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PubMed1980

Restitution of myocardial adenine nucleotides: acceleration by administration of ribose.

In the present study, the influence of ribose on the biosynthesis of myocardial adenine nucleotides was examined in rats in vivo utilizing two experimental models which are characterized by a reduction in the adenine nucleotide content; recovery from oxygen deficiency and application of isoproterenol. 1. The biosynthesis (= de novo synthesis) of cardiac adenine nucleotides was enhanced by 90% during the first 60 min of recovery from five intermittent periods of asphyxia of 4.5 min duration (Table I). 2. Isoproterenol induced a stimulation of myocardial adenine nucleotide synthesis in a dose-dependent manner amounting to 640% three hours after s.c. administration of 25 mg/kg (Fig. 1). 3. Ribose which bypasses the hexose monophosphate shunt in the myocardium and which leads to an elevation of the available pool of 5-phosphoribosyl-1-pyrophosphate (PRPP), stimulated the biosynthesis of adenine nucleotides in the heart, but not in liver and kidney, of rats one hour after i.v. application of a single dose of 100 mg/kg from 6 nmoles/g/h to 27 nmoles/g/h (Fig. 2). 4. When ribose was constantly infused during the first 60 min of recovery from asphyxia, the enhancement of cardiac adenine nucleotide biosynthesis was further stimulated from 12.6 nmoles/g/h to 20.5 nmoles/g/h (with 500 mg ribose/kg/h) and to 43.4 nmoles/g/h (with 1 000 mg ribose/kg/h) (Fig. 3). 5. Continuous I.V. infusion of ribose (200 mg/kg/h) for 24 hours in isoproterenol-treated rats during a 13-fold increase in myocardial adenine nucleotide biosynthesis compared with the control (Fig. 4). In this condition, the isoproterenol-induced decline in the adenine nucleotide level did not occur (Table II).

Adenine Nucleotides↗

PubMed1983

Metabolism of exogenous adenine in a pyruvate kinase-deficient patient.

The metabolism of adenine, administered intravenously and orally, was studied in a patient with red cell pyruvate kinase (PK)-deficiency. Intravenous infusion of 58.5 mumol of adenine per kg containing 6475 Bq of 8-14C adenine caused an initial decrease in red cell ATP level and the gradual return to the pre-infusion level coincided with an increase in radiolabelled ATP. Of the infused adenine, 1.3% had entered the red cell pool after 2 h. The route of administration affected the urinary excretion of adenine and its oxymetabolites; after 6 h, 10.7% of the infused adenine had been excreted, compared with 7.4% after ingestion of a similar dose. In 10 control subjects given 45.1-55.5 mumol/kg, excretion of adenine plus oxymetabolites accounted for 13.5 +/- 2.9% of the ingested dose. Differences were also observed in the urinary purine excretion patterns for the two routes of administration. This study has shown that adenine can be incorporated into ATP in the red cells of a PK-deficient patient.

Adenine↗

PubMed1983

Adenine binding sites of the lectin from lima beans (Phaseolus lunatus).

A single high-affinity binding site for adenine and related compounds was identified in the lima bean lectin (LBL) component III tetramer. This site is identical with the high affinity site for 2,6-toludinyl-naphthalenesulfonate described previously (Roberts, D. D., and Goldstein, I. J. (1982) J. Biol. Chem. 257, 11274-11277). [14C]Adenine was bound with high affinity (Kd = 1.2 +/- 0.1 X 10(-5) M, T = 25 degrees C) and a high degree of specificity in that hypoxanthine and guanine were very poor ligands for this site. Specificity was also observed for free purine bases relative to nucleosides or nucleotides. A number of N6 derivatives of adenine with cytokinin activity were found to bind to LBL, with relative affinities decreasing in the order: N6 - benzyladenine greater than kinetin greater than zeatin greater than N6 - [delta 2-isopentenyl]adenine greater than dihydrozeatin greater than zeatin riboside. Evidence was also obtained for heterotropic interaction between the adenine binding site and a second class of hydrophobic sites present on each subunit of LBL. Binding of adenine and N6-benzyladenine to LBL was found to produce a 2.3- and 3.8-fold increase, respectively, in the affinity of the lectin subunit hydrophobic sites for 1,8-anilinonaphthalenesulfonate. 1,8-Anilinonaphthalenesulfonate, in turn, enhanced the affinity of LBL for adenine, demonstrating that binding of ligands to the two classes of hydrophobic sites is thermodynamically linked. Equilibrium dialysis also revealed high affinity binding sites for [14C]adenine on the lectins from Dolichos biflorus, Phaseolus vulgaris, and soybean (Glycine max).

