| Research Abstract |
1. Human, porcine and rat prepepsinogens and calf preprochymosin were isolated and identified, and the complete amino acid sequences of their N-terminal signal sequences determined.
2. Various pepsinogens and related aspartic proteases were isolated and their molecular and enzymatic properties investigated, including human pepsinogens A, human pepsinogen C, human slow moving protease, monkey prostate pepsinogen C-like zymogen, and turtle, frog and tuna pepsinogens.
3. By using the protein-chemistry techniques, the complete primary structures were determined for monkey pepsinogens A and C, tuna pepsinogen, rhizopuspepsin, and Aspergillus niger Type A acid protease, and partial primary structures for human pepsinogens A and C, frog pepsinogens and Scytalidium lignicolum acid protease. In addition, the complete primary structures of human and rat pepsinogens C and turtle pepsinogen were deduced from the nucleotide sequence analyses of their corresponding DNAs.
4. The activation mechanisms of
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human pepsinogens A and C, monkey and porcine pepsinogens, and various other pepsinogens were investigated and compared in detail.
5. Various mutants of porcine pepsinogen were prepared by site-directed mutagenesis, and expressed in E. coli. The change of either one of the two active-site aspartic acids to glutamic acid abolished completely the potential pepsin activity.
6. The nucleotide sequences of all the nine exons and the 5'- and 3'-flanking regions of human and rat pepsinogens C were determined, and human pepsinogen C was located on the chromosome 6. Further, analyses of human pepsinogens A showed that there exist at least three different genes and that at least two of them are tandemly aligned on a human chromosomal DNA.
7. A positive correlation was found between the expression of rat and human pepsinogens C genes and their level of demethylation. In addition, a 25-Da transcriptional regulation factor was found in the nuclear fraction of rat gastric mucosa cells, which binds 5'-upstream region of the gene. Less
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