Kinetics of calcium(2+) ion carrier in rat liver mitochondria

Abstract

The rate of aerobic Ca2+ transport is limited by the rate of the H+ pump rather than by the Ca2+ carrier. The kinetics of the Ca2+ carrier has therefore been studied by using the K+ diffusion potential as the driving force. The apparent Vmax of the Ca2+ carrier is, at 20 degrees C, about 900 nmol (mg of protein)-1 min-1, more than twice the rate of the H+ pump. The apparent Vmax is depressed by Mg2+ and Li+. This supports the view that the electrolytes act as noncompetitive inhibitors of the Ca2+ carrier. The degree of sigmoidicity of the kinetics of Ca2+ transport increases with the lowering of the temperature and proportionally with the concentration of impermeant electrolytes such as Mg2+ and Li+ but not choline. The effects of temperature and of electrolyte do not support the view that the sigmoidicity is due to modifications of the surface potential. Rather, they suggest that Ca2+ transport occurs through a multisubunit carrier, where cooperative phenomena are the result of ligand-induced conformational changes due to the interaction of several allosteric effectors with the carrier subunits. In contrast with La3+ which acts as a competitive inhibitor, Ruthenium Red affects the kinetics by inducing phenomena both of positive and of negative cooperativity. The Ruthenium Red induced kinetics has been reproduced through curve-fitting procedures by applying the Koshland sequential interaction hypothesis to a four-subunit Ca2+ carrier model.

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].…”

A comparison between the responses of neutral red and acridine orange: Acridine orange should be preferential and alternative to neutral red as a dye for the monitoring of contaminants by means of biological sensors

“…The mitochondria from rat liver were prepared following the usual procedure [12]. The mitochondrial protein content was determined using the method of Lowry and coworkers [13].…”

TCMS inhibits ATP synthesis in mitochondria: A systematic analysis of the inhibitory mechanism

“…Mitochondria were prepared from livers of fasted albino Wistar rats, weighing about 300 g (Bragadin et al, 1980). The mitochondrial protein concentration was determined using the Lowry procedure (Lowry et al, 1951).…”