SUMMARYIn liie activated complement system, vitronectin (complement S-protcin) occupies ihe metastabie membrane binding site of the nascent precursor compiex C5b-7, so that the newly formed SC5b-7 is unable to insert into celi membranes. Some evidence also indicates that vitronectin limits on-going membrane-associated pore ibrmation by inhibiting C9 poiynierization. It has been assumed that these two stages of terminal complement complex (TCC) inhibition taice piace through charge interactions between the heparin-binding region of vitronectin and homoiogous cysteine-rich sequences of the late complement proteins C6, C7, C8 and C9. We examined SC5b-7 formation and inhibition of C9 binding in the TCC using separate haemolylie assays. The mode of action of vitronectin in these assays was compared with iwo 15mer peptides which span residues .148-379 of ihe heparin-binding region, and a heparin-atlinity polypeptide. protamine sulphate. The results showed that vitronectin acts predominantly through SC5b-7 production with a lesser effect on the inhibition of C9 lytic pore formation, in contrast, protamine suiphate did not prevent C5b-7 membrane attachment, but was a potent inhiiiitor of C9-mediated lysis. The peptides did not inhibit C5b-7 membrane insertion and only one aiTeeted C9 binding. These data suggest that the two stages of TCC inhibition involve separate binding sites on the vitronectin molecule. The site for association with nascent C5b-7 is unknown, whereas inhibition of C9 binding and pore formation taices place through the heparin-binding region.

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SUMMARYVitronectin (complement S-protein)., a multifunctional glycoprotein, inhibits complementmediated cytolysis at two identified stages of terminal complement complex (TCC) formation: blocking of C5b-7 membrane binding, and prevention of C9 polymerization. However, the functional domain(s) of vitronectin involved in these reactions remains incompletely defined. In order to identify the complement inhibition site, a 12-kD heparin binding fragment and two other internal fragments (53 kD and 43 kD) of vitronectin were isolated after cyanogen bromide (CNBr) treatment of the native molecule. Potent inhibition of guinea pig erythrocyte (GPE) reactive lysis was demonstrated with native vitronectin, total CNBr digest and the 53-kD and 43-kD fragments, but only very poorly by the heparin binding 12-kD peptide. Sitnilarly. the 43-kD fragment blocked the binding of C5b-7 to immobilized vitronectin. whereas the 12-kD fragment had no effect. These data localize the C5b-7 binding site to a 4.VkD internal region. Further characterization of the fragments was carried out in an assay which detected C9 polymerization in the presence of C5b-8. Polymerized material was separated by PAGE, detected by autoradiography and quantified after excision from the gels. Results showed that polymerization did not occur in the presence of the 53-kD and 43-kD fragments. However, the 12-kD heparin binding fragment had no effect. It is proposed that prevention of C5b-8-induced C9 polymerization resides at a site in an internal region of the vitronectin molecule.

Two groups off hordein-reactive antibodies (cross-reactive in barley with B/C monomers or subunits from the B/D-hordein aggregates) were used for the analysis of malt, wort and beer. A number of additional lower molecular weight hordein-derived polypeptides were present in water extracts of malts and worts, and protease inhibitors or higher mash-in temperature had a stabilising effect on certain hordein-derived polypeptides.The solubilisation profiles of hordein polypeptides monitored by specific immunological methods were clearly different from total wort polypeptides monitored by the Bradford method. There were also differences between the profiles for the two groups of hordein-derived polypeptides with the solubilisation of polypeptides recognised by antibody with specificity for the subunits from the B/D aggregates being especially temperature dependent.Beer samples mainly contained hordein-derived polypeptides of lower molecular weight than barley hordeins with polypeptides in the B and C molecular weight regions being lost during the brewing process. Beer polypeptides (Mr > 50,000) were recognised by antibodies cross-reactive with the subunits from the B/D aggregates in barley. A number of lower molecular weight polypeptides (Mr < 30,000) were recognised by antibodies with specificity for B and C hordeins; a subset of the latter antibody group bound to a specific Mr 23,000 beer polypeptide.