Calpain from rat intestinal epithelial cells: Age-dependent dynamics during cell differentiation

Micromolar and millimolar Ca²⁺-requiring neutral protease (calpain I and calpain II) along with their endogenous inhibitor calpastatin were isolated and partially purified from the same preparation of rat intestinal epithelial cells. Calpain I and II were partially purified by 1300 and 900-fold with 57 and 53 per cent yield, respectively. The optimum assay conditions revealed pH 7.5, 20 min incubation at 25° C and 0.24% casein substrate for both calpains. The optimum calcium concentration obtained for calpain I and II were 25 M and 4 mM, respectively. Distribution of rat intestinal epithelial cells calpain I and II along with calpastatin during cell differentiation stages in weanling to senescence age were studied. Calpain I in weanling rats was in an increasing order from villus to crypt regions. Adult rats indicated well expressed consistent calpain I throughout the differentiation stages. Whereas, significant lowering towards crypt region cells were evident in old rats. Calpain II in weanling and adult rats was found to be consistent throughout the differentiation stages. Old animals revealed an increasing trend from villus to crypt region with insignificant activity present in upper villus cells. Concomitantly, different concentrations of calpastatin were observed throughout the differentiation stages in all the age groups. Moreover, the levels of calpains exceeded that of calpastatin in most of the epithelial cell populations during developmental stages. In addition to casein, intestinal epithelial cell membranes were found to be equally good substrates for calpains. Proteolytic susceptibility of weanling, adult and old rat membrane proteins varied significantly all along the ageing process in rats. Simultaneous age-dependent calpastatin response were also evident. Taken together the results obtained provided strong evidence that calpain plays significant role in rat intestinal cell differentiation and ageing process with calpastatin as its specific regulatory protein.Abbreviations DEAE-cellulose O-(Diethylaminoethyl)-cellulose - EDTA Ethylene Diamine Tetra Acetic Acid - Tris Tris (hydroxymethyl) amino methane - KH₂PO₄ potassium dihydrogen orthophosphate - Na₂HPO₄ disodium hydrogen phosphate - CaCl₂ Calcium Chloride - TCA Trichloroacetic Acid - PMSF Phenylmethylsulfonyl Fluoride