CHYMOTRYPSIN GENE-EXPRESSION IN RAT PERIPHERAL ORGANS

Prior studies have revealed the presence of chymotrypsinlike protease in peripheral organs, although no definitive evidence for the synthesi s of this enzyme in tissue other than the pancreas is available. In an attempt to detect chymotrypsinogen mRNA in peripheral organs, a fragm ent of the pancreatic chymotrypsin mRNA from rat was amplified using P CR. The sequence was identified as a portion of the rat chymotrypsin B gene overlapping exon 5 through exon 7. It was subcloned into the pGE M-4Z vector and used as a template for the vitro transcription of an a ntisense riboprobe. Using ribonuclease protection and Northern blot an alyses,chymotrypsin mRNA was detected in the rat pancreas, stomach, du odenum, ovary, and spleen. Monoclonal and polyclonal antisera against chymotrypsin detected chymotrypsinlike immunoreactivity in rat and hum an pancreas, rat stomach, duodenum and jejunum. Electrophoresis and im munoblotting revealed chymotrypsin-chymotrypsinogen bands (25-29 kDa) in the stomach and duodenum. Synthesis of a potent protease such as ch ymotrypsin in tissue other than pancreas is significant, suggesting a potential physiological and/or pathological role in these tissues. hig h enzyme activity of this organ. Subsequently, numerous studies have d emonstrated the presence of chymotrypsinlike proteases elsewhere in th e periphery (e.g., Kaiser and Hoskin 1992; King et al. 1987). All of t hese chymotrypsinlike proteases had the common property of cleaving ch ymotrypsin substrates, but were unaffected by chymotrypsin-specific in hibitors. No evidence has been presented to indicate that chymotrypsin itself is expressed in any tissue other than the exocrine glands of t he pancreas. The field of molecular biology has dramatically expanded the array of techniques available for detection of gene expression. In the present paper we surveyed peiripheral tissues from rats for chymo trypsin mRNA using a sensitive ribonuclease protection analysis (RPA) and Northern blot analysis. Chymotrypsin mRNA was detected in the panc reas, stomach, intestine, ovary and spleen. Immunohistochemistry confi rmed the presence of chymotrypsin immunoreactivity in cells of the pan creas and gastrointestinal tract. In addition, immunoprecipitation Wes tern blot analysis identified chymotrypsin proteins in the pancreas, s tomach and intestine.