Journal of Free Radicals in Biology & Medicine. Vol. I. pp. 319-325. 1985 0748-5514/85 $3.00+ .00 Printed in the USA. All rights reserved. ~ 1986 Pergamon Press Ltd. BIOSYNTHESIS OF SUPEROXIDE DISMUTASE IN SACCHAROMYCES CEREVISIAE: EFFECTS OF PARAQUAT AND COPPER FANG-JEN LEE and HOSNf M. HASSAN Departments of Food Scienceand Microbiology. North Carolina State University, Raleigh, NC 27695, U.S.A. (Received 15 July 1985; Revised 14 October 1985; Accepted 18 October 1985) Abstract--Growth of Saccharomyces cerevisiae in the presence of paraquat caused an increase the intracellular fluxof superoxide radicals and in superoxide dismutase biosynthesis. The addition of copper to the growth medium also elicited an increase in superoxide dismutase levels. A cytochrome c deficient mutant strain was found to be more responsive than the wild type strain to paraquat and/or copper by increasing its copper-zinc superoxide dismutase. Catalase activity in both strains was not significantly affected by paraquat and/or copper. Keywords--Saccharomyces cerevisiae, Paraquat, Copper, Superoxide Dismutase, Catalase, Cyt. c. deficiency INTRODUCTION The partially reduced oxygen intermediates: superoxide radical (02-), hydrogen peroxide (H202) and the hy- droxyl radical (OH-) are dangerously reactive. About 2-5% of the total oxygen consumed by aerobic orga- nisms is reduced by the univalent pathway and gener- ates O2-, H202 and OH.. I-3 These partially reduced species of oxygen are known to be toxic and destructive to living cells. They can inactivate enzymes, cause lipid peroxidation, destroy the integrity of cell membranes, cause DNA breaks and cause mutations.l'3-6 Aerobic survival is only possible by virtue of a unique set of protective enzymes: superoxide dismutase (SOD) and catalase which dismutase 02- and H202, respectively. These enzymes lower the steady state concentrations of O2- and H202, and minimize the likelihood of their interaction to generate OH" via the iron catalyzed Ha- ber-weiss reaction. 3.7 The application of superoxide dismutase and catalase as antioxidants in the food industry, 8-1° and as anti- inflammatory and radioprotective drugs in medicine 1'-13 has prompted its commercial production from bovine blood and from genetically engineered microorga- nisms. 14 In a previous study we have shown that oxygen induces the copper-zinc superoxide dismutase (Cu- Paper Number 10008 of the Journal Series of the North Carolina AgriculturalResearchService, Raleigh,NC 27695. The use of trade names in this publicationdoes not implyendorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned. ZnSOD) in Saccharomyces cerevisiae and that cyto- chrome c deficiency results in higher enzyme yields. 15 Previous studies in E. coil and other procaryotes 2"6"16-2° have demonstrated that increasing the intracellular flux of O2-, via cyclic oxidation-reduction of several redox active compounds, induces the synthesis of the man- ganese-containing SOD (MnSOD). The objective of this study was to determine the optimum conditions for maximal biosynthesis of superoxide dismutase in S. cerevisiae. We demonstrate that both paraquat and cop- per increase the specific activity of CuZnSOD in S. cerevisiae, while having no effect on catalase. MATERIALS AND METHODS Organisms Saccharomyces cerevisiae vat. ellipsoideus ATCC 18790 (wild type) and ATCC 18789 (cytochrome c de- ficient mutant) were obtained from the American Type Culture Collection. MeSa The composition of the growth medium was (per liter): MgSO4 - 7H20, 0.5 g; KH2PO,, 1.0 g; (NH4)2 S04, 5.0 g; CaCI2, 0.1 g; NaCI, 0.1 g; yeast extract, 7.0 g (BBL). Glucose was added at 5% (glucose-rich medium, GRM) or at 0.5% (glucose-limited medium, GLM). Yeast extracted was reduced to 2.0 g per liter in studies dealing with the kinetics of enzyme biosyn- thesis. 319