Comp. Biochem. PhysioL, 1975, Vol. 5013,pp. 61 to 64. Pergamon Press. Printed in Great Britain
KINETIC STUDIES ON THE ERYTHROCYTE HEXOKINASE OF THE DOMESTIC PIG, SUS SCROFA EARL DIXON, JR. Department of Physiology and Pharmacology, School of Veterinary Medicine, Tuskegee Institute, Tuskegee Institute, Alabama 36088, U.S.A. (Received 4 December 1973)
Abstract--1. Pig erythrocyte hexokinase (ATP: D-hexose-6-phosphotransferase E.C. 2.7.1.1) was isolated in a partially purified preparation by Sephadex G-100 gel filtration. 2. In the mature pig (> 1 year of age), a high affinity component exhibits a maximum velocity at 0.2--0.4 mM glucose. This component seems to be inhibited by excessive glucose concentrations (> 0.5 raM). 3. The young, adult pig (3--6 months of age) possesses a hexokinase which exhibits a maximum velocity at 1.0 g M glucose. 4. 2,3-Diphosphoglycerate inhibits the enzyme at low concentrations of M g s+ (0.05 raM), but the inhibition is relieved as the concentration of M g ~+ is increased beyond I-0 raM. 5. These findings indicate that the mature erythrocyte of the pig may possess unique biochemical properties that have not been observed in other mammalian red blood cells.
INTRODUCTION
pig, studies have been designed to determine the kinetic properties of hexokinase in respect to substrate, co-factors and effector concentrations. The present study was designed to determine the relationship between the enzyme and its substrate and to determine the influence o f 2,3-diphosphoglycerate (DPG) on the activity of the enzyme. The results obtained from a partially purified enzyme preparation indicate that the pig erythrocyte possesses a hexokinase that exhibit unique properties in respect to substrate requirements. That is, the data presented suggest that the pig red cell may possess two isoenzymes of hexokinase. Furthermore, the relationship between the enzyme and substrate is related to the age of the animal. D P G was found to be an inhibitor of the enzyme at the in vivo concentrations of the glycolytic intermediate. The implications of these findings arc discussed as they relate to glycolyticcontrol in the mature erythrocyte of the pig.
ERYTHROCYTE hexokinases (ATP: D-hexose-6-phosphotransferase E.C. 2.7.1.1) have not been extensively studied at the comparative level. While there have been many studies on the human, rabbit and rat red cell enzyme, there has been an absence of literature on the kinetic properties of hexokinases isolated from the red cells of other mammals (Rose & O'Connell, 1964; Rose et al., 1964; de Verdier & Garby, 1964; Rapoport, 1966). Since hexokinases represent one of the key enzymes in glucose utilization, information obtained from kinetic studies on this enzyme can be utilized to evaluate metabolic control of the intact erythrocyte (Rapoport, 1966). The mature erythrocyte of pig possesses one of the slowest glycolytic rates observed in any mammal (McManus, 1976; Kim and McManus, 1972). Studies designed to estimate the glycolytic capacity by quantitating the activities of the glycolytic enzymes in stroma-free hemolysates have revealed that these cells possess an adequate enzymatic apparatus that would allow a more rapid glycolytic rate (Dixon et al., 1966). However, hexokinase was found to possess the lowest specific activity of any enzyme observed. Other studies have indicated that glucose permeability was the rate-limiting factor of glucose utilization in the pig erythrocyte (Widdas, 1955). Recent evidence suggests that glucose permeability as well as the ability of these cells to utilize ribose in vitro was related to the age of the animal (McManns & Kim, 1968). In an attempt to elucidate the mechanisms that regulate glycolysis in the mature erythrocyte of the
MATERIALS AND METHODS Chemicals
ATP (disodium salt from equine muscle), NADP and G6PD (NADP oxidoreductase E.C. 1.1.1.49) were purchased from the Boehringer-Mannheim Chemical Company. 2,3-Diphosphoglycerate (pentacyclohexylammonium salt),D-glucose, Scphadex G-100 and MgCl~ were purchased from the Sigma Chemical Company. Partial purification of the pig red-cell hexokinase
Pig blood was collected from the albatroit in 5 units of heparin per ml of whole blood and processed within 6 hr of collection. The red cells were separated from the 61
62
EARL DIXON, JR.
plasma by centrifugation at 5000rev/min for 10min (International Model No. HT). The plasma and buffy coat were removed by suction. The packed red cells were resuspended in an adequate volume of 0.17 M NaCI and spun at 5000 rev/min for 10 min. After the third wash, the white cells had been reduced to a small percentage of the total cell population. The cells were mixed with an equal volume of cold distilled H~O and Saponin was added to equal a final concentration of 0.1%. This suspension was allowed to stand for 30 rain with intermittent mixing. The stroma was removed by centrifugation at 10,000rev/min for 20min. The supernate fluid was separated from the stroma pellet for further enzyme processing. Ten to fifteen milliliters of hemolysate was added to a Sephadex G-100 column (30 x 400 mm) previously equilibrated with 0"02 M Tris-Cl buffer at pH 7"4. The gel was prepared according to the instructions supplied by the Sigma Chemical Company. The hemolysate was allowed to flow through the column at a rate of t "0 ml per 2 rain and the eluant was collected in 4-ml fractions. The eluant tubes exhibiting hexokinase activity were subsequently used to determine the kinetic properties of the enzyme.
Assay o f hexokinase activity Hexokinase was assayed at 37°C by a modification of previously reported methods (Chapman et al., 1962; Dixon et al., 1966). The standard assay procedure (total volume, 1'0 ml) contained 200 mM Tris-C1 buffer (pH 7-40), 4 mM MgClz, 1.0 mM ATP, 0'1 mM NADP, 6-2 mM o-glucose, G6PD and a sufficient volume of the enzyme material. Alterations in the concentrations of glucose were made as indicated. Reactions were followed in a Beckman Du spectrophotometer equipped with a Gilford Model 2000 Multiple sampling accessory and a Haake constant temperature circulator. The enzymatic activity was determined by utilizing a Gilford Model 6040 recorder. Units of hexokinase were expressed as /~M N A D P H formed per min using the extinction coefficient for the compound at 340 nm. All assays were performed utilizing co-factor and substrate concentrations that exceeded the maximum velocity for the enzyme under the conditions of the assay. The commercial preparation of G6PD was free of hexokinase at the dilution of the enzyme used in these experiments. Hemoglobin concentrations were determined using the cyanmethemoglobin method. The reaction pH was ascertained using a Radiometer pH meter (Model 26) equipped with a micro pH unit (Type E5021a). RESULTS Fig. 1 represents a velocity vs substrate concentration plot of the hexokinase isolated f r o m the mature erythrocytes of an adult pig (approximately 1 year of age). The m a x i m u m velocity of the enzyme occurred at 0-2-0.4 m M of glucose. Additional increases in substrate concentration caused a decrease in enzyme activity. At glucose concentrations in excess of 1'0 m M , there was a plateau in the velocity of the enzyme. Some preparations exhibited a secondary increase in velocity at concentrations o f glucose exceeding 1.0 m M . These findings were consistent regardless of the species or the source in which the blood samples were obtained.
hexokinase
40
~ 3.0 0
2'0 ::L >
I~0
I.'0 Glucose,
2 i0 mM
3~ 0
a
Fig. 1. Plot of initial reaction velocity (v) vs. glucose concentration. The concentrations of ATP and Mg ~+ were maintained constant at 1.0 and 5.0 mM respectively. This enzyme preparation was obtained from the erythrocytes of an adult pig (> 1 year). The data in Fig. 2 demonstrate the velocitysubstrate profile obtained on a typical hexokinase preparation isolated from the red cells o f a young adult pig (3-6 months). It can be observed that there is a shift of the high velocity c o m p o n e n t to the left on the substrate axis. The high velocity component occurs at approximately 0.001 m M of
~.5
Pig
hexokinase
o. I.O
.i ::L >
05
0
2
4
Glucose,
mM
6
Fig. 2. Plot of initial velocity (v) vs. glucose concentration. The enzyme preparation was obtained from a young adult pig that was approximately 6 months of age. glucose. There is a decrease in activity as the substrate concentration approaches 1.0 m M . Further elevations in substrate concentration causes a secondary increase in the velocity of the enzyme. T h o u g h the m a x i m u m velocities of the enzyme occur at different concentrations of substrate in the young adult and adult pig, the relative activities of the two preparations are approximately equal. T h a t is, the data would suggest that the enzyme obtained from the red cells o f the young and the mature pig exhibit
Kinetics of pig red cell hexokinase
63
The intracellular concentrations of glucose in the pig red cell has been reported to be below detection by routine chemical procedures (Kim & McManus, 1971). The data presented here would indicate that the low substrate component would be active at low glucose concentrations. Since the permeability of the adult pig red cell membrane is low, assigning a physiological role to the high substrate enzyme component at this time would be futile. The data also indicate that the young, adult pig I00 (2-3 months) has a low substrate enzyme component ~, Pig hexok i n a s e that exhibits a maximum velocity at 0.001 mM. Since there is a paucity of data on the biochemical +',>_ 7:5 and physiological activity of the pig, the period of transition cannot be correlated with other mechan-)isms operating concurrently. The inability to consistently obtain a secondary increase in velocity N 5o in the adult pig's hexokinase may be due to our method of isolation. The enzyme was not protected by glucose or K + during the isolation procedures 25 which may have resulted in inactivation of this particular enzyme component (Salas et al., 1965; Grossbard & Schimke, 1966; Parry & Walker, 0 I"0 a*'O 31"0 41.0 5"0 1966). (t)PG) x io ~ Experiments are in progress to isolate these enzyme components and determine the kinetic and Fig. 3. Plot of initial reaction velocity (v) vs DPG con centration. The Mg~+ and glucose concentrations in these electrophoretic properties of each enzyme respecexperiments were maintained at 0.05 and 0"33 mM tively. respectively. D P G concentrations in the pig erythrocyte are approximately 5-8 mM/1. of RBC (Bartlett, 1970). approximately 70 per cent. The inhibition of hexo- The data presented here indicated that DPG inhibits kinase was found to be related to the concentration the enzyme at the in rive concentration of the of Mg 2+. In these experiments, 0.5 and 0.2 mM of intermediate. These results are consistent with the Mg ~+ and glucose respectively, were utilized. previously unpublished data mentioned elsewhere Studies are in progress to determine the specific (McManus, 1967). Recently, other reports have relationship between ATP and Mg 2+ in respect to the been published on the human red cell hexokinase that supports these data (Brewer, 1969; Ponce et al., inhibition of pig red cell hexokinase by DPG. 1971; Srivastava & Beutler, 1972). These investigators found that the inhibition of hexokinase was DISCUSSION dependent on the concentrations of Mg ~+ and ATP. The data presented here suggest that the partially The large concentration of D P G found in the pig purified hexokinase isolated from the mature red cell would suggest that this compound would be erythrocyte of the pig possess kinetic propcrti¢~ in another mechanism for scaling down the activity of respect to substrate requirements that are markedly glucose utilization by this cell. different when compared to other mammals. That is, These findings would indicate that pig red cell the enzyme exhibits both low and high substrate hexokinase does not possess the type of biochemical components of hexokinase activity. It was reported properties that have been previously reported for that rabbit reticulocytes possessed two hexokinase other mammalian red cells. Furthermore, the components but the low affinity component was not enzyme exhibits a substrate-velocity profile that observed in the mature erythrocyte of the rabbit varies as a function of age. Since these cells were (Gerber et al., 1970). Other reports have not obtained from animals that are 3-6 months of age, indicated this type of finding in the red cell of the rat these findings cannot be attributed to the presence or human (de Verdier & Garby, 1965; Rapoport, of nee-natal red cells in the circulation. These data 1966; Rose et al., 1969). In these studies we have further suggest the importance of comparative attempted to identify reticulocytes in our pack cells studies in evaluating metabolic control processes that were utilized to prepare the stroma-free hemoly- in the mature red cell at the enzymatic level. The sate. Significant numbers of reticulocytes could not implications of these data regarding the absence be demonstrated in these red blood cells ( < 0.5 per of glucose utilization by the intact red cell in vitro cent). are still unknown. similar specific activities of hexokinase in spite of the apparent variations in glucose affinity. Since it was earlier observed that D P G inhibited pig red cell hexokinase (McManus, 1967), experiments were designed to determine the percentage of inhibition by the intermediate at its in vivo concentration. Figure 3 shows that at the concentration of 5.0 mM DPG, hexokinase is inhibited by a factor of
64
EARL DIXON, JR.
Acknowlegement--This investigation was supported by General Research Support Grant No. 42510, National Institutes of Health, U.S. Public Health Service. REFERENCES
BARTLETTG. R. (1970) Patterns of phosphate compounds in red cells of man and animals. In Red Cell Metabolism and Functions (Edited by BREWER G. J.), pp. 245256. Plenum Press, New York. BREWER G. J. (1966) Erythrocyte metabolism and function: hexokinase inhibition by 2,3-diphosphoglycerate and interaction with ATP and Mg ~+. Biochim. biophys. Acta 192, 157-161. CHAPMAN R. G., HENNESSEYM. A., WALTERSDOLPHA. M., HUENNEKENN F. N., & GABRIO B. W. (1962) Erythrocyte metabolism--V. Levels of glycolytic enzymes and regulation of gylcolysis. J. Clin. Invest. 41, 1249-1256. D~XON E., MCMANUS T. J. & TOSTESOND. C. (1966) Glycolytic enzymes in pig and sheep red cells (an abstract) Fedn Proc. Fedn Am. Socs exp. Biol. 25, 641. GERBER G., SCHULTZE J. • RAPOPORT S. M. (1970) Occurrence and function of high K m hexokinase in immature red blood cells. Fur. J. Biochem. 17, 445-449. GROSSBAROL. & SCr~IMKER. T. (1966) Multiple forms of rat tissue hexokinase. J. biol. Chem. 241, 3546-3560. KIM H. D. & MCMANUS T. J. (1971) Studies on the energy metabolism of pig red cells--1. The limiting role of membrane permeability in glycolysis. Biochim. biophys. Acta 230, I-11. MCMANUS T. J. (1967) Comparative biology of red cells. Fedn. Proc. Fedn Am. Sots exp. Biol. 26, 1821-1826. MCMANUS T. J. & KIM H. D. (1968) Energy metabolism in the pig red cell. In Stroffwechsel und Membrane Permeability yon Erythrocyten und Thrombocyten (Edited by DEUTSCHE., GERLACH E. & MOSER K.), pp. 43-49. George Thieme Verlag, Stuttgart.
PARRY M. & WALKER D. G. (1966) Purification and properties of adenosine-5'-triphosphate-D-glucose-6phosphotransferase from rat liver. Biochem. J. 99, 266-274. PONCE J., ROTH S. & HARKNESS D. R. (1971) Kinetic studies on the inhibition of glycolytic kinases of human erythrocytes by 2,3-diphosphoglyceric acid. Biochim. biophys. Acta 250, 63-74. RAPOPORT S. (1966) The regulation of glycolysis in mammalian erythrocytes. In Essays in Biochemistry (Edited by CAMPBELLP. N. & GREENVILLEG. D.), Vol. 4, 69-103. Academic Press, London. ROSE I. A. & O'CONNELLE. L. (1964) The role of glucose6-phosphate in the regulation of glucose metabolism in human erythrocytes. J. biol. Chem. 239, 12-17. Rose I. A., WARMSJ. V. B. & O'CoNNELL E. L. (1964) Role of inorganic phosphate in stimulating the glucose utilization of human red blood cells. Biochem. biophys. Res. Commun. 15, 33-37. SALAS J., SALAS M., VINVELA E. & SOLS A. 0965) Glucokinase of rabbit livers. J. biol. Chem. 240, 1014-1020. SRIVASTAVAS. K. & BEUTLER E. (1972) The effects of normal red cell constituents on the activities of red cell enzymes. Archs Biochem. Biophys. 146, 249-255. DE VERDIERC. H. & GARBYL. (1965) Glucose metabolism in normal erythrocytes II. Factors influencing the hexokinase step. Scand. J. Hematol. 2, 305-317. WIDDAS W. F. (1955) Hexose permeability of foetal erythrocytes. J. Physiol., Lond. 127, 318-327. Key word index--Erythrocyte metabolism; red cell hexokinase; isoenzymes of hexokinase; pig; Sus scrofa.
KINETIC STUDIES ON THE ERYTHROCYTE HEXOKINASE OF THE DOMESTIC PIG, SUS SCROFA EARL DIXON, JR. Department of Physiology and Pharmacology, School of Veterinary Medicine, Tuskegee Institute, Tuskegee Institute, Alabama 36088, U.S.A. (Received 4 December 1973)
Abstract--1. Pig erythrocyte hexokinase (ATP: D-hexose-6-phosphotransferase E.C. 2.7.1.1) was isolated in a partially purified preparation by Sephadex G-100 gel filtration. 2. In the mature pig (> 1 year of age), a high affinity component exhibits a maximum velocity at 0.2--0.4 mM glucose. This component seems to be inhibited by excessive glucose concentrations (> 0.5 raM). 3. The young, adult pig (3--6 months of age) possesses a hexokinase which exhibits a maximum velocity at 1.0 g M glucose. 4. 2,3-Diphosphoglycerate inhibits the enzyme at low concentrations of M g s+ (0.05 raM), but the inhibition is relieved as the concentration of M g ~+ is increased beyond I-0 raM. 5. These findings indicate that the mature erythrocyte of the pig may possess unique biochemical properties that have not been observed in other mammalian red blood cells.
INTRODUCTION
pig, studies have been designed to determine the kinetic properties of hexokinase in respect to substrate, co-factors and effector concentrations. The present study was designed to determine the relationship between the enzyme and its substrate and to determine the influence o f 2,3-diphosphoglycerate (DPG) on the activity of the enzyme. The results obtained from a partially purified enzyme preparation indicate that the pig erythrocyte possesses a hexokinase that exhibit unique properties in respect to substrate requirements. That is, the data presented suggest that the pig red cell may possess two isoenzymes of hexokinase. Furthermore, the relationship between the enzyme and substrate is related to the age of the animal. D P G was found to be an inhibitor of the enzyme at the in vivo concentrations of the glycolytic intermediate. The implications of these findings arc discussed as they relate to glycolyticcontrol in the mature erythrocyte of the pig.
ERYTHROCYTE hexokinases (ATP: D-hexose-6-phosphotransferase E.C. 2.7.1.1) have not been extensively studied at the comparative level. While there have been many studies on the human, rabbit and rat red cell enzyme, there has been an absence of literature on the kinetic properties of hexokinases isolated from the red cells of other mammals (Rose & O'Connell, 1964; Rose et al., 1964; de Verdier & Garby, 1964; Rapoport, 1966). Since hexokinases represent one of the key enzymes in glucose utilization, information obtained from kinetic studies on this enzyme can be utilized to evaluate metabolic control of the intact erythrocyte (Rapoport, 1966). The mature erythrocyte of pig possesses one of the slowest glycolytic rates observed in any mammal (McManus, 1976; Kim and McManus, 1972). Studies designed to estimate the glycolytic capacity by quantitating the activities of the glycolytic enzymes in stroma-free hemolysates have revealed that these cells possess an adequate enzymatic apparatus that would allow a more rapid glycolytic rate (Dixon et al., 1966). However, hexokinase was found to possess the lowest specific activity of any enzyme observed. Other studies have indicated that glucose permeability was the rate-limiting factor of glucose utilization in the pig erythrocyte (Widdas, 1955). Recent evidence suggests that glucose permeability as well as the ability of these cells to utilize ribose in vitro was related to the age of the animal (McManns & Kim, 1968). In an attempt to elucidate the mechanisms that regulate glycolysis in the mature erythrocyte of the
MATERIALS AND METHODS Chemicals
ATP (disodium salt from equine muscle), NADP and G6PD (NADP oxidoreductase E.C. 1.1.1.49) were purchased from the Boehringer-Mannheim Chemical Company. 2,3-Diphosphoglycerate (pentacyclohexylammonium salt),D-glucose, Scphadex G-100 and MgCl~ were purchased from the Sigma Chemical Company. Partial purification of the pig red-cell hexokinase
Pig blood was collected from the albatroit in 5 units of heparin per ml of whole blood and processed within 6 hr of collection. The red cells were separated from the 61
62
EARL DIXON, JR.
plasma by centrifugation at 5000rev/min for 10min (International Model No. HT). The plasma and buffy coat were removed by suction. The packed red cells were resuspended in an adequate volume of 0.17 M NaCI and spun at 5000 rev/min for 10 min. After the third wash, the white cells had been reduced to a small percentage of the total cell population. The cells were mixed with an equal volume of cold distilled H~O and Saponin was added to equal a final concentration of 0.1%. This suspension was allowed to stand for 30 rain with intermittent mixing. The stroma was removed by centrifugation at 10,000rev/min for 20min. The supernate fluid was separated from the stroma pellet for further enzyme processing. Ten to fifteen milliliters of hemolysate was added to a Sephadex G-100 column (30 x 400 mm) previously equilibrated with 0"02 M Tris-Cl buffer at pH 7"4. The gel was prepared according to the instructions supplied by the Sigma Chemical Company. The hemolysate was allowed to flow through the column at a rate of t "0 ml per 2 rain and the eluant was collected in 4-ml fractions. The eluant tubes exhibiting hexokinase activity were subsequently used to determine the kinetic properties of the enzyme.
Assay o f hexokinase activity Hexokinase was assayed at 37°C by a modification of previously reported methods (Chapman et al., 1962; Dixon et al., 1966). The standard assay procedure (total volume, 1'0 ml) contained 200 mM Tris-C1 buffer (pH 7-40), 4 mM MgClz, 1.0 mM ATP, 0'1 mM NADP, 6-2 mM o-glucose, G6PD and a sufficient volume of the enzyme material. Alterations in the concentrations of glucose were made as indicated. Reactions were followed in a Beckman Du spectrophotometer equipped with a Gilford Model 2000 Multiple sampling accessory and a Haake constant temperature circulator. The enzymatic activity was determined by utilizing a Gilford Model 6040 recorder. Units of hexokinase were expressed as /~M N A D P H formed per min using the extinction coefficient for the compound at 340 nm. All assays were performed utilizing co-factor and substrate concentrations that exceeded the maximum velocity for the enzyme under the conditions of the assay. The commercial preparation of G6PD was free of hexokinase at the dilution of the enzyme used in these experiments. Hemoglobin concentrations were determined using the cyanmethemoglobin method. The reaction pH was ascertained using a Radiometer pH meter (Model 26) equipped with a micro pH unit (Type E5021a). RESULTS Fig. 1 represents a velocity vs substrate concentration plot of the hexokinase isolated f r o m the mature erythrocytes of an adult pig (approximately 1 year of age). The m a x i m u m velocity of the enzyme occurred at 0-2-0.4 m M of glucose. Additional increases in substrate concentration caused a decrease in enzyme activity. At glucose concentrations in excess of 1'0 m M , there was a plateau in the velocity of the enzyme. Some preparations exhibited a secondary increase in velocity at concentrations o f glucose exceeding 1.0 m M . These findings were consistent regardless of the species or the source in which the blood samples were obtained.
