ABSTRACT
We have previously reported that the urine of febrile humans contained large quantities of an inhibitor of IL-1-induced murine thymocyte proliferation that was a glycoprotein between 30 and 40 kD in size. In the present study this factor has been purified to homogeneity using a sequence of eight purification steps (ammonium sulfate precipitation, ion exchange chromatography, molecular sieve chromatography, hydrophobic affinity chromatography, hydroxylapatite chromatography, fast protein liquid chromatography, and two HPLC steps). SDS-PAGE analysis indicates that the purified material is a 38-kD molecule. Evidence based on a partial amino acid sequence analysis as well as enzyme studies indicates that this inhibitor is a type of human DNase I.
Subject(s)
Glycoproteins/urine , Interleukin-1/antagonists & inhibitors , Proteins/isolation & purification , Sialoglycoproteins , Amino Acid Sequence , Deoxyribonuclease I/metabolism , Humans , Interleukin 1 Receptor Antagonist Protein , Molecular Sequence Data , Molecular Weight , Sequence Homology, Nucleic AcidABSTRACT
The generation of leukotrienes and histamine release by the mouse mastocytoma cell line MMC-16 was investigated. These cells produced leukotriene C4 (LTC4) and released histamine upon calcium ionophore A23187 and antigen stimulation. The ionophore also stimulated the biosynthesis of leukotriene B4 (LTB4) by MMC-16. Generation of LTC4 was confirmed by its characteristic UV absorption spectrum, fast atom bombardment-MS, equivalent HPLC retention time with an authentic standard and radioimmunoassay. Leukotriene B4 was characterized by its distinctive UV spectrum and HPLC retention time compared with synthetic material. IgE-mediated LTC4 generation was also observed in a dose dependent fashion with MMC-16 cells passively sensitized with monoclonal IgE specific for ovalbumin. LTC4 biosynthesis was effectively inhibited by the lipoxygenase inhibitor NDGA.
