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The 2,3-diphosphoglyceric acid phosphatase activity of phosphoglyceric acid mutase purified from human erythrocytes
ARCHIVES
OF
BIOCHEMISTRY
AND
138, 208-219 (1970)
BIOPHYSICS
The 2,3-Diphosphoglyceric Phosphoglyceric
Acid
Acid
Mutase
Phosphatase Purified
Activity
from
of
Human
Erythrocytes’ D. R. HARKNESS,2
WILLIAM AND
Departmenls
of Medicine
THOMPSON,3
VICTORIA
SANDRA
ROTH,
GRAYSON
and Biochemistry, University of Miami School of Medicine, Administration Hospital, Miami, Florida
Received
December
1, 1969; accepted
February
Veterans
10, 1970
The partial purification of phosphoglyceric acid mutase from human erythrocytes is described. This enzyme has an absolute requirement for 2,3-diphosphoglyceric acid under the conditions used for the assay. 2,3-Diphosphoglyceric acid phosphatase activity copurified with the mutase, and evidence that both of these activities are carried out by the same enzyme is reported. The phosphatase activity is markedly enhanced by inorganic pyrophosphate, 20- to 50-fold by 20 mM concentrations. In experiments utilizing 32P-labeled pyrophosphate, direct involvement in the reaction by pyrophosphate was excluded. Intracellular compounds in physiological concentrations failed to augment significantly the phosphatase activity of phosphoglycerie acid mutase. A second 2,3diphosphoglyceric acid phosphatase which is stimulated by sodium bisulfite and unassociated with mutase activity was separated from the pyrophosphate-stimulated enzyme. The relative significance of these two enzymes in the metabolism of 2,3diphosphoglyceric acid in the erythrocyte is discussed.
Phosphoglyceric acid (PGA) mutases purified from several different sources have been found to possess 2,3-diphosphoglyceric acid (2,3-DPG) phosphatase activity (1). Zancan, Recondo, and Leloir (2) described a 2,3-DPG phosphatase in rabbit muscle which was stimulated by inorganic pyrophosphate (PP,). Although they were unable to separate this activity from PGA mutase, they concluded that it was a separate enzyme (3). We found that PPi also enhanced 2,3-DPG phosphatase activity in hemol-
ysates of human erythrocytes and in preliminary studies were unable to separate this activity from PGA mutase (4). On the contrary, we found that the ratio of the activities of this PPi-stimulated phosphatase to PGA mutase in the erythrocytes of nine different mammals was nearly constant, an observation consistent with their being the same enzyme (4). Because of the known importance of 2,3DPG in the mammalian erythrocyte and the likelihood that the PPi-stimulated phosphatase and mutase are the same enzyme, we have undertaken this study to clarify their relationship. In this communication we report a procedure for the partial puri-fication of PGA mutase from the human red cell. We present evidence that it does possess2,3-DPG phosphatase activity and that it is the activity of this enzyme that is dramatic-
1 This research was supported by United States Public Health Service Grant No. AM-09001-05, the United States Veterans Administration, and the Rayne Haber Karp Laboratories for Hematology Research. 2 John and Mary Markle Scholar in Academic Medicine. 3 Supported by the Helen Bell Sadoff Research Fund. 208
2,3-DPG
PHOSPHATASE
ACTIVITY
ally enhanced by PPi. Separation of the PPi-stimulated phosphatase from a second 2,3-DPG phosphatase, which is activated by sodium bisulfite, was accomplished during the purification. Some properties of the mutase are described, and its possible role as a 2,3-DPG phosphatase within the cell is discussed. A sensitive radioactive assay suitable for measuring the small amounts of 2,3-DPG phosphatase activity in theerythrocyte is described. MATERIALS
AND
METHODS
The disodiumsalt of ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane (Tris), diethylaminoethyl cellulose (DEAE-cellulose), coarse mesh, 0.84 mEq/g, and DEAESephadex A-50 were obtained from Sigma. The DEAE-cellulose was successively washed with 1 N NaOH, 1 N HCl, 1 N NaOH, and distilled water before use (5). Hydroxylapatite (Bio-Gel HTP) was purchased from Bio-Rad Laboratories, Dowex 1 X 8, chloride form, 100-200 mesh, from CalBiochem, and sodium pyrophosphate from Fisher Scientific Company. The substrates, cofactors, and purified enzymes used in the assays for the enzymes of glycolysis and the hexose monophosphate shunt were purchased from CalBiochem. 3-Phosphoglyceric acid was prepared free of contaminating 2,3-diphosphoglyceric acid by anion exchange chromatography (6). Carrier-free 32P-labeled inorganic phosphate came from Tracer Lab. Hyamine hydroxide, 2,5diphenyloxazole (PPO), and 1,4-bis[2-(4-methyl5-phenyloxazolyl)]benzene (dimethyl POPOP) were purchased from Packard Instrument Company, Inc. The sodium salt of heparin USP came from Fellows Testagar Medical Manufacturing Co., Inc. The 32P-labeled sodium pyrophosphate, prepared by the method of Berg (7), was supplied by Dr. Karl Muench. Other chemicals, all of reagent from Fisher Scientific grade, were purchased Company. Preparation of 2,3-Dg2PG. Radioactive 2,3-DPG was prepared by incubating in a total volume of 30 ml the following mixture for 3 hr at 37’: Trischloride buffer, pH 7.5, 200 pmoles; MgClz , 100 pmoles; NaF, 100 Nmoles; 3-PGA, 20 pmoles; Na pyruvate, 20 r*moles; inosine, 100 pmoles; 2 mCi carrier-free 32P: and 25 ml of a 1:l distilled water lysate of washed human erythrocytes. The reaction was terminated by the addition of one volume 10% trichloracetic acid. The precipitate was removed by centrifugation and the supernatant fluid shaken with approximately 1 g acid-washed Norit A to remove the nucleotides. After filtration, the trichloroacetic acid was removed by extraction, and
OF PGA MUTASE
209
50 rmoles carrier 2,3-DPG was added. The resulting neutralized material was applied to a 2 X 20cm Dowex-1 Cl- column. The column was washed with 0.02 N HCl and then developed with a linear gradient from 0.02 to 0.12 N HCl. Two well separated peaks of radioactivity were eluted, fructose diphosphate and 2,3-DPG. The latter material was concentrated by flash evaporation, neutralized with NaOH, and quantitated by the chromotropic acid assay (8). Enzyme assays. Phosphoglyceromutase was measured at ambient temperature (2324.5”) in a Cary recording spectrophotometer by the enolase-coupled assay described by Grisoiia (9). The reaction mixtures contained in a total volume of 1.0 ml: 25 mM Tris-chloride buffer (pH 7.0)) 50 mM 3-PGA, 0.25 mM 2,3-DPG, 3 mM MgCl, , and 20 fig of enolase. The extinction coefficient for phosphoenolpyruvate (PEP) at 240 rnp under the conditions of this assay was determined to be 1100 by enzymatic conversion of known amounts of 2-PGA to PEP and also from standard solutjions of PEP. The changes in absorbancy at 240 rn/* per minute were converted to Mmoles by dividing by 1.1. One unit of mutase activity represents the formation of 1 pmole PEP per minute. 2,3-Diphosphoglyceric acid phosphatase (DPG phosphatase) was measured by a radioactive assay in the presence of optimal concentrations of an activator, sodium pyrophosphate. Reaction mixtures, 0.5 ml, containing 100 mM Tris-acetate buffer (pH 6.5), 20 mM sodium pyrophosphate, 0.2-1.5 rnM 2,3-D32PG, and enzyme were incubated at 37” for 60 min. Reactions were stopped by adding 1.0 ml 67, perchloric acid. After centrifugation, 1.0 ml of the supernat,ant fluid was transferred to a tube containing 2.0 ml of the following mixture: H*O:l N HISOI: 570 (NH&Mo~O~~.~H~O:~Y NaCl (10:3:3:6). After mixing, the phosphomolybdate complex was extracted into 3.0 ml water-saturated 2-butanol as by Berenblum and Chain (10). The tubes were centrifuged lightly to facilitate phase separation, and then 2.0 ml of the upper butanol phase was washed with an equal volume of centrifugation, 1.0 ml of the 1N HzSOa . After washed butanol phase was t,ransferred to scintillation vials, dried, and 0.2 ml hyamine hydroxide added. Ten milliliters of counting solution (5 g PPO and 0.3 g dimethyl POPOP per liter toluene) were added, and the radioactivity measured in a Packard liquid scintillation spectrometer. With more purified enzyme preparations, prot,ein concentration was low enough to combine the perchloric acid with the complexing mixture, eliminating one centrifugation and transfer. This assay is linear with respect to time and enzyme concentration (Figs. 1 and 2).
210
ET AL.
HARKNESS
71 a, * 0 L al D .-
4-
40
60
Time
Minutes
FIG. 1. Time course of 2,3-DPG phosphatase taining 50 mr.r Tris-acetate buffer, pH 6.5; 20 mM rmole) ; and 9 units of PGA mutase (58.5 units/mg 0.5-ml aliquots were removed at the times indicated described under Methods. I
I
120
100
80
in
assay. The 15-ml reaction mixture conPPi; 1.5 mM 2,3-DzePG (1.5 X lo6 cpm/ protein) was incubated at 37”. Duplicate and the Pi extracted and quantitated as
I
I
1
02
025
400 b * E 3.0a ” -0 m 0L 20. .-I .-
0.1
0 15
Enzyme,
ml
FIG. 2. Enzyme concentration curve for 2,3-DPG phosphatase activity. Duplicate 0.5ml reaction mixtures containing the same concentrations of buffer, substrate, and PPi , described under Fig. 1, and from 0 to 10 rg enzyme (58.5 units/mg protein) were incubated at 37” for 60 min. Reaction mixtures were stopped and prepared for counting as described under Met.hods.
