Prevalence of Cobalt in the Environment and Its Role in Biological Processes.

Cobalt (Co) is an essential trace element for humans and other animals, but high doses can be harmful to human health. It is present in some foods such as green vegetables, various spices, meat, milk products, seafood, and eggs, and in drinking water. Co is necessary for the metabolism of human beings and animals due to its key role in the formation of vitamin B12, also known as cobalamin, the biological reservoir of Co. In high concentrations, Co may cause some health issues such as vomiting, nausea, diarrhea, bleeding, low blood pressure, heart diseases, thyroid damage, hair loss, bone defects, and the inhibition of some enzyme activities. Conversely, Co deficiency can lead to anorexia, chronic swelling, and detrimental anemia. Co nanoparticles have different and various biomedical applications thanks to their antioxidant, antimicrobial, anticancer, and antidiabetic properties. In addition, Co and cobalt oxide nanoparticles can be used in lithium-ion batteries, as a catalyst, a carrier for targeted drug delivery, a gas sensor, an electronic thin film, and in energy storage. Accumulation of Co in agriculture and humans, due to natural and anthropogenic factors, represents a global problem affecting water quality and human and animal health. Besides the common chelating agents used for Co intoxication, phytoremediation is an interesting environmental technology for cleaning up soil contaminated with Co. The occurrence of Co in the environment is discussed and its involvement in biological processes is underlined. Toxicological aspects related to Co are also examined in this review.

The Effects of Cadmium Toxicity.

Cadmium (Cd) is a toxic non-essential transition metal that poses a health risk for both humans and animals. It is naturally occurring in the environment as a pollutant that is derived from agricultural and industrial sources. Exposure to cadmium primarily occurs through the ingestion of contaminated food and water and, to a significant extent, through inhalation and cigarette smoking. Cadmium accumulates in plants and animals with a long half-life of about 25-30 years. Epidemiological data suggest that occupational and environmental cadmium exposure may be related to various types of cancer, including breast, lung, prostate, nasopharynx, pancreas, and kidney cancers. It has been also demonstrated that environmental cadmium may be a risk factor for osteoporosis. The liver and kidneys are extremely sensitive to cadmium's toxic effects. This may be due to the ability of these tissues to synthesize metallothioneins (MT), which are Cd-inducible proteins that protect the cell by tightly binding the toxic cadmium ions. The oxidative stress induced by this xenobiotic may be one of the mechanisms responsible for several liver and kidney diseases. Mitochondria damage is highly plausible given that these organelles play a crucial role in the formation of ROS (reactive oxygen species) and are known to be among the key intracellular targets for cadmium. When mitochondria become dysfunctional after exposure to Cd, they produce less energy (ATP) and more ROS. Recent studies show that cadmium induces various epigenetic changes in mammalian cells, both in vivo and in vitro, causing pathogenic risks and the development of various types of cancers. The epigenetics present themselves as chemical modifications of DNA and histones that alter the chromatin without changing the sequence of the DNA nucleotide. DNA methyltransferase, histone acetyltransferase, histone deacetylase and histone methyltransferase, and micro RNA are involved in the epigenetic changes. Recently, investigations of the capability of sunflower (Helianthus annuus L.), Indian mustard (Brassica juncea), and river red gum (Eucalyptus camaldulensis) to remove cadmium from polluted soil and water have been carried out. Moreover, nanoparticles of TiO2 and Al2O3 have been used to efficiently remove cadmium from wastewater and soil. Finally, microbial fermentation has been studied as a promising method for removing cadmium from food. This review provides an update on the effects of Cd exposure on human health, focusing on the cellular and molecular alterations involved.

Nickel: Human Health and Environmental Toxicology.

Nickel is a transition element extensively distributed in the environment, air, water, and soil. It may derive from natural sources and anthropogenic activity. Although nickel is ubiquitous in the environment, its functional role as a trace element for animals and human beings has not been yet recognized. Environmental pollution from nickel may be due to industry, the use of liquid and solid fuels, as well as municipal and industrial waste. Nickel contact can cause a variety of side effects on human health, such as allergy, cardiovascular and kidney diseases, lung fibrosis, lung and nasal cancer. Although the molecular mechanisms of nickel-induced toxicity are not yet clear, mitochondrial dysfunctions and oxidative stress are thought to have a primary and crucial role in the toxicity of this metal. Recently, researchers, trying to characterize the capability of nickel to induce cancer, have found out that epigenetic alterations induced by nickel exposure can perturb the genome. The purpose of this review is to describe the chemical features of nickel in human beings and the mechanisms of its toxicity. Furthermore, the attention is focused on strategies to remove nickel from the environment, such as phytoremediation and phytomining.

