516
Biochimica et Biophysica Acta, 496 (1977) 516-520 © Elsevier/North-Holland Biomedical Press
BBA 28151
ELEVATED ERYTHROCYTE GLUTATHIONE ASSOCIATED WITH ELEVATED SUBSTRATE IN HIGH- AND LOW-GLUTATHIONE SHEEP * JOSEPH E. SMITH
Department of Pathology, College of Veterinary Medicine, Kansas Sate University, Manhattan, Kan. 66506 (U.S.A.) (Received June 15th, 1976)
Summary Erythrocyte glutathione concentration increases dramatically in sheep when they become anemic. To determine the mechanism of this change in glutathione control, we measured the enzymes and substrates necessary for glutathione synthesis after acute blood loss in both low- (-y-glutamylcysteine synthetase deficient) and high-glutathione sheep. Erythrocyte glutamate, ATP, and glycine increased dramatically in all sheep. Erythrocyte -y-glutamylcysteine synthetase increased slowly and seemed unrelated to changes in glutathione. Erythrocyte glutathione synthetase and cysteine and plasma cysteine, glutamate and glycine did not change significantly. Apparently substrate concentrations may be important in regulating erythrocyte glutathione levels.
Introduction Erythrocyte glutathione is synthesized from its three constituent amino acids in two reactions. In the first -y-glutamylcysteine (Glu-Cys) is formed from glutamic acid and cysteine in a reaction catalyzed by Glu-Cys synthetase (Lglutamate: L-cysteine -y-ligase (ADP-forming), EC 6.3.2.2); in the second reaction glycine is added to Glu-Cys forming glutathione that is catalyzed by glutathione synthetase (-Y-L-glutamyl-L-cysteine : glycine ligas8 (ADP-forming), EC 5.3.2.3). Both reactions require adenosine-5'-triphosphate (ATP) and magnesium or manganese and both yield adenosine-5'-diphosphate (ADP) and inorganic phosphate [1] . Normally erythrocyte glutathione exists predominantly as the reduced form (GSH) and is maintained fairly constant [2]. In sheep GSH concentration is * Contributions
No. 396. of the Department ()f Pathology, College of Veterinary Medicine. AES, Kansas State University, Manhattan, Kan. 66506, U.S.A.
517
genetically controlled. Levels of GSH (> 1 mg/g of hemoglobin) in some sheep are similar to red cell levels in other mammals; in others it is lower « 1 mg/g of hemoglobin) [3,4]. There are two distinct types of low-glutathione sheep. One type has dimished Glu-Cys synthetase activity; the other has a defect cysteine transport system [6]. Acute blood loss in either low- or high-GSH sheep disturbs mechanisms that control glutathione and allows glutathione to increase [7-9] . To understand the change in GSH control, we measured the enzymes and substrates required for GSH synthesis during and after phlebotomy of highand low-glutathione sheep. Materials and Methods Two sheep with high erythrocyte glutathione and two with low erythrocyte glutathione (GC synthetase-deficient type) were selected for each experiment. This paper describes the results of two experiments. During the experiment they were fed alfalfa hay and a mixed grain ration with no additional supplement (e.g. iron). Blood (approximately 10 ml/lb of body weight) was taken daily from each animal. After three phlebotomies, packed cell volumes decreased to 1/3 of the pre-phlebotomy levels, so the procedure was stopped. Samples were taken at regular intervals before and after phlebotomy for substrate and enzymatic activities. We determined reduced glutathione as the yellow DTNB anion [10]; ATP by using the luciferin-luciferase method [11]; hemoglobin as the cyanmethemoglobin derivative [12] ; and packed cell volume (PCV), by microhematocrit; counted reticulocytes on blood smears stained supravitally with new methylene blue; and measured Glu-Cys synthetase and GSH synthetase using the radioisotopic method of Minnich et al. [13] with minor modifications [5]. For amino acid analysis we mixed a sample of blood or plasma with equal parts of a 0.6 M perchloric acid and determined glycine in the protein free filtrate by degrading it to formaldehyde [14]. We determined glutamate using glutamate dehydrogenase [15], and cysteine by the noradenochrome method [16] after neutralizing the sample with potassium phosphate to pH 7.5. We tested effects of acute blood loss on various measurements by analysis of variance. When differences were significant, we compared the control period and individual days using orthogonal contrasts [17] . We calculated erythrocyte concentration of ATP and the various amino acids from the plasma concentration, whole blood concentration, and packed cell volume. Erythrocyte glutathione was calculated using the blood hemoglobin and assuming that the contribution by plasma and other formed elements were negligible. No correction was made for cysteine and Glu-Cys contribution to the glutathione level. Results Erythrocyte glutathione, ATP, glutamate, and glycine increased dramatically in all sheep after phlebotomy (Figs. 1 and 2). Glutathione increased significantly on day 3, peaked between days 5 and 7, then returned to normal. occasionally the glutathione in high-glutathione sheep decreased below pre-
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Biochimica et Biophysica Acta, 496 (1977) 516-520 © Elsevier/North-Holland Biomedical Press
BBA 28151
ELEVATED ERYTHROCYTE GLUTATHIONE ASSOCIATED WITH ELEVATED SUBSTRATE IN HIGH- AND LOW-GLUTATHIONE SHEEP * JOSEPH E. SMITH
Department of Pathology, College of Veterinary Medicine, Kansas Sate University, Manhattan, Kan. 66506 (U.S.A.) (Received June 15th, 1976)
Summary Erythrocyte glutathione concentration increases dramatically in sheep when they become anemic. To determine the mechanism of this change in glutathione control, we measured the enzymes and substrates necessary for glutathione synthesis after acute blood loss in both low- (-y-glutamylcysteine synthetase deficient) and high-glutathione sheep. Erythrocyte glutamate, ATP, and glycine increased dramatically in all sheep. Erythrocyte -y-glutamylcysteine synthetase increased slowly and seemed unrelated to changes in glutathione. Erythrocyte glutathione synthetase and cysteine and plasma cysteine, glutamate and glycine did not change significantly. Apparently substrate concentrations may be important in regulating erythrocyte glutathione levels.
Introduction Erythrocyte glutathione is synthesized from its three constituent amino acids in two reactions. In the first -y-glutamylcysteine (Glu-Cys) is formed from glutamic acid and cysteine in a reaction catalyzed by Glu-Cys synthetase (Lglutamate: L-cysteine -y-ligase (ADP-forming), EC 6.3.2.2); in the second reaction glycine is added to Glu-Cys forming glutathione that is catalyzed by glutathione synthetase (-Y-L-glutamyl-L-cysteine : glycine ligas8 (ADP-forming), EC 5.3.2.3). Both reactions require adenosine-5'-triphosphate (ATP) and magnesium or manganese and both yield adenosine-5'-diphosphate (ADP) and inorganic phosphate [1] . Normally erythrocyte glutathione exists predominantly as the reduced form (GSH) and is maintained fairly constant [2]. In sheep GSH concentration is * Contributions
No. 396. of the Department ()f Pathology, College of Veterinary Medicine. AES, Kansas State University, Manhattan, Kan. 66506, U.S.A.
517
genetically controlled. Levels of GSH (> 1 mg/g of hemoglobin) in some sheep are similar to red cell levels in other mammals; in others it is lower « 1 mg/g of hemoglobin) [3,4]. There are two distinct types of low-glutathione sheep. One type has dimished Glu-Cys synthetase activity; the other has a defect cysteine transport system [6]. Acute blood loss in either low- or high-GSH sheep disturbs mechanisms that control glutathione and allows glutathione to increase [7-9] . To understand the change in GSH control, we measured the enzymes and substrates required for GSH synthesis during and after phlebotomy of highand low-glutathione sheep. Materials and Methods Two sheep with high erythrocyte glutathione and two with low erythrocyte glutathione (GC synthetase-deficient type) were selected for each experiment. This paper describes the results of two experiments. During the experiment they were fed alfalfa hay and a mixed grain ration with no additional supplement (e.g. iron). Blood (approximately 10 ml/lb of body weight) was taken daily from each animal. After three phlebotomies, packed cell volumes decreased to 1/3 of the pre-phlebotomy levels, so the procedure was stopped. Samples were taken at regular intervals before and after phlebotomy for substrate and enzymatic activities. We determined reduced glutathione as the yellow DTNB anion [10]; ATP by using the luciferin-luciferase method [11]; hemoglobin as the cyanmethemoglobin derivative [12] ; and packed cell volume (PCV), by microhematocrit; counted reticulocytes on blood smears stained supravitally with new methylene blue; and measured Glu-Cys synthetase and GSH synthetase using the radioisotopic method of Minnich et al. [13] with minor modifications [5]. For amino acid analysis we mixed a sample of blood or plasma with equal parts of a 0.6 M perchloric acid and determined glycine in the protein free filtrate by degrading it to formaldehyde [14]. We determined glutamate using glutamate dehydrogenase [15], and cysteine by the noradenochrome method [16] after neutralizing the sample with potassium phosphate to pH 7.5. We tested effects of acute blood loss on various measurements by analysis of variance. When differences were significant, we compared the control period and individual days using orthogonal contrasts [17] . We calculated erythrocyte concentration of ATP and the various amino acids from the plasma concentration, whole blood concentration, and packed cell volume. Erythrocyte glutathione was calculated using the blood hemoglobin and assuming that the contribution by plasma and other formed elements were negligible. No correction was made for cysteine and Glu-Cys contribution to the glutathione level. Results Erythrocyte glutathione, ATP, glutamate, and glycine increased dramatically in all sheep after phlebotomy (Figs. 1 and 2). Glutathione increased significantly on day 3, peaked between days 5 and 7, then returned to normal. occasionally the glutathione in high-glutathione sheep decreased below pre-
518 c
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m
41 Glutathione
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Glutathione
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