Influence of buffer composition, membrane lipids and proteases on the kinetics of reconstituted cytochrome-c oxidase from bovine liver and heart

Abstract

Isolated cytochrome-c oxidases from bovine heart and liver were reconstituted in liposomes with asolectin and the kinetics of cytochrome c oxidation were measured under various uncoupled conditions. With 40 m M KC1, 10 mM Hepes, pH 7.4, the liver enzyme showed a higher Vmax in the polarographic but a lower V,,, in the photometric assay. With 125 mM phosphate buffer at pH 6.0 both enzymes revealed identical kinetics. Reconstitution with pure phosphatidylcholine leads to a low activity, which is specifically stimulated for the heart enzyme by inclusion of 10% cardiolipin. Proteoliposomes of both enzymes prepared with asolectin have a high activity, which is unaffected by cardiolipin. Exchanging the intraliposomal buffer, Hepes, for phosphate causes an opposite change of the Vma, and a similar change of the K, for both enzymes suggesting a conformational change of the extraliposomal binding domain for cytochrome c through the membrane. Proteases change the kinetics of both enzymes, but to a different degree. The data indicate a complex and tissue-specific influence of nucleus-coded subunits on the catalytic activity of cytochrome-c-oxidase.

Adaptive changes in the expression of nuclear and mitochondrial encoded subunits of cytochrome c oxidase and the catalytic activity during hypoxia

“…In organelle RNA synthesis with isolated mitochondrial particles from cells grown under control (141 Torrr O 2 ) and hypoxia (1 Torr O 2 ) for 10 h was carried out using [α‐ 32 P]UTP as described in Materials and methods. Rate of 32 P incorporation in the RNA fraction was determined as described before [21]. RNA isolated from in vitro incubated mitochondria (5 µg each) was hybridized to CytOX subunit I and II encoding DNA from the mouse mitochondrial genome by slot blot hybridization.…”

Tissue-specific regulation of bovine heart cytochrome-c oxidase activity by ADP via interaction with subunit VIa.

“…The homology between subunits VIc and VIa-H concerns only the N-terminal part, whereas the homology between subunits VIa-H and VIa-L (liver-type) is mainly found in the C-terminal region (Fig. 6) differences in the kinetics of reconstituted COX from bovine liver and heart have been studied in detail previously (45).…”

Mitochondria-targeted heme oxygenase-1 induces oxidative stress and mitochondrial dysfunction in macrophages, kidney fibroblasts and in chronic alcohol hepatotoxicity

“…Sodium ascorbate (10–20 mg) was added to one of the cuvettes and after 10 min of incubation, the reduced minus oxidized difference spectra from 400 to 700 nm were recorded at room temperature (25 °C). The heme aa3 content was calculated from the difference spectra (ascorbate reduced minus air oxidized) using an absorption coefficient of 164 mM −1  cm −1 at 445 nm [38].…”