Oxidative stress and neurodegeneration: the involvement of iron.

Many evidences indicate that oxidative stress plays a significant role in a variety of human disease states, including neurodegenerative diseases. Iron is an essential metal for almost all living organisms due to its involvement in a large number of iron-containing proteins and enzymes, though it could be also toxic. Actually, free iron excess generates oxidative stress, particularly in brain, where anti-oxidative defences are relatively low. Its accumulation in specific regions is associated with pathogenesis in a variety of neurodegenerative diseases (i.e., Parkinson's disease, Alzheimer's disease, Huntington's chorea, Amyotrophic Lateral Sclerosis and Neurodegeneration with Brain Iron Accumulation). Anyway, the extent of toxicity is dictated, in part, by the localization of the iron complex within the cell (cytosolic, lysosomal and mitochondrial), its biochemical form, i.e., ferritin or hemosiderin, as well as the ability of the cell to prevent the generation and propagation of free radical by the wide range of antioxidants and cytoprotective enzymes in the cell. Particularly, ferrous iron can act as a catalyst in the Fenton reaction that potentiates oxygen toxicity by generating a wide range of free radical species, including hydroxyl radicals (·OH). The observation that patients with neurodegenerative diseases show a dramatic increase in their brain iron content, correlated with the production of reactive oxigen species in these areas of the brain, conceivably suggests that disturbances in brain iron homeostasis may contribute to the pathogenesis of these disorders. The aim of this review is to describe the chemical features of iron in human beings and iron induced toxicity in neurodegenerative diseases. Furthermore, the attention is focused on metal chelating drugs therapeutic strategies.

Mercury Exposure and Heart Diseases.

Environmental contamination has exposed humans to various metal agents, including mercury. It has been determined that mercury is not only harmful to the health of vulnerable populations such as pregnant women and children, but is also toxic to ordinary adults in various ways. For many years, mercury was used in a wide variety of human activities. Nowadays, the exposure to this metal from both natural and artificial sources is significantly increasing. Recent studies suggest that chronic exposure, even to low concentration levels of mercury, can cause cardiovascular, reproductive, and developmental toxicity, neurotoxicity, nephrotoxicity, immunotoxicity, and carcinogenicity. Possible biological effects of mercury, including the relationship between mercury toxicity and diseases of the cardiovascular system, such as hypertension, coronary heart disease, and myocardial infarction, are being studied. As heart rhythm and function are under autonomic nervous system control, it has been hypothesized that the neurotoxic effects of mercury might also impact cardiac autonomic function. Mercury exposure could have a long-lasting effect on cardiac parasympathetic activity and some evidence has shown that mercury exposure might affect heart rate variability, particularly early exposures in children. The mechanism by which mercury produces toxic effects on the cardiovascular system is not fully elucidated, but this mechanism is believed to involve an increase in oxidative stress. The exposure to mercury increases the production of free radicals, potentially because of the role of mercury in the Fenton reaction and a reduction in the activity of antioxidant enzymes, such as glutathione peroxidase. In this review we report an overview on the toxicity of mercury and focus our attention on the toxic effects on the cardiovascular system.

Mercury toxicity and neurodegenerative effects.

