Involvement of cys298 in the interaction of PGA1 with AKR1B1.

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Involvement of cys298 in the interaction of PGA1 with AKR1B1. Involvement of cys298 in the interaction of PGA1 with AKR1B1. (A) Interaction of PGA1 with an AKR1B1 synthetic peptide. The synthetic peptide 297VCALLSCTSHK307 was incubated with vehicle or PGA1 and the peaks corresponding to the unmodified peptide (m/z 1161.5, upper panel) and the PGA1-peptide adduct (m/z 1498.1, lower panel) were fragmented by MALDI-TOF-TOF MS-MS and the sequences obtained are shown. Asterisks and pound symbols denote the ions for which peaks compatible with the addition of PGA1 on Cys303 and Cys298, respectively, were observed. (B) AKR1B1 was incubated with or without 100 μM GSNO for 30 minutes at RT. The reaction mixtures were then incubated with 1 μM PGA1-B for 90 minutes at RT. AKR1B1 modification was analyzed by SDS-PAGE and blot with HRP-streptavidin and protein levels assessed with an anti-AKR antibody. Lower panel depicts the quantitation of biotin signal corrected by AKR1B1 levels. Results shown are average values of three determinations ± S.E.M. *P < 0.05 versus the absence of GSNO. Beatriz Díez-Dacal et al. Mol Pharmacol 2016;89:42-52 Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics