Inhibition of mitochondrial creatine kinase activity by D-2-hydroxyglutaric acid in cerebellum of young rats

D-2-Hydroxyglutaric aciduria (DHGA) is a neurometabolic disorder biochemically characterized by tissue accumulation and excretion of high amounts of D-2-hydroxyglutaric acid (DGA). Although the affected patients have predominantly severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In previous studies we have demonstrated that DGA, at concentrations as low as 0.25 mM, significantly decreased creatine kinase activity and other parameters of energy metabolism in cerebral cortex of young rats. In the present study, we investigated the effect of DGA (0.25-5 mM) on total creatine kinase (tCK) activity, as well as on CK activity in cytosolic (Cy-CK) and mitochondrial (Mi-CK) preparations from cerebellum of 30-day-old Wistar rats in order to test whether the inhibitory effect of DGA on CK was tissue specific. We verified that tCK (22% inhibition) and Mi-CK (40% inhibition) activities were moderately inhibited by DGA at concentrations of 2.5 mM and higher, in contrast to Cy-CK, which was not affected by the acid. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by preincubation of the homogenates with reduced glutathione, suggesting that the inhibition of CK activity by DGA is possibly mediated by modification of essential thiol groups of the enzyme. Our present results therefore demonstrate a relatively weak inhibitory effect of DGA on cerebellum Mi-CK activity, as compared to that provoked in cerebral cortex, and may possibly be related to the neuropathology of DHGA, characterized by cerebral cortex abnormalities.     

Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro

D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.     

Antioxidant activity of butane type lignans, secoisolariciresinol, dihydroguaiaretic acid, and 7,7'-oxodihydroguaiaretic acid

The antioxidant activity of butane-type lignans was evaluated. Secoisolariciresinol (SECO) and dihydroguaiaretic acid (DGA) showed higher radical scavenging activity than that of 7,7'-dioxodihydroguaiaretic acid (ODGA). SECO and DGA inhibited the oxidation of unsaturated fatty acid. Both enantiomers of DGA were also lipoxygenase inhibitors, but neither enantiomer of SECO inhibited the lipoxygenase activity.     

In vitro biosynthesis of ether-type glycolipids in the methanoarchaeon Methanothermobacter thermautotrophicus

The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the beta-D-glucosyl-(1-->6)-D-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose beta-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose beta-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose --> MGA + UDP, and (ii) MGA + UDP-glucose --> DGA + UDP. Both enzymes required the addition of K(+) ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl(2), in contrast to MGA synthase, which did not require Mg(2+). It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg(2+) requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg(2+) requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg(2+) increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.     

Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by D-2-hydroxyglutaric acid in vitro.

L-2-Hydroxyglutaric (LGA) and D-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO(2) production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO(2) production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35-95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by D-2-hydroxyglutaric aciduria.     

Morphogenesis of the respiratory bronchiole in rhesus monkey lungs

The epithelium of the respiratory bronchiole in the adult rhesus monkey consists of two populations: a pseudostratified epithelium with basal, mucous goblet, and ciliated cells located near the pulmonary artery (PA); and a simple cuboidal epithelium composed only of nonciliated bronchiolar epithelial (or Clara) cells in areas away from the PA. This study describes the pattern of differentiation of these two epithelial populations, and their relationship to the PA and to the time of appearance of alveoli in the respiratory bronchiole of the rhesus monkey during the period of 90-125 days gestational age (DGA). These events were related to changes in the adjacent parenchyma. Dissected airways of infusion-fixed, critical-point-dried lungs were evaluated by scanning microscopy followed by light microscopy of the same airways. At 54% of gestation (90 DGA), the distal airway was lined by a mixture of ciliated and nonciliated cells. By 67% of gestation (110 DGA), the ciliated cells were confined to the epithelium over the PA. The underlying connective tissue initially was cellular containing few fibers but was fibrous by 76% of gestation (125 DGA). Alveolarization began near the most distal cartilage at 57% of gestation (95 DGA), the same period at which secondary septation occurred in the distal acinus. Thus, alveolarization occurred simultaneously in two centers: 1) the proximal centriacinar region in the vicinity of the most distal cartilage and 2) the distal lung parenchyma. The duration of centriacinar alveolarization was short, approximately 5 days.     

Purification and characterization of a glycolic acid (GA) oxidase active toward diglycolic acid (DGA) produced by DGA-utilizing Rhodococcus sp. No. 432

Diglycolic acid (DGA) oxidizing activity was found in crude extracts of Rhodococcus sp. no. 432 grown in DGA. Glycolic acid (GA) oxidase was purified approximately 80 times by treatment with streptomycin sulfate, precipitation with (NH₄)₂SO₄, chromatographies with DEAE-cellulose, DEAE-Toyopearl and Butyl-Toyopearl, and gel filtration on Toyopearl HW-55. The purified GA oxidase was almost homogeneous on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The purity was calculated to be more than 95%. The molecular weight of the enzyme, which appeared to consist of three identical units, was 158,000. Each subunit of GA oxidase included one molecule of FAD as a cofactor. The isoelectric point of the enzyme was around 5.3. GA oxidase was stable below 30°C and at the pH range of 6.0–8.5. The optimum pH and temperature were around 7.5 and 40°C, respectively. Oxygen, cytochrome c, ferricyanide and 2,6-dichlorophenol indophenol (DCIP) acted as an electron acceptor. The activity of GA oxidase was strongly inhibited by potassium cyanide, quinine, quinacrine, monoiodoacetate, 1,4-benzoquinone and some heavy metal ions. GA oxidase also had activity in DGA, GA, glyoxylic acid (GOA), methoxy acetate, ethoxy acetate and l-malate. Alcohols and other organic acids were not oxidized by the enzyme. The apparent K_m values for DGA, GA and GOA were about 26.7, 0.5 and 4.4 mM, respectively. The reaction products from DGA were supposed to be GOA and GA by the enzymatic assays. The reaction mechanism of GA oxidase in oxidation of DGA was supposed to be as follows: HOOCH₂COCH₂COOH+H₂O+acceptor→HOOCCHO+HOOCCH₂OH+ reduced acceptor.     

Antioxidant activity and cytoprotective effect of kappa-carrageenan oligosaccharides and their different derivatives

Antioxidant activity of kappa-carrageenan oligosaccharides (OM) and their chemical modification derivatives was investigated employing various established in vitro systems, such as reducing power, iron ion chelation, and total antioxidant activity using beta-carotene-linoleic acid system. The oversulfated (SD), lowly (LAD), and highly acetylated derivatives (HAD) in reducing power assay, the phosphorylated derivative (PD) in metal chelating assay, and oversulfated and phosphorylated derivatives in total antioxidant activity assay exhibited antioxidant activity higher than that of carrageenan oligosaccharides. The results indicated that the chemical modification of carrageenan oligosaccharides can enhance their antioxidant activity in vitro. The protective effects of the carrageenan oligosaccharides and their chemically modified derivatives against H(2)O(2) and UVA (long-wave ultraviolet radiation) induced oxidative damage on rat thymic lymphocyte were investigated by measuring cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Thymic lymphocyte exposure to H(2)O(2) and UVA, a marked reduction in cell survival was observed, which was significantly prevented by carrageenan oligosaccharides and their derivatives (preincubated for 2h) at 66.7-2000 microg/mL. But both the carrageenan oligosaccharides and their different derivatives showed the similar protective effects on intracellular level. Taken together, these results suggest that carrageenan oligosaccharides and their derivatives show relevant antioxidant activity both in vitro and in a cell system.     

Biological activities and structure-activity relationship of substitution compounds of N-[2-(3-indolyl)ethyl]succinamic acid and N-[2-(1-naphthyl)ethyl]succinamic acid, derived from a new category of root-promoting substances, N-(phenethyl)succinamic acid analogs

In a previous study, it was demonstrated that N-(phenethyl)succinamic acid (PESA) derivatives form a new category of root-promoting substances which do not exhibit auxin-like activities, such as stem elongation and leaf epinasty (Soejima et al., 2000 [Plant Cell Physiol. 41s: 197]). In this study, N-[2-(3-indolyl)ethyl]succinamic acid (IESA) and N-[2-(1-naphthyl)ethyl]succinamic acid (NESA) were synthesized, and their biological activities were evaluated. In an adzuki root-promoting assay, IESA and NESA exhibited root-promoting activity equivalent to PESA. In adzuki stem elongation assays, elongation activity was not observed in the stem segments soaked in either an IESA or NESA aqueous solution, whereas the stem segments immersed in Indole-3-acetic acid (IAA) or 1-naphthylacetic acid (NAA) aqueous solution were clearly elongated. In an epinastic bending study, IAA and NAA exhibited leaf epinasty, whereas IESA and NESA did not, suggesting that the IESA and NESA derivatives belong to the same category of root-promoting substances as PESA derivatives and are different from auxin-like substances. In addition, eleven kinds of IESA derivatives and nineteen kinds of NESA derivatives were synthesized, and their root-promoting activities were measured. The activities of methyl ester derivatives were approximately three times higher than that of the acid compounds, with exceptions for some compounds. The partition coefficient (P) between 1-octanol and water for each IESA, NESA, and PESA derivative was measured in order to evaluate the hydrophobicity of their molecules and to determine their structure–activity relationship. The results indicate that the root-promoting activity of the acid compounds was significantly correlated with their hydrophobicity, whereas that of ester derivatives was not correlated.     

