Modification of the Neurospora crassa plasma membrane [H+]-ATPase with N,N'-dicyclohexylcarbodiimide.
The [H+]-ATPase of the Neurospora plasma membrane is composed of a single Mr = 104,000 polypeptide (B. J. Bowman, F. Blasco, and C. W. Slayman, J. Biol. Chem. (1981) 256, 12343-12349). The carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the ATPase with pseudo-first order kinetics, suggesting that one site on the enzyme is involved. The rate constant for inactivation at pH 7.5 and 30 degrees C is approximately 1000 M-1 min-1, similar to values reported for the DCCD-binding proteolipid of F0-F1-type [H+]-ATPases and for the sarcoplasmic reticulum [Ca+2]-ATPase. Although hydrophobic carbodiimides are inhibitory at micromolar concentrations, a hydrophilic analogue, 1-ethyl-3-(dimethylaminopropyl)-carbodiimide, is completely inactive even at millimolar concentrations. This result implies that the DCCD-reactive site is located in a lipophilic environment. [14C]DCCD is incorporated into the Mr = 104,000 polypeptide at a rate similar to the rate of inactivation. There is no evidence for a separate low molecular weight DCCD-binding proteolipid. Using quantitative amino acid analysis, we established that complete inhibition occurs at a stoichiometry of 0.4 mol of DCCD/mol of polypeptide. Overall, the results are consistent with the idea that DCCD reacts with a single amino acid residue of the Neurospora [H+]-ATPase, thereby blocking ATP hydrolysis and proton translocation.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D002234:Carbodiimides / Q000494:pharmacology*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007700:Kinetics; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D006180:Proton-Translocating ATPases
D002234:Carbodiimides; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases