RENAL EPIDERMAL GROWTH-FACTOR PRECURSOR - PROTEOLYTIC PROCESSING IN AN IN-VITRO CELL-FREE SYSTEM

The enzymatic processing of the membrane-bound renal epidermal growth factor precursor (proEGF) could be an important step in the control of nephrogenic repair consecutive to kidney insult. The enzyme machinery responsible for that processing was examined in a cell-free system co nsisting of renal membranes isolated from kidney homogenates by differ ential centrifugation, and incubated in vitro. After a 24-h incubation at 37 degrees C, 6-14% of membrane-bound proEGF was processed and sol uble products with EGF immunoreactivity were released. As revealed by HPLC and Western blotting analysis, the products of proEGF proteolysis consisted of 6 kDa EGF (the molecular weight of mature EGF) and two p olypeptides with molecular weights around 45 kDa. Interestingly the 45 kDa EGF forms, like the 6 kDa EGF, exhibited mitogenic activity towar d growth-arrested NRK-52E renal cell line. The kinetic study of proEGF degradation gave data consistent with the 45 kDa product(s) being pro cessing intermediate(s) between proEGF and 6 kDa EGF. The enzymatic ac tivity responsible for proEGF nicking was inhibited by divalent heavy metal ions (Cu2+ Or Zn2+) and several protease inhibitors (aprotinin, PMSF, leupeptin, soybean trypsin inhibitor), suggesting that proEGF is processed by kallikrein-like serine proteases present in the membrane preparations. Along with previous studies, the current observations s uggest that renal kallikreins might play a role in renal tubular regen eration by promoting the release of soluble EGF in renal tissue.