[14C]N-ethylmaleimide labeling of the plasma membrane [H+]-ATPase of Neurospora crassa.

R. J. Brooker, C. W. Slayman

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)222-226
Number of pages5
JournalJournal of Biological Chemistry
Volume258
Issue number1
StatePublished - Jan 10 1983

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Neurospora crassa
Ethylmaleimide
Proton-Translocating ATPases
Cell membranes
Labeling
Cell Membrane
Adenosine Triphosphatases
Adenosine Diphosphate
Cysteine
Sulfhydryl Reagents
Peptides
Peptide Fragments
Enzymes
Autoradiography
Nucleotides
Kinetics

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[14C]N-ethylmaleimide labeling of the plasma membrane [H+]-ATPase of Neurospora crassa. / Brooker, R. J.; Slayman, C. W.

In: Journal of Biological Chemistry, Vol. 258, No. 1, 10.01.1983, p. 222-226.

Research output: Contribution to journalArticle

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