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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Cell Metab. 2014 Apr 1;19(4):605–617. doi: 10.1016/j.cmet.2014.03.014

Figure 3. SIRT5 catalyzes lysine deglutarylation reactions in vitro and in vivo.

Figure 3

(A) Screening of HDAC lysine deglutarylation activity in vitro. Fluorometric assay to detect in vitro lysine deglutarylation activities using recombinant SIRT1-7.

(B) 32P-NAD+ consumption monitored by thin layer chromatography after an in vitro SIRT3, SIRT4, or SIRT5 enzymatic assay using chemically acylated BSA as a substrate; o-glutaryl-ADP ribose, OG-ADPR; o-succinyl-ADP ribose, OS-ADPR; o-acetyl-ADP ribose, OA-ADPR.

(C) HPLC trace of a glutarylated peptide, VKSKgluATNLWW, before and after in vitro deglutarylation reaction. The assays were carried out without hSIRT5, with hSIRT5, with enzymatically-inactive mutant of hSIRT5 (H158Y), without NAD+, with SIRT5 in the presence of nicotinamide (25 mM), or sirtinol (200 µM), or TSA (2 µM), or sodium butyrate (NaBu) (25mM). A triangle and a diamond indicate modified (HRMS, m/z, 673.8588 Da) and unmodified (HRMS, m/z, 616.8439 Da) peptides, respectively.