PURIFICATION OF RAT INTESTINAL MALTASE-GLUCOAMYLASE AND ITS ANOMALOUS DISSOCIATION EITHER BY HEAT OR BY LOW PH
PURIFICATION OF RAT INTESTINAL MALTASE-GLUCOAMYLASE AND ITS ANOMALOUS DISSOCIATION EITHER BY HEAT OR BY LOW PH
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DOI：
10.1042/bj1730553
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发表时间：
1978-01-01
影响因子：
4.1
通讯作者：
FORSTNER, GG
中科院分区：
文献类型：
--
作者：
FLANAGAN, PR;FORSTNER, GG
麦芽糖酶[EC 3.2.1.20]/葡萄糖淀粉酶[EC 3.2.1.3]是一种微绒毛膜胞外酶，用木瓜酶从大鼠肠黏膜中溶解，纯化至均一，总收率为10-20%。纯化酶的抗体在肠匀浆的免疫扩散实验中形成了一条单一的沉淀线。该酶是一种酸性糖蛋白，含少量半胱氨酸，不含唾液酸。在pH为3-6时，麦芽糖酶活性缓慢丧失，但通过重新调节pH至中性，该酶被重新激活。但在十二烷基硫酸钠存在下，酸性pH值使麦芽糖酶不可逆地失活，同时将麦芽糖酶(相对分子质量约为50万)转化为5种新的麦芽糖酶。经聚丙烯酰胺凝胶电泳法测定，表观分子量在134,000-48,000之间。同样的5个片段也是在有或没有还原剂的情况下，在十二烷基硫酸钠或尿素的存在下短暂煮沸酶而形成的。解离后的菌种在再电泳法上是稳定的，氨基酸分析表明它们彼此之间非常相似，而且与原酶非常相似。这些谱带在不同浓度的聚丙烯酰胺凝胶上发生了异常迁移，从而阻碍了精确分子量的分配。这是有可能的，5个种。可以代表酶的一种常见单体的稳定聚集体。
Maltase[EC 3.2.1.20]/glucoamylase [EC 3.2.1.3], a microvillous membrane ectoenzyme, was solubilized from rat intestinal mucosa by digestion with papain and subsequently purified to homogeneity with an overall yield of 10-20%. An antibody [rabbit] to the purified enzyme formed a single precipitin line in immunodiffusion experiments with an intestinal homogenate. The enzyme was shown to be an acidic glycoprotein (20% sugar by wt) which contained low amounts of cysteine and no sialic acid. At pH 3-6, maltase activity was slowly lost, but the enzyme was re-activated by re-adjustment of the pH to neutrality. However, in the presence of sodium dodecyl sulfate, acid pH values inactivated maltase irreversibly, and at the same time converted the enzyme (MW 500,000 approximately) into 5 new spp. with apparent MW ranging from 134,000-480,000 as judged by polyacrylamide-gel electrophoresis. The same 5 fragments were also formed by boiling the enzyme for brief periods in the presence of sodium dodecyl sulfate or urea either with or without reducing agents. The dissociated species were stable on re-electrophoresis, and amino acid analysis showed them to be very similar to each other and to the original enzyme. The bands migrated anomalously on polyacrylamide gels of different concentration, thereby preventing the assignment of precise MW. It is possible that the 5 spp. may represent stable aggregates of a common monomer of the enzyme.