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Stabilization of the adenylate energy charge in erythrocytes of rats and humans at high altitude hypoxia
Camp. Biochem. Physiol. Printed in Great Britain
Vol. IOIA,
No. 1, pp. 6548, 1992
0300-9629/92 $5.00+ 0.00 0 1991Pergamon Pressplc
STABILIZATION OF THE ADENYLATE ENERGY CHARGE IN ERYTHROCYTES OF RATS AND HUMANS AT HIGH ALTITUDE HYPOXIA MASATAKA
YosHtNo,*t
CHIKASHI YAMAMOTO,~
KEIKO MURAKAMI,$
YOSHINAO
KATSUMATA~[
and SHIGEO MORI$ *Department of Genetics, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan; fResearch Institute for Environmental Medicine, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan; §Inazawa Junior College, Inazawa, Aichi 492, Japan; IIDepartment of Legal Medicine, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan (Received 23 April 1991) Abstract-l.
Stabilization of adenylate energy charge and control of adenylate pool were analysed in the erythrocytes of the rat and the human exposed to highly hypoxic conditions. 2. Red cell energy charge was decreased in the rats exposed to a simulated altitude of 5000-8000 m, and then recovered to the normal value with the depletion of adenylate pool. 3. The energy charge and the adenylate pool size of the human erythrocytes did not show any change under highly hypoxic conditions. 4. Anaerobic incubation of rat erythrocytes caused a marked decrease in the energy charge, and its recovery was accompanied by the depletion of total adenylates. 5. The energy charge and total adenylates of human red cells did not change under the anaerobic incubation of erythrocytes. 6. These results suggest that the energy charge of rat erythrocytes can be controlled by depletion of the adenylate pool, but the adenylate degradation is not responsible for the stabilization of the energy charge in human erythrocytes.
INTRODUCIION
Stabilization of adenylate energy charge can be colttrolled by adenylate depletion (Chapman and Atkinson, 1973; Solano and Coffee, 1978; Yoshino ani Murakami, 1981) and glycolytic stimulation (Yoshino and Murakami, 1982). Adenylate degradation, which can be controlled principally by AMP deitminase (EC 3.5.4.6) in most tissues, is closely coyrelated with the stimulation of glycolysis causing AI’P regeneration (Yoshino and Murakami, 1982). Mitmmalian erythrocytes lack mitochondrial respiration, and therefore give a good model for the study of the control of the energy charge and glycolysis. Red cell AMP deaminase activity varies widely depending on the species (Kruckeberg and Chilson, 19 ‘3) but the role of the enzyme in erythrocytes remains unclear. In this paper we deal with the rel#ationship between the control of the energy charge and stabilization of adenylate pool in rat and human erl’throcytes under the in vivo and the in vitro condiiions. The energy charge of the rat erythrocytes cat be stabilized by active reaction of adenylate degradation, which is unlikely to be operative in human erythrocytes.
tcorrespondence should be addressed to: Masataka Yoshino, Department of Genetics, Institute for Developmental Research, Aichi Prefectural Colony, Kamiya-cho 713-8, Kasugai, Aichi 480-03, Japan.
MATERIALS
AND METHODS
All chemicals were obtained from commercial sources as described previously (Yoshino and Murakami, 1981, 1982). Exposure of rats to high altitude
Male Sprague-Dawley rats weighing 120-150 g were starved overnight. They were permitted free access to water. Groups of four rats were submitted to an altitude chamber at the Research Institute for Environmental Medicine (Nagoya University) as described previously (Yoshino et al., 1986a). The rate of decompression was 120-150m/min. The rats remained at the simulated altitude for I hr, and returned to sea-level at the same rate. Rats were killed by cervical dislocation at appropriate intervals and blood was collected in heparinized test tubes. Exposure of humans to high altitude
Ten healthy unacclimatized male students, aged 20-25 years, were subjected to simulated altitudes in the altitude chamber and blood was collected from the antecubital vein in heparinized syringes at appropriate intervals. In vitro incubation of human and rat erythrocytes Rat and human erythrocytes were incubated anaerobically in Thumberg tubes. Isolated erythrocytes of 0.5 ml were added to 0.425 ml of 12 mM potassium phosphate buffer (pH 7.1) containing 0.9% NaCl in the main-chamber of each tube. Glucose (40 mM) in 0.9% NaCl was placed in the side chamber of each tube. Air in the tube was replaced completely by nitrogen gas. The reaction was started by adding the glucose in the side chamber to the erythrocyte solution, and incubating it at 37°C. The mixture of each tube was deproteinized by 1.3 ml of 10% perchloric acid
MASATAKA Y~~HINO et al.
