Inhibition of the coated vesicle proton pump and labeling of a 17,000-dalton polypeptide by N,N'-dicyclohexylcarbodiimide
N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits 100% of proton transport and 80-85% of (Mg2+)-ATPase activity in clathrin-coated vesicles. Half-maximum inhibition of proton transport is observed at 10 microM DCCD after 30 min. Although treatment of the coated vesicle (H+)-ATPase with DCCD has no effect on ATP hydrolysis in the detergent-solubilized state, sensitivity of proton transport and ATPase activity to DCCD is restored following reconstitution into phospholipid vesicles. In addition, treatment of the detergent-solubilized enzyme with DCCD followed by reconstitution gives a preparation that is blocked in both proton transport and ATP hydrolysis. These results suggest that although the coated vesicle (H+)-ATPase can react with DCCD in either a membrane-bound or detergent-solubilized state, inhibition of ATPase activity is only manifested when the pump is present in sealed membrane vesicles. To identify the subunit responsible for inhibition of the coated vesicle (H+)-ATPase by DCCD, we have labeled the partially purified enzyme with (/sup 14/C)DCCD. A single polypeptide of molecular weight 17,000 is labeled. The extremely hydrophobic nature of this polypeptide is indicated by its extraction with chloroform:methanol. The 17,000-dalton protein can be labeled to a maximum stoichiometry of 0.99 mol of DCCD/mol of protein with 100% inhibition of proton transport occurring at a stoichiometry of 0.15-0.20 mol of DCCD/mol of protein. Amino acid analysis of the chloroform:methanol extracted 17,000-dalton polypeptide reveals a high percentage of nonpolar amino acids. The similarity in properties of this protein and the DCCD-binding subunit of the coupling factor (H+)-ATPases suggests that the 17,000-dalton polypeptide may function as part of a proton channel in the coated vesicle proton pump.
- Research Organization:
- Tufts Univ. School of Medicine, Boston, MA
- OSTI ID:
- 6375590
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 262:23; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
AMINO ACID SEQUENCE
ANIMALS
ATP-ASE
BARYONS
BIOLOGICAL FUNCTIONS
BODY
BRAIN
CARBON 14 COMPOUNDS
CATTLE
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
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ELEMENTARY PARTICLES
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