Jennifer Moyle

Cytochrome c oxidase is not a proton pump.

We conclude that the reduction of O2 to 2 H2O by cytochrome c oxidase of rat liver mitochondria involves the translocation of 4-from cytochrome c at the outer surface of the cristae membrane per O2 reduced and protonated by 4 H+ ions that enter the reaction domain from the inner aqueous phase. This net electron-translocating function of cytochrome c oxidase plugged through the mitochondrial cristae membrane is not linked to a proton-pumping function, such as that proposed by Wikström [7,8].

Active/inactive state transitions of mitochondrial ATPase molecules influenced by Mg2+, anions and aurovertin.

The complete proton-translocating ATPase of mitochondria is activated by certain anions, such as phosphate, sulphate and chromate, but is inhibited by other anions, such as azide and thiocyanate [l-3]. Similar anion activations and inhibitions have also been observed in preparations of the F1 component of the ATPase [4-81. These activations and inhibitions have generally been attributed to effects of the anions on the reaction velocity V, characteristic of all the ATPase catalytic units in the ATPase preparations. However, the relatively slow time-dependence of some kinetic properties of the ATPase [ 1,2,7,8,14], and the fact that oligomycin inhibits the ATPase reaction by almost the same factor, independently of the degree of anion activation or inhibition [ 1,2], suggested to us recently that the enzyme population might consist df a dynamic mixture of active and virtually inactive catalytic units, and that the equilibrium ratio of active to inactive ATPase units or molecules might be influenced by the ionic composition of the medium, and possibly by other agents, such as aurovertin. The experiments described in this paper show that the complete ATPase of rat liver mitochondria is activated in a KC1 medium at low Mg2+ concentration, and that 5 mM MgC12 induces inactivation at a rate that is slow compared with the rate of the ATPase reaction. This inactivation is enhanced by azide. The converse process of anion activation proceeds at a rate that depends on the activating anion and may take as long as ten minutes to reach equilibrium.

The Glycerophospho-protein Complex Envelope of Micrococcus pyogenes

SUMMARY: The glycerophospho-compound which has been shown to account for more than one-quarter of the total organic phosphate of Micrococcus pyogenes is present almost exclusively in the particulate fractions of mechanically disintegrated cell suspensions. The main particulate fraction corresponds to the cell envelope material of Dawson (1949). Three-quarters of the weight of this material is made up of a glycerophospho-protein complex of which the protein moiety resembles silk fibrom in amino-acid composition and in its general properties. The amount of hydroxylamino-acid in the protein of the envelope would be sufficient to bind the polyolphosphoric acids covalently, but the possibility that the polyolphosphoric acids form a polymer which is adsorbed on to a protein matrix is not excluded. A subsidiary particulate fraction, made up of very small particles containing a high proportion of phospholipid, was found to be otherwise similar in composition to the envelope fraction. It is suggested that in the intact cell, the material of the small particle fraction may form a continuous layer (lipid membrane?) lying beneath the glycerophospho-protein complex envelope (cell wall?).

The lanthanide-sensitive calcium phosphate porter of rat liver mitochondria

The import of calcium (and of some other bivalent cations including strontium) into respiring mitochondria from rat liver occurs electrophoretically via a lanthanide-sensitive porter system that is insensitive to thiol reactors. We recently obtained evidence that only one electric charge is translocated through the lanthanide-sensitive porter system per Ca* imported; and we proposed the hypothesis that the so-called calcium porter may actually be a calcium phosphate porter, more precisely described as a (Ca2)4+-HPO

Electric charge stoicheiometry of calcium translocation in rat liver mitochondria

symporter [ 1 ] . Two other systems that catalyse phosphate translocation in rat liver mitochondria are known [2-41. One is the NEM-sensitive and mersalyl-sensitive phosphoric acid uniporter (or phosphate/hydroxylion antiporter). The other is the mersalyl-sensitive phosphate/dicarboxylate antiporter. Our hypothesis that there is a (Ca2)4+-HP0

Proton-translocating pyrophosphatase of Rhodospirillum rubrum.

symporter therefore predicts that it should be possible to observe a lanthanide-sensitive, but NEMand mersalyl-insensitive, import of phosphate that is dependent on and stoicheiometric with the import of Cap or Sr*. In the present paper we verify this prediction.

Activation and inhibition of mitochondrial adenosine triphosphatase by various anions and other agents.

It has been shown by a variety of methods that calcium ions enter respiring mitochondria electrophoretically via a specific lanthanide-sensitive porter in the cristae membrane [l-l 21. When a small amount of calcium chloride is added to a suspension of rat liver mitochondria respiring in State 4, there is a transient respiratory stimulation and a net export of H’ ions, while virtually all the calcium ions are imported. In such experiments it is generally observed that the number of H’ ions exported per extra oxygen atom reduced (the +H’/O ratio) is about 2 per conventional coupling site in operation [2,7]. The number of calcium ions imported per extra oxygen atom reduced (the +calcium/O ratio) is likewise about 2 per conventional coupling site in operation [2,7]. In other words, each calcium ion appears to carry only one positive charge as it is taken up electrophoretically through the porter system. The simple interpretation would be that the calcium-specific porter is not a Ca’+ uniporter, but is a calcium-anion symporter or a calcium/cation antiporter through which the net calcium and charge translocation stoicheiometry can be represented as <a’. As far as we know, this interpretation is not irreconcilable with other experimental data [ 1131 concerning the calcium-specific porter. However,

Measurements of mitochondrial ←H+/O quotients: Effects of phosphate and N-ethylmaleimide

The membrane of chromatophores of Rhodospirillum rubrum contains an oligomycin-insensitive reversible pyrophosphatase system [l] that is involved in energy transduction by a pathway separate from that of the oligomycin-sensitive ATPase system [2-61. Pyrophosphate hydrolysis by chromatophores induces a change of carotenoid absorbance [7], a change in the fluorescence of added 8-anilinonaphthalene-1-sulphonic acid [8] and an uptake of phenyl dicarbaundecaborane anion [9]. Therefore it has been inferred that pyrophosphate hydrolysis by the pyrophosphatase system is coupled to (electrogenic) ion translocation across the chromatophore membrane; but the species of ion involved has not yet been identified. In this paper we describe measurements of changes of the pH and pK of the outer medium (pH, and pK,) during the hydrolysis of pulses of inorganic pyrophosphate (PPi) by suspensions of chromatophores from R. rubrum under various conditions, and we show that the pyrophosphatase system translocates protons, as suggested earlier [ 10, 111.

Alternative hypotheses of proton ejection in cytochrome oxidase vesicles. Transmembrane proton pumping or redox-linked deprotonation of phospholipid-cytochrome c complex(es).

The activating or inhibiting actions of a variety of anion species and of oligomycin, aurovertin and Dio-9 on the ATPase of a sonic particle preparation of rat liver mitochondria have been characterized by measurements of the relevantVmax,Ki andKm values.The normalVmax was increased by a factor near 7 by the anions: dichromate, chromate, pyrophosphate, orthophosphate, orthoarsenate and sulphate. The fully activating concentration varied from about 2 mM for dichromate to 150 mM for sulphate. The increase inVmax was accompanied by a time-dependent decrease in (Ki)ADP, but there was no change in (Km)ATP. The increase inVmax by the activating anions was abolished by aurovertin; but in presence of oligomycin, the lowVmax was increased by the activating anions by the same factor as theVmax in absence of oligomycin.Certain anions, notably azide, decreasedVmax, but did not affect (Ki)ADP or (Km)ATP. The decrease inVmax by azide and oligomycin were approximately additive. Even at high concentration, Dio-9 was without detectable effect on the ATPase, but it had a gramicidinlike effect on the intact mitochondria.The specificity of the ATPase for ATP relative to GTP was found to be attributable to the high value of (Vmax)ATP compared with (Vmax)GTP. The values of (Km)ATP and (Km)GTP were virtually the same.Some rationalization of these and other supporting observations is attempted in terms of present knowledge of the constitution of the ATPase complex.

Aurovertin-sensitive phosphate activation of mitochondrial adenosine triphosphatase

Some years ago [l] we discovered that the +H/O value of 6, measured by the respiratory-pulse technique [2] in suspensions of rat liver mitochondria in a 150 mM KC1 medium without added substrate, could be increased to 8 by preincubating the mitochondria with NEM. The action of the NEM appeared to be explained by our observation that ‘it largely inactivated succinate dehydrogenase and the NAD-linked enzymes but did not inactivate the NADP-linked isocitrate dehydrogenase (or the NADH oxidase and NAD(P) transhydrogenase). The conditions, after NEM treatment, thus favoured the involvement of the complete redox chain from NADPH to oxygen in the respiratory pulses’ ([l] and see [3] ). It is noteworthy that the maximum quantity of oxygen normally injected as air-saturated saline in the respiratory-pulse experiments (1 pg atom 0 per g protein) is equivalent to only about 12% of the total intramitochondrialNAD t,NADP t isocitrate t citrate (2.6 E.tmol NAD, 3.5. bmol NADP and about 3 pmol