The adenine nucleotide carrier

The other kinetic method [153], in which the rate constant of the first order approach to equilibrium is determined, provides ambiguous results because of uncertainty in the size of the exchangeable pool of nucleotides. The natural log plot of the percentage equilibrated is not linear regardless of the value chosen as 100% equilibrated. This is probably due to heterogeneity of the exchangeable in-tramitochondrial pool. The intramitochondrial adenine nucleotide pool is composed of ATP, ADP and AMP. AMP is not a substrate of the carrier, yet radioactive ATP 

Recently, the effect of internal substrate level has been systematically studied at different concentrations of external nucleotide using intact rat liver mitochondria 

In order to circumvent the problems inherent in measuring kinetics in intact mitochondria, Kramer and Klingenberg [159,160] measured adenine nucleotide 

The heteroexchanges ATP o for ADP,j j, and ADP m) for ATP,j ), were also studied, but under conditions where both substrates were present in equimolar concentration on both sides of the membrane. The rate of entry of radiolabelled ADP under these conditions was studied at saturating substrate levels at variable Aif/ from -Hl20 to —180 mV with respect to the external side of the membrane. The ratio of the rate of ADP entry to ATP entry was plotted as a function of Atp and the ratio varied from 5 to 0.2 as a linear function of Aip from -1-120 to —90 mV. 

The data can perhaps be reconciled with a sequential model if one assumes that, under all conditions, both steps in the sequence are partially rate controlling. Thus, in the presence of an electrical potential across the membrane favoring ATP efflux, ATP/ATP exchange could be faster than the exchange of external ATP for internal ADP, if the return of the carrier to the outward facing direction is partially rate 

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Joseph-Iiauzun, E., Farges, R., Delmas, P., Ferrara, P. Loison, G. (1997). The Mr 18,000 subunit of the peripheral-type benzodiazepine receptor exhibits both benzodiazepine and isoquinoline carboxamide binding sites in the absence of the voltage-dependent anion channel or of the adenine nucleotide carrier. J. Biol. Chem. 272, 28102-6. 

McEnery, M.W., Snowman, A.M., Trifiletti, R.R., and Snyder, S.H., 1992, Isolation ofthe mitochondrial benzodiazepine receptor association with the voltage-dependent anion channel and the adenine nucleotide carrier, Proc.Natl.Acad.Sci. U.S.A. 89 3170-3174. 

A much slower transport process which catalyzes the net uptake of ATP has been demonstrated in liver [42,43] and heart mitochondria [44,45], and shown to be physiologically important in neonatal liver mitochondria [46,47]. The transporter s activity may be affected by hormones since it has been shown that glucagon treatment increases the total nucleotide content of subsequently isolated liver mitochondria [48]. Although initially thought to be due to rather nonspecific leakiness of mitochondrial membranes or unidirectional transport on the adenine nucleotide carrier, its lack of sensitivity to membrane potential, and its almost absolute dependence on the presence of Mg and phosphate suggest that the observed activity is due to a separate transporter [42]. Net nucleotide uptake in plant mitochondria is much more marked but has a similar dependence on Mg " and phosphate in the media [49]. 

The biosynthesis of the adenine nucleotide carrier has been studied most extensively in Neurospora crassa. The carrier isolated from Neurospora mitochondria is very similar to the carrier isolated from mammalian mitochondria [78]. Its molecular weight, subunit structure, amino acid composition, hydrophobicity and inhibitor specificity are remarkably similar to the mammalian heart and liver carriers. Specific antibodies to the Neurospora carrier have been raised in rabbits [79]. 

Although a few subunits of mitochondrial membrane proteins are coded by mitochondrial DNA and synthesized in the mitochondrial matrix, most membrane proteins including the adenine nucleotide carrier are coded by nuclear genes and synthesized on cytoplasmic ribosomes [80,81], Chloramphenicol, an inhibitor of mitochondrial protein synthesis, does not inhibit incorporation of radioactive leucine into the carrier in growing Neurospora crassa, but cycloheximide, an inhibitor of cytoplasmic protein synthesis, does inhibit leucine incorporation [78]. Also, a yeast nuclear respiratory mutant has been shown to cause a defect in adenine nucleotide transport [81], and the nuclear gene responsible for coding the carrier in yeast is currently being cloned for further studies [82]. 

Deviation from expected kinetic behavior for the gated pore model has also been observed in the case of the two electrogenic transporters, the glutamate/aspartate and the adenine nucleotide carriers. 

Fig. 8.1. Two-dimensional schematic representation of the structure of the adenine nucleotide carrier. The line represents the amino acid chain of the protein and all numbers on or within the line represent the number of the amino acids in the linear sequence. The black dots are cysteine residues, about which there is significant sequence homology [186]. The helical regions are segments of hydrophobic amino acids thought to span the membrane [186]. The CAT arrow represents the site of photoaffinity labelling of an azido derivative of atractyloside [189]. The open circles are lysine residues which react with pyridoxal phosphate in intact mitochondria or submitochondrial particles [190,191].

At the present time, it is the opinion of the present reviewers that Wilson and collaborators have not provided sufficient evidence to conclude that the adenine nucleotide carrier is electroneutral and near equilibrium, nor have they conclusively shown that the first two sites of oxidative phosphorylation are in near equilibrium, especially in view of the likelihood that the P/O ratio of the first two sites is 1.5 rather than 2 [230,231]. Therefore, we conclude that their studies do not convincingly exclude a role for the adenine nucleotide translocator in the control of mitochondrial respiration. 

In general, mechanistic studies of the mitochondrial metabolite transporters carried out in the last five years have provided evidence supporting a gated-pore, sequential model for transport. Structural studies of the adenine nucleotide carrier are more advanced than studies of the other transporters, and these provide the most clear cut evidence. 

Mans AM, De Joseph MR, Hawkins RA. Metabolic abnormalities and grade of encephalopathy in acute hepatic failure. J. Neurochem., 63, 1829-1838, 1994 McEnery MW, Snowman AM, Trifiletti RR, Snyder SH. Isolation of the mitochondrieil benzodiazepine receptor Association with the voltage-dependent anion channel tuid the adenine nucleotide carrier. Proc. Natl. Acad. Sci. USA, 89, 3170-3174, 1992 Mena EE, Cotman CW. Pathologic concentrations of ammonium ions block 1-gluttunate uptake. Exp. Neurol, 59, 259-263, 1985... 

The adenine nucleotide carrier or translocase is specifically inhibited by atractyloside (competitive with respect to adenine nucleotide) and bongkrekic acid (noncompetitive). Atractyloside has been known for some time to be highly poisonous and its mechanism of action attests to the importance of the adenine nucleotide translocase. Other nucleotides, such as GTP, must first be converted to ATP by nucleoside diphosphokinase prior to transport out of the mitochondria. The outer mitochondrial compartment also contains nucleoside diphosphokinase for the conversion of ATP to GTP. 

Pantothenic acid, sometimes called vitamin B3, is a vitamin that makes up one part of a complex coenzyme called coenzyme A (CoA) (Figure 18.23). Pantothenic acid is also a constituent of acyl carrier proteins. Coenzyme A consists of 3, 5 -adenosine bisphosphate joined to 4-phosphopantetheine in a phosphoric anhydride linkage. Phosphopantetheine in turn consists of three parts /3-mercaptoethylamine linked to /3-alanine, which makes an amide bond with a branched-chain dihydroxy acid. As was the case for the nicotinamide and flavin coenzymes, the adenine nucleotide moiety of CoA acts as a recognition site, increasing the affinity and specificity of CoA binding to its enzymes. 

Tire important adenine nucleotide carrier takes ADP into the mitochondrial matrix for phosphorylation in a 1 1 ratio with ATP that is exported into the cytoplasm.299 300b This is one of the major rate-determining processes in respiration. It has been widely accepted that the carrier is electrogenic,300... 

