Adenine nucleotide translocator isolation

Ciapaite, J., Van Eikenhorst, G., Bakker, S.J.L., Diamant, M., Heine, R.J., Wagner, M.J., Westerhofif, H.V. and Krab, K. (2005) Modular kinetic analysis of the adenine nucleotide translocator-mediated effects of palmitoyl-CoA on the oxidative phosphorylation in isolated rat liver mitochondria. Diabetes 54, 944-951. 

Secondly, the transport inhibitor must be able to pass the cell membrane. The inability of benzene-1,2,3-tricarboxylate to inhibit gluconeogenesis from lactate in perfused pigeon liver, a tissue in which mitochondrial efflux of phosphoenolpyruvate is obligatory for glucose synthesis, is presumably due to lack of penetration through the plasma membrane [16], This observation is interesting since it shows that the ability of an inhibitor to penetrate the cell membrane may vary from tissue to tissue benzene-1,2,3-tricarboxylate does inhibit lipogenesis in hepatocytes of neonatal chicks, as discussed above. Another example is the apparent relative impermeability of the plasma membrane of isolated foetal rat hepatocytes, as compared with that from adult rats, for atractyloside, the inhibitor of the adenine nucleotide translocator [17]. 

According to Klingenberg and Rottenberg [9] the ratio (ATP/ADP) / (ATP/ADP)in in isolated liver mitochondria, incubated under conditions of zero net flux through the adenine nucleotide translocator, varies linearly with the mitochondrial membrane potential. According to their results a membrane potential... 

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]. 

Freshly isolated, intact mitochondria contain considerable amounts of adenine nucleotides which are resistant to removal by repeated washings with isotonic sucrose. This indicates that these compounds are in a compartment—presumably within the inner mitochondrial membrane—which is inaccessible to the sucrose solution. When exogenous adenine nucleotide is added to the mitochondria, there is a rapid exchange with endogenous adenine nucleotides with no net increase in the concentration of adenine nucleotides in the mitochondria. ADP exchanges most rapidly, followed by ATP and then by AMP, which is relatively impermeable. It is the inner mitochondrial membrane through which the adenine nucleotides do not permeate and which contains the specific adenine-nucleotide transporting system. The movement of ATP across the inner mitochondrial membrane (and hence out of the mitochondria) depends directly on the translocation of ADP in the presence of adenylate kinase in the outer compartment of the mitochondria. 

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