Identification of a cross-linked peptide of a covalent complex between adrenodoxin reductase and adrenodoxin.
A cross-linked complex between bovine NADPH-adrenodoxin reductase (AR) and adrenodoxin (AD) was prepared with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and purified, as described previously [Hara, T. & Kimura, T. (1989) J. Biochem. 105, 594-600]. The covalent complex was S-pyridylethylated and digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase HPLC to identify the cross-linked peptide. Comparison of the HPLC chromatograms of the peptides showed that (i) two tandem peptides (K-4 and K-5) from AD and a peptide (K-1) from AR were missing in the chromatogram of the peptides of the covalent complex and (ii) a single new peak was observed in the chromatogram of the peptides from the covalent complex. Amino acid composition and sequence analyses showed that the newly observed peptide was a covalently cross-linked peptide formed between a peptide K-4-K-5 (Ile-25-Lys-98) derived from AD and a peptide K-1 (Ser-1-Lys-27) derived from AR, in which an amide bond had been formed between the epsilon-amino group of Lys-66 in AD and the gamma-carboxyl group of Glu-4 in AR. These results indicate that the binding site of AR with AD is localized in the amino-terminal part of AR and that of AD with AR is localized around Lys-66 of AD. The six clustered basic amino acid residues (His-24, Lys-27, His-28, His-29, Arg-31, and His-33) present in the amino-terminal portion of AR and the eight clustered acidic amino acid residues (Glu-65, Glu-68, Asp-72, Glu-73, Glu-74, Asp-76, Asp-79, and Asp-86) present in the middle part of AD may play an important role in the complex formation.
Hara
T
T
Section of Biochemistry, Graduate School of Health and Nutrition Sciences, Nakamura Gakuen College, Fukuoka.
Miyata
T
T
D000325:Adrenodoxin / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives / Q000737:chemistry; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D008969:Molecular Sequence Data
D000596:Amino Acids; D003432:Cross-Linking Reagents; D000325:Adrenodoxin; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; D005287:Ferredoxin-NADP Reductase; D005022:Ethyldimethylaminopropyl Carbodiimide