Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Plant J. 2009 Dec;60(5):894-906. doi: 10.1111/j.1365-313X.2009.03997.x. Epub 2009 Aug 13.
Traditional methods to localize beta-glycosidase activity in tissue sections have been based on incubation with the general substrate 6-bromo-2-naphthyl-beta-d-glucopyranoside. When hydrolysed in the presence of salt zinc compounds, 6-bromo-2-naphthyl-beta-d-glucopyranoside affords the formation of an insoluble coloured product. This technique does not distinguish between different beta-glycosidases present in the tissue. To be able to monitor the occurrence of individual beta-glycosidases in different tissues and cell types, we have developed a versatile histochemical method that can be used for localization of any beta-glycosidase that upon incubation with its specific substrate releases a reducing sugar. Experimentally, the method is based on hydrolysis of the specific substrate followed by oxidation of the sugar released by a tetrazolium salt (2,3,5-triphenyltetrazolium chloride) that forms a red insoluble product when reduced. The applicability of the method was demonstrated by tissue and cellular localization of two beta-glucosidases, amygdalin hydrolase and prunasin hydrolase, in different tissues and cell types of almond. In those cases where the analysed tissue had a high content of reducing sugars, this resulted in strong staining of the background. This interfering staining of the background was avoided by prior incubation with sodium borohydride. The specificity of the devised method was demonstrated in a parallel localization study using a specific antibody towards prunasin hydrolase.
传统的组织切片β-葡糖苷酶活性定位方法基于与一般底物 6-溴-2-萘基-β-d-吡喃葡萄糖苷孵育。当在盐锌化合物存在下水解时，6-溴-2-萘基-β-d-吡喃葡萄糖苷会形成不溶性有色产物。该技术无法区分组织中存在的不同β-葡糖苷酶。为了能够监测不同组织和细胞类型中个体β-葡糖苷酶的发生，我们开发了一种通用的组织化学方法，可用于定位用其特定底物孵育后释放还原糖的任何β-葡糖苷酶。在实验中，该方法基于特定底物的水解，然后用四唑盐（2,3,5-三苯基氯化四氮唑）氧化释放的糖，当被还原时，四唑盐形成红色不溶性产物。该方法的适用性通过杏仁中两种β-葡糖苷酶（苦杏仁苷酶和李苷酶）在不同组织和细胞类型中的组织和细胞定位得到了证明。在分析组织中含有大量还原糖的情况下，这会导致背景强烈染色。通过事先用硼氢化钠孵育，可以避免这种背景的干扰染色。在使用针对李苷酶的特异性抗体进行的平行定位研究中，证明了设计方法的特异性。