66
at appropriate intervals. Aerobic incubation in uifro was carried out without replacing air by nitrogen gas. Determination of adenylates
Whole blood and erythrocyte mixture of rats and humans were deproteinized by perchloric acid, and neutralized supematant was used for determining adenylates. ATP was analysed by luciferin-luciferase reaction, and ADP and AMP were measured as the difference after enzymatic conversion to ATP (Yoshino and Murakami, 1982). RESULTS
Changes in the energy charge in rat erythrocytes are shown in Fig. lA, when the rats were exposed to each simulated altitude. The energy charge was decreased at the exposure to a 5000 m altitude, and further decreased gradually during the exposure. The energy charge was returned immediately to sea-level. The degree of the decrease in the energy charge appears to be dependent on the altitude to which the rats were exposed: the value was decreased to 0.6 on the exposure to an 8000m altitude. Adenylate pool was depleted on an exposure to high altitude hypoxia, and the extent of the depletion was also dependent on the altitude exposed (Fig. 1B). The energy charge of the human erythrocytes was not at all affected by the exposure to high altitude I.0 A 1
1
hypoxia: the value remained constant during and after the exposure to a 6000m altitude (Fig. 2A). Total adenylates were also unaffected by the exposure (Fig. 2B). Rat erythrocytes were anaerobically incubated under the in vitro conditions. The energy charge was rapidly dropped, and gradually recovered to the original value after 90 min incubation (Fig. 3A). The total adenylates were decreased before the energy charge began to rise (Fig. 3B), suggesting a possible role of adenylate degradation in the recovery of the energy charge. On the other hand, aerobic incubation did not affect the value of the energy charge and the level of total adenylates in rat erythrocytes. Human erythrocytes were incubated under the anaerobic and aerobic conditions. No difference in the erythrocyte energy charge values was observed under the aerobic and anaerobic conditions, and higher value of the energy charge was maintained throughout the incubation period under both conditions (Fig. 4A). Total adenylates also remained at the original values under the aerobic conditions. Anaerobic incubation caused only a little decline of the total adenylates (Fig. 4B). DISCUSSION
Acute exposure of animals to high altitude hypoxia causes a series of physiological and biochemical adaptation to lower oxygen availability (Frisancho, 1975). Increased production of lactate and uric acid can be explained by the stimulation of anaerobic glycolysis with the enhanced adenylate degradation
Oi.
0
120 180 60 Time (min) Fig. 1. Changes in the adenylate energy charge (A) and total adenylates (B) in erythrocytes of rats exposed to 5000, 6000, and 8000 m simulated altitude. Rats were starved overnight and submitted to an altitude chamber. Blood was collected by decapitation and the erythrocytes were immediately deproteinized by perchloric acid. The neutralized supernatant was utilized for the determination of adenylates. Adenylate energy charge (EC) was calculated as follows: EC = ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP)]. Total adenylates including ATP, ADP and AMP were shown as the concentration in erythrocytes. The barometric pressure is presented as the equivalent altitude in (C). Each point shows the mean k SD with four rats. 0, 5OOOm;l ,6000 m; A, 8000m.
