J. Biol. Chem., Vol. 263, Issue 4, 1861-1867, 02, 1988
Utilization of arylazido-ATP by sarcoplasmic reticulum ATPase in the absence of calcium
RG Oliveira, C Coan and S Verjovski-Almeida
Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Brazil.
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.
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