Radioenzymatic determination of adenosine

Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U-14C]ribose-labeled inosine via the following reactions: adenosine + H2O----adenine + ribose (adenosine nucleosidase); adenine + [U-14C]ribose 1-phosphate in equilibrium with T[U-14C]ribose-adenosine + Pi (adenosine phosphorylase); [U-14C]ribose-adenosine + H2O----[U-14C]ribose-inosine + NH3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.     

A sensitive fluorimetric procedure for the determination of aliphatic epoxides under physiological conditions

Ribose 1-phosphate has been measured in rat tissues by an enzymatic radioactive assay. The sugar phosphate is converted into [¹⁴C]inosine via the two following combined reactions: ribose 1-phosphate + [¹⁴C]adenine ? [¹⁴C]adenosine + phosphate (adenosine phosphorylase); [¹⁴C]adenosine + H₂O → [¹⁴C]inosine + NH₃ (adenosine deaminase). Tissue extracts are incubated in the presence of excess [¹⁴C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of ribose 1-phosphate present in tissue extracts. Liver was found to contain the highest level of ribose 1-phosphate (ca. 800 nmol/g wet wt).     

Hypoxanthine-guanine exchange by intact human erythrocytes

The uptake and release of [14C]hypoxanthine by human erythrocytes, suspended in a tris(hydroxymethyl)aminomethane (Tris)-glucose-NaCl isotonic medium (pH 7.4), have been studied at 37 degrees C. The uptake of hypoxanthine, mediated by its incorporation into inosine 5'-monophosphate (IMP), was markedly stimulated by preincubating the cells in phosphate-buffered saline. After a lag time, [14C]IMP-enriched erythrocytes released [14C]hypoxanthine in the medium. Formycin B, at concentrations known to inhibit purine nucleoside phosphorylase in intact erythrocytes, affected hypoxanthine uptake and release and led to an increase in the intracellular concentration of inosine, suggesting that the main catabolic path of IMP is the sequential degradation of the nucleotide to inosine and hypoxanthine. The addition of guanine to a suspension of [14C]IMP-enriched erythrocytes led to an increase in the rate of [14C]hypoxanthine release, which was unaffected by the presence of formycin B. During the guanine-induced hypoxanthine release, guanine was taken up by the cells as GMP. These results suggest that the presence of guanine in the incubation medium activates a catabolic path in human erythrocytes leading to IMP degradation without formation of inosine.     

Metabolism of nicotinamide, adenine and inosine in developing microspore-derived canola (Brassica napus) embryos

The metabolic fate of [carbonyl-(14)C]nicotinamide, [8-(14)C]adenine and [8-(14)C]inosine was examined in microspore-derived canola (Brassica napus) embryos at different developmental stages: globular stage (day 10, stage 1), early cotyledonary stage (day 20, stage 2), late cotyledonary stage (day 25, stage 3), and fully developed stage (day 35, stage 4). Uptake of [8-(14)C]nicotinamide by the embryos was always rapid. A lower uptake rate was found for [8-(14)C]adenine and [8-(14)C]inosine, especially at stages 1 and 2. [Carbonyl-(14)C]nicotinamide was converted to nicotinic acid and further metabolized to pyridine nucleotides (NAD/NADP). Some radioactivity was also associated to nicotinic acid glucoside. [8-(14)C]adenine was efficiently utilized for the synthesis of adenine nucleotides and RNA. A small fraction of adenine was degraded to CO(2) via ureides. Up to 40% of [8-(14)C]inosine was salvaged to nucleotides and RNA, although degradation of [8-(14)C]inosine to CO(2) was pronounced. At stage 1, highest salvage activities of nicotinamide, adenine and inosine were observed. In contrast, the lowest purine salvage and highest purine catabolism were found in stage 3 embryos. These results suggest that both nicotinamide and purine salvage for NAD/NADP and purine nucleotides synthesis are extremely high in the globular stage (stage 1). These activities decrease gradually until the late cotyledonary stage (stage 3), before increasing again in the fully developed embryos (stage 4). Overall it appears that nicotinamide and purine salvage are required in support of active growth during the initial phases of embryogenesis and at the end of the maturation period, in preparation for post-embryonic growth.     

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.     

Metabolism of adenosine, AMP and ATP in rats

Metabolism of [14C]adenosine in a dose of 100 mg per 1 kg of mass and [14C]ATP in the equimolar quantity was studied in rats after intraperitoneal administration. Adenosine is shown to enter tissues of the liver, spleen, thymus, heart and erythrocytes where it phosphorylates into adenine nucleotides (mainly ATP) and deaminates into inosine. The content of adenosine increases for a short period in the above tissues, except for erythrocytes and plasma. The latter accumulates a considerable amount of inosine and hypoxanthine, but only traces of uric acid, xanthine and adenine nucleotides. ATP administered to rats catabolizes through the adenosine formation. The exogenic adenosine and ATP replace in tissues and erythrocytes only a slight part (1-12%) of their total adenine nucleotide pool. The content of these metabolites and ADP in the blood plasma does not change essentially under the effect of adenosine, ATP and AMP. It is shown on rats whose adenine nucleotide pool of cells is marked by the previous administration of [14C]adenine that injections of adenosine, ATP and inosine do not accelerate catabolism of adenine nucleotides in tissues and erythrocytes as well as do not increase the level of catabolism products in the blood plasma. Adenosine enhances and ATP lowers the content of cAMP in spleen and myocardium, respectively.     

A new isotopic assay for purine nucleoside phosphorylase

We have developed a new assay for purine nucleoside phosphorylase which is based on the release of tritium when [2-3H]inosine is used as the substrate and the reaction is coupled with xanthine oxidase. After the reaction is terminated, residual [2-3H]inosine is adsorbed on charcoal and the supernatant solution is assayed for radioactivity by liquid scintillation spectrometry. The new method gave results indistinguishable from those obtained by spectrophotometric determination of uric acid produced by the phosphorylase-xanthine oxidase-coupled reaction or by radioassay of chromatographically isolated [8-14C]hypoxanthine when [8-14C]inosine was used as substrate. The new method is faster than those involving chromatographic isolation of products. In comparison with spectrophotometric methods, it not only requires less manual time, but it also has the advantage that it can be used to study inhibitors whose ultraviolet absorption might interfere with spectrophotometric determination of uric acid.     

Inhibition of caffeine biosynthesis in tea (Camellia sinensis) and coffee (Coffea arabica) plants by ribavirin

The effects of ribavirin, an inhibitor of inosine-5'-monophosphate (IMP) dehydrogenase, on [8-(14)C]inosine metabolism in tea leaves, coffee leaves and coffee fruits were investigated. Incorporation of radioactivity from [8-(14)C]inosine into purine alkaloids, such as theobromine and caffeine, guanine residues of RNA, and CO(2) was reduced by ribavirin, while incorporation into nucleotides, including IMP and adenine residues of RNA, was increased. The results indicate that inhibition of IMP dehydrogenase by ribavirin inhibits both caffeine and guanine nucleotide biosynthesis in caffeine-forming plants. The use of IMP dehydrogenase-deficient plants as a potential source of good quality caffeine-deficient tea and coffee plants is discussed.     

Effect of papaverine on the absorption and metabolism of 14C- adenosine and 14C-adenine in thymocytes

Some peculiarities of adenosine and adenine nucleotide metabolism in rat thymocytes were investigated. It was shown that the uptake of labelled adenosine or adenine by thymocytes is markedly inhibited by papaverine due to the decrease of the adenylate kinase activity, on the one hand, and to the acceleration of ATP catabolism and inosine and hypoxanthine release into the environment, on the other. ATP catabolism occurs in a special compartment which in [14C] adenosine and [14C] adenine prelabelled thymocytes has a higher specific radioactivity as compared with the whole cell. In [14C] adenine-prelabelled thymocytes and extracellular medium, papaverine does not influence the content but increases the specific radioactivity of adenosine.     

The catabolism of endogenous adenine nucleotides in rat liver mitochondria

Summary The degradation of intramitochondrial adenine nucleotides to nucleosides and bases was investigated by incubating isolated rat liver mitochondria at 37°C under non-phosphorylating conditions in the presence of oligomycin and carboxyatractyloside. Within 30 min the adenine nucleotides were degraded by about 25 per cent. The main products formed were adenosine and inosine the contents of which increased five- to sevenfold.Compartmentation studies revealed that about 50 to 60 per cent of the adenosine formed remained inside the organelles whereas inosine was almost completely released into the surrounding medium. Outside the mitochondria only very small amounts of adenine nucleotides were detected. Similar incubations in the presence of [¹⁴C]-adenosine yielded no [¹⁴C]-inosine ruling out extramitochondrial adenosine deamination.It is concluded that endogenous adenine nucleotides can be degraded in mitochondria via AMP dephosphorylation and subsequent adenosine deamination. A purine nucleoside transport system mediating at least the efflux of inosine from the mitochondria is suggested.     

