Article Title: FAMIN Is a Multifunctional Purine Enzyme Enabling the Purine Nucleotide Cycle
Figure Lengend Snippet: FAMIN Metabolizes Purine Nucleosides, Related to Figure 1 (A) Coomassie SDS-PAGE of recombinant human FAMIN 254I and FAMIN 254V following Strep-Tactin affinity purification. Lanes indicate ladder (L), FAMIN 254I or FAMIN 254V transfected HEK293T lysate input, column flow-through and concentrated protein eluate. (B) Left, size exclusion chromatogram of affinity purified FAMIN that has undergone TEV-cleavage to remove Strep-tag. Blue trace corresponds to A280 (protein) and purple trace to A260 (DNA) signal. Fractions C6-C8 were collected, concentrated, and subjected to Coomassie SDS-PAGE. Inset depicts entire chromatogram. Right, Coomassie SDS-PAGE of fractions obtained from size exclusion chromatography. Lanes indicate ladder (L) and fractions B12, C5, C6, C7, C8 and C9, corresponding to the size exclusion chromatogram, and the concentrated protein from fractions C6-C8. (C) Differential scanning fluorimetry (DSF) of recombinant human FAMIN. (D) Cell proliferation of HepG2 cells silenced for FAMIN (si FAMIN ) or transfected with scrambled siRNA (siCtrl) as measured by CyQUANT assay (n = 12). (E) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of HepG2 cells 48 h after transfection with FAMIN or control siRNA. Basal OCR measurement was followed by sequential treatment (dotted vertical lines) with oligomycin A (Oligo), FCCP, and rotenone plus antimycin A (Rot + ant). Basal ECAR measurement was followed by sequential treatment with oligomycin (Oligo) and 2-deoxyglucose (2-DG) (n = 3). (F) Representative mass spectra and extracted chromatograms for putative FAMIN-catalyzed metabolites and corresponding standards for inosine, hypoxanthine and guanine. (G) Guanosine and guanine levels following incubation of HepG2 cell aqueous extract with 10 μg recombinant FAMIN 254I in 100 μL PBS. (n = 3). (H) Left, Representative extracted chromatograms for FAMIN-catalyzed compound ‘f’ and corresponding standards for ribose-1-phosphate, ribose-5-phosphate, ribulose-5-phosphate and xylulose-5-phosphate. All measurements performed using a BEH amide HILIC column and TSQ Quantiva triple quadrupole. Right, Ratio of selected reaction monitoring (SRM) daughter ions with nominal m/z values of 79 and 97. (I) Inosine, guanosine, cytidine, uridine and ATP levels following incubation of 0.1, 1.0, 10.0 or 100.0 μg of recombinant FAMIN 254I with the complete metabolomic library (aqueous phase of methanol:chloroform extract of FAMIN -silenced HepG2 cells) in 100 μL PBS (n = 3). (J) LC-MS peaks putatively identified as adenine, hypoxanthine, inosine, or ribose-1-phosphate with nominal m/z values of 136, 137, 269 and 229, respectively, were selectively targeted and fragmented using a higher-energy collision dissociation (HCD) collision voltage of 25 eV to give the fragments shown. Data are represented as mean ± SEM or representative of at least 3 independent experiments. ∗ p
Article Snippet: For targeted analysis, samples were analyzed using a Quantiva triple stage quadrupole mass spectrometer coupled to a Vanquish Horizon (all analytical instrument combinations supplied by Thermo Fisher Scientific).
Techniques: SDS Page, Recombinant, Affinity Purification, Transfection, Strep-tag, Size-exclusion Chromatography, CyQUANT Assay, Incubation, Hydrophilic Interaction Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy