Journal of Bioenergetics and Biomembranes

, Volume 17, Issue 5, pp 269–282 | Cite as

Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

  • Samuel H. K. Ho
  • Uttam Das Gupta
  • John S. Rieske
Research Articles
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Revised:

Abstract

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K i =ca. 0.5 µM) was considerably less than that of antimycin (K i ≤3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [3H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [3H]DAA, respectively. The labeling of band 7 by [3H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [3H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.

Key Words

Ubiquinol-cytochromec reductase Complex III subunits polyacrylamide gel electrophoresis antimycin-binding site photoaffinity labeling deformamidoazidoantimycin A 
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Copyright information

© Plenum Publishing Corporation 1985

Authors and Affiliations

  • Samuel H. K. Ho
    • 1
  • Uttam Das Gupta
    • 1
  • John S. Rieske
    • 1
  1. 1.Department of Physiological Chemistry, College of MedicineThe Ohio State UniversityColumbus

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