Characterization of TAT-mediated transport of detachable kinase substrates

Biochemistry
Joseph S SoughayerNancy L Allbritton

Abstract

The conjugation of peptides derived from the HIV TAT protein to membrane-impermeant molecules has gained wide acceptance as a means for intracellular delivery. Numerous studies have addressed the mechanism of uptake and kinetics of TAT translocation, but the cytosolic concentrations and bioavailability of the transported cargo have not been well-characterized. The current paper utilizes a microanalytical assay to perform quantitative single-cell measurements of the concentration and accessibility of peptide-based substrates for protein kinase B (PKB) and Ca(2+)/calmodulin-activated kinase II. The substrate peptide and TAT were conjugated through a releasable linker, either a disulfide or photolabile bond. Free substrate peptide concentrations of approximately 10(-20)-10(-18) moles were attainable in a cell when substrates were delivered utilizing these conjugates. The substrate peptides delivered as a disulfide conjugate were often present in the cytosol as several oxidized forms. Brief exposure of cells loaded with the photolabile conjugates to UVA light released free substrate peptide into the cytosol. Substrate peptide delivered by either conjugate was accessible to cytosolic kinase as demonstrated by the efficient phosphory...Continue Reading

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Citations

Feb 11, 2005·Analytical and Bioanalytical Chemistry·Melissa Gayton-ElyLisa A Holland
Jun 2, 2005·Analytical and Bioanalytical Chemistry·Karen J OlsonEdgar A Arriaga
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Nov 26, 2009·Biochimica Et Biophysica Acta·Soline AubryFabienne Burlina
Jun 12, 2013·Chemical Communications : Chem Comm·Kenji UsuiHisakazu Mihara
May 16, 2019·Chemical Reviews·Patrick G DoughertyDehua Pei

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