Eceptors, the incubation samples have been mixed with 50 Al of 40 six (wt/vol) PEG 6000 and placed on ice for 20 min prior to filtration, to precipitate the proteins. The filters were washed three instances with ice-cold phosphate-buffered saline containing 0.1 bovine serum albumin (membranes) or 9o PEG 6000 (solubilized receptors), and dried, and radioBRPF2 Inhibitor Species activity was measured. The amount of binding internet sites (60,000-90,000 receptors per cell; 2-7 X 109 receptors per ,ug of membrane protein; 0.7-1.four x 109 receptors per ,ug of solubilized protein), the dissociation constants, as well as the nonspecific binding parameters had been determined by laptop or computer modeling as described (17). Nonspecific binding didn’t exceed 3 (with complete cells), 5 (with membranes), and 11 (with solubilized proteins) from the respective absolutely free ligand concentrations. Cross-Linking Experiments. The protocol for labeling of neutrophil receptors on complete cells with iodinated IL-8 has been described in detail (17) and was applied in cross-linking experiments with iodinated GROa(Y) and NAP-2(Y). For cross-linking studies with soluble receptors, membranes have been freshly solubilized as described above and 60-100 Ag of soluble proteins was incubated in a total volume of 370 IlI with 0.3-4 nM of iodinated IL-8, GROa(Y), or NAP-2(Y) within the presence or absence of unlabeled ligands at 21 for 90 min. After cross-linking with 1 mM disuccinimidyl suberateProc. Natl. Acad. Sci. USA 89 (1992)for 15 min at 21 , 40 ul of 1 M Tris HCl (pH 7.4) was added plus the soluble proteins had been sedimented by incubation with 140 ,ul of 40 o PEG 6000 for five min on ice and centrifugation at 15,000 x g for ten min. The proteins inside the pellets had been analyzed by SDS/PAGE and autoradiography as described above. Elastase Release Assay and HIV Antagonist custom synthesis Protein Determination. The biological activity of GROa, NAP-2, and analogs was assessed by measuring the release of elastase from human neutrophils pretreated with cytochalasin B (8, 12). Protein was determined working with the kit Micro BCA assay (Pierce).Benefits Tyrosine-Substituted Ligands. The tyrosine-substituted peptides GROa(Y) and NAP-2(Y) were compared with organic GROa and NAP-2 for activation of human neutrophils and binding to cellular receptors. As shown in Fig. 1A, GROa and GROa(Y) were equally active in induction of elastase release, whereas NAP-2(Y) was slightly far more potent than NAP-2. Each, the organic and modified cytokines competed together with the same efficiency with their iodinated counterparts for binding to neutrophils (Fig. 1B). In agreement with our former observations (17), GROa, NAP-2, along with the tyrosinesubstituted derivatives didn’t displace 1251-labeled IL-8 as efficiently as unlabeled IL-8. In addition, in contrast to displacement with unlabeled IL-8, the competitors curves obtained with unlabeled GROa(Y), NAP-2(Y), and their organic forms had been not sigmoidal (Fig. 1C). Binding to Neutrophils. Because incorporation of tyrosine residues didn’t substantially have an effect on competitors for IL-8 binding or biological activity, radioiodinated GROa(Y) and NAP-2(Y) have been applied for direct binding studies. Human neutrophils had been incubated for 90 min at four with increasing concentrations of 125 I-labeled GROa(Y) or 125I-labeled NAP2(Y) within the presence or absence of an excess of unlabeled ligand, as well as the binding information have been analyzed. Finest fitting as assessed by the least values on the sum of squares of residuals was achieved by applying a two-binding-site model. Fig. two shows Scatchard plots of representative bindi.