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  Substrate Translocation Kinetics of Excitatory Amino Acid Carrier 1 Probed with Laser-Pulse Photolysis of a New Photolabile Precursor of d-Aspartic Acid

Grewer, C., Mobarekeh, S. A. M., Watzke, N., Rauen, T., & Schaper, K. (2001). Substrate Translocation Kinetics of Excitatory Amino Acid Carrier 1 Probed with Laser-Pulse Photolysis of a New Photolabile Precursor of d-Aspartic Acid. Biochemistry, 40(1), 232-240. doi:10.1021/bi0015919.

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 Creators:
Grewer, Christof1, Author           
Mobarekeh, Sayed Abdollah Madani 2, Author
Watzke, Natalie1, Author           
Rauen, Thomas3, Author
Schaper, Klaus2, Author
Affiliations:
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society, ou_2068289              
2Institut für Organische Chemie und Makromolekulare Chemie I, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany, ou_persistent22              
3Max-Planck-Institut für Hirnforschung, Deutschordenstrasse 46, D-60528 Frankfurt, Germany , ou_persistent22              

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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.

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Language(s): eng - English
 Dates: 2000-10-122000-07-102000-12-092001-01-01
 Publication Status: Published in print
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/bi0015919
 Degree: -

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Title: Biochemistry
Source Genre: Journal
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Publ. Info: Columbus, Ohio : American Chemical Society
Pages: 304 Volume / Issue: 40 (1) Sequence Number: - Start / End Page: 232 - 240 Identifier: ISSN: 0006-2960
CoNE: https://pure.mpg.de/cone/journals/resource/954925384103