Abstract
The shape and overall dimensions of the oxidized and reduced form of the V(1) ATPase from Manduca sexta were investigated by synchrotron radiation x-ray solution scattering. The radius of gyration of the oxidized and reduced complex differ noticeably, with dimensions of 6. 20 +/- 0.06 and 5.84 +/- 0.06 nm, respectively, whereas the maximum dimensions remain constant at 22.0 +/- 0.1 nm. Comparison of the low resolution shapes of both forms, determined ab initio, indicates that the main structural alteration occurs in the head piece, where the major subunits A and B are located, and at the bottom of the stalk. In conjunction with the solution scattering data, decreased susceptibility to tryptic digestion and tryptophan fluorescence of the reduced V(1) molecule provide the first strong evidence for major structural changes in the V(1) ATPase because of redox modulation.
Highlights
The shape and overall dimensions of the oxidized and reduced form of the V1 ATPase from Manduca sexta were investigated by synchrotron radiation x-ray solution scattering
Studies on the bovine clathrin-coated V-ATPase have shown that disulfide bond formation between the conserved residues Cys254 and Cys532 of the catalytic A subunit leads to reversible inactivation of the enzyme [12, 13]
The fraction of larger distances is significantly diminished in the p(r) function upon reduction by DTT, suggesting that the particle becomes more compact in the reduced state than in the oxidized state of the enzyme
Summary
Studies on the bovine clathrin-coated V-ATPase have shown that disulfide bond formation between the conserved residues Cys254 and Cys532 of the catalytic A subunit leads to reversible inactivation of the enzyme [12, 13]. Such a reversibly inactivated, disulfidebonded state has been found in significant fractions of V-ATPase in native clathrin-coated vesicles [12]. Disulfide bonds at the catalytic site of the V-ATPase are induced by the nitric oxide-generating reagent S-nitrosoglutathione [14] This is consistent with the finding that in Neurospora crassa oxidizing and reducing agents have inhibitory and stabilizing effects, respectively, on the enzyme [15]. Altered mobility of the V1 subunits in polyacrylamide gels, decreased tryptophan fluorescence, and trypsin susceptibility are discussed in the light of these structural changes
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