TIIE JOURNAL OP ~SIOLOGICAL CIIE~IISTRY Vol. 238, No. 7, July 1963

I’rinled in U.S. A.

Human Erythrocyte Glucose 6-Phosphate Dehydrogenase

I. ISOLATIOX ASD PROPERTIES OF THE EiYZl-ME*

ALBERT E. CHuxGf' AND ROBERT G. LANGDON

From the Department of Physiological Chemistry, The Johns Hopkins School of Medicine, Baltimore 5, Naryland

(Received for publication, October 23, 1962)

Glucose 6-phosphate dehydrogenase was first described b) Warburg and Christian (1, 2). Since then the enzyme has been purified to varying degrees from yeast (3-6), Aspergillus niger (7), Neurospora crassa (8)) Escherichia coli (9), human erythro- cyte (lo-12), mammary gland (13), adrenal gland (14), and liver (15).

This enzyme has aroused considerable interest because of its central position in the pentose phosphate pathway (16), its in- volvement in various hemolytic disorders (17), the variation of its activity under different hormonal and nutritional states (18, 19), and its potential as a regulator for the availability of the reduced nicotinamide adenine dinuclrotide phosphate required for various biosynthetic processes.

In this communication, the preparation of a highly purified enzyme from human erythrocytes is described. Studies have been performed to determine some of its physical constants and its NHZ-terminal amino acids. In the accompanying communi- cation (20), studies on its reversible inactivation, the complex relationship with its cocnzyme, and the effect of various reagents on its activity will be described.

EXPERIMENTAL PROCI<I)URES AND MATERIALS

.Vaterials-TPN was purchased from Pabst Laboratories. Tris (Sigma 121) and glucose B-phosphate dipotassium salt were obtained from the Sigma Chemical Company. Ethylenedia- minetetraacetic acid was obtained from the J. T. Baker Chemical Company. Sephades G--25 was purchased from Pharmacia, Uppsala. Selectacel DE.4E-cellulosr and carborymethyl cellu- lose were obtained from t.he 13rown Company and the Carl Schleicher and Schuell Company. Calcium phosphate gel was prepared according to the method of Kcilin and Hartree (21). Bovine plasma albumin was a product of Armour Research Lab- oratories. The dinitrophenyl derivatives of the amino acids, alanine, asparagine, aspartic acid, cystinr, glutamic acid, gluta- mine, glycine, histidine, isoleucine, leucine, lysine, mrthionine, phenylalaninc, prolinc, serine, tryptophan, thrconine, tyrosine, and valine, were obtained from Mann Research Laboratories. C”-Dinitrofluorobenzene, 1.23 mc per mmole, was supplied by Nichem, Inc., Bethesda, Maryland. Hydrolyzed starch was obtained from Connaught Laboratories, and Nitro-BT and

* This work was supported by a grant (H-1713 (03)) from the United States Public Health Service. The data presented were taken from a dissertation submitted by A. E. Chung to the Faculty of Philosophy of the Johns Hopkins University in partial fulfill- ment of the reuuirements for the degree of I>octor of Philosophy.

t Fellow of t’he Inter-University Council for Higher Ia:ducation Overseas. Present address, Ijepartment of Chemistry, Harvard University, Cambridge, Massachusetts.

phenazine methosulfate were obtained from the Dajac Labora- tories, Monomer-Polymer, Inc., Leominster, Massachusetts.

Human blood was obtained from the Johns Hopkins Hospital Blood Bank and the Baltimore Division of the Red Cross 1Zlood Bank. The blood had been stored at 4” for periods of 3 to 5 weeks. Clotting was prevented by mixing 4 parts of blood with 1 part of a solution containing 1.32 g of sodium citrate, 0.44 g of citric acid, and 1.47 g of dextrose per 100 ml.

Jfethods-Ion exchange cellulose for chromatography was pre- pared as previously described (22). Protein was determined by absorption at 260 to 280 rnp (23). The salt content of fractions from the columns was determined by measuring the resistance of the fractions on a conductivity bridge; standardiza- tion of the bridge was effected with KC1 solutions in 0.005 M

potassium phosphate buffer, pH 6.0, containing lop4 M EDTA, under conditions similar to those used when measuring the re- sistance of column fractions.

