通过在酵母中表达新克隆的人amy3 cDNA鉴定一种新型α-淀粉酶。

Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast.

作者信息

Shiosaki K, Takata K, Omichi K, Tomita N, Horii A, Ogawa M, Matsubara K

机构信息

Institute for Molecular and Cellular Biology, Osaka University, Japan.

出版信息

Gene. 1990 May 14;89(2):253-8. doi: 10.1016/0378-1119(90)90013-h.

DOI:10.1016/0378-1119(90)90013-h

PMID:2197187

Abstract

A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes.

摘要

已经描述了一种新的淀粉酶基因（amy3），其核苷酸序列与唾液淀粉酶基因（amy1）和胰腺淀粉酶基因（amy2）不同[富田等人，《基因》76（1989年）11 - 18]，但该基因是否能编码一种活性酶尚未得到证实。我们从肺类癌组织获得的mRNA中制备了该基因的cDNA，并在酸性磷酸酶启动子的控制下在酿酒酵母中进行表达。产物分泌到培养基中，并显示出酶活性，这表明这种新的α -淀粉酶基因（amy3）可以编码一种功能性同工酶。我们纯化了这种酶，并将其生物学特性与在酵母中类似表达的人唾液淀粉酶和胰腺淀粉酶的生物学特性进行了比较。我们观察到这种新的淀粉酶同工酶比其他同工酶对热更敏感，并且其底物特异性与其他两种同工酶不同。