Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, 2000, Rosario, Argentina.
Mol Biol Rep. 2012 Jan;39(1):585-91. doi: 10.1007/s11033-011-0774-6. Epub 2011 May 17.
The metabolic pathways leading to the synthesis of bacterial glycogen involve the action of several enzymes, among which glycogen synthase (GS) catalyzes the elongation of the α-1,4-glucan. GS from Agrobacterium tumefaciens uses preferentially ADPGlc, although UDPGlc can also be used as glycosyl donor with less efficiency. We present here a continuous spectrophotometric assay for the determination of GS activity using ADP- or UDPGlc. When ADPGlc was used as the substrate, the production of ADP is coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH). With UDPGlc as substrate, UDP was converted to ADP via adenylate kinase and subsequent coupling to PK and LDH reactions. Using this assay, we determined the kinetic parameters of GS and compared them with those obtained with the classical radiochemical method. For this purpose, we improved the expression procedure of A. tumefaciens GS using Escherichia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as sugar-nucleotide donors.
导致细菌糖原合成的代谢途径涉及几种酶的作用，其中糖原合酶 (GS) 催化α-1,4-葡聚糖的延伸。根瘤农杆菌中的 GS 优先使用 ADPGlc，但 UDPGlc 也可以作为糖基供体，但效率较低。我们在这里提出了一种使用 ADP-或 UDPGlc 测定 GS 活性的连续分光光度法。当 ADPGlc 作为底物时，通过丙酮酸激酶 (PK) 和乳酸脱氢酶 (LDH) 将 ADP 的产生与 NADH 氧化偶联。当 UDPGlc 作为底物时，UDP 通过腺苷酸激酶转化为 ADP，随后与 PK 和 LDH 反应偶联。使用该测定法，我们确定了 GS 的动力学参数，并将其与经典放射性化学方法获得的参数进行了比较。为此，我们使用大肠杆菌 BL21(DE3)-RIL 细胞改进了根瘤农杆菌 GS 的表达程序。该测定法允许使用 ADPGlc 或 UDPGlc 作为糖核苷酸供体连续监测糖基转移酶活性。
