Comp. Biochem. PhysioL Vol. 95B,No. 2, pp. 287-294, 1990 Printed in Great Britain
0305-0491/90$3.00+ 0.00 © 1990PergamonPress pie
REGULATION A N D RATE OF THE HEXOSE MONOPHOSPHATE SHUNT IN RANA RIDIBUNDA ERYTHROCYTES MARTHA KALOYIANNI* and MARIAKALOMENOPOULOU Laboratory of Animal Physiology, Department of Zoology, Science School, University of Tbessaloniki, Thessaloniki 54006, Greece (Received 19 June 1989) Abstract--1. Resting rates of Rana ridibunda erythrocyte glucoseconsumption and ~4CO2production from IJ4C-glucose were found to be significantly lower than the respective values in human erythrocytes. 2. In the presence of 1-t4C-glucose Methylene Blue stimulated t4CO2 production 7-fold, while in the presence of 6-14C-glucose Methylene Blue stimulated 14CO2 production 1.2-fold. 3. The Km of G-6-PD for G-6-P and NADP were 29 and 12/~M, respectivelywhile the K~ of 6-PGD for 6-PG and NADP were 83 and 32 aM, respectively. The Ki of G-6-PD and 6-PGD for NADPH were 80 and 12 laM, respectively. 4. Excess amounts of NADP resulted in a significantdecrease of ~4CO2production from 1-~4C-glucose in total haemolysates. 5. ATP, ADP and fructose diphosphate inhibited both G-6-PD and 6-PGD, the latter being more sensitive than G-6-PD to their inhibitory effect. 2,3-DPG and reduced and oxidized glutathione showed a marked inhibitory effect on 6-PGD, while the phosphorylated trioses inhibited only G-6-PD. 6. Physiological concentrations of oxidized glutathione decreased the inhibition exercised by NADPH on G-6-PD. 7. The possible role of the two dehydrogenases in the regulation of the HMS is discussed.
INTRODUCTION Glucose metabolism in intact mammalian red blood cells is accomplished through the Embden-Meyerhoff Pathway (EMP) and the hexose monophosphate shunt (HMS), the relative importance of which, as assessed by employing l-~4C-glucose, is secondary in terms of average glucose consumption (Herman et aL, 1962; Gaetani et al., 1974, 1976; Magnani et al., 1980). The adequate operation of the HMS, however, is as important to red cell survival as that of the EMP, since it was shown that severe restriction of the HMS is associated with a shortened red cell survival and susceptibility to oxidative injury (Yoshida, 1973; Harvey and Kaneko, 1977; Valentine et al., 1985). The available data concerning amphibian red blood cells seem to indicate that their overall partition of glucose metabolism differs from that of mammalian red blood cells (Herman et al., 1962). Amphibian red blood cells are typical of non-mammalian vertebrates in that they are nucleated and are expected to metabolize 1-14C-glucose more actively than non-nucleated red blood cells. However, it was shown by Herman et al. (1962) that Rana pipiens red cells produced little 14CO2 from 1-14C-glucose, being intermediate between avian and human red blood cells in the amount of 14CO2 produced. This raised questions as to whether frog red cells, presumably having an active TCA cycle, would metabolize glucose via the HMS. *Author to whom all correspondence should be addressed. caP,B)95/2--F
The proper function of the HMS requires the action of two dehydrogenases, the availability of glutathione, as well as the capacity to reduce the oxidized glutathione generated during detoxification from harmful peroxides and molecular oxygen (Jacob et al., 1965; Jacob and Jandle, 1966). The activity of the HMS is believed to reflect the relative amount of NADP (Cahill et al., 1958) and if so, the rate of its metabolism should be linked to the rate of oxidation and reduction of glutathione wherein NADPH is oxidized. Since the requirements for reducing equivalents are variable, depending on the amount of oxidant stress to which the red cell is exposed, regulatory mechanisms must be expected to adjust the rate of the shunt to changing needs. These are likely to operate at the initial steps, i.e. the reactions mediated by the two dehydrogenases (Dische, 1964). In this study we have attempted to evaluate the relative flux of the HMS in frog erythrocytes as well as to approach the problem of the regulation of the oxidative phase of the HMS, by which the two molecules of NADPH are generated per glucose molecule consumed. Intact red blood cells were examined in order to more closely approximate their usual physiological state. In addition, some of the enzymes which are considered to be of primary importance in protecting erythrocytes against oxidative attack were assaye~ in total haemolysate preparations. The kinetic properties of these enzymes as well as their catalytic capacities in the presence of various intracellular metabolites were further studied in order to investigate the flux control mechanisms of the HMS operating in the amphibian erythrocyte in vivo.
287
288
MARTHA KALOYIANNIand MARIAKALOMENOPOULOU MATERIALS AND METHODS
Animals Frogs (Rana ridibunda) were supplied by a local dealer. They were caught in the vicinity of Thessaloniki, kept in containers in the laboratory in freshwater and used a week after arrival. Chemicals and enzymes The substrates, enzymes and coenzymes were purchased from Sigma Chemical (St Louis, USA). All other chemicals were obtained either from Serva or Merck. The radioactive glucose (1J4C-gtucose and 6-14C-glucose) were from Amersham Radiochemical Center. Sampling o f red cells; determination o f enzymatic activities Blood samples were obtained by heart puncture from anaesthetized frogs using heparinized syringes. Immediately after collection the blood was centrifuged at 500 g for 10 min and the plasma and the upper one-fifth of the cells were removed by aspiration. The erythrocytes were washed three times with 0.12mM NaC1 and haemolyzed with ice-cold distilled water and consequent sonication for 3 × 10 sec with a MSE Soniprep 150 sonicator. The haemolysate was used for the determination of enzymatic activities at 25~C using a Hitachi recording spectrophotometer. The change in A340 caused by oxidation or reduction of NADP(H) was followed. Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was measured according to Glock and McLean (1953), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) according to Rodriguez-Segade et al. (1978) and glutathione reductase (E.C. 1.6.4.2) according to Racker (1955). Intracellular oxidized and reduced glutathione were measured according to Bernt and Bergmeyer (1974) in perchloric acid extracts of Rana ridibunda erythrocytes. The effect of NADPH, oxidized glutathione (GSSG), reduced glutathione (GSH), adenine nucleotides and various intracellular metabolites on the activities of glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD) was determined by adding varying concentrations of the above substances to the reaction mixture used for the determination of the catalytic activities of these dehydrogenases under suboptimal substrate concentrations. ZnSO 4 (0.015 raM) was used in order to completely inhibit glutathione reductase activity, when G-6-PD and 6-PGD were assayed in the presence of oxidized glutathione and NADPH. The percentage activation exercised by GSSG against the NADPH inhibition of G-6-PD or 6-PGD activity was calculated by the following expression: % increase in activity= 100 ( x / y - 1 ) , where x = v a l u e obtained for the enzyme activity in the presence of NADPH and GSSG and y = value obtained for the enzyme activity in the presence of NADPH. Metabolic studies All metabolic studies were performed with washed erythrocytes drawn from three to five animals to give one pool of cells, which were subsequently suspended in an imidazole-glycylglycine buffer, pH 7.4, containing I00 mM NaCI, 5.9mM KC1, 1.25mM CaC12, 2.4mM MgSO4,
4.2 mM imidazole, 7.6 mM glycylglycine, 1.2 mM KHEPO4 and 3 mM glucose. The suspensions were equilibrated at 25°C, their haematocrit measured and shaken in air to get a complete haemoglobin oxygenation. Glucose utilization and 14CO2 production from [1-t4C]- or [6-14C]-glucose were determined by incubating the 2 ml erythrocyte suspension with 0.3ml [l°t4C]- or [6-14C]-glucose (about 0.5 pCi in 0.3 ml) in Erlenmeyer flasks (25 ml) fitted with a rubber cup and equipped with a disposable plastic centre well. The flasks were protected from the light and incubated in a metabolic shaker for 60 min at 25°C at 120 oscillations per rain. At the end of the incubation 0.2 ml of 2.0 M KOH were injected through the rubber stopper into the centre wells, followed by 0.7 ml of 3.7 M perchloric acid into the incubation mixture. The flasks were shaken thoroughly and incubated again for 30 min. The radioactivity trapped in the KOH of the centre well was counted (cpm ~4COE). A sample from the cell extract was then neutralized, assayed for glucose by the method of Bergmeyer et al. (1963) and applied to a Dowex-I (acetate form 1 × 8) anion exchange resin in a pasteur pipette. Glucose was eluted with double distilled water until 15 ml were collected. The anions were eluted from the columns by addition of 10M HCI. The glucose and anion fractions (0.5 ml of each) were then counted in a liquid scintillation counter. The scintillation fluid of Welt et al. (1971) was used. The glucose concentration of the first eluate was measured enzymatically according to Bergmeyer et al. (1963) and the total glucose consumption was considered as the sum of #mol of ~4COz evolution and ~4C-anions production. Glucose and ~4C-anions were, in each case, also determined in blanks, in which 3.7 M perchloric acid was added in the incubation flasks at the beginning of the incubation. Experiments with normal human erythrocyte suspensions were simultaneously conducted and used as controls. The calculation of the share of the EMP and the HMS in glucose consumption in Rana ridibunda erythrocytes is based on the following considerations: the actual value of the ~4CO2 formed from l-laC-glucose indicates the sum of the glucose oxidized via the HMS and the fraction entering the EMP and finally oxidized in the mitochondria. On the other hand, the ~4CO2 formed from 6-~4C-glucose indicates the proportion of pyruvate oxidation in the mitochondria, if the total glucose utilized and the counting yield are known; in this case, the pyruvate may come either from glucose entering the EMP or the HMS. The Methylene Blue stimulatory effect on the HMS was tested by adding 0.25ml of Methylene Blue solution (18.5 mg in 2 ml glycine-imidazole buffer) to the incubation mixture at the beginning of the incubation. The effect of NADP on the flux of the HMS in the intact Rana ridibunda erythrocyte and haemolysate was tested by adding NADP in the incubation mixture at a final concentration of 2 mM. RESULTS Resting rates o f Rana ridibunda erythrocyte glucose c o n s u m p t i o n and 14CO2 p r o d u c t i o n from
Table 1. Glucose consumptionand '4CO2production in intact red cells of frog and man, incubated with 124C-glucose,with or without MethyleneBlue (MB). Experimentswere conducted with three to five animals to give one pool of cells. Values are the mean of five experiments_+SEM Frog Man Effector added None MB None MB Glucose consumption 0.766 +_0.058 1.359 + 0.150 1.515 +_0.048 (#molhr ~ml ~ RBC) 1.060" 1.4" CO2 production 0.021 + 0.003 0.151 ___0.019 0.078 + 0.0007 (/lmolhr -I ml i RBC) 1.160" 3.4* Percentage (%) 2.90 + 0.41 11.10 +_0.55 5.18 + 0.21 15.0" 242.8* *Values obtained from Albrecht et al. (1971).
Hexose monophosphate shunt in frog RBCs
289
Table 2. Glucoseconsumptionand ~4CO2production in intact frog cells incubatedwith 6:4C-glucose,with or without MethyleneBlue(MB). Each experimentwas conductedwiththree to fiveanimalsto giveone poolof cells. Values are the mean of four experiments+ SEM Effector added None MB Glucose consumption 0.8240+ 0.0160 1.4010_+0.0570 (gmol hr-t ml-I RBC) ~4CO2 production 0.0050_+0.0005 0.0060 + 0.0006 (pmolhr i ml-i RBC) Percentage (%) 0.600+ 0.0057 0.40 + 0.03
1-~4C-glucose were found to be significantly lower than the respective values in the human erythrocyte (Table 1), for which it is known that the only source of CO2 is the decarboxylation of glucose carbon-1 in the hexose monophosphate shunt (HMS). In order to test whether nucleated frog red cells have active mitochondrial metabolism and if so to evaluate the extent of the contribution of the pyruvate oxidation to 14CO2 production from 1-14C-glucose (Table 1), 6-~4C-glucose was used; the production of ~4CO2was found to be very low (0.005 + 0.0005, Table 2) but still measurable. In contrast, the activities of several TCA cycle enzymes (pyruvate carboxylase, citrate synthase, aconitase and isocitrate dehydrogenase) were non-detectable (activity less than 0. l #mol/min) as assayed in Rana ridibunda haemolysates. Methylene Blue stimulates glucose consumption in Rana ridibunda erythrocytes (Tables l, 2). When 1-14C-glucose was used, Methylene Blue resulted in a 7-fold increase of ~4CO2 production (Table 1), whereas when 6-~4C-glucose was used, Methylene Blue resulted in a 1.2-fold increase of 14CO2 production. Total glucose consumption was in both cases increased by approximately 2-fold (Tables 1, 2) compared to that of the resting rate. The maximum catalytic activities of G-6-PD, 6-PGD and glutathione reductase are shown in Table 3. The study of the kinetic properties of the two dehydrogenases showed that the Km of G-6-PD for NADP is lower (12/aM) than the resp.ective value of Table 3. Maximum catalytic activities of G-6-PD, 6-PGD and glutathionereductasein Rana ridibunda haemolysates.Experiments were conductedwith two animalsto giveone pool of cells. Values are the mean of five experiments+ SEM Activity Enzyme (#mol/ml RBC) Glucose-6-phosphatedehydrogenase 4.140+ 0.058 6-Phosphogluconatedehydrogenase 0.790_+_+0.180 Glutathione reductase 0.340+ 0.090
6-PGD (32pM, Fig. 1). NADPH strongly inhibits both 6-PGD and G-6-PD activities, its effect being more prominent on 6-PGD, since the Kj values were found to be 8 0 # M for G-6-PD and 12/aM for 6-PGD (Fig. 2). The Km values of G-6-PD and 6-PGD for glucose-6-phosphate and 6-phosphogluconate were 29/aM and 8 3 v M , respectively (Fig. 3). The effect of excess NADP (2raM) on total haemolysates and intact erythrocytes with respect to glucose metabolism from 1-14C-glucose was examined (Table 4). In the absence of NADP, glucose consumption and 14CO2production were not statistically significant in total haemolysates compared to intact erythrocytes. On the other hand, NADP resulted in a significant decrease of 14CO2 production in total haemolysates (Table 4). In order to elucidate the nature of the HMS regulation, the effects of various intermediate metabolites, adenine nucleotides, oxidized and reduced glutathione on the activities of G-6-PD and 6-PGD were examined (Table 5). According to this table, both dehydrogenases are inhibited by ATP, ADP and fructose diphosphate, 6-PGD being more sensitive than G-6-PD to the inhibitory effect of these metabolites. Furthermore, 2,3-diphosphoglycerate (2,3-DPG), GSH and GSSG have a marked inhibitory effect on 6-PGD, while their effect on G-6-PD is weaker or absent. Finally, the trioses (dehydroxyacetone phosphate and glyceraldehyde phosphate) inhibit only the first dehydrogenase of the HMS, while 6-PGD is not affected by them. Since the proper function of the HMS in protecting red cells is accomplished through glutathionemediated mechanisms, the intracellular levels of both GSSG and GSH in Rana ridibunda erythrocytes were determined and found to be 0.179+0.05 and 0.68 _4-0.008, respectively. Subsequently, the effect of various GSSG concentrations on both dehydrogenases was studied in the presence of NADPH, the
3°°I 50
100 150 I/NADP (mM 1)
200
Fig. 1. Lineweaver-Burk plots of G-6-PD (O) and 6-PGD (O) activity at various NADP concentrations.
290
M A R T H A KALOYIANN1 a n d
MARIA KALOMENOPOULOU
300
"iE 200 100 [
I
50
I
I
I
I
100 150 1/S (raM -1) Fig. 2. Lineweaver Burk plots of G-6-PD (Q) and 6-PGD (C)) activity at various glucose-6-phosphate and 6-phosphogluconate concentrations. value of N A D P H / N A D P being kept at 4 (Table 6).Preliminary experiments showed that 0.015mM ZnSO4 completely inhibited glutathione reductase,
100 '~ "- !
•
•
51
/
|
-Ki
0.1
.o
,- ! 200
_
_
-Ki
0.2
0.3
I>
~
0.05 0.10 NADPH(mM)
although hardly affected the activities of G-6-PD and 6-PGD. According to Table 6, GSSG at physiological concentrations decreases the inhibition exercised by N A D P H on G-6-PD, Similarly, although GSSG inhibits 6-PGD in the absence of N A D P H (Table 5), it decreases the inhibition exercised by N A D P H on 6-PGD. With this starting point, the percentage activation of the two dehydrogenases by 50/~M GSSG at various N A D P H / N A D P ratios was determined (Tables 7, 8). The activating effect of GSSG on G-6-PD increases as the N A D P H / N A D P ratio increases (Table 7). Similarly, when the value of the N A D P H / N A D P ratio is higher than 0.5 an activating effect of GSSG on 6-PGD is observed (Table 8). Since Mg 2+ seems to be an activator of the oxidative enzymes of the HMS, the effect of varying concentrations of free Mg 2+ ions on the two dehydrogenases at different states of substrate saturation was studied (Fig. 4). According to Fig. 4, when the enzymes are saturated with respect to NADP, Mg 2÷ does not significantly affect the activities of the enzymes. On the other hand, Mg 2+ seems to play an important role on both dehydrogenases at suboptimal N A D P and substrate concentrations. DISCUSSION
(b)
Fig. 3. (a) Inhibition of G-6-PD by NADPH at [NADP] = 0.010 rnM ( 0 ) and at [NADP] = 0.040 mM (A). (b) Inhibition of 6-PGD by NADPH at [NADP] = 0.020 mM (©) and at [NADP] = 0.080 mM,(A).
The data presented in Tables 1 and 2 imply that the mature Rana ridibunda erythrocyte oxidizes glucose mainly through the anaerobic glycolytic pathway and the aerobic HMS, in order to meet its requirements for ATP and reducing equivalents, The presence of glutathione reductase activity and the higher intracellular concentration of GSH compared to that of GSSG suggest that the N A D P H produced by the two dehydrogenases of the oxidative phase of the shunt is used to keep glutathione in the reduced state within the red cell. GSH may have the same critical role in amphibian erythrocytes as in mammalian erythrocytes, i.e. to maintain various thiol groups of the cell in the reduced state through oxidation-reduction interaction (Allen and Jandl, 1961; Jacob and Jandl, 1962). Furthermore, the reduction of GSSG to GSH
H e x o s e m o n o p h o s p h a t e s h u n t in frog R B C s
291
Table 4. Glucose consumption and ~4CO~production in Rana ridibunda intact erythrocytes and haemolysates incubated with or without 2 mM NADP. Experiments were conducted with three to five animals to give one pool of cells. Values are the mean of five experiments + SEM Haemolysate Nucleotide added Glucose consumption (#mol hr -I, ml -I RBC) CO 2 production (~molhr i, ml I RBC) Percentage (%)
Intact RBCs
None
NADP
None
NADP
0.788 ± 0.033
0.824 ± 0.067
0.766 ± 0.058
0.781 ± 0.165
0.021 ± 0.002
0.014 ± 0.002
0.021 ± 0.003
0.023 ± 0.001
2.660 ± 0.080
1.690 ± 0.400
2.900 ± 0.410
2.990 ± 0.320
Table 5. Inhibitory effect of various substances on the activities of G-6-PD and 6-PGD. G-6-PD and 6-PGD were measured at suboptimal substrate concentrations (G6P or 6PG, 0.05 mM; NADP ÷, 0.04 mM) Metabolite ATP
ADP
2,3-DPG
FDP DHAP GAP GSSG GSH
Concentration (mM)
Inhibition (%) G-6-PD 6-PGD
1 5 10 20 30 0.5
4 3 19 51 66 0
1.0
0
3.0 4.0 0.8 1.6 3.2 3.8 0.02 0.10 0.20 0.05 0.25 0.50 0.05 0.25 0.50 0.05 0.20 0.40 0.75 1.50 3.00
12 10 0 0 0 6.7 11.7 9.2 10.0 0 0 64.0 15.0 15.0 56.2 0 0 0 7.9 8.1 6.8
3.2 6.5 32.2 80.6 100 9.6 7.8 9.6 25.8 26.0 34.7 47.8 47.8 10.3 17.1 17.4 0 0 0 0 0 0 29.0 29.0 36.9 4.3 17.4 26.0
O.03q 0.02 0.01(
K)"° i
GSSG (~ M) 0.0 12.5 25.0 50.0 100.0 200.0
Inhibition (%) G-6-PD 6-PGD 26 ± 4 26±8 18 + 4 10 ± 3 10 ± 2 10 ± 3
66 ± 66460 ± 60 ± 60 ± 60 ±
15 15 8 7 8 8
i
._~
0.03 C) 0.02,
0305-0491/90$3.00+ 0.00 © 1990PergamonPress pie
REGULATION A N D RATE OF THE HEXOSE MONOPHOSPHATE SHUNT IN RANA RIDIBUNDA ERYTHROCYTES MARTHA KALOYIANNI* and MARIAKALOMENOPOULOU Laboratory of Animal Physiology, Department of Zoology, Science School, University of Tbessaloniki, Thessaloniki 54006, Greece (Received 19 June 1989) Abstract--1. Resting rates of Rana ridibunda erythrocyte glucoseconsumption and ~4CO2production from IJ4C-glucose were found to be significantly lower than the respective values in human erythrocytes. 2. In the presence of 1-t4C-glucose Methylene Blue stimulated t4CO2 production 7-fold, while in the presence of 6-14C-glucose Methylene Blue stimulated 14CO2 production 1.2-fold. 3. The Km of G-6-PD for G-6-P and NADP were 29 and 12/~M, respectivelywhile the K~ of 6-PGD for 6-PG and NADP were 83 and 32 aM, respectively. The Ki of G-6-PD and 6-PGD for NADPH were 80 and 12 laM, respectively. 4. Excess amounts of NADP resulted in a significantdecrease of ~4CO2production from 1-~4C-glucose in total haemolysates. 5. ATP, ADP and fructose diphosphate inhibited both G-6-PD and 6-PGD, the latter being more sensitive than G-6-PD to their inhibitory effect. 2,3-DPG and reduced and oxidized glutathione showed a marked inhibitory effect on 6-PGD, while the phosphorylated trioses inhibited only G-6-PD. 6. Physiological concentrations of oxidized glutathione decreased the inhibition exercised by NADPH on G-6-PD. 7. The possible role of the two dehydrogenases in the regulation of the HMS is discussed.
INTRODUCTION Glucose metabolism in intact mammalian red blood cells is accomplished through the Embden-Meyerhoff Pathway (EMP) and the hexose monophosphate shunt (HMS), the relative importance of which, as assessed by employing l-~4C-glucose, is secondary in terms of average glucose consumption (Herman et aL, 1962; Gaetani et al., 1974, 1976; Magnani et al., 1980). The adequate operation of the HMS, however, is as important to red cell survival as that of the EMP, since it was shown that severe restriction of the HMS is associated with a shortened red cell survival and susceptibility to oxidative injury (Yoshida, 1973; Harvey and Kaneko, 1977; Valentine et al., 1985). The available data concerning amphibian red blood cells seem to indicate that their overall partition of glucose metabolism differs from that of mammalian red blood cells (Herman et al., 1962). Amphibian red blood cells are typical of non-mammalian vertebrates in that they are nucleated and are expected to metabolize 1-14C-glucose more actively than non-nucleated red blood cells. However, it was shown by Herman et al. (1962) that Rana pipiens red cells produced little 14CO2 from 1-14C-glucose, being intermediate between avian and human red blood cells in the amount of 14CO2 produced. This raised questions as to whether frog red cells, presumably having an active TCA cycle, would metabolize glucose via the HMS. *Author to whom all correspondence should be addressed. caP,B)95/2--F
The proper function of the HMS requires the action of two dehydrogenases, the availability of glutathione, as well as the capacity to reduce the oxidized glutathione generated during detoxification from harmful peroxides and molecular oxygen (Jacob et al., 1965; Jacob and Jandle, 1966). The activity of the HMS is believed to reflect the relative amount of NADP (Cahill et al., 1958) and if so, the rate of its metabolism should be linked to the rate of oxidation and reduction of glutathione wherein NADPH is oxidized. Since the requirements for reducing equivalents are variable, depending on the amount of oxidant stress to which the red cell is exposed, regulatory mechanisms must be expected to adjust the rate of the shunt to changing needs. These are likely to operate at the initial steps, i.e. the reactions mediated by the two dehydrogenases (Dische, 1964). In this study we have attempted to evaluate the relative flux of the HMS in frog erythrocytes as well as to approach the problem of the regulation of the oxidative phase of the HMS, by which the two molecules of NADPH are generated per glucose molecule consumed. Intact red blood cells were examined in order to more closely approximate their usual physiological state. In addition, some of the enzymes which are considered to be of primary importance in protecting erythrocytes against oxidative attack were assaye~ in total haemolysate preparations. The kinetic properties of these enzymes as well as their catalytic capacities in the presence of various intracellular metabolites were further studied in order to investigate the flux control mechanisms of the HMS operating in the amphibian erythrocyte in vivo.
