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Inhibition and labeling of the plant plasma membrane ATPase with N-ethyl maleimide

Conference · · Plant Physiol.; (United States)
OSTI ID:5499433
;

H/sup +/-ATPase activity in plasma membranes isolated from oat root cells is inhibited by N-ethyl maleimide (NEM), a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced by ADP and ATP. Even at high ADP concentrations, the rate of inactivation is not zero, leading us to hypothesize that there are at least two reactive cysteines, only one of which is affected by ADP binding. When plasma membranes are treated with /sup 3/H-NEM and analyzed by SDS-PAGE, prominent radioactive bands appear at M/sub r/ = 100,000 and several other positions. However only radioactivity in the M/sub r/ = 100,000 protein is reduced by the presence of ADP. These results provide independent evidence that the M/sub r/ = 100,000 polypeptide which is observed in purified preparations of the enzyme is the catalytic subunit. Tryptic peptides were produced from /sup 3/H-NEM labeled M/sub r/ = 100,000 protein and separated by reverse phase HPLC. Three major radioactive peaks were observed, one of which was absent when labeling was performed in the presence of ADP.

Research Organization:
Univ. of Wisconsin, Madison
OSTI ID:
5499433
Journal Information:
Plant Physiol.; (United States), Journal Name: Plant Physiol.; (United States) Vol. 80:4; ISSN PLPHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201 -- Biochemistry-- Tracer Techniques
560301* -- Chemicals Metabolism & Toxicology-- Cells-- (-1987)
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ACID ANHYDRASES
ADP
AMINO ACIDS
ATP
ATP-ASE
BIOLOGICAL EFFECTS
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CEREALS
CYSTEINE
ENZYME ACTIVITY
ENZYME INHIBITORS
ENZYMES
GRASS
HYDROLASES
INHIBITION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LABELLING
MEMBRANES
NUCLEOTIDES
OATS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHOHYDROLASES
PLANT CELLS
PLANTS
PROTEINS
ROOTS
STRUCTURAL CHEMICAL ANALYSIS
THIOLS
TRACER TECHNIQUES
TRITIUM COMPOUNDS