Benau D A, Schumacher W G, McGuire E J, Fitzpatrick-McElligott S, Storey B T, Roth S
Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia 19104.
J Histochem Cytochem. 1990 Jan;38(1):23-30. doi: 10.1177/38.1.2104633.
We describe an assay for light microscopic visualization of specific glycosyltransferases on tissue sections or on cells. The assay uses a sequence of enzyme reactions that yields two moles of NADH for each mole of the uridine-5'-diphosphate (UDP) released during transfer of a monosaccharide from a UDP sugar to an acceptor. When diaphorase and tetrazolium salts are present in the incubation mixture, the tetrazolium salts are reduced to colored diformazans, which precipitate at the sites of glycosyltransferase activity. The validity of the assay was established by applying the technique to spermatozoa and liver, in which some glycosyltransferases have previously been localized. When suspensions of mouse spermatozoa were assayed for galactosyltransferase (GalTase) activity, diformazan precipitates appeared on the plasma membranes overlying the anterior heads of the spermatozoa, in agreement with immunochemical localizations. In mouse liver slices assayed with bilirubin as acceptor for glucuronyltransferase (GluTase) activity, dense diformazan deposits appeared on the hepatocytes but not on endothelial cells, also in agreement with immunochemical data. In the absence of acceptor or UDP sugar donor, diformazan deposits were minimal and random in all tissues tested. The assay's versatility was tested by incubating tissues with different sugar donors and acceptors to localize other sites of transferase activity. In mouse frozen liver sections, GalTase activity occurred in both hepatocytes and endothelial cells; in sections of rat submaxillary glands, GalTase activity was detected in mast cells. In liver sections, GlcuTase activity with o-aminophenol as acceptor was located primarily on the endothelial cells. With the appropriate sugar donor and acceptor, this assay should detect any transferase, other than the glucosyltransferases, that utilizes UDP sugars.
我们描述了一种用于在组织切片或细胞上对特定糖基转移酶进行光学显微镜观察的检测方法。该检测方法使用一系列酶促反应，每摩尔从尿苷 - 5'-二磷酸（UDP）糖转移到受体的单糖释放过程中会产生两摩尔的NADH。当在孵育混合物中存在黄递酶和四氮唑盐时，四氮唑盐会被还原为有色的双甲臜，其在糖基转移酶活性位点沉淀。通过将该技术应用于精子和肝脏来确定该检测方法的有效性，此前已在其中定位了一些糖基转移酶。当检测小鼠精子悬液中的半乳糖基转移酶（GalTase）活性时，双甲臜沉淀出现在覆盖精子头部前端的质膜上，这与免疫化学定位结果一致。在用胆红素作为葡萄糖醛酸基转移酶（GluTase）活性受体检测的小鼠肝脏切片中，致密的双甲臜沉积物出现在肝细胞上，而在内皮细胞上未出现，这也与免疫化学数据一致。在没有受体或UDP糖供体的情况下，在所有测试组织中双甲臜沉积物极少且分布随机。通过用不同的糖供体和受体孵育组织来定位转移酶活性的其他位点，测试了该检测方法的通用性。在小鼠冷冻肝脏切片中，GalTase活性在肝细胞和内皮细胞中均有出现；在大鼠下颌下腺切片中，在肥大细胞中检测到GalTase活性。在肝脏切片中，以邻氨基酚作为受体的GluTase活性主要位于内皮细胞上。使用合适的糖供体和受体，该检测方法应能检测除利用UDP糖的葡糖基转移酶之外的任何转移酶。
