Ligand-dependent structural changes in the V(1) ATPase from Manduca sexta.
The response of V(1) ATPase of the tobacco hornworm Manduca sexta to Mg(2+) and nucleotide binding in the presence of the enhancer methanol has been studied by CuCl(2)-induced disulfide formation, fluorescence spectroscopy, and small-angle X-ray scattering. When the V(1) complex was supplemented with CuCl(2) nucleotide-dependence of A-B-E and A-B-E-D cross-linking products was observed in absence of nucleotides and presence of MgADP+Pi but not when MgAMP.PNP or MgADP were added. A zero-length cross-linking product of subunits D and E was formed, supporting their close proximity in the V(1) complex. The catalytic subunit A was reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP.PNP, -ATP, -ADP+Pi, or -ADP. Differences in the fluorescence emission of these nucleotide-binding states were monitored using the intrinsic tryptophan fluorescence. The structural composition of the V(1) ATPase from M. sexta and conformational alterations in this enzyme due to Mg(2+) and nucleotide binding are discussed on the basis of these and previous observations.
Coskun
Unal
U
Universität des Saarlandes, Fachrichtung 2.5 - Biophysik, D-66421 Homburg, Germany.
Rizzo
Vincenzo F
VF
Koch
Michel H J
MH
Grüber
Gerhard
G
D000818:Animals; D003300:Copper; D003432:Cross-Linking Reagents / Q000378:metabolism; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D018402:Manduca / Q000201:enzymology*; D000432:Methanol / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D025262:Vacuolar Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism*; D014965:X-Rays
D003432:Cross-Linking Reagents; D008024:Ligands; D009711:Nucleotides; D003300:Copper; D025262:Vacuolar Proton-Translocating ATPases; D008274:Magnesium; C029892:cupric chloride; D000432:Methanol