Partial purification and characterization of beta-mannosyltransferase from suspension-cultured soybean cells.
Partial purification and characterization of beta-mannosyltransferase from suspension-cultured soybean cells.
复制标题
悬浮培养大豆细胞中 β-甘露糖基转移酶的部分纯化和表征。
DOI：
10.1021/bi00398a061
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Elbein,AD
中科院分区：
文献类型：
--
作者：
Kaushal,GP;Elbein,AD
德克萨斯大学健康科学中心生物化学系，圣安东尼奥，德克萨斯州78284接收日期：1987年2月5日;修订后的手册，接收日期：1987年7月13日摘要：通过用1.5%的甘露糖处理，从悬浮培养的大豆细胞的微粒体中溶解催化由GDP-甘露糖和长甘油-PP-GlcNAc-GlcNAc合成Man-β-GlcNAc-PP-长甘油的β-甘露糖基转移酶。Triton X-100，并通过在DEAE-纤维素、羟基磷灰石和GDP亲和柱上的色谱法纯化约700倍。纯化的酶在20%甘油和0.5 mM二硫苏糖醇存在下相当稳定。该酶需要去污剂（Triton X-100或NP-40）或磷脂才能获得最大活性，但这两种作用不是相加的。因此，无论是磷脂酰胆碱或Triton X-100可以提供最大的刺激。在磷脂刺激方面，头基和酰基链似乎都很重要，因为具有18个碳不饱和脂肪酸的磷脂酰胆碱最有效。纯化的酶具有6.9-7.0的最适pH值，并且需要二价阳离子。Mg 2+是最佳金属离子，最佳活性发生在6 mM，但Mn 2+是合理有效的，而Ca 2+是轻微的刺激。GDP-甘露糖的Km计算为1.7 × 10 - 6 M，长羟甲基-PP-GlcNAc-GlcNAc的Km计算为约9 × 10 - 6 M。该酶被一些鸟苷酸如GDP-葡萄糖、GDP、GMP和GTP抑制，但各种尿苷和腺苷酸不起作用。纯化的酶显然不含α-1，3-甘露糖基转移酶（以及可能的其它甘露糖基转移酶）和长酚-β-甘露糖合酶，因为从长酚-PP-GlcNAc-GlcNAc和GDP-甘露糖看到的唯一产物是Man-β-GlcNAc-GlcNAc-PP-长酚。当长叶醇-β-甘露糖代替GDP-甘露糖时，没有观察到活性。
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas78284 Received February 5, 1987; Revised Manuscript Received July 13, 1987 abstract: The/3-mannosyltransferase that catalyzes the synthesis of Man-/3-GlcNAc-GlcNAc-PP-dolichol from GDP-mannoseand dolichyl-PP-GlcNAc-GlcNAc was solubilized from microsomes of suspension-cultured soybean cells by treatment with 1.5% Triton X-100 and was purified about 700-fold by chromatography on DEAE-cellulose, hydroxylapatite, and a GDP affinity column. The purified enzyme was reasonably stable in the presence of 20% glycerol and 0.5 mM dithiothreitol. The enzyme required either detergent (Triton X-100 or NP-40) or phospholipid for maximum activity, but the effects of these two were not additive. Thus, either phosphatidylcholine or Triton X-100 could give maximum stimulation. In terms of phospholipid stimulation, both thehead group and the acyl chain appeared to be important since phosphatidylcholines with 18-carbon unsaturated fatty acids were most effective. The purified enzyme had a sharp pH optimum of 6.9-7.0 and required a divalent cation. Mg2+ was the best metal ion with optimum activity occurring at 6 mM, but Mn2+ was reasonably effective while Ca2+ was slightly stimulatory. The Km for GDP-mannose was calculated to be 1.7 X 10 “6 M and that for dolichyl-PP-GlcNAc-GlcNAc about 9 X 10" 6 M. The enzyme was inhibited by a number of guanosine nucleotides such as GDP-glucose, GDP, GMP, and GTP, but various uridine and adenosine nucleotides were without effect. The purified enzyme was apparently free of a-1, 3-mannosyltransferase (and perhaps other mannosyltransferases) and dolichyl-P-mannose synthase since the only product seen from dolichyl-PP-GlcNAc-GlcNAc and GDP-mannose was Man-/3-GlcNAc-GlcNAc-PP-dolichol. No activity was observed when dolichyl-P-mannose replaced the GDP-mannose.