Characterization of cytochrome‐c oxidase mutants in human fibroblasts

Abstract

Skin fibroblasts were selected as having cytochrome-c oxidase deficiency by activity mesurements in whole cells. Each cell line was cultured in sufficient amount to isolate mitochondria for biochemical characterization. Cytochrome-c oxidase was then examined by activity measurements, by heme determination and by polypeptide analysis using antibodies specific to the enzyme subunits. The cytochrome-c ox&se activity in the different cell lines ranged from 9% to 54% of that of normal fibroblasts. Heme determinations and polypeptide analysis established that the lowered cytochrome-c oxidase activity was due to reduced amounts of the complex in the mitochondrial inner membrane. In all cases, there was defective assembly of the enzyme, with the amounts of mitochondrially coded and nuclear coded subunits being reduced proportionally. These studies show that fibroblasts can be used for prenatal diagnosis of mitochondrial diseases and are a useful system in which to study mitochondrial biogenesis.

The Use of Skin Fibroblast Cultures in the Detection of Respiratory Chain Defects in Patients with Lacticacidemia

“…However, the cytosolic coup le remains much more oxidized because of electrogenic expu lsion of aspartate from the mitochondrial compartme nt. oxidase in the deficient fibroblast cultures (9,20). The majority of those with severe cytochrome oxidase deficiency did not assemble the complex.…”

“…When the pyruvate deh ydrogenase complex is deficient, higher rates of lactate and pyruvate production are seen, with an L/P ratio that is norm al or low (19). When the mitochondrial respiratory chain is defective, the lactate output is increased, whereas the pyruvate output is decreased, giving an elevated L/P ratio (6,7,9). Th ose patients with elevated L/P ratios can be divided int o two basic groups, those with defects in the cytochrom e oxidase complex (complex IV of the respiratory chain) and those with defects in NADHcoenzymeQ oxidoreductase (complex I of the respiratory chain).…”

Mitochondrial complex I deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase.

“…Northern blotting was carried out using total RNA prepared from fibroblasts as described previously (15) and electrophoresed in 1% (wt/vol) agarose containing 1.8% (wt/vol) formaldehyde. The RNA was transferred to a Hybond-N ϩ support membrane (Amersham Corp., Arlington Heights, IL).…”

“…The RNA was transferred to a Hybond-N ϩ support membrane (Amersham Corp., Arlington Heights, IL). This was then incubated with a 32 P-labeled cDNA probe obtained by random priming a MnSOD cDNA template followed by washing in standard buffers (15). Total RNA loading was monitored by simultaneously probing on the same blot for mitochondrial EFTU (mitochondrial translation factor, 1.7 kb) (Merante, F., M. Ling, H.-S. Chen, and B.H.…”

Mitochondrial Dysfunction in Congenital Nephrotic Syndrome

“…The rotenone-sensitive NADH-cytochrome c reductase activity (complexes I and III) was measured by the method of Moreadith et al (1984). COX activity (complex IV) was measured as described by Glerum et al (1988), and succinate cytochrome reductase activity (complexes II and III) by the method of Fujii et al (1990). The protein concentrations were determined by the method of Lowry et al (1951).…”