Mercury is among the most toxic heavy metals and has no known physiological role in humans. Three forms of mercury exist: elemental, inorganic and organic. Mercury has been used by man since ancient times. Among the earliest were the Chinese and Romans, who employed cinnabar (mercury sulfide) as a red dye in ink (Clarkson et al. 2007). Mercury has also been used to purify gold and silver minerals by forming amalgams. This is a hazardous practice, but is still widespread in Brazil's Amazon basin, in Laos and in Venezuela, where tens of thousands of miners are engaged in local mining activities to find and purify gold or silver. Mercury compounds were long used to treat syphilis and the element is still used as an antiseptic,as a medicinal preservative and as a fungicide. Dental amalgams, which contain about 50% mercury, have been used to repair dental caries in the U.S. since 1856.Mercury still exists in many common household products around the world.Examples are: thermometers, barometers, batteries, and light bulbs (Swain et al.2007). In small amounts, some organo mercury-compounds (e.g., ethylmercury tiosalicylate(thimerosal) and phenylmercury nitrate) are used as preservatives in some medicines and vaccines (Ballet al. 2001).Each mercury form has its own toxicity profile. Exposure to Hg0 vapor and MeHg produce symptoms in CNS, whereas, the kidney is the target organ when exposures to the mono- and di-valent salts of mercury (Hg+ and Hg++, respectively)occur. Chronic exposure to inorganic mercury produces stomatitis, erethism and tremors. Chronic MeHg exposure induced symptoms similar to those observed in ALS, such as the early onset of hind limb weakness (Johnson and Atchison 2009).Among the organic mercury compounds, MeHg is the most biologically available and toxic (Scheuhammer et a!. 2007). MeHg is neurotoxic, reaching high levels of accumulation in the CNS; it can impair physiological function by disrupting endocrine glands (Tan et a!. 2009).The most important mechanism by which mercury causes toxicity appears to bemitochondrial damage via depletion of GSH (Nicole et a!. 1998), coupled with binding to thiol groups ( -SH), which generates free radicals. Mercury has a high affinity for thiol groups ( -SH) and seleno groups ( -SeH) that are present in amino acids as cysteine and N-acetyl cysteine, lipoic acid, proteins, and enzymes. N-acetylcysteine and cysteine are precursors for the biosynthesis of GSH, which is among the most powerful intracellular antioxidants available to protect against oxidative stress and inflammation.Mercury and methylmercury induce mitochondrial dysfunction, which reduces ATP synthesis and increases lipid, protein and DNA peroxidation. The content of metallothioneines, GSH, selenium and fish high in omega-3 fatty acids appear to be strongly related with degree of inorganic and organic mercury toxicity, and with the protective detoxifying mechanisms in humans. In conclusion, depletion of GSH,breakage of mitochondria, increased lipid peroxidation, and oxidation of proteins and DNA in the brain, induced by mercury and his salts, appear to be important factors in conditions such as ALS and AD (Bains and Shaw 1997; Nicole eta!. 1998;Spencer eta!. 1998; Alberti et a!. 1999).

Localization of nerve growth factor (NGF) receptors in the mitochondrial compartment: characterization and putative role.

BACKGROUND: The neurotrophin NGF receptors trkA and p75NTR are expressed in the central and peripheral nervous system as well as in non-neuronal tissues; originally described to localize to the plasma membrane, recent studies have suggested other intracellular localizations for both NGF receptors. SCOPE OF REVIEW: In order to determine whether NGF receptors localize to the mitochondrial compartment mitochondria isolated from human kidney, rat tissues and a human podocyte as cell line before and after differentiation were used. MAJOR CONCLUSIONS: Our results demonstrate that NGF receptors are localized in the mitochondrial compartment of undifferentiated human podocytes and in all tissues analyzed including rat central nervous system. In mitochondria p75NTR, but not trkA, co-immunoprecipitates with the adenine nucleotide translocator (ANT) and the phosphodiesterase 4 isoform A5 (PDE4A5). Moreover, NGF, via trkA, protects isolated mitochondria of rat brain cortex from mitochondrial permeability transition induced by Ca(2+). GENERAL SIGNIFICANCE: Although NGF receptors have been described as mainly citoplasmatic so far, we proved evidence of their expression at the mitochondrial level and their interaction with specific proteins. Our results demonstrating the expression of NGF receptors in the mitochondria provide new insights into the role of NGF at subcellular level, in different areas of the organism, including CNS.

Bid as a potential target of apoptotic effects exerted by low doses of PPARγ and RXR ligands in breast cancer cells.

