Cytochrome b560 (QPs1) of mitochondrial succinate-ubiquinone reductase. Immunochemistry, cloning, and nucleotide sequencing.
Mitochondrial succinate-ubiquinone reductase is composed of two parts, a water-soluble succinate dehydrogenase and a two-polypeptide membrane-anchoring protein fraction (QPs). The larger polypeptide of QPs is believed to be associated with cytochrome b560 (QPs1). The structure of QPs1 was studied by immunochemistry and molecular cloning and sequencing. Antibodies against QPs1 were raised in rabbits, purified, and characterized by enzyme-linked immunosorbent assay and Western blotting. The purified antibodies inhibited 75% of the reconstitutive activity of QPs and reacted with both submitochondrial particles (SMP) and mitoplasts. The binding of these antibodies to SMP was greatly increased when succinate dehydrogenase was removed from SMP by alkaline treatment, indicating that QPs1 is a transmembranous protein and that some of its specific epitopes are covered by succinate dehydrogenase. Anti-QPs1 antibodies were used to screen one cDNA clone encoding QPs1 from a bovine heart cDNA lambda gt11 expression library. The cDNA insert is 946 base pairs with an open reading frame of 396 base pairs that encodes for 132 amino acid residues. The molecular weight of QPs1, calculated from the deduced amino acid sequence, is 14,320. Although the apparent molecular weight of QPs1, estimated by high resolution SDS-polyacrylamide gel electrophoresis, is approximately 11,000, the existence of a presequence was ruled out by mass spectrometric analysis of protein fragments. QPs1 is a very hydrophobic protein. Three probable membrane-spanning segments were revealed by a hydropathy plot of the sequence. QPs1 has a higher sequence similarity to the sdhC peptide of Escherichia coli than to the sdhC peptide (cytochrome b558) of Bacillus subtilis. Like the bacterial proteins, QPs1 has 2 conserved histidines at positions 34 and 90. The conserved nature and similar location of these 2 histidines, on the matrix-side surface of the membrane, suggest that they are involved in heme ligation of cytochrome b560.
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Department of Biochemistry, Oklahoma State University, Stillwater 74078-0450.
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Usui
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D003573:Cytochrome b Group / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D042963:Electron Transport Complex II; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D007140:Immunoglobulin Fab Fragments; D007150:Immunohistochemistry; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D019255:NADPH Oxidases; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013367:Submitochondrial Particles / Q000201:enzymology*; D013385:Succinate Dehydrogenase / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D003573:Cytochrome b Group; D007140:Immunoglobulin Fab Fragments; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C025355:cytochrome b560; D004247:DNA; C036888:cytochrome b558; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase; D019255:NADPH Oxidases