A novel 14-kDa V-ATPase subunit in the tobacco hornworm midgut
1994
Graf, R. | Lepier, A. | Harvey, W.R. | Wieczorek, H.
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.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
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