Assay of Enzyme-Glucose-B-l’ dehydrogenase was assayed spectrophotometrically (24). The assay was performed by following the change in absorbancy at 340 mu in a Beckman model B spectrophotometer adapted for recording. TPN re- duction was measured as a function of time in a system con- taining 100 pmoles of Tris buffer, pH 7.5, 20 kmoles of MgC12, 2.5 pmoles of glucose-6-P, 0.2 pmole of TPN, water, and enzyme to a total of 3 ml; the light path was 1 cm. The reaction was effected at room temperature (25’). One enzyme unit is defined as that quantity of enzyme which reduces 1 pmole of TPN per minute under the above assay conditions.

Purification Procedures

Preparation of Erythrocytes-For each purification procedure, 18 pints of blood were used. The supernatant plasma was re- moved by a pipette, to which was attached a rubber bulb. The red cells were collected by centrifugation and were washed four times with 0.15 M KC1 in 0.005 M potassium phosphate buffer, pH 7.0, containing 10m4 M EDT.4. The washed cells were stored at 4” until required.

Hemolysis of Red Cells-The washed crythrocytes were lysed by mixing them with an equal volurne of water in a flask with a round bottom, freezing by immersion in a bath consisting of a mixture of solid carbon dioxide and ethylene glycol monomethyl ether, and thawing in a bath of tap water at 10”. All subsequent operations were performed at 4” unless otherwise stated.

DEA I&cellulose Fractionation-‘l’he hrmolyzed r.:d cells from 6 pints of blood were diluted to 2000 ml with water and mixed with an equal volume of a suspension of 150 g of DE.Q-cellulose in a buffer of 0.005 M potassium phosphate and lop4 M EDTA

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2310 Erythrocyte Glucose-6-P Dehydrogenase. I Vol. 238, n-o. 7

at pH 7.0. The mixture was stirred thoroughly at first and then intermittently for 30 minutes.

The suspension was then poured into an 8- x 60.cm column with a stopcock; a cellulose sponge at the base of the column retained the DEAF-cellulose. The cellulose column was washed with a buffer of 4000 ml of 0.005 M potassium phosphate and 1OW M EDTA at pH 7.0. This procedure removed most of the hemoglobin while the enzyme was retained on the DEAE-cellu- lose. The enzyme was Fluted with a solution of 0.3 M KC1 in the same buffer.

.lmmonium Sulfate Fractionation-The enzymatically active fractions from the DEhE-cellulose column were pooled and mixed with 351 g of solid (lu’Hd)zS04 per liter of enzyme solution. The precipitate, which contained the enzyme, was collected by centrifugation. The precipitate was redissolved in a buffer of 0.005 N potassium phosphate, pH 7.0-10-4 M EDTA, and re- precipitated with (NH,)804 as before.

Calcium Phosphate Gel l+actionation-The precipitate from the second ammonium sulfate treatment was dissolved in a buffer of 0.005 M potassium phosphate, pH 6.0-10e4 M EDTA, and the (NHJsSOl was removed by passage through a Sephadex G-25 column (25). The enzyme was elutc?d in 0.005 M potassium phosphate, pH 6.0-10-4 M EDTA. The desalted solution was treated with successive quantities of calcium phosphate gel, at a gel to protein ratio of 1:2, until the enzyme was almost com- pletely adsorbed. After each treatment, the gel was collected by centrifugation at approximately 650 X g for 5 minutes, and the supernatant solution was assayed for enzymatic activity. Elution of the enzyme was carried out by mixing the gel with an equal volume of a buffer of 0.12 N potassium phosphate, pH 7.0, allowing equilibration for 30 minutes, and then collecting the supernatant solution by centrifugation. The elution was repeated twice. The cluates were pooled, and the enzyme was concentrated by precipitating with 390 g of (NHd)zSOd per 1000 ml of enzyme solution; the precipitate was dissolved in a small volume of 0.005 M potassium phosphate buffer and lo-’ M EDTA, pH 6.0.

Carboxymethyl Cellulose F’ractionation-The concentrated cn- zyme was desalted on a Sephades G-25 column and eluted in 0.005 x potassium phosphate buffer-l0-4 M EDTA at pH 6.0. This solution was diluted to a protein concentration of 2 to 5 mg per ml of buffer. TPN was added to a final concentration of 2 x 10e6 M. The diluted enzyme was applied to a CM-cellu- lose column which had been previously equilibrated with the same buffer in which the enzyme was dissolved. The column, which was 4.5 x 40 cm, was packed under gravity and contained 60 g of dried ion exchange cellulose. After the protein solution was added, the column was washed with 1000 ml of buffer. De- velol)mcnt of the column was achieved by using a linear ionic strength gradient with 2000 ml of buffer and 2000 ml of 1 M

KC1 in buffer. The flow rate of the column was 6 ml per min- ute; fractions were collected at 2-minute intervals and assayed for ljrotein, enzyme activity, and salt concentration. The most active fractions were pooled.

Second Calcium Phosphate Gel Fractionation-The pooled en- zyme from the CiWcellulose column was adsorbed on calcium phosljhate gel at a gel to protein ratio of 6:l. The gel was col- lccted by centrifugation and eluted with small volumes of 0.12 M potassium phosphate buffer, pH 7.0, until all the enzyme was cluted. The eluted enzyme fractions were pooled for further fractionation.

Second DEA E-cellulose Fractionation-The calcium phosphate gel eluate was diluted to a buffer concentration of 0.05 M potas- sium phosphate, 1O-4 M EDTA, and 2 X lo-” M TPN. This was applied to a DEXE-cellulose column (1.2 x 22 cm) which had been packed under a pressure of 120 mm of Hg and equili- brated with 100 ml of 0.05 M potassium phosphate, 10e4 M EDTA, and 2 x 1OV M TPN. After the enzyme was adsorbed, the column was washed with 50 ml of this buffer solution; it was then rluted with either a linear ionic strength gradient with the use of 100 ml of buffer and 100 ml of 0.5 M KC1 in buffer or at constant ionic strength with 0.2 M potassium chloride in buffer. Two-milliliter fractions were collected and analyzed for enzyme activity and protein concentration.

Second CKcellulose Fractionation-Thr: active fractions from the DEAE-cellulose column were pooled and the KC1 removed by a Sephades G-25 column; the enzyme was eluted in 0.005 M

potassium phosphate buffer and 1O-4 M EDTA at pH 6.0. The solution was made 2 x 1OV M with respect to TPN, and applied to a 1.2- x 22.cm CM-cellulose column which had been packed under a pressure of 90 mm of Hg and equilibrated with buffer. After a washing with 50 ml of buffer, the column was developed with a linear ionic strength gradient with the use of 100 ml of buffer and 100 ml of 0.5 M KC1 in buffer. Two-milliliter frac- tions were collected and assayed for protein concentration and enzyme activity.

Concentration of Enzyme with Calcium Phosphate Gel-The protein concentration in the eluates from the DEAF-cellulose or CX-cellulose columns was usually low, and to carry out fur- ther studies on the enzyme, concentration of the protein was necessary. If the eluate was obtained from a DEAF-cellulose fractionation, the buffer composition was changed to 0.005 M

potassium phosphate-lo-4 M EDTLA at pH 6.0 with the aid of a Sephades G-25 column, and the effluent enzyme was adsorbed on calcium phosphate gel at a gel to protein ratio of 6: I. I f the eluate was obtained from a CM-cellulose fract,ionation the en- zymc was adsorbed directly on the gel. In each case, the gel was treated with several small volumes of 0.12 M potassium phosphate buffer, pH 7.0, until all the enzyme was eluted.

The outline for the purification of the enzyme was slightly modified from one preparation to the nest, depending on the starting material and the purification obtained in each step. .A summary of a tyljical purification procedure is presented in Table 1. A specific activity of 113 enzyme units per mg of protein was obtained, which represents a purification of over 40,000-fold above the crude hemolysate. The purification pro- cedure was quite reproducible, and yields of 5 to 10yO were usually obtained.

The pattern of enzymatic activity and of 280 rnp absorbancy eluted from the CYI-cellulose column (Step 6 in Table I) is pre- sented in Fig. 1.

The KC1 concentration, as determined by the conductivity bridge, is also presented in Fig. 1. A considerable quantity of protein was rluted in the pregradient washing.

In Fig. 2 are ljresented the patterns of emergence of enzy- matic activity and of material absorbing at 280 rnp from a DEAF-cellulose column (Step 9 in Table I). Elution of the enzyme was achieved with 0.2 M KC1 in a buffer of 0.15 M po-

tassium phosphate-10-4~ EDTA at pH 7.0, and 2 X lo6 M TPN. Purity of Enzyme-Ultracentrifugal analyses of the purified

enzyme were performed with a Sl)inco model E ultracentrifuge. A typical sedimentation pattern is presented in Fig. 3. The

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July 1963 A. E. Chung and R. G. Langdon 2311

TABLE I

Glucose &phosphate dehydrogenase purification

steps

1. Hemolysate.............................. 2. Activity recovered after DEAE-cellulose fractionation. 3. Activity recovered after (?jH,)zSOd fractionation.. 4. Activity recovered after calcium phosphate gel fractiona-

tion........................... 5. Activity recovered after (NH4)2S04 fractionation.. 6. Activity recovered after CM-cellulose fractionation.. 7. Activity recovered after calcium phosphate gel fractiona-

tion .._.............. 8. Activity recovered after second CM-cellulose fractionation. 9. Activity recovered after concentration with calcium phos-

phate gel and DEAE-cellulose fractionation. 10. Act,ivity recovered after calcium phosphate gel fractiona-

tion.....................................................

.4

.2

TUBE# 20 40 60 80 100 120 140 , ‘6s I”,0 ZOO 220

KCI CONC. .05 .IO .15 M

FIG. 1. Simultaneous emergence of enzyme activity and protein from a CM-cellulose column. The figure represents the appear- ance of 280.rnr absorption (O--O) and enzyme activity (o--e) from the CM-cellulose column represented as Step 6 in Table I. The KC1 concentrations indicated correspond to the tube numbers on the abscissa.

sedimentation pattern indicates that there are two components. The major component, which represents approximately 80% of

the total protein, has a sedimentation constant of 7.0 S. In a

separate experiment, the sedimentation constant of the catalyt- ically active component of this preparation was determined by the moving partition cell method described by Yphantis and Waugh (26), and found to bc 6.9 S. The close agreement of the sedimentation constants for the major protein component and the enzymatic activity suggest that the enzyme protein repre- sents 80% of the total protein. Details of these studies in the ultracentrifuge are presented in a subsequent section.

The purity of the enzyme was confirmed by electrophoresis on cellulose acetate paper and in starch gel.’ After electrophoresis in starch, the gel slab was sliced longitudinally, the enzyme was

1 We are greatly indebted to Dr. S. H. Boyer, Department of Medicine, for making available to us his methods and apparatus for starch gel electrophoresis of glucose-6-P dehydrogenase before publication.

Protein Enzyme activity

Enzyme specific

activity

-- Puritica- tion per

step Accumulative purification Yield

I- g ZlXilJ units/mg

1,990 5,170 0.0026

14.7 1,810 0.123 6.8 1,450 0.21

0 0

47.2 47.2 1.8 81

9%

100 35 28

5.4 1,230 0.31 1.5 119 24 2.3 970 0.60 1.9 230 19 0.127 955 7.55 12.6 2,900 18

0.049 0.026

538 571

434

390

11.1 23.1

0.005

0.0035

93

113

1.5 2.1

4.0

1.2

4,270 10 8,900 11

35,800 8.4

43 ) 500 7.5

ioo-

go-

80-

70-

-; 60-

< r 5O- 5

ti 40- z

30-

20 -

IO -

I ,x-x-Y *,x,

IO 30 50 70 VOLUME (ml.)

- .90

- .80

- .70

- .60

- .50 s Z

-.40

FIG. 2. Emergenceof highly purified glucose-6-P dehydrogenase and of protein from a DEAE-cellulose column. The figure cor- responds to Step 9, Table I. X- -X, Absorbancy at 280 m/l; O--O, enzyme units per ml of cluate.

located in the top portion of the gel according to the method of Bayer,’ while the bottom portion was stained for protein ac- cording to the method of Smithies (27). The catalytic activity corresponded to the major protein component as shown in Fig. 4.

These experiments, although not conclusive, indicate that the enzyme is approaching a state of high purity.

PHYSICAL CONSTaNTS

Diffusion Constant-The diffusion constant of the enzyme was determined with a porous diaphragm diffusion cell as originally described by Northrop and Anson (28) and more recently by Mize, Thompson, and Langdon (29). The cell constant K was

calculated from the formula

D= Va x co

KXTX&

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Erythrocyte Glucose-6-P Dehydrogenase. I Vol. 238, No. 7

FIG. 3. Sedimentation pattern of glucose-6-P dehydrogenase. The experiment was carried out at 10” in 0.15 M potassium phos- phate buffer, pH 7.0. The picture was taken after centrifugation for 64 minutes at 50,740 r.p.m. The specific activity of the enzyme was 113 enzyme units per mg of protein, and the protein concen- tration was 2.23 mg per ml.

in which VO is the volume of the solvent in the outer compart- ment, Co is the final concentration of solute in the outer com- partment, T is the diffusion time in seconds, Ci is the mean con- centration of the solute in the diffusion cell over the time of diffusion, and D is the diffusion constant of the calibrating solute.

The cell constant was determined by using bovine plasma albumin as the calibrating protein. The temperature of the experiment was lo’, and the albumin was dissolved in 0.15 M

potassium phosphate buffer at pH 7.0. The results of one ex- periment are given in Table II. Protein concentration was de- termined by the 260:280 rnp absorbancy method. The time interval for diffusion was selected so that less than 1 $$a of solute diffused out of the diffusion cell. The mean concentration, Ci, was determined by finding the mean of the concentration of the solute at the end of the experiment, and the concentration of the solute at the beginning of the experiment; correction was made for the quantity of material which diffused out of the cell during each interval. The value used for the diffusion constant of bovine serum albumin was 4.63 x lo-’ cm2 per second (30). The cell constant determined was 8.2.

The results for an experiment with glucose-6-P dehydrogenase are presented in Table III. The specific activity of the enzyme was 113 enzyme units per mg. The enzyme was dissolved in 0.15 M potassium phosphate buffer, pH 7.0, with 4 X 10e6 M

TPN. The temperature of the experiment was 10”. Because of the low concentration of enzyme in the outer compartment of the cell, activity measurements were aided by use of a scale expansion device on the Brown Recorder so that full scale de-

flection was obtained between 90 and 100% transmission. The diffusion constant determined was 3.4 x 10e7 cm2 per second.

Sedimentation Constant-The sedimentation constant of the enzyme was determined in a Spinco model E ultracentrifuge either with the use of a moving partition cell as described by

FIG. 4. Starch gel electrophoresis of glucose-6-P dehydrogenase. The starch was prepared at a concentration of 12.2 g per 100 ml of 0.070 M Tris-citrate at pH 8.6. The electrode chambers contained 0.5 M borate buffer at pH 8.4. Electrophoresis was performed at 4’ for 15 hours at a constant voltage of 3.5 volts per cm. The protein concentration was 2.2 mg per ml, and the specific activity was 113 enzyme units per mg of protein. The enzyme was located in the ton section of the gel by overlaying it with 110 ml of solution containing 100 ml of 0.5~ Tris-HCi, pH 8.6, 10e2 M MgC12, 2 X 10W4 M TPN. 5 X 10e3 M alucose-6-P. and 10 ma of Nitro-BT in 10 ml of water; 8. mg of phenazine methosulfat; were added just before using. Development was carried out in the dark until the enzyme was located by a purple spot. The left section of the photograph shows the location of the enzyme by this method. The protein was located in the bottom section of the gel according to the method of Smithies (27) with Amido schwarz stain. The result is shown in the right section of the figure.

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July 1963 A. E. Chunq and R. C. Langdon 2313

Yphantis and Waugh (26) or by the moving boundary method. Since the sedimentation constant determined by the moving partition cell was based on enzymatic activity, it could be de- termined which of the two components observed in the purest preparation was associated with enzymatic activity. The sedi- mentation constant determined by the moving boundary method also provided a check for the sedimentation constant determined by the Ijartition cell.

In the moving partition cell method, the value of si w2 dt was determined graphically as suggested in the original article (26). The times at which predetermined speeds were reached during acceleration and the deceleration were recorded, w2 was then plotted against time and

+ s

t d dt

t2 deceleration

determined. The experiments were run at a top speed of 50,740 r.p.m. deceleration was accomplished by applying rapid braking to a speed of 5,000 r.p.m. at which time medium braking was applied; this allowed the moving partition to return to its rest position slowly and smoothly. The volumes of the solutions were determined by weighing and assuming that unit weight corresponded to unit volume.

The cell and rotor constants were measured under static conditions and used without making corrections for the small changes occurring during operation of the centrifuge. The position of the meniscus was obtained by taking photographs of the ccl1 and rotor during operation of the centrifuge and using a microcomparator to determine the relative positions of the meniscus and the two reference points on the rotor.

The concentrations or activities of the initial solution and of the solution in the top compartment of the cell after centrifuga- tion were determined; from the various measurements, the sedi- mentation constant was calculated.

Sufficient quantities of highly purified enzyme were available to allow determination of its sedimentation constant by optical methods. All the sedimentation experiments were done at ap- prosimatrly 10” and in 0.10 M or 0.15 M potassium phosphate buffer, pH 7.0.

The results of the sedimentation experiments arc summarized in Table IV. The mean sedimentation constant determined by the separation cell method is 6.5 S; by the moving boundary method the sedimentation constant of the major component is 7.1 S. The close correspondence of these values indicates that the enzyme activity is associated with the major protein com- ponent. The sedimentation constant of the slower moving component in Experiment 3 in Table V was determined and found to bc 3.9 S. Subsequent purification by carboxymethyl ccllulosc increased the specific activity of this preparation by a factor of 2, while at the same time the slower moving component was almost completely removed as indicated by subsequent analysis in the ultracentrifuge (Experiment 4 in Table IV).

Calculation of Molecular U-eight-The molecular weight of the enzyme was calculated from the diffusion and sedimentation constants of the enzyme by the Svedberg equation. The partial specific volume of the enzyme was assumed to be 0.74. The value used for the sedimentation constant was 7.1 S, the mean of the values obtained by the moving boundary method. This

TABLE II

Determination of diffusion cell constant

1 2 3

SEC

7200

7200 7200

I

mg fvolein/ml

0.04’2 14.71 0.040 14.61 0.038 14.51

8.6 8.2 7.8

8.2 f 0.4t

* The diffusion constant used for bovine serum albumin was 4.63 x 10-T cm* per second (30).

t Mean.

TABIX III

Determination oj difusion constant Jar glucose 6-P dehydrogenase

Sample No.

1 2 3

T

Se‘

7200 7200 7200

VO

ml

10 10 10

CO / a

enzyme wni1s/ml

0.0088 4.03 0.0075 4.01 0.0076 3.99

D X 10’

cm=/sec

3.7 3.2 3.2 3.4*

* Mean.

value was considered to be more reliable than the value obtained by the moving partition method. The molecular weight thus calculated was 190,000.

Determination of Frictional Coeficient and .I xial Ratio-From

the nomogram of Wyman and Ingalls (31), the frictional ratio of the enzyme was found to be 1.6 and its axial ratio 11 when its shape was assumed to be that of a prolate ellipsoid of revolu- tion. These values are unusually high and indicate a high de- gree of asymmetry for the enzyme molecule.

NH&erminal . I mine Acid ;lnaZysis-NH&crminal amino acid analysis was performed according to the method of Fraenkel- Conrat et al. (32) with the modification that C’4-fluorodinitro- benzene was employed. The 2,4-dinitrofluorobenzene had a specific activity of 1.23 mc per mmole, and the enzyme solution had a specific activity of 113 e.u. per mg of protein at a con- centration of 2.23 mg per ml of 0.15 M potassium phosphate buffer, pH 7.0. Protein solution, 0.5 ml, was mixed with 5 mg of NaH03 and 8.0 pmoles of Cl*-2,4-dinitrofluorobenzene in 1.5 ml of ethanol. The mixture was shaken for 3 hours at room temperature, acidified with concentrated hydrochloric acid, and estracted several times with peroxide-free ether to free the dinitrophenylated protein of escess fluorodinitrobenzene and dinitrobenzene. The ether-extracted residue was suspended in constant boiling HCl and approximately 10 pmoles each of the DNP2 derivatives of isoleucine, glutamic acid, alanine, threonine, proline, methionine, and tyrosine (di-DNP), added. The mix- ture was heated at 105” for 16 hours in a sealed tube.

After acid hydrolysis, the hydrolysate was diluted to 1.0 N

HCl and extracted several times with peroxide-free ether. The ether extracts were pooled and evaporated to dryness under a stream of nitrogen. The DNP-amino acids were separated by two-dimensional paper chromat,ography. The solvent system in the first dimension was tertiary amyl alcohol saturated with

2 The zkbreviation used is: DNP, dinitrophenyl.

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2314 Erythrocyte Glucose-6-P Dehydrogenase. I

TABLE IV

Sedimentation constant of glucose 6-phosphate dehydrogenase

No.

1 2

3 4 5

* Mean.

Method Specific activity Enzyme Protein

!

Separation cell Separation cell

Moving boundary Moving boundary Moving boundary

enzyme units/ my prohz

"0 113

50 150 95 209

113 260

TABLE V

DNP-AVHz-terminal amino acids of glucose-6-P dehydrogenase

-

Compound Predicted* Experimental

7nJmoles

DNP-alanine Di-DNP-tyrosinet

4.4

/

2.5 4.4 1.3

* The predicted values for the derivatives were based on the assumptions that (a) the Cl*-dinitrofluorobenzene reacted quan- titatively with the NHs-terminal amino acids, (b) the hydrolysis of the protein was complete, (c) the DNP-amino acid of the pro- tein behaved in the same way to acid hydrolysis as the added carrier, (d) there was no exchange of the CY4-dinitrophenyl radical with other amino acids, (e) the enzyme preparation was SOO10 pure, and (f) there was only one of each species of peptide chain per 200,000 g of protein. The experimental value, a, was calculated from the formula a = n/c X b/s in which n was the total counts recovered from the chromatogram, a, b, and c the number of millimicromoles of NHa-terminal amino acid, the amount of added carrier and the amount of carrier recovered from the chro- matogram, respect.ively, and s the specific activity of the Cl4- DNP-NHz-terminal amino acid.

t The specific activity of the di-DNP-tyrosine was assumed to be twice that for the mono-DNP amino acids.

0.05 M potassium phthalatc buffer, pH 6.0; the second dimension was run in 1.5 M potassium phosphate buffer, pH 6.0.

The radioactive spots were located by autoradiography. There were blackened areas on the autoradiogram which exactly corresponded to spots on the chromatogram identified as di- DNP-tyrosine, DNP-alanine, and dinitrophenol. Minor spots were observed, which from DNP-amino acid maps obtained in the same solvent systems, corresponded to DNP-glycine and DNP-aspartic acid.

The major radioactive spots corresponding to DNP-alanine and di-DNP-tyrosine were eluted in ethanol and evaporated to dryness, and each was redissolved in 1 ml of acetone. The con- centration of DNP-amino acid in each solution was determined spectrophotometrically; the C%ontent was determined by dry- ing aliquots on planchets and counting in a windowless gas flow counter. From the data obtained, the specific activity of each DNP-amino acid was calculated. The identity of each com- pound was confirmed by rechromatography, after mixing with the appropriate authentic derivative.

-

ntglml

0.5 0.04

3.0 2.2 2.2

--

Concentration of potassium phos- phate buffer, pH

7.0

M

0.1 0.15

9.15 0.15 0.15

-I-

Temperature s

70.0” 10.0

9.8 10.4 10.0

Vol. 238, 10. 7

6.1 6.9

(6.5 h 0.4)* 7.3 6.9 7.0

(7.1 + 0.2)*

I f it is assumed that the enzyme preparation was SO<% pure, then there were 4.4 mpmoles of enzyme in the reaction misture. For each peptide chain, there were also 4.4 mpmoles of SH2- terminal amino acid. If there were more than one of the same peptide chain per enzyme molecule, then there would be the corresponding multiple of 4.4 mpmoles of its NHQ-terminal amino acid.

The results of the analysis and identification of the NH-ter- minal amino acids are recorded in Tables V and VI, respectively. In Table V, the predicted value for the quantity of NH*-terminal amino acid from one peptide chain is compared with the quan- tity calculated from the isotope dilution. The theoretical spe- cific activities were based on the assumptions that NHz-terminal amino acids were completely dinitrophenylated, that complete hydrolysis occurred, and that rates of hydrolysis of the dini- trophenyl amino bonds of the substituted peptides were identical with those of the carrier DNP-amino acids. The values ob- tained and presented in Table V are thus minimal ones. Taken at their face value, these results indicate that there is one KH2- terminal alanine and one NHz-terminal tyrosine residue per 200,000 g of enzyme protein.

A method for purification of human erythrocyte glucose-6-P dehydrogenase has been described which reproducibly gave enzyme preparations with specific activities of up to 113 enzyme

TABLE VI

Rechromatography of radioactive compounds eluted from chromatogram*

Specilic activity

Compound Refore After

rechromatography rechromstography

DNP-alanine. Di-DNP-tyrosine Dinitrophenol

C.p.W+W?SOlL?

3,000 3,000 11,680 18,000

3,506 1,296

* The radioactive compounds were mixed with the appropriate authentic compounds and rechromatographed in a potassium phosphate buffer system. The spots obtained were excised and eluted, and the specific activities were compared with the initial materials.

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July 1963 A. E. Chung and R. G. Langdon

units per mg of protein in yields of 5 to 10%. This is the most highly purified preparation from mammalian tissue so far re- ported.

This purified enzyme is not homogeneous in the ultracentrifuge but appears to be approaching a state of high purity. The major sedimentable component represents approximately 80% of the total protein as estimated by determining the areas of the sedimenting peaks in the ultracentrifuge. That this component is the enzyme is strongly suggested by the close agreement be- tween its sedimentation constant and the sedimentation con- stant of the enzymatic activity determined by measurements in a moving partition cell. Further confirmatory evidence has been obtained from its electrophoretic behavior, in starch gel. The enzymatic activity is associated with the major protein component. When an enzyme preparation with a specific ac- tivity of 50 enzyme units per mg of protein was examined in the ultracentrifuge, there were two sedimenting peaks of equal mag- nitude. A Z-fold purification resulted in a preparation which was predominantly composed of a fast moving component, the sedimentation constant of which was characteristic of the en- zyme.

Recently, Noltmann, Gubler, and Kuby (6) have succeeded in crystallizing glucose-6-P dehydrogenase from yeast. The crystallized enzyme had a specific activity of 667 enzyme units per mg of protein, a value considerably higher than that of the purified erythrocyte enzyme or of the crystalline enzyme from cow mammary gland, which had a specific activity of 67 enzyme units per mg of protein (13). The difference in the specific activities of the yeast enzyme and these mammalian preparations may be due to differences in their turnover numbers. Although the erythrocyte enzyme appears to be approaching a state of purity by the criteria used, the possibility also exists that the preparation may be contaminated with other proteins and, in particular, by catalytically inactive glucose-6-P dehydrogenase (20). At the same time, since the activity of this enzyme is so sensitive to variations in its environment, the yeast enzyme may have been activated during its purification. The yeast enzyme could be crystallized only in the presence of TPN.

There is evidence that the yeast enzyme also differs in other respects from the erythrocyte enzyme. The Michaelis constants for TPN were 2.0 X lop5 M (5) and 2.1 X low6 M (10) for the yeast and erythrocyte enzymes, respectively. While the eryth- rocyte enzyme is inhibited by certain steroids, the yeast enzyme is not (11). Until the two enzymes have been more thoroughly characterized with respect to their chemical and physical prop- erties, the problem will remain unresolved.

A similar situation exists with crystallized yeast and purified rat liver glutathione reductase (32). While the yeast enzyme catalyzed the oxidation of 27.4 pmoles of TPNH per minute per mg of protein, the purified mammalian enzyme oxidized 839 pmoles of TPNH per minute per mg of protein.

Although glucose-6-P dehydrogenase was first identified in 1931 (1, Z), only recently has its physical characterization been attempted. Kirkman (33, 34) has very recently purified this enzyme from erythrocytes to a specific activity of 64 units per mg and has examined its sedimentation behavior in a sucrose den- sity gradient by the method of Martin and Ames (35); he has estimated that its molecular weight in a dimeric form is 105,000. By more direct methods, which agree well, we have determined the sedimentation and diffusion constants for the catalytically active species and the major protein component of a preparation

with a specific activity of 113 units per mg; based on these measurements, we have calculated for this enzyme a molecular weight of 190,000 and an axial ratio of 11. Whether the differ- ences are related to differences in methods of purification, in purity, or in methods of determination of the physical properies must be determined by further research.

The enzyme has 1 mole each of NH*-terminal alanine and tyrosine per 200,000 g of protein, which indicates that there are at least 2 peptide chains per molecule of enzyme. This number represents a minimal value, since other peptide chains may be present in the protein in which the NH*-terminal amino acids may be acetylated or otherwise unavailable for reaction with fluorodinitrobenzene.

The low value obtained experimentally for the NHz-terminal amino acids may be due to incomplete reaction of the C14- fluorodinitrobenzene with the enzyme, incomplete hydrolysis of the DNP-protein, instability of the DNP-NHpterminal amino acid compared with added carrier, or exchange of the labeled dinitrophenyl radical with other DNP-amino acids. Moreover, the value for tyrosine is that for the di-DNP derivative; the mono-DNP derivative was not extracted into the ether phase, and its loss could account for the low value obtained.

Since there were difficulties in obtaining stoichiometric re- coveries of the labeled NH&erminal amino acid derivatives, duplication of the peptide chains could not be ascertained.

SUMMARY

Human erythrocyte glucose 6-phosphate dehydrogenase has been purified to a specific activity of up to 113 enzyme units per milligram of protein. This is the highest purification so far reported for the enzyme from mammalian tissues. The enzyme appears to be approximately 80% pure as indicated by ultracentrifugal and electrophoretic measurements.

Sedimentation and diffusion constants for the enzyme have been determined, and from these a molecular weight of 190,000 has been obtained. The molecule is highly asymmetrical, the axial ratio being 11.

The enzyme has at least two peptide chains in which the NHY terminal amino acids have been tentatively identified as tyrosine and alanine.

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Albert E. Chung and Robert G. LangdonAND PROPERTIES OF THE ENZYME

Human Erythrocyte Glucose 6-Phosphate Dehydrogenase: I. ISOLATION

1963, 238:2309-2316.J. Biol. Chem. 

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