloside, a high affinity inhibitor of the translocase which is noncompetitive with ADP, were carried out to assess the dependence of the respiratory rate on translocase activity. Plots of respiratory rate versus [carboxyatractyloside] were all strongly sigmoidal. In liver mitochondria, 40%-70% and in heart mitochondria 66% of the sites could be blocked with carboxyatractyloside before a 10% decrease in the respiratory rate was observed. Further analysis showed that liver and heart mitochondria have translocase/cytochrome a ratios of 1.52 and 3.20, respectively, and that at 23 degrees C the maximal turnover numbers for the translocases were 65 s-1 and 23 s-1. In all states of controlled respiration (no added inhibitor), a substantial excess of translocase activity was present, suggesting that the translocase was not normally rate-limiting in oxidative phosphorylation.     

Adenine nucleotide translocation in yeast mitochondria. Effect of inhibitors of mitochondrial biogenesis on the ADP translocase

1. Optimal test conditions for adenine nucleotide translocation in Candida utilis mitochondria are a standard medium, consisting of 0.63 M mannitol, 2 mM EDTA (or ethylene glycol tetraacetic acid, EGTA), 10 mM morpholinopropane sulfonic acid (pH 6.8), and a temperature of 0 °C.

2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the K_m value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The K_m value for ADP is 2 μM. The Q₁₀ is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process.

3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole.

4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent K_d for the binding of [³⁵S]-carboxyatractyloside and [¹⁴C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [³⁵S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa.

5. Binding of [¹⁴C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg²⁺ at 20 °C. The amount of bound [¹⁴C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein.

6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa₃. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [³⁵S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities.     

Interaction between aurovertin and adenine nucleotide binding sites on mitochondrial F1-ATPase and the isolated beta subunit

The effect of aurovertin on the binding parameters of ADP and ATP to native F1 from beef heart mitochondria in the presence of EDTA has been explored. Three exchangeable sites per F1 were titrated by ADP and ATP in the absence or presence of aurovertin. Curvilinear Scatchard plots for the binding of both ADP and ATP were obtained in the absence of aurovertin, indicating one high affinity site (Kd for ADP = 0.6-0.8 microM; Kd for ATP = 0.3-0.5 microM) and two lower affinity sites (Kd for ADP = 8-10 microM; Kd for ATP = 7-10 microM). With a saturating concentration of aurovertin capable of filling the three beta subunits of F1, the curvilinearity of the Scatchard plots was decreased for ATP binding and abolished for ADP binding, indicating homogeneity of ADP binding sites in the F1-aurovertin complex (Kd for ADP = 2 microM). When only the high affinity aurovertin site was occupied, maximal enhancement of the fluorescence of the F1-aurovertin complex was attained with 1 mol of ADP bound per mol of F1 and maximal quenching for 1 mol of ATP bound per mol of F1. When the F1-aurovertin complex was incubated with [3H]ADP followed by [14C]ATP, full fluorescence quenching was attained when ATP had displaced the previously bound ADP. In the case of the isolated beta subunit, both ADP and ATP enhanced the fluorescence of the beta subunit-aurovertin complex. The Kd values for ADP and ATP in the presence of EDTA were 0.6 mM and 3.7 mM, respectively; MgCl2 decreased the Kd values to 0.1 mM for both ADP and ATP. It is postulated that native F1 possesses three equivalent interacting nucleotide binding sites and exists in two conformations which are in equilibrium and recognize either ATP (T conformation) or ADP (D conformation). The negative interactions between the nucleotide binding sites of F1 are strongest in the D conformation. Upon addition of aurovertin, the site-site cooperativity between the beta subunits of F1 is decreased or even abolished.     

Aspects of energy-linked calcium accumulation by rat heart mitochondria.

When intact rat heart mitochondria were pulsed with 150 nmol of CaCl2/mg of mitochondrial protein, only a marginal stimulation of the rate of oxygen consumption was observed. This result was obtained with mitochondria isolated in either the presence or absence of nagarse. In contrast, rat liver mitochondria under similar conditions demonstrated a rapid, reversible burst of respiration associated with energy-linked calcium accumulation. Direct analysis of calcium retention using 45Ca and Millipore filtration indicated that calcium was accumulated by heart mitochondria under the above conditions via a unique energy-dependent process. The rate of translocation by heart mitochondria was less than that of liver mitochondria; likewise the release of bound calcium back into the medium was also retarded. These results suggest that the slower accumulation and release of calcium is characteristic of heart mitochondria. The amound of calcium bound was independent of penetrant anions at low calcium concentrations. Above 100 nmol/mg of mitochondrial protein, the total calcium bound was increased by the presence of inorganic phosphate. Under nonrespiring conditions, a biphasic Scatchard plot indicative of binding sites with different affinities for Ca2+ was observed. The extrapolated constants are 7.5 nmol/mg bound with an apparent half-saturation value of 75 muM and 42.5 nmol/mg bound with half-saturation at 1.15 mM. The response of the reduced State 4 cytochrome b to pulsed additions of Ca2+ was used to calculate an energy-dependent half-saturation constant of 40 muM. When the concentration of free calcium was stabilized at low levels with Ca2+-EGTA buffers, the spectrophotometrically determined binding constant decreased two orders of magnitude to an apparent affinity of 4.16 X 10(-7) M. Primary of calcium transport over oxidative phosphorylation was not observed with heart mitochondria. The phosphorylation of ADP competed with Ca2+ accumulation, depressed the rates of cation transport, and altered the profile of respiration-linked H+ movements. Consistent with these result was the observation that with liver mitochondrial the magnitude of the cytochrome b oxidation-reduction shift was greater for Ca2+ than for ADP, whereas calcium responses never surpassed the ADP response in heart mitochondria. Furthermore, Mg2+ ingibited calcium accumulation by heart mitochondria while having only a slight effect upon calcium transport in liver mitochondria. The unique energetics of heart mitochondrial calcium transport are discussed relative to the regulated flux of cations during the cardiac excitation-relaxation cycle.     

Kinetic, binding and ultrastructural properties of the beef heart adenine nucleotide carrier protein after incorporation into phospholipid vesicles

1. ADP/ATP transport has been reconstituted by incorporation of the purified carrier protein in liposomes filled with ATP. The transport was assayed by uptake of [14C]ADP into the liposomes, and by release of ATP as determined by a luminescence technique. [14C]ADP uptake was strictly dependent on internal ATP. 2. The simplest phospholipid system capable of yielding high rates of ADP/ATP transport was a mixture of phosphatidylethanolamine and cariolipin (92: 8, w/w). 3. ADP/ATP transport in the reconstituted system proceeded by exchange-diffusion with a 1/1 stoichiometry. The specificity for aDP and ATP was absolute. The capacity and the rate of exchange depended on the concentration of ATP present in liposomes. The rate of transport at 20 degrees C, at 20 mM internal ATP, routinely ranged between 300 and 1000 nmol of nucleotide exchanged per min/mg of added carrier protein. The apparent Km value for external ADP was around 10 microM. 4. The ADP/ATP exchange in the reconstituted system was rather stable to ageing. It dropped by only 20% after 1 day of ageing at 20 degrees C. Divalent cations (Mg2+, Mn2+, Ca2+) at concentrations higher than 1 to 2 mM had a deleterious effect on ADP/ATP transport, concomitant with the release of internal ATP and accumulation of multilamellar vesicles. 5. Atractyloside behaved as a competitive inhibitor and carboxyatractyloside as a non-competitive inhibitor. Bongkrekic acid required a slightly acidic pH to be inhibitory. The data concerning atractyloside, carboxyatractyloside and bongkrekic acid were similar to those obtained with whole mitochondria, suggesting that the carrier protein in liposomes has the same asymmetrical arrangement as in the mitochondria. 6. The percentage of competent carrier protein in liposomes was calculated from dose-response data concerning the inhibition of ADP/ATP transport by atractyloside or carboxyatractyloside, and from the amount of bound [3H]-atractyloside removable by ADP. By both methods, 3 to 6% of the added carrier protein was found to be competent in ADP/ATP transport, based on the assumption that the binding of one atractyloside or carboxyatractyloside molecule per 30000 molecular weight carrier unit results in complete inhibition of transport. 7. Freeze-fracture electron microscopy showed that the ADP/ATP carrier protein-lipid preparations are formed by small vesicles, most of which give rise to smooth fracture faces (probably pure lipid vesicles). Only a small percentage of the vesicles (2 to 4% depending on the amount of carrier protein added) were clearly particulated. About 90% of the particulated vesicles showed no more than 2 particles per vesicle and only 5% more than 5 particles per vesicle. The distribution of the particles between convex and concave fracture faces was asymmetric; about 2/3 of the protein molecules were anchored at the external surface of the vesicles and only 1/3 at the internal one...     

