Oxygen-pulse curves in rat liver mitochondrial suspensions. Some observations and deductions

Abstract

1. The inference, implicit in the chemiosmotic hypothesis, that protons move into the bulk phase during ATP synthesis was investigated. 2. Incubation of rat liver mitochondria in the presence of the cation exchanger CM-Sephadex C-50 caused alkalinization in the medium, though total ATP synthesis remained unchanged. The addition of N-ethylmaleimide prevented the alkalinization, but there was still no indication of protons passing into the medium. The expected proton movement [Mitchell & Moyle (1967) Biochem. J. 105, 1147--1162] was readily detected when as an equivalent acid pulse. 3. Analysis of delta H+ decay curves after O2 pulses (3 micrograms-atoms of O/g of protein) indicated the presence of fast and slow components of decay, with first-order rate constants (k) of 0.24s-1 and 0.032s-1. The fast decay was finite and was eliminated in the presence of N-ethylmaleimide. 4. These observations are interpreted as evidence for the development of unmasking of fixed charges on the outer surface of the mitochondrial inner membrane during energization and for the existence of proton-retentive electrical fields (rho-zones) on this surface. The charge concentration is calculated as about 1 charge/10nm2. 5. A cycle of changes in a single fixed-charge molecule is proposed which mediates both Ca2+ uptake and the first step in the utilization of the rho-zone protonmotive force, delta p rho.

Determination of the Proton Electrochemical Gradient across Biological Membranes

“…One difficulty is that when bacteria and mitochondria receive an oxygen pulse, no protons are detected in the aqueous phase unless permeant ions are present (Pozzan and Azzone, 1976;Gould and Cramer, 1977;Gould, 1979;Archbold et al, 1979;Conover and Azzone, 1981;Hitchens and Kell, 1982b). Although bulk-to-bulk Δ/Ι Η is generally assumed as the coupling intermediate, its kinetic and thermodynamic competence is far from established.…”

Anionic lipid headgroups as a proton-conducting pathway along the surface of membranes: a hypothesis.

“…Other reports (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) have described proton movements in restricted domains or in isolated open membrane sheets not capable of sustaining a transmembrane pH gradient (which nevertheless can be energized, ostensibly by generation of an electrochemical H+ gradient for ATP synthesis) or have demonstrated that the pH may not account for the activities attributed to membrane "energization." Taken together, the observations suggest that there may be specific proton-conducting pathways in or on such energy-transducing membranes that allow them to carry out energy transduction without necessarily involving a pH gradient across the membrane.…”

The protonmotive force and ATP synthesis/membrane-fixed-charge relationships

“…hypothesis, Archbold et al (1979) have recently summarized their view that this hypothesis has resulted from an unjustified application of experimental data to the conditions of synthesis in between the chemiosmotic hypothesis and the p-zone interpretation, the mechanism of mitochondria1 energy transduction The critical difference lies in the acknowledgement, or not, of Department of Biochemistry, The Queen's University of Belfast, OiOO. Table 1 shows the most important points Of distinction Table 1.…”

“…Starting from the initial speculations of Azzi (1969), the evidence comes from an increasing number of sources: from cytopherometer and surface-potential studies (Kamo et al, 1976;Aiuchi et al, 1977); from the pre-emptive uptake of Ca2+ over protons in the initial aerobic phase of 0,-pulse experiments, and the kinetics of Ca2+ release in an ensuing anaerobic phase when mitochondria are given limited 0,-pulse injections (Archbold et al, 1979); and from the effect of positively and negatively charged inhibitors on energy transduction in mitochondria and submitochondrial particles (Higuti et al, 1978a(Higuti et al, ,b, 1979.…”

“…(mv), is localized in, and limited to, (Archbold et al, 1979) is characterized by the importance it these surface p-zone areas. The attribution of a bulk-phase gives to the generation, or unmasking, of negative fixed charges protonmotive force (Mitchell & Moyle, 1969) is considered to be on the outer surface of the coupling membrane during electron an error arising from the delocalization of &;, in the 'rationale'…”