Development of prostaglandin endoperoxide synthase expression in the ovine fetal central nervous system and pituitary

In this study, we tested the hypothesis that prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and PGHS-2) are expressed throughout the latter half of gestation in ovine fetal brain and pituitary. Hypothalamus, pituitary, hippocampus, brainstem, cortex and cerebellum were collected from fetal sheep at 80, 100, 120, 130, 145 days of gestational age (DGA), 1 and 7 days postpartum lambs, and from adult ewes (n = 4–5 per group). mRNA and protein were isolated from each region, and expression of prostaglandin synthase-1 (PGHS-1) and -2 (PGHS-2) were evaluated using real-time RT-PCR and western blot. PGHS-1 and -2 were detected in every brain region at every age tested. Both enzymes were measured in highest abundance in hippocampus and cerebral cortex, and lowest in brainstem and pituitary. PGHS-1 and -2 mRNA’s were upregulated in hypothalamus and pituitary after 100 DGA. The hippocampus exhibited decreases in PGHS-1 and increases in PGHS-2 mRNA after 80 DGA. Brainstem PGHS-1 and -2 and cortex PGHS-2 exhibited robust increases in mRNA postpartum, while cerebellar PGHS-1 and -2 mRNA’s were upregulated at 120 DGA. Tissue concentrations of PGE₂ correlated with PGHS-2 mRNA, but not to other variables. We conclude that the regulation of expression of these enzymes is region-specific, suggesting that the activity of these enzymes is likely to be critical for brain development in the late-gestation ovine fetus.     

Synthesis and antiviral activity of N9-[3-fluoro-2-(phosphonomethoxy)propyl] analogues derived from N6-substituted adenines and 2,6-diaminopurines

An efficient method for the synthesis of N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases has been developed. Both (R)- and (S)-enantiomers of the N(6)-substituted FPMP derivatives of adenine and 2,6-diaminopurine were prepared and their anti-human immunodeficiency virus (HIV) and anti-Moloney murine sarcoma virus (MSV) activity was evaluated. Whereas none of the 6-substituted FPMPA derivatives showed any antiviral activity, several FPMPDAP derivatives had a moderate antiretroviral activity. Moreover, the data obtained from the study of the substrate activity of the active derivatives towards N(6)-methyl-AMP aminohydrolase support the notion that the studied N(6)-substituted FPMPDAP derivatives act as prodrugs of the antiretroviral FPMPG analogues.     

Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by d-2-hydroxyglutaric acid in vitro

l-2-Hydroxyglutaric (LGA) and d-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as l-2-hydroxyglutaric aciduria and d-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO₂ production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO₂ production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35–95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by d-2-hydroxyglutaric aciduria.     

基于与木糖进行美拉德反应的低聚壳聚糖衍生物的抗氧化性能研究

研究低聚壳聚糖与木糖的美拉德反应,考察了两种体系(低聚壳聚糖与木糖的质量比分别为1∶1和1∶3)反应过程中pH、吸光度及荧光值的变化,醇沉法提取4 h和8 h的低聚壳聚糖美拉德反应衍生物,分别为CX11-4、CX13-4、CX11-8和CX13-8。对衍生物进行红外表征和分子量测定,并研究其对羟基自由基.OH和DPPH的清除能力以及还原能力。结果显示:壳聚糖衍生物的抗氧化能力都明显优于低聚壳聚糖,抗氧化活性顺序为CX13-4>CX11-4,CX11-8>CX13-8。可见,壳聚糖美拉德衍生物的抗氧化活性不仅与反应物的比例有关,还与反应的时间有关。     

Antimicrobial and antiviral screening of novel indole carboxamide and propanamide derivatives

A few series of indole derivatives were screened for antimicrobial, antifungal and anti-HBV activities. The compounds were tested for their in vitro antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and for their antifungal activity against Candida albicans using a disc diffusion method, which measures the diameter of the inhibition zone around a paper disc soaked in a solution of the test compounds. The antimicrobial activity results showed that all compounds are as a active as the standard compound ampicillin against Staphylococcus aureus. It was also found that indole carboxamide derivatives, substituted at 3-position with several benzyl groups, showed better inhibition of Bacillus subtilis than their congeners substituted at 2-position. Activity patterns of the compounds against Escherichia coli and Staphylococcus aureus were found slightly different by the same method. In this case, there was no correlation between structure and activity of the compounds. The antifungal activity of carboxamide derivatives was found higher compared to that of the propanamide derivatives. The minimum inhibitory concentration (MIC) values of some indole derivatives were also determined by the tube dilution technique. The MIC values of the compounds were found nearly 20- to 100-fold smaller compared to the standard compounds ciprofloxacin and ampicillin (1.56-3.13 microg/ml and 1.56-12.5 microg/ml, respectively) against Staphylococcus aureus, Bacillus subtilis and Escherichia coli. The MIC values of the tested compounds showed that these are better inhibitors for Candida albicans. Indole derivatives were screened by the anti-HBV susceptibility test. No compound showed good inhibition against the HBV virus.     

Synthesis of feruloyl-myo-insitol derivatives and their inhibitory effects on phorbol ester-induced superoxide generation and Esptein-Barr virus activation

We prepared 14 feruloyl-myo-inositol derivatives, and evaluated the relationships between their stereostructure and inhibitory activity toward the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced superoxide (O(2)(-)) generation. And further, their suppressive effect on the TPA-induced Epstein-Barr virus (EBV) activation was examined in order to estimate their anti-carcinogenic potentials. Among the derivatives tested, 1,6-O-bis[3-(4'-hydroxy-3'-methoxyphenyl)-2-propenoyl]-myo-inositol (6b) showed an excellent suppressive activity on the O(2)(-) generation at a concentration of 20 microM. For the suppressive effects on the EBV activation, 2,4,6-O-tris[3-(4'-hydroxy-3'-methoxyphenyl)-2-propenoyl]-myo-inositol 1,3,5-orthoformate (9b) showed the highest activity at a concentration of 100 microM among the derivatives tested. These results suggest that the inhibitory potencies of feruloyl-myo-inositol derivatives depend on the stereostructure of molecules rather than the hydrophobicity of molecules.     

Daily ethanol exposure during late ovine pregnancy: physiological effects in the mother and fetus in the apparent absence of overt fetal cerebral dysmorphology

High levels of ethanol (EtOH) consumption during pregnancy adversely affect fetal development; however, the effects of lower levels of exposure are less clear. Our objectives were to assess the effects of daily EtOH exposure (3.8 USA standard drinks) on fetal-maternal physiological variables and the fetal brain, particularly white matter. Pregnant ewes received daily intravenous infusions of EtOH (0.75 g/kg maternal body wt over 1 h, 8 fetuses) or saline (8 fetuses) from 95 to 133 days of gestational age (DGA; term ～145 DGA). Maternal and fetal arterial blood was sampled at 131-133 DGA. At necropsy (134 DGA) fetal brains were collected for analysis. Maternal and fetal plasma EtOH concentrations reached similar maximal concentration (～0.11 g/dl) and declined at the same rate. EtOH infusions produced mild reductions in fetal arterial oxygenation but there were no changes in maternal oxygenation, maternal and fetal Pa(CO(2)), or in fetal mean arterial pressure or heart rate. Following EtOH infusions, plasma lactate levels were elevated in ewes and fetuses, but arterial pH fell only in ewes. Fetal body and brain weights were similar between groups. In three of eight EtOH-exposed fetuses there were small subarachnoid hemorrhages in the cerebrum and cerebellum associated with focal cortical neuronal death and gliosis. Overall, there was no evidence of cystic lesions, inflammation, increased apoptosis, or white matter injury. We conclude that daily EtOH exposure during the third trimester-equivalent of ovine pregnancy has modest physiological effects on the fetus and no gross effects on fetal white matter development.     

Synthesis and Biological Activity of the Functional Derivatives of 3- and 4-(Dimethyl-aminomethyl)-1,2-dithiolanes

A variety of derivatives of 3- and 4-(dimethylaminomethyl)-1,2-dithiolanes were prepared as their biological equivalents in an analogous way to that for the nereistoxin derivatives. Most of them showed insecticidal and acaricidal activity. High potency against Chilo suppressalis was displayed by S,S′-[2-(dimethylaminomethyl)trimethylene] bis(thiobenzoate), and a broad spectrum of activity was observed for 5-(dimethylaminomethyl)-1,2,3-trithiane. The stereochemistry of the 1,3-dithianes is also discussed.     

Synthesis of 3-(1-alkyl/aminoalkyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-ones and their 2-methylene derivatives as potential spermicidal and microbicidal agents

A series of twenty two derivatives of 3-(1-alkyl/aminoalkyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-one and their 2-methylene derivatives were synthesized from naturally abundant cinchonine (I). Tartarate salts of these compounds were prepared and evaluated for spermicidal activity. The most active compounds (24, 27, 34, 36, and 38) showing potent spermicidal activity were further evaluated against different strains of Trichomonas vaginalis, for antimicrobial activity, in HeLa cell lines for cytotoxicity and against Lactobacillus jensenii for eco-safety. The tartarate of 3-(1-pentyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-one (27) was found to be more active than N-9 in spermicidal activity.     

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.