Adenine↗

PubMed1982

Human adenine phosphoribosyltransferase. Immunochemical quantitation and protein blot analysis of mutant forms of the enzyme.

We have studied the catalytic, immunochemical, and electrophoretic properties of adenine phosphoribosyltransferase in hemolysates from 30 patients with a deficiency of this enzyme in six unrelated families. We have found that: 1) the level of adenine phosphoribosyltransferase enzyme activity and immunoreactive protein in the four homozygous deficient patients was less than 1% of control values; 2) adenine phosphoribosyltransferase enzyme activity was uniformly decreased to approximately 25% of normal in all 26 heterozygotes studied while the level of adenine phosphoribosyltransferase immunoreactive protein was consistent within each kindred but ranged in value from 22% to 112% of control; and 3) protein blot analysis revealed a single isoelectric form of the adenine phosphoribosyltransferase subunit in hemolysate from normal controls and from every heterozygote except for patient M.R. who exhibited both a normal and a more acidic adenine phosphoribosyltransferase subunit species. These studies provide the first evidence for the existence of a variety of different mutations in the structural gene for adenine phosphoribosyltransferase in patients exhibiting a deficiency of enzyme activity. We further conclude from our data that: 1) the variant enzymes are more labile in vivo and/or catalytically nonfunctional, and 2) heterozygotes express only 25% of normal enzyme activity because the normal and variant enzyme subunits form a hybrid dimer which is either more labile or less catalytically active than the normal adenine phosphoribosyltransferase dimer.

Adenine Phosphoribosyltransferase↗

PubMed1994

[The meiosis arrest of mouse oocytes induced by adenine].

The effect of purine on the meiosis of mouse oocytes was studied. When adenine and benzyladenine, a similar substance of adenine, was microinjected into the germinal vesicle of oocytes, the meiotic maturation of oocytes could be obviously arrested depending on the dosage of injection. The stimulator of adenylate cyclase, NaF, could enhance the arrest effect of adenine. It indicated that cAMP played a important role in mouse oocytes meiotic resumption. Hypoxanthine and adenine had different effects in different medium. Hypoxanthine could obviously arrest the maturation of mouse cumulus oocyte complex (COC) and cumulus denuded oocytes (DO) in DMEM or EMEM. But only in DMEM adenine could obviously arrest the maturation of COC, and had co-effect with hypoxanthine in DMEM. They could almost arrest the maturation of COC and DO. Adenine in EMEM had little arrest effect on maturation of COC and DO. The different effects may result from the different adenine absorption by oocyte in DMEM and EMEM. Adding glutamine into EMEM could enhance the arrest effect of adenine to COC and DO.

Adenine↗

PubMed1981

5-Methylnicotinamide-adenine dinucleotide. Kinetic investigation with major and minor isoenzymes of liver alcohol dehydrogenase and structural determination of its binary complex with alcohol dehydrogenase.

5-Methylnicotinamide-adenine dinucleotide and 3-cyano-5-methylpyridine-adenine dinucleotide was prepared from 5-methylthionicotinamide-adenine dinucleotide by chemical conversion. The 5-methylthionicotinamide-adenine dinucleotide was obtained by enzymic transglucosidation. Model compounds ascertained the structure. None of the dinucleotides methylated at C-5 was active with the major isoenzyme EE of horse liver alcohol dehydrogenase, but activity with 5-methylnicotinamide-adenine dinucleotide was measured with the minor isoenzymes. The binding of 5-methylnicotinamide-adenine dinucleotide to liver alcohol dehydrogenase, investigated by X-ray diffraction methods to 0.37-nm resolution, occurs with the pyridinium ring away from the active site as previously described for 3-iodopyridine-adenine and pyridine-adenine dinucleotides. A general conclusion on the use of inhibitors as tools for exploration of the active site is drawn.

Alcohol Oxidoreductases↗

PubMed1977

Labeling of the releasable adenine nucleotides of washed human platelets.

In rabbit platelets, the metabolically active ATP pool equilibrates with the releasable ATP pool within 1 day. The studies showing this have now been extended to human platelets. Human platelets labeled with 14C-adenosine or 14C-adenine were incubated for up to 10 hr in vitro at 37 degrees C. After 10 hr, about 12% of the total platelet 14C-ATP and 14C-ADP had become releasable with thrombin (4.2 units/ml). Lysis of platelets did not occur, since less than 1% of the platelet-bound 51Cr from platelets labeled with this radioisotope appeared in the ambient fluid upon thrombin treatment. The 14C-ATP/14C-ADP ratio of the released adenine nucleotides (7.6) was similar to the 14C-ATP/14C-ADP ratio of the nonreleasable adenine nucleotides (7.1) 2 hr after the labeling with 14C-adenosine. However, upon prolonged incubation (10 hr) in vitro, the 14C-ATP/14C-ADP ratio of the releasable adenine nucleotides decreased to 2.7. The adenylate energy charge and the 14C-ATP/14C-ADP ratio of the metabolic adenine nucleotide pool did not change significantly during the time of observation. The 14C-ATP content of the platelets decreased by less than 1% hr of incubation at 37 degrees C. These observations are interpreted to mean that the 14C is transferred from the metabolically active, nonreleasable adenine nucleotide pool of human platelets into the releasable adenine nucleotide pool as ATP and is partially hydrolyzed there to yield ADP. The transfer of ATP across the storage organelle membrane of platelets may be similar to transport processes in the chromaffin cells of the adrenal medulla and may represent a general phenomenon in cells that possess storage organelles containing adenine nucleotides.

Adenosine Diphosphate↗

PubMed1985

Gas chromatographic-mass spectrometric determination of isotopic enrichment of 6-15NH2 in adenine nucleotides.

A gas chromatographic-mass spectrometric method for the determination of isotopic abundance in [6-15NH2]adenine nucleotides is described. The method involves formation of the di-t-butyldimethylsilyl (TBDMS) derivative of adenine following isolation of the nucleotide fraction with solid-phase ion-exchange chromatography and subsequent acid hydrolysis of nucleotides to free base. Mass spectra for both adenine-diTBDMS and [6-15NH2]adenine-diTBDMS were obtained to identify those ions containing the 6-NH2 moiety. The base peak (m/z 306) was formed by loss of C4H9 (57) and constitutes approximately one-third of the total ion current. Using selected ion monitoring of the m/z 306/m/z 307 ratio, levels of isotopic abundance of 1.0-50.0 mol% excess could be measured reproducibly with the injection of 10-20 pmol of the adenine-diTBDMS derivative obtained from isolated rat hepatocytes. Confirmation that measured isotopic abundance was referable to labeling of the 6-15NH2 group was obtained by oxidation of adenine to hypoxanthine and determination of enrichment in the hypoxanthine-diTBDMS derivative. The method was used to study the formation of [6-15NH2]adenine nucleotides during the incubation of isolated rat hepatocytes with [15N]alanine. A level of approximately 6.0 mol% excess was observed at 60 min incubation.

Adenine↗

PubMed2001

Structure of the N6-adenine DNA methyltransferase M.TaqI in complex with DNA and a cofactor analog.

The 2.0 A crystal structure of the N6-adenine DNA methyltransferase M.TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the methyl group from the cofactor S-adenosyl-l-methionine to the 6-amino group of adenine within the double-stranded DNA sequence 5'-TCGA-3', the target nucleoside is rotated out of the DNA helix. Stabilization of the extrahelical conformation is achieved by DNA compression perpendicular to the DNA helix axis at the target base pair position and relocation of the partner base thymine in an interstrand pi-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine is specifically recognized in the active site, and the 6-amino group of adenine donates two hydrogen bonds to Asn 105 and Pro 106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These hydrogen bonds appear to increase the partial negative charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the methyl group of the cofactor.

