Induction of lysyl oxidase with copper. Properties of an in vitro system.

Aortic tissue from chicks raised 8 to 11 days on diets lacking copper contains diminished levels of the enzyme lysyl oxidase, a copper metalloenzyme. By incubating the deficient aortic tissue in fully oxygenated Waymouth growth medium (MB 751/2) supplemented with 3 to 5 pg/ml of CuSO4, it was possible to restore lysyl oxidase activity to the aorta. Enzyme activation required copper supplements to the serum-free medium. When a partially purified preparation of serum copper proteins supplied the copper, activation was also achieved but at a much lower copper concentration (0.2 pg/ml). Homogenizing the tissue or incubating it under Nz or in the cold blocked the appearance of the copper-induced enzyme activity. Incubating the tissue in a simple solution containing buffer salts and CuS04 did not result in activation. A time course analysis showed that a 3to 5-h lag period preceded the appearance of enzyme activity in the tissue. Further studies with [3H]lysine and ‘%u revealed that the metal became bound to a newly synthesized protein component which affixed to a collagen-derivatized Sepharose 4B column and eluted with 6 M urea, typical of the behavior of lysyl oxidase. On polyacrylamide gel in the presence of sodium dodecyl sulfate, this component migrated with a molecular weight of about 60,000, approximately the molecular weight of chick aorta lysyl oxidase. Cycloheximide, but not actinomycin D, completely inhibited the incorporation of both [3H]lysine and ‘%u into the 60,000 molecular weight component. These data obtained with aortic tissue in a defined medium suggest that activation of lysyl oxidase with copper is a property of metabolically active tissues and proceeds with the binding of copper to newly synthesized protein. The findings strongly support the possibility that induction involves enzyme synthesis rather than enzyme activation.