PURIFICATION AND CHARACTERIZATION OF AMPHIPHILIC LACTASE-PHLORIZIN HYDROLASE FROM HUMAN SMALL-INTESTINE
PURIFICATION AND CHARACTERIZATION OF AMPHIPHILIC LACTASE-PHLORIZIN HYDROLASE FROM HUMAN SMALL-INTESTINE
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DOI：
10.1111/j.1432-1033.1981.tb05193.x
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
NOREN, O
中科院分区：
文献类型：
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作者：
SKOVBJERG, H;SJOSTROM, H;NOREN, O
人肠道乳糖酶/根皮苷水解酶(EC 3.2.1.23/62)经免疫吸附层析纯化为两亲性蛋白。纯化因子为.apprx。600美元，回收率为14%。该酶基本上不含其他已知的刷状缘肽酶和二糖酶，在十二烷基硫酸钠的交叉免疫电泳法和聚丙烯酰胺凝胶电泳法中均一。纯化后的酶对乳糖(最适pH为5.8~6.0，Km为21 mM)、根茎苷(Km为0.44 mM)等β-半乳糖苷和β-葡萄糖苷有较强的水解性。Tris抑制乳糖的水解，而根茎苷的水解率几乎不受影响。对这两种底物的活性也表现出不同的热稳定性。推测人的酶有两种不同的酶切位点：一种是乳糖水解酶，被根皮苷抑制，另一种是根皮苷水解酶。经UltroGel ACA 34凝胶过滤，两亲型酶的相对分子质量为32万，而亲水型(木瓜酶处理)的相对分子质量为28万。这表明锚链段(S)加上结合的洗涤剂具有.apprx的分子量。4万美元。在十二烷基硫酸钠的聚丙烯酰胺凝胶电泳法中，完全变性的酶的表观分子量为160,000。显然，人乳糖酶/根皮苷水解酶由两个单体组成，每个单体的相对分子质量为16万。新兴市场的情况为这一观点提供了进一步的证据。此外，还讨论了合成高相对分子质量1多肽链的可能性。
Human intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purification factor was .apprx. 600 and the recovery 14%. The enzyme was essentially free from other known brush-border peptidases and disaccharidases and appeared homogeneous in crossed immunoelectrophoresis and polyacrylamide gel electrophoresis in sodium dodecylsulphate. The purified enzyme hydrolyzed lactose (pH optimum 5.8-6.0, Km 21 mM), phlorizin (Km 0.44 mM) and other .beta.-galactosides and .beta.-glucosides. Tris inhibited the hydrolysis of lactose whereas phlorizin hydrolysis was almost unaffected. The activity against these 2 substrates also showed different thermal stability. It is suggested that the human enzyme has 2 different enzymatic sites: one for lactose hydrolysis, inhibited by phlorizin, and one for phlorizin hydrolysis. By gel filtration on Ultrogel AcA 34 the amphiphilic form of the enzyme had a MW of 320,000 while the hydrophilic form (papain-treated) had a MW of 280,000. This indicates that the anchoring segment(s) plus the bound detergent has a MW of .apprx. 40,000. In polyacrylamide gel electrophoresis in sodium dodecylsulphate the fully denatured enzyme had an apparent MW of 160,000. Apparently the human lactase/phlorizin hydrolase is composed of 2 monomers each with a MW of 160,000. The EM picture gives further evidence for this suggestion. In addition the possibility of a high MW, 1 polypeptide chain is discussed.