Structural organisation of the rat genes encoding liver- and heart-type of cytochrome c oxidase subunit VIa and a pseudogene related to the COXVIa-L cDNA.

To study the tissue-specific expression of the heart(H)- and liver(L)-type of rat cytochrome-c oxidase subunit VIa (rCOXVIa), we have screened and sequenced the genes for the two isoforms. Both genes contain three exons and two introns, spanning 880 bp (rCOXVIa-H) and 3089 bp (rCOXVIa-L), respectively. In both genes, exon I codes for the whole leader sequence comprising 12 (rCOXVIa-H) or 26 (rCOXVIa-L) amino acids and for 12 (rCOXVIa-H) or 10 (rCOXVIa-L) amino acids of the corresponding mature protein, while the remaining amino acids for the mature proteins are encoded by exons II and III. The 5' region of the genes lack both TATA and CAAT boxes, but show a high G+C content in the early 5'-upstream region. We have identified in upstream regions and in the introns of both genes several putative binding sites associated with respiratory function, muscle gene activation and housekeeping function. In rCOXVIa-H, we identified a CCAC/Myo-D motif, known to be required for muscle-specific expression of the human myoglobin-encoding gene, which is not present in rCOXVIa-L. In addition, we have analyzed a pseudogene, showing 84% homology to the COXVIa-L cDNA sequence.

Respiratory chain proteins.

Mammalian mitochondrial DNA codes for 13 proteins, which are all components of energy transducing enzyme complexes of the respiratory chain, i.e. the complexes which translocate protons across the inner mitochondrial membrane. The number of subunits of these enzyme complexes increase with increasing evolutionary stage of the organism. The additional nuclear coded subunits of the enzyme complexes from higher organisms are involved in the regulation of respiration, as demonstrated by the influence of intraliposomal ATP and ADP on the reconstituted cytochrome c oxidase (COX) from bovine heart. This regulation is not found with the reconstituted enzyme from P. denitrificans, which lacks the nuclear coded subunits. Some of the nuclear coded subunits occur in tissue-specific isoforms, as reported for COX and NADH dehydrogenase. Tissue-specific regulation of COX activity is also demonstrated by the differential effects of intraliposomal ADP on the kinetics of reconstituted COX from bovine liver and heart, which differ in subunits VIa, VIIa and VIII. At least 3 different COX isozymes occur in bovine liver, heart or skeletal muscle and smooth muscle. An evolutionary relationship between COX subunits VIa and VIc and between VIIa and VIIb is suggested based on the crossreactivity of monoclonal antibodies, amino acid sequence homology and hybridization at low stringency of PCR-amplified cDNAs for subunits VIa-1, VIa-h and VIc from the rat.

Cross reactivity of monoclonal antibodies and cDNA hybridization suggest evolutionary relationships between cytochrome c oxidase subunits VIa and VIc and between VIIa and VIIb.

Monoclonal antibodies to subunits of bovine heart cytochrome c oxidase were prepared by immunizing mice with the isolated enzyme. The majority of antibody-producing cell lines were found to react with two different subunits of similar molecular mass, as shown by Western blotting and ELISA titrations with the HPLC-purified subunits. The affinities of the monoclonal antibodies to the subunits were determined by ELISA titrations with increasing concentrations of NH4SCN. Two monoclonal antibodies with a low affinity to subunit VIa had a high affinity to subunit VIc, whereas two other antibodies showed the same affinity to subunits VIIa and VIIb. The same affinity of monoclonal antibodies suggested an evolutionary relationship of subunits VIIa and VIIb, which was further supported by reactivity of these antibodies to subunits VIIa and VIIb of cytochrome c oxidase from different species and tissues. Also the evolutionary relationship between subunit VIa and VIc was shown by hybridization at low stringency of cDNAs for rat cytochrome c oxidase subunits VIc and VIa-h (heart-type), after amplification by the polymerase chain reaction, with a probe of VIa-l (liver-type).