Enzymes of glycolysis and the hexose monophosphate shunt were assayed by published methods or slight alterations of these as previously described (11). Protein was measured by the method of Lowry el al. (12) and phospho-
glyceric acid with lett (8).
chromotropic
acid after
Bart-
RESULTS
Enzyme purification. Blood was withdrawn from the anteeubital vein of volun-
2,3-DPG
PHOSPHATASE
ACTIVITY
teem using heparin, 10 units/ml blood, as anticoagulant. The cells were washed three times with three volumes of cold 0.154 M NaCl. The saline wash and buffy coat were removed by aspiration. The packed cell volumes were measured, and the washed packed cells were either frozen and stored at -20” or used immediately. Outdated bank blood has also been used but contains less enzyme activity than freshly obtained cells. The preparation of purified PGA mutase described below was performed on freshly obtained blood which had been stored 2 weeks prior to use. One hundred milliliters washed frozen
211
OF PGA MUTASE
cells (packed cell volume 77 %) was thawed and mixed with 100 ml each of 5 rn$r Trischloride buffer, pH 7.5, 1 mM EDTA, and 1 mM /Smercaptoethanol. DEAE-cellulose, approximately 10 g, was added and the slurry stirred at 3” for 4 hr. The DEAE-cellulose was collected by centrifugation and washed free of hemoglobin with 10 m&r Tris-chloride buffer, pH 7.5, containing 1 mnI EDTA and 1 rnpr /?-mercaptoethanol. The adsorbed enzymes were eluted from the DEAEcellulose by stirring with 300 ml and then 250 ml of 0.25 JI KC1 in 10 rn$r Tris-chloride buffer, pH 7.5, 1 rnyr EDTA and 1 rnbr Pmercaptoethanol. 2
10.0’
80 I!
E \6.0 2 .I
s
x .; .-
4 0
.
I
i
G 2 0
55
:
-04
s= 20
-03 -02 -0.1 75
Fraction
Number
FIG. 3. Chromatography of 2,3-DPG phosphatase on hydroxylapatite. Approximately 156 mg protein was applied to a 2 X l&cm column of hydroxylapatite. The column was developed with a linear gradient consisting of 500 ml 10 mM potassium phosphate buffer, pH 6.8, containing 1 mM p-mercaptoethanol and 1 mM EDTA in the mixing chamber and 500 ml 150 m potassium phosphate buffer, pH 6.8, containing 1 mM EDTA and 1 mM fl-mercaptoethanol in the reservoir. The flow rate was 0.5 ml/minute. Six-milliliter fractions were collected and assayed for PGA mutase (O--O), and 2,3-DPG phosphatase in the presence of 20 mM PPi U-w) or 20 mM sodium bisulfite (o-0). The absorbance at 280 rnM was measured (A--A).
212
HARKNESS
The protein in the hemoglobin-free combined eluate (534 ml) was precipitated by two volumes of acetone (- 20’) in a 4-l. glass cylinder. The precipitate was allowed to aggregate and settle for several hours or overnight at -20”. After removing much of the clear supernatant fluid by siphoning, the precipitate was collected by centrifugation at -20’. The acetone precipitate was suspended in 10 mM potassium phosphate buffer, pH 6.8, containing 1 m&f P-mercaptoethanol and 1 mM EDTA. The insoluble materials were removed by centrifugation and extracted with additional buffer. The combined solution, 100 ml, was then applied to a 2 X 15cm column of hydroxylapatite which had been thoroughly washed with starting buffer (10 rnM potassium phosphate, pH 6.8; 1 mM EDTA; 1 rnM /3-mercaptoethanol). The protein was eluted from the column with a linear gradient consisting of 500 ml starting buffer in the mixing chamber and 500 ml 0.15 M potassium phosphate buffer, pH 6.8, containing 1 mM EDTA and 1 mM P-mercaptoethanol in the reservoir. A tiow rate of 0.5 ml/min was maintained with a Sigmamotor peristaltic pump. Six-milliliter fractions were collected. The elution profile of protein, PGA mutase, and the PPi-stimulated and bisulfite-stimulated 2,3-DPG phosphatases are shown in Fig. 3. Fractions 3048 containing most, of the PGA mutase activity were combined and dialyzed against 10 mM Tris-chloride buffer, pH 7.5, containing 1 mM B-mercaptoethanol. The dialyzed preparation, 117 ml, was applied to a 1 X 25-cm column of DEAESephadex A-50 which had been thoroughly equilibrated with starting buffer (10 mM Tris-chloride buffer, pH 7.5, containing 1 mM fl-mercaptoethanol and 1 rnhr EDTA). The column was developed with a linear gradient consisting of 500 ml of the starting buffer in the mixing chamber and 500 ml of the same buffer containing 0.25 M KC1 in the reservoir. The flow rate was adjusted to 1.0 ml/min with a Sigmamotor peristaltic pump; 9.0 ml fractions were collected. The fractions containing the mutase activity (51-70) were combined. Sometimes these were concentrated by dialysis against buffered 30% solutions of polyethylene glycol
ET AL. TABLE PURIFICATION
Hemolysate DEAE eluate Acetone precipitate Hydroxylapatite chromatography 5. DEAE-Sephadex chromatography 1. 2. 3. 4.
I
OF PGA MUTASE ERYTHROCYTES
40028,400 534 379 100 152 117 41 175
FROM HUMAN
704 ,024 507 1.34 462 3.04 336 8.20
6.7 264 39.7
72 66 48 38
(mol wt 20,000). The enzyme is quite stable, even in dilute solution, when kept at 3’ in 10 mM Tris-chloride buffer, pH 7.5, but only when dialyzed to remove the high salt concentrations required for elution from the column. The above preparative procedure gave a 1656-fold purification with 38 % recovery (Table I). These steps have consistently yielded similar recoveries of enzyme of 1500-2000-fold purification. A final step of gel filtration on G-200 Sephadex has given preparations as high as 3000-fold purified, but the recoveries were quite low. This was possibly due to high concentrations of salt, used in the eluting fluid since we have noted instability of the enzyme under such conditions. The enzyme preparation described above is estimated to be about one-fourth as pure as that of the crystalline preparations from yeast, rabbit muscle, and chicken muscle (13). As an indication of the stepwise separation of the PGA mutase from the other erythrocytic enzymes in the preparation described above, all of the enzymes of glycolysis, glucose-6-phosphate dehydrogenase, 6-phosphogluconic acid dehydrogenase, and glutathione reductase were assayed at, each stage of purification. The only activities other than PGA mutase remaining after the final stage of purification were trace quantities of triose isomerase and phosphoglyceric acid kinase. Characteristics of PGA mutase. The pH of optimal enzyme activity for the mutase was 7.0. In the presence of 0.25 mM 2,3-DPG the K, for 3-PGA was found to be 0.8 rnbl.
2,3-DPG
PHOSPHATASE
ACTIVITY
Under the conditions used here, when assayed with 3-PGA that had been completely freed of contaminating 2,3-DPG, enzyme activity was entirely dependent upon the addition of 2,3-DPG. The K, for 2,3-DPG in the presence of 50 ma 3-PGA as determined from the double reciprocal plot of Lineweaver and Burk (14) was 6 mM. These values are similar to those for PGA mutases purified from caprine erythrocytes (15) and yeast (1). Identity of PGA-mu&se and 2,3-DPG phosphatase activities. The PPi-stimulated 2,3-DPG phosphatase and the PGA mutase activities purified together. The elution profiles of the two activities were identical upon column chromatography on hydroxylapatite
OF PGA MUTASE
(Fig. 3) and DEAESephadex (Fig. 4), gel filtration on Sephadex G-200, and by sucrose gradient centrifugation. The other 2,3DPG phosphatase, which is stimulated by sodium bisulfite and not by PPi, was clearly separated on hydroxylapatite (Fig. 3). When the partially purified PGA mutase was heated at 45“ at pH 8.0, both the mutase and phosphatase activities were lost at the same rate with all activity destroyed after heating for 1 hr. The presence of 2 mM MgC% during heating had no protective effect. Both enzymatic activities were protected against inactivation by heating at this temperature in the presence of 20 mu 2,3-DPG or 3-PGA, or 40 mM PPi. With these additions there was no significant loss
4.1
r > .?I 2 3. x .> ." 4 2. Q) E; ; 1.
Fraction
213
Number
FIG. 4. Chromatography of 2,3-DPG phosphatase on DEAE-Sephadex. Forty milligrams of protein were applied to a 1 X 25-cm column of DEAE-Sephadex. The column was then developed at 3” with a linear gradient composed of 500 ml 10 mu Tris-chloride buffer, pH 7.5, containing 1 mu p-mercaptoethanol and 1 mu EDTA in the mixing vessel and 500 ml of the same mixture containing 0.25 M KC1 in the reservoir. The flow rate was 1.0 ml/ min; 9.0~ml fractions were collected. Fractions were assayed for PGA mutase activity (O-O) and for pyrophosphate-stimulated 2,3-DPG phosphatase activity m.4).
214
HARKNESS
>k 5 4 60Q F z40t z E a
20-
00
64.5', pH 8.0
15
30
45
60
TIME (minutes)
FIG. 5. The effect of heating PGA mutase in the presence of 2,3-DPG, 3-PGA, or PPi at 64.5’. Incubation mixtures contained approximately 2 mg enzyme (20 units/mg protein); 106 mM Trischloride buffer, pH 8.0; and 20 mu 2,3-DPG (m), 20 mM 3-PGA (A), or 40 mM PPi (a). These were heated at 64.5”. Samples were removed at the intervals noted and frozen until assayed for PGA mutase (---) and PPi-stimulated phosphatase (- - -) activities.
of either phosphatase or mutase activity in 1 hr of heating up to 60”. The results of heating the enzyme at 64.5’ are shown in Fig. 5. At this temperature there was no protection of either activity by 3-PGA or PPi and only moderate protection against heat inactivation by 2,3-DPG. There was no appreciable disparity between the effects on mutase and phosphatase activities. The e$ect of PP; on the phosphatase activity. The phosphatase activity of the purified erythrocytic PGA mutase (Fig. 6) is markedly enhanced by PPi. We have used 20 m&r PPi in our routine phosphatase assays, a level giving nearly maximal stimulation. This gives a 20- to 50-fold increase in activity over reaction mixtures containing no PPi. A K, of approximately 3 mM is obtained by replotting the results of the experiment represented in Fig. 6 after Lineweaver and Burk (14). The pH optimum for
ET AL.
the phosphataxe activity is 6.5 with or without PPi. Inorganic pyrophosphate effects the phosphatase activity by altering the I’,,,-, but not the affinity of the enzyme for substrate. A double reciprocal plot of phosphatase activity versus substrate concentration in the presence of PPi appears in Fig. 7. The K, for 2,3-DPG is 0.2 m&i. Nearly the same K, is obtained in experiments with no added PPi. To investigate the possible direct participation of pyrophosphate in the reaction, 16 units mutase in 100 ml1 Tris-acetate buffer, pH 6.5, 5 m&i 2,3-D32PG, and 50 m&z PPi, and a control reaction mixture without enzyme were incubated at 37” for 90 min. The reaction mixtures mere boiled for 5 min, diluted to 100 ml, and 25 pmoles each of carrier 3-PGA and 2,3-DPG were added. The samples were applied to 1 X 15-cm columns of Dowex-1 Cl- which were successively washed with 100 ml distilled water, 300 ml 0.01 N HCl, and 200 ml 0.02 N HCl. Then a 500-ml linear gradient from 0.02 to 0.1 N HCl was pumped over the columns, and lo-ml fractions were collected. A similar reaction mixture containing half as much PPi and 50 rnkr Pi was handled in identical manner. Total phosphate, radioactivity, and glyceric acid were measured on each fraction from the gradients. The PPi was eluted in fractions 23 through 30 and 2,3DPG from 34-43. No radioactivity appeared in the PPi. Similar experiments were performed in which 32PPi was incubated together with enzyme, buffer, and either 2,3-DPG or 3PGA. Carrier 2,3-DPG and 3-PGA were added, and the monophosphoglyceric acid, PPi, and 2,3-DPG separated from one another as in the previous experiment. nTo radioactivity was found in either PGA or 2,3-DPG. It was concluded that pyrophosphate does not enter into any exchange or phosphoryl transfer reaction with either 2,3-DPG or 3-PGA. The e$ect of intracellular metabolites on the phosphatase activity of PGA mutase. Since the human erythrocytes contain no measurable inorganic pyrophosphate, a survey of various intracellular metabolic intermediates as
2,3-DPG PHOSPHATASE
I
ACTIVITY
I
OF PGA MUTASE
I
I
I
.““,M
4o
5o
215
IO “[PPi]
FIG. 6. Stimulation of 2,3-DPG phosphatase activity by inorganic pyrophosphate. Reaccontained 100 mu Tris-acetate buffer, pH 6-5; tion mixtures, in quadruplicate, 1 mu 2,3-D3*PG (3.6 X lOa cpm/gmoIe); enzyme, 160 Mg (spec. act, 0.9); and from 0 to 50 rnM PPi . Duplicates were stopped at 0 time and after 60 min at 37” and prepared for counting as described under Methods.
FIG. 7. Effect of substrate concentration upon velocity of the reaction. Reaction mixtures contained 100 mu Tris-acetate buffer, pH 6.5; 20 mM pyrophosphate; PGA mutase, 0.5 units; and from 2.3 to 55 PM 2,3-D3*PG (lo6 cpm/pmole). K, = 2 x 10e4M.
possible activators of the phosphatase activity of this enzyme was carried out. Compounds were tested at 20 and 2 rnM concentrations. At 20 m&f concentrations, a few
compounds had more than 10% of the activity of PPi. These are listed in Table II. At 2 mM concentrations, the activity with these compounds was little more than with
216
ET AL.
HARKNESS TABLE
II
SURVEYOFCELLULARMET~BOLITESFORPOSSIBLE STIMULATION OF 2,3-DPG PHOSPHBTSSE ACTIVITY"
20mM (%I PPi No addition G 6-P Fru-1,6-di-P PEP 6-PGA TPN ATP CTP UTP GTP ITP ADP UDP
100 2 11.0 14.8 13.4 15.0 23.8 14.0 12.2 13.2 15.8 14.5 10.9 10.3
2 m (%‘c) 5 2 1.1 0.8 3.0 2.4 5.0 2.5 2.2 3.7 6.9 5.1 0.7 2.3
a Reaction mixtures contained 100 mM Trisacetate buffer, pH 6.5; 1 mM 2,3-Da2PG; 0.5 units PGA mutase; and either 20 or 2 mM concentration of the other additions indicated. All activities are expressed relative to the activity in 20 mM PPi.
enzyme alone. At 20 rnM concentrations, the following compounds gave from 5 to 10 % of the stimulation noted with PPi : fructose 6-P, glyceraldehyde 3-P, 2-phosphoglyceric acid, ribose 5-P, cytidine diphosphate, and guanosine diphosphate. Glucose, pyruvate, lactate, glycerol, &glycerophosphate, DPN, 3-phosphoglyceric acid, adenylic acid, cytidylic acid, inosinic acid, uridine, inosine, and cytidine effected less than a 5 % stimulation. Hexametaphosphate stimulated the enzyme 17 and 26% when added at 2 and 20 rnM concentrations. Several metal chelators such as EDTA, citrate, ar,~dipyridyl, and o-phenanthroline all failed to activate the phosphatase. Inhibitor studies. The effects of several compounds upon the activity of 2,3-DPG phosphatase activity in the presence of pyrophosphate were tested (Table III). No significant inhibition was seen with 20 mM citrate, EDTA, NaF, or NaCl. There was moderate inhibition by 20 rnwl iodoacetate and 20 mu Pi. At 2 m&I concentration, there was some inhibition by CX,CY-dipyridyl and
o-phenanthroline and somewhat more by p-hydroxymercuribenzoate. Both 3-PGA and 2-PGA inhibit the PPistimulated phosphatase activity of PGA mutase. However, Lineweaver and Burk plots of this data do not give straight lines (Fig. 8). The probable explanation is that rapid exchange and mutase reactions take place, thereby lowering the specific activity of the substrate. Hence, the greater the ratio TABLE
III
EFFECTS OF VARIOUS COMPOUNDS ON PHOSPHATASE ACTIVITY OF PGA MUTASE~
-.
20ml.5(%I 2mu (%)
Addition None Citrate EDTA NaF p-Hydroxymercuribenzoate Iodoacetate 01,or-Dipyridyl o-Phenanthroline Pi NaCl
100 82 104 95 65 70 98
85 92 90 105 88 66 72 95 -
a Incubation mixtures contained 100 mM Tris-acetate buffer, pH 6.5; 1 mM 2,3-DaaPG; 0.5 units of PGA mutase; 20 mM PPi; and either 20 or 2 mM of the other additions as indicated. 3
I
I
50uM
3-PGA
FIG. 8. Inhibition of 2,3-DPG phosphatase by 3-PGA. Reaction mixtures contained from 4.6 to 46 PM substrate; 20 rnM PPi; 100 mM Tris-acetate, pH 6.5; enzyme and 50 PM, 5 @M, or no 3-PGA.
2,3-DPG PHOSPHATASE
ACTIVITY
of inhibitor to substrate, the greater the apparent inhibition. Attempt at reversing the phosphatase activity. Mixtures of radioactive inorganic phosphate, 100 rnM (90,000 cpm/pmole) ; 3-PGA, 10 rn>r i; Tris-acetate buffer, pH 6.5, 100 mM; 4 units of enzyme! with and without 20 m&l PPi in a total volume of 4.0 ml were incubated for 3 hr at 37”. The reaction mixtures were then diluted to 50 ml with water and, after addition of 25 pmoles carrier 2,3-DPG, applied to Dowex-1 Cl- columns. The 2, X-DPG, isolated free of Pi and 3-PGA by gradient elution with dilute HCl, contained no radioactivity. DISCUSSION
Zancan, Recondo, and Leloir (2) reported studies of a search for enzymes capable of adenylyl diphosphoglyceric catabolizing acid, a nucleotide reportedly isolated from porcine and human erythrocytes (16, 17). They described an enzymatic activity in rabbit muscle which hydrolyzed the free phosphate in the 2-position of both this nucleotide and free 2,3-DPG. The hydrolysis was stimulated by inorganic pyrophosphate with optimal concentration around 20 mnI. The pH of optimal activity was between 6.5 and 7.0. In a second paper from Leloir’s laboratory, the authors reported their inability to separate this 2,3-DPG phosphatase activity from PGA mutase (3). However, after comparing phosphatase, transferase, and mutase activities at various stages of purification of the phosphatase in rabbit muscle, it was concluded that the transferase and phosphatase activities were catalyzed by the same enzyme and that PGA mutase was probably a separate enzyme. Grisolia and co-workers (1) have reported that crystalline PGA mutases possess 2,3DPG phosphatase activity and that at least one highly purified 2,3-DPG phosphatase exhibited the mutase activity (18). Immunological studies with yeast enzyme supported the contention that the two activities resided on the same protein (19). However, similar studies with muscle enzymes gave conflicting results. The anti-PGA mutase inhibited the mutase activity but not the phosphatase activity (13).
OF PGA MUTASE
217
The data presented in this paper on an enzyme purified from yet another source, the, human erythrocyte, also indicate that PGA mutase has inherent 2,3-DPG phosphatase activity. The identical elution profiles of the PPi-stimulated phosphatase and mutase activities from hydroxylapatite and DEAESephadex columns and the failure to dissociate these activities by gel filtration on Sephadex G-200 or by sucrose gradient centrifugation are strong evidence that one enzyme, or an enzyme complex, is responsible for both activities. The results of the heat denaturation studies and the parallel protection of both activities by PPi, 3-PGA, and 2,3-DPG support this conclusion. It seems most unlikely that if there were two separate proteins, all three compounds would interact with both to afford similar degrees of protection against inactivation by heating. Grisolia and Tecson (20) induced a reversible transformation of PGA mutase into 2,3-DPG phosphatase with Hg2+. This interconversion occurred in all animal PGA mutases tested, all of which contain SH groups and which require 2,3-DPG as cofactor. This interconversion was not possible with yeast PGA mutase, which contains no SH groups or with plant PGA mutases which do not require 2,3-DPG for activity. They interpreted their data as further support that in the case of animal mutases both enzymatic activities are associated with the same protein. It is of interest that the phosphatase of chicken muscle purified after Joyce and Grisolia (18) was active ivithout PPi and that added PPi gave no enhancement of activity (2). During preparation of that enzyme, it is treated with Hg2+ and apparently is already optimally “activated.” Using radioactive PPi or 2,3-DPG we were unable to obtain any evidence that PPi enters directly into the enzymatic reaction. It seems likely that the PPi, which affects the V,,, but not the K, for substrate, alters the conformation of the enzyme in such a way as to enhance phosphatase activity without appreciably decreasing its mutase activity. As noted above, Hg2+, on the other hand, stimulates the phosphatase activity but inhibits the mutase activity. Stimulation by PPi does not appear to be the
21s
HARKNESS
result of metal chelation since other chelators had little effect on the phosphatase activity. Considerable interest in the cellular mechanisms regulating the levels of 2,3DPG in the mammalian erythrocyte has been generated by the recent demonstration of the effect of this compound on the position of the oxygen dissociation curve of hemoglobin (20, 21). 2,3-Diphosphoglyceric acid is synthesized from 1 ,3-DPG and 3-PGA by an enzyme first described by Rapoport and Luebering (23) and recently studied in detail by Rose (24). This enzyme, diphosphoglycerate mutase, is extremely sensitive to inhibition by product having a Ki of 0.85 ~~11(24). Several findings suggest that a plausible mechanism for regulation of 2,3DPG synthesis is its exclusive binding to deoxyhemoglobin (25) with consequent protection of the enzyme from inhibition. Hypoxia from many causes is associated with elevated intraerythrocytic levels of 2,3-DPG (26, 27, 28). In red cell hemolysate;, glycolysis and 2,3-DPG synthesis are increased under nitrogen but not under carbon monoxide or oxygen (29, 30). 2,3DPG synthesis is not inhibited by the addition of fetal deoxyhemoglobin which does not significantly bind 2,3-DPG (31, 32, 33). A second means of controlling intracellular concentrations of 2,3-DPG is regulation of its catabolic rate. Rapoport and Luebering (34) showed that 2,3-DPG is degraded by a p’rosphatase which hydrolyzes it to Pi and phosphomonoglycerate. We were intrigued by the possibility that the erythrocyte, a cell which is unique in its high content of 2,3DPG and also in deriving all of its energy from glycolysis, might utilize the same enzyme for catabolizing 2,3-DPG and for the mutase reaction which requires 2,3-DPG as a cofactor. It suggested that there might be interesting control mechanisms for regulating the act,iTvlties of such an enzyme. The fact that a small molecule such as PPi could so remarkably enhance the phosphatase activity made it seem plausible that the cell might indeed use this enzyme in a dual way. Erythrocytes contain no- measureable PPi; this may be attributable to the presence of a potent pyrophosphatase (25). A search for
ET AL.
other possible activators was therefore conducted but failed to reveal significant enhancement of enzymatic activity by physiological concentrations of any intracellular compounds studied. Acid extracts and extracts of boiled erythrocytes also had no effect. The presence of a second 2,3-DPG phosphatase in mammalian erythrocytes was first demonstrated by MBnyai and VBrady (36). Studies on some properties of this enzyme have been published by deverdier (37) and by us (38). It is stimulated approximately 40-fold by 20 m&r sodium bisulfite. Rose has demonstrated that inorganic phosphate and chloride together can effect a similar degree of stimulation (39). In contrast to the PPistimulated enzyme, this enzyme is inhibited by sulfhydryl reagents. The erythrocyte of the goat has very low concentrations of 2,3DPG, shows minimal capacity for its synthesis, and contains almost none of this enzyme (15). It can be argued that the erythrocytes of the goat have no need for a cofactor for hemoglobin deoxygenation since caprine hemoglobin has such a low affinity for oxygen that it dissociates sufficiently at capillary PO2 for adequate tissue oxygenation (40). On the other hand, we have purified PGA mutase from caprine erythrocytes and found that it has considerable PPi-stimulated phosphatase activity and is similar in all other respects to the enzyme from human erythrocytes described in this paper (15). In conclusion, we have demonstrated that under the influence of PPi, PGA mutase purified from human erythrocytes possesses 2,3-DPG phosphatase activity. There is no evidence to support a physiological role for this activity. Our inability to identify any plausible intracellular activator for this enzyme and the presence of a second enzyme in the erythrocyte with the same activity causes us to believe that PGA mutase may not function as a phosphatase within the cell. The almost complete absence of this second phosphatase in some mammalian erythrocytes such as those of the goat, which also contain little 2,3-DPG or diphosphoglyceric acid mutase and yet possess PPi-stimulated phosphatase activity proportionate to mutase activity, supports this conclusion.
2,3-DPG
PHOSPHATASE
ACTIVITY
REFERENCES V. W., TOWNE, J. C., .~ND GRISOLIA, S., J. Biol. Chem. 228, 875 (1957). 2. ZANCAN, G. T., RECONDO, E. F., AND LELOIR, L. F., Biochim. Biophys. Acta 92, 125 (1964). 3. ZANCAN, G. T., KRISMAN, C. R., MORDOH, J., .~ND LELOIR, L. F., Biochim. Biophys. Acla 110, 348 (1965). 4. HARKNESS, D. R., PONCE, J., AND GRAYV., Comp. Biochem. Physiol. 28, 129 SON, (1969). 5. PETERSON, E. A., AND SOBER, H. A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. V, p. 6. Academic Press, New York (1962). 6. TOWNE, J. C., RODWELL, V. W., AND GRISOLIA, S., J. Biol. Chem. 226, 777 (1957). 7. BERG, P., J. Biol. Chem. 233, 601 (1958). 8. BARTLETT, G. R., J. Riol. Chem. 234, 466 (1959). 9. GRISOLIS, S., in “Methods in Enzymology” S. P. Colowick and N. 0. Caplan, eds.), Vol V, p. 236. Academic Press, New York (1962). I., AND CH-\IN, E., Biochem. J. 10. BERENBLUM, J. 32, 295 (1938). 11. HARKNESS, D. R., AND GRAYSON, V., Comp. Biochem. Physiol. 28, 1289 (1969). 12. LOWRY, 0. N., ROSEBROUGH, N. J., F~RR, A. L., .~ND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 13. GRISOLIA, S., in “Homologus Enzymes and Biochemical Evolution” (N. Van Thoai and J. Roche, eds.), pp. 167-198. Gordon and Breach, New York (1968). 14. LINEWEAVER, H., AND BURK, P., J. Chem. Sot. 66, 658 (1934). 15. HARKNESS, D. R., AND PONCE, J., Arch. Biothem. Biophys. 134, 113 (1969). H., Biochem. 16. HASHIMOTO, T., AND YOSHIKAW.~, Biophys. Res. Commun. 6,71 (1961). 17. HASHIMOTO, T., ISHII, Y., TATIB.INA, M., AND YOSHIKAWA, H., J. Biochem. Tokyo 60, 471 (1961). 18. JOYCE, B. K., AND GRISOLU, S., J. Biol. Chem. 233, 350 (1958). 1. RODWELL,
OF PGA MUTASE
219
19. GRISOLI~, S., AND DETTER, J. C., Biochem. 2. 342, 239 (1965), S., .~ND TECSON, J., Biochim. Bio20. GRISOLI.~, phys. Acta 132, 56 (1967). 21. CHANUTIN, A., .~ND CUI~UISH, R. P., Arch. Biochem. Biophys. 121, 96 (1967). 22, BENESCH, R., AND BENESCH: R. E., Biochem. Biophys. Res. Commun. 26, 162 (1967). 23. RAPOPORT, S., AND LUERERING, J., J. Biol. Chem. 196, 583 (1952). 24. ROSE, Z. B., J. Biol. Chem. 243, 4810 (1968). R., AND BENESCH, R. E., Nature 25. BENESCH, London 221, 618 (1969). 26. LENFANT, C., TORRANCE, J., ENGLISH, E., FINCH, C. A., REYNBFARJE, C., RAMOS, J., AND FAURA, J., J. Clin. Invest. 47, 2652 (1968). 27. OSKI, F. A., GOTTLIEB, A. J., DELIVORLP~PADOPOULOS, M., AND MILLER, M., ,V. Engl. J. Med. 280, 1165 (1969). 28. EATON, J. W., AND BREWER, G. J., Proc. Nat. .4cad. Sci. U.S. 61, 756 (1968). 29. ASAKURA, T., SATO, Y., MINAKaMI, S., AND YOSHIKAWA, H., J. Biochem. Tokyo 6, 524 (1966). J., TROTMAN, B., GOTTLIEB, A., 30. WEISSHERG, AND OSKI, F., C&n. Res. 16, 543 (1968). 31. OSKI, F., MILLER, W., DELIVORIA-PAPADOPOULOS, M., AND GOTTLIER, A., Pediat. Res. 3, 376 (1969). 32. BAUER, CH., LUDWIG, I., AND LUDWIG, M., Life Sci. 7, 1339 (1968). I., AND SHIMIZU, K., ilrch. Biochem. 33. TYUMA, 129, 404 (1969). S., AND LUEHERING, J., J. Biol. 34. RAPOPORT, Chem. 189, 683 (1951). 35. PYNES, G. B., AND YOUNATHAN, E. S., J. Biol. Chem. 242, 2119 (1967). S., AND VhRADY, Zs., Acta Physiol. 36. M~NYAI, 14, 103 (1957). 37. DEVERDIER, C. H., Folia Haematol. Leipzig 90, 215 (1968). 38. HARKNESS, D. R., AND ROTH, S., Biochem. Biophys. Res. Commun. 34, 849 (1969). 39. ROSE, Z. B., Fed. Proc. Fed. Amer. Sot. Exp. Biol. 28, 727 (1969). 40. RIEGEL, K., HILPERT, P., AND BARTELS, H., Acta Haematol. 26, 164 (1961).
