The purified amino-terminal fragment (ATF) of human urokinase plasminogen activator (residues 1-135), which is not (1,6). This possibility has been strengthened by the recent report of Vassalli et al. (7), who showed that urokinase binds specifically to freshly isolated blood monocytes and to the U937 monocyte line.Urokinase (8) is synthesized as a single-chain prepropolypeptide (9), secreted as an inactive single-chain prourokinase zymogen (411 residues) (10-12), and activated by proteolysis, which removes lysine-158 (10) to generate a two-chain urokinase molecule (residues 1-157 and 159-411; Mr = 50,000) (13,14). This form of active urokinase is referred to as high molecular weight (HMW) urokinase. Another active form of the enzyme (the Mr 33,000 urokinase) contains only the carboxyl-terminal two-thirds of HMW urokinase (residues 136-157 and 159-411) (13). Thus, the amino-terminal portion of HMW urokinase (residues 1-135) is not required for catalytic activity.We have prepared specific fragments of urokinase (the amino-terminal peptide, which lacks proteolytic activity, and the carboxyl-terminal peptide, which contains the catalytic domain) and have examined the binding of these fragments to urokinase receptors. We report that (i) the purified aminoterminal fragment of urokinase (ATF; residues 1-135) is totally sufficient to bind specifically to the urokinase receptor on U937 monocytes; (ii) the receptor-bound ligand remains on the cell surface, possibly to stimulate localized proteolysis through the catalytic, carboxyl-terminal domain; and (iii) differentiation of the monocytes into macrophage-like cells results in a 10-to 20-fold increase in ATF binding.MATERIALS AND METHODS Materials. Human urinary urokinase was purified to homogeneity at Lepetit Spa Laboratories (specific activity, 120,000 international units/mg). Epidermal growth factor was purified from male mouse submaxillary glands similarly to the procedure described (15). 1251I-labeled insulin was purchased from New England Nuclear. Phorbol 12-myristate 13-acetate (PMA; LC Service, Woburn, MA) was dissolved at 1 mg/ml in dimethyl sulfoxide and diluted to working concentrations with RPMI 1640 medium (GIBCO) containing 10% heat-inactivated fetal bovine serum (Biofluids, Rockville, MD). Purified tissue plasminogen activator (80,000 international units/mg) was the gift of Keith Marotti (Upjohn).Purification of ATF. Purified HMW urokinase (30 mg in 1 ml) was incubated for 8 hr in 50 mM sodium phosphate buffer, pH 8/0.2 M NaCl. Reaction products were separated by gel filtration at a flow rate of 3 ml/hr on a column (1.5 x 100 cm) of Sephadex G-100 (superfine) equilibrated with 50 mM sodium phosphate buffer, pH 8.0/0.2 M NaCl. Fractions containing ATF were pooled and directly subjected to ionexchange chromatography using a fast protein liquid chromatography (FPLC) apparatus (Pharmacia) and a Mono S HR5/5 column equilibrated with 50 mM sodium acetate buffer, pH 4.8. A sodium chloride gradient (0-1.0 M in 35 min) was used to elute bound proteins. F...

Measurement of sTNF-Rs, in addition to antigenic and bioactive TNF-alpha, is essential for evaluation of the activation of this cytokine in CHF. Both sTNF-Rs increase in preterminal patients with severe CHF and might inhibit the in vitro cytotoxicity of TNF-alpha. Antigenic TNF-alpha also increases in severe CHF. The increased levels of sTNF-RII independently correlate with poor short-term prognosis.

No abstract

The stability of oligomeric human tumour necrasis factor alpha (TNF) at bioactive levels has been studied by two immunoenzymatic assays: one able to specifically detect oligomeric and not monomeric TNF (O-e.l.i.s.a.) and the other able to detect both forms (OM-e.l.i.s.a.). The selectivity of O-e.l.i.s.a. and OM-e.l.i.s.a. for oligomeric and monomeric TNF was demonstrated with isolated forms prepared by partial dissociation of recombinant TNF with 10% (v/v) dimethyl sulphoxide and gel-filtration h.p.l.c. Evidence for instability of oligomeric TNF were obtained in physiological buffers, as well as in serum and cell-culture supernatants, as a function of TNF concentration. In particular, only a half of the TNF antigen was recovered in the oligomeric form after 72 h incubation (37 degrees C) at 0.12 nM, whereas no apparent dissociation was detected at 4 nM. The structural changes observed at picomolar concentrations were rapidly reversed by raising the concentration of TNF to about 2 nM by ultrafiltration, suggesting that subunit dissociation and reassociation reactions occur in the picomolar and nanomolar range respectively. The cytolytic activity of L-M cells correlates with oligomeric-TNF levels after incubation at picomolar concentrations. Moreover, isolated oligomeric TNF was cytotoxic towards L-M cells, whereas monomeric TNF was virtually inactive. In conclusion, the results suggest that bioactive oligomeric TNF is unstable at picomolar levels and slowly converts into inactive monomers, supporting the hypothesis that quaternary-structure changes in TNF may contribute to the fine regulation of TNF cytotoxicity.