A study on the exchange of tightly bound nucleotides on the membrane-associated chloroplast ATP synthase complex

Light-induced exchange of tightly bound ADP on the membrane-associated chloroplast coupling factor 1 (CF₁) was concluded to be a two-step mechanism involving a loose enzyme-ADP complex (Strotmann, H., Bickel-Sandkötter, S. and Shoshan, V. (1979) FEBS Lett. 101, 316–320). Rapid binding of [¹⁴C]ADP to the coupling factor after deenergization of thylakoids which were illuminated in the presence of [¹⁴C]ADP was suggested to reflect the conversion of loosely bound to tightly bound ADP. Experimental data of the present paper support the assumption of an intermediate enzyme form with loosely bound ADP: (a) the amplitude of the rapid binding phase is independent on the concentration of uncoupler added in the light; (b) the amplitude is virtually unaffected by dilution of the medium [¹⁴]CADP concentration; (c) high concentrations of unlabeled ADP are required to reduce the rapid binding phase while binding of medium [¹⁴C]ADP is inhibited by unlabeled ADP in the micromolar range. These results exclude the possibility that the rapid initial formation of tightly bound [¹⁴C]ADP on deenergization might be caused by an energized nucleotide-free enzyme form which is able to pick up [¹⁴C]ADP from the medium at a higher rate than the deenergized nucleotide-free form. At saturating [¹⁴C]ADP concentrations in the light, the amount of the loose enzyme-ADP complex is about 35%, while 65% of the coupling factors contain a tightly bound ADP. Dissociation of the loose complex is slow in the absence of medium nucleotides but accelerated if ADP is present, suggesting that ADP binding to another site of the enzyme promotes release of the former ADP molecule. The significance of the loosely bound nucleotide in the catalytic mechanism is discussed.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO₂-dependent oxygen evolution by isolated pea chloroplasts. Both α, β- and β, γ-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO₂-dependent oxygen evolution. The concentration of adenylyl-imidodiphosphate required for 50% inhibition of CO₂-dependent oxygen evolution was 50 μM.2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation.3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [¹⁴C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator.4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO₂ fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F₁ with Mg-ATP was required for the binding of the natural inhibitor, IF₁, to F₁ to form the inactive F₁-IF₁ complex. When F₁ was incubated in the presence of [¹⁴C]ATP and MgCl₂, about 2 mol ¹⁴C-labeled adenine nucleotides were found to bind per mol of F₁; the bound ¹⁴C-labeled nucleotides consisted of [¹⁴C]ADP arising from [¹⁴C]ATP hydrolysis and [¹⁴C]ATP. The ¹⁴C-labeled nucleotide binding was not prevented by IF₁. These data are in agreement with the idea that the formation of the F₁-IF₁ complex requires an appropriate conformation of F₁. (2) The ¹⁴C-labeled adenine nucleotides bound to F₁ following preincubation of F₁ with Mg-[¹⁴C]ATP could be exchanged with added [³H]ADP or [³H]ATP. No exchange occurred between added [³H]ADP or [³H]ATP and the ¹⁴C-labeled adenine nucleotides bound to the F₁-IF₁ complex. These data suggest that the conformation of F₁ in the isolated F₁-IF₁ complex is further modified in such a way that the bound ¹⁴C-labeled nucleotides are no longer available for exchange. (3) ³²P_i was able to bind to isolated F₁ with a stoichiometry of about 1 mol of P_i per mol of F₁ (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891–2899). There was no binding of ³²P_i to the F₁-IF₁ complex. Thus, not only the nucleotides sites, but also the P_i site, are masked from interaction with external ligands in the isolated F₁-IF₁ complex.     

ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase

A modified ‘cold chase’ technique was used to study tight [¹⁴C]ADP and [¹⁴C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF₀F₁). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF₀F₁ or CF₁ reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k _d of about 5 min⁻¹. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF₀F₁. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF₁ dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF₁ becomes bound to a thylakoid membrane.     

The interaction of phenylglyoxal with soluble and membrane-bound chloroplast coupling factor 1

The rate of inhibition of cyclic photophosphorylation in chloroplast thylakoids by the arginine reagent phenylglyoxal was enhanced in the light, i.e., under conditions where membrane energization occurred. Uncouplers, but not energy-transfer inhibitors, prevented the effect of light. Chemical modification of chloroplast thylakoids by phenylglyoxal under dark or in light conditions affected differently the light-induced exchange of tightly bound ADP. In both cases the exchange was less inhibited than photophosphorylation. Complete inhibition of ATPase activity of soluble CF₁ was correlated with the incorporation of 8 mol [¹⁴C]phenylglyoxal per mol enzyme. About 50% of the incorporated radioactivity was lost at different rates depending on the buffer present and suggesting a change in the stoichiometry of the adduct from 2:1 to 1:1. Inhibition of ATPase and photophosphorylating activities of chloroplasts by modification with [¹⁴C]phenylglyoxal in the dark was associated with the incorporation of 1 and 2 mol reagent per mol membrane-bound CF₁, respectively. In the light the rate of incorporation was enhanced and both reactions were inactivated when 2 mol $\text{[}{}^{14}\text{C]phenylglyoxal}\text{CF}{}_1$ were bound. In all the labelling experiments the radioactivity was mainly recovered from the α- and β-subunits.     

pH dependent changes in ADP and ATP affinity for the tight nucleotide-binding site of chloroplast coupling factor 1

The pH-dependence of ADP and ATP affinity for CF₁ tight nucleotide-binding sites was studied under conditions of equilibrium between bound and free labeled nucleotides. With the nucleotide/CF₁ ratio>1, the ATP content in tightly bound nucleotides depended only slightly on medium pH. With the nucleotide/CF₁ ratio approaching 1, tightly bound ATP content grew rapidly with decreasing pH. Calculations of ADP/ATP ratio in free and tightly bound nucleotides showed that decreasing the pH from 8.0 to 6.0 induced a 150 times greater affinity of the nucleotide-binding site for ATP than for ADP. The data indicates that ATP-ADP equilibrium at the CF₁ tight nucleotide-binding site depends on protonation of specific acid-base groups of the enzyme.Abbreviations CF₁, BF₁, and MF₁ coupling factors of chloroplasts, bacteria, and mitochondria, respectively - AdN adenine nucleotide     

Loose and tight binding of adenine nucleotides by membrane-associated chloroplast ATPase

Steady-state binding of adenine nucleotides by thylakoid membranes is measured by employing a centrifugation technique. By this method tightly bound nonexchangeable nucleotides can be discriminated from loosely bound, exchangeable nucleotides. Nucleotide binding requires membrane energization and is highly specific for medium ADP. In illuminated chloroplasts almost no exogenous AMP and only some ATP are incorporated, most being recovered as tightly bound nucleotides. In light-triggered chloroplasts, however, which are capable of hydrolyzing ATP, a high level of exchangeable nucleotides is found on the membranes. The sum of tightly bound and loosely bound nucleotides originating from medium ADP is about one per CF₁. The ratio between them decreases with increasing proton-motive force. Exchangeable nucleotides most probably represent the ligands involved in the catalytic process, as suggested from substrate specificity and the effect of a competitive inhibitor of photophosphorylation, naphthoyl ADP. This compound in a low concentration range supresses loose binding but not tight binding of medium ADP. Under phosphorylating conditions (presence of ADP, P_i and light), some of the tightly bound nucleotides exist as ATP even in the presence of a hexokinase system. The results are discussed in the context of the regulation of chloroplast ATPase by tight nucleotide binding.     

Energy-dependent changes in the ATP/ADP ratio at the tight nucleotide binding site of chloroplast ATP synthase

Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [¹⁴C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [¹⁴C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m^–2, ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.Abbreviations AdN adenine nucleotide - Chl chlorophyll - DTT dithiothreitol - FCCP carbonylcianide p-trifluoromethoxyphenilhydrazone - P_i inorganic orthophosphate - PMS phenazine methosulfate - TLC thin-layer chromatography - Tricine N-[tris(hydroxymethyl)methyl] glycine     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F1 with Mg-ATP was required for the binding of the natural inhibitor, IF1, to F1 to form the inactive F1-IF1 complex. When F1 was incubated in the presence of [14C]ATP and MgCl2, about 2 mol 14C-labeled adenine nucleotides were found to bind per mol of F1; the bound 14C-labeled nucleotides consisted of [14C]ADP arising from [14C]ATP hydrolysis and [14C]ATP. The 14C- labeled nucleotide binding was not prevented by IF1. These data are in agreement with the idea that the formation of the F1-IF1 complex requires an appropriate conformation of F1. (2) The 14C-labeled adenine nucleotides bound to F1 following preincubation of F1 with Mg-[14C] ATP could be exchanged with added [3H]ADP or [3H]ATP. No exchange occurred between added [3H]ADP or [3H]ATP and the 14 C-labeled adenine nucleotides bound to the F1-IF1 complex. These data suggest that the conformation of F1 in the isolated F1-IF1 complex is further modified in such a way that the bound 14C-labeled nucleotides are no longer available for exchange. (3) 32Pi was able to bind to isolated F1 with a stoichiometry of about 1 mol of Pi per mol of F1 (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891-2899). There was no binding of 32Pi to the F1-IF1 complex. Thus, not only the nucleotides sites, but also the Pi site, are masked from interaction with external ligands in the isolated F1-IF1 complex.     

Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C-labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐¹⁴C]adenine and [8‐¹⁴C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐¹⁴C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐¹⁴C]adenosine, [8‐¹⁴C]adenine, and [8‐¹⁴C]inosine was also detected in ureide, especially allantoic acid, and CO₂. Among these 3 precursors, the radioactivity from [8‐¹⁴C]inosine was predominantly incorporated into CO₂. These results suggest the operation of a conventional degradation pathway. Both [2‐¹⁴C]uracil and [2‐¹⁴C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐¹⁴C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐¹⁴C]uracil was recovered as CO₂ and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO₂.     

Effect of Escherichia coli endotoxin on adenine nucleotide translocation in canine heart mitochondria

The in vitro effect of Escherichia coli endotoxin on the translocation of adenine nucleotides in dog heart mitochondria was studied. Mitochondrial adenine nucleotides were labeled with ¹⁴C by incubating mitochondrial preparations in the presence of [¹⁴C]ADP. The exchange reaction was initiated by addition of unlabeled ADP, proceeded for 5 to 60 s at 4 °C, and was terminated by addition of atractyloside. The results showed that preincubation of mitochondria with endotoxin (50 μg/mg protein) for 10 min at 23 °C decreased the exchange reaction by 21.2% (P < 0.05). The inhibitory effect of endotoxin was increased with increasing concentrations of endotoxin with an I₅₀ value of 45 μg/mg protein. The initial rate and the total extent of exchange were both affected. Double reciprocal plots showed that only the V but not the K_m for ADP was affected by endotoxin, indicating that the inhibition was noncompetitive in nature. The exchange of adenine nucleotide remained depressed by endotoxin in the presence of either oligomycin or antimycin A, indicating that the inhibitory effect of endotoxin was independent of the action of endotoxin on oxidative phosphorylation. The leakage of labeled adenine nucleotides from mitochondria at 23 °C was increased by 100% by endotoxin (100 μg/mg protein) in the absence of added unlabeled ADP, and this increase in the leakage could not be blocked by atractyloside. The endotoxin-induced changes in adenine nucleotide exchange and leakage were either partially or completely prevented by hydrocortisone, heparin, dibucaine, or EDTA. Since most of these agents have in common an effect on lipid metabolism, it is suggested that endotoxin-induced alterations in the exchange and leakage of adenine nucleotides in heart mitochondria are protected through a mechanism involving membrane lipid reorganization.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport.

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO2-dependent oxygen evolution by isolated pea chloroplasts. Both alpha, beta- and beta, gamma-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO2-dependent oxygen evolution. The concentration of adenylylimidodiphosphate required for 50% inhibition of CO2-dependent oxygen evolution was 50 micronM. 2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation. 3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [14C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator. 4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO2 fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

Secretory mechanisms. Behaviour of adenine nucleotides during the platelet release reaction induced by adenosine diphosphate and adrenaline