OF
BIOCHEMISTRY
AND
138, 208-219 (1970)
BIOPHYSICS
The 2,3-Diphosphoglyceric Phosphoglyceric
Acid
Acid
Mutase
Phosphatase Purified
Activity
from
of
Human
Erythrocytes’ D. R. HARKNESS,2
WILLIAM AND
Departmenls
of Medicine
THOMPSON,3
VICTORIA
SANDRA
ROTH,
GRAYSON
and Biochemistry, University of Miami School of Medicine, Administration Hospital, Miami, Florida
Received
December
1, 1969; accepted
February
Veterans
10, 1970
The partial purification of phosphoglyceric acid mutase from human erythrocytes is described. This enzyme has an absolute requirement for 2,3-diphosphoglyceric acid under the conditions used for the assay. 2,3-Diphosphoglyceric acid phosphatase activity copurified with the mutase, and evidence that both of these activities are carried out by the same enzyme is reported. The phosphatase activity is markedly enhanced by inorganic pyrophosphate, 20- to 50-fold by 20 mM concentrations. In experiments utilizing 32P-labeled pyrophosphate, direct involvement in the reaction by pyrophosphate was excluded. Intracellular compounds in physiological concentrations failed to augment significantly the phosphatase activity of phosphoglycerie acid mutase. A second 2,3diphosphoglyceric acid phosphatase which is stimulated by sodium bisulfite and unassociated with mutase activity was separated from the pyrophosphate-stimulated enzyme. The relative significance of these two enzymes in the metabolism of 2,3diphosphoglyceric acid in the erythrocyte is discussed.
Phosphoglyceric acid (PGA) mutases purified from several different sources have been found to possess 2,3-diphosphoglyceric acid (2,3-DPG) phosphatase activity (1). Zancan, Recondo, and Leloir (2) described a 2,3-DPG phosphatase in rabbit muscle which was stimulated by inorganic pyrophosphate (PP,). Although they were unable to separate this activity from PGA mutase, they concluded that it was a separate enzyme (3). We found that PPi also enhanced 2,3-DPG phosphatase activity in hemol-
ysates of human erythrocytes and in preliminary studies were unable to separate this activity from PGA mutase (4). On the contrary, we found that the ratio of the activities of this PPi-stimulated phosphatase to PGA mutase in the erythrocytes of nine different mammals was nearly constant, an observation consistent with their being the same enzyme (4). Because of the known importance of 2,3DPG in the mammalian erythrocyte and the likelihood that the PPi-stimulated phosphatase and mutase are the same enzyme, we have undertaken this study to clarify their relationship. In this communication we report a procedure for the partial puri-fication of PGA mutase from the human red cell. We present evidence that it does possess2,3-DPG phosphatase activity and that it is the activity of this enzyme that is dramatic-
1 This research was supported by United States Public Health Service Grant No. AM-09001-05, the United States Veterans Administration, and the Rayne Haber Karp Laboratories for Hematology Research. 2 John and Mary Markle Scholar in Academic Medicine. 3 Supported by the Helen Bell Sadoff Research Fund. 208
2,3-DPG
PHOSPHATASE
ACTIVITY
ally enhanced by PPi. Separation of the PPi-stimulated phosphatase from a second 2,3-DPG phosphatase, which is activated by sodium bisulfite, was accomplished during the purification. Some properties of the mutase are described, and its possible role as a 2,3-DPG phosphatase within the cell is discussed. A sensitive radioactive assay suitable for measuring the small amounts of 2,3-DPG phosphatase activity in theerythrocyte is described. MATERIALS
AND
METHODS
The disodiumsalt of ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane (Tris), diethylaminoethyl cellulose (DEAE-cellulose), coarse mesh, 0.84 mEq/g, and DEAESephadex A-50 were obtained from Sigma. The DEAE-cellulose was successively washed with 1 N NaOH, 1 N HCl, 1 N NaOH, and distilled water before use (5). Hydroxylapatite (Bio-Gel HTP) was purchased from Bio-Rad Laboratories, Dowex 1 X 8, chloride form, 100-200 mesh, from CalBiochem, and sodium pyrophosphate from Fisher Scientific Company. The substrates, cofactors, and purified enzymes used in the assays for the enzymes of glycolysis and the hexose monophosphate shunt were purchased from CalBiochem. 3-Phosphoglyceric acid was prepared free of contaminating 2,3-diphosphoglyceric acid by anion exchange chromatography (6). Carrier-free 32P-labeled inorganic phosphate came from Tracer Lab. Hyamine hydroxide, 2,5diphenyloxazole (PPO), and 1,4-bis[2-(4-methyl5-phenyloxazolyl)]benzene (dimethyl POPOP) were purchased from Packard Instrument Company, Inc. The sodium salt of heparin USP came from Fellows Testagar Medical Manufacturing Co., Inc. The 32P-labeled sodium pyrophosphate, prepared by the method of Berg (7), was supplied by Dr. Karl Muench. Other chemicals, all of reagent from Fisher Scientific grade, were purchased Company. Preparation of 2,3-Dg2PG. Radioactive 2,3-DPG was prepared by incubating in a total volume of 30 ml the following mixture for 3 hr at 37’: Trischloride buffer, pH 7.5, 200 pmoles; MgClz , 100 pmoles; NaF, 100 Nmoles; 3-PGA, 20 pmoles; Na pyruvate, 20 r*moles; inosine, 100 pmoles; 2 mCi carrier-free 32P: and 25 ml of a 1:l distilled water lysate of washed human erythrocytes. The reaction was terminated by the addition of one volume 10% trichloracetic acid. The precipitate was removed by centrifugation and the supernatant fluid shaken with approximately 1 g acid-washed Norit A to remove the nucleotides. After filtration, the trichloroacetic acid was removed by extraction, and
OF PGA MUTASE
209
50 rmoles carrier 2,3-DPG was added. The resulting neutralized material was applied to a 2 X 20cm Dowex-1 Cl- column. The column was washed with 0.02 N HCl and then developed with a linear gradient from 0.02 to 0.12 N HCl. Two well separated peaks of radioactivity were eluted, fructose diphosphate and 2,3-DPG. The latter material was concentrated by flash evaporation, neutralized with NaOH, and quantitated by the chromotropic acid assay (8). Enzyme assays. Phosphoglyceromutase was measured at ambient temperature (2324.5”) in a Cary recording spectrophotometer by the enolase-coupled assay described by Grisoiia (9). The reaction mixtures contained in a total volume of 1.0 ml: 25 mM Tris-chloride buffer (pH 7.0)) 50 mM 3-PGA, 0.25 mM 2,3-DPG, 3 mM MgCl, , and 20 fig of enolase. The extinction coefficient for phosphoenolpyruvate (PEP) at 240 rnp under the conditions of this assay was determined to be 1100 by enzymatic conversion of known amounts of 2-PGA to PEP and also from standard solutjions of PEP. The changes in absorbancy at 240 rn/* per minute were converted to Mmoles by dividing by 1.1. One unit of mutase activity represents the formation of 1 pmole PEP per minute. 2,3-Diphosphoglyceric acid phosphatase (DPG phosphatase) was measured by a radioactive assay in the presence of optimal concentrations of an activator, sodium pyrophosphate. Reaction mixtures, 0.5 ml, containing 100 mM Tris-acetate buffer (pH 6.5), 20 mM sodium pyrophosphate, 0.2-1.5 rnM 2,3-D32PG, and enzyme were incubated at 37” for 60 min. Reactions were stopped by adding 1.0 ml 67, perchloric acid. After centrifugation, 1.0 ml of the supernat,ant fluid was transferred to a tube containing 2.0 ml of the following mixture: H*O:l N HISOI: 570 (NH&Mo~O~~.~H~O:~Y NaCl (10:3:3:6). After mixing, the phosphomolybdate complex was extracted into 3.0 ml water-saturated 2-butanol as by Berenblum and Chain (10). The tubes were centrifuged lightly to facilitate phase separation, and then 2.0 ml of the upper butanol phase was washed with an equal volume of centrifugation, 1.0 ml of the 1N HzSOa . After washed butanol phase was t,ransferred to scintillation vials, dried, and 0.2 ml hyamine hydroxide added. Ten milliliters of counting solution (5 g PPO and 0.3 g dimethyl POPOP per liter toluene) were added, and the radioactivity measured in a Packard liquid scintillation spectrometer. With more purified enzyme preparations, prot,ein concentration was low enough to combine the perchloric acid with the complexing mixture, eliminating one centrifugation and transfer. This assay is linear with respect to time and enzyme concentration (Figs. 1 and 2).
