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 protein M(r) = 25,000 (p25) from human granulocytes. In this study, the protein p25 was characterized by its: (a) ability to bind DNA, (b) subunit association, (c) partial protein sequencing, (d) subcellular localization, (e) cellular and species specificity and (f) stability in the presence of released granulocytic proteinases. For the detection of p25 in various extracts, fractions and types of human or animal hematopoietic cells, SDS-PAGE/Western blotting and immunohistochemical staining were used. The protein p25 was subjected to N-terminal amino acid sequence analysis. Protein p25-DNA interactions were monitored using Southwestern 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 sequence of p25 was: RLNYNKPHAA. Binding capacity for double stranded DNA without significant sequence specificity was revealed and nuclear localization of some fraction of p50 dimer was established. The data concerning the cell and species specificity demonstrated that the protein is expressed only in normal human granulocytes. In summary, protein p25 originates from splitting of the p50 dimer. This subunit shows no identity with proteins already sequenced. DNA-binding of p25 is not sequence specific. It is concluded that the protein p50 is localized in the nuclei and cytoplasmic granules of mature human polymorphonuclear leukocytes or granulocytes of species high on the evolutionary tree. The functions of this protein remain to be determined.
Cabart
P
P
Department of Cell Biochemistry, Institute of Hematology and Blood Transfusion, Prague, Czech Republic.
Kalousek
I
I
Jandová
D
D
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D000090266:Chromobox Protein Homolog 5; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D006098:Granulocytes / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D012016:Reference Values; D013045:Species Specificity; D013347:Subcellular Fractions / Q000737:chemistry*
D000911:Antibodies, Monoclonal; C000715594:CBX5 protein, human; D004268:DNA-Binding Proteins; D007976:Leupeptins; D010436:Pepstatins; D011480:Protease Inhibitors; C103843:p25 protein (protein M subunit); D000090266:Chromobox Protein Homolog 5; C014195:Streptomyces pepsin inhibitor; C032854:leupeptin; C031375:pepstatin