Purification and properties of four species of lysyl oxidase from bovine aorta

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Abstract
Lysyl oxidase of bovine aorta was resolved into 4 enzymically active species by elution from DEAE-cellulose with a salt gradient in 6 M urea, consistent with purification results obtained with enzyme of other tissues. Each of the 4 peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6 M urea. Each enzyme is eluted as a species with MW approximately 30,000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with MW ranging up to 1,000,000 in the absence of urea. The apparent subuit MW of each enzyme species determined by electrophoresis in sodium dodecyl sulfate and 8 M urea is approximately 32,000-33,000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of .alpha.-toluenesulfonyl fluoride into the purification scheme does not alter the resolution of enzyme into 4 spp., suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu2+, and of a carbonyl compound for enzyme function. A method for the purification of multiple species of lysyl oxidase is described. Significant chemical differences exist between the different enzyme forms.