210
ET AL.
HARKNESS
71 a, * 0 L al D .-
4-
40
60
Time
Minutes
FIG. 1. Time course of 2,3-DPG phosphatase taining 50 mr.r Tris-acetate buffer, pH 6.5; 20 mM rmole) ; and 9 units of PGA mutase (58.5 units/mg 0.5-ml aliquots were removed at the times indicated described under Methods. I
I
120
100
80
in
assay. The 15-ml reaction mixture conPPi; 1.5 mM 2,3-DzePG (1.5 X lo6 cpm/ protein) was incubated at 37”. Duplicate and the Pi extracted and quantitated as
I
I
1
02
025
400 b * E 3.0a ” -0 m 0L 20. .-I .-
0.1
0 15
Enzyme,
ml
FIG. 2. Enzyme concentration curve for 2,3-DPG phosphatase activity. Duplicate 0.5ml reaction mixtures containing the same concentrations of buffer, substrate, and PPi , described under Fig. 1, and from 0 to 10 rg enzyme (58.5 units/mg protein) were incubated at 37” for 60 min. Reaction mixtures were stopped and prepared for counting as described under Met.hods.
Enzymes of glycolysis and the hexose monophosphate shunt were assayed by published methods or slight alterations of these as previously described (11). Protein was measured by the method of Lowry el al. (12) and phospho-
glyceric acid with lett (8).
chromotropic
acid after
Bart-
RESULTS
Enzyme purification. Blood was withdrawn from the anteeubital vein of volun-
2,3-DPG
PHOSPHATASE
ACTIVITY
teem using heparin, 10 units/ml blood, as anticoagulant. The cells were washed three times with three volumes of cold 0.154 M NaCl. The saline wash and buffy coat were removed by aspiration. The packed cell volumes were measured, and the washed packed cells were either frozen and stored at -20” or used immediately. Outdated bank blood has also been used but contains less enzyme activity than freshly obtained cells. The preparation of purified PGA mutase described below was performed on freshly obtained blood which had been stored 2 weeks prior to use. One hundred milliliters washed frozen
211
OF PGA MUTASE
cells (packed cell volume 77 %) was thawed and mixed with 100 ml each of 5 rn$r Trischloride buffer, pH 7.5, 1 mM EDTA, and 1 mM /Smercaptoethanol. DEAE-cellulose, approximately 10 g, was added and the slurry stirred at 3” for 4 hr. The DEAE-cellulose was collected by centrifugation and washed free of hemoglobin with 10 m&r Tris-chloride buffer, pH 7.5, containing 1 mnI EDTA and 1 rnpr /?-mercaptoethanol. The adsorbed enzymes were eluted from the DEAEcellulose by stirring with 300 ml and then 250 ml of 0.25 JI KC1 in 10 rn$r Tris-chloride buffer, pH 7.5, 1 rnyr EDTA and 1 rnbr Pmercaptoethanol. 2
10.0’
80 I!
E \6.0 2 .I
s
x .; .-
4 0
.
I
i
G 2 0
55
:
-04
s= 20
-03 -02 -0.1 75
Fraction
Number
FIG. 3. Chromatography of 2,3-DPG phosphatase on hydroxylapatite. Approximately 156 mg protein was applied to a 2 X l&cm column of hydroxylapatite. The column was developed with a linear gradient consisting of 500 ml 10 mM potassium phosphate buffer, pH 6.8, containing 1 mM p-mercaptoethanol and 1 mM EDTA in the mixing chamber and 500 ml 150 m potassium phosphate buffer, pH 6.8, containing 1 mM EDTA and 1 mM fl-mercaptoethanol in the reservoir. The flow rate was 0.5 ml/minute. Six-milliliter fractions were collected and assayed for PGA mutase (O--O), and 2,3-DPG phosphatase in the presence of 20 mM PPi U-w) or 20 mM sodium bisulfite (o-0). The absorbance at 280 rnM was measured (A--A).
212
HARKNESS
The protein in the hemoglobin-free combined eluate (534 ml) was precipitated by two volumes of acetone (- 20’) in a 4-l. glass cylinder. The precipitate was allowed to aggregate and settle for several hours or overnight at -20”. After removing much of the clear supernatant fluid by siphoning, the precipitate was collected by centrifugation at -20’. The acetone precipitate was suspended in 10 mM potassium phosphate buffer, pH 6.8, containing 1 m&f P-mercaptoethanol and 1 mM EDTA. The insoluble materials were removed by centrifugation and extracted with additional buffer. The combined solution, 100 ml, was then applied to a 2 X 15cm column of hydroxylapatite which had been thoroughly washed with starting buffer (10 rnM potassium phosphate, pH 6.8; 1 mM EDTA; 1 rnM /3-mercaptoethanol). The protein was eluted from the column with a linear gradient consisting of 500 ml starting buffer in the mixing chamber and 500 ml 0.15 M potassium phosphate buffer, pH 6.8, containing 1 mM EDTA and 1 mM P-mercaptoethanol in the reservoir. A tiow rate of 0.5 ml/min was maintained with a Sigmamotor peristaltic pump. Six-milliliter fractions were collected. The elution profile of protein, PGA mutase, and the PPi-stimulated and bisulfite-stimulated 2,3-DPG phosphatases are shown in Fig. 3. Fractions 3048 containing most, of the PGA mutase activity were combined and dialyzed against 10 mM Tris-chloride buffer, pH 7.5, containing 1 mM B-mercaptoethanol. The dialyzed preparation, 117 ml, was applied to a 1 X 25-cm column of DEAESephadex A-50 which had been thoroughly equilibrated with starting buffer (10 mM Tris-chloride buffer, pH 7.5, containing 1 mM fl-mercaptoethanol and 1 rnhr EDTA). The column was developed with a linear gradient consisting of 500 ml of the starting buffer in the mixing chamber and 500 ml of the same buffer containing 0.25 M KC1 in the reservoir. The flow rate was adjusted to 1.0 ml/min with a Sigmamotor peristaltic pump; 9.0 ml fractions were collected. The fractions containing the mutase activity (51-70) were combined. Sometimes these were concentrated by dialysis against buffered 30% solutions of polyethylene glycol
ET AL. TABLE PURIFICATION
Hemolysate DEAE eluate Acetone precipitate Hydroxylapatite chromatography 5. DEAE-Sephadex chromatography 1. 2. 3. 4.
I
OF PGA MUTASE ERYTHROCYTES
40028,400 534 379 100 152 117 41 175
FROM HUMAN
704 ,024 507 1.34 462 3.04 336 8.20
6.7 264 39.7
72 66 48 38
(mol wt 20,000). The enzyme is quite stable, even in dilute solution, when kept at 3’ in 10 mM Tris-chloride buffer, pH 7.5, but only when dialyzed to remove the high salt concentrations required for elution from the column. The above preparative procedure gave a 1656-fold purification with 38 % recovery (Table I). These steps have consistently yielded similar recoveries of enzyme of 1500-2000-fold purification. A final step of gel filtration on G-200 Sephadex has given preparations as high as 3000-fold purified, but the recoveries were quite low. This was possibly due to high concentrations of salt, used in the eluting fluid since we have noted instability of the enzyme under such conditions. The enzyme preparation described above is estimated to be about one-fourth as pure as that of the crystalline preparations from yeast, rabbit muscle, and chicken muscle (13). As an indication of the stepwise separation of the PGA mutase from the other erythrocytic enzymes in the preparation described above, all of the enzymes of glycolysis, glucose-6-phosphate dehydrogenase, 6-phosphogluconic acid dehydrogenase, and glutathione reductase were assayed at, each stage of purification. The only activities other than PGA mutase remaining after the final stage of purification were trace quantities of triose isomerase and phosphoglyceric acid kinase. Characteristics of PGA mutase. The pH of optimal enzyme activity for the mutase was 7.0. In the presence of 0.25 mM 2,3-DPG the K, for 3-PGA was found to be 0.8 rnbl.
