Characterization and purification of a mycoplasma membrane-derived macrophage-activating factor

A highly hydrophobic component derived from the membrane of Mycoplasma capricolum has been characterized, purified and assessed for its ability to activate macrophages to tumor cytotoxicity. Initially, crude membranes were evaluated for their solubility in a wide range of solvents. Despite differential solubility in the various solvents, the mycoplasma membranes retained their ability to potentiate macrophage tumor cytotoxicity. Mycoplasma membranes were further characterized by appraising their macrophage-activating ability subsequent to various chemical treatments: cleavage of ester and thioester bonds, oxidation of vicinal hydroxyl groups, and exposure to a broad range of pH. Only strong alkaline treatment (pH>12) caused a reduction in mycoplasma membrane activity: all other chemical treatments were inconsequential. With potential therapeutic applications in mind, mycoplasma membranes were subjected to various physical treatments including heating, freezing/thawing, sonication, lyophilization and storage. The ability of the membranes to induce macrophage activation was stably maintained following all these treatments. Purification of membranes was initiated by a chloroform/methanol lipid extraction. Macrophage-activating ability was found predominantly in the interphase. Proteolytic cleavage with trypsin increased specific activity at least sixfold. Trypsinized fractions were solubilized in 2-chloroethanol and gel filtration was performed on a hydroxylated Sephadex LH-60 column. The active fraction from this column had a further tenfold increase in specific activity. Subsequent rounds of reverse-phase HPLC on this fraction yielded three to four peaks absorbing at 280 nm, of which only one had macrophage-activating ability.