NEUTROPHIL-ACTIVATING PEPTIDE-2 AND MELANOMA GROWTH-STIMULATORY ACTIVITY ARE FUNCTIONAL AS MONOMERS FOR NEUTROPHIL ACTIVATION

Neutrophil-activating peptide-2 (NAP-2) and melanoma growth stimulator y activity (MGSA) are members of the chemokine family of inflammatory proteins. The structures of NAP-2, determined by x-ray crystallography , and MGSA, elucidated by NMR spectroscopy, revealed a tetramer and di mer, respectively. In order to address the relevance of multimeric spe cies to their activities on neutrophils, analogs of NAP-2 and MGSA wer e synthesized in which the backbone amide proton of Leu-22 in NAP-2, a nd Val-26 in MGSA, was substituted with the bulky methyl group (NH --> NCH3). These analogs were shown to be monomeric by sedimentation equi librium ultracentrifugation studies and were similar to the correspond ing native protein in assays for neutrophil elastase release and Ca2mobilization from IL-8R1 and IL-8R2 transformed cells. Sedimentation e quilibrium studies of the native NAP-2 and MGSA were also carried out to address the association behavior. For NAP-2, there was no evidence for the tetramer, but an equilibrium between monomers and dimers and t he dissociation constant was calculated to be 50-100 mu M. Similarly, MGSA showed a monomer-dimer equilibrium with a K-d of similar to-5 mu M. The data from the monomeric analogs and also the calculation of dis sociation constants indicate that NAP-2 and MGSA have a tendency to as sociate above the concentrations required for maximal activity or for receptor activation, but at functional concentrations they are predomi nantly monomers.