Dataset Information

Regulation of aortic CuZn-superoxide dismutase with copper. Caeruloplasmin and albumin re-activate and transfer copper to the enzyme in culture.

ABSTRACT: Caeruloplasmin and albumin were compared as potential donors of copper to Cu2Zn2-superoxide dismutase (CuZn-SOD) in culture. Aortas from 15-day copper-deficient chicks were suspended in oxygenated, serum-free, Waymouth medium (752/1) for 24 h. SOD activity was restored when the medium was supplemented with CuCl2, a copper-albumin complex or caeruloplasmin, all present at a level equivalent to 5 microM-copper. Activation did not occur at 4 degrees C or with Cu-EDTA as the supplement. Mn2+ and Zn2+, alone or in combination, did not activate nor enhance the activation achieved by CuCl2. The activation with CuCl2 was not inhibited by cycloheximide or cordycepin. [67Cu]Caeruloplasmin and albumin when added to the medium transferred radioactive copper to at least three cytosolic protein fractions, one of which was determined by immunoprecipitation to be CuZn-SOD. The transfer of 67Cu from caeruloplasmin was inhibited by increasing amounts of unlabelled caeruloplasmin; disodium EDTA (1.0 mM) had no effect on the transfer of copper from caeruloplasmin. These data show that aortic SOD activity, suppressed in copper deficiency, can be restored by incubating the aortas in culture medium supplemented with copper salts. In this system, caeruloplasmin and Cu-albumin appear equally capable of activating aortic CuZn-SOD. Moreover, the transfer of copper into the enzyme structure appears to be the primary event restoring catalytic activity to the enzyme.

SUBMITTER: Dameron CT

PROVIDER: S-EPMC1148601 | biostudies-other | 1987 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:BackgroundCuZn-Superoxide dismutase (SOD) is a unique enzyme, which can catalyzes the dismutation of inevitable metabolic product i.e.; superoxide anion into molecular oxygen and hydrogen peroxide. The enzyme has gained wide interest in pharmaceutical industries due to its highly acclaimed antioxidative properties. The recombinant expression of this protein in its enzymatically active and stable form is highly desired and hence optimization of culture conditions and characterization of the related biochemical properties are essential to explore the significance of the enzyme in physiological, therapeutic, structural and transgenic research.ResultsHigh-level expression of the chloroplastic isoform of Pisum sativum CuZn-SOD was achieved at 18°C, upon isopropyl β-D-1-thiogalactopyranoside induction and the process was optimized for maximum recovery of the protein in its soluble (enzymatically active) form. Both crude and purified protein fractions display significant increase in activity following supplementation of defined concentration Cu (CuSO4) and Zn (ZnSO4). Yield of the purified recombinant protein was ~ 4 mg L(-1) of culture volume and the bacterial biomass was ~ 4.5 g L(-1). The recombinant pea chloroplastic SOD was found to possess nearly 6 fold higher superoxide dismutase activity and the peroxidase activity was also 5 fold higher as compared to commercially available CuZn-superoxide dismutase. The computational, spectroscopic and biochemical characterization reveals that the protein harbors all the characteristics features of this class of enzyme. The enzyme was found to be exceptionally stable as evident from pH and temperature incubation studies and maintenance of SOD activity upon prolonged storage.ConclusionsOverexpression and purification strategy presented here describes an efficient protocol for the production of a highly active and stable CuZn-superoxide dismutase in its recombinant form in E. coli system. The strategy can be utilized for the large-scale preparation of active CuZn-superoxide dismutase and thus it has wide application in pharmaceutical industries and also for elucidating the potential of this protein endowed with exceptional stability and activity.

Dataset Information

Regulation of aortic CuZn-superoxide dismutase with copper. Caeruloplasmin and albumin re-activate and transfer copper to the enzyme in culture.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets