Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast.
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.
Shiosaki
K
K
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Takata
K
K
Omichi
K
K
Tomita
N
N
Horii
A
A
Ogawa
M
M
Matsubara
K
K
D000135:Acid Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D007700:Kinetics; D008175:Lung Neoplasms / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D007527:Isoenzymes; D000135:Acid Phosphatase; D000516:alpha-Amylases