hexokinase
40
~ 3.0 0
2'0 ::L >
I~0
I.'0 Glucose,
2 i0 mM
3~ 0
a
Fig. 1. Plot of initial reaction velocity (v) vs. glucose concentration. The concentrations of ATP and Mg ~+ were maintained constant at 1.0 and 5.0 mM respectively. This enzyme preparation was obtained from the erythrocytes of an adult pig (> 1 year). The data in Fig. 2 demonstrate the velocitysubstrate profile obtained on a typical hexokinase preparation isolated from the red cells o f a young adult pig (3-6 months). It can be observed that there is a shift of the high velocity c o m p o n e n t to the left on the substrate axis. The high velocity component occurs at approximately 0.001 m M of
~.5
Pig
hexokinase
o. I.O
.i ::L >
05
0
2
4
Glucose,
mM
6
Fig. 2. Plot of initial velocity (v) vs. glucose concentration. The enzyme preparation was obtained from a young adult pig that was approximately 6 months of age. glucose. There is a decrease in activity as the substrate concentration approaches 1.0 m M . Further elevations in substrate concentration causes a secondary increase in the velocity of the enzyme. T h o u g h the m a x i m u m velocities of the enzyme occur at different concentrations of substrate in the young adult and adult pig, the relative activities of the two preparations are approximately equal. T h a t is, the data would suggest that the enzyme obtained from the red cells o f the young and the mature pig exhibit
Kinetics of pig red cell hexokinase
63
The intracellular concentrations of glucose in the pig red cell has been reported to be below detection by routine chemical procedures (Kim & McManus, 1971). The data presented here would indicate that the low substrate component would be active at low glucose concentrations. Since the permeability of the adult pig red cell membrane is low, assigning a physiological role to the high substrate enzyme component at this time would be futile. The data also indicate that the young, adult pig I00 (2-3 months) has a low substrate enzyme component ~, Pig hexok i n a s e that exhibits a maximum velocity at 0.001 mM. Since there is a paucity of data on the biochemical +',>_ 7:5 and physiological activity of the pig, the period of transition cannot be correlated with other mechan-)isms operating concurrently. The inability to consistently obtain a secondary increase in velocity N 5o in the adult pig's hexokinase may be due to our method of isolation. The enzyme was not protected by glucose or K + during the isolation procedures 25 which may have resulted in inactivation of this particular enzyme component (Salas et al., 1965; Grossbard & Schimke, 1966; Parry & Walker, 0 I"0 a*'O 31"0 41.0 5"0 1966). (t)PG) x io ~ Experiments are in progress to isolate these enzyme components and determine the kinetic and Fig. 3. Plot of initial reaction velocity (v) vs DPG con centration. The Mg~+ and glucose concentrations in these electrophoretic properties of each enzyme respecexperiments were maintained at 0.05 and 0"33 mM tively. respectively. D P G concentrations in the pig erythrocyte are approximately 5-8 mM/1. of RBC (Bartlett, 1970). approximately 70 per cent. The inhibition of hexo- The data presented here indicated that DPG inhibits kinase was found to be related to the concentration the enzyme at the in rive concentration of the of Mg 2+. In these experiments, 0.5 and 0.2 mM of intermediate. These results are consistent with the Mg ~+ and glucose respectively, were utilized. previously unpublished data mentioned elsewhere Studies are in progress to determine the specific (McManus, 1967). Recently, other reports have relationship between ATP and Mg 2+ in respect to the been published on the human red cell hexokinase that supports these data (Brewer, 1969; Ponce et al., inhibition of pig red cell hexokinase by DPG. 1971; Srivastava & Beutler, 1972). These investigators found that the inhibition of hexokinase was DISCUSSION dependent on the concentrations of Mg ~+ and ATP. The data presented here suggest that the partially The large concentration of D P G found in the pig purified hexokinase isolated from the mature red cell would suggest that this compound would be erythrocyte of the pig possess kinetic propcrti¢~ in another mechanism for scaling down the activity of respect to substrate requirements that are markedly glucose utilization by this cell. different when compared to other mammals. That is, These findings would indicate that pig red cell the enzyme exhibits both low and high substrate hexokinase does not possess the type of biochemical components of hexokinase activity. It was reported properties that have been previously reported for that rabbit reticulocytes possessed two hexokinase other mammalian red cells. Furthermore, the components but the low affinity component was not enzyme exhibits a substrate-velocity profile that observed in the mature erythrocyte of the rabbit varies as a function of age. Since these cells were (Gerber et al., 1970). Other reports have not obtained from animals that are 3-6 months of age, indicated this type of finding in the red cell of the rat these findings cannot be attributed to the presence or human (de Verdier & Garby, 1965; Rapoport, of nee-natal red cells in the circulation. These data 1966; Rose et al., 1969). In these studies we have further suggest the importance of comparative attempted to identify reticulocytes in our pack cells studies in evaluating metabolic control processes that were utilized to prepare the stroma-free hemoly- in the mature red cell at the enzymatic level. The sate. Significant numbers of reticulocytes could not implications of these data regarding the absence be demonstrated in these red blood cells ( < 0.5 per of glucose utilization by the intact red cell in vitro cent). are still unknown. similar specific activities of hexokinase in spite of the apparent variations in glucose affinity. Since it was earlier observed that D P G inhibited pig red cell hexokinase (McManus, 1967), experiments were designed to determine the percentage of inhibition by the intermediate at its in vivo concentration. Figure 3 shows that at the concentration of 5.0 mM DPG, hexokinase is inhibited by a factor of
64
EARL DIXON, JR.
Acknowlegement--This investigation was supported by General Research Support Grant No. 42510, National Institutes of Health, U.S. Public Health Service. REFERENCES
BARTLETTG. R. (1970) Patterns of phosphate compounds in red cells of man and animals. In Red Cell Metabolism and Functions (Edited by BREWER G. J.), pp. 245256. Plenum Press, New York. BREWER G. J. (1966) Erythrocyte metabolism and function: hexokinase inhibition by 2,3-diphosphoglycerate and interaction with ATP and Mg ~+. Biochim. biophys. Acta 192, 157-161. CHAPMAN R. G., HENNESSEYM. A., WALTERSDOLPHA. M., HUENNEKENN F. N., & GABRIO B. W. (1962) Erythrocyte metabolism--V. Levels of glycolytic enzymes and regulation of gylcolysis. J. Clin. Invest. 41, 1249-1256. D~XON E., MCMANUS T. J. & TOSTESOND. C. (1966) Glycolytic enzymes in pig and sheep red cells (an abstract) Fedn Proc. Fedn Am. Socs exp. Biol. 25, 641. GERBER G., SCHULTZE J. • RAPOPORT S. M. (1970) Occurrence and function of high K m hexokinase in immature red blood cells. Fur. J. Biochem. 17, 445-449. GROSSBAROL. & SCr~IMKER. T. (1966) Multiple forms of rat tissue hexokinase. J. biol. Chem. 241, 3546-3560. KIM H. D. & MCMANUS T. J. (1971) Studies on the energy metabolism of pig red cells--1. The limiting role of membrane permeability in glycolysis. Biochim. biophys. Acta 230, I-11. MCMANUS T. J. (1967) Comparative biology of red cells. Fedn. Proc. Fedn Am. Sots exp. Biol. 26, 1821-1826. MCMANUS T. J. & KIM H. D. (1968) Energy metabolism in the pig red cell. In Stroffwechsel und Membrane Permeability yon Erythrocyten und Thrombocyten (Edited by DEUTSCHE., GERLACH E. & MOSER K.), pp. 43-49. George Thieme Verlag, Stuttgart.
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