The combined treatment with nanomolar doses of the PPARγ ligand Rosiglitazone (BRL) and the RXR ligand 9-cis­retinoic acid (9RA) induces a p53-dependent apoptosis in MCF7, SKBR3 and T47D human breast cancer cells. Since MCF7 cells express a wild-type p53 protein, while SKBR3 and T47D cells harbor endogenous mutant p53, we elucidated the mechanism through which PPARγ and RXR ligands triggered apoptotic processes independently of p53 transcriptional activity. We showed an upregulation of Bid expression enhancing the association between Bid/p53 in both cytosol and mitochondria after the ligand treatment. Particularly in the mitochondria, the complex involves the truncated Bid that plays a key role in the apoptotic process induced by BRL and 9RA, since the disruption of mitochondrial membrane potential, the induction of PARP cleavage and the percentage of TUNEL-positive cells were reversed after knocking down Bid. Moreover, PPARγ and RXR ligands were able to reduce mitochondrial GST activity, which was no longer noticeable silencing Bid expression, suggesting the potential of Bid in the regulation of mitochondrial intracellular reactive oxygen species scavenger activity. Our data, providing new insight into the role of p53/Bid complex at the mitochondria in promoting breast cancer cell apoptosis upon low doses of PPARγ and RXR ligands, address Bid as a potential target in the novel therapeutical strategies for breast cancer.

Proliferative and anti-proliferative effects of retinoic acid at doses similar to endogenous levels in Leydig MLTC-1/R2C/TM-3 cells.

BACKGROUND: Vitamin A is suggested to be protective against oxidative stress. However, different authors observed pro-oxidant effects of retinoids both in experimental works and clinical trials. These discordances are the bases for the investigation of the proliferative and anti-proliferative properties of retinoic acid (RA) in biological systems. METHODS: Cell viability is determined with the MTT assay. Oxidative stress parameters are detected measuring catalase (CAT) and glutathione S-transferase (GST) enzymatic activities. FABP5 mRNA levels are measured by RT-PCR. Autophagy and apoptosis are analyzed by Monodansylcadaverine (MDC) staining and TUNEL assay, respectively. RESULTS AND CONCLUSIONS: RA, at nutraceutic/endogenous doses (10-200 nM), increases cell viability of testes tumor Leydig cell lines (MLTC-1 and R2C) and modulates antioxidant enzyme activities, as CAT and GST. RA is able to induce proliferation through non-classical and redox-dependent mechanisms accompanied by increased levels of FABP5 mRNA. The redox environment of the cell is currently thought to be extremely important for controlling either apoptosis or autophagy. Apoptosis occurs at pharmacological doses, while autophagy, which plays a critical role in removing damaged or surplus organelles in order to maintain cellular homeostasis, is triggered at the critical concentration of 500 nM RA, both in normal and tumoral cells. Slight variations of RA concentrations are evaluated as a threshold value to distinguish between the proliferative or anti-proliferative effects. GENERAL SIGNIFICANCE: Although retinoids have a promising role as antineoplastic agents, physiological levels of RA play a key role in Leydig cancer progression, fostering proliferation and growth of testicular tumoral mass.

miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro.

The mammalian nervous system exerts essential control on many physiological processes in the organism and is itself controlled extensively by a variety of genetic regulatory mechanisms. microRNA (miR), an abundant class of small non-coding RNA, are emerging as important post-transcriptional regulators of gene expression in the brain. Increasing evidence indicates that miR regulate both the development and function of the nervous system. Moreover, deficiency in miR function has also been implicated in a number of neurological disorders. Expression profile analysis of miR is necessary to understand their complex role in the regulation of gene expression during the development and differentiation of cells. Here we present a comparative study of miR expression profiles in neuroblastoma, in cortical development, and in neuronal differentiation of embryonic stem (ES) cells. By microarray profiling in combination with real time PCR we show that miR-7 and miR-214 are modulated in neuronal differentiation (as compared to miR-1, -16 and -133a), and control neurite outgrowth in vitro. These findings provide an important step toward further elucidation of miR function and miR-related gene regulatory networks in the mammalian central nervous system.

Combined low doses of PPARgamma and RXR ligands trigger an intrinsic apoptotic pathway in human breast cancer cells.

