The interaction of carboxyl group reagents with the Rhodospirillum rubrum F1-ATPase and its isolated beta-subunit.
The carboxyl group reagents dicyclohexylcarbodiimide (DCCD) and N-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) inactivate the soluble Rhodospirillum rubrum F1-ATPase (RrF1). The inactivation is both time- and concentration-dependent and also pH-dependent, being more marked at acid pH. Under the same conditions, N-ethyl-5-phenylisoxazolium 3'-sulfonate causes almost no inactivation of the RrF1-ATPase. Complete inhibition of the enzyme activity requires the binding of 1 mol of DCCD/mol of RrF1. The isolated, reconstitutively active, beta-subunit of RrF1 is affected by the three carboxyl group reagents in a very similar manner to the RrF1-ATPase. Incubation of the beta-subunit with DCCD and EEDQ eliminates its capacity to rebind to beta-less chromatophores. Consequently the DCCD or EEDQ-modified beta-subunit cannot restore ATP synthesis or hydrolysis activities to the beta-less chromatophores. The interaction of the isolated beta-subunit with DCCD and EEDQ is both time and concentration dependent. The elimination of the reconstitutive activity of the beta-subunit by DCCD is accompanied with a covalent binding of about 1 mol of [14C]DCCD/mol of beta and is pH-dependent, showing a half-maximal effect at about pH 7.4. Divalent cations, inorganic phosphate, and to a lesser extent ATP and ADP decrease the binding stoichiometry of DCCD to the beta-subunit. Pretreatment of either RrF1 or its isolated beta-subunit with EEDQ reduces drastically their ability to bind [14C]DCCD, suggesting that in both RrF1 and the beta-subunit, EEDQ and DCCD might react at the same site. The similar effect of the carboxyl group reagents on RrF1 and on its isolated beta-subunit is in accord with the suggestion that DCCD and EEDQ affect the F1-ATPases by interacting with their beta-subunits.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002234:Carbodiimides / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007555:Isoxazoles / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology; D010785:Photophosphorylation / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011804:Quinolines / Q000494:pharmacology*; D012247:Rhodospirillum rubrum / Q000201:enzymology*
D002234:Carbodiimides; D007555:Isoxazoles; D046911:Macromolecular Substances; D010080:Oxazoles; D010710:Phosphates; D011804:Quinolines; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D004024:Dicyclohexylcarbodiimide; C000470:EEDQ; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium; D002118:Calcium