Purpose. Recoverin (Rv) can extend the lifetime of activated rhodopsin by inhibiting rhodopsin kinase (RK). Elevated Ca2+ levels in dark-adapted photoreceptors favor binding of Rv to RK and inhibition of RK activity. We applied Surface Plasmon Resonance (SPR) technology (BIAcore Instrument) to dissect this mechanism by focusing directly on the protein-protein interaction between Rv and RK. Methpd. SPR monitors interactions between a protein immobilized on a sensor chip and a ligand delivered in solution. The interaction is recorded in real time so binding rate constants can be measured directly. We coupled Rv to the sensor chip while solutions of RK were delivered under various conditions. Results. Binding of RK to Rv is fast. The kass is > 10s Ms-1 and k-4ss is O.ls '. The apparent Kd, 1 jiM, determined as kjjss lkass was in agreement with the concentration of RK required for half-saturation under equilibrium conditions. No significant difference in affinity or kinetics was observed between myristoylated and non-acylated forms of Rv. The Ca2+-dependence had an EC;o of 500 nM for myristoylated and 100 nM for non-acylated Rv. ATP inhibited binding of RK to Rv 5-fold while ADP stimulated the interaction by 50%. The effect was specific for adenine nucleotides. The ECso's for both ATP and ADP are 10'6 M. Conclusions. (1) SPR analysis demonstrated that dissociation of RK from Rv is significantly faster than for interactions between other visual cascade proteins such as Ta-PDE or Tβy-phosducin. The moderate affinity and fast on/off kinetics make RK-Rv system very sensitive to Ca2+ fluctuations. (2) RK-Rv binding is regulated by physiological concentrations of free Ca2+. (3) Acylation of Rv does not affect the RvRK binding directly. Instead it appears to enhance the RK-Rv interaction by increasing the local concentration of Rv at the disc membrane surface. (4) The new assay of RKRv binding on BIAcore revealed the effects of adenine nucleotides which would be difficult to detect and interpret using a standard phosphorylation test with [32P]ATP. The molecular mechanism of ATP action will be discussed.
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Dec 1 1997|
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