Time (min) Fig. 2. Changes in the adenylate energy charge (A) and total adenylates (B) in erythrocytes of human exposed to 6000 m simulated altitude. Ten men were submitted to an altitude chamber. Blood was collected from the antecubital vein in heparinized tubes, and was treated as described in the rat experiments. The barometric pressure is represented as the equivalent altitude in (C). Each point shows the mean k SD.
61
Adenylate energy charge in RBC
Time (mm) Fig,. 3. Changes in the adenylate energy charge (A) and tot II adenylates (B) in rat erythrocytes under the aerobic ant. anaerobic conditions. Isolated rat erythrocytes were incllbated aerobically and anaerobically with Thumberg tutles as described in Materials and Methods. 0, Aerobic incubation; 0, anaerobic incubation.
(Yijshino and Murakami, 1982; Yoshino et al., 19+6a,b). The concentration of intracellular ATP va ies with a variety of metabolic stress; however, themadenylate energy charge is a good indicator of metabolic regulation (Chapman and Atkinson, 1971). In most tissues and organisms the energy charge ha,< a value of approximately 0.90 (Chapman and Attinson, 1971). When organisms are subjected to
go.31
Bl
;o.2f=-==== 1 g
OoW
Time (min) Fig. 4. Changes in the adenylate energy charge (A) and total adenylates (B) in human erythrocytes under the aerobic and anaerobic conditions. Isolated human erythrocytes were incubated aerobically and anaerobically with Thumberg tubes as described in Materials and Methods. 0, Aerobic incubation; 0, anaerobic incubation.
metabolic stress such as hypoxia, the energy charge decreases. Decreased energy charge can be recovered at the expense of the concentrations of the adenylate pool. Adenylates are degraded principally by AMP deaminase in eukaryotes (Chapman and Atkinson, 1973). In the physiological range of the energy charge the rate of AMP deamination catalysed by AMP deaminase increases sharply with decreasing energy charge (Chapman and Atkinson, 1971, 1973; Solano and Coffee, 1978; Yoshino and Murakami, 1981). This response serves to protect against transient decrease in the energy charge: when the charge decreases the resulting removal of AMP will oppose the decrease in charge. Furthermore, ammonium ion produced by the AMP deamination can participate in the activation of phosphofructokinase resulting in the stimulation of glycolysis (Yoshino and Murakami, 1982). Glycolytic enhancement with ATP depletion can act as a principal role in the stabilization of the energy charge through the ATP replenishment. Red cell adenylates are degraded by AMP deaminase but little by S-nucleotidase (Schauer et al., 1981a,b). Thus, degradation of red cell adenylates of the rats exposed to a high altitude may be carried out by AMP deaminase. However, AMP deaminase activity in rat red cells is considerably lower than that of human red cells (Kruckeberg and Chilson, 1973). Of particular interest is the finding that the adenylate pool can be readily depleted in the rat erythrocytes with lower AMP deaminase activity, but that the adenylate pool size remains constant in human red cells with considerably higher activity of AMP deaminase under both in vivo and in vitro conditions. The present data are in good agreement with the results that the biological half-life of adenine moiety is about IO-fold longer in humans than in other mammals such as rabbit erythrocytes (Mager et al., 1966, 1967). Turnover rate of adenine nucleotides in human erythrocytes is markedly lower than the value estimated from the AMP deaminase activity (Kruckeberg and Chilson, 1973) and thus, human red cell AMP deaminase is likely to be kept under highly inhibited conditions (Sasaki et al., 1976). Inherited complete deficiency of AMP deaminase was reported in human erythrocytes (Ogasawara et al., 1984). No hematological disorders and no clinical symptoms in the erythrocyte AMP deaminase-deficient individuals also indicate that the reaction of AMP deaminase itself is not physiologically operative or is inhibited in human erythrocytes (Ogasawara et al., 1987). Adenylate degradation, which is essentially due to the AMP deaminase reaction in eukaryotic cells including erythrocytes, may play a principal role in the stabilization of the energy charge in rat red cells, but not in human erythrocytes: the energy charge in human red cells may be controlled by ATP regeneration through glycolytic enhancement without adenylate depletion. REFERENCES
Chapman A. G. and Atkinson D. E. (1971) Adenine nucleotide concentrations and turnover rates. Their correlation with biological activity in bacteria and yeast. Adv. Microbial Physiol. 15, 253-306.
