Mechanisms of Regulation for Serine Protease Activity in the Rat Ileocecal Junction /

Digestive enzymes in the gut have recently been implicated as primary factors in the process of inflammatory shock. Pancreatic serine proteases have been determined as key players in causing damage to an ischemic intestine. Most studies have focused on the pancreas and small intestine when investigating enzymatic activity in the digestive tract. They are fully activated in the small intestine, but their activity in the large intestine is reduced by a mechanism that is largely unknown. This study is designed to investigate proteolytic activity at the transition from the small to the large intestine. Segments of rat small intestine, cecum, and large intestine were harvested. Using zymographic techniques, the protease activity of intestinal homogenates was measured with specific substrates of trypsin, chymotrypsin, and elastase. We tested whether cecal wall contains serine protease inhibitors that result in reduction of proteolytic activity in the large intestine using reverse zymography. Results indicate that there is a significant reduction in protease activity, specifically trypsin, in the cecum and large intestine when compared to the small intestine. Reverse zymography results suggest that a previously unknown inhibitor of about 30 kDa for trypsin may be present in the cecal wall but absent in the small or large intestinal homogenates. When eluted and unpurified cecal wall homogenate sample was mixed in equal volume with trypsin, there was a noticeable reduction in trypsin activity. Mass spectroscopy results suggest an immunoglobulin heavy chain antibody may be the source of the unknown inhibitor, but further studies are neede