2,3-DPG
PHOSPHATASE
ACTIVITY
Under the conditions used here, when assayed with 3-PGA that had been completely freed of contaminating 2,3-DPG, enzyme activity was entirely dependent upon the addition of 2,3-DPG. The K, for 2,3-DPG in the presence of 50 ma 3-PGA as determined from the double reciprocal plot of Lineweaver and Burk (14) was 6 mM. These values are similar to those for PGA mutases purified from caprine erythrocytes (15) and yeast (1). Identity of PGA-mu&se and 2,3-DPG phosphatase activities. The PPi-stimulated 2,3-DPG phosphatase and the PGA mutase activities purified together. The elution profiles of the two activities were identical upon column chromatography on hydroxylapatite
OF PGA MUTASE
(Fig. 3) and DEAESephadex (Fig. 4), gel filtration on Sephadex G-200, and by sucrose gradient centrifugation. The other 2,3DPG phosphatase, which is stimulated by sodium bisulfite and not by PPi, was clearly separated on hydroxylapatite (Fig. 3). When the partially purified PGA mutase was heated at 45“ at pH 8.0, both the mutase and phosphatase activities were lost at the same rate with all activity destroyed after heating for 1 hr. The presence of 2 mM MgC% during heating had no protective effect. Both enzymatic activities were protected against inactivation by heating at this temperature in the presence of 20 mu 2,3-DPG or 3-PGA, or 40 mM PPi. With these additions there was no significant loss
4.1
r > .?I 2 3. x .> ." 4 2. Q) E; ; 1.
Fraction
213
Number
FIG. 4. Chromatography of 2,3-DPG phosphatase on DEAE-Sephadex. Forty milligrams of protein were applied to a 1 X 25-cm column of DEAE-Sephadex. The column was then developed at 3” with a linear gradient composed of 500 ml 10 mu Tris-chloride buffer, pH 7.5, containing 1 mu p-mercaptoethanol and 1 mu EDTA in the mixing vessel and 500 ml of the same mixture containing 0.25 M KC1 in the reservoir. The flow rate was 1.0 ml/ min; 9.0~ml fractions were collected. Fractions were assayed for PGA mutase activity (O-O) and for pyrophosphate-stimulated 2,3-DPG phosphatase activity m.4).
214
HARKNESS
>k 5 4 60Q F z40t z E a
20-
00
64.5', pH 8.0
15
30
45
60
TIME (minutes)
FIG. 5. The effect of heating PGA mutase in the presence of 2,3-DPG, 3-PGA, or PPi at 64.5’. Incubation mixtures contained approximately 2 mg enzyme (20 units/mg protein); 106 mM Trischloride buffer, pH 8.0; and 20 mu 2,3-DPG (m), 20 mM 3-PGA (A), or 40 mM PPi (a). These were heated at 64.5”. Samples were removed at the intervals noted and frozen until assayed for PGA mutase (---) and PPi-stimulated phosphatase (- - -) activities.
of either phosphatase or mutase activity in 1 hr of heating up to 60”. The results of heating the enzyme at 64.5’ are shown in Fig. 5. At this temperature there was no protection of either activity by 3-PGA or PPi and only moderate protection against heat inactivation by 2,3-DPG. There was no appreciable disparity between the effects on mutase and phosphatase activities. The e$ect of PP; on the phosphatase activity. The phosphatase activity of the purified erythrocytic PGA mutase (Fig. 6) is markedly enhanced by PPi. We have used 20 m&r PPi in our routine phosphatase assays, a level giving nearly maximal stimulation. This gives a 20- to 50-fold increase in activity over reaction mixtures containing no PPi. A K, of approximately 3 mM is obtained by replotting the results of the experiment represented in Fig. 6 after Lineweaver and Burk (14). The pH optimum for
ET AL.
the phosphataxe activity is 6.5 with or without PPi. Inorganic pyrophosphate effects the phosphatase activity by altering the I’,,,-, but not the affinity of the enzyme for substrate. A double reciprocal plot of phosphatase activity versus substrate concentration in the presence of PPi appears in Fig. 7. The K, for 2,3-DPG is 0.2 m&i. Nearly the same K, is obtained in experiments with no added PPi. To investigate the possible direct participation of pyrophosphate in the reaction, 16 units mutase in 100 ml1 Tris-acetate buffer, pH 6.5, 5 m&i 2,3-D32PG, and 50 m&z PPi, and a control reaction mixture without enzyme were incubated at 37” for 90 min. The reaction mixtures mere boiled for 5 min, diluted to 100 ml, and 25 pmoles each of carrier 3-PGA and 2,3-DPG were added. The samples were applied to 1 X 15-cm columns of Dowex-1 Cl- which were successively washed with 100 ml distilled water, 300 ml 0.01 N HCl, and 200 ml 0.02 N HCl. Then a 500-ml linear gradient from 0.02 to 0.1 N HCl was pumped over the columns, and lo-ml fractions were collected. A similar reaction mixture containing half as much PPi and 50 rnkr Pi was handled in identical manner. Total phosphate, radioactivity, and glyceric acid were measured on each fraction from the gradients. The PPi was eluted in fractions 23 through 30 and 2,3DPG from 34-43. No radioactivity appeared in the PPi. Similar experiments were performed in which 32PPi was incubated together with enzyme, buffer, and either 2,3-DPG or 3PGA. Carrier 2,3-DPG and 3-PGA were added, and the monophosphoglyceric acid, PPi, and 2,3-DPG separated from one another as in the previous experiment. nTo radioactivity was found in either PGA or 2,3-DPG. It was concluded that pyrophosphate does not enter into any exchange or phosphoryl transfer reaction with either 2,3-DPG or 3-PGA. The e$ect of intracellular metabolites on the phosphatase activity of PGA mutase. Since the human erythrocytes contain no measurable inorganic pyrophosphate, a survey of various intracellular metabolic intermediates as
2,3-DPG PHOSPHATASE
I
ACTIVITY
I
OF PGA MUTASE
I
I
I
.““,M
4o
5o
215
IO “[PPi]
FIG. 6. Stimulation of 2,3-DPG phosphatase activity by inorganic pyrophosphate. Reaccontained 100 mu Tris-acetate buffer, pH 6-5; tion mixtures, in quadruplicate, 1 mu 2,3-D3*PG (3.6 X lOa cpm/gmoIe); enzyme, 160 Mg (spec. act, 0.9); and from 0 to 50 rnM PPi . Duplicates were stopped at 0 time and after 60 min at 37” and prepared for counting as described under Methods.
FIG. 7. Effect of substrate concentration upon velocity of the reaction. Reaction mixtures contained 100 mu Tris-acetate buffer, pH 6.5; 20 mM pyrophosphate; PGA mutase, 0.5 units; and from 2.3 to 55 PM 2,3-D3*PG (lo6 cpm/pmole). K, = 2 x 10e4M.
possible activators of the phosphatase activity of this enzyme was carried out. Compounds were tested at 20 and 2 rnM concentrations. At 20 m&f concentrations, a few
compounds had more than 10% of the activity of PPi. These are listed in Table II. At 2 mM concentrations, the activity with these compounds was little more than with
216
ET AL.
HARKNESS TABLE
II
SURVEYOFCELLULARMET~BOLITESFORPOSSIBLE STIMULATION OF 2,3-DPG PHOSPHBTSSE ACTIVITY"
20mM (%I PPi No addition G 6-P Fru-1,6-di-P PEP 6-PGA TPN ATP CTP UTP GTP ITP ADP UDP
100 2 11.0 14.8 13.4 15.0 23.8 14.0 12.2 13.2 15.8 14.5 10.9 10.3
2 m (%‘c) 5 2 1.1 0.8 3.0 2.4 5.0 2.5 2.2 3.7 6.9 5.1 0.7 2.3
a Reaction mixtures contained 100 mM Trisacetate buffer, pH 6.5; 1 mM 2,3-Da2PG; 0.5 units PGA mutase; and either 20 or 2 mM concentration of the other additions indicated. All activities are expressed relative to the activity in 20 mM PPi.
enzyme alone. At 20 rnM concentrations, the following compounds gave from 5 to 10 % of the stimulation noted with PPi : fructose 6-P, glyceraldehyde 3-P, 2-phosphoglyceric acid, ribose 5-P, cytidine diphosphate, and guanosine diphosphate. Glucose, pyruvate, lactate, glycerol, &glycerophosphate, DPN, 3-phosphoglyceric acid, adenylic acid, cytidylic acid, inosinic acid, uridine, inosine, and cytidine effected less than a 5 % stimulation. Hexametaphosphate stimulated the enzyme 17 and 26% when added at 2 and 20 rnM concentrations. Several metal chelators such as EDTA, citrate, ar,~dipyridyl, and o-phenanthroline all failed to activate the phosphatase. Inhibitor studies. The effects of several compounds upon the activity of 2,3-DPG phosphatase activity in the presence of pyrophosphate were tested (Table III). No significant inhibition was seen with 20 mM citrate, EDTA, NaF, or NaCl. There was moderate inhibition by 20 rnwl iodoacetate and 20 mu Pi. At 2 m&I concentration, there was some inhibition by CX,CY-dipyridyl and
o-phenanthroline and somewhat more by p-hydroxymercuribenzoate. Both 3-PGA and 2-PGA inhibit the PPistimulated phosphatase activity of PGA mutase. However, Lineweaver and Burk plots of this data do not give straight lines (Fig. 8). The probable explanation is that rapid exchange and mutase reactions take place, thereby lowering the specific activity of the substrate. Hence, the greater the ratio TABLE
III
EFFECTS OF VARIOUS COMPOUNDS ON PHOSPHATASE ACTIVITY OF PGA MUTASE~
-.
