Ca(2+)-modulated phosphorylation of a low-molecular-mass polypeptide in rat liver mitochondria: evidence that it is identical with subunit c of F(0)F(1)-ATPase

A 3.5-kDa polypeptide associated with the inner membrane of rat liver was found to be phosphorylated by [gamma-(32)P]ATP, presumably via a cAMP-dependent kinase. The phosphorylation was modulated by [Ca(2+)] in the physiological range, with a minimum at 1 microM and rising fourfold toward lower (10 nM) and higher (10 microM) concentrations. Further characterization of the 3.5-kDa component showed that the polypeptide has the same electrophoretic mobility as subunit c of F(0)F(1)-ATPase and that it selectively binds to antibodies against subunit c.     

Interference of calmidazolium with measurement of mitochondrial membrane potential using the tetraphenylphosphonium electrode or the fluorescent probe rhodamine 123

Calmidazolium (CMZ) is a positively charged, hydrophobic compound used as a calmodulin antagonist. It may cause unspecific effects in mitochondria, e.g., a decrease in membrane potential (deltapsi), swelling, and uncoupling. Several groups have advised against use of CMZ in studying signal transduction in mitochondria. We report here that it interferes with measurement of deltapsi in rat liver mitochondria (RLM) when using the tetraphenyl phosphonium (TPP+) electrode. We also found that CMZ reduces the signal, indicating an apparent drop in deltapsi. CMZ itself gave a signal with the TPP+ electrode in the absence of RLM. At high concentrations, > 10 microM, it also reduced the fluorescence quenching of the probe rhodamine 123. This may be due to an interference with mitochondrial uptake and binding of this positively charged probe or to an uncoupling effect. It is concluded that CMZ and similar positively charged calmodulin antagonists such as trifluoperazine may be used in mitochondria if these interferences are controlled and calibration is carried out under the experimental conditions used.     

Isolation of calcium-transporting lipid from the mitochondrial glycolipoprotein

Summary From the mitochondrial Ca²⁺-transporting glycolipoprotein (GLP) the lipid was isolated which induced Ca²⁺-translocation through bilayer lipid membranes. Electroconductivity of modified phospholipid membranes in the presence of CaCl₂ is increased 150-200 times. At 10-fold CaCl₂ gradient a generation of membrane potential is observed close to its theoretical value. It is shown that the lipid forms separate conductivity channels of 10 and 20 pS in the bilayer. The mode of action of GLP in the membrane is proposed It is assumed that the carbohydrate part of GLP is a selective receptor-accumulator for Ca²⁻, whereas the function of the lipid component consists in forming channels in the bilayer.     

Isolation of calcium-transporting lipid from the mitochondrial glycolipoprotein

From the mitochondrial Ca2+-transporting glycolipoprotein (GLP) the lipid was isolated which induced Ca2+-translocation through bilayer lipid membranes. Electroconductivity of modified phospholipid membranes in the presence of CaCl2 is increased 150-200 times. At 10-fold CaCl2 gradient a generation of membrane potential is observed close to its theoretical value. It is shown that the lipid forms separate conductivity channels of 10 and 20 pS in the bilayer. The mode of action of GLP in the membrane is proposed. It is assumed that the carbohydrate part of GLP is a selective receptor-accumulator for Ca2+, whereas the function of the lipid component consists in forming channels in the bilayer.