Kinetics of nucleotide transport in rat heart mitochondria studied by a rapid filtration technique

A rapid filtration technique has been used to measure at room temperature the kinetics of ADP and ATP transport in rat heart mitochondria in the millisecond time range. Transport was stopped by cessation of the nucleotide supply, without the use of a transport inhibitor, thus avoiding any quenching delay. The mitochondria were preincubated for 30 s either in isotonic KCl containing succinate, MgCl2, and Pi (medium P) or in isotonic KCl supplemented only with EDTA and Tris (medium K); they were referred to as energized and resting mitochondria, respectively. The kinetics of [14C]ADP transport in energized mitochondria were apparently monophasic. The plateau value for [14C]ADP uptake reached 4-5 nmol of nucleotide.(mg of protein)-1. Vmax values for [14C]ADP transport of 400-450 nmol exchanged.min-1.(mg of protein)-1 with Km values of the order of 13-15 microM were calculated, consistent with rates of phosphorylation in the presence of succinate of 320-400 nmol of ATP formed.min-1.(mg of protein)-1. The rate of transport of [14C]ATP in energized mitochondria was 5-10 times lower than that of [14C]ADP. Upon uncoupling, the rate of [14C]ATP uptake was enhanced, and that of [14C]ADP uptake was decreased. However, the two rates did not equalize, indicating that transport was not exclusively electrogenic. Transport of [14C]ADP and [14C]ATP by resting mitochondria followed biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists

Heterogeneity of the muscarinic receptor population in the rat central and peripheral lung was found in competition binding experiments against [3H]quinuclidinyl benzilate [( 3H]QNB) using the selective antagonists pirenzepine, AF-DX 116 and hexahydrosiladifenidol (HHSiD). Pirenzepine displaced [3H]QNB with low affinity from preparations of central airways indicating the absence of M1 receptors in the trachea and bronchi. Muscarinic receptors in the central airways are comprised of both M2 and M3 receptors since AF-DX 116, an M2-selective antagonist, bound with high affinity to 70% of the available sites while HHSiD, an M3-selective antagonist bound with high affinity to the remaining binding sites. In the peripheral lung, pirenzepine bound with high affinity to 14% of the receptor population, AF-DX 116 bound with high affinity to 79% of the binding sites while HHSiD bound with high affinity to 18% of the binding sites. The presence of M1 receptors in the peripheral airways but not in the central airways was confirmed using [3H]telenzepine, an M1 receptor ligand. [3H]Telenzepine showed specific saturable binding to 8% of [3H]QNB labeled binding sites in homogenates of rat peripheral lung, while there was no detectable specific binding in homogenates of rat trachea or heart. The results presented here demonstrate that there are three muscarinic receptor subtypes in rat lungs, and that the distribution of the different subtypes varies within the lungs. Throughout the airways, the dominant muscarinic receptor subtype is M2. In the trachea and bronchi the remaining receptors are M3, while in the peripheral lungs, the remaining receptors are both M1 and M3.     

6'-O-dansyl-gamma-aminobutyryl atractyloside, a fluorescent probe of the ADP/ATP carrier: exploration of conformational changes of the membrane-bound ADP/ATP carrier elicited by substrates and inhibitors

A fluorescent atractyloside analogue, the 6'-O-dansyl-gamma-aminobutyryl atractyloside (DGA), has been used to probe the binding of the inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BA) and nucleotide substrates to the membrane-bound ADP/ATP carrier protein in beef heart mitochondria. Binding and release of DGA were followed by fluorescence responses. Specifically bound DGA was fully released by CATR alone, or by BA in the presence of micromolar amounts of ADP. In the absence of the inhibitors, ADP increased the rate of the specific binding of DGA. The effect of ADP was shared by transportable nucleotides. Non transportable nucleotides were ineffective. These data are consistent with the previously described CATR and BA conformations of the ADP/ATP carrier that are able to bind CATR and BA respectively, the transition between the two conformations being accelerated by micromolar concentrations of transportable nucleotides.     

High-affinity binding sites for PK 11195, but not for RO5-4864, in porcine aortic smooth muscle

Peripheral benzodiazepine (BZ) binding sites were characterized in porcine aortic smooth muscle membrane preparation. [3H]PK11195 bound with high affinity to the membranes (Kd = 8.6 + 0.9 nM), whereas [3H]Ro5-4864 bound slightly to the membranes. The Ki value of Ro5-4864 obtained from the inhibition of [3H]PK 11195 binding was 1200 + 200 nM, which was 480 times weaker than that obtained in rat kidney. Furthermore, the Ro5-4864 effect was temperature-insensitive. When [3H]PK 11195 binding was examined in porcine, human and rat platelets, Ro5-4864 inhibited the binding in porcine and human platelets one order of magnitude less potently than that in rat platelets. These results suggest that low affinity for Ro5-4864 in porcine aorta smooth muscle originates in porcine tissue, but not in smooth muscle.     

Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria: I. General Characteristics and Effect of Inhibitors

Corn mitochondria show respiration-linked net accumulation of [³H]ADP in the presence of phosphate and magnesium, especially if the formation of ATP is blocked with oligomycin. Inhibition of ADP-ATP exchange by carboxyatractyloside also activates ADP accumulation, and addition of carboxyatractyloside or palmitoyl-coenzyme A to oligomycin-blocked mitochondria produces additional ADP uptake. With carboxyatractyloside the accumulated ADP is phosphorylated to ATP. With oligomycin, only a little ATP is formed. Millimolar concentrations of ADP are required for maximum uptake, and the K_m (3.77 millimolar) for ADP translocation is independent of whether oligomycin or carboxyatractyloside is used. This is not true for ADP concentrations in the 0.05 to 0.25 millimolar range. Accumulated [³H]ADP rapidly exchanges with unlabeled AMP, ADP, or ATP, but not with other diphosphate nucleotides or 2 millimolar substrate anions. [³H]AMP is not accumulated, but [³H]ATP is accumulated to about one-half the extent of [³H]ADP. Tricarboxylate substrates inhibit ADP net uptake, and inhibition by citrate is competitive with K_i = 10 millimolar. The evidence suggests the presence of a pathway, carboxyatractyloside-insensitive and different from the translocase, which operates to maintain adenine nucleotides in the matrix.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[alpha-32P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[alpha-32P]ADP in the dark with a Kd value of congruent to 8 microM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[alpha-32P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[alpha-32P]ADP, both the ADP/ATP carrier and the beta subunit of the membrane-bound F1-ATPase are covalently labeled. The binding specificity of 2-azido-[alpha-32P]ADP for the beta subunit of F1-ATPase is ascertained by prevention of photolabeling of isolated F1 by preincubation with an excess of ADP.     

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

Prostaglandin moieties that determine receptor binding specificity in the bovine corpus luteum.

This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2 alpha' [3H]PGE2' [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2 alpha and [3H]PGD2 was inhibited by non-radioactive PGF2 alpha (IC50 values of 21 and 9 nmol l-1, respectively), PGD2 (35 and 21 nmol l-1), and PGE2 (223 and 81 nmol l-1), but not by PGE1 (> 10,000 and 5616 nmol l-1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l-1) and PGE2 (7 nmol l-1), but minimally by PGD2 (2316 nmol l-1) and PGF2 alpha (595 nmol l-1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2 alpha required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1' PGE2' agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2 alpha' PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.     

Multiple binding sites for phencyclidine on the nicotinic acetylcholine receptor from Torpedo ocellata electric organ

Binding of [3H]-phencyclidine ( [3H]-PCP) to acetylcholine-receptor enriched membrane from Torpedo ocellata electric organ was studied over a ligand concentration range of 1 to 200 microM. The results indicate that [3H]-PCP is bound to two classes of sites: high affinity (Kd = 6-9 microM) and low affinity (Kd = 85 microM) binding sites. In the absence of cholinergic drugs the ratio of high affinity [3H]-PCP binding sites to 125I-alpha-bungarotoxin (alpha-Bgt) binding sites is 0.37, and that of low affinity [3H]-PCP binding sites to 125I-alpha-Bgt is 1.06. Low affinity [3H]-PCP binding can be completely inhibited by alpha-bungarotoxin (alpha-Bgt), carbamylcholine and d-tubocurarine. This inhibition, together with the one to one stoichiometry with 125I-alpha-Bgt, suggests that the sites to which [3H]-PCP binds with low affinity are the acetylcholine (AcCho) binding sites. In the presence of 1 microM alpha-Bgt which blocks binding of [3H]-PCP to the AcCho binding sites, the ratio of high affinity [3H]-PCP sites to 125I-alpha-Bgt sites is 0.5, indicating the existence of one high affinity PCP site per receptor molecule, The toxin, however, decreases the apparent affinity of [3H]-PCP towards the AcCho receptor as well as the potency of tetracaine or dibucaine in inhibiting [3H]-PCP binding to that receptor. In the latter case the effect involves changes from a biphasic to a simple inhibition curve. The results suggest that non-competitive blockers to the AcCho receptors may affect their own sites as well, and that they do this also by binding to the AcCho binding sites. This is also inferred from the accelerated dissociation of [3H]-PCP from its high affinity binding sites by unlabeled PCP in the concentration range of 10(-3) to 10(-4) M, at which the drug occupies AcCho binding sites as well.     

