Fig. 5.

³¹P-NMR in the presence of L-arginine amide identifies InsP₅ [1-OH] as the XopH reaction product. a Upper trace: Mixture of InsP₅ [1-OH] (A) and InsP₅ [3-OH] (B) in different ratios in ammonium acetate buffer (pH 7.1) in the absence of L-arginine amide (L-Arg-N). No peak separation was observed and integration gives the expected values 1:1:2:1 (from left to right). Addition of L-Arg-N in excess (ca. 100-fold) leads to separation of the resonances of InsP₅ [1-OH] (A) or InsP₅ [3-OH] (B) in all three different ratios studied (1:1.5, 1:1, and 1.5:1). Asterisks mark an impurity. b Digest of 600 nmol InsP₆ by XopH in ammonium acetate buffer (pH 7.1), spiking with 400 nmol InsP₅ [1-OH] and addition of L-Arg-N in excess (ca. 100-fold). No additional peaks can be seen in a proton-decoupled spectrum (upper trace). A proton-coupled spectrum identifies all resonances belonging to inositol-bound phosphates (A). c Digest of 600 nmol InsP₆ by XopH, spiking with InsP₅ [1-OH] and addition of L-Arg-N in excess (ca. 100-fold). The mixture was heated to 95 °C for 15 min to denature residual XopH. Integration identifies the phosphate resonance (expected ratio is 1.0:0.6) and shows that no decomposition after boiling takes place. d XopH digest of 600 nmol and excess L-Arg-N (ca. 100-fold) boiled at 95 °C for 15 min. Subsequent addition of InsP₅ [3-OH] (B) in different ratios leads to appearance of new resonances