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Modification of the Neurospora crassa plasma membrane (H/sup +/)-ATPase with N,N'-dicyclohexycarbodiimide

Journal Article · · J. Biol. Chem.; (United States)
OSTI ID:5932288
;
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/sup 0/C is approximately 1000 M/sup -1/ min/sup -1/, similar to values reported for the DCCD-binding proteolipid of F/sub 0/-F/sub 1/-type (H/sup +/)-ATPases and for the sarcoplasmic reticulum (Ca/sup +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. (/sup 14/C)DCCD is incorporated into the M/sub r/ = 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 the DCCD reacts with a single amino acid residue of the Neuspora (H/sup +/)-ATPase, thereby blcoking ATP hydrolysis and proton translocation. 21 references, 5 figures, 2 tables.
Research Organization:
Yale Univ. School of Medicine, New Haven, CT
OSTI ID:
5932288
Journal Information:
J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 258:3; ISSN JBCHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ATP
ATP-ASE
BARYONS
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
BIOLOGICAL EFFECTS
CARBON 14 COMPOUNDS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL BONDS
CHEMISTRY
DATA
ELEMENTARY PARTICLES
ENZYME ACTIVITY
ENZYMES
EXPERIMENTAL DATA
FERMIONS
FUNGI
HADRONS
HYDROLASES
IMIDES
INFORMATION
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MEMBRANES
MODIFICATIONS
NEUROSPORA
NUCLEONS
NUCLEOTIDES
NUMERICAL DATA
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDES
PHOSPHOHYDROLASES
PLANTS
POLYPEPTIDES
PROTEINS
PROTONS
REACTION KINETICS
TRACER TECHNIQUES
TRANSLOCATION