Structural and functional characterization of bovine adrenodoxin reductase by limited proteolysis.
Previously, we have proposed that bovine adrenocortical mitochondrial adrenodoxin reductase may possess a domain structure, based upon the generation of two major peptide fragments from limited tryptic proteolysis. In the present study, kinetic characterization of the NADPH-dependent ferricyanide reductase activity of the partially proteolyzed enzyme demonstrates that Km(NADPH) increases (from 1.2 microM to 2.7 microM), whereas Vmax remains unaltered at 2100 min-1. The two proteolytic fragments have been purified to homogeneity by reverse-phase HPLC, and amino-acid sequence analysis unambiguously demonstrates that the 30.6 kDa fragment corresponds to the amino terminal portion of the intact protein, whereas the 22.8 kDa fragment is derived from the carboxyl terminus of the reductase. Trypsin cleavage occurs at either Arg-264 or Arg-265. Covalent crosslinking experiments using a water-soluble carbodiimide show that adrenodoxin crosslinks exclusively to the 30.6 kDa fragment, thus implicating the N-terminal region of adrenodoxin reductase in binding to the iron-sulfur protein. Our inability to detect covalent carbohydrate on either intact or proteolyzed adrenodoxin reductase prompted a re-examination of the previously reported requirement of an oligosaccharide moiety for efficient electron transfer from the reductase to adrenodoxin. Treatment of adrenodoxin reductase with a highly purified preparation of neuraminidase demonstrates that neither the adrenodoxin-independent ferricyanide reductase activity nor the adrenodoxin-dependent cytochrome c reductase activity of the enzyme is affected by neuraminidase treatment.
Warburton
R J
RJ
Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282.
Seybert
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism*
D002241:Carbohydrates; D010446:Peptide Fragments; D005287:Ferredoxin-NADP Reductase; D009439:Neuraminidase; D014357:Trypsin