[14C]N-ethylmaleimide labeling of the plasma membrane [H+]-ATPase of Neurospora crassa.
Treatment of the purified plasma membrane [H+]-ATPase of Neurospora crassa with the sulfhydryl reagent N-ethylmaleimide (NEM) leads to a marked inhibition of ATPase activity. Several lines of evidence indicate that inhibition is caused by the reaction of NEM with a single cysteine residue on the Mr = 104,000 polypeptide. (1) Inhibition by NEM follows pseudo-first order kinetics. (2) MgADP protects against NEM inactivation and at the same time decreases the incorporation of [14C]NEM into the Mr = 104,000 polypeptide. When "protectable" [14C]NEM incorporation is plotted as a function of inhibition of ATPase activity, a linear relationship is observed with a slope of 0.8. (3) Labeling of the ATPase with [14C]NEM can be restricted to the protectable site by pretreatment with cold NEM in the presence of MgADP, followed by an incubation with [14C]NEM in the absence of nucleotides. When this is done, and the enzyme is subjected to tryptic mapping and autoradiography, a single radioactive peptide fragment is found. The protectable site may yield information about the role of cysteine in the ATPase mechanism, and should also serve as a useful point of reference in enzyme mapping studies.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000201:enzymology; D005033:Ethylmaleimide / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D006180:Proton-Translocating ATPases; D014357:Trypsin
D002250:Carbon Radioisotopes; D010446:Peptide Fragments; D014357:Trypsin; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide