Eceptors, the incubation samples had been mixed with 50 Al of 40 six (wt/vol) PEG 6000 and placed on ice for 20 min before filtration, to precipitate the proteins. The filters were washed 3 times with ice-cold phosphate-buffered saline containing 0.1 bovine serum albumin (membranes) or 9o PEG 6000 (solubilized receptors), and dried, and radioactivity was measured. The number of binding websites (60,000-90,000 receptors per cell; 2-7 X 109 receptors per ,ug of Caspase Inhibitor Compound membrane protein; 0.7-1.4 x 109 receptors per ,ug of solubilized protein), the dissociation constants, along with the nonspecific binding parameters have been determined by pc modeling as described (17). Nonspecific binding did not exceed 3 (with complete cells), 5 (with membranes), and 11 (with solubilized proteins) of the respective free of charge 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 D1 Receptor Inhibitor Biological Activity soluble receptors, membranes had been freshly solubilized as described above and 60-100 Ag of soluble proteins was incubated inside 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.four) was added plus the soluble proteins have been sedimented by incubation with 140 ,ul of 40 o PEG 6000 for 5 min on ice and centrifugation at 15,000 x g for ten min. The proteins within the pellets have been analyzed by SDS/PAGE and autoradiography as described above. Elastase Release Assay and 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).Final results Tyrosine-Substituted Ligands. The tyrosine-substituted peptides GROa(Y) and NAP-2(Y) have been 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) have been equally active in induction of elastase release, whereas NAP-2(Y) was slightly additional potent than NAP-2. Both, the organic and modified cytokines competed using the same efficiency with their iodinated counterparts for binding to neutrophils (Fig. 1B). In agreement with our former observations (17), GROa, NAP-2, and 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 natural forms were not sigmoidal (Fig. 1C). Binding to Neutrophils. Considering the fact that incorporation of tyrosine residues didn’t substantially impact competition for IL-8 binding or biological activity, radioiodinated GROa(Y) and NAP-2(Y) had been applied for direct binding research. Human neutrophils had been incubated for 90 min at four with escalating concentrations of 125 I-labeled GROa(Y) or 125I-labeled NAP2(Y) inside the presence or absence of an excess of unlabeled ligand, and also the binding data have been analyzed. Most effective fitting as assessed by the least values from the sum of squares of residuals was achieved by applying a two-binding-site model. Fig. two shows Scatchard plots of representative bindi.
