Purification and properties of four species of lysyl oxidase from bovine aorta.
Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49-60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000-33000. 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-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, 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 Cu(2+), and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.
D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001011:Aorta / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008249:Protein-Lysine 6-Oxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
D000596:Amino Acids; D002241:Carbohydrates; D014508:Urea; D000594:Amino Acid Oxidoreductases; D008249:Protein-Lysine 6-Oxidase