287
288
MARTHA KALOYIANNIand MARIAKALOMENOPOULOU MATERIALS AND METHODS
Animals Frogs (Rana ridibunda) were supplied by a local dealer. They were caught in the vicinity of Thessaloniki, kept in containers in the laboratory in freshwater and used a week after arrival. Chemicals and enzymes The substrates, enzymes and coenzymes were purchased from Sigma Chemical (St Louis, USA). All other chemicals were obtained either from Serva or Merck. The radioactive glucose (1J4C-gtucose and 6-14C-glucose) were from Amersham Radiochemical Center. Sampling o f red cells; determination o f enzymatic activities Blood samples were obtained by heart puncture from anaesthetized frogs using heparinized syringes. Immediately after collection the blood was centrifuged at 500 g for 10 min and the plasma and the upper one-fifth of the cells were removed by aspiration. The erythrocytes were washed three times with 0.12mM NaC1 and haemolyzed with ice-cold distilled water and consequent sonication for 3 × 10 sec with a MSE Soniprep 150 sonicator. The haemolysate was used for the determination of enzymatic activities at 25~C using a Hitachi recording spectrophotometer. The change in A340 caused by oxidation or reduction of NADP(H) was followed. Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was measured according to Glock and McLean (1953), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) according to Rodriguez-Segade et al. (1978) and glutathione reductase (E.C. 1.6.4.2) according to Racker (1955). Intracellular oxidized and reduced glutathione were measured according to Bernt and Bergmeyer (1974) in perchloric acid extracts of Rana ridibunda erythrocytes. The effect of NADPH, oxidized glutathione (GSSG), reduced glutathione (GSH), adenine nucleotides and various intracellular metabolites on the activities of glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD) was determined by adding varying concentrations of the above substances to the reaction mixture used for the determination of the catalytic activities of these dehydrogenases under suboptimal substrate concentrations. ZnSO 4 (0.015 raM) was used in order to completely inhibit glutathione reductase activity, when G-6-PD and 6-PGD were assayed in the presence of oxidized glutathione and NADPH. The percentage activation exercised by GSSG against the NADPH inhibition of G-6-PD or 6-PGD activity was calculated by the following expression: % increase in activity= 100 ( x / y - 1 ) , where x = v a l u e obtained for the enzyme activity in the presence of NADPH and GSSG and y = value obtained for the enzyme activity in the presence of NADPH. Metabolic studies All metabolic studies were performed with washed erythrocytes drawn from three to five animals to give one pool of cells, which were subsequently suspended in an imidazole-glycylglycine buffer, pH 7.4, containing I00 mM NaCI, 5.9mM KC1, 1.25mM CaC12, 2.4mM MgSO4,
4.2 mM imidazole, 7.6 mM glycylglycine, 1.2 mM KHEPO4 and 3 mM glucose. The suspensions were equilibrated at 25°C, their haematocrit measured and shaken in air to get a complete haemoglobin oxygenation. Glucose utilization and 14CO2 production from [1-t4C]- or [6-14C]-glucose were determined by incubating the 2 ml erythrocyte suspension with 0.3ml [l°t4C]- or [6-14C]-glucose (about 0.5 pCi in 0.3 ml) in Erlenmeyer flasks (25 ml) fitted with a rubber cup and equipped with a disposable plastic centre well. The flasks were protected from the light and incubated in a metabolic shaker for 60 min at 25°C at 120 oscillations per rain. At the end of the incubation 0.2 ml of 2.0 M KOH were injected through the rubber stopper into the centre wells, followed by 0.7 ml of 3.7 M perchloric acid into the incubation mixture. The flasks were shaken thoroughly and incubated again for 30 min. The radioactivity trapped in the KOH of the centre well was counted (cpm ~4COE). A sample from the cell extract was then neutralized, assayed for glucose by the method of Bergmeyer et al. (1963) and applied to a Dowex-I (acetate form 1 × 8) anion exchange resin in a pasteur pipette. Glucose was eluted with double distilled water until 15 ml were collected. The anions were eluted from the columns by addition of 10M HCI. The glucose and anion fractions (0.5 ml of each) were then counted in a liquid scintillation counter. The scintillation fluid of Welt et al. (1971) was used. The glucose concentration of the first eluate was measured enzymatically according to Bergmeyer et al. (1963) and the total glucose consumption was considered as the sum of #mol of ~4COz evolution and ~4C-anions production. Glucose and ~4C-anions were, in each case, also determined in blanks, in which 3.7 M perchloric acid was added in the incubation flasks at the beginning of the incubation. Experiments with normal human erythrocyte suspensions were simultaneously conducted and used as controls. The calculation of the share of the EMP and the HMS in glucose consumption in Rana ridibunda erythrocytes is based on the following considerations: the actual value of the ~4CO2 formed from l-laC-glucose indicates the sum of the glucose oxidized via the HMS and the fraction entering the EMP and finally oxidized in the mitochondria. On the other hand, the ~4CO2 formed from 6-~4C-glucose indicates the proportion of pyruvate oxidation in the mitochondria, if the total glucose utilized and the counting yield are known; in this case, the pyruvate may come either from glucose entering the EMP or the HMS. The Methylene Blue stimulatory effect on the HMS was tested by adding 0.25ml of Methylene Blue solution (18.5 mg in 2 ml glycine-imidazole buffer) to the incubation mixture at the beginning of the incubation. The effect of NADP on the flux of the HMS in the intact Rana ridibunda erythrocyte and haemolysate was tested by adding NADP in the incubation mixture at a final concentration of 2 mM. RESULTS Resting rates o f Rana ridibunda erythrocyte glucose c o n s u m p t i o n and 14CO2 p r o d u c t i o n from
Table 1. Glucose consumptionand '4CO2production in intact red cells of frog and man, incubated with 124C-glucose,with or without MethyleneBlue (MB). Experimentswere conducted with three to five animals to give one pool of cells. Values are the mean of five experiments_+SEM Frog Man Effector added None MB None MB Glucose consumption 0.766 +_0.058 1.359 + 0.150 1.515 +_0.048 (#molhr ~ml ~ RBC) 1.060" 1.4" CO2 production 0.021 + 0.003 0.151 ___0.019 0.078 + 0.0007 (/lmolhr -I ml i RBC) 1.160" 3.4* Percentage (%) 2.90 + 0.41 11.10 +_0.55 5.18 + 0.21 15.0" 242.8* *Values obtained from Albrecht et al. (1971).
Hexose monophosphate shunt in frog RBCs
289
Table 2. Glucoseconsumptionand ~4CO2production in intact frog cells incubatedwith 6:4C-glucose,with or without MethyleneBlue(MB). Each experimentwas conductedwiththree to fiveanimalsto giveone poolof cells. Values are the mean of four experiments+ SEM Effector added None MB Glucose consumption 0.8240+ 0.0160 1.4010_+0.0570 (gmol hr-t ml-I RBC) ~4CO2 production 0.0050_+0.0005 0.0060 + 0.0006 (pmolhr i ml-i RBC) Percentage (%) 0.600+ 0.0057 0.40 + 0.03
1-~4C-glucose were found to be significantly lower than the respective values in the human erythrocyte (Table 1), for which it is known that the only source of CO2 is the decarboxylation of glucose carbon-1 in the hexose monophosphate shunt (HMS). In order to test whether nucleated frog red cells have active mitochondrial metabolism and if so to evaluate the extent of the contribution of the pyruvate oxidation to 14CO2 production from 1-14C-glucose (Table 1), 6-~4C-glucose was used; the production of ~4CO2was found to be very low (0.005 + 0.0005, Table 2) but still measurable. In contrast, the activities of several TCA cycle enzymes (pyruvate carboxylase, citrate synthase, aconitase and isocitrate dehydrogenase) were non-detectable (activity less than 0. l #mol/min) as assayed in Rana ridibunda haemolysates. Methylene Blue stimulates glucose consumption in Rana ridibunda erythrocytes (Tables l, 2). When 1-14C-glucose was used, Methylene Blue resulted in a 7-fold increase of ~4CO2 production (Table 1), whereas when 6-~4C-glucose was used, Methylene Blue resulted in a 1.2-fold increase of 14CO2 production. Total glucose consumption was in both cases increased by approximately 2-fold (Tables 1, 2) compared to that of the resting rate. The maximum catalytic activities of G-6-PD, 6-PGD and glutathione reductase are shown in Table 3. The study of the kinetic properties of the two dehydrogenases showed that the Km of G-6-PD for NADP is lower (12/aM) than the resp.ective value of Table 3. Maximum catalytic activities of G-6-PD, 6-PGD and glutathionereductasein Rana ridibunda haemolysates.Experiments were conductedwith two animalsto giveone pool of cells. Values are the mean of five experiments+ SEM Activity Enzyme (#mol/ml RBC) Glucose-6-phosphatedehydrogenase 4.140+ 0.058 6-Phosphogluconatedehydrogenase 0.790_+_+0.180 Glutathione reductase 0.340+ 0.090
6-PGD (32pM, Fig. 1). NADPH strongly inhibits both 6-PGD and G-6-PD activities, its effect being more prominent on 6-PGD, since the Kj values were found to be 8 0 # M for G-6-PD and 12/aM for 6-PGD (Fig. 2). The Km values of G-6-PD and 6-PGD for glucose-6-phosphate and 6-phosphogluconate were 29/aM and 8 3 v M , respectively (Fig. 3). The effect of excess NADP (2raM) on total haemolysates and intact erythrocytes with respect to glucose metabolism from 1-14C-glucose was examined (Table 4). In the absence of NADP, glucose consumption and 14CO2production were not statistically significant in total haemolysates compared to intact erythrocytes. On the other hand, NADP resulted in a significant decrease of 14CO2 production in total haemolysates (Table 4). In order to elucidate the nature of the HMS regulation, the effects of various intermediate metabolites, adenine nucleotides, oxidized and reduced glutathione on the activities of G-6-PD and 6-PGD were examined (Table 5). According to this table, both dehydrogenases are inhibited by ATP, ADP and fructose diphosphate, 6-PGD being more sensitive than G-6-PD to the inhibitory effect of these metabolites. Furthermore, 2,3-diphosphoglycerate (2,3-DPG), GSH and GSSG have a marked inhibitory effect on 6-PGD, while their effect on G-6-PD is weaker or absent. Finally, the trioses (dehydroxyacetone phosphate and glyceraldehyde phosphate) inhibit only the first dehydrogenase of the HMS, while 6-PGD is not affected by them. Since the proper function of the HMS in protecting red cells is accomplished through glutathionemediated mechanisms, the intracellular levels of both GSSG and GSH in Rana ridibunda erythrocytes were determined and found to be 0.179+0.05 and 0.68 _4-0.008, respectively. Subsequently, the effect of various GSSG concentrations on both dehydrogenases was studied in the presence of NADPH, the
3°°I 50
100 150 I/NADP (mM 1)
200
Fig. 1. Lineweaver-Burk plots of G-6-PD (O) and 6-PGD (O) activity at various NADP concentrations.
