Alteration Of NAMPT In Aged Mice Brain And Serum And Preparation And Enzymatic Detection Of Recombined Human NAMPT

Nicotinomide phosphoribosyltransferase (NAMPT) is a key enzyme for NAD biosynthesis. NAMPT is involved in age-related disorders, such as cancers, diabetes. In brain, NAMPT is expressed in neurons and plays a protective role against ischemia. However, the detailed expression of NAMPT in brain, its changes during aging, and its correlation with behavioral performance are not clear. In this study, we found a region-specific change of NAMPT expression during aging-NAMPT level dropped in cortex and hippocampus whereas increased in serum; we also found that NAMPT was expressed in microglia of aged mice but not in young mice, and NAD level decreased in hippocampus and cerebellum while remained constant in cortex and striatum. In behavioral tests, aged mice showed attenuated motor activity, central exploration and memory. The expression of NAMPT in cortex was positively correlated with motor activity, while that in cerebellum and serum was negatively correlated. Thus, we have demonstrated an age-related expression, distribution and activity pattern of NAMPT in brain and serum, which could be responsible for the declined motor activity during aging. Objective:To prepare and purify recombinant human NAMPT and NAMPT (H247A) protein, and detection their enzymatic activity.Methods:Using pcDNA3.1-hnampt as template, full-length hnampt was sub-cloned into pET-11a(+) plasmid. The hnampt (H247A) muatant was obtained by site-directed mutagenesis. The plasmids were introduced in Escherichia coli BL21star for protein expression. The recombined NAMPT and NAMPT(H247A) were purified by flowing through nickel column and size-exclusion column. The target proteins were confirmed by SDS-PAGE and mass spectrometry detection. The enzymatic activities were assessed by solution NMR.Results:The DNA sequences showed that hnampt (wild type) and hnampt (H247A) (mutation) were cloned into pET-11a(+). The recombinant proteins were expressed in Escherichia coli BL21star in soluble form. The purified protein was confirmed to be NAMPT with a molecular weight of 56 KD. The enzyme activity of NAMPT (H247A) was dramatically decreased relative to wildtype NAMPT.Conclusion:The recombinant hNAMPT and hNAMPT (H247A) were successful prepared and purified. The H247A mutation dramatically decreased the enzymatic activity of NAMPT. This work provides a basis for further research on the NAMPT protein.