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Urokinase-related proteins in human urine. Isolation and characterization of single-chain urokinase (pro-urokinase) and urokinase-inhibitor complex.

1986; Elsevier BV; Volume: 261; Issue: 3 Linguagem: Inglês

10.1016/s0021-9258(17)36085-4

1083-351X

D C Stump, M Thienpont, Désiré Collen,

Peptidase Inhibition and Analysis

Urokinase-related proteins were purified from 60liter batches of human urine collected into the protease inhibitor aprotinin to prevent proteolytic degradation.Three homogeneous species were obtained by chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, Sephadex G-100, benzamidine-Sepharose, and immunoad~rption on a murine anti-human urokinase monoclonal antibody.One urokinase-related protein with M, 95,000 representing a complex of two-chain urokinase with an inhibitor accounts for about 70% of the total urokinase-related antigen in urine.Nucleophilic agents dissociate the complex into active twochain urokinase and a protein with M, 45,000-50,O~~ which is immunologically distinct from urokinase.Approximately 25% of the urinary urokinase-related antigen represents a single-chain molecule with Mr 54,000.This highly purified single-chain molecule was obtained with a yield of 5 gglliter of urine.Only trace amounts (<5%) of the uroklnase-related antigen were recovered as free two-chain urokinase.The urinary single-chain urokinase-related protein has no specific affinity for fibrin.It has a very low activity on Pyroglu-Gly-Arg-p-nitroanilide, a urokinase-specific synthetic substrate, but directly activates plasminogen following Michaelis-Menten kinetics with K,,, = 0.7 p~ and kcat = 0.0011 s -I .The single-chain molecule is rapidly converted to active two-chain urokinase by plasmin.Active two-chain urinary urokinase has a very high amidolytic activity and activates plasminogen with K,,, = 60 g~ and kc, = 1.4s-'.It is concluded that the urokinase-related proteins in human urine consist of about 25% of single-chain urokinase (10-20 pg/liter) and of about 75% two-chain urokinase (40-50 pg/liter), the bulk of which is complexed to an inhibitor.Because even in freshly voided urine most of the urokinase-related antigen is already converted to two-chain urokinase, urine does not seem to be a suitable source for the large-scale purification of single-chain urokinase.In view of the very significant intrinsic plasminogenactivating properties of single-chain urokinase, it should not be considered to be a proenzyme form of urokinase.The dramatic differences of its kinetic constants from those of urokinase render the designation single-chain urokinase equally inadequate.Conse-