Klebl B M, Pette D
Fakultät für Biologie, Universität Konstanz, Konstanz, D-78434, Germany.
Anal Biochem. 1996 Aug 1;239(2):145-52. doi: 10.1006/abio.1996.0309.
Using 1,N6-etheno NAD, a fluorescent analog of NAD, we extended an existing assay for NAD glycohydrolase to the measurement of mono-ADP-ribosyltransferase (mADP-RT) activity using agmatine as acceptor for ADP-ribose. The reaction products were analyzed by reversed-phase chromatography. In the presence of agmatine two newly formed fluorescent products were tentatively identified as ADP-ribosylagmatine anomers. Fluorescence intensity increased upon splitting the N-glycoside bondage of 1,N6-etheno NAD. Therefore, 1, N6-etheno AMP could be used for calibration. The nonradioactive assay yielded values nearly identical to those obtained with the [carbonyl-14C]NAD method. It proved to be highly reproducible, rapid, and suitable for an improved purification protocol yielding a 76,000-fold enriched mADP-RT preparation from rabbit skeletal muscle. The identity and high purity of the enzyme were confirmed immunochemically. The assay served to determine the pH optimum of the enzyme (pH 9.0) and its KM for 1,N6-etheno NAD (287 microM).
我们使用NAD的荧光类似物1,N6-乙烯基NAD，将现有的NAD糖水解酶检测方法扩展为以胍丁胺作为ADP-核糖受体来测量单ADP-核糖基转移酶（mADP-RT）活性。通过反相色谱分析反应产物。在胍丁胺存在的情况下，两种新形成的荧光产物初步鉴定为ADP-核糖基胍丁胺异头物。1,N6-乙烯基NAD的N-糖苷键断裂时荧光强度增加。因此，1,N6-乙烯基AMP可用于校准。该非放射性检测方法得到的值与用[羰基-14C]NAD方法获得的值几乎相同。它被证明具有高度可重复性、快速，并且适用于改进的纯化方案，从兔骨骼肌中获得了富集76000倍的mADP-RT制剂。通过免疫化学方法确认了该酶的同一性和高纯度。该检测方法用于确定该酶的最适pH（pH 9.0）及其对1,N6-乙烯基NAD的米氏常数（287 microM）。