Table 7.2. Effects of various metabolites on [a32P]ADP uptake into E. coli cells expressing several adenine nucleotide carriers. For uptake experiments, effectors were always present in a fivefold higher concentration than the given uptake substrates. [a32P]ADP uptake by AAC2(A.t.), AACl(N.sp.), HMP31(T.g.), and ANTl( ./z.) was measured at a substrate concentration of 10, 100, 50, and 200 pM, respectively (Voncken et al. 2002 Tjaden et al. 2004 Leroch et al. 2005 Leroch 2006) ...

The control of the respiration process and ATP synthesis shifts as the metabolic state of the mitochondria changes. In an isolated mitochondrion, control over the respiration process in state 4 is mainly due to the proton leak through the mitochondrial inner membrane. This type of control decreases from state 4 to state 3, while the control by the adenine nucleotide and the dicarboxylate carriers, cytochrome oxidase, increases. ATP utilizing reactions and transport activities also increase. Therefore, in state 3, most of the control is due to respiratory chain and substrate transport. 

Biosynthesis has been studied only in the case of the adenine nucleotide exchange carrier, which has been isolated in pure form from several sources [72-74]. Antibodies have been raised to some of these pure proteins [75], and the availability of antibodies as well as the availability of inhibitors [76] which bind specifically to the nucleotide carrier have permitted studies of its biosynthesis not possible for the other carriers. The immunological work is important since the antibodies can detect the carrier in precursor forms, not yet assembled in the membrane. 

A pool of adenine nucleotide carrier precursor protein has been identified in the... 

Additional studies [212,218,219,242,243] to quantitate the role of the adenine nucleotide translocator in the control of mitochondrial respiration have been performed utilizing inhibitor titrations with carboxyatractyloside. The results indicated that in State 4 (no ADP), no control was exerted by the translocator. However, as the rate of respiration was increased up to State 3 (excess ADP), the control strength of the carrier increased to a maximum value of 30%, at 80% of State 3 respiration. These studies indicate that the adenine nucleotide translocator cannot be considered to be the only rate-controlling step in oxidative phosphorylation. However, they do provide experimental support for a controlling role for the carrier at intermediate to maximal levels of respiration. An important corollary of these studies is that the reaction rate may be altered by a change in substrate concentration (elasticity). It is also clear that to confirm these studies quantitatively, they must be extended to intact cells. Although such studies have been more difficult, the results are compatible with the conclusion reached by Tager et al. [212]. 

In recent years several attempts have been made to isolate some of the translocator proteins. Major progress has been achieved with the purification of the adenine nucleotide translocator. This protein has been isolated in its native form and its molecular weight and immunological properties have been characterised [57,104]. The carrier protein from beef heart and rat liver is a dimer. The of each of the subunits is 30000. In heart, the carrier protein makes up 10% of the total mitochondrial protein. The amino acid composition of the yeast protein has been determined recently [105]. 

The mitochrondrial peripheral-type benzodiazepine receptor (PTBR) is a multimeric complex comprising three snbnnits, namely an 18 KDa isoqninoline crrrboxamine-binding protein (IBP), a 34 KDa voltage-dependent anion channel and a 30 KDa adenine nucleotide carrier (McEnery et al., 1992). 

Figure 6 compares the amino acid sequence of M2 loops of 22 sources. In these sequences, more then 40% amino acides are conserved. These amino acid residues may be essential for exhibition of the transport activity. In the bovine heart mitochondrial ADP/ATP carrier, the conserved amino acids are located at both ends of loop M2 connected to the membrane segments (Asp -Asp " and Gly -Gln " ), and the middle part of the loop (Arg -Lys ). As the conserved hydorphobic amino acids (Phe , Leu , Ile °, Ile and Phe " ) are located in the middle segment, and as the adenine nucleotide binding site should be close to Cys , the middle peptide segment in loop M2 may constitute the primary binding site of ADP and ATP, and also of BKA and fluorescein derivatives including EMA. Possibly, the... 

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