Hooy Richard, Sohn Jungsan
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, United States.
Methods Enzymol. 2019;625:77-86. doi: 10.1016/bs.mie.2019.06.005. Epub 2019 Jul 5.
Cyclic GMP-AMP synthase, cGAS, converts ATP and GTP into a cyclic dinucleotide second messenger, cyclic GMP-AMP or cGAMP, through its enzymatic, nucleotidyl transferase (NTase) activity. Although many methods are available to directly measure cGAMP production, these assays often have high cost of implementation and/or experimental limitations. This chapter details how to implement an alternative approach that is relatively inexpensive, accurate and medium-throughput. The assay measures cGAS NTase activity by quantifying pyrophosphate production, a byproduct of the cGAS reaction. A coupling enzyme, pyrophosphatase, catalyzes the hydrolysis of pyrophosphate into inorganic phosphate, which enables facile detection of cGAS activity through conventional phosphomolybdate-malachite green absorbance methodology. This method is amenable for conventional steady-state kinetic measurements as well as high-throughput compound screening.
环状GMP-AMP合酶（cGAS）通过其酶促核苷酸转移酶（NTase）活性，将ATP和GTP转化为环状二核苷酸第二信使——环状GMP-AMP或cGAMP。虽然有许多方法可直接测量cGAMP的产生，但这些检测方法往往实施成本高昂和/或存在实验局限性。本章详细介绍如何实施一种相对廉价、准确且具有中等通量的替代方法。该检测方法通过量化焦磷酸的产生来测量cGAS NTase活性，焦磷酸是cGAS反应的副产物。一种偶联酶——焦磷酸酶，催化焦磷酸水解为无机磷酸盐，这使得能够通过传统的磷钼酸-孔雀石绿吸光度方法轻松检测cGAS活性。该方法适用于传统的稳态动力学测量以及高通量化合物筛选。
