396
Biochimica et Biophysica A cta, 444 (1976) 396--406
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 28023 COPPER DEFICIENCY AND ERYTHROCUPREIN (2Cu,2Zn-SUPEROXIDE DISMUTASE)
WOLFRAM BOHNENKAMP and ULRICH WESER Physiologisch-Chemisches Institut der Universitiit, Hoppe-Seyler-Strasse 1, D-7400 Tiibingen (G.F.R.)
(Received March 4th, 1976)
Summary The activities of 2Cu,2Zn-superoxide dismutase, ferroxidase (ceruloplasmin), catalase and glutathione peroxidase were measured in the blood of rats during copper depletion. Two control groups of animals were used; one received the regular diet containing all essential components including copper and the other group was maintained on a diet, containing 1% the a m o u n t of copper in normal diet, copper being supplied as Cu(Leu)2 in the drinking water. Both groups showed no detectable differences, either in the copper content of blood or in the measured four enzymic activities. Excessive copper (injected intraperitoneally) caused only an insignificant rise in the enzymic activities (0---10%) compared to either control. After starting copper depletion ferroxidase activity decreases to 15% on the 15th day, while the 2Cu,2Zn-superoxide dismutase activity decreases to 40% on the 45th day. Ferroxidase activity shows rapid but transient changes immediately after perturbation in plasma copper levels. By contrast, the 2Cu,2Znsuperoxide dismutase activity more closely parallels the overall copper deficiency. Dietary repletion with copper raises the 2Cu,2Zn-superoxide dismutase activity to 94% and the ferroxidase activity to 80% of the control values within 36 h. Apart from the copper-dependent anemia catalase activity was decreased. However, 15 days after the start of the copper depletion catalase activity rises again and reaches the control value on the 40th day and a 30% stimulation was even seen on the 58th day. Upon copper repletion catalase activity reaches 166% of the control within 14 days. No copper-dependent differences of glutathione peroxidase activity were seen regardless whatever copper level was present in the rats.
397 Introduction Erythrocyprein, a 2Cu,2Zn-protein which displays superoxide dismutase activity, belongs at present to the most studied of the metal-proteins [ 1--5], even though it remains unclear whether or n o t the superoxide dismutase activity represents the genuine physiological role of this enzyme and to what extent 2Cu, 2Zn-superoxide dismutase is essential for an aerobic organism. As it is possible to measure the 2Cu,2Zn-superoxide dismutase activity in limited amounts of crude biological material [6], this enzyme turned out to be suitable for measurements over a longer period under changing conditions. Measurements with radioactive copper ions revealed that 2Cu,2Zn-superoxide dismutase obtains its Cu 2÷ from ceruloplasmin (ferroxidase) [7,8]. Thus, it is obvious that both copper-proteins are related to each other. The determination of ceruloplasmin levels measured by the ferroxidase activity is c o m m o n l y employed to study s y m p t o m s of a copper deficiency on a molecular level [9-14]. In this context it excited great interest to examine the correlation of copper deficiency and the 2Cu,2Zn-superoxide dismutase activity. Both enzymic assays would be most convenient for the determination of a copper deficiency at an early state. However, it must be ascertained that in the case of 2Cu,2Znsuperoxide dismutase measurement disturbing superoxide dismutase activities derived from other components including the Fe- and/or the Mn-enzymes are successfully excluded. Provided the 2Cu,2Zn-superoxide dismutase is an important enzyme in singlet oxygen [ 5], superoxide and/or peroxide metabolism, the organism must take care that the enzymic activity of erythrocuprein does not decrease too much or this activity drop ought to be compensated by other enzymes involved in the biochemistry of oxygen. Measurements of the 2Cu,2Zn-superoxide dismutatase, ferroxidase, and glutathione peroxidase activities during copper deficiency promised to bring some light to this field. Materials and Methods Chemicals used in the electrophoresis were from Serva (Heidelberg). All other chemicals were from Merck (Darmstadt) and of reagent grade quality. Deionized water which was additionally distilled over quartz was used throughout. Animals: White male Wistar rats (40 g) were obtained from Versuchstieranstalt (Tuttlingen) and were maintained in all-plastic cages at 21°C and 60% humidity. Diet and drinking water were supplied ad libitum. For the cleaning of the cages only quartz-distilled water was employed. Normal (cat. No. 1320) and copper-deficient (cat. No. C1040) diets were obtained from Altromin (Lage). The powder was transferred into pellets to minimize urinary or fecal deterioration. The copper c o n t e n t was less than 0.25 mg/ kg, i.e. two orders of magnitude lower than the normal diet. Drinking water was tap water for the control rats and quartz-distilled for the copper
Biochimica et Biophysica A cta, 444 (1976) 396--406
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 28023 COPPER DEFICIENCY AND ERYTHROCUPREIN (2Cu,2Zn-SUPEROXIDE DISMUTASE)
WOLFRAM BOHNENKAMP and ULRICH WESER Physiologisch-Chemisches Institut der Universitiit, Hoppe-Seyler-Strasse 1, D-7400 Tiibingen (G.F.R.)
(Received March 4th, 1976)
Summary The activities of 2Cu,2Zn-superoxide dismutase, ferroxidase (ceruloplasmin), catalase and glutathione peroxidase were measured in the blood of rats during copper depletion. Two control groups of animals were used; one received the regular diet containing all essential components including copper and the other group was maintained on a diet, containing 1% the a m o u n t of copper in normal diet, copper being supplied as Cu(Leu)2 in the drinking water. Both groups showed no detectable differences, either in the copper content of blood or in the measured four enzymic activities. Excessive copper (injected intraperitoneally) caused only an insignificant rise in the enzymic activities (0---10%) compared to either control. After starting copper depletion ferroxidase activity decreases to 15% on the 15th day, while the 2Cu,2Zn-superoxide dismutase activity decreases to 40% on the 45th day. Ferroxidase activity shows rapid but transient changes immediately after perturbation in plasma copper levels. By contrast, the 2Cu,2Znsuperoxide dismutase activity more closely parallels the overall copper deficiency. Dietary repletion with copper raises the 2Cu,2Zn-superoxide dismutase activity to 94% and the ferroxidase activity to 80% of the control values within 36 h. Apart from the copper-dependent anemia catalase activity was decreased. However, 15 days after the start of the copper depletion catalase activity rises again and reaches the control value on the 40th day and a 30% stimulation was even seen on the 58th day. Upon copper repletion catalase activity reaches 166% of the control within 14 days. No copper-dependent differences of glutathione peroxidase activity were seen regardless whatever copper level was present in the rats.
397 Introduction Erythrocyprein, a 2Cu,2Zn-protein which displays superoxide dismutase activity, belongs at present to the most studied of the metal-proteins [ 1--5], even though it remains unclear whether or n o t the superoxide dismutase activity represents the genuine physiological role of this enzyme and to what extent 2Cu, 2Zn-superoxide dismutase is essential for an aerobic organism. As it is possible to measure the 2Cu,2Zn-superoxide dismutase activity in limited amounts of crude biological material [6], this enzyme turned out to be suitable for measurements over a longer period under changing conditions. Measurements with radioactive copper ions revealed that 2Cu,2Zn-superoxide dismutase obtains its Cu 2÷ from ceruloplasmin (ferroxidase) [7,8]. Thus, it is obvious that both copper-proteins are related to each other. The determination of ceruloplasmin levels measured by the ferroxidase activity is c o m m o n l y employed to study s y m p t o m s of a copper deficiency on a molecular level [9-14]. In this context it excited great interest to examine the correlation of copper deficiency and the 2Cu,2Zn-superoxide dismutase activity. Both enzymic assays would be most convenient for the determination of a copper deficiency at an early state. However, it must be ascertained that in the case of 2Cu,2Znsuperoxide dismutase measurement disturbing superoxide dismutase activities derived from other components including the Fe- and/or the Mn-enzymes are successfully excluded. Provided the 2Cu,2Zn-superoxide dismutase is an important enzyme in singlet oxygen [ 5], superoxide and/or peroxide metabolism, the organism must take care that the enzymic activity of erythrocuprein does not decrease too much or this activity drop ought to be compensated by other enzymes involved in the biochemistry of oxygen. Measurements of the 2Cu,2Zn-superoxide dismutatase, ferroxidase, and glutathione peroxidase activities during copper deficiency promised to bring some light to this field. Materials and Methods Chemicals used in the electrophoresis were from Serva (Heidelberg). All other chemicals were from Merck (Darmstadt) and of reagent grade quality. Deionized water which was additionally distilled over quartz was used throughout. Animals: White male Wistar rats (40 g) were obtained from Versuchstieranstalt (Tuttlingen) and were maintained in all-plastic cages at 21°C and 60% humidity. Diet and drinking water were supplied ad libitum. For the cleaning of the cages only quartz-distilled water was employed. Normal (cat. No. 1320) and copper-deficient (cat. No. C1040) diets were obtained from Altromin (Lage). The powder was transferred into pellets to minimize urinary or fecal deterioration. The copper c o n t e n t was less than 0.25 mg/ kg, i.e. two orders of magnitude lower than the normal diet. Drinking water was tap water for the control rats and quartz-distilled for the copper