Adenine nucleotide synthesis from inosine during normoxia and after ischaemia in the isolated perfused rat heart

[14C]inosine in a range of concentrations of 20 microM to 1 mM was administered to the isolated perfused rat heart for 30 min. The incorporation of the nucleoside into myocardial adenine nucleotides increased for extracellular concentrations of the precursor up to 50 microM, reaching a plateau at 60 nmol . g-1 X 30 min-1 with concentrations ranging between 50 and 200 microM. The supply of 500 microM and 1 mM of inosine induced a further increase in cardiac adenine nucleotide synthesis to about 200 nmol . g-1 X 30 min-1. When supplied during low flow ischaemia (0.5 mL . min-1, 30 min.), 1 mM of inosine protected the heart against ATP degradation, while 100 microM of inosine was inefficacious. In the presence of 1 mM of inosine on reperfusion the adenine nucleotide content of the heart was similar to that observed in the absence of the nucleoside. The incorporation of [14C]inosine into adenine nucleotides was, in this last condition, below the value measured before ischaemia. Inosine administration was effective in protecting the heart against ischaemic breakdown of glycogen and favoured postischaemic restoration of glycogen stores.     

Vectorial production of adenosine by 5'-nucleotidase in the perfused rat heart

Rat hearts were perfused simultaneously with [8-3H] AMP and [8-14C]adenosine. [8-3H] AMP was hydrolzyed by 5'-nucleotidase to produce intra- and extracellular [8-3H] adenosine. Comparison of the specific activities of [3H]- and [14C]adenosine in the heart cells with the specific activities of [3H]- and [14C]adenosine in the effluent perfusate showed that much more [3H]adenosine accumulated in the tissue than would be expected if extracellular adenosine were the immediate precursor of intracellular adenosine. Conversely, perfusion of rat hearts with [8-14C]AMP and [8-3H]adenosine led to a much greater accumulation of intracellular [14C]adenosine than would be expected from an uptake of adenosine from the perfusate. These results are interpreted to be due to hydrolysis of extracellular AMP by 5'-nucleotidase, located in the plasma membrane, and release of the resulting adenosine inside the cell. Measurements of the specific activities of 3H and 14C in ATP, ADP, AMP, and inosine support this interpretation.     

Hypoxanthine Transport and Metabolism in the Central Nervous System

The mechanisms by which hypoxanthine, the principal purine in plasma and CSF, enters and leaves rabbit brain, choroid plexus, and CSF were investigated in the isolated choroid plexus in vitro and by injecting [14C]hypoxanthine intraventricularly and [3H]hypoxanthine intravenously. The isolated choroid plexus accumulated and extensively metabolized [14C]hypoxanthine; however, 14C was readily released from choroid plexus principally as [14C]-hypoxanthine. After infusion of [3H]hypoxanthine intravenously, [3H]hypoxanthine entered CSF and brain slowly and was converted in brain to nucleotides. Fewer than 5% of the acid-soluble purine nucleotides in brain entered rabbit brain from plasma hypoxanthine (and inosine) per 24 h. After intraventricular injection of [14C]hypoxanthine, the [14C]hypoxanthine was cleared from the CSF into the blood or accumulated by brain and largely converted into 14C-nucleotides. Little [14C]xanthine and no [14C]uric acid or allantoin were formed. These studies show that brain, unlike most other tissues, rapidly recycles hypoxanthine and converts it into purine nucleotides, and not unsalvageable purines.     

Effects of probenecid on transport and metabolism of cyclic AMP by isolated rabbit renal tubules

The effects of probenecid on the transport and metabolism of cyclic [14C]-AMP were studied in isolated rabbit kidney cortex tubules. Incubation in a medium with 10-400 microM probenecid for 30 min caused a 30-70% decrease in the tubular uptake of labeled material from a medium containing 0.1 mM cyclic [14C]AMP. The radioactivity in the tubules, after 30 min incubation, with or without probenecid, was mostly in the form of inosine and hypoxanthine. The disappearance of external cyclic [14C]AMP was retarded by probenecid and the concentration ratio of cyclic AMP to inosine + hypoxanthine was increased. Cyclic AMP phosphodiesterase activities, from both the soluble and particulate fractions of the kidney, were inhibited by probenecid. These findings indicate that the changes caused by probenecid on the renal disposal of extracellular cyclic AMP can be accounted for by a decrease in the accumulation of the products of cyclic AMP metabolism secondary to inhibition of extracellular cyclic AMP phosphodiesterase activity.     

