Oligovanadate binding to sarcoplasmic reticulum ATPase. Evidence for substrate analogue behavior.
Solutions of vanadate were controlled through concentration and pH adjustment to give specific compositions of mono- and oligovanadates. By monitoring the EPR spectrum of iodoacetamide spin-labeled ATPase, it is shown that decavanadate and the oligovanadate species present at neutral pH exhibit behavior typical of a substrate analogue. This is seen in terms of Ca2+ binding site affinity (microM), outward Ca2+ site orientation, and conformational effects on the enzyme normally associated with enzyme activation. In contrast, monovanadates exhibit behavior identical to that observed with Pi, with one exception: the vanadoenzyme is stable to Ca2+ in the concentration range of high affinity binding at the vanadate concentrations used here (200 microM). It is further demonstrated that Ca2+ binding in the 100 microM range directly induces enzyme devanadation of the monovanadate enzyme complex through Ca2+ binding to internal sites. Extensive array formation of dimeric ATPase units is found only with decavanadate in the absence of Ca2+, and then stoichiometric amounts are sufficient. Electron micrographs of dimeric arrays show evidence of increased penetration into the lipid bilayer, including freeze-fracture replicas which show evidence of corresponding "pits" in the inner leaflet of the bilayer. In turn, EPR spectra provide a means of following vanadate binding to the ATPase per se, as well as monitoring Ca2+-induced changes in the vanadoenzyme conformation, as only binding to specific sites on the enzyme affect the EPR spectrum.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005614:Freeze Fracturing; D007460:Iodoacetamide; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013113:Spin Labels; D014638:Vanadates; D014639:Vanadium / Q000378:metabolism*
D046911:Macromolecular Substances; D013113:Spin Labels; D014639:Vanadium; D000266:Adenylyl Imidodiphosphate; D014638:Vanadates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium; D007460:Iodoacetamide