Adenine↗

PubMed1977

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.

Adenine↗

PubMed1999

On the substrate specificity of DNA methyltransferases. adenine-N6 DNA methyltransferases also modify cytosine residues at position N4.

Methylation of DNA is important in many organisms and essential in mammals. Nucleobases can be methylated at the adenine-N6, cytosine-N4, or cytosine-C5 atoms by specific DNA methyltransferases. We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam methyltransferases as well as the N- and C-terminal domains of the M. FokI enzyme, which were formerly all classified as adenine-N6 DNA methyltransferases, also methylate cytosine residues at position N4. Kinetic analyses demonstrate that the rate of methylation of cytosine residues by M.EcoRV and the M.FokI enzymes is reduced by only 1-2 orders of magnitude in relation to methylation of adenines. This result shows that although these enzymes methylate DNA in a sequence specific manner, they have a low substrate specificity with respect to the target base. This unexpected finding has implications on the mechanism of adenine-N6 DNA methyltransferases. Sequence comparisons suggest that adenine-N6 and cytosine-N4 methyltransferases have changed their reaction specificity at least twice during evolution, a model that becomes much more likely given the partial functional overlap of both enzyme types. In contrast, methylation of adenine residues by the cytosine-N4 methyltransferase M.BamHI was not detectable. On the basis of our results, we suggest that adenine-N6 and cytosine-N4 methyltransferases should be grouped into one enzyme family.

Adenine↗

PubMed1998

Restoring adenine nucleotides in a brain slice model of cerebral reperfusion.

Tissue adenine nucleotides are depleted during cerebral ischemia, impeding recovery after reperfusion. Although prior studies have attempted to prevent the initial loss of adenylates, the present study tests the hypothesis that stimulating synthesis of adenine nucleotides, through either adenosine kinase or adenine phosphoribosyltransferase, would result in significant cerebroprotection. To study the effects on neurons and glia directly while avoiding the influence of the cerebral vasculature, hippocampal brain slices were used for the model of transient ischemia with reperfusion. The standard brain slice insult of brief exposure to anoxia with aglycemia was modified based on studies which showed that a 30-minute exposure to air with 1 mmol/L glucose produced a stable, moderate reduction in ATP during the insult and that, 2 hours after return to normal conditions, there was moderate depletion of tissue adenine nucleotides and histologic injury. Treatments with 1 mmol/L adenosine, AMP, or adenine were equivalent in partially restoring adenine nucleotides. Despite this, only adenosine afforded histologic protection, suggesting a protective role for adenosine receptors. There also was evidence for metabolic cycling among adenine nucleotides, nucleosides, and purines. Adenosine may exert direct cerebroprotective effects on neural tissue as well as indirect effects through the cerebral vasculature.

Adenine↗

PubMed1981

Dual control of the gua operon of Escherichia coli K12 by adenine and guanine nucleotides.

The gua operon of Escherichia coli K12 comprises structural genes for the two enzymes, IMP dehydrogenase and GMP synthetase, required for the biosynthesis of GMP from IMP. The specific activities of these enzymes were measured in various purine auxotrophs. GuaA and guaB mutants (guanine-specific) were depressed under conditions of growth limitation by guanine but were repressed by excess guanine. This suggests that formation of the enzymes is normally controlled by a guanine nucleotide. Derepression of the operon in purine-starved pur mutants depended on the type of mutant and on whether adenine or guanine was provided. A purA strain (adenine-specific) and strains with early blocks in purine biosynthesis (purF and purD) did not derepress. PurE or purC strains [5'-phosphoribosyl-5-aminoimidazole (AIR)-accumulating] derepressed only 4-fold. The operon was repressed in purH strains [5'-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR)-accumulating] grown with limiting guanine or hypoxanthine, but derepressed by growth with limiting adenine. Two mutants (purA guaA and purA guaB) which can neither synthesize AMP and GMP de novo, nor interconvert them, were isolated. The specific activity of IMP dehydrogenase in one of these strains grown with different concentrations of guanine and adenine revealed that adenine induces tha gua operon whereas guanine represses it. Intracellular purine nucleotide pools wee measured in a purH mutant repressed (guanine-grown) and derepressed (adenine-grown) for IMP dehydrogenase. The guanylate pool was similar under the two growth conditions; however the adenylate pool of the adenine-grown bacteria was two to three times greater than that of the guanine-grown cells. A dual mechanism for regulating expression of the gua operon, involving induction by AMP and repression by GMP, is proposed.

