Zymograph profiling reveals a divergent evolution of sirtuin that may originate from class III enzymes.

Sirtuins are a group of NAD+-dependent deacylases that conserved in three domains of life and comprehensively involved in the regulation of gene transcription, chromosome segregation, RNA splicing, apoptosis, and aging. Previous studies in mammalian cells have revealed that sirtuins not only exist as multiple copies, but also show distinct deacylase activities in addition to deacetylation. However, the understanding of sirtuin zymographs in other organisms with respect to molecular evolution remains at an early stage. Here, we systematically analyze the sirtuin activities in representative species from archaea, bacteria, and eukaryotes, using both the HPLC assay and a 7-amino-4-methylcoumarin-based fluorogenic method. Global profiling suggests that the deacylase activities of sirtuins could be divided into three categories and reveals undifferentiated zymographs of class III sirtuins, especially for those from bacteria and archaea. Nevertheless, initial differentiation of enzymatic activity was also observed for the class III sirtuins at both paralog and ortholog levels. Further phylogenetic analyses support a divergent evolution of sirtuin that may originate from class III sirtuins. Together, this work demonstrates a comprehensive panorama of sirtuin zymographs and provides new insights into the cellular specific regulation and molecular evolution of sirtuins.

Zymography assisted quick purification, characterization and inhibition analysis of K. pneumoniae alkaline phosphatase by mercury and thiohydroxyal compounds.

In-gel hydrolysis of para-nitrophenyl phosphate (p-NPP) to yellow colored para-nitrophenol was used to locate precisely the K. pneumoniae alkaline phosphatase (Kp-ALKP) on 7% native PAGE. Subsequent removal of the yellow-stained band and electroelution yielded a 54 kDa, Kp-ALKP with Km, Vmax and kcat values of (0.7 ± 0.02) mM, (80 ± 4.5) µmol min-1 and (39.2 ± 2.2) × 104 s-1 respectively for p-NPP. Kp-ALKP was optimally active at 70 °C and pH 7.2 that was activated by Mg2+, Ca2+, Co2+ and inhibited by EDTA, PO4, Pb2+, Cu2+ and Hg2+. The enzyme was trypsin resistant and retained 75% activity in presence of 10 mM PO4 and 65% activity at 3 mM Hg2+ showing it's PO43- irrepressibility and Hg2+-tolerance. Molecular dynamics simulation revealed increased structural stability of Kp-ALKP at 70 °C that accounts for it's optimal temperature. Zymography revealed that both DTT and ß-mercaptoethanol induced activity loss accompanied by mobility retardation of Kp-ALKP on 7% native PAGE. These results and in Silico analysis shows that both DTT and ßME reduce the C308-C358 disulfide bond, leading to an open conformation of the enzyme. However, Hg2+ had negligible effect on the in-gel mobility of Kp-ALKP indicating it's plausible non-covalent interaction with surface-accessible amino-acids without significant conformational change. For the first time our study reveals the zymography as an easy, inexpensive and convenient tool for quick purification, characterization and conformational analysis of K. pneumoniae alkaline phosphatase.