title: Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts, Cynops pyrrhogaster.
abstract: Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians.
authors.affiliation: Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554;
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Nagasawa
authors.affiliation: Department of Chemistry, Faculty of Science, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295; and.
authors.forename: Kaori
authors.initials: K
authors.lastname: Sano
authors.affiliation: Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554;
authors.forename: Mari
authors.initials: M
authors.lastname: Kawaguchi
authors.affiliation: Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554;
authors.forename: Ken-Ichiro
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554;
authors.forename: Shigeki
authors.initials: S
authors.lastname: Yasumasu
authors.affiliation: Department of Biology, Faculty of Education, Utsunomiya University, 350 Mine, Utsunomiya, Tochigi 321-8505, Japan inokuchi@cc.utsunomiya-u.ac.jp.
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Inokuchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057055:Aspartic Acid Proteases / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D020059:Cathepsin E / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D004792:Enzyme Precursors / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D005753:Gastric Mucosa / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010434:Pepsin A / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D010435:Pepsinogens / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D010436:Pepstatins / Q000494:pharmacology; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D012455:Salamandridae / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D004792:Enzyme Precursors; D010435:Pepsinogens; D010436:Pepstatins; D011480:Protease Inhibitors; D057055:Aspartic Acid Proteases; C067581:procathepsin E; D010434:Pepsin A; C020557:gastricsin; D020059:Cathepsin E; C031375:pepstatin
keywords: newt; pepsinogen; pro-cathepsin E; purification; stomach