title: Purification and partial biochemical characterization of a Mycoplasma fermentans-derived substance that activates macrophages to release nitric oxide, tumor necrosis factor, and interleukin-6.
abstract: Mycoplasmal products may exert a number of diverse in vitro effects on cells of the immune system. A macrophage-activating substance from Mycoplasma fermentans was described in this laboratory and named mycoplasma-derived high-molecular-weight material (MDHM). Using synthesis of nitric oxide by peritoneal cells from endotoxin low-responder mice as an assay system, MDHM was purified as follows. After freeze-thawing of M. fermentans, MDHM activity was sedimented with the membrane fraction. Membranes were delipidated with chloroform-methanol, and MDHM activity was extracted with octyl glucoside. Coextracted proteins were degraded by proteinase K. MDHM was further purified by reversed-phase high-pressure liquid chromatography and eluted in one major and one minor peak of activity. Neither carbohydrates nor amino acids were found as constituents. MDHM had the following properties: it partitioned into the phenol phase upon phenol-water extraction and into the Triton phase after extraction with Triton X-114. MDHM was not inactivated by either phospholipase A2 or triglyceride lipases. However, mild periodate treatment led to a > 95% loss of activity. Also, alkaline hydrolysis at 25 degrees C completely abolished MDHM activity with a half-life of 2 min. MDHM activity was spread out over a wide molecular weight range upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes, whereas after proteinase treatment MDHM activity migrated close to the front. These features of MDHM, taken together, speak in favor of an amphiphilic molecule with a lipid moiety carrying fatty acids in ester linkage and a polyol moiety of unknown character. MDHM was active in the nanogram-per-milliliter range, activating macrophages to release nitric oxide, interleukin-6, and tumor necrosis factor.
authors.affiliation: Immunology Research Group, Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany.
authors.forename: P F
authors.initials: PF
authors.lastname: Mühlradt
authors.forename: M
authors.initials: M
authors.lastname: Frisch
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005960:Glucosides / Q000494:pharmacology; D015850:Interleukin-6 / Q000096:biosynthesis*; D008262:Macrophage Activation / Q000187:drug effects*; D008563:Membrane Lipids / Q000302:isolation & purification; D051379:Mice; D008809:Mice, Inbred C3H; D008970:Molecular Weight; D016858:Mycoplasma fermentans / Q000737:chemistry*; D009569:Nitric Oxide / Q000096:biosynthesis*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D005960:Glucosides; D015850:Interleukin-6; D008563:Membrane Lipids; D014409:Tumor Necrosis Factor-alpha; C018619:octyl-beta-D-glucoside; D009569:Nitric Oxide