GRANULOCYTIC PROTEIN P25 IS A DNA-BINDING SUBUNIT OF PROTEIN M(R)=50,000 - SUBCELLULAR-LOCALIZATION, CELL AND SPECIES-SPECIFICITY

We have previously reported the presence and isolation of the novel pr otein M(r) = 25,000 (p25) from human granulocytes. In this study, the protein p25 was characterized by its: (a) ability to bind DNA, (b) sub unit association, (c) partial protein sequencing, (d) subcellular loca lization, (e) cellular and species specificity and (f) stability in th e presence of released granulocytic proteinases. For the detection of p25 in various extracts, fractions and types of human or animal hemato poietic cells, SDS-PAGE/Western blotting and immunohistochemical stain ing were used. The protein p25 was subjected to N-terminal amino acid sequence analysis. Protein p25-DNA interactions were monitored using S outhwestern blotting. Selective inhibition of granulocytic proteinases was performed. Granulocytic protein p25 was found to be a product of oxidative cleavage of disulfide bridges in the p50 dimer. It was shown that neither protein p50 nor the p25 subunit is a degradation product of a protein of higher molecular weight. The N-terminal amino acid se quence of p25 was: RLNYNKPHAA. Binding capacity for double stranded DN A without significant sequence specificity was revealed and nuclear lo calization of some fraction of p50 dimer was established. The data con cerning the cell and species specificity demonstrated that the protein is expressed only in normal human granulocytes. In summary, protein p 25 originates from splitting of the p50 dimer. This subunit shows no i dentity with proteins already sequenced. DNA-binding of p25 is not seq uence specific. It is concluded that the protein p50 is localized in t he nuclei and cytoplasmic granules of mature human polymorphonuclear l eukocytes or granulocytes of species high on the evolutionary tree. Th e functions of this protein remain to be determined. Copyright (C) 199 6 Elsevier Science Ltd.