Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes.
Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics--molecular mass, basic isoelectric point, and dye column binding--were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.
Yoshimura
T
T
Immunopathology Section, National Cancer Institute, Frederick, MD 21701.
Robinson
E A
EA
Tanaka
S
S
Appella
E
E
Leonard
E J
EJ
D000596:Amino Acids / Q000302:isolation & purification*; D002630:Chemotactic Factors / Q000302:isolation & purification* / Q000502:physiology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007525:Isoelectric Focusing; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008213:Lymphocyte Activation; D008970:Molecular Weight; D009000:Monocytes / Q000502:physiology*; D009504:Neutrophils / Q000502:physiology; D010835:Phytohemagglutinins
D000596:Amino Acids; D002630:Chemotactic Factors; D010835:Phytohemagglutinins; C058918:glioma-derived monocyte chemotactic factor 2