Molecular cloning and characterization of chymopasin, a novel serine protease from rat pancreas.
BACKGROUND
Pancreas secretes many enzymes for food digestion into the pancreatic juice. We cloned a novel serine protease, chymopasin, from rat pancreas.


OBJECTIVE
To know the localization of this enzyme in the pancreas and to analyze the enzymatic characteristics.


METHODS
We cloned chymopasin cDNA using 3' and 5' RACEs. Northern blot and in situ hybridization were used to study the expression of this enzyme. Recombinant chymopasin protein produced by was analyzed by Western blot using specific antibody, and its enzymatic characteristics were examined using commercially available synthetic substrates, fibrin and gelatin.


RESULTS
The open reading frame of rat chymopasin consisted of 792 bp encoding 264 amino acid residues. The deduced amino acid sequence contained the essential catalytic triad characteristic of the serine protease family. There was no putative N-glycosylation site. The amino acid sequence of rat chymopasin showed 54.5% identity to rat chymotrypsin B. Northern blot analysis showed that the transcript was strongly expressed in the pancreas. In situ hybridization with digoxigenin-labeled cRNA probe showed that the positive signals were observed in the acinar cells, but not in the islet or duct cells. Chymopasin protein was detected in the pancreas homogenate and bile-pancreatic juice. Further, cerulein stimulated the secretion of rat chymopasin into bile-pancreatic juice.


CONCLUSIONS
These results suggested that rat chymopasin might be a digestive enzyme secreted from the acinar cells. From the enzyme assay using synthetic substrates, the purified recombinant chymopasin expressed in showed chymotrypsin-like activity. In addition, rat recombinant chymopasin showed fibrinolytic and gelatinolytic activities. These results suggested a role in the pathogenesis of pancreatic damage.
Sogame
Yoshio
Y
Third Department of Internal Medicine, Research Institute for Neurological Disease and Geriatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Kataoka
Keisho
K
Kato
Masato
M
Sakagami
Junichi
J
Osawa
Saori
S
Takatera
Ami
A
Mitsuyoshi
Mayuko
M
Usui
Noriko
N
Mitsui
Shinichi
S
Yamaguchi
Nozomi
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002918:Chymotrypsin / Q000235:genetics; D003001:Cloning, Molecular; D008297:Male; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D014018:Tissue Distribution
D012333:RNA, Messenger; D012697:Serine Endopeptidases; C469375:chymopasin, rat; D002918:Chymotrypsin; C117609:chymotrypsin B