Characterization of proteases and protease inhibitors in the rat
stomach.
Nagy, Lajos, Brett R. Johnson, Peter Hauschka, and Sandor Szabo.
Chemical Pathology Research Division, Departments of Pathology,
Brigham & Women's Hospital and Harvard Medical School, 75 Francis
Street, Boston, MA 02115
APStracts 3:0269G, 1996.
Previously our laboratory reported increased activity of the thiol
proteinase cathepsin B in gastric juice after ethanol-induced mucosal
injury. In this study we measured proteinase activity (PA) and
proteinase inhibitory activity (PIA) with general substrates
hemoglobin, azocasein and bovine serum albumin (BSA) at optimal pH
(2.0, 5.6, 7.4) of aspartic, cysteine and serine proteinases.
Homogenates of glandular stomach mucosa and gastric juice from fasted
rats were incubated in the presence or absence of specific inhibitors
and sulfhydryl (SH) alkylators N-ethylmaleimide (NEM) or iodoacetate
(IA). PIA was measured after acid and heat inactivation of endogenous
proteinases and addition of 20 [mu]g/ml pepsin, 20 or 100 [mu]g/ml
thiol proteinase papain, or 20 [mu]g/ml trypsin for 5 min before
digestion at 37oC. The highest proteolytic activity was found at pH
2.0 (pepsin) in both juice and mucosal homogenate, but proteases were
also found at pH 5.6 and 7.4 where pepsin was inactive. Pepstatin
inhibited most proteolytic activity at pH 2.0. SH protease inhibitor
leupeptin diminished PA mainly at pH 5.6. NEM or IA substantially
reduced the PA in acidic milieu with maximum effect at pH 5.6.
Endogenous PIA expressed as inhibition (%) of the effect of 1 [mu]g
of pepsin, papain and trypsin on BSA was 13.1, 1.4 and 9.2 in the
gastric mucosa, and 15.3, 22.5 and 6.2 in gastric juice, at pH 2.0,
5.6 and 7.4, respectively. Conclusions: 1) Endogenous proteinases and
inhibitors in rat stomach were measured using BSA and hemoglobin as
substrates. 2) Of the proteinases found in the stomach 98% was pepsin
at pH 2.0, and up to 27% or 17% was SH-sensitive at pH 5.6 or 7.4,
respectively. 3) Proteinases and their specific endogenous inhibitors
may play a role in gastric mucosal injury and protection.
Received 5 June 1995; accepted in final form 9 December 1996.
APS Manuscript Number G237-5.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 31 December 1996