Does 2-hydroxy-5-nitrobenzyl bromide react with the epsilon-subunit of the mitochondrial F1-ATPase?
The incubation of bovine mitochondrial F1-ATPase with 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent toward tryptophan residues in proteins, produced a concentration dependent inactivation of the enzyme and the covalent binding of 0.88 mol reagent/mol F1. Although HNB is highly specific for tryptophan it has also some reactivity toward cysteine, then a pre-treatment of F1 with several sulphydryl reagents has been performed to make the site of reaction clearer. This pre-treatment had neither effects in the binding stoichiometry nor in the extent of catalytic inhibition, suggesting that readly accessible thiol groups are not involved in the reaction with HNB. Since the only tryptophan bearing polypeptide of the bovine mitochondrial F1-ATPase complex is its smallest subunit, subunit-epsilon, this is the most probable candidate for HNB reaction. Therefore it may be inferred that the intactness and/or the correct conformation of this subunit could be important factor(s) for the multisite ATP hydrolytic activity of the enzyme.
Baracca
A
A
Department of Biology, University of Bologna, Italy.
Menegatti
D
D
Parenti Castelli
G
G
Rossi
C A
CA
Solaini
G
G
D015077:2-Hydroxy-5-nitrobenzyl Bromide / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D008929:Mitochondria, Heart / Q000201:enzymology*; D010446:Peptide Fragments / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000187:drug effects* / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014364:Tryptophan
D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D015077:2-Hydroxy-5-nitrobenzyl Bromide; D014364:Tryptophan; D006180:Proton-Translocating ATPases