Hyperbolic Kinetics of the Electrophoretic Carrier of Ca²⁺ Uptake in Liver Mitochondria

Abstract

The use of a fast-responding Ca2 +-selective electrode has allowed the simultaneous mcasurenient of the true Ca' +-activity in the medium and of the Ca' +-transport rate, eliminating the uncertainties connected with the use of indirect methods of estimating Ca2+ transport rates (e.g. 0,-stimulation, K + -efflux). The results on the kinetics of Ca2+ uptake by rat liver mitochondria obtained in this study are qualitatively similar to those obtained by Bragadin et al. [Bragadin, M., Pozzan, T.&Azzone, G. F. (1979) Biochemistry, 18, 5973 -59781 by monitoring the K+-efflux from mitochondria. In the valinomycin-driven Ca2 + uptake, the Ca2+ translocase is the limiting factor, whereas in the system driven by succinate extraneous limiting factors play a role. Thus, liver mitochondria, when studied under appropriate non-limiting conditions, exhibit a hyperbolic dependence of the Ca' + uptake rate on Ca' ' -activity analogous to that of mitochondria from other tissues. The sigmoidal dependence of the uptake rate on Ca' +-activity in liver mitochondria, observed under conditions which produce hyperbolic kinetics in mitochondria from other tissues, can thus be attributed to factors which are cxtraneous to the Ca2+ transport systcm per .ye.

Slow Ca2+‐induced inactive/active transition of the energy‐dependent Ca2+ transporting system of rat liver mitochondria: clue for Ca2+ influx cooperativity

“…Our findings are in line with previous reports on Ca2+-induced Ca 2+ release from de-energized mitochondria [10,11] and those on external Ca2+-induced modification of the transport kinetic parameters [9]. In addition to providing new information on the slow interconversion between de-activated and active forms of Ca2+-uniporter our data seem to explain the contradictory reports on the sigmoidal [4,5] or simple hyperbolic [7,8] dependence of the reaction rate on Ca 2+ concentration. In addition to obvious differences in experimental conditions employed in different laboratories (mitochondria from different tissues, ionic composition of the reaction mixture, the Ca 2+ measurement methods) the initial state of the uniporter appears to be a crucial factor.…”

“…In addition to obvious differences in experimental conditions employed in different laboratories (mitochondria from different tissues, ionic composition of the reaction mixture, the Ca 2+ measurement methods) the initial state of the uniporter appears to be a crucial factor. The latter is expected to be dependent, in particular, on protein concentration: when a relatively high content of the mitochondria is used in the experiments [7,8] the release of endogenous Ca 2+ before membrane energization would result in activation of the uniporter and hyperbolic kinetics would be observed.…”

“…The model for the mitochondrial bivalent cation transporting system with an 'activating' site was proposed to explain the kinetic cooperativity of Ca 2+ and Mn 2+ uptake [5]. The methodological difficulties of in vitro studies of Ca 2+ transport [6] have prompted several authors to reinvestigate kinetic parameters of Ca 2+ uptake by liver mitochondria by monitoring the valinomycin2.induced K + efflux-driven Ca 2+ uptake, in order to exclude AgH+ generation as the limiting factor [7,8]. It was concluded that sigmoidal dependence of the uptake rate on Ca 2+ concentration 'can be attributed to factors which are extraneous to the Ca 2+ transport system per se' [8].…”

“…The methodological difficulties of in vitro studies of Ca 2+ transport [6] have prompted several authors to reinvestigate kinetic parameters of Ca 2+ uptake by liver mitochondria by monitoring the valinomycin2.induced K + efflux-driven Ca 2+ uptake, in order to exclude AgH+ generation as the limiting factor [7,8]. It was concluded that sigmoidal dependence of the uptake rate on Ca 2+ concentration 'can be attributed to factors which are extraneous to the Ca 2+ transport system per se' [8]. However, further evidence for an activating site have come independently from three different laboratories.…”

Pathways of Ca2+ efflux from liver and heart mitochondria

“…The number of Na' exchan ed er Caz+ is not known, 1976, 1977Crompton & Heid, 1978;Affolter & Carafoli, 1980;Hayat & Crompton, 1982). In liver electroneutral…”