Ligand activation of peroxisome proliferator-activated receptor (PPAR)gamma and retinoid X receptor (RXR) induces antitumor effects in cancer. We evaluated the ability of combined treatment with nanomolar levels of the PPARgamma ligand rosiglitazone (BRL) and the RXR ligand 9-cis-retinoic acid (9RA) to promote antiproliferative effects in breast cancer cells. BRL and 9RA in combination strongly inhibit of cell viability in MCF-7, MCF-7TR1, SKBR-3, and T-47D breast cancer cells, whereas MCF-10 normal breast epithelial cells are unaffected. In MCF-7 cells, combined treatment with BRL and 9RA up-regulated mRNA and protein levels of both the tumor suppressor p53 and its effector p21(WAF1/Cip1). Functional experiments indicate that the nuclear factor-kappaB site in the p53 promoter is required for the transcriptional response to BRL plus 9RA. We observed that the intrinsic apoptotic pathway in MCF-7 cells displays an ordinated sequence of events, including disruption of mitochondrial membrane potential, release of cytochrome c, strong caspase 9 activation, and, finally, DNA fragmentation. An expression vector for p53 antisense abrogated the biological effect of both ligands, which implicates involvement of p53 in PPARgamma/RXR-dependent activity in all of the human breast malignant cell lines tested. Taken together, our results suggest that multidrug regimens including a combination of PPARgamma and RXR ligands may provide a therapeutic advantage in breast cancer treatment.

Adrenal glands and testes as steroidogenic tissue are affected by retinoylation reaction.

This study was undertaken to better understand the physiological role of the retinoylation process in steroidogenic tissues. In adrenal gland mitochondria, the retinoylation extent was found equal to that of testes mitochondria but without ATP in the incubation buffer. We pointed out that the endogenous mitochondrial ATP in adrenal glands is much higher than in testes, about 1.3 x 10(-2) M and 5.2 x 10(-8) M, respectively. In addition, less CoASH is required for the maximal acylation activity of the retinoyl moiety to protein(s) compared to testes. The fatty acid analysis revealed a different composition of mitochondrial membranes of these two tissues. Among the different values of fatty acids, it is important to note that adrenal glands contain a much higher amount of C18:0 and a much lower amount of C22:5 omega6 and C22:6 omega3 than testes in the mitochondrial membranes. In addition, there were also differences in arachidonic acid (ARA, C20:4 omega6) content between adrenal glands and testes mitochondria. These different values in the fatty acids composition should explain the different extent of the retinoylation process between the two organs.

Retinoic acid-induced testosterone production and retinoylation reaction are concomitant and exhibit a positive correlation in Leydig (TM-3) cells.

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. The effects of RA are thought to be mediated by the nuclear retinoid receptors; however, not all the effects of RA can be explained by the nuclear receptor pathways. Indeed, retinoylation is another mechanism of action elicited by RA. In growing TM-3 Leydig cell cultures, the extent of retinoylation depends in a saturable manner on the initial concentration of 3H-RA, time and cell number. In addition, dose-response curves for RA-induced testosterone production and retinoylation are concomitant and exhibit a positive correlation. In the present study we demonstrate that RA is able to influence a retinoylation reaction on protein(s) probably involved on steroidogenesis.

Identification and kinetic characterization of HtDTC, the mitochondrial dicarboxylate-tricarboxylate carrier of Jerusalem artichoke tubers.

Jerusalem artichoke (Helianthus tuberosus L.) tubers were reported to be tolerant to cold and freezing. The aim of this study was to perform a kinetic characterization of the mitochondrial dicarboxylate-tricarboxylate carrier (HtDTC) and to assess a possible involvement of this carrier in the cold tolerance of tubers. The HtDTC was purified from isolated mitochondria by sequential chromatography on hydroxylapatite/celite and Matrex Gel Orange A. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31.6 kDa. A polyclonal antibody raised against the tobacco DTC cross-reacted with the purified protein on Western blot analysis. In gel trypsin, digestion of the purified HtDTC yielded peptides that exhibited strong amino acid sequence similarity to previously identified plant DTCs. Furthermore, using degenerate primers, a portion of the Htdtc cDNA was amplified and sequenced; this cDNA encoded for a protein with high sequence similarity to known plant homolog DTCs. When reconstituted in liposomes loaded with dicarboxylate (2-oxoglutarate, malate, malonate, succinate, and maleate) or tricarboxylate anions (citrate, trans-aconitate, and isocitrate), the purified HtDTC transported all these anions in exchange with external [14C]2-oxoglutarate. A kinetic characterization of HtDTC was performed: (a) the half-saturation constant Km and the Vmax at 25 degrees C of the 2-oxoglutarate/2-oxoglutarate exchange by reconstituted HtDTC were found to be 360 microM and 10.9 micromol/(min mg protein), respectively; (b) the activation energy Ea of the succinate/2-oxoglutarate exchange by the reconstituted HtDTC was found to be 50.7 kJ/mol constant between -5 and 35 degrees C. Similarly, the activation energy Ea of succinate respiration of isolated Jerusalem artichoke mitochondria, measured between -2 and 35 degrees C, was shown to be constant (65.3 kJ/mol). The physiological relevance of kinetic properties and temperature dependence of transport activities of HtDTC is discussed with respect to the cold tolerance ability of Jerusalem artichoke tubers.

