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PMID: 212021 Published · ppublish English Journal Article

The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential.

The Biochemical journal ·Vol. 174 ·No. 1 ·1978-07-15 ·Pages 237-56

Sorgato MC, Ferguson SJ, Kell DB, John P

Abstract

1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0' + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N'-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.

MeSH Terms
Adenosine Triphosphate/biosynthesis Animals Ascorbic Acid/metabolism Cattle Electrochemistry In Vitro Techniques Membrane Potentials Mitochondria/metabolism Mitochondria, Heart/metabolism NAD/metabolism Osmolar Concentration Phosphorylation Protons Submitochondrial Particles/metabolism Succinates/metabolism
Chemicals
Protons Succinates NAD Adenosine Triphosphate Ascorbic Acid
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Sorgato M C
Ferguson S J
Kell D B
John P
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj1740237
Published
1978-07-15
Pages
237-56
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1185904
Subset
IM
External Links
PubMed source DOI: 10.1042/bj1740237 PMC Full Text View journal details
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