The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential.

The Biochemical journal ·Vol. 174 ·No. 1 ·1978-07-15 ·Pages 257-66

Kell DB, John P, Ferguson SJ

Abstract

1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S(14)CN(-), and the transmembrane pH gradient component from the uptake of [(14)C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN'N'-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (DeltaG(p)=DeltaG(0)'+RTln[ATP]/ [ADP][P(i)]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO(3) the protonmotive force was not detectable (<60mV) but DeltaG(p) was not altered. This result may indicate either that there is no relationship between the protonmotive force and DeltaG(p), or that for an unidentified reason the equilibration of SCN(-) or methylamine with the membrane potential and the pH gradient is prevented by NO(3) (-) in this system.

MeSH Terms

Adenosine Triphosphate/biosynthesis Ascorbic Acid/metabolism Cell Membrane/metabolism,ultrastructure Electrochemistry Membrane Potentials NAD/metabolism Paracoccus denitrificans/metabolism Phosphorylation Protons Succinates/metabolism Thiocyanates/metabolism

Chemicals

Protons Succinates Thiocyanates NAD Adenosine Triphosphate Ascorbic Acid

Authors & Affiliations

3 authors, click to expand affiliations / ORCID

Kell D B

John P

Ferguson S J

References (43)

43 references, click to expand

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Article Info

Journal

The Biochemical journal

Abbr.

Biochem J

ISSN

0264-6021

DOI

10.1042/bj1740257

Published

1978-07-15

Pages

257-66

Language

English

Region

England

NLM ID

2984726R

PMCID

PMC1185905

Subset

External Links

PubMed source DOI: 10.1042/bj1740257 PMC Full Text View journal details

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The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential.

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