Bioluminescent determination of 0.1 picomole amounts of guanine nucleotides

Abstract

A bioluminescence procedure for the determination of the guanylates has been optimized to allow measurement of 0.1 pmol amounts. Modifications of the Karl procedure include the use of purified firefly luciferase and nucleoside diphosphate kinase instead of a crude extract of firefly tails, the use of Tricine buffer instead of the inhibitory arsenate buffer, and optimization of the amounts of reagents and incubation times for each of the partial reactions. In the determination of GMP, background values varied widely with different lots of bovine guanylate kinase. Careful selection of a suitable lot of bovine brain guanylate kinase was essential for determination of lower amounts of guanylates. This establishes that selection of guanylate kinase must be based on experimental determination and not reported adenylate kinase activity. The wide variation in background was not eliminated by the inclusion of adenylate kinase inhibitors.

Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling

“…This, in turn, should drive nucleotide exchange in the G α i 3 binding pocket with CCR3 functioning as the guanine nucleotide exchange factor (GEF). The remaining GTP is then converted to a luminescent signal after conversion to ATP ( Figure 6A ) ( Ford et al, 1994 ; Ford and Leach, 1998a ; Ford and Leach, 1998b ). In the absence of a suitable GEF, 20 mM EDTA can be used to stimulate intrinsic activity, verifying construct activity ( Supplementary Figure S4 ).…”