The 23-kilodalton protein, a substrate of protein kinase C in bovine neutrophil cytosol is a member of the S100 family

Abstract

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 Ca(2+)-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 Ca(2+)-binding proteins belonging to the S100 family.(ABSTRACT TRUNCATED AT 250 WORDS)

The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils

“…Because of its high concentration in neutrophils, the S100A8/A9 protein is effectively a potential reservoir of arachidonic acid of high capacity. Its translocation to the plasma membrane [13] together with p67phox, p47phox and Rac at the onset of the oxidase activation would be consistent with the delivery of arachidonic acid to the membrane-bound flavocytochrome b.…”

“…Peptide masses were assigned and used for database searching (Swiss Prot) using the program MS-Fit at the University of California San Francisco (http://prospector.ucsf.edu/). Eight peptides with masses ranging from 715.4 to 2184.9 were found to fully match with peptide fragments corresponding to the sequence of the bovine S100A9, previously called p23 [13] and also referred as calgranulin B. In addition, the mass spectrum indicated the absence of protein contaminant that could have comigrated with S100A9 during SDS/PAGE.…”

“…The S100A8/A9 was in large excess with respect to the cytosolic phox proteins, the disproportion in these two categories of proteins reflecting probably their disproportion in crude neutrophil cytosol. In fact S100A8/ A9 represents 10% to 20% of the cytosolic protein content of bovine neutrophil [13], compared to less than 0.5% for p47phox and p67phox [19]. The second pool (HTP II fractions 18-21) consisted of the remainder of S100A8/A9, accompanied by a majority of p67phox, p47phox and Rac.…”

“…The preincubation step is required for the transition of the flavocytochrome from a dormant state to an active state [12]. In an early study of the bovine S100A8/A9 (called at that time p7/p23) [13], it was found by an immunofluorescent approach that in resting neutrophils the S100A8/A9 protein was evenly distributed within the cytoplasm and that after activation of the cells by phorbol ester, it concentrated under the plasma membrane, together with the cytosolic phox proteins. A similar behaviour was reported in the case of human neutrophil S100A8 [14].…”

Regulation of superoxide production in neutrophils: role of calcium influx

“…Subsequent to Ca 2+ fixation, a heterocomplex is formed by a noncovalent association of Ca 2+ -binding proteins S100A8 and S100A9 [154, 155]. Using an immunofluorescence approach, it has been reported that in bovine neutrophils stimulated by PMA [156], these S100 proteins are concentrated under the plasma membrane with cytosolic phox proteins. A similar observation is made for S100A8 in human neutrophils [155].…”