Purification and physical properties of hexokinase from human erythrocytes

Biochimica et Biophysica Acta 873 (1986) 335-339 Elsevier

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BBA 32544

Purification and physical properties of hexokinase from human erythrocytes Athanassios A. Haritos * and Michael A. Rosemeyer Department of Biochemistry, University College London, Gower Street, London WCIE 6BT (U.K.) (Received 10 July 1986)

Key words: Hexokinase purification; Reversible dimerization; Glucose 6-phosphate; Enzyme-product interaction; (Human erythrocytes)

A bulk purification is described for hexokinase (ATlP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from human erythrocytes. Following a l l0000-fold purification from 40 litres of blood, 5 mg protein with a specific activity of 22 units/mg were obtained. On application of various separation techniques, the enzyme activity co-migrated with the main protein component. The physical properties, such as the relative molecular mass of 108000 and sedimentation coefficient of 5.5 S, are similar to those of the enzyme from human heart. In particular, there is a correspondence in the conformational response to glucose 6-phosphate as shown by an association of the enzyme promoted by this metabolite.

Introduction

Materials and Methods

Owing to the low levels of hexokinase in erythrocytes, it is extremely difficult to purify enough material to study its physical properties. Nevertheless, the enzyme is of fundamental importance to this tissue that is totally dependent on the glycolytic flux, which is limited by the level of hexokinase [1]. Despite the low levels of hexokinase, the erythrocyte has a notably long life span of 120 days [2], which implies that the functionally important properties of the enzyme are retained over this period. In this work, we report a bulk purification of hexokinase from human erythrocytes, and show that the enzyme in aged red cells retains physical properties similar to those found for hexokinase from human heart [3].

Separation techniques

* Present address: Zoological Laboratory, Faculty of Science, University of Athens, GR 157 84 Athens, Greece. Correspondence address: Dr. M.A. Rosemeyer, Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K.

Electrophoresis and ultracentrifugation were carried out as before [3]. Relative molecular masses were estimated by gel-filtration on Sephadex G-200 as given for the purification below, using bovine y-globulin, lactate dehydrogenase, bovine serum albumin and ovalbumin as protein markers [4.5].

Assays Protein concentration was estimated on the assumption that a 1 m g / m l solution has an absorbance of 1 at 280 nm [6]. At the final purification step, it was assumed that the absorbance would be 0.6, the same as the value for hexokinase from human heart [3]. Enzyme activity was measured in 130 mM TesK O H buffer, pH 7.2 0.05 I, with 75 mM KC1, 5 mM glucose, 5 mM ATP, 10 mM MgC12, 0.6 mM NADP and 0.025 units/ml glucose-6-phosphate dehydrogenase. A unit of enzyme activity is taken as the amount that phosphorylates 1/~mol glucose per min at 25 ° C.

0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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Purification of hexokinase from human erythrocytes The purification procedure required large volumes (40 1) of human blood, which had been stored at 0-4°C for 3 weeks in acid-citrate-dextrose to prevent clotting. Residual plasma was removed, and the cells were washed with isotonic saline. Following previously published procedures [7-9]: (1) half the total volume of erythrocytes was lysed and the stroma removed by centrifugation; (2) the haemolysate was diluted and applied to two 24 cm Buchner funnels, each with a 2 litre bed of DEAE-Sephadex equilibrated with phosphate buffer, pH 6.5 0.05 I, and eluted with acetate buffer, pH 5.8 0.05 I, containing 0.35 M KC1; the eluate was then (3) diluted and applied to a single 24 cm Buchner funnel containing CMSephadex equilibrated with acetate buffer, pH 5.8 0.012 I, containing 0.088 M KC1, and eluted with phosphate buffer, pH 7.0 0.1 I, and 0.3 M KC1. These steps were repeated on the second half of the blood, and the eluates were combined. Subsequent steps were carried out at 4 ° C, and the buffers routinely contained 10 mM glucose, 1 mM EDTA and 2-mercaptoethanol. The next three steps reduced the volume following the procedure used previously [3] and involved: (4) salt fractionation with ammonium sulphate at 45-60% saturation; (5) anion-exchange on a 30 × 2.7 cm column of DEAE-Sephadex in 0.1 I phosphate buffer (pH 7.0) with a 500 ml linear gradient of 0-0.3 M KCI; and (6) cation-exchange on a 30 × 2.7 cm column of P-11 phosphocellulose in 0.025 I acetate buffer (pH 5.8) with 0.125 M KCI, developed with a 500 ml gradient of 0-0.4 M KC1. The three final steps were: (7) gel-filtration on a 150 × 2 cm column of Sephadex G-200, equilibrated with phosphate buffer, pH 7.0 0.1 I, containing 10 mM glucose, 1 mM EDTA and 0.1% 2-mercaptoethanol; (8)salt fractionation with ammonium sulphate at 40-55% saturation; and, after dialysis to remove salt and residual glucose, (9) affinity chromatography. This last step was carried out on an 18 × 2.8 cm column of the affinity matrix, made by attaching glucosamine to Sepharose 4B through an aminocaproate spacer [3]. The column was equilibrated with triethanolamine buffer (pH 7.0, 20 mM and without glucose), eluted with the addition of 50 mM KC1 and of 200 mM glucose, followed by concentra-

tion of the enzyme by vacuum dialysis against 0.1 I phosphate buffer (pH 7.0) and storage at 4°C under nitrogen [3].

Heart hexokinase The enzyme was purified from human heart as reported previously [3]. Results and Discussion

Purification The purification procedure contained nine steps and was carried out in 3 weeks. From 40 litres blood, 5 mg enzyme were obtained, representing a 12% yield and a purification of 110000 (Table I). The yield was low, but provided sufficient material for further study of physical properties. The purification combines previous strategies for the isolation of erythrocyte enzymes [7-9] with steps developed specifically for the enzyme from heart [3]. Thus, the initial bulk steps using anionand cation-exchangers reduced the total protein from 4 kg to 11 g, removed the haemoglobin, and achieved a 250-fold purification. The ion-exchanges were repeated on a small scale, followed by gel-filtration and salt fractionation. Finally, affinity chromatography was used as previously for the specific isolation of hexokinase, using glu: cosamine attached to Sepharose 4B [3]. This step was very effective and gave a final 6-fold purification. Two previous purifications of hexokinase from human erythrocytes have been reported [1,10], of which the more successful used about 2 litres blood to obtain 0.4 mg enzyme with a specific activity of 14 units/mg [10]. However, this activity was measured at 0.06 I and a temperature of 37 ° C, and this combination would be expected to boost the apparent specific activity by a factor of 2.8 compared to the present conditions. In this connection, the assay conditions used in the present purification were not optimal, being those used by Gerber et al. [1] and approximating physiological ionic conditions. While keeping the temperature to 25°C, the activity of the present preparations could be doubled by using the more favourable assay conditions of Tes-KOH or TrisHC1 buffers at pH 8 and 0.05 I. This is anticipated from the increase in activity with pH shown by

337 TABLE I RESULTS OF PURIFICATION OF HEXOKINASE FROM HUMAN ERYTHROCYTES Step

Volume (ml)

Protein a (mg)

Activity (units)

Spec. Act. (units/rag)

Purification (-fold)

Yield (%)

1. Haemolysate 2. DEAE-Sephadex (Buchner) 3. CM-Sephadex (Buchner) 4. Ammonium sulphate 45-60% satd. 5. DEAE-Sephadex (column) 6. P-11 phosphocellulose (column) 7. G-200 Sephadex 8. Ammonium sulphate 40-55% satd. 9. Affinity chromatography

23000 14000 8 000 80 130 215 97 2.7 285

4.0-106 _b 11.103 2.3.103 580 150 69 46 4.8

860 770 580 336 355 270 250 165 106

0.0002 0.052 0.15 0.61 1.8 3.6 3.6 22.2

1 250 714 2900 8 500 17 300 17 000 106000

100 90 68 39 41 31 29 19 12

During the purification an absorbance of 1 at 280 nm was assumed for a 1 mg/ml solution, except for the final step when an absorbance of 0.6 was used on the basis of the-value obtained for the heart enzyme [3]. b Protein concentration was not obtained owing to the high absorbance at 260 nm, indicating the presence of endogenous nucleotides and other non-protein contaminants.

a

the h e a r t e n z y m e t o g e t h e r with that arising f r o m the lower ionic s t r e n g t h [3].

Physical properties A f t e r e l e c t r o p h o r e s i s o n s t a r c h gel in TrisE D T A - b o r a t e at p H 8.6 [11], a single b a n d s t a i n e d for activity, that h a d m o v e d t o w a r d s the a n o d e w i t h a m o b i l i t y slightly less t h a n that of a c a r b o n m o n o x y h a e m o g l o b i n marker. I n p h o s p h a t e b u f f e r at p H 6 the e n z y m e activity r e m a i n e d at the origin, i n d i c a t i n g that this is the isoelectric p H for the enzyme. T h e result differs f r o m the p H of 4.7 o b t a i n e d b y isoelectric focussing of the m a i n p r o tein c o m p o n e n t in a p r e v i o u s p r e p a r a t i o n of the h u m a n e r y t h r o c y t e e n z y m e [10]. It is close to values for the e n z y m e f r o m p i g h e a r t [12] a n d f r o m rat b r a i n [13]. E l e c t r o p h o r e s i s of the p u r i f i e d e n z y m e on p o l y a c r y l a m i d e gels gave essentially one c o m p o n e n t w i t h a trace o f a m i n o r c o m p o n e n t (Fig. 1). E l e c t r o p h o r e s i s o n p o l y a c r y l a m i d e in the presence of S D S s h o w e d one m a i n c o m p o n e n t a n d several m i n o r species t h a t c o u l d have arisen f r o m p r o t e o l y s i s (Fig. 2). This c o n t r a s t s with the p r e p a r a t i o n f r o m h e a r t which shows two m a i n c o m p o n e n t s that are similar in size, b u t shows less o f the s m a l l e r c o m p o n e n t s . T h e m a i n e r y t h r o c y t e c o m p o n e n t is in a l i g n m e n t w i t h the slower b a n d seen f r o m the h e a r t p r e p a r a t i o n a n d which, on c o m p a r i s o n with m a r k e r s , c o r r e s p o n d e d to a rela-

Fig. 1. Spectrophotometric trace after electrophoresis on polyacrylamide. The polyacrylamide concentration was 7.5%, and 50 /~g hexokinase purified from erythrocytes were applied. O represents the origin, and M the position of the bromophenol blue marker dye after electrophoresis.

338

Fig. 3. The effect of glucose 6-phosphate on the sedimentation pattern of human hexokinase from erythroeytes (A, B) and

from heart (C, D). The controls were in phosphate buffer, pH 7.0 0.1 I, with 10 mM glucose (A, C). The solutions containing 1 mM glucose 6-phosphate were in Tris-HCI, pH 8.0 0.1 I, with KC1 at 0.1 M (B) or 0.2 M (D). From absorbance measurements, the protein concentration was approximately 3 mg/ml, and the schlieren plate angles were 45* (A and D) or 40 ° (B and C). All solutions contained 1 mM EDTA and 0.1% 2-mercaptoethanol, and the temperature was 10°C. Photographs were taken 90 min after reaching a speed of 59780 rev/min. Fig. 2. Electrophoresis on polyacrylamide gel in the presence of SDS of human hexokinase purified from erythrocytes (A) and from heart (B). Migration was from top to bottom.

tive molecular mass of 124000 [3]. Gel-filtration gave a value of 108000 for the relative molecular mass of the enzyme. A similar value of 106000 was obtained for the enzyme from heart [3]. Previous values for the h u m a n erythrocyte enzyme were also measured by gelfiltration and ranged from 104000 [14] to 132000 [10]. Rijksen and Staal [10] suggested that the high value for the relative molecular mass and also the low value for the isoelectric point for their preparation were due to bound lipid. Sedimentation of purified hexokinase showed one main component with a trace of a faster sedimenting species (Fig. 3A). The main component had an s20,w of 5.45 S, which agrees with the value of 5.5 S found for the enzyme from h u m a n heart [3].

The traces of impurities seen on electrophoresis and sedimentation confirm the difficulties in achieving a preparation of hexokinase of the highest purity from erythrocytes. However, it is possible to obtain information on the enzyme while it remains the major species present and the proportion of contaminants is low. The type I isoenzyme of hexokinase is known to be homogeneous in various tissues [15], and previous investigations have shown the predominance of this isoenzyme in h u m a n erythrocytes [14,16]. Although it is the main isoenzyme of erythrocytes, it has been separated into several subtypes by ion-exchange chromatography [17,18], etectrophoresis [18] and isoelectric focussing [17]. The heterogeneity, which depends on the age of the cell [17-20], is related mainly to differences in charge, while the size and kinetic properties of the subtypes do not differ significantly [17,18]. This background of post-

