Human Small Intestinal Maltase-glucoamylase cDNA Cloning
Human Small Intestinal Maltase-glucoamylase cDNA Cloning
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DOI：
10.1074/jbc.273.5.3076
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发表时间：
1998-01
期刊：
影响因子：
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通讯作者：
B. Nichols;J. Eldering;S. Avery;D. Hahn;A. Quaroni;E. Sterchi
中科院分区：
文献类型：
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作者：
B. Nichols;J. Eldering;S. Avery;D. Hahn;A. Quaroni;E. Sterchi
据推测，当管腔α-淀粉酶活性因不成熟或营养不良而降低时，人粘膜葡糖淀粉酶（EC3.2.1.20和3.2.1.3）活性可作为淀粉消化的替代途径，麦芽糖酶-葡糖淀粉酶在食品生产中使用的麦芽膳食低聚糖的消化中发挥独特作用。作为测试这一假设的第一步，我们克隆了人小肠麦芽糖酶-葡糖淀粉酶cDNA，以允许在随后的表达实验中研究麦芽糖和淀粉酶水解酶活性的个体催化和结合位点。通过免疫分离纯化人麦芽糖酶-葡萄糖淀粉酶并进行部分测序。采用简并引物和基因特异性引物，通过逆转录-聚合酶链反应，从人肠道RNA中扩增麦芽糖酶-葡萄糖淀粉酶cDNA。6，513个碱基对的cDNA含有一个开放阅读框，编码一个1，857个氨基酸的蛋白质（分子量209，702 Da）。麦芽糖酶-葡萄糖淀粉酶与蔗糖酶-异麦芽糖酶有两个相同的催化位点，但蛋白质的同源性仅为59%。两者都是糖基水解酶家族31的成员，其具有多种底物特异性。我们的研究结果表明，在碳水化合物结合序列的分歧必须确定共享一个保守的催化位点的四种不同的酶活性的底物特异性。
It has been hypothesized that human mucosal glucoamylase (EC 3.2.1.20 and 3.2.1.3) activity serves as an alternate pathway for starch digestion when luminal α-amylase activity is reduced because of immaturity or malnutrition and that maltase-glucoamylase plays a unique role in the digestion of malted dietary oligosaccharides used in food manufacturing. As a first step toward the testing of this hypothesis, we have cloned human small intestinal maltase-glucoamylase cDNA to permit study of the individual catalytic and binding sites for maltose and starch enzyme hydrolase activities in subsequent expression experiments. Human maltase-glucoamylase was purified by immunoisolation and partially sequenced. Maltase-glucoamylase cDNA was amplified from human intestinal RNA using degenerate and gene-specific primers with the reverse transcription-polymerase chain reaction. The 6,513-base pair cDNA contains an open reading frame that encodes a 1,857-amino acid protein (molecular mass 209,702 Da). Maltase-glucoamylase has two catalytic sites identical to those of sucrase-isomaltase, but the proteins are only 59% homologous. Both are members of glycosyl hydrolase family 31, which has a variety of substrate specificities. Our findings suggest that divergences in the carbohydrate binding sequences must determine the substrate specificities for the four different enzyme activities that share a conserved catalytic site.