Figure 1: HPLC and NMR analysis of the products of Ins(1,4,5)P3 and InsP6 phosphorylation by EhIP6KA. | Nature Communications

Figure 1: HPLC and NMR analysis of the products of Ins(1,4,5)P3 and InsP6 phosphorylation by EhIP6KA.

From: IP6K structure and the molecular determinants of catalytic specificity in an inositol phosphate kinase family

Figure 1

(a) HPLC analysis (Partisphere SAX, Gradient 1; see Methods) of InsP6 phosphorylation by either EhIP6KA (57 ng, 20 min; solid line) or HsIP6K2 (7.6 ng, 20 min; dotted line). (b) NMR analysis of the products of the reaction between InsP6 and ATP catalysed by EhIP6KA; the figure depicts an overlay of the 1D 1H and 2D 1H/31P HSQC spectra. The acquisition parameters are given under Methods. (c) HPLC analysis (Partisphere SAX, Gradient 2; see Methods) of the phosphorylation of ~\n9,000 d.p.m. [14C]Ins(1,3,4,5,6)P5 by EhIP6KA (6 μg, 20 min, 50 μl; filled circles). Also shown are the elution of [3H]-labelled internal standards of PP-InsP4 and [PP]2-InsP3, as well as InsP6 (open circles). (d,e) depict representative HPLC assays (90 min; 200 μl) that contained ~\n10,000 d.p.m. [3H]Ins(1,4,5)P3 plus 2 μg EhIP6KA. After quenching and neutralization, products (filled circles) were analysed by Q100 SAX HPLC (Gradient 3); d includes the elution positions of standards of [3H]Ins(1,2,4,5)P4 and [3H]InsP6 (open circles), determined in parallel HPLC runs; e zooms in on the InsP4 region of the chromatograph; elution of internal [14C] standards of Ins(1,3,4,5)P4 and Ins(1,4,5,6)P4 are also shown (open circles).

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