Open AccessA Kinetic Study of Mitochondrial Calcium Transport
Author(s) -
REED Ken C.,
BYGRAVE Fyfe L.
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb02187.x
Subject(s) - chemistry , ruthenium red , egta , calcium , nitrilotriacetic acid , biophysics , divalent , ion transporter , quenching (fluorescence) , analytical chemistry (journal) , inorganic chemistry , fluorescence , nuclear chemistry , biochemistry , chromatography , chelation , membrane , organic chemistry , physics , quantum mechanics , biology
This report describes a kinetic analysis of energy‐linked Ca 2+ transport in rat liver mitochondria, in which a ruthenium red/EGTA [ethanedioxy‐bis(ethylamine)‐tetraacetic acid] quenching technique has been used to measure rates of 45 Ca 2+ transport. Accurately known concentrations of free 45 Ca 2+ were generated with Ca 2+ /nitrilotriacetic acids buffers for the determination of substrate/velocity relationships. The results show that the initial velocity of transport is a sigmoidal function of Ca 2+ concentration (Hill coefficient = 1.7), the K m being 4 μM Ca 2+ at 0°C and pH 7.4. These values for the Hill coefficient and the K m remain constant in the presence of up to 2 mM phosphate, but with 10 mM acetate both parameters are increased slightly. Both permeant acids increase the maximum velocity to an extent dependent on their concentration. The Ca 2+ ‐binding site(s) of the carrier contains a group ionizing at pH approximately 7.5 at 0°C, which is functional in the dissociated state. The stimulatory effect of permeant acids is ascribed to their facilitating the release of Ca 2+ from the carrier to the internal phase, an interpretation which is strengthened by the lack of effect of the permeant anion SCN − on Ca 2+ transport. Studies on the time‐course of Ca 2+ uptake and of EGTA‐induced Ca 2+ efflux from pre‐loaded mitochondria demonstrate the reversibility of the carrier in respiring mitochondria and the extent to which this property is influenced by permeant acids. These data are accommodated in a carrier mechanism based on electrophoretic transport of Ca 2+ bound to pairs of interacting acidic sites.