Efficient extra-mitochondrial aerobic ATP synthesis in neuronal membrane systems.

The nervous system displays high energy consumption, apparently not fulfilled by mitochondria, which are underrepresented therein. The oxidative phosphorylation (OxPhos) activity, a mitochondrial process that aerobically provides ATP, has also been reported also in the myelin sheath and the rod outer segment (OS) disks. Thus, commonalities and differences between the extra-mitochondrial and mitochondrial aerobic metabolism were evaluated in bovine isolated myelin (IM), rod OS, and mitochondria-enriched fractions (MIT). The subcellular fraction quality and the absence of contamination fractions have been estimated by western blot analysis. Oxygen consumption and ATP synthesis were stimulated by conventional (pyruvate + malate or succinate) and unconventional (NADH) substrates, observing that oxygen consumption and ATP synthesis by IM and rod OS are more efficient than by MIT, in the presence of both kinds of respiratory substrates. Mitochondria did not utilize NADH as a respiring substrate. When ATP synthesis by either sample was assayed in the presence of 10-100 µM ATP in the assay medium, only in IM and OS it was not inhibited, suggesting that the ATP exportation by the mitochondria is limited by extravesicular ATP concentration. Interestingly, IM and OS but not mitochondria appear able to synthesize ATP at a later time with respect to exposure to respiratory substrates, supporting the hypothesis that the proton gradient produced by the electron transport chain is buffered by membrane phospholipids. The putative transfer mode of the OxPhos molecular machinery from mitochondria to the extra-mitochondrial structures is also discussed, opening new perspectives in the field of neurophysiology.

Hypothesis of lipid-phase-continuity proton transfer for aerobic ATP synthesis.

The basic processes harvesting chemical energy for life are driven by proton (H(+)) movements. These are accomplished by the mitochondrial redox complex V, integral membrane supramolecular aggregates, whose structure has recently been described by advanced studies. These did not identify classical aqueous pores. It was proposed that H(+) transfer for oxidative phosphorylation (OXPHOS) does not occur between aqueous sources and sinks, where an energy barrier would be insurmountable. This suggests a novel hypothesis for the proton transfer. A lipid-phase-continuity H(+) transfer is proposed in which H(+) are always bound to phospholipid heads and cardiolipin, according to Mitchell's hypothesis of asymmetric vectorial H(+) diffusion. A phase separation is proposed among the proton flow, following an intramembrane pathway, and the ATP synthesis, occurring in the aqueous phase. This view reminiscent of Grotthus mechanism would better account for the distance among the Fo and F1 moieties of FoF1-ATP synthase, for its mechanical coupling, as well as the necessity of a lipid membrane. A unique active role for lipids in the evolution of life can be envisaged. Interestingly, this view would also be consistent with the evidence of an OXPHOS outside mitochondria also found in non-vesicular membranes, housing the redox complexes.

Structural modification of proteins by direct electric current from low voltage.

The interaction of direct electric current (dc) and proteins is a little explored topic. We had reported that exposure of Crotalus atrox venom to dc caused irreversible inactivation of phospholipase A(2) and metalloprotease and that the eukaryote adenylate kinases (AK) precipitate in nondenaturing gel electrophoresis. AK1 displays an elevated percent difference of CHarged versus POlar amino acid content (CH-PO 14). Commercial AK1 and other 17 enzymes with various CH-PO values were exposed in solution to dc (0-0.7 mA) from low voltage (0-10 V), then enzymatic activity was assayed. The enzymes with CH-PO higher than 10.0 were irreversibly inactivated by current exposure; those with CH-PO between +3 and -5 were not. Inactivation was dependent on the ionic strength of the medium and not on the net charge of the protein. Circular dichroic spectroscopy showed a structural modification in some of the inactivated enzymes. CH-PO could be a crucial, although rough, parameter for predicting protein inactivation by low-voltage exposure. The observed effect seems due to the current density. Enzymatic activity maybe a more accurate sensor of conformational changes than circular dichroism spectroscopy. A better understanding of efficacy of many electrical devices utilized in medical practice may follow.