A bovine neutrophil protein termed p23 because of an apparent molecular mass of 23 kDa in SDS-PAGE is present in large amounts both in a soluble form in the cytosolic fraction of bovine neutrophil homogenates and associated to the cytoskeleton. P23 is accompanied during the first steps of the purification procedure by a smaller size protein termed p7 on the basis of a rate of migration in SDS-PAGE corresponding to a 7-kDa protein [Stasia, M. J., Dianoux, A. C., & Vignais, P. V. (1989) Biochemistry 28, 9659-9667]. The two proteins, p23 and p7, have been purified to homogeneity by an improved procedure consisting of two chromatographic steps. The electrospray mass spectrometry technique applied to p23 and p7 indicated molecular masses close to 17 and 10 kDa, respectively, significantly different from the masses derived by SDS-PAGE. Bovine neutrophil p23 and p7 presented large primary structure homologies with two human proteins, MRP14 and MRP8, which are expressed in large amounts in macrophages under conditions of chronic inflammation. In addition, p23 and p7 cross-reacted with monoclonal antibodies specific of MRP14 and MRP8. Bovine p23 and p7 bound Ca2+, and their amino acid sequences contained two Ca2+-binding domains per protein, largely identical to those of human MRP14 and MRP8. Bovine p23 and p7 associated together to form a heterodimeric complex, which largely escaped attack by trypsin, whereas the isolated p23 and p7 components were readily digested. These features are typical of Ca2+-binding proteins belonging to the S100 family. Despite similarities, bovine p23 differed from human MRP14 by a more extended C-terminal region which most likely contained the PKC-specific site of phosphorylation of p23. By an immunofluorescent technique, the p23,p7 complex was found to form clusters evenly distributed in the cytoplasm of resting neutrophils and to concentrate under the plasma membrane in phorbol-activated neutrophils, suggesting possible association with cytoskeleton components. Whereas the p23 subunit in the p23,p7 complex is phosphorylated in bovine neutrophils activated by phorbol esters or opsonized zymosan, chemotaxis was hardly affected by added p23,p7 complex, and there was no significant effect of p23 antibodies on oxidase activation in a cell-free system.
