McGovern-Gooch Kayleigh R, Rodrigues Trevor, Darling Joseph E, Sieburg Michelle A, Abizaid Alfonso, Hougland James L
Department of Chemistry (K.R.M.-G., J.E.D., M.A.S., J.L.H.), Syracuse University, Syracuse, New York 13244; and Department of Neuroscience (T.R., A.A.), Carleton University, Ottawa, Ontario, Canada K1S5B6.
Endocrinology. 2016 Nov;157(11):4330-4338. doi: 10.1210/en.2016-1657. Epub 2016 Sep 13.
Ghrelin is a peptide hormone involved in multiple physiological processes related to energy homeostasis. This hormone features a unique posttranslational serine octanoylation modification catalyzed by the enzyme ghrelin O-acyltransferase, with serine octanoylation essential for ghrelin to bind and activate its cognate receptor. Ghrelin deacylation rapidly occurs in circulation, with both ghrelin and desacyl ghrelin playing important roles in biological signaling. Understanding the regulation and physiological impact of ghrelin signaling requires the ability to rapidly protect ghrelin from deacylation in biological samples such as blood serum or cell lysates to preserve the relative concentrations of ghrelin and desacyl ghrelin. In in vitro ghrelin O-acyltransferase activity assays using insect microsomal protein fractions and mammalian cell lysate and blood serum, we demonstrate that alkyl fluorophosphonate treatment provides rapid, complete, and long-lasting protection of ghrelin acylation against serine ester hydrolysis without interference in enzyme assay or ELISA analysis. Our results support alkyl fluorophosphonate treatment as a general tool for stabilizing ghrelin and improving measurement of ghrelin and desacyl ghrelin concentrations in biochemical and clinical investigations and suggest current estimates for active ghrelin concentration and the ghrelin to desacyl ghrelin ratio in circulation may underestimate in vivo conditions.
胃饥饿素是一种参与能量稳态相关多种生理过程的肽类激素。这种激素具有一种独特的翻译后丝氨酸辛酰化修饰，由胃饥饿素O-酰基转移酶催化，丝氨酸辛酰化对于胃饥饿素结合并激活其同源受体至关重要。胃饥饿素脱酰化在循环中迅速发生，胃饥饿素和去酰基胃饥饿素在生物信号传导中都发挥着重要作用。要了解胃饥饿素信号传导的调节和生理影响，需要有能力在生物样品（如血清或细胞裂解物）中快速保护胃饥饿素不被脱酰化，以保持胃饥饿素和去酰基胃饥饿素的相对浓度。在使用昆虫微粒体蛋白组分、哺乳动物细胞裂解物和血清进行的体外胃饥饿素O-酰基转移酶活性测定中，我们证明烷基氟膦酸盐处理能快速、完全且持久地保护胃饥饿素酰化不被丝氨酸酯水解，且不会干扰酶测定或酶联免疫吸附测定分析。我们的结果支持将烷基氟膦酸盐处理作为一种稳定胃饥饿素以及改善生化和临床研究中胃饥饿素和去酰基胃饥饿素浓度测量的通用工具，并表明目前对循环中活性胃饥饿素浓度以及胃饥饿素与去酰基胃饥饿素比率的估计可能低估了体内情况。