68
MASATAKA Yosmdo el al.
Chapman A. G. and Atkinson D. E. (1973) Stabilization of adenylate energy charge by the adenylate deaminase reaction. J. biol. Chem. 248, 8309-8312. Frisancho A. R. (1975) Functional adaptation to high altitude hypoxia. Changes occurring during growth and development are of major importance in man’s adapting to high altitudes. Science 187, 313-319. Kruckeberg W. C. and Chilson 0. P. (1973) Red cell AMP deaminase: Levels of activity in hemolysates from twenty different vertebrate species. Comp. Biochem. Physiol. 46B, 653-660. Mager J., Dvilansky A., Razin A., Wind E. and Izak G. (1966) Turnover of purine nucleotides in human red blood cells. Israel J. Med. Sci. 2, 297-301. Mager J., Hershko A., Zeitlin-Beck R., Shoshani T. and Razin A. (1967) Turnover of purine nucleotides in rabbit erythrocytes. I. Studies in vitro. Biochim. biophys. Acra 149, 50-58. Ogasawara N., Goto H., Yamada Y., Nishigaki I., Ito T. and Hasegawa I. (1984) Complete deficiency of AMP deaminase in human erythrocytes. Biochem. biophys. Res. Commun. 122, 1344-1349. Ogasawara N., Goto H., Yamada Y., Nishigaki I., Hasegawa I. and Parkes K. S. (1987) Deficiency of AMP deammase in erythrocytes. Human Genet. 75, i5-18. Sasaki R., Ikura K., Katsura S. and Chiba H. (1976) Regulation of human erythrocyte AMP deaminase by ATP and 2,3-bisphosphoglycerate. Agr. Biol. Chem. 40, 1797-1803. Schauer M., Heinrich R. and Rapoport S. M. (1981a) Mathematische Modellierung der Glykolyse und des Adeninnukleotidstoffwechsels menschlicher Erythro-
zyten. I. Reaktionskinetsche Ansatze, Analyse des in vivoZustandes und Bestimmung der Anfagsbedingungen fur der in vitro-Experimente. Acta Biol. Med. Germ. 40, 1659-1682. Schauer M., Heinrich R. and Rapoport S. M. (1981b) Mathematische Modellierung der Glykolyse und des Adeninnukleotidstoffwechsels menschicher Erythrozyten. II. Simulation des Adeninnukleotidabbaus bei Glukoseverarmung. Acta Biol. Med. Germ. 40, 1683-1497. Solano C. and Coffee C. J. (1978) Differential response of AMP deaminase isozymes to changes in the adenylate energy charge. Biochem. biophys. Res. Commun. 85, 564-571. Yoshino M. and Murakami K. (1981) In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts. Biochim. biophys. Acta 672, 16-20. Yoshino M. and Murakami K. (1982) AMP deaminase reaction as a control system of glycolysis in yeast. Activation of phosphofructokinase and pyruvate kinease by the AMP deaminase-ammonia system. J. biol. Chem. 257, 2822-2828. Yoshino M., Murakami K., Katsumata Y., Takabayashi A. and Mori S. (1986a) Stimulation of glycolysis with hyperuricemia in rats at high altitude hypoxia. Biomed. Res. 7, 113-117. Yoshino M., Murakami K., Katsumata Y., Takabayashi A. and Mori S. (1986b) Changes in plasma phosphate with the stimulation of anaerobic metabolism in rats during hypoxic-anoxic states. Comp. Biochem. Physiol. 85A, 455-457.
