Purification and characterization of two forms of urokinase.

Affinity chromatography on agmatine-Sepharose was used for the separation of two active forms of urokinase (EC 3.4.99.26) from partially purified human urinary urokinase. The approximate molecular weight of the heavier form was 47 000 and of the lighter 33 400. Both forms were homogeneous by sodium dodecyl sulfate gel electrophoresis and by 3H-labeled diisopropylphosphorofluoridate and 14C-labeled p-nitrophenyl-p'-guanidinobenzoate incorporation studies. The 33 400 mol. wt. form had a single chain, and the 47 000 mol. wt. form had two chains (33 100 and 18 600 mol. wt.) linked by disulfide bonds. The specific activity of the heavier form was 104 000 CTA units/mg protein, compared with 226 000 units/mg for the lighter form but the activities per mmol of active site (molar activities) of the two forms were almost identical (9.6-10(9) and 10.2-10(9) CTA units/mmol). Isoelectric focusing on gels showed that the 47 000 material contained one major subform with a pI of 8.60 and a minor subform with a pI of 8.90, while the 33 400 material had three major subforms with pI values of 8.35, 8.60 and 8.70, respectively, and a minor subform with a pI of 8.05. 3H-labeled diisopropylphosphorofluoridate incorporation studies revealed an active-site serine residue in the heavy chain.

Identification of an active site histidine in urokinase.

Two forms of urokinase (EC 3.4.99.26) with apparent molecular weights of 33 400 and 47 000 purified by affinity chromatography have been modified specifically with newly synthesized peptide chloroketones by affinity labeline. Rapid inactivation of the enzyme preparations was observed with Ac-Gly-Lys-CH2 Cl and Nle-Gly-Lys-CH2 Cl which might be associated with a change in which a histidine residue is lost. After performic acid oxidation, an equivalent amount of 3-carboxymethyl histidine could be recovered, indicating alkylation at the N-3 of a histidine residue. In the case of the norleucine derivative, norleucine was concomitantly incorporated into the protein. It is thus likely that urokinase belongs in the class of enzymes utilizing the Asp..His..Ser triad for their catalytic action. The two active site residues so far identified, serine and histidine, were located in the heavy chain (33 100 mol. wt) of the 47 000 molecular weight form and in the 33 400 molecular weight form, the molecular weight of which remained constant.