A novel 14-kDa V-ATPase subunit in the tobacco hornworm midgut.
A cDNA clone encoding a hydrophilic protein with a calculated molecular mass of 13,839 Da was isolated by shotgun screening with an anti-V-ATPase holoenzyme serum. The deduced amino acid sequence showed no significant homology to any other known protein. Southern blots revealed the existence of only one gene encoding the 14-kDa protein. Monospecific antibodies purified by affinity to the recombinant protein demonstrated the presence of a 14-kDa protein in the highly purified goblet cell apical membrane and inhibited ATP-dependent proton transport as well as V-ATPase activity to the same extent. Thus, the 14-kDa protein was shown to be a part of the V-ATPase holoenzyme. Binding of the monospecific antibodies to the ATPase seemed to require an ATP-dependent conformational change of the enzyme, since inhibition only occurred when ATP was present during the antibody binding step. The 14-kDa subunit could be stripped from the membrane by treatment with the chaotropic agent KI, confirming it to be part of the soluble complex of the V-ATPase. In immunoblots, the 14-kDa-specific antibodies showed no cross-reaction with several xenic V-ATPases.
Gräf
R
R
Zoological Institute, University of Munich, Germany.
Lepier
A
A
Harvey
W R
WR
Wieczorek
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004064:Digestive System / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D007700:Kinetics; D007814:Larva; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D014617:Vacuoles / Q000201:enzymology
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D006180:Proton-Translocating ATPases