Purification and characterization of cathepsin E type acid proteinase from gastric mucosa of bullfrog, Rana catesbeiana.
An acid proteinase different from pepsin was purified from bullfrog (Rana catesbeiana) gastric mucosa by chromatography on hydroxyapatite, Q-Sepharose, Con A-Sepharose 4B, and Mono Q columns. Its molecular weight after purification was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 45 kDa under reducing conditions and about 90 kDa under nonreducing conditions. Thus, it is a dimer of two identical subunits. On acid treatment, the molecular weight of the subunit decreased from 45 to 42 kDa, showing a similar change to that of pepsinogen in its activation under acidic conditions. Therefore, the enzyme was thought to have both a proform and a mature form. It preferred hemoglobin to other protein substrates examined and showed broad optimal activity in the range of pH 2.0 to 3.5 towards hemoglobin. Its proteolytic activity, like that of porcine pepsin, was strongly inhibited by pepstatin. Its amino acid composition was similar to those of other aspartic proteinases. From these results, the enzyme was identified as a cathepsin E type acid proteinase of bullfrog, and cathepsin E type enzyme was purified from anuran for the first time.
Inokuchi
T
T
Life Science Institute, Sophia University, Tokyo.
Kobayashi
K
K
Horiuchi
S
S
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D020059:Cathepsin E; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid; D005753:Gastric Mucosa / Q000201:enzymology*; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010436:Pepstatins / Q000494:pharmacology; D011892:Rana catesbeiana; D013379:Substrate Specificity
D000596:Amino Acids; D006454:Hemoglobins; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D002403:Cathepsins; D016282:Aspartic Acid Endopeptidases; D020059:Cathepsin E; C031375:pepstatin