Purification and characterization of rat liver glycosylasparaginase.
Purification and characterization of rat liver glycosylasparaginase.
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大鼠肝糖基天冬酰胺酶的纯化和表征。
DOI：
10.1042/bj2600101
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
AronsonJr,NN
中科院分区：
文献类型：
--
作者：
Tollersrud,OK;AronsonJr,NN
1.通过盐分级分离、CM-纤维素和DEAE-纤维素层析、Ultrogel AcA-54凝胶过滤、伴刀豆球蛋白A-Sepharose亲和层析、70 ℃热处理和制备SDS/聚丙烯酰胺凝胶电泳，将大鼠肝糖基天冬酰胺酶[N4-（β-N-乙酰葡糖胺）-L-天冬酰胺酶，EC 3.5.1.26]纯化至均一。纯化的酶以N4-（β-N-乙酰葡糖胺）-L-天冬酰胺为底物，比活为3.8 μ mol N-乙酰葡糖胺/min/mg。2.天然酶的分子量为49 kDa，由两个不同的亚基组成，通过强非共价力连接，并通过SDS/聚丙烯酰胺凝胶电泳测定分子量为24和20 kDa。3. 20 kDa亚基含有一条高甘露糖型寡糖链，24 kDa亚基含有一条高甘露糖型寡糖链和一条复合型寡糖链。4.每个亚基的N-末端序列分析显示24 kDa亚基的N-末端有磨损，同一亚基中的Asn-15有明显的N-糖基化。5.该酶表现出高于7的宽pH最大值。在pH 6.4和6.6下发现两种主要的等电形式。6.糖基天冬酰胺酶在75 ℃和pH 7.0的5%（w/v）SDS中稳定。
1. Rat liver glycosylasparaginase [N4-(beta-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. 2. The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS/polyacrylamide-gel electrophoresis. 3. The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain. 4. N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit. 5. The enzyme exhibited a broad pH maximum above 7. Two major isoelectric forms were found at pH 6.4 and 6.6. 6. Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0.