Affinity modification of creatine kinase and ATP-ADP translocase in heart mitochondria: determination of their molar stoichiometry

Oxidized dialdehyde analogs of ADP or ATP (oADP and oATP) were shown to inhibit irreversibly adenine nucleotide translocator (T) and creatine kinase (CK) in heart mitochondria. Inactivation of T and CK was parallel with carboxyatractyloside - sensitive and (ADP + phosphocreatine) - sensitive incorporation of o[3H]ADP into mitochondria, respectively. o[3H]ADP incorporation sensitive to CAT or ADP+phosphocreatine was used to determine T and CK contents in mitochondria. T content in cardiac mitochondria from rat, rabbit, dog, and chicken was calculated to be 2.6 - 2.9 moles/mole cyt.aa3. The same value of T/cyt.aa3 ratio was found in liver mitochondria with lower cytochrome aa3 content. In all types of cardiac mitochondria CK content was found to be 2.4 - 2.6 moles/mole cyt.aa3. The data show that T and CK are present in molar ratio 1:1 in all types of cardiac mitochondria.     

Binding of malonyl-CoA to isolated mitochondria. Evidence for high- and low-affinity sites in liver and heart and relationship to inhibition of carnitine palmitoyltransferase activity.

[14C]Malonyl-CoA bound to intact mitochondria isolated from rat liver and heart in a manner consistent with the presence of two independent classes of binding sites in each tissue. The binding characteristics for mitochondria obtained from fed male rats were: for heart, KD(1) = 11-18nM, KD(2) = 30 microM, N1 = 7pmol/mg of protein, N2 = approx. 660pmol/mg of protein; for liver, KD(1) = 0.1 microM, KD(2) = 5.6 microM, N1 = 11pmol/mg of protein, N2 = 165pmol/mg of protein. In the presence of 40 microM-palmitoyl-CoA the characteristics of binding at the high-affinity sites were changed, so that for heart KD(1) = 0.26 microM, with no change in N1 and for liver KD(1) = approx. 2 microM, with N1 increased to approx. 40pmol/mg of protein. Differences between the two tissues in tightness of malonyl-CoA binding at the high-affinity sites explains the considerably greater sensitivity of heart CPT1 (overt form of carnitine palmitoyltransferase) to inhibition by malonyl-CoA [Saggerson & Carpenter, (1981) FEBS Lett. 129, 229-232; McGarry, Mills, Long & Foster (1983) Biochem. J. 214, 21-28]. Starvation (24h) did not change the characteristics of [14C]malonyl-CoA binding to liver mitochondria and did not alter the I50 (concentration giving 50% inhibition) for displacement of [14C]malonyl-CoA by palmitoyl-CoA. Therefore the decreased sensitivity of liver CPT1 to inhibition by malonyl-CoA in starvation [Saggerson & Carpenter (1981) FEBS Lett. 129, 225-228; Bremer (1981) Biochim. Biophys. Acta 665, 628-631] is not explained by differences in malonyl-CoA binding. Percentage occupancy of the high-affinity sites in heart mitochondria by malonyl-CoA correlated closely with percentage inhibition of CPT1 measured under similar conditions. This finding supports the proposal that the high-affinity binding sites are the functional sites mediating inhibition of CPT1 by malonyl-CoA. Similar experiments with liver mitochondria also suggested that the occupancy of high-affinity sites by malonyl-CoA regulates CPT1 activity. 5,5'-Dithiobis-(2-nitrobenzoic acid), which decreased the sensitivity of heart or liver CPT1 to inhibition by malonyl-CoA [Saggerson & Carpenter (1982) FEBS Lett. 137, 124-128], also decreased [14C]malonyl-CoA binding to the high-affinity sites of heart mitochondria. N1 values for [14C]malonyl-CoA binding to high-affinity sites in liver mitochondria were determined in various physiological states which encompassed a 7-fold range of CPT1 maximal activity (fed, starved, pregnant, hypothyroid, foetal). The N1 value did not change in these states.(ABSTRACT TRUNCATED AT 400 WORDS)