Characterization of a metalloprotease which cleaves with high site-specificity the Glu(143)-Leu(144) bond of urokinase

Abstract

An enzyme which cleaves the Glu(143)-Leu(144) bond of pro-urokinase has been partially purified from the conditioned media of cultured human kidney cells. The products of the reaction catalyzed by this enzyme are forms of pro-urokinase and urokinase with Leu(144) as the amino-terminal residue. The protease has been purified using three chromatographic steps: (a) S-sepharose; (b) zinc-sepharose; and (c) gel filtration. The enzyme is a metalloprotease, requiring calcium or zinc ions, and is inhibited by EDTA. The activity is not affected by serine or thiol protease inhibitors. Although the enzyme has been purified more than 1000-fold from the culture media, a preparation of homogeneous protein has not yet been obtained. Completion of the isolation of the protease will allow determination of whether the urokinase cleaving activity is the property of a novel enzyme. The activity is assayed by the specific cleavage of r-pro-urokinase into two fragments, as determined by SDS-PAGE of unreduced samples of the reaction products of pro-urokinase with the enzyme. We have used the cleaving enzyme to prepare, in a single digestion, milligram quantities of both an amino-terminal fragment of urokinase, comprised of the growth factor and kringle domains, and low molecular weight pro-urokinase. The low molecular weight zymogen can be converted to the active enzyme by careful treatment with plasmin. Low molecular weight urokinase with Leu(144) as its amino-terminal residue is produced by culture of human kidney cells. This is in contrast to the low molecular weight urokinase isolated from urine, which begins at Lys(136). Since the amino-terminal fragment Ser(1) to Glu(143) has been shown to bind with high affinity to the urokinase receptor, and also to be active in stimulating the growth or differentiation of certain cells in culture, it is possible this metalloprotease has a role in certain cell regulatory functions.