Topology of subunits of the mammalian cytochrome c oxidase: Relationship to the assembly of the enzyme complex
- Univ. of Oregon, Eugene (USA)
The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane({sup 35}S)sulfonate and sodium methyl 4-({sup 3}H)formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C-domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage produce from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.
- OSTI ID:
- 5596845
- Journal Information:
- Biochemistry; (United States), Vol. 30:15; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CYTOCHROME OXIDASE
MOLECULAR STRUCTURE
CATTLE
ELECTROPHORESIS
HEART
MONOCLONAL ANTIBODIES
PEPTIDE HYDROLASES
SULFUR 35
TOPOLOGY
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARDIOVASCULAR SYSTEM
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ENZYMES
EVEN-ODD NUCLEI
HAEM DEHYDROGENASES
HYDROLASES
ISOTOPES
LIGHT NUCLEI
MAMMALS
MATHEMATICS
NUCLEI
ORGANS
OXIDOREDUCTASES
RADIOISOTOPES
RUMINANTS
SULFUR ISOTOPES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques