Purification and amino acid analysis of a human macrophage cytotoxicity-inducing factor (MCF).
Recently we have described a CD4+ human T-cell hybridoma Ft.F3 (ATCC HB 9713). This hybridoma produces two proteins having molecular weights of 29 kd (P29) and 14.7 kd (P14.7) that function as activators of human monocyte tumor cytotoxicity and interleukin 1 (IL-1) synthesis (macrophage cytotoxicity-inducing factors, MCFs). Both MCF species were purified to apparent homogeneity, as assessed by two-dimensional (2D) gel electrophoresis, by a combination of dye ligand, ion exchange, and hydrophobic interaction chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Exhaustive treatment of P29 by endoglycosidase F, performic acid, and 40x molar excess 2-mercaptoethanol failed to generate P14.7 from P29. Antisera raised to P29 recognized only P29 in immunoblots of 2D gels of crude hybridoma supernatant. Amino acid composition analyses of both species are similar but not identical. These MCFs appear to be distinct but possibly related proteins important in the inflammatory response, whereas N-terminal analysis of P29 reveals it to be a previously undescribed cytokine.
Jones
C M
CM
Department of Medicine, University of Texas Medical School, Houston.
Prince
C A
CA
Williams
J S
JS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D002848:Chromatography, DEAE-Cellulose; D016207:Cytokines / Q000032:analysis* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005561:Formates; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D015151:Immunoblotting; D008264:Macrophages / Q000502:physiology*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D013601:T-Lymphocytes / Q000378:metabolism
D000596:Amino Acids; D016207:Cytokines; D005561:Formates; C008817:peroxyformic acid; D008623:Mercaptoethanol; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase