Evidence of a calcium-ion-transport system in mitochondria isolated from flight muscle of the developing sheep blowfly Lucilia cuprina.
The EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-Ruthenium Red-quench technique (Reed & Bygrave, 1974a) was used to measure initial rates of Ca-2+ transport in mitochondria from flight muscle of the blowfly Lucilia cuprina. Evidence is provided for the existence in these mitochondria of a Ca-2+-transport system that has many features in common with that known to exist in rat liver mitochondria. These include requirement for energy, saturation at high concentrations of Ca-2+, a sigmoidal relation between initial rates of Ca-2+ transport and Ca-2+ concentration, a high affinity for free Ca-2+ (Km approx. 5 muM) and high affinity for the Ca-2+-transport inhibitoy, Ruthenium Red (approx. 0.03 nmol of carrier-specific binding-sites/mg of protein; Ki approx. 1.6 x 10- minus 8 M). Controlled respiration can be stimulated by Ca-2+ after a short lag-period provided the incubation medium contains KCl and not sucrose. The ability of Lucilia mitochondria to transport Ca-2+ critically depends on the stage of mitochondrial development; Ca-2+ transport is minimal in mitochondria from pharate adults, is maximal between 0 and 2h post-emergence and thereafter rapidly declines to reach less than 20% of the maximum value by about 2-3 days post-emergence. Respiration in mitochondria from newly emerged flies does not respond to added Ca-2+; that from 3-5-day-old flies is stimulated approx. 50%. Whereas very low concentrations of Ca-2+ inhibit ADP-stimulated respiration and oxidative phosphorylation in mitochondria from newly emerged flies (Ki approx. 60 ng-ions of Ca-2+/mg of protein); much higher concentrations (approx. 200 ng-ion/mg of protein) are needed to inhibit these processes in those from older flies. The potential of this system for studying the function and development of metabolite transport systems in mitochondria is discussed.
D000244:Adenosine Diphosphate; D000367:Age Factors; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D004175:Diptera; D004533:Egtazic Acid / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008930:Mitochondria, Liver / Q000378:metabolism; D008931:Mitochondria, Muscle / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D010085:Oxidative Phosphorylation / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects; D011189:Potassium Chloride; D051381:Rats; D012430:Ruthenium Red; D014475:Uncoupling Agents
D014475:Uncoupling Agents; D012430:Ruthenium Red; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D002118:Calcium