scholarly journals A heart mitochondrial Ca2+-dependent pore of possible relevance to re-perfusion-induced injury. Evidence that ADP facilitates pore interconversion between the closed and open states

1990 ◽  
Vol 266 (1) ◽  
pp. 33-39 ◽  
Author(s):  
M Crompton ◽  
A Costi
Keyword(s):  
Arsenazo Iii ◽  
Heart Mitochondria ◽  
Ionophore A23187 ◽  
Relative Permeabilities ◽  
Pulsed Flow ◽  
Pore Closure ◽  
Open States ◽  
Matrix Free ◽  
Pore Number

The permeability properties of a putative Ca2(+)-activated pore in heart mitochondria, of possible relevance to re-perfusion-induced injury, have been investigated by a pulsed-flow solute-entrapment technique. The relative permeabilities of [14C]mannitol, [14C]sucrose and arsenazo III are consistent with permeation via a pore of about 2.3 nm diameter. Ca2+ removal with EGTA induced pore closure, and the mitochondria became ‘resealed’. The permeability of the unresealed mitochondria during resealing was markedly stimulated by 200 microM-ADP, and the relative permeabilities to solutes of different size were stimulated equally, indicating an increase in open-pore number, rather than an increase in pore dimensions. This is paradoxical, since ADP also stimulated the rate of resealing. The rate of EGTA-induced resealing was also stimulated by the Ca2+ ionophore A23187, which indicates that the rate of removal of matrix free Ca2+ is limiting for pore closure. An explanation for the paradox is suggested in which ADP facilitates pore interconversion between the closed and open states in permeabilized mitochondria, and pore closure in Ca2(+)-free mitochondria occurs much faster than previously thought.

scholarly journals The effects of Mg2+ and adenine nucleotides on the sensitivity of the heart mitochondrial Na+-Ca2+ carrier to extramitochondrial Ca2+. A study using arsenazo III-loaded mitochondria

1987 ◽  
Vol 244 (3) ◽  
pp. 533-538 ◽  
Author(s):  
L H Hayat ◽  
M Crompton
Keyword(s):  
Adenine Nucleotides ◽  
Mitochondrial Protein ◽  
Arsenazo Iii ◽  
Heart Mitochondria ◽  
Small Decrease ◽  
Regulatory Site ◽  
Normal Heart ◽  
Partial Inhibition ◽  
Regulatory Sites

The technique of reversible Ca2+-induced permeabilization [Al Nasser & Crompton (1986) Biochem. J. 239, 19-29, 31-40] has been applied to the preparation of heart mitochondria loaded with the Ca2+ indicator arsenazo III (2 nmol of arsenazo III/mg of mitochondrial protein). The loaded mitochondria (‘mitosomes’) were used to study the control of the Na+-Ca2+ carrier by extramitochondrial Ca2+ mediated by putative regulatory sites. The Vmax. of the Na+-Ca2+ carrier and the degree of regulatory-site-mediated inhibition were similar to normal heart mitochondria. Ca2+ occupation of the sites in mitosomes yields partial inhibition, which is half-maximal with 0.8 microM external free Ca2+. The inhibition consists of a small decrease in Vmax. and a relatively large increase in apparent Km for internal Ca2+. Mg2+ also appears to interact with the sites, but this is largely abolished by ATP and ADP (but not AMP) under conditions in which the free [Mg2+] is maintained constant. The results indicate that the regulatory sites are effective in controlling the Na+-Ca2+ carrier at physiological concentrations of adenine nucleotides, Mg2+, intra- and extra-mitochondrial free Ca2+.

scholarly journals The entrapment of the Ca2+ indicator arsenazo III in the matrix space of rat liver mitochondria by permeabilization and resealing. Na+-dependent and -independent effluxes of Ca2+ in arsenazo III-loaded mitochondria

1986 ◽  
Vol 239 (1) ◽  
pp. 31-40 ◽  
Author(s):  
I Al-Nasser ◽  
M Crompton
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Arsenazo Iii ◽  
Rat Liver Mitochondria ◽  
Matrix Space ◽  
The Matrix ◽  
Matrix Compartment ◽  
Matrix Free ◽  
Regulatory Sites

The permeabilization-resealing technique [Al-Nasser & Crompton, Biochem. J. (1986) 239, 19-29] has been applied to the entrapment of arsenazo III in the matrix compartment of rat liver mitochondria. The addition of 10 mM-arsenazo III to mitochondria permeabilized with Ca2+ partially restores the inner-membrane potential (delta psi) and leads to the recovery of 3.9 nmol of arsenazo III/mg of protein in the matrix when the mitochondria are washed three times. The recovery of entrapped arsenazo III is increased 2-fold by 4 mM-Mg2+, which also promotes repolarization. ATP with or without Mg2+ decreased arsenazo III recovery. Under all conditions, less arsenazo III than [14C]sucrose is entrapped, in particular in the presence of ATP. The amount of arsenazo III entrapped is proportional to the concentration of arsenazo III used as resealant, and is equally distributed between heavy and light mitochondria. Arsenazo III-loaded permeabilized and resealed (PR) mitochondria develop delta psi values of 141 +/- 3 mV. PR mitochondria retain arsenazo III and [14C]sucrose for more than 2 h at 0 degrees C. At 25 degrees C, and in the presence of Ruthenium Red, PR mitochondria lose arsenazo III and [14C]sucrose at equal rates, but Ca2+ efflux is more rapid; this indicates that Ca2+ is released by an Na+-independent carrier in addition to permeabilization. The Na+/Ca2+ carrier of PR mitochondria is partially (60%) inhibited by extramitochondrial free Ca2+ stabilized with Ca2+ buffers; maximal inhibition is attained with 2 microM free Ca2+. A similar inhibition occurs in normal mitochondria with 3.5 nmol of matrix Ca2+/mg of protein, but the inhibition is decreased by increased matrix Ca2+. The data suggest the presence of Ca2+ regulatory sites on the Na+/Ca2+ carrier that change the affinity for matrix free Ca2+.

scholarly journals On the relationship between matrix free Mg2+ concentration and total Mg2+ in heart mitochondria

1997 ◽  
Vol 1320 (3) ◽  
pp. 310-320 ◽  
Author(s):  
Dennis W Jung ◽  
Edward Panzeter ◽  
Kemal Baysal ◽  
Gerald P Brierley
Keyword(s):  
Heart Mitochondria ◽  
The Relationship ◽  
Matrix Free

Effects of intracellular free Ca and rate of Ca influx on the Ca pump

1989 ◽  
Vol 256 (6) ◽  
pp. C1138-C1144 ◽  
Author(s):  
Y. C. Yang ◽  
D. R. Yingst
Keyword(s):  
Rate Constant ◽  
Arsenazo Iii ◽  
Ionophore A23187 ◽  
Peak Activity ◽  
Ca Pump ◽  
Human Red Blood Cells ◽  
Column Method ◽  
Ca Uptake ◽  
New Finding ◽  
Resealed Ghosts

The activity of the Ca pump of human red blood cells was studied in resealed ghosts as a function of intracellular free Ca (fCai). Resealed ghosts were made by the agarose column method to contain, in addition to other constituents, less than 0.1 microM fCai, 100 microM arsenazo III, and either 1 mM ATP plus an ATP regenerating system (active ghosts) or no added ATP and no regenerating system (passive ghosts). The rate of Ca influx into these ghosts was manipulated by suspending them in solutions containing various combinations of free Ca (1-30 microM) and the Ca ionophore A23187 (0.1-0.7 microM). Entering Ca increased the fCai and stimulated the pump in active ghosts. In passive ghosts, all the Ca movement could be described by a single rate constant. The activity of the Ca pump was calculated from the rate of net Ca uptake in the active ghosts, using the rate constant for passive Ca movement as determined in the passive ghosts. fCai and the rates of Ca transport in both active and passive ghosts were calculated from the absorbance of entrapped arsenazo III. In general, increasing fCai from 1 to 10 microM activated the pump. Higher fCai caused an inhibition compared with peak activity. The maximum rate of pumping was 80 microM/min. The major new finding is that the rate of active transport at a given fCai appeared to vary with the rate of fCai accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Determination of the matrix free Ca2+ concentration and kinetics of Ca2+ efflux in liver and heart mitochondria.

1982 ◽  
Vol 257 (15) ◽  
pp. 8696-8704 ◽  
Author(s):  
K E Coll ◽  
S K Joseph ◽  
B E Corkey ◽  
J R Williamson
Keyword(s):  
Heart Mitochondria ◽  
The Matrix ◽  
Matrix Free ◽  
Kinetics Of

scholarly journals The mechanism of palmitoyl-CoA inhibition of Ca2+ uptake in liver and heart mitochondria

1981 ◽  
Vol 194 (1) ◽  
pp. 71-77 ◽  
Author(s):  
M C Beatrice ◽  
D R Pfeiffer
Keyword(s):  
Energy Source ◽  
Electron Transport ◽  
Membrane Permeability ◽  
Uptake Rate ◽  
Competitive Inhibitor ◽  
Heart Mitochondria ◽  
Ionophore A23187 ◽  
Inner Membrane

The mechanism by which palmitoyl-CoA inhibits Ca2+ uptake in liver and heart mitochondria was examined. At a given concentration of palmitoyl-CoA, the extent of inhibition is inversely related to the concentration of the respiratory substrate succinate. Palmitoyl-CoA inhibition of uncoupler-stimulated respiration and respiration stimulated by ionophore-A23187-induced Ca2+ cycling is also relieved by high succinate concentrations. These effects of palmitoyl-CoA and succinate concentration are distinct from the increase in inner-membrane permeability, which can be produced by palmitoyl-CoA and Ca2+ [Beatrice, Palmer & Pfeiffer (1980) J. Biol. Chem. 255, 8663-8671]. The apparent K0.5 of the mitochondrial Ca2+ pump is not altered by palmitoyl-CoA. No or negligible effects of palmitoyl-CoA on the Ca2+-uptake rate are observed when ascorbate replaces succinate as an energy source. These findings, together with the known activity of palmitoyl-CoA as a competitive inhibitor of the dicarboxylate carrier [Morel, Lauquin, Lunardi, Duszynski & Vignais (1974) FEBS Lett. 39, 133-138], indicate that palmitoyl-CoA inhibits energy-linked Ca2+ transport by limiting the rate of electron transport through limitation of succinate entry into the mitochondria rather than by directly inhibiting the Ca2+ carrier.

scholarly journals Measurement of the matrix free Ca2+ concentration in heart mitochondria by entrapped fura-2 and quin2

1987 ◽  
Vol 248 (2) ◽  
pp. 609-613 ◽  
Author(s):  
G L Lukács ◽  
A Kapus
Keyword(s):  
Heart Mitochondria ◽  
Fura 2 ◽  
Isolated Mitochondria ◽  
The Matrix ◽  
Matrix Free

A method was developed to monitor continuously the matrix free Ca2+ concentration ([Ca2+]m) of heart mitochondria by use of the fluorescent Ca2+ indicators, fura-2 and quin2. The acetoxymethyl esters of fura-2 and quin2 were accumulated in and hydrolysed by isolated mitochondria. An increase of the mitochondrial Ca content from 0.3 nmol/mg of protein to 6 nmol/mg corresponded to a rise of [Ca2+]m from 30 to 1000 nM. The results indicate that physiological fluctuations of the mitochondrial Ca content elicit changes of [Ca2+]m in that range which regulates the matrix dehydrogenases.

Matrix free magnesium changes with metabolic state in isolated heart mitochondria

Biochemistry ◽  
1990 ◽  
Vol 29 (17) ◽  
pp. 4121-4128 ◽  
Author(s):  
Dennis W. Jung ◽  
Lynn Apel ◽  
Gerald P. Brierley
Keyword(s):  
Isolated Heart ◽  
Heart Mitochondria ◽  
Metabolic State ◽  
Matrix Free ◽  
Free Magnesium

scholarly journals The stoichiometry of the exchange catalysed by the mitochondrial calcium/sodium antiporter

1985 ◽  
Vol 229 (1) ◽  
pp. 161-166 ◽  
Author(s):  
M D Brand
Keyword(s):  
Steady State ◽  
Thermodynamic Analysis ◽  
Rat Heart ◽  
Ruthenium Red ◽  
Heart Mitochondria ◽  
Mitochondrial Calcium ◽  
Ionophore A23187 ◽  
Obvious Change ◽  
Rat Heart Mitochondria

Rat heart mitochondria respiring on succinate in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) released Ca2+ on the calcium/sodium antiporter until a steady state was reached. Addition of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) did not perturb this steady state. Thermodynamic analysis showed that if a Ca2+/nNa+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would cause an obvious change in extramitochondrial free [Ca2+]. Therefore the endogenous calcium/sodium antiporter of mitochondria catalyses electroneutral Ca2+/2Na+ exchange.

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