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 abil ity to activate macrophages to tumor cytotoxicity. Initially, crude me mbranes were evaluated for their solubility in a wide range of solvent s. Despite differential solubility in the various solvents, the mycopl asma membranes retained their ability to potentiate macrophage tumor c ytotoxicity. Mycoplasma membranes were further characterized by apprai sing their macrophage-activating ability subsequent to various chemica l treatments: cleavage of ester and thioester bonds, oxidation of vici nal 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 pot ential therapeutic applications in mind, mycoplasma membranes were sub jected to various physical treatments including heating, freezing/thaw ing, sonication, lyophilization and storage. The ability of the membra nes to induce macrophage activation was stably maintained following al l these treatments. Purification of membranes was initiated by a chlor oform/methanol lipid extraction. Macrophage-activating ability was fou nd predominantly in the interphase. Proteolytic cleavage with trypsin increased specific activity at least sixfold. Trypsinized fractions we re solubilized in 2-chloroethanol and gel filtration was performed on a hydroxylated Sephadex LH-60 column. The active fraction from this co lumn had a further tenfold increase in specific activity. Subsequent r ounds of reverse-phase HPLC on this fraction yielded three to four pea ks absorbing at 280 nm, of which only one had macrophage-activating ab ility.