Binding of all-trans-retinoic acid to MLTC-1 proteins.

The covalent incorporation of [(3)H]all-trans-retinoic acid into proteins has been studied in tumoural Leydig (MLTC-1) cells. The maximum retinoylation activity of MLTC-1 cell proteins was 710+/-29 mean+/-SD) fmoles/8 x 10(4) cells at 37 degrees C. About 90% of [(3)H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion and about 65--75% after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation reaction was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC(50) values of 0.9 microM and 0.65 microM, respectively. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (250 microM); but there was an increase of the binding activity of about 25% and 130%, respectively. On the other hand, the retinoylation reaction was inhibited (about 40%) by 250 microM lauric acid. After pre-incubation of the cells with different concentrations of unlabeled RA, the retinoylation reaction with 100 nM [(3)H]RA involved first an increase at 100 nM RA and then a decrease of retinoylation activity between 200 and 600 nM RA. After cycloheximide treatment of the tumoural Leydig cells the binding activity of [(3)H]RA was about the same as that in the control, suggesting that the bond occurred on proteins in pre-existing cells.

Characterization of rat testes mitochondrial retinoylating system and its partial purification.

Retinoylation (retinoic acid acylation), a posttranslational modification of proteins occurring in a variety of eukariotic cell lines both in vivo and in vitro, was studied in rat testes mitochondria. all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins of rat testes mitochondria. The maximum retinoylation activity of rat testes mitochondrial proteins was 21.6 pmoles mg protein(-1) 90 min(-1) at 37 degrees C. The activation energy was 44 kJ mol(-1) from 5 to 37 degrees C. The retinoylation activity had a pH optimum of 7.5. The retinoylation process was specific for the presence of ATP, ADP, and GTP (even if only 30% of the control). The half saturation constant (Km) was 0.69 microM for all-trans-retinoic acid, while the inhibition constant (Ki) was 1.5 microM for 13-cis-retinoic acid. Retinoylation was not inhibited by high concentrations of myristic acid (MA) and palmitic acid (PA), indicating that retinoylation and acylation reactions involved different rat testes mitochondrial proteins. The ATP or CoASH saturation curves of retinoylation reaction showed sigmoidal behavior with apparent half saturation constants (K0.5) of 6.5 mM ATP and 40.6 microM CoASH. On SDS-gel electrophoresis, the hydroxylapaptite/celite eluate showed various protein bands between 25 and 80 kDa. This retinoylated protein was purified 17-fold with respect to the mitochondrial extract.

Purification and characterization of the reconstitutively active adenine nucleotide carrier from mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers.

The adenine nucleotide carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 33 kDa. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5'-phosphate-sensitive ATP/ATP exchange. It was purified 75-fold with a recovery of 15% and a protein yield of 0.18% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ATP, ADP, and GTP and was inhibited by bongkrekate, phenylisothiocyanate, pyridoxal 5'-phosphate, mersalyl and p-hydroxymercuribenzoate (but not N-ethylmaleimide). Atractyloside and carboxyatractyloside (at concentrations normally inhibitory in animal and plant mitochondria) were without effect in Jerusalem artichoke tubers mitochondria. Vmax of the reconstituted ATP/ATP exchange was determined to be 0.53 micromol/min per mg protein at 25 degrees C. The half-saturation constant Km and the corresponding inhibition constant Ki were 20.4 microM for ATP and 45 microM for ADP. The activation energy of the ATP/ATP exchange was 28 KJ/mol between 5 and 30 degrees C. The N-terminal amino acid partial sequence of the purified protein showed a partial homology with the ANT protein purified from mitochondria of maize shoots.