Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase.
The organotin complex dibutyltin-3-hydroxyflavone bromide [Bu2Sn(of)Br] has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H(+)-ATPase. Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein. Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid. The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide. Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity. Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid. The fluorescent enhancement of affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity. Interaction of the probe with mutant proteolipid was unaffected by DCCD. These data suggest an overlap in the binding sites of organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the facuolar H(+)-ATPase.
Hughes
G
G
Department of Biochemistry and Molecular Biology, University of Leeds, UK.
Harrison
M A
MA
Kim
Y I
YI
Griffiths
D E
DE
Finbow
M E
ME
Findlay
J B
JB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004024:Dicyclohexylcarbodiimide; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D008099:Liver / Q000737:chemistry; D009154:Mutation; D008121:Nephropidae / Q000737:chemistry / Q000235:genetics; D009947:Organotin Compounds / Q000494:pharmacology*; D010179:Pancreas / Q000737:chemistry; D011510:Proteolipids / Q000187:drug effects*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000378:metabolism; D011522:Protons; D011994:Recombinant Proteins / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017186:Titrimetry; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology*
D004791:Enzyme Inhibitors; D005419:Flavonoids; D009947:Organotin Compounds; D011510:Proteolipids; D011522:Protons; D011994:Recombinant Proteins; C087027:proteolipid, Nephrops; C084071:dibutyltin 3-hydroxyflavone; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases