The calcium conductance of the inner membrane of rat liver mitochondria and the determination of the calcium electrochemical gradient

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.

Section: Resultssupporting

confidence: 76%

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

Affolter¹,

Carafoli²

1981

“…The sucroseimpermeable space of the mitochondrial matrix was determined as previously described [12]. A value of 0.74 pl/mg was obtained.…”

Section: Materials a N D Methodsmentioning

confidence: 99%

“…Rat liver mitochondria were prepared as previously described [12]. Mitochondria1 protein was determined by the biuret method [13].…”

Section: Materials a N D Methodsmentioning

confidence: 99%

The Role of Phosphate in the Regulation of the Independent Calcium‐Efflux Pathway of Liver Mitochondria

Zoccarato

Nicholls

1982

120

The rate of spontaneous efflux of Ca from liver mitochondria incubated in the absence of ATP and Mg increases with time and is associated with a synchronous collapse of membrane potential and with Pi efflux. In the presence of Mg and ATP the ruthenium-red-induced Ca efflux does not change with time. The activity of the Ca efflux pathway in Pi-depleted mitochondria is 15-fold greater than in mitochondria equilibrated with 3.3 m M Pi. 50 % inhibition is caused by 0.3 mM Pi. The membrane potential is not affected by changes in Pi concentration, although the steadystate extra-mitochondria1 free Ca concentration reflects the alterations in efflux rate. In the presence of Pi, the ruthenium-red-induced efflux rate is independent of the total matrix Ca content; however in P,-depleted mitochondria, with acetate substituting as permeant anion, the efflux rate increases with total matrix Ca content. The lowered efflux rate in the presence of Pi is not due to a limitation in the rate of dissociation of the matrix Ca-phosphate complex. The efflux pathway is activated by a lowered membrane potential, but the relative effect of Pi is retained. Under the present conditions Na slightly inhibits the efflux rate. The lack of an effect of total matrix Ca content on the efflux rate in the presence of Pi is used as the basis of a highly accurate determination of the activity of the Ca uniporter as a function of external free Ca concentration.The role of Pi in liver mitochondrial Ca transport has been the subject of considerable debate [for reviews see 1-31. It is generally agreed that Pi crosses the inner membrane electroneutrally, by nominal proton symport [4], and that it therefore allows Ca to be accumulated in the matrix without alkalinization of the matrix [I -31. It is also generally [I -31 accepted that Ca and Pi form a complex in the matrix and that the extensive acidification of the medium which can be observed under these conditions [5] is a consequence of the expulsion by the respiratory chain of the protons generated in the matrix during the formation of this complex [6].In addition to these effects, however, Pi has variously been postulated to activate the Ca uniporter [7,8], to be involved in a Ca-Pi symport for the uptake [8] or release [9] of Ca and to activate the independent efflux pathway [lo], while we have reported the converse, namely that Pi inhibits the efflux pathway [Ill. Superimposed upon this complexity is the long established ability of Pi to potentiate the spontaneous efflux of Ca from mitochondria incubated in media lacking Mg and The purpose of the present paper is to resolve some of these complexities, firstly by determining the precise temporal sequence of Ca efflux, Pi release and All/ collapse during spontaneous Ca efflux. Secondly we investigate the interaction of Pi concentration, membrane potential and monovalent cations on the activity of the efflux pathway. Finally we confirm and attempt to explain why the Ca efflux rate appears not to depend upon the matrix Ca content in P,-containing mitocho...

“…Rat liver mitochondria were isolated as previously described (Heaton and Nicholls, 1976). Final pellet was resuspended in a buffer containing 250 mM sucrose, 5 mg/ml albumin, 10 mM Tes, pH 7.4.…”

Section: Isolation Of Mitochondria and Determination Of Permeability mentioning

confidence: 99%

Permeabilization of the Mitochondrial Inner Membrane by Short Cecropin‐A–Melittin Hybrid Peptides

Díaz‐Achirica

Prieto

Ubach

et al. 1994

A number of cecropin‐A–melittin hybrid peptides have previously been shown to be potent antibacterial agents [Andreu, D., Ubach, J., Boman, A., Wahlin, B., Wade, D., Merrifield, R. B. & Boman, H. G. (1992) FEBS Lett. 296, 190–1941. In the present report we analyze their action on biological systems using rat liver mitochondria as a test system. We demonstrate that the longest peptide, cecropin‐A‐(1–8)‐melittin(l–18) permeabilizes the mitochondrial inner membrane allowing the movement of both charged and non‐charged solutes. Concentrations used have already been shown to be bactericidal. This effect is also demonstrated under respiring conditions where succinate oxidation is uncoupled. Shorter analogs also permeabilize mitochondria although at tenfold higher concentrations. Heparin potentiates the peptide effects at low concentrations, while at high concentration it becomes inhibitory. We propose that the cecropinmelittin analogs disrupt the mitochondrial membrane in a detergent‐like mode rather than by creating selective channels as had been previously suggested.