title: In vitro biosynthesis of complement protein D by U937 cells.
abstract: Preliminary studies demonstrating the secretion of antigenic D by blood monocytes/macrophages led us to study the biosynthesis of D by U937 cells, a human monocyte cell line. The kinetics of secretion of D into cell culture supernatants were followed by a solid-phase radioimmunoassay and by hemolytic assay. Daily synthesis of antigenic D was nearly linear (mean +/- 1 SD = 5.3 +/- 2.2 ng D/10(6) cells) over a 6-day period. The D produced after day 2 was hemolytically active, with a specific hemolytic activity greater than (although in the same range as) D in normal serum. Cycloheximide (10(-7) M) inhibited D synthesis, which returned to the levels found in untreated cells after removal of the inhibitor. Supernatants and lysates of cells grown in the presence of [35S]methionine were incubated with rabbit anti-D serum or FD10-1, a monoclonal anti-D antibody, bound to protein A-agarose. Autoradiograms of SDS-PAGE analysis of the precipitates demonstrated a main band of an approximate m.w. of 24,000, co-migrating with purified 125I-D. Identity of this band with D was established by blocking with excess purified D. Pulse-chase studies with the use of [35S]cysteine demonstrated a single D band both intra and extracellularly. Both forms of D had the same apparent m.w. which was approximately 3000 heavier than control 125I-D. These data demonstrate that U937 cells synthesize functionally active D, which appears to be structurally and antigenically similar to D in serum.
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003166:Complement Activating Enzymes / Q000096:biosynthesis*; D011416:Complement Factor D / Q000096:biosynthesis* / Q000502:physiology; D003470:Culture Media; D005312:Fetal Blood / Q000502:physiology; D006461:Hemolysis; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D016403:Lymphoma, Large B-Cell, Diffuse / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D011817:Rabbits
chemical_list: D000596:Amino Acids; D003470:Culture Media; D007531:Isoflurophate; D003166:Complement Activating Enzymes; C493488:CFD protein, human; D011416:Complement Factor D