UPTAKE AND RELEASE OF [14C]ADENINE DERIVATIVES AT BEDS OF MAMMALIAN CORTICAL SYNAPTOSOMES IN A SUPERFUSION SYSTEM

(1) Synaptosomal fractions from guinea pig neocortical dispersions prepared in sucrose solutions were deposited from saline media as ‘beds’ on nylon bolting cloth. When incubated with 0.5–10 μm -[¹⁴C]adenine or adenosine in glucose bicarbonate salines, uptake of ¹⁴C from adenosine proceeded at about four times the rate of uptake of [¹⁴C]adenine. This contrasted with the relative uptake of the two compounds to neocortical tissue slices or to beds made from mitochondrial fractions, where uptake was similar with the two precursors. Uptake of both precursors to synaptosome beds was much greater than uptake of inosine. (2) Synaptosome beds, [¹⁴C]adenosine-loaded, contained 88 per cent of the ¹⁴C as 5′-adenine nucleotides, the remainder being present as cyclic AMP, inosine, hypoxanthine and adenosine. When superfused, the ¹⁴C output consisted mainly of adenosine, inosine and hypoxanthine, with some 7 per cent of 5′-nucleotides and 4 per cent of cyclic AMP. (3) Electrical pulses and the addition of 50 mm -KCl each increased the efflux of ¹⁴C from superfused [¹⁴C]adenosine-loaded beds. The superfusates issuing after excitation contained the same ¹⁴C-labelled compounds as issued before, with a small increase in the proportional yield of adenosine. The additional output of ¹⁴C following electrical pulses was diminished by about 50 per cent by 0.5 μm -tetrodotoxin while that following KCl was not affected; it was however prevented when the superfusing fluids were free of Ca²⁺.     

Properties of the penicillin-binding proteins of Escherichia coli K12,.

Benzyl[14C]penicillin binds to six proteins with molecular weights between 40000 and 91000 in the inner membrane of Escherichia coli. Two additional binding proteins with molecular weights of 29000 and 32000 were sometimes detected. All proteins were accessible to benzyl[14C]penicillin in whole cells. Proteins 5 and 6 released bound benzyl[14C]penicillin with half times of 5 and 19 min at 30 degrees C but the other binding proteins showed less than 50% release during a 60-min period at 30 degrees C. The rate of release of bound penicillin from some of the proteins was greatly stimulated by 2-mercaptoethanol and neutral hydroxylamine. Release of benzyl[14C]penicillin did not occur if the binding proteins were denatured in anionic detergent and so was probably enzymic. No additional binding proteins were detected with two [14C]cephalosporins. These beta-lactams bound to either all or some of those proteins to which benzyl[14C]penicillin bound. No binding proteins have been detected in the outer membrane of E coli with any beta-[14C]lactam. The binding of a range of unlabelled penicillins and cephalosporins were studied by measuring their competition for the binding of benzyl[14C]penicillin to the six penicillin-binding proteins. These results, together with those obtained by direct binding experiments with beta-[14C]lactams, showed that penicillins bind to all six proteins but that at least some cephalosporins fail to bind, or bind very slowly, to proteins 2, 5 and 6, although they bind to the other proteins. Since these cephalosporins inhibited cell division and caused cell lysis at concentrations where we could detect no binding to proteins 2, 5 and 6, we believe that these latter proteins are not the target at which beta-lactams bind to elicit the above physiological responses. The binding properties of proteins 1, 3, and 4 correlate reasonably well with those expected for the above killing targets.     

Human red-blood-cell Ca2+-antagonist binding sites. Evidence for an unusual receptor coupled to the nucleoside transporter

The human red blood cell ghost Ca2+-antagonist binding sites were characterized with (+/-)-[3H]nimodipine. The labelled 1,4-dihydropyridine bound in a non-cooperative, reversible manner with a Kd of 52 nM at 25 degrees C to 9.65 pmol sites/mg ghost protein. The stereochemistry of the binding domain was evaluated with the optically pure enantiomers of chiral 1,4-dihydropyridines. In contrast to the 1,4-dihydropyridine-selective receptors on Ca2+ channels in electrically excitable tissues, the (+) enantiomer of nimodipine and the (-) enantiomer of the benzoxadiazol 1,4-dihydropyridine (PN 200-110) were bound with higher affinity than the respective optical antipodes. The human red blood cell ghost [3H]nimodipine-labelled sites also interacted with the inorganic Ca2+-antagonist La3+ (increase in the number of binding sites), and were allosterically regulated by the optical enantiomers of the phenylalkylamine-type Ca2+-antagonists (e.g. verapamil, desmethoxyverapamil, methoxyverapamil). The benzothiazepines d- or l-cis-diltiazem were without effect. Nucleosides (adenosine approximately equal to inosine greater than cytidine) were inhibitory at the nimodipine-labelled site, as were the nucleoside uptake inhibitors dipyridamole, hexobendine, dilazep, nitrobenzylthioinosine and nitrobenzylthioguanosine. The binding sites have essential sulfhydryl groups, show trypsin sensitivity, but are relatively heat stable. When nitrobenzylthioinosine was employed as a covalent probe to inactivate the red blood cell ghost nucleoside carrier, [3H]nimodipine binding was irreversibly lost. (+)-Nimodipine greater than (-)-nimodipine inhibited [14C]adenosine transport into human red blood cells. A good correlation between IC50 values for inhibition of [3H]nimodipine binding and IC50 values for inhibition of [14C]adenosine uptake was found for 18 compounds. Sheep red blood cells (which lack the nucleoside transporter) had no detectable [3H]nimodipine binding sites. It is concluded that the Ca2+-antagonist receptor sites of the human erythrocyte are coupled to the nucleoside transporter.     

A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.     

A general method of alpha-aminoaldehyde synthesis using alcohol dehydrogenase

A method for measuring ribose 1-phosphate in cell extracts is described. Cell extracts are first fractionated on polyethyleneimine-impregnated cellulose columns to remove nucleoside and base components which otherwise interfere with the enzymatic assay. Ribose 1-phosphate in the eluate is made limiting for the conversion of [¹⁴C]hypoxanthine to [¹⁴C]inosine in the presence of purine nucleoside phosphorylase. Labeled substrate and product are then easily separated on boronate gel columns or by paper chromatography.     

Interactions of [14C]phosphonoformic acid with renal cortical brush-border membranes. Relationship to the Na+-phosphate co-transporter

Since phosphonoformic acid (PFA) acts as a specific competitive inhibitor of Na+-Pi co-transport across renal brush-border membrane (BBM), we employed the [14C]PFA as a probe to determine the mechanism of its interaction with rat renal BBM. The binding of [14C]PFA to BBM vesicles (BBMV), with Na+ present in extravesicular medium (Na+o), was time- and temperature-dependent. The replacement of Na+o with other monovalent cations reduced the PFA binding by -80%. Cl- was the most effective accompanying monovalent anion as NaCl for maximum PFA binding. The Na+o increased the apparent affinity of BBMV for [14C]PFA binding, but it did not change the maximum binding capacity. The maximum [14C]PFA binding was achieved at Na+o approximately equal to 50 mM. The extent of Na+-dependent [14C]PFA binding correlated (r = 0.98; p less than 0.01) with percent inhibition by an equimolar dose of PFA of the (Na+o greater than Na+i)-dependent BBMV uptake of 32Pi. Intravesicular Na+ (Na+i) decreased [14C]PFA binding, on BBMV, and this inhibition by Na+i was dependent on the presence of Na+o. The increase in Na+i, at constant [Na+]o, decreased the Vmax, but not the Km, for [14C]PFA binding on BBMV. Bound [14C]PFA was displaced from BBMV by phosphonocarboxylic acids proportionally (r = 0.99; p less than 0.05) to their ability to inhibit (Na+o greater than Na+i)-gradient-dependent Pi transport, whereas other monophosphonates, diphosphonates, L-proline, or D-glucose did not influence the [14C]PFA binding. The Na+-dependent binding of [14C]PFA and of [3H]phlorizin by BBMV was 10 times higher than binding of these ligands to renal basolateral membranes and to mitochondria. [14C]PFA probably binds onto the same locus on the luminal surface of BBM, where Pi and Na+ form a ternary complex with the Na+-Pi co-transporter.