Utilization of arylazido-ATP by sarcoplasmic reticulum ATPase in the absence of calcium.
The ATP analog arylazido-ATP 5'-triphosphate) (3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate) was shown to phosphorylate the calcium-ATPase from sarcoplasmic reticulum in the absence of calcium. Levels of 0.6 nmol of phosphoenzyme/mg of protein were attained. Calcium either at micromolar or millimolar concentrations did not affect the level of phosphoenzyme. A non-Michaelian dependence of the hydrolytic activity as a function of analog concentration was obtained in the absence of calcium. Calcium addition did not modify either the analog concentration dependence for the activation of hydrolysis or the maximal rate of hydrolysis. In the presence of micromolar calcium, arylazido-ATP promoted calcium accumulation inside the vesicles, and a steady-state level of 100 nmol of calcium/mg of protein was maintained. ESR spectra of spin-labeled ATPase showed that addition of the analog in the absence of calcium caused a spectral change, and the resulting spectral parameters were different from those obtained for ATP under similar conditions. Calcium addition did not cause any further modification of the spectra, which was clearly distinct from the change when ATP was used. The partition coefficient of the analog from a water medium into an organic phase was found to be 1 order of magnitude higher than that of ATP. It is suggested that it might be the hydrophobic nature of the analog which makes it bypass the calcium requirement for utilization of the substrate by the ATPase.
Oliveira
R G
RG
Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Brazil.
Coan
C
C
Verjovski-Almeida
S
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
C026397:3'(O)-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)-8-azidoadenosine 5'-triphosphate; D000345:Affinity Labels; D001386:Azides; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium