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Chemical cross-linking studies of chloroplast coupling factor 1.

Publication ,  Journal Article
Baird, BA; Hammes, GG
Published in: J Biol Chem
November 25, 1976

Cross-linking reagents have been used to link covalently adjacent subunits of solubilized spinach chloroplast coupling factor 1, which is a latent ATPase. 1,5-Difluoro-2,4-dinitrobenzene, dimethyl-3,3'-dithiobispropionimidate, and dimethylsuberimidate are able to form bridges of 3 to 11 A between amino groups, and hydrogen peroxide and the o-phenanthroline-cupric ion complex catalyze the oxidation of intrinsic sulfhydryl groups. The five individual subunit bands (alpha, beta, gamma, delta, and epsilon) and several new aggregate bands can be separated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same four fastest moving aggregate bands, as characterized by their mobilities, migrate more slowly than the heaviest subunit band and appear with all of the cross-linkers employed. The subunit composition of the aggregate bands has been determined through the use of the reversible cross-linkers, dimethyldithiobispropionimidate, (o-phenanthroline)2Cu(II), and H2O2, and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which aggregates are separated in the first dimension, the disulfide cross-links are cleaved, and the individual subunits present in the aggregates are separated in the second dimension. The subunits are detected by Coomassie brilliant blue staining and by labeling some of the sulfhydryl groups of the gamma and epsilon subunits with radioactive N-ethylmaleimide. The results obtained indicate that the alpha and beta subunits can cross-link directly with each of the other subunits, that two beta subunits are adjacent, and that gamma epsilon, gamma epsilon 2, alpha delta, and beta delta aggregates are present. A minimal subunit stoichiometry consistent with these results is alpha 2 beta 2 gamma delta epsilon 2. A possible structural model of the coupling factor is derived from the data. Similar, but less extensive, experiments have been carried out with the heat-activated coupling factor (which is an ATPase); no differences in the spatial arrangement of subunits are detected from the two-dimensional gel electrophoresis analysis of the cross-linked aggregates.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

November 25, 1976

Volume

251

Issue

22

Start / End Page

6953 / 6962

Location

United States

Related Subject Headings

  • Spectrometry, Fluorescence
  • Protein Conformation
  • Protein Binding
  • Plants
  • Plant Proteins
  • Molecular Weight
  • Macromolecular Substances
  • Ethylmaleimide
  • Dinitrofluorobenzene
  • Chloroplasts
 

Citation

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Baird, B. A., & Hammes, G. G. (1976). Chemical cross-linking studies of chloroplast coupling factor 1. J Biol Chem, 251(22), 6953–6962.
Baird, B. A., and G. G. Hammes. “Chemical cross-linking studies of chloroplast coupling factor 1.J Biol Chem 251, no. 22 (November 25, 1976): 6953–62.
Baird BA, Hammes GG. Chemical cross-linking studies of chloroplast coupling factor 1. J Biol Chem. 1976 Nov 25;251(22):6953–62.
Baird, B. A., and G. G. Hammes. “Chemical cross-linking studies of chloroplast coupling factor 1.J Biol Chem, vol. 251, no. 22, Nov. 1976, pp. 6953–62.
Baird BA, Hammes GG. Chemical cross-linking studies of chloroplast coupling factor 1. J Biol Chem. 1976 Nov 25;251(22):6953–6962.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

November 25, 1976

Volume

251

Issue

22

Start / End Page

6953 / 6962

Location

United States

Related Subject Headings

  • Spectrometry, Fluorescence
  • Protein Conformation
  • Protein Binding
  • Plants
  • Plant Proteins
  • Molecular Weight
  • Macromolecular Substances
  • Ethylmaleimide
  • Dinitrofluorobenzene
  • Chloroplasts