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Free keywords:
Photodissociation; Kinetic parameters; Peptides and proteins; Monomers; Kinetics
Abstract:
Here we report the synthesis and photochemical and biological characterization of a new photolabile precursor of d-aspartic acid, α-carboxynitrobenzyl-caged d-aspartate (α-CNB-caged d-aspartate), and its application for studying the molecular mechanism of the neuronal excitatory amino acid carrier 1 (EAAC1). Investigation of the photochemical properties of α-CNB-caged d-aspartate by transient absorption spectroscopy of the aci-nitro intermediate revealed that it photolyzes with a quantum yield of 0.19 at pH 7.0. The major component of the aci-nitro intermediate (77% of the total absorbance) decays with a time constant of 26 μs. This decay is slowed by only a factor of 2 when increasing the pH to 10. A minor component (21%) decays with a time constant of 410 μs and is pH insensitive. The compound was tested with respect to its biological activity with the glutamate transporter EAAC1 expressed in HEK293 cells. Whole-cell current recordings from these cells in the presence and absence of α-CNB-caged d-aspartate demonstrated that the compound neither activates nor inhibits EAAC1. Upon photolysis, d-aspartate-mediated whole-cell currents were generated. In contrast to laser-pulse photolysis experiments with α-CNB-caged l-glutamate, only a minor and much slower transient current component was observed. These results indicate that the substrate translocation step, which is not rate-limiting for the overall turnover of the transporter with l-glutamate, becomes rate-limiting when d-aspartate is translocated. The results demonstrate that the new caged d-aspartate derivative is a useful tool for the investigation of the molecular mechanism of glutamate transporters and probably other aspartate translocating systems using rapid chemical kinetic techniques.