Adenine↗

PubMed2001

The cytosine N4-methyltransferase M.PvuII also modifies adenine residues.

Methylation of DNA occurs at the C5 and N4 positions of cytosine and N6 of adenine. The chemistry of methylation is similar among methyltransferases specific for cytosine-N4 and adenine-N6. Moreover these enzymes have similar structures and active sites. Previously it has been demonstrated that the DNA-(adenine-N6)-methyltransferases M.EcoRV, M.EcoRI, E. coli dam and both domains of M.FokI also modify cytosine residues at the N4 position [Jeltsch et al., J. Biol. Chem. 274 (1999), 19538-19544]. Here we show that the cytosine-N4 methyltransferase M.PvuII, which modifies the second cytosine in CAGCTG sequences, also methylates adenine residues in CAGATG/CAGCTG substrates in which the target cytosine is replaced by adenine in one strand of the recognition sequence. Therefore, adenine-N6 and cytosine-N4 methyltransferases have overlapping target base specificities. These results demonstrate that the target base recognition by N-specific DNA methyltransferases is relaxed in many cases. Furthermore, it shows that the catalytic mechanisms of adenine-N6 and cytosine-N4 methyltransferases are very similar.

Adenine↗

PubMed1982

Adenine phosphoribosyltransferase deficiency in cultured mouse mammary tumor FM3A cells resistant to 4-carbamoylimidazolium 5-olate.

4-Carbamoylimidazolium 5-olate (CIO), the aglycone of the nucleoside antibiotic, bredinin (4-carbamoyl-1-beta-D-ribofuranosylimidazolium 5-olate), exhibited potent cytotoxic effects of subclonal line F28-7 of C3H mouse mammary carcinoma FM3A cells in culture. We isolated 11 cell lines resistant to CIO from wild-type F28-7 cells mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine. These resistant (cio') lines were 160- to 400-fold less sensitive to CIO than were the wild-type cells and inherited the resistant phenotypes during subculture for more than 3 months in the drug-free medium. They were cross-resistant to an adenine analog, 2,6-diaminopurine, while 2,6-diaminopurine-resistant (dap') lines, isolated independently, were cross-resistant to CIO. Neither of the cio' lines tested were able to form colonies in agar medium containing azaserine and adenine, nor were they able to incorporate tritiated adenine into the macromolecular fraction, indicating that they could not utilize exogenous adenine for growth. Enzyme assays using cell-free extracts revealed that all the cio' lines had undetectable levels of adenine phosphoribosyltransferase (EC 2.4.2.7) activity, but they, except one, had normal levels of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20) activities. These results demonstrate that the CIO resistance in these lines is attributed to deficient adenine phosphoribosyltransferase activity and therefore that CIO is activated by adenine phosphoribosyltransferase to form a cytotoxic nucleotide within the drug-sensitive cells.

Adenine↗

PubMed1998

New modular delivery system for diagnostic and therapeutic pre-targeting using tautomer-specific monoclonal antibody EM-6-47 and 3-substituted adenines.

