Vanadate-catalyzed, conformationally specific photocleavage of the Ca2+-ATPase of sarcoplasmic reticulum

Distinct Topologies of Mono- and Decavanadate Binding and Photo-oxidative Cleavage in the Sarcoplasmic Reticulum ATPase

“…At any rate, Ser 186 is the main residue undergoing photo-oxidation by decavanadate in the absence of Ca 2ϩ and ADP. Cleavage of its oxidized product is consistent with the size (88 and 21 kDa) of the cleaved fragments and is reasonably near the site (T2: Arg 198 ) predicted by previous studies (11,12).…”

“…As reported by Vegh et al (11) and Molnar et al (12), exposure of the ATPase-vanadate complex to UV light at neutral pH yields a pattern of peptide cleavage that is dependent on the presence or the absence of Ca 2ϩ : two fragments of 88-and 21-kDa electrophoretic mobility in the absence of Ca 2ϩ , and 71-and 38-kDa in the presence of Ca 2ϩ ( Fig. 1).…”

“…Cleavage of SR ATPase by UV irradiation in the presence of vanadate was previously reported by Vegh et al (11) and Molnar et al (12), who, at neutral pH, observed different cleavage sites in the absence as opposed to the presence of Ca 2ϩ . We find that experimentation at neutral pH produces rapid equilibration of mono-and oligovanadate species, and demonstration of a selective monovanadate effect requires alkaline pH.…”

“…It is not entirely clear whether vanadate monomers and oligomers, which are known to coexist in various experimental conditions (9), occupy the phosphorylation and/or distinct sites in the SR ATPase (10). We considered that this uncertainty may be clarified by studies of vanadate-dependent ATPase cleavage, which was reported to occur following UV irradiation at different sites depending on the presence or the absence of Ca 2ϩ (11,12). However, the cleavage sites and the mechanism of cleavage could not be characterized in previous studies on the SR ATPase, due to amino terminus blockage in the cleaved fragments (11,12).…”

“…We considered that this uncertainty may be clarified by studies of vanadate-dependent ATPase cleavage, which was reported to occur following UV irradiation at different sites depending on the presence or the absence of Ca 2ϩ (11,12). However, the cleavage sites and the mechanism of cleavage could not be characterized in previous studies on the SR ATPase, due to amino terminus blockage in the cleaved fragments (11,12).…”