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Biomedical subjects

E Carafoli

Publications and source records attributed to E Carafoli.

At least 271 records · Page 15Linked to original sources
PubMed1979

The charge stoichiometry of cytochrome c oxidase in the reconstituted system.

Purified cytochrome c oxidase was reconstituted into phospholipid vesicles having high internal pH buffering capacity. In the presence of valinomycin, 2 K+ ions were taken up by the vesicles per electron transferred from cytochrome c to oxygen. The charge stoichiometry of 2 was obtained from simultaneous measurement of changes of K+, H+, and oxygen in the medium after addition of the reductant ascorbate/TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine). The changes in oxygen concentration were measured with a fast responding oxygen electrode (90% response time, 0.4 s). The existence of a proton pump in cytochrome c oxidase could thus be confirmed, and its charge stoichiometry measured, in a reconstituted system uncomplicated by other respiratory chain components.

Animals↗
PubMed1979

Purification of the (Ca2+-Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column.

The (Ca2+-Mg2+)-ATPase from human erythrocyte membranes has been solubilized in Triton X-100 and purified on a calmodulin affinity chromatography column in the presence of phosphatidylserine, to limit the inactivation of the enzyme. The enzyme was purified at least 150 times when compared with the original ghosts and showed a specific activity of 3.8 mumol.mg-1.min-1. In sodium dodecyl sulfate-polyacrylamide gels, a single major band was visible at a position corresponding to a molecular weight of about 125,000; a minor band (11% of the total protein) was present at a position corresponding to Mr = 205,000. Upon incubation of the purified preparation with [32P]ATP, both bands were phosphorylated in proportion to their mass, suggesting that both were active forms of purified ATPase.

Calcium-Transporting ATPases↗
PubMed1979

Hydroperoxides can modulate the redox state of pyridine nucleotides and the calcium balance in rat liver mitochondria.

When rats are fed a selenium-deficient diet, the glutathione peroxidase activity in liver mitochondria decreases within 5 weeks to 0-6% of that of control animals fed on a diet supplemented with 0.5 ppm of selenium as sodium selenite. Analysis of the temperature dependence of energy-linked Ca(2+) uptake by means of Arrhenius plots reveals two breaks (at around 11 degrees C and 24 degrees C) in mitochondria isolated from selenium-supplemented animals, whereas in selenium-deficient rats the break at 11 degrees C is absent. Ca(2+)-loaded mitochondria of selenium-supplemented rats-i.e., with active glutathione peroxidase in the matrix-lose Ca(2+) rapidly, with a concomitant oxidation of endogenous NAD(P)H, when exposed to t-butyl hydroperoxide or H(2)O(2). In contrast, in selenium deficiency, t-butyl hydroperoxide and H(2)O(2) induce neither a release of Ca(2+) nor an oxidation of NAD(P)H. The peroxide-induced oxidation of NAD(P)H is reversible in the presence of succinate when no Ca(2+) has been taken up. When Ca(2+) has previously been accumulated, however, the oxidation of NAD(P)H is irreversible. Enzymatic analysis of mitochondrial pyridine nucleotides reveals that the peroxide-induced oxidation of NAD(P)H in Ca(2+)-loaded mitochondria leads to a loss of NAD(+) and NADP(+). It is proposed that the redox state of mitochondrial pyridine nucleotides can be or is in part controlled by glutathione peroxidase and glutathione reductase and is a factor in the balance of Ca(2+) between mitochondria and medium.

Animals↗
PubMed1979

Calcium transport and translocation of adenine nucleotides in mitochondria from Morris hepatoma 3924A.

The interaction of Ca2+ with Morris hepatoma 3924A mitochondria and its effect on the adenine nucleotide translocation have been studied. The characteristics of the Ca2+ transport process in mitochondria from Morris hepatoma are not significantly different from those of normal liver mitochondria. The Km for Ca2+ is 2 to 3 microM, and the rate versus concentration curve exhibits hyperbolic kinetics. A lower activity of the adenine nucleotide translocation was found, probably due to the high endogenous Ca2+ content of Morris hepatoma mitochondria (123 +/- 15 nmol Ca2+ per mg protein). No further inhibition of the translocase activity was observed after isolated mitochondria had accumulated extra amounts of Ca2+. The total amount of adenine nucleotides in tumor mitochondria is one-half those present in control liver, and a significantly lower percentage of the pool is present as adenosine 5'-monophosphate.

Adenine Nucleotides↗
PubMed1978

The proton pump of cytochrome c oxidase and its stoichiometry.

The operation of cytochrome c oxidase with ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine as substrate in antimycin-A-inhibited rat liver mitochondria is coupled to proton ejection. Measurements of the initial rate of valinomycin-dependent K+ uptake have shown that nearly 4 K+ are taken up as 2 electrons are transferred from cytochrome c to oxygen. This proves directly that a charge separation of nearly 4 occurs across the inner mitochondrial membrane each time 2 electrons are transferred to oxygen. Measurements of the initial rate of proton movement after addition of the reductant show that about 1.6 protons are released by the mitochondria as 2 electrons are transferred from cytochrome c to oxygen. The data support the suggestion of a proton pump coupled to the operation of cytochrome c oxidase [Wikström, M. F. K. (1977) Nature (Lond.) 266, 271--273].

Animals↗