The interaction of ferredoxin and glutamate synthase: cross-linking and immunological studies.
The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) serves as an effective reagent for cross-linking spinach leaf ferredoxin and the ferredoxin-dependent spinach leaf enzyme, glutamate synthase. The cross-linked complex was functional in the absence of added ferredoxin, suggesting that ferredoxin is cross-linked to glutamate synthase at the physiological binding site on the enzyme for this iron-sulfur protein electron donor. The ferredoxin:glutamate synthase stoichiometry of the cross-linked complex was estimated to be 2:1. The absorbance spectrum of the oxidized, cross-linked complex was very similar to that of an electrostatically stabilized, noncovalent, 2:1 complex of the two proteins. An antibody raised against spinach NADP+ reductase, which recognizes a ferredoxin-binding site on glutamate synthase, does not recognize the cross-linked ferredoxin-glutamate synthase complex. This implies that the ferredoxin-binding sites on the two enzymes are structurally similar enough so that an antibody raised against one of these ferredoxin-dependent enzymes recognizes an epitope at the ferredoxin-binding site of the second enzyme. Cross-linking of ferredoxin to its binding site on glutamate synthase renders this epitope inaccessible to the antibody.
Hirasawa
M
M
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409-1061.
Chang
K T
KT
Knaff
D B
DB
D000906:Antibodies; D001666:Binding Sites, Antibody; D002942:Circular Dichroism; D005288:Ferredoxins / Q000276:immunology / Q000378:metabolism*; D005970:Glutamate Synthase / Q000276:immunology / Q000378:metabolism*; D005779:Immunodiffusion; D007074:Immunoglobulin G; D007700:Kinetics; D009572:Nitrite Reductases / Q000378:metabolism; D010944:Plants / Q000378:metabolism; D011487:Protein Conformation; D013053:Spectrophotometry / Q000379:methods
D000906:Antibodies; D005288:Ferredoxins; D007074:Immunoglobulin G; D005970:Glutamate Synthase; D009572:Nitrite Reductases