绿豆幼苗 UDP-GlcNAc：Dolichyl-Pyrophosphoryl-GlcNAc GlcNAc 转移酶的纯化和性质。
Purification and Properties of UDP-GlcNAc:Dolichyl-Pyrophosphoryl-GlcNAc GlcNAc Transferase from Mung Bean Seedling.
机构信息
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284.
出版信息
Plant Physiol. 1986 Aug;81(4):1086-91. doi: 10.1104/pp.81.4.1086.
The N-acetylglucosamine (GlcNAc) transferase that catalyzes the formation of dolichyl-pyrophosphoryl-GlcNAc-GlcNAc from UDP-GlcNAc and dolichyl-pyrophosphoryl-GlcNAc was solubilized from the microsomal enzyme fraction of mung beans with 1.5% Triton X-100, and was purified 140-fold on columns of DE-52 and hydroxylapatite. The partially purified enzyme preparation was quite stable when stored in 20% glycerol and 0.5 millimolar dithiothreitol, and was free of GlcNAc-1-P transferase and mannosyl transferases. The GlcNAc transferase had a sharp pH optimum of 7.4 to 7.6 and the K(m) for dolichyl-pyrophosphoryl-GlcNAc was 2.2 micromolar and that for UDP-GlcNAc, 0.25 micromolar. The enzyme showed a strong requirement for the detergent Triton X-100 and was stimulated somewhat by the divalent cation Mg(2+). Uridine nucleotides, especially UDP and UDP-glucose inhibited the enzyme as did the antibiotic, diumycin. However, a variety of other antibiotics including tunicamycin were without effect. The product of the reaction was characterized as dolichyl-pyrophosphoryl-GlcNAc-GlcNAc.
从绿豆微粒体酶部分用 1.5%Triton X-100 溶解，催化从 UDP-GlcNAc 和焦磷酸化多萜醇-GlcNAc-GlcNAc 形成焦磷酸化多萜醇-GlcNAc-GlcNAc 的 N-乙酰氨基葡萄糖（GlcNAc）转移酶，并在 DE-52 和羟磷灰石柱上纯化 140 倍。部分纯化的酶制剂在 20%甘油和 0.5 毫摩尔二硫苏糖醇中储存时非常稳定，且不含 GlcNAc-1-P 转移酶和甘露糖转移酶。GlcNAc 转移酶的 pH 最适值为 7.4 至 7.6，对焦磷酸化多萜醇-GlcNAc 的 K(m)为 2.2 微摩尔，对 UDP-GlcNAc 的 K(m)为 0.25 微摩尔。该酶对去污剂 Triton X-100 有强烈的需求，二价阳离子 Mg(2+)对其有一定的刺激作用。尿苷核苷酸，尤其是 UDP 和 UDP-葡萄糖，以及抗生素二霉素都抑制该酶，但其他多种抗生素，包括衣霉素，均无影响。反应产物被鉴定为焦磷酸化多萜醇-GlcNAc-GlcNAc。
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