290
M A R T H A KALOYIANN1 a n d
MARIA KALOMENOPOULOU
300
"iE 200 100 [
I
50
I
I
I
I
100 150 1/S (raM -1) Fig. 2. Lineweaver Burk plots of G-6-PD (Q) and 6-PGD (C)) activity at various glucose-6-phosphate and 6-phosphogluconate concentrations. value of N A D P H / N A D P being kept at 4 (Table 6).Preliminary experiments showed that 0.015mM ZnSO4 completely inhibited glutathione reductase,
100 '~ "- !
•
•
51
/
|
-Ki
0.1
.o
,- ! 200
_
_
-Ki
0.2
0.3
I>
~
0.05 0.10 NADPH(mM)
although hardly affected the activities of G-6-PD and 6-PGD. According to Table 6, GSSG at physiological concentrations decreases the inhibition exercised by N A D P H on G-6-PD, Similarly, although GSSG inhibits 6-PGD in the absence of N A D P H (Table 5), it decreases the inhibition exercised by N A D P H on 6-PGD. With this starting point, the percentage activation of the two dehydrogenases by 50/~M GSSG at various N A D P H / N A D P ratios was determined (Tables 7, 8). The activating effect of GSSG on G-6-PD increases as the N A D P H / N A D P ratio increases (Table 7). Similarly, when the value of the N A D P H / N A D P ratio is higher than 0.5 an activating effect of GSSG on 6-PGD is observed (Table 8). Since Mg 2+ seems to be an activator of the oxidative enzymes of the HMS, the effect of varying concentrations of free Mg 2+ ions on the two dehydrogenases at different states of substrate saturation was studied (Fig. 4). According to Fig. 4, when the enzymes are saturated with respect to NADP, Mg 2÷ does not significantly affect the activities of the enzymes. On the other hand, Mg 2+ seems to play an important role on both dehydrogenases at suboptimal N A D P and substrate concentrations. DISCUSSION
(b)
Fig. 3. (a) Inhibition of G-6-PD by NADPH at [NADP] = 0.010 rnM ( 0 ) and at [NADP] = 0.040 mM (A). (b) Inhibition of 6-PGD by NADPH at [NADP] = 0.020 mM (©) and at [NADP] = 0.080 mM,(A).
The data presented in Tables 1 and 2 imply that the mature Rana ridibunda erythrocyte oxidizes glucose mainly through the anaerobic glycolytic pathway and the aerobic HMS, in order to meet its requirements for ATP and reducing equivalents, The presence of glutathione reductase activity and the higher intracellular concentration of GSH compared to that of GSSG suggest that the N A D P H produced by the two dehydrogenases of the oxidative phase of the shunt is used to keep glutathione in the reduced state within the red cell. GSH may have the same critical role in amphibian erythrocytes as in mammalian erythrocytes, i.e. to maintain various thiol groups of the cell in the reduced state through oxidation-reduction interaction (Allen and Jandl, 1961; Jacob and Jandl, 1962). Furthermore, the reduction of GSSG to GSH
H e x o s e m o n o p h o s p h a t e s h u n t in frog R B C s
291
Table 4. Glucose consumption and ~4CO~production in Rana ridibunda intact erythrocytes and haemolysates incubated with or without 2 mM NADP. Experiments were conducted with three to five animals to give one pool of cells. Values are the mean of five experiments + SEM Haemolysate Nucleotide added Glucose consumption (#mol hr -I, ml -I RBC) CO 2 production (~molhr i, ml I RBC) Percentage (%)
Intact RBCs
None
NADP
None
NADP
0.788 ± 0.033
0.824 ± 0.067
0.766 ± 0.058
0.781 ± 0.165
0.021 ± 0.002
0.014 ± 0.002
0.021 ± 0.003
0.023 ± 0.001
2.660 ± 0.080
1.690 ± 0.400
2.900 ± 0.410
2.990 ± 0.320
Table 5. Inhibitory effect of various substances on the activities of G-6-PD and 6-PGD. G-6-PD and 6-PGD were measured at suboptimal substrate concentrations (G6P or 6PG, 0.05 mM; NADP ÷, 0.04 mM) Metabolite ATP
ADP
2,3-DPG
FDP DHAP GAP GSSG GSH
Concentration (mM)
Inhibition (%) G-6-PD 6-PGD
1 5 10 20 30 0.5
4 3 19 51 66 0
1.0
0
3.0 4.0 0.8 1.6 3.2 3.8 0.02 0.10 0.20 0.05 0.25 0.50 0.05 0.25 0.50 0.05 0.20 0.40 0.75 1.50 3.00
12 10 0 0 0 6.7 11.7 9.2 10.0 0 0 64.0 15.0 15.0 56.2 0 0 0 7.9 8.1 6.8
3.2 6.5 32.2 80.6 100 9.6 7.8 9.6 25.8 26.0 34.7 47.8 47.8 10.3 17.1 17.4 0 0 0 0 0 0 29.0 29.0 36.9 4.3 17.4 26.0
O.03q 0.02 0.01(
K)"° i
GSSG (~ M) 0.0 12.5 25.0 50.0 100.0 200.0
Inhibition (%) G-6-PD 6-PGD 26 ± 4 26±8 18 + 4 10 ± 3 10 ± 2 10 ± 3
66 ± 66460 ± 60 ± 60 ± 60 ±
15 15 8 7 8 8
i
._~
0.03 C) 0.02,
