Three forms of monocyte-derived neutrophil chemotactic factor (MDNCF) distinguished by different lengths of the amino-terminal sequence.
Human monocyte-derived neutrophil chemotactic factor (MDNCF) was purified from culture supernatant of lipopolysaccharide-stimulated human peripheral blood mononuclear leukocytes on a column of Sepharose-bound murine monoclonal anti-MDNCF. About 65% of the culture fluid chemotactic activity was bound to the column. The unbound 35% probably represents chemotactic activity of other cytokines in the culture fluid. More than 85% of the bound activity was eluted by pH 2.5 glycine buffer. When this material was applied to an HPLC-CM column, gradient elution produced four well-separated A280 peaks, each of which had chemotactic activity. N-terminal amino acid analysis of the four peaks revealed three different sequences. One (MDNCF-c) was identical to the sequence that we reported previously. The other two (MDNCF-a and -b) had seven and five additional amino acids, respectively, at the N-terminus. MDNCF-a, -b and -c accounted for 8, 47 and 45% of the total MDNCF peptide. Alignment with the MDNCF cDNA sequence shows that MDNCF-a results from cleavage of a 20 residue signal peptide. MDNCF-c results from culture fluid proteolytic cleavage of the N-terminal sequences of MDNCF-a and -b at an R-S bond. The three peptides occurred in the four HPLC-CM peaks in different ratios. The bulk of any one peptide was distributed in two adjacent HPLC-CM peaks. This suggests that each peptide exists in a minimum of two states. In contrast to our previous multi-step purification, the immunoaffinity and HPLC-CM column sequence resulted in complete purification of MDNCF in two steps and led to identification of two additional MDNCF peptides, one of which has not heretofore been detected.
Yoshimura
T
T
Laboratory of Immunobiology, National Cancer Institute, Frederick, MD 21701.
Robinson
E A
EA
Appella
E
E
Matsushima
K
K
Showalter
S D
SD
Skeel
A
A
Leonard
E J
EJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002630:Chemotactic Factors / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016209:Interleukin-8; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000276:immunology*
D000596:Amino Acids; D002630:Chemotactic Factors; D016209:Interleukin-8