1. Platelets containing adenine nucleotides labelled with ³H and ¹⁴C in vitro were aggregated biphasically with ADP and adrenaline. Amounts of ATP and ADP as well as the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine and adenine were determined in platelets and plasma at different stages of aggregation. 2. ATP and ADP were released during the second aggregation phase and had a low specific radioactivity compared with the ATP and ADP retained by the cells. The specific radioactivity of intracellular nucleotides increased during release. The parameters observed with ADP and adrenaline as release inducers were the same as for collagen and thrombin. 3. Release induced by all four inducers was accompanied by conversion of cellular [³H]ATP into extracellular [³H]-hypoxanthine. By variation of temperature, inducer concentration, time after blood withdrawal and use of acetylsalicylic acid, the aggregation pattern caused by adrenaline and ADP could be made mono- or bi-phasic. Release or second-phase aggregation was intimately connected with the ATP–hypoxanthine conversion, whereas first phase aggregation was not. 4. The [³H]ATP–hypoxanthine conversion started immediately after ADP addition. With adrenaline it usually started with the appearance of the second aggregation phase. The conversion was present during first phase of ADP-induced aggregation only if a second phase were to follow. 5. When secondary aggregation took place while radioactive adenine was being taken up by the platelets, increased formation of labelled hypoxanthine still occurred, but there was either no change or an increase in the concentration of labelled ATP. 6. Biphasically aggregated platelets converted [³H]adenine more rapidly into [³H]-ATP and -hypoxanthine than non-aggregated platelets. Addition of [³H]adenine at different stages of biphasic aggregation showed that more [³H]hypoxanthine was formed during than after the release step. 7. We conclude that ADP and adrenaline, like thrombin and collagen, cause extrusion of non-metabolic granula-located platelet adenine nucleotides. During release metabolic ATP breaks down to hypoxanthine, and this process might reflect an ATP-requiring part of the release reaction.     

The incorporation of 32Pi into intramitochondrial ADP fraction dependent on the substrate-level phosphorylation

1. A significant amount of ³²P_i is incorporated into ADP fraction if mitochondrial phosphorylation is allowed to proceed solely dependent on the endogenous adenine nucleotides even in the absence of uncouplers or inhibitors of oxidative phosphorylation. This formation of [³²P]ADP is accompanied by a significant labelling of the GTP fraction as well as by a decrease in mitochondrial AMP.2. A good correlation, highly significant on a statistical basis, is obtained between the incorporation of ³²P_i into ADP on the one hand and the oxidation of [1-¹⁴C]glutamate to ¹⁴CO₂ on the other, under a wide variety of conditions of respiration, suggesting that the substrate-level phosphorylation linked to the oxidation of 2-oxoglutarate leads to the phosphorylation of AMP in rat liver mitochondria.3. Since intramitochondrial GTP is not directly labelled by the [³²P]ATP added, it is concluded that neither nucleoside diphosphokinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) nor adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is functioning in such an EDTA-containing medium as employed in the present study because of lack of the enzymes inside the inner membrane. This not only indicates that ATP never serves as a phosphate donor for the observed phosphorylation of AMP, but also, along with several other lines of evidence, lends strong support to the view that [³²P]GTP generated as a result of the substrate-level phosphorylation is a direct precursor of [³²P]ADP through the mediation of GTP:AMP phosphotransferase, which has been verified to be located inside the inner membrane by the significant labelling of GTP by [³²P]ADP.     

Tightly bound nucleotides of the energy-transducing ATPase,and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations.2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300 000. An inhibitor protein is bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment.3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one.4. Under de-energised conditions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate ³²P_i. ³²P_i is incorporated into the β and γ positions of the bound nucleotides, but β-labelling probably does not occur on the coupling ATPase.5. Uncouplers inhibit the exchange of the free nucleotides or ³²P_i into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange.6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.     

Studies of adenosine incorporation in Langendorff rat heart and rat heart mitochondria

The acid-insoluble product isolated from well-oxygenated Langendorff rat heart after perfusion with [¹⁴C]adenosine was purified by phenol extraction and subjected to specific phosphorolysis by pure polynucleotide phosphorylase. TLC analysis of the reaction mixture showed that ADP was the only radioactive product, proving that the original substance was a polyribonucleotide. Studies of the time course of labelling and of the distribution of the acid-insoluble product between the mitochondrial and nuclear fractions showed that both are labelled even after 1 min at 25 °C, but at short times and low temperature more radioactivity is found in the mitochondria. The kinetics of adenosine incorporation resemble those expected for the labelling of hnRNA and mRNA. Isolated, respiring mitochondria incorporate adenosine and adenine nucleotides into acid insoluble form by a process dependent on oxidative phosphorylation and the adenine nucleotide translocase that is specific for adenine derivatives. The results are discussed in terms of the hypothesis that the polyribonucleotide might be a storage form of adenine nucleotides: it is concluded that the bulk of the labelled product is unlikely to play a major role in energy metabolism.     

