Inhibition and labeling of the plant plasma membrane H+-ATPase with N-ethylmaleimide

• Published in: Plant physiology (Bethesda) Vol. 83; no. 4; pp. 977 - 981
• Main Authors: Katz, D.B, Sussman, M.R
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01.04.1987
• Subjects: ADENOSINDIFOSFATO; ADENOSINE DIPHOSPHATE; Adenosine triphosphatases; Analytical, structural and metabolic biochemistry; AVENA SATIVA; Biological and medical sciences; Cell membranes; CELL STRUCTURE; Electrophoresis; ENZYME INHIBITORS; Enzymes; Enzymes and enzyme inhibitors; ESTRUCTURA CELULAR; ETIQUETADO; Fundamental and applied biological sciences. Psychology; Gels; HIDROLASAS; HYDROLASE; HYDROLASES; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; LABELLING; MAGNESIO; MAGNESIUM; MARQUAGE; Nucleotides; Oats; PEPTIDE; PEPTIDES; PEPTIDOS; Plants; RACINE; RADIACTIVIDAD; Radioactive decay; RADIOACTIVITE; RADIOACTIVITY; RAICES; Room temperature; ROOTS; STRUCTURE CELLULAIRE; Avena sativa; Monocotyledones; Cysteine; Chemical modification; Vegetals; Gramineae; Enzyme; Angiospermae; Plasma membrane; Molecular interaction; Spermatophyta
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.83.4.977

H+-ATPase activity in plasma membranes isolated from Avena sativa root cells is inhibited by N-ethylmaleimide, a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced by ADP, MgADP, and MgATP, but even at 40 millimolar ADP the enzyme is only partially protected against inactivation. When plasma membranes are treated wth N-[2-_{3}\text{H}$]ethylmaleimide and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, prominent radioactive bands appear at Mr=100,000 and several other positions. However, only radioactivity in the Mr=100,000 protein is reduced by the presence of MgADP. These results provide independent evidence that the Mr=100,000 polypeptide which is observed in purified preparations of the enzyme is the catalytic subunit of the H+-ATPase. When tryptic peptides are produced from N-[2-_{3}\text{H}$]ethylmaleimide labeled Mr=100,000 protein and separated by reverse phase high performance liquid chromatography, two radioactive peaks are observed for which N-[2-3H]ethylmaleimide incorporation is reduced in the presence of MgADP.