Vol. IOIA,
No. 1, pp. 6548, 1992
0300-9629/92 $5.00+ 0.00 0 1991Pergamon Pressplc
STABILIZATION OF THE ADENYLATE ENERGY CHARGE IN ERYTHROCYTES OF RATS AND HUMANS AT HIGH ALTITUDE HYPOXIA MASATAKA
YosHtNo,*t
CHIKASHI YAMAMOTO,~
KEIKO MURAKAMI,$
YOSHINAO
KATSUMATA~[
and SHIGEO MORI$ *Department of Genetics, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan; fResearch Institute for Environmental Medicine, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan; §Inazawa Junior College, Inazawa, Aichi 492, Japan; IIDepartment of Legal Medicine, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan (Received 23 April 1991) Abstract-l.
Stabilization of adenylate energy charge and control of adenylate pool were analysed in the erythrocytes of the rat and the human exposed to highly hypoxic conditions. 2. Red cell energy charge was decreased in the rats exposed to a simulated altitude of 5000-8000 m, and then recovered to the normal value with the depletion of adenylate pool. 3. The energy charge and the adenylate pool size of the human erythrocytes did not show any change under highly hypoxic conditions. 4. Anaerobic incubation of rat erythrocytes caused a marked decrease in the energy charge, and its recovery was accompanied by the depletion of total adenylates. 5. The energy charge and total adenylates of human red cells did not change under the anaerobic incubation of erythrocytes. 6. These results suggest that the energy charge of rat erythrocytes can be controlled by depletion of the adenylate pool, but the adenylate degradation is not responsible for the stabilization of the energy charge in human erythrocytes.
INTRODUCIION
Stabilization of adenylate energy charge can be colttrolled by adenylate depletion (Chapman and Atkinson, 1973; Solano and Coffee, 1978; Yoshino ani Murakami, 1981) and glycolytic stimulation (Yoshino and Murakami, 1982). Adenylate degradation, which can be controlled principally by AMP deitminase (EC 3.5.4.6) in most tissues, is closely coyrelated with the stimulation of glycolysis causing AI’P regeneration (Yoshino and Murakami, 1982). Mitmmalian erythrocytes lack mitochondrial respiration, and therefore give a good model for the study of the control of the energy charge and glycolysis. Red cell AMP deaminase activity varies widely depending on the species (Kruckeberg and Chilson, 19 ‘3) but the role of the enzyme in erythrocytes remains unclear. In this paper we deal with the rel#ationship between the control of the energy charge and stabilization of adenylate pool in rat and human erl’throcytes under the in vivo and the in vitro condiiions. The energy charge of the rat erythrocytes cat be stabilized by active reaction of adenylate degradation, which is unlikely to be operative in human erythrocytes.
tcorrespondence should be addressed to: Masataka Yoshino, Department of Genetics, Institute for Developmental Research, Aichi Prefectural Colony, Kamiya-cho 713-8, Kasugai, Aichi 480-03, Japan.
MATERIALS
AND METHODS
All chemicals were obtained from commercial sources as described previously (Yoshino and Murakami, 1981, 1982). Exposure of rats to high altitude
Male Sprague-Dawley rats weighing 120-150 g were starved overnight. They were permitted free access to water. Groups of four rats were submitted to an altitude chamber at the Research Institute for Environmental Medicine (Nagoya University) as described previously (Yoshino et al., 1986a). The rate of decompression was 120-150m/min. The rats remained at the simulated altitude for I hr, and returned to sea-level at the same rate. Rats were killed by cervical dislocation at appropriate intervals and blood was collected in heparinized test tubes. Exposure of humans to high altitude
Ten healthy unacclimatized male students, aged 20-25 years, were subjected to simulated altitudes in the altitude chamber and blood was collected from the antecubital vein in heparinized syringes at appropriate intervals. In vitro incubation of human and rat erythrocytes Rat and human erythrocytes were incubated anaerobically in Thumberg tubes. Isolated erythrocytes of 0.5 ml were added to 0.425 ml of 12 mM potassium phosphate buffer (pH 7.1) containing 0.9% NaCl in the main-chamber of each tube. Glucose (40 mM) in 0.9% NaCl was placed in the side chamber of each tube. Air in the tube was replaced completely by nitrogen gas. The reaction was started by adding the glucose in the side chamber to the erythrocyte solution, and incubating it at 37°C. The mixture of each tube was deproteinized by 1.3 ml of 10% perchloric acid
MASATAKA Y~~HINO et al.
66
at appropriate intervals. Aerobic incubation in uifro was carried out without replacing air by nitrogen gas. Determination of adenylates
Whole blood and erythrocyte mixture of rats and humans were deproteinized by perchloric acid, and neutralized supematant was used for determining adenylates. ATP was analysed by luciferin-luciferase reaction, and ADP and AMP were measured as the difference after enzymatic conversion to ATP (Yoshino and Murakami, 1982). RESULTS
Changes in the energy charge in rat erythrocytes are shown in Fig. lA, when the rats were exposed to each simulated altitude. The energy charge was decreased at the exposure to a 5000 m altitude, and further decreased gradually during the exposure. The energy charge was returned immediately to sea-level. The degree of the decrease in the energy charge appears to be dependent on the altitude to which the rats were exposed: the value was decreased to 0.6 on the exposure to an 8000m altitude. Adenylate pool was depleted on an exposure to high altitude hypoxia, and the extent of the depletion was also dependent on the altitude exposed (Fig. 1B). The energy charge of the human erythrocytes was not at all affected by the exposure to high altitude I.0 A 1
1
hypoxia: the value remained constant during and after the exposure to a 6000m altitude (Fig. 2A). Total adenylates were also unaffected by the exposure (Fig. 2B). Rat erythrocytes were anaerobically incubated under the in vitro conditions. The energy charge was rapidly dropped, and gradually recovered to the original value after 90 min incubation (Fig. 3A). The total adenylates were decreased before the energy charge began to rise (Fig. 3B), suggesting a possible role of adenylate degradation in the recovery of the energy charge. On the other hand, aerobic incubation did not affect the value of the energy charge and the level of total adenylates in rat erythrocytes. Human erythrocytes were incubated under the anaerobic and aerobic conditions. No difference in the erythrocyte energy charge values was observed under the aerobic and anaerobic conditions, and higher value of the energy charge was maintained throughout the incubation period under both conditions (Fig. 4A). Total adenylates also remained at the original values under the aerobic conditions. Anaerobic incubation caused only a little decline of the total adenylates (Fig. 4B). DISCUSSION
Acute exposure of animals to high altitude hypoxia causes a series of physiological and biochemical adaptation to lower oxygen availability (Frisancho, 1975). Increased production of lactate and uric acid can be explained by the stimulation of anaerobic glycolysis with the enhanced adenylate degradation
Oi.
0
120 180 60 Time (min) Fig. 1. Changes in the adenylate energy charge (A) and total adenylates (B) in erythrocytes of rats exposed to 5000, 6000, and 8000 m simulated altitude. Rats were starved overnight and submitted to an altitude chamber. Blood was collected by decapitation and the erythrocytes were immediately deproteinized by perchloric acid. The neutralized supernatant was utilized for the determination of adenylates. Adenylate energy charge (EC) was calculated as follows: EC = ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP)]. Total adenylates including ATP, ADP and AMP were shown as the concentration in erythrocytes. The barometric pressure is presented as the equivalent altitude in (C). Each point shows the mean k SD with four rats. 0, 5OOOm;l ,6000 m; A, 8000m.
Time (min) Fig. 2. Changes in the adenylate energy charge (A) and total adenylates (B) in erythrocytes of human exposed to 6000 m simulated altitude. Ten men were submitted to an altitude chamber. Blood was collected from the antecubital vein in heparinized tubes, and was treated as described in the rat experiments. The barometric pressure is represented as the equivalent altitude in (C). Each point shows the mean k SD.
61
Adenylate energy charge in RBC
Time (mm) Fig,. 3. Changes in the adenylate energy charge (A) and tot II adenylates (B) in rat erythrocytes under the aerobic ant. anaerobic conditions. Isolated rat erythrocytes were incllbated aerobically and anaerobically with Thumberg tutles as described in Materials and Methods. 0, Aerobic incubation; 0, anaerobic incubation.
(Yijshino and Murakami, 1982; Yoshino et al., 19+6a,b). The concentration of intracellular ATP va ies with a variety of metabolic stress; however, themadenylate energy charge is a good indicator of metabolic regulation (Chapman and Atkinson, 1971). In most tissues and organisms the energy charge ha,< a value of approximately 0.90 (Chapman and Attinson, 1971). When organisms are subjected to
go.31
Bl
;o.2f=-==== 1 g
OoW
Time (min) Fig. 4. Changes in the adenylate energy charge (A) and total adenylates (B) in human erythrocytes under the aerobic and anaerobic conditions. Isolated human erythrocytes were incubated aerobically and anaerobically with Thumberg tubes as described in Materials and Methods. 0, Aerobic incubation; 0, anaerobic incubation.
metabolic stress such as hypoxia, the energy charge decreases. Decreased energy charge can be recovered at the expense of the concentrations of the adenylate pool. Adenylates are degraded principally by AMP deaminase in eukaryotes (Chapman and Atkinson, 1973). In the physiological range of the energy charge the rate of AMP deamination catalysed by AMP deaminase increases sharply with decreasing energy charge (Chapman and Atkinson, 1971, 1973; Solano and Coffee, 1978; Yoshino and Murakami, 1981). This response serves to protect against transient decrease in the energy charge: when the charge decreases the resulting removal of AMP will oppose the decrease in charge. Furthermore, ammonium ion produced by the AMP deamination can participate in the activation of phosphofructokinase resulting in the stimulation of glycolysis (Yoshino and Murakami, 1982). Glycolytic enhancement with ATP depletion can act as a principal role in the stabilization of the energy charge through the ATP replenishment. Red cell adenylates are degraded by AMP deaminase but little by S-nucleotidase (Schauer et al., 1981a,b). Thus, degradation of red cell adenylates of the rats exposed to a high altitude may be carried out by AMP deaminase. However, AMP deaminase activity in rat red cells is considerably lower than that of human red cells (Kruckeberg and Chilson, 1973). Of particular interest is the finding that the adenylate pool can be readily depleted in the rat erythrocytes with lower AMP deaminase activity, but that the adenylate pool size remains constant in human red cells with considerably higher activity of AMP deaminase under both in vivo and in vitro conditions. The present data are in good agreement with the results that the biological half-life of adenine moiety is about IO-fold longer in humans than in other mammals such as rabbit erythrocytes (Mager et al., 1966, 1967). Turnover rate of adenine nucleotides in human erythrocytes is markedly lower than the value estimated from the AMP deaminase activity (Kruckeberg and Chilson, 1973) and thus, human red cell AMP deaminase is likely to be kept under highly inhibited conditions (Sasaki et al., 1976). Inherited complete deficiency of AMP deaminase was reported in human erythrocytes (Ogasawara et al., 1984). No hematological disorders and no clinical symptoms in the erythrocyte AMP deaminase-deficient individuals also indicate that the reaction of AMP deaminase itself is not physiologically operative or is inhibited in human erythrocytes (Ogasawara et al., 1987). Adenylate degradation, which is essentially due to the AMP deaminase reaction in eukaryotic cells including erythrocytes, may play a principal role in the stabilization of the energy charge in rat red cells, but not in human erythrocytes: the energy charge in human red cells may be controlled by ATP regeneration through glycolytic enhancement without adenylate depletion. REFERENCES
Chapman A. G. and Atkinson D. E. (1971) Adenine nucleotide concentrations and turnover rates. Their correlation with biological activity in bacteria and yeast. Adv. Microbial Physiol. 15, 253-306.
68
MASATAKA Yosmdo el al.
Chapman A. G. and Atkinson D. E. (1973) Stabilization of adenylate energy charge by the adenylate deaminase reaction. J. biol. Chem. 248, 8309-8312. Frisancho A. R. (1975) Functional adaptation to high altitude hypoxia. Changes occurring during growth and development are of major importance in man’s adapting to high altitudes. Science 187, 313-319. Kruckeberg W. C. and Chilson 0. P. (1973) Red cell AMP deaminase: Levels of activity in hemolysates from twenty different vertebrate species. Comp. Biochem. Physiol. 46B, 653-660. Mager J., Dvilansky A., Razin A., Wind E. and Izak G. (1966) Turnover of purine nucleotides in human red blood cells. Israel J. Med. Sci. 2, 297-301. Mager J., Hershko A., Zeitlin-Beck R., Shoshani T. and Razin A. (1967) Turnover of purine nucleotides in rabbit erythrocytes. I. Studies in vitro. Biochim. biophys. Acra 149, 50-58. Ogasawara N., Goto H., Yamada Y., Nishigaki I., Ito T. and Hasegawa I. (1984) Complete deficiency of AMP deaminase in human erythrocytes. Biochem. biophys. Res. Commun. 122, 1344-1349. Ogasawara N., Goto H., Yamada Y., Nishigaki I., Hasegawa I. and Parkes K. S. (1987) Deficiency of AMP deammase in erythrocytes. Human Genet. 75, i5-18. Sasaki R., Ikura K., Katsura S. and Chiba H. (1976) Regulation of human erythrocyte AMP deaminase by ATP and 2,3-bisphosphoglycerate. Agr. Biol. Chem. 40, 1797-1803. Schauer M., Heinrich R. and Rapoport S. M. (1981a) Mathematische Modellierung der Glykolyse und des Adeninnukleotidstoffwechsels menschlicher Erythro-
zyten. I. Reaktionskinetsche Ansatze, Analyse des in vivoZustandes und Bestimmung der Anfagsbedingungen fur der in vitro-Experimente. Acta Biol. Med. Germ. 40, 1659-1682. Schauer M., Heinrich R. and Rapoport S. M. (1981b) Mathematische Modellierung der Glykolyse und des Adeninnukleotidstoffwechsels menschicher Erythrozyten. II. Simulation des Adeninnukleotidabbaus bei Glukoseverarmung. Acta Biol. Med. Germ. 40, 1683-1497. Solano C. and Coffee C. J. (1978) Differential response of AMP deaminase isozymes to changes in the adenylate energy charge. Biochem. biophys. Res. Commun. 85, 564-571. Yoshino M. and Murakami K. (1981) In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts. Biochim. biophys. Acta 672, 16-20. Yoshino M. and Murakami K. (1982) AMP deaminase reaction as a control system of glycolysis in yeast. Activation of phosphofructokinase and pyruvate kinease by the AMP deaminase-ammonia system. J. biol. Chem. 257, 2822-2828. Yoshino M., Murakami K., Katsumata Y., Takabayashi A. and Mori S. (1986a) Stimulation of glycolysis with hyperuricemia in rats at high altitude hypoxia. Biomed. Res. 7, 113-117. Yoshino M., Murakami K., Katsumata Y., Takabayashi A. and Mori S. (1986b) Changes in plasma phosphate with the stimulation of anaerobic metabolism in rats during hypoxic-anoxic states. Comp. Biochem. Physiol. 85A, 455-457.