Characterization of exchangeable and nonexchangeable bound adenine nucleotides in F1-ATPase from Escherichia coli

The total amount of bound exchangeable and nonexchangeable adenine nucleotides in Escherichia coli F1-ATPase (BF1) was determined; three exchangeable nucleotides were assessed by equilibrium dialysis in a [14C]ADP-supplemented medium. When BF1 was purified in a medium supplemented with ATP, a stoichiometry of nearly 6 mol of bound nucleotides/mol of enzyme was found; three of the bound nucleotides were ATP and the others ADP. When BF1 was filtered on Sephadex G-50 in a glycerol medium (Garrett, N.E., and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647), bound ADP was rapidly released, in contrast to bound ATP which remained firmly attached to the enzyme. Upon incubation of BF1 with [14C]ADP, the bound ADP rather than the bound ATP was exchanged. Of the three [14C]ADPs which have bound to BF1 by exchange after equilibrium dialysis, one was readily lost by gel filtration on Sephadex G-50; the loss of bound [14C]ADP was markedly reduced by incubation of BF1 with aurovertin, a specific ligand of the beta subunit which is known to increase the affinity of the beta subunit for nucleotides (Issartel, J.-P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). Upon photoirradiation of BF1 with [alpha-32P]2-azido-ADP, only the beta subunit was labeled; concomitantly, bound ADP was released, but the content in bound ATP remained stable. These results suggest that specific sites located on the three beta subunits bind nucleotides in a reversible manner. Consequently, the tightly bound ATP of native BF1 would be located on the alpha subunits.     

Exchange transamination and the metabolism of glutamate in brain

1. Experiments were performed to throw light on why the incorporation of ¹⁴C from labelled carbohydrate precursors into glutamate has been found to be more marked in brain than in other tissues. 2. Rapid isotope exchange between labelled glutamate and unlabelled α-oxoglutarate was demonstrated in brain and liver mitochondrial preparations. In the presence but not in the absence of α-oxoglutarate the yield of ¹⁴CO₂ from [1-¹⁴C]glutamate exceeded the net glutamate removal, and the final relative specific activities of the two substrates indicated that complete isotopic equilibration had occurred. Also, when in a brain preparation net glutamate removal was inhibited by malonate, isotope exchange between [1-¹⁴C]glutamate and α-oxoglutarate and the formation of ¹⁴CO₂ were unaffected. 3. The time-course of isotope exchange between labelled glutamate and unlabelled α-oxoglutarate was followed in uncoupled brain and liver mitochondrial fractions, and the rate of exchange calculated by a computer was found to be 3–8 times more rapid than the maximal rate of utilization of the two substrates. 4. The physiological situation was imitated by the continuous infusion of small amounts of α-oxo[1-¹⁴C]glutarate into brain homogenate containing added glutamate. The fraction of ¹⁴C infused that was retained in the glutamate pool depended on the size of the latter, and the final relative specific activities of the two substrates indicated almost complete isotope exchange. Isotopic equilibration also occurred when α-oxoglutarate was generated from pyruvate through the tricarboxylic acid cycle in a brain mitochondrial preparation containing [1-¹⁴C]glutamate. 5. The differences in the incorporation of ¹⁴C from labelled glucose into the glutamate of brain and liver are discussed in terms of the rates of isotope exchange, the glutamate pool sizes and the rates of formation of labelled α-oxoglutarate in the two tissues. It is concluded that the differences between tissues in the incorporation of glucose carbon into glutamate reflect features of their metabolism largely unrelated to that of glutamate.     

UTILIZATION OF ACETATE AND PYRUVATE FOR THE SYNTHESIS OF'TOTAL','BOUND'AND'FREE'ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

—Slices of tissue of the electric organ of Torpedo marmorata were incubated in vitro in a salineurea-sucrose solution containing a labelled precursor of the acetyl moiety of ACh ([1-¹⁴C]glucose, [2-¹⁴C]pyruvate, or [1-¹⁴C]acetate) either alone or in the presence of another unlabelled precursor. The incorporation of ¹⁴C from [1-¹⁴C]acetate into ACh was considerably higher than from the other two substrates. The specific radioactivities (SRA) of the‘total',‘bound’and‘free’ACh were compared in experiments with [2-¹⁴C]pyruvate and [1-¹⁴C]acetate. With both precursors, the SRA of the‘bound’ACh were lower than those of‘total’ACh; consequently, the‘free’ACh pool was more labelled than the‘bound’pool. After short incubations with [2-¹⁴C]pyruvate the SRA of'bound’ACh were closer to the SRA of‘total’ACh than with [1-¹⁴C]acetate. A simple method is described for the labelling of ACh and its separation from other labelled compounds in experiments with the electric organ using [¹⁴C]acetate as the labelled precursor.