We have developed a new modular affinity system for the 2-step delivery of functional molecules to target cells. The system is based on the tautomer-specific monoclonal antibody (MAb) EM-6-47, which binds to 3- and 3,8-substituted adenines with high affinity (Ka > 10(9) l/mol) without cross-reacting with naturally occurring purine derivatives. This MAb serves as the hapten-specific fusion partner to produce bispecific MAbs (bs-MAbs) recognizing a target cell antigen and a low-m.w. hapten as carrier molecule for, e.g., radionuclides. Either the C-8 or the N-3 position of adenines can be used for conjugation with effector molecules; the remaining position may be substituted with different moieties to modulate the pharmacokinetics of the haptens. Different 3- and 3,8-substituted adenines conjugated to the chelates DOTA and DTPA or to the drug daunomycin were synthesized. Adenine-chelate derivatives were efficiently labeled with (111)In and 90Y, while high-affinity binding of 3-substituted adenines to MAb EM-6-47 remained almost unaffected by the conjugation to radiochelates. To confirm the validity of the delivery system, a prototype bs-MAb, EM-168-47, was generated by somatic cell fusion of MAb EM-6-47 and MAb EM-168-2, the latter recognizing a surface antigen on canine hematopoietic cells. Two-step targeting assays in vitro verified the bs-MAb-mediated, dose-dependent delivery of (111)In-labeled adenine-chelate derivatives to myeloid cells. This system represents a powerful tool for new pre-targeting approaches relying on bs-MAbs and low-m.w. haptens. Suitable cellular antigens can be targeted by fusing the appropriate MAbs with hapten-specific MAb EM-6-47, and tailor-made 3-substituted adenines may be labeled with diagnostic or therapeutic radionuclides, cytotoxic drugs or other functional molecules.

Adenine↗

PubMed2001

Adenine toxicity and transport in the moderately halophilic eubacterium Halomonas elongata.

The presence of adenine in the L-alanine defined medium substantially inhibited the growth of the moderately halophilic eubacterium Halomonas elongata. Extensive attempts to reverse the adenine toxicity for growth were made using a variety of purine and pyrimidine compounds, vitamins, and amino acids. Of the compounds tested, only cytosine was found to reverse the adenine growth inhibition. This indicates a mechanism similar to that found for some strains of Escherichia coli in which the presence of exogenous purines (e.g. adenine) was found to stop purine de novo synthesis and repress the synthesis of the pyrimidine salvage enzyme cytosine deaminase. H. elongata was found to possess an active adenine uptake system that was sodium dependent with only lithium having a considerable capacity to replace the sodium. A competition study indicated that the adenine transport system was quite specific. This paper represents the initial study of purine and pyrimidine salvage pathways and adenine uptake for the moderately halophilic eubacteria.

Adenine↗

PubMed1993

Net adenine nucleotide transport in rat kidney mitochondria.

This study investigated the hypothesis that changes in the adenine nucleotide (ATP + ADP + AMP) content of kidney mitochondria can occur by a transport mechanism that catalyzes net transfer of adenine nucleotides across the inner mitochondrial membrane. The adenine nucleotide content of isolated kidney mitochondria was 8.23 +/- 0.85 nmol/mg mitochondrial protein. This amount increased or decreased as a function of the external [ATP-Mg] when mitochondria were incubated in phosphate-containing medium. The increases and decreases were inhibited to different extents by 100 microM EGTA (ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid) or 5 microM carboxyatractyloside (CAT), suggesting two transport mechanisms. The unidirectional components (influx and efflux) of net flux were examined separately for the CAT-insensitive (EGTA-sensitive) and CAT-sensitive (EGTA-insensitive) mechanisms. CAT-insensitive adenine nucleotide influx and efflux were stimulated by [Ca2+]free up to 2 microM; for ATP influx, Km was 1.7 mM, Vmax was 3.5 nmol/min/mg protein, and Mg2+ was required. Efflux varied as a function of both the external and matrix [ATP] and was completely inhibited by mersalyl. ATP was a better substrate than ADP, and ADP transport did not require Mg2+. The CAT-sensitive mechanism was characterized by studying phosphate-induced adenine nucleotide efflux. Efflux varied with external [Pi] and with matrix [ATP] and was not inhibited by cyclosporin. The amount of CAT required for maximal inhibition was 800 pmol/mg protein. In contrast to CAT-insensitive efflux, this pathway was only partially inhibited by mersalyl and showed no preference for ATP vs ADP. In conclusion, two distinct mechanisms for net adenine nucleotide transport were demonstrated. Both exchange adenine nucleotides (ATP-Mg or ADP) for Pi. One mechanism is identical to the CAT-insensitive ATP-Mg/Pi carrier known in liver mitochondria; the other is a CAT-sensitive mechanism that is not present in liver and may represent a novel function of the ADP/ATP translocase or another CAT-sensitive carrier.

Adenine Nucleotides↗