Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.
Anal Biochem. 2011 Jul 15;414(2):278-81. doi: 10.1016/j.ab.2011.04.001. Epub 2011 Apr 6.
Here we report a new fluorescence-based assay for measuring MshB (N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase) activity. The current assay for measuring MshB activity requires the fluorescent labeling of reaction mixtures and subsequent analysis using high-performance liquid chromatography (HPLC), resulting in a significant amount of processing time per sample. Here we describe a more rapid fluorescnce-based assay for the measurement of MshB activity that does not require HPLC analysis and can be carried out in multiwell plates. This fluorescamine (FSA)-based assay was used to determine the steady-state parameters for the deacetylation of N-acetyl-glucosamine (GlcNAc) by MshB, and the results from these experiments support the hypothesis that the inositol moiety primarily contributes to the affinity of GlcNAc-Ins (N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside) for MshB. The rapid nature of this assay will aid efforts toward a more detailed biochemical characterization of MshB. Furthermore, because this assay relies on the formation of a primary amine, it could be adapted to measure the activity of mycothiol-S-conjugate amidase, a metal-dependent amidase that is a potential drug target involved in the mycothiol detoxification pathway.
我们在此报告了一种新的荧光测定法，用于测量 MshB（N-乙酰基-1-d-肌醇基-2-氨基-2-脱氧-α-d-吡喃葡萄糖苷脱乙酰酶）活性。目前测量 MshB 活性的方法需要对反应混合物进行荧光标记，然后使用高效液相色谱法（HPLC）进行分析，导致每个样品的处理时间都很长。在这里，我们描述了一种更快速的基于荧光的 MshB 活性测量方法，该方法不需要 HPLC 分析，可以在多孔板中进行。该荧光胺（FSA）测定法用于确定 MshB 对 N-乙酰葡萄糖胺（GlcNAc）脱乙酰化的稳态参数，这些实验的结果支持了这样的假设，即肌醇部分主要有助于 GlcNAc-Ins（N-乙酰基-1-d-肌醇基-2-氨基-2-脱氧-α-d-吡喃葡萄糖苷）与 MshB 的亲和力。该测定法的快速性质将有助于更详细地对 MshB 进行生化特征分析。此外，由于该测定法依赖于伯胺的形成，因此可以对其进行修改，以测量巯基乙胺-S-结合物酰胺酶的活性，巯基乙胺-S-结合物酰胺酶是一种依赖于金属的酰胺酶，是参与巯基乙胺解毒途径的潜在药物靶标。
