Evidence linking ceruloplasmin with lysyl oxidase activation

Abstract

The hypothesis that ceruloplasmin transports copper from the liver to peripheral tissues has been evaluated and extended. Activity levels or aortic lysyl oxidase, a copper metalloenzyme, have been compared with ceruloplasmin (p-phenylenediamine oxidase activity) concentration in copper deficient and normal chicks. The marked decrease in lysyl oxidase activity in copper deficiency and this enzyme's rapid return following CuSO(,4) injection have been correlated with similar behavior in ceruloplasmin concentrations. Administering estradiol-17(beta) to deficient birds enhanced the subsequent activation of lysyl oxidase by CuSO(,4). The heightened response correlated with an increase in ceruloplasmin levels but not serum copper. The hormone without copper had no effect on either lysyl oxidase activity or ceruloplasmin. L-ascorbic acid, vitamin C, also enhanced lysyl oxidase activation, but this compound was effective only when copper levels in the system were adequate. In both cases ceruloplasmin levels correlated with aortic enzyme activity. In addition, L-ascorbic acid increased both lysyl oxidase activity and ceruloplasmin in copper-fed chicks. Thus, lysyl oxidase activity and ceruloplasmin levels behaved in a similar fashion under a number of circumstances suggesting a metabolic link between these two proteins. A scheme has been devised for preparing highly purified chicken ceruloplasmin which is needed for a more direct evaluation of this protein's role in lysyl oxidase activation. Ceruloplasmin was isolated from pooled chick serum by precipitation with ethanol:chloroform, ion exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and ion exchange on CM-Sephadex. The purified product, an intense blue protein, migrated as a single band on polyacrylamide gel electrophoresis and was 1300-fold purer than ceruloplasmin in serum. The protein gave a A(,610 nm)/A(,280 nm) of 0.069 (indicative of a high state of purity) and was found to be immunochemically distinct from human ceruloplasmin. The highly purified protein did not increase lysyl oxidase activity in either copper deficient or normal chicks when injected intraveneously (heart puncture) at a dose of 10 (mu)g Cu/kg body wt. Optimum conditions for evaluating a possible role for chicken ceruloplasmin in lysyl oxidase activation are still under investigation.