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translational modification underlies the difficulty in isolating the enzyme in quantity. However, the present work indicates that a single, active component persists, despite the age of the cells and the time taken for the preparation. Although the specific activity is relatively low, the properties of the erythrocyte preparation correspond to those seen for the enzyme isolated from human heart [3]. Hence the low specific activity could arise from the presence of some inactivated hexokinase. Type I mammalian hexokinases associate in the presence of glucose 6-phosphate [3,21,22] and this property supports the proposal of allosteric control of the enzyme by this metabolite [23]. The behaviour of human erythrocyte hexokinase is similar to the enzyme from heart, examined under equivalent experimental conditions (Fig. 3). The phenomenon is dearly present in both cases, but the extent of dimerization was less for the erythrocyte preparation compared to that from heart, which suggests a correlation between the activity of hexokinase and its conformational response to glucose 6-phosphate. Estimates of the dissociation constant of the enzyme-glucose 6-phosphate complex range from 10 to 70/xM [24,1], and the concentration of this metabolite in the red cell was within this range at 40 #M [24]. A concentration of 1 mM was used in the present experiments to provide an excess of the metabolite. The ensuing conformational change is revealed by an accompanying concentration-dependent dimerization. In tissues such as heart the control of hexokinase activity also involves interaction of the enzyme with mitochondria [23,25]. However, as the mature erythrocyte has no mitochondria, this consideration does not apply. It would appear that the enzyme retains its characteristic allosteric property despite the removal of the matrix on which this type of control could be amplified.

Acknowledgements We are indebted to Dr. T.E. Cleghorn at the North London Transfusion Centre and Professor E.R. Huehns at University College School of Medicine for generous supplies of outdated red cell residues. A.A.H. was supported by fellowships

from N.A.T.O. and E.M.B.O. during the conduct of this research.

References 1 Gerber, G., Preissler, H., Heinrich, R. and Rapoport, S.M. (1974) Eur. J. Biochem. 45, 39-52 2 Berlin, N.I. (1964) in The Red Blood Cell (Bishop, C. and Surgenor, D.M., eds.), pp. 423-450, Academic Press, New York 3 Haritos, A.A. and Rosemeyer, M.A. (1985) Biochim. Biophys. Acta 830, 113-119 4 Determann, H. and Mattner, U. (1969) FEBS Lett. 2, 163-166 5 Andrews, P. (1965) Biochem. J. 96, 595-606 6 Warburg, O. and Christian, W. (1941) Biochem. Z. 310, 384-421 7 Cohen, P. and Rosemeyer, M.A. (1969) Eur. J. Biochem. 8, 1-7 8 Pearse, B.M.F. and Rosemeyer, M.A. (1974) Eur. J. Biochem. 42, 213-223 9 Worthington, D.J. and Rosemeyer, M.A. (1974) Eur. J. Biochem. 48, 167-177 10 Rijksen, G. and Staal, G.E.J. (1976) Biochim. Biophys. Acta 445, 330-341 11 Rosemeyer, M.A. and Huehns, E.R. (1967) J. Mol. Biol. 25, 253-273 12 Easterby, J.S. and O'Brien, M.J. (1973) Eur. J. Biochem. 38, 201-211 13 Chou, A.C. and Wilson, J.E. (1972) Arch. Biochem. Biophys. 151, 48-55 14 Rogers, P.A., Fisher, R.A. and Harris, H. (1975) Biochem. Gen. 13, 857-866 15 Rijksen, G., Staal, G.E.J., Beks, P.J., Streefkerk, M. and Akkerman, J.W.N. (1982) Biochim. Biophys. Acta 719, 431-437 16 Neumann, S., Falkenberg, F. and Pfleiderer, G. (1974) Biochim. Biophys. Acta 334, 328-342 17 Stocchi, V., Magnani, M., Canestrari, F., Dacha, M. and Fornaini, G. (1982) J. Biol. Chem. 257, 2357-2364 18 Rijksen, G., Jansen, G., Kraaijenhagen, R.J., van der Vlist, M.J.J., Vlug, A.M.C. and Staal, G.E.J. (1981) Biochim. Biophys. Acta 659, 292-301 19 Rogers, P.A., Fisher, R.A. and Harris, H. (1975) Clin. Chim. Acta 65, 291-298 20 Rijksen, G., Schoop, I. and Staal, G.E.J. (1977) Clin. Chim. Acta 80, 193-202 21 Chakrabarti, U. and Kenkare, U.W. (1974) J. Biol. Chem. 249, 5984-5988 22 Easterby, J.S. (1975) Eur. J. Biochem. 58, 231-235 23 Wilson, J.E. (1980) in Current Topics in Cellular Regulation (Horecker, B.L. and Stadtman, E.R., eds.), Vol. 16, pp. 2-54, Academic Press, New York 24 Rijksen, G. and Staal, G.E.J. (1977) Biochim. Biophys Acta 485, 75-86 25 Bessman, S.P. and Geiger, P.J. (1980) in Current Topics in Cellular Regulation (Horecker, B.L. and Stadtman, E.R., eds.), Vol. 16, pp. 55-86, Academic Press, New York