Effects of vanadate on the rotational dynamics of spin-labeled calcium adenosinetriphosphatase in sarcoplasmic reticulum membranes.
We have studied the effects of vanadate on the rotational motion of the calcium adenosine-triphosphatase (Ca-ATPase) from sarcoplasmic reticulum (SR), using saturation-transfer electron paramagnetic resonance (ST-EPR). Vanadate has been proposed to act as a phosphate analogue and produce a stable intermediate state similar to the phosphoenzyme. This study provides evidence about the physical state of this intermediate. In particular, since ST-EPR provides a sensitive measure of microsecond protein rotational mobility, and hence of protein-protein association, these studies allowed us to ask (a) whether the vanadate-induced protein association observed in electron micrographs of SR vesicles also occurs under physiological (as opposed to fixed, stained, or frozen) conditions and (b) whether vanadate-induced changes in protein association also occur under conditions sufficient for enzyme inhibition but not for the production of large arrays detectable by electron microscopy (EM). At 5 mM decavanadate, a concentration sufficient to crystallize the ATPase on greater than 90% of the membrane surface area in EM, ST-EPR showed substantial immobilization of the spin-labeled protein, indicating protein-protein association in the unstained vesicles. Conventional EPR spectra of lipid probes showed that lipid hydrocarbon chain mobility is unaffected by decavanadate-induced protein crystallization in SR, suggesting that changes in protein-protein contacts do not involve the lipid hydrocarbon region. At 5 mM monovanadate, a concentration sufficient to inhibit the ATPase but not to form crystals detectable by EM, no changes were observed in ST-EPR or conventional EPR spectra of either protein or lipid.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D008854:Microscopy, Electron; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D013053:Spectrophotometry; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology*
D014639:Vanadium; D014638:Vanadates; D000252:Calcium-Transporting ATPases