PURIFICATION AND CHARACTERIZATION OF AN INDUCIBLE METALLOPROTEASE INHIBITOR FROM THE HEMOLYMPH OF GREATER WAX MOTH LARVAE, GALLERIA-MELLONELLA

In this paper, we report the detection, purification and characterizat ion of the first metalloprotease inhibitor (IMPI) from invertebrates. IMPI was purified from the hemolymph of last-instar larvae of Galleria mellonella by precipitation with trichloroacetic acid and heat follow ed by affinity chromatography on a thermolysin-Sepharose column and ge l filtration or reverse-phase high-performance liquid chromatography. For the detection of inhibitor activity, a new azocoll assay was estab lished. IMPI was only detectable in larvae that had been injected with bacterial or fungal provocators, suggesting that it is induced nonspe cifically during the humoral immune response. Injection of larvae with IMPI rendered them resistant to thermolysin, in quantities that norma lly would be lethal for them. IMPI was shown to be specific for metall oproteases. The molecular mass of IMPI was determined by mass spectrom etry to be 8360 Da. Purified IMPI was heterogeneous, owing to differen t degrees of glycosylation with hexose/hexosamine and deoxyhexose resi dues. Ten cysteine residues were found in the molecule, and these are presumed to form five disulfide bridges. The amino terminus was blocke d, but a partial amino-acid sequence starting from the thermolysin cle avage site was determined; this sequence exhibited no similarity with other known proteins, suggesting that the IMPI represents a new type o f protease inhibitor.