20ml.5(%I 2mu (%)
Addition None Citrate EDTA NaF p-Hydroxymercuribenzoate Iodoacetate 01,or-Dipyridyl o-Phenanthroline Pi NaCl
100 82 104 95 65 70 98
85 92 90 105 88 66 72 95 -
a Incubation mixtures contained 100 mM Tris-acetate buffer, pH 6.5; 1 mM 2,3-DaaPG; 0.5 units of PGA mutase; 20 mM PPi; and either 20 or 2 mM of the other additions as indicated. 3
I
I
50uM
3-PGA
FIG. 8. Inhibition of 2,3-DPG phosphatase by 3-PGA. Reaction mixtures contained from 4.6 to 46 PM substrate; 20 rnM PPi; 100 mM Tris-acetate, pH 6.5; enzyme and 50 PM, 5 @M, or no 3-PGA.
2,3-DPG PHOSPHATASE
ACTIVITY
of inhibitor to substrate, the greater the apparent inhibition. Attempt at reversing the phosphatase activity. Mixtures of radioactive inorganic phosphate, 100 rnM (90,000 cpm/pmole) ; 3-PGA, 10 rn>r i; Tris-acetate buffer, pH 6.5, 100 mM; 4 units of enzyme! with and without 20 m&l PPi in a total volume of 4.0 ml were incubated for 3 hr at 37”. The reaction mixtures were then diluted to 50 ml with water and, after addition of 25 pmoles carrier 2,3-DPG, applied to Dowex-1 Cl- columns. The 2, X-DPG, isolated free of Pi and 3-PGA by gradient elution with dilute HCl, contained no radioactivity. DISCUSSION
Zancan, Recondo, and Leloir (2) reported studies of a search for enzymes capable of adenylyl diphosphoglyceric catabolizing acid, a nucleotide reportedly isolated from porcine and human erythrocytes (16, 17). They described an enzymatic activity in rabbit muscle which hydrolyzed the free phosphate in the 2-position of both this nucleotide and free 2,3-DPG. The hydrolysis was stimulated by inorganic pyrophosphate with optimal concentration around 20 mnI. The pH of optimal activity was between 6.5 and 7.0. In a second paper from Leloir’s laboratory, the authors reported their inability to separate this 2,3-DPG phosphatase activity from PGA mutase (3). However, after comparing phosphatase, transferase, and mutase activities at various stages of purification of the phosphatase in rabbit muscle, it was concluded that the transferase and phosphatase activities were catalyzed by the same enzyme and that PGA mutase was probably a separate enzyme. Grisolia and co-workers (1) have reported that crystalline PGA mutases possess 2,3DPG phosphatase activity and that at least one highly purified 2,3-DPG phosphatase exhibited the mutase activity (18). Immunological studies with yeast enzyme supported the contention that the two activities resided on the same protein (19). However, similar studies with muscle enzymes gave conflicting results. The anti-PGA mutase inhibited the mutase activity but not the phosphatase activity (13).
OF PGA MUTASE
217
The data presented in this paper on an enzyme purified from yet another source, the, human erythrocyte, also indicate that PGA mutase has inherent 2,3-DPG phosphatase activity. The identical elution profiles of the PPi-stimulated phosphatase and mutase activities from hydroxylapatite and DEAESephadex columns and the failure to dissociate these activities by gel filtration on Sephadex G-200 or by sucrose gradient centrifugation are strong evidence that one enzyme, or an enzyme complex, is responsible for both activities. The results of the heat denaturation studies and the parallel protection of both activities by PPi, 3-PGA, and 2,3-DPG support this conclusion. It seems most unlikely that if there were two separate proteins, all three compounds would interact with both to afford similar degrees of protection against inactivation by heating. Grisolia and Tecson (20) induced a reversible transformation of PGA mutase into 2,3-DPG phosphatase with Hg2+. This interconversion occurred in all animal PGA mutases tested, all of which contain SH groups and which require 2,3-DPG as cofactor. This interconversion was not possible with yeast PGA mutase, which contains no SH groups or with plant PGA mutases which do not require 2,3-DPG for activity. They interpreted their data as further support that in the case of animal mutases both enzymatic activities are associated with the same protein. It is of interest that the phosphatase of chicken muscle purified after Joyce and Grisolia (18) was active ivithout PPi and that added PPi gave no enhancement of activity (2). During preparation of that enzyme, it is treated with Hg2+ and apparently is already optimally “activated.” Using radioactive PPi or 2,3-DPG we were unable to obtain any evidence that PPi enters directly into the enzymatic reaction. It seems likely that the PPi, which affects the V,,, but not the K, for substrate, alters the conformation of the enzyme in such a way as to enhance phosphatase activity without appreciably decreasing its mutase activity. As noted above, Hg2+, on the other hand, stimulates the phosphatase activity but inhibits the mutase activity. Stimulation by PPi does not appear to be the
21s
HARKNESS
result of metal chelation since other chelators had little effect on the phosphatase activity. Considerable interest in the cellular mechanisms regulating the levels of 2,3DPG in the mammalian erythrocyte has been generated by the recent demonstration of the effect of this compound on the position of the oxygen dissociation curve of hemoglobin (20, 21). 2,3-Diphosphoglyceric acid is synthesized from 1 ,3-DPG and 3-PGA by an enzyme first described by Rapoport and Luebering (23) and recently studied in detail by Rose (24). This enzyme, diphosphoglycerate mutase, is extremely sensitive to inhibition by product having a Ki of 0.85 ~~11(24). Several findings suggest that a plausible mechanism for regulation of 2,3DPG synthesis is its exclusive binding to deoxyhemoglobin (25) with consequent protection of the enzyme from inhibition. Hypoxia from many causes is associated with elevated intraerythrocytic levels of 2,3-DPG (26, 27, 28). In red cell hemolysate;, glycolysis and 2,3-DPG synthesis are increased under nitrogen but not under carbon monoxide or oxygen (29, 30). 2,3DPG synthesis is not inhibited by the addition of fetal deoxyhemoglobin which does not significantly bind 2,3-DPG (31, 32, 33). A second means of controlling intracellular concentrations of 2,3-DPG is regulation of its catabolic rate. Rapoport and Luebering (34) showed that 2,3-DPG is degraded by a p’rosphatase which hydrolyzes it to Pi and phosphomonoglycerate. We were intrigued by the possibility that the erythrocyte, a cell which is unique in its high content of 2,3DPG and also in deriving all of its energy from glycolysis, might utilize the same enzyme for catabolizing 2,3-DPG and for the mutase reaction which requires 2,3-DPG as a cofactor. It suggested that there might be interesting control mechanisms for regulating the act,iTvlties of such an enzyme. The fact that a small molecule such as PPi could so remarkably enhance the phosphatase activity made it seem plausible that the cell might indeed use this enzyme in a dual way. Erythrocytes contain no- measureable PPi; this may be attributable to the presence of a potent pyrophosphatase (25). A search for
ET AL.
other possible activators was therefore conducted but failed to reveal significant enhancement of enzymatic activity by physiological concentrations of any intracellular compounds studied. Acid extracts and extracts of boiled erythrocytes also had no effect. The presence of a second 2,3-DPG phosphatase in mammalian erythrocytes was first demonstrated by MBnyai and VBrady (36). Studies on some properties of this enzyme have been published by deverdier (37) and by us (38). It is stimulated approximately 40-fold by 20 m&r sodium bisulfite. Rose has demonstrated that inorganic phosphate and chloride together can effect a similar degree of stimulation (39). In contrast to the PPistimulated enzyme, this enzyme is inhibited by sulfhydryl reagents. The erythrocyte of the goat has very low concentrations of 2,3DPG, shows minimal capacity for its synthesis, and contains almost none of this enzyme (15). It can be argued that the erythrocytes of the goat have no need for a cofactor for hemoglobin deoxygenation since caprine hemoglobin has such a low affinity for oxygen that it dissociates sufficiently at capillary PO2 for adequate tissue oxygenation (40). On the other hand, we have purified PGA mutase from caprine erythrocytes and found that it has considerable PPi-stimulated phosphatase activity and is similar in all other respects to the enzyme from human erythrocytes described in this paper (15). In conclusion, we have demonstrated that under the influence of PPi, PGA mutase purified from human erythrocytes possesses 2,3-DPG phosphatase activity. There is no evidence to support a physiological role for this activity. Our inability to identify any plausible intracellular activator for this enzyme and the presence of a second enzyme in the erythrocyte with the same activity causes us to believe that PGA mutase may not function as a phosphatase within the cell. The almost complete absence of this second phosphatase in some mammalian erythrocytes such as those of the goat, which also contain little 2,3-DPG or diphosphoglyceric acid mutase and yet possess PPi-stimulated phosphatase activity proportionate to mutase activity, supports this conclusion.
2,3-DPG
PHOSPHATASE
ACTIVITY
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19. GRISOLI~, S., AND DETTER, J. C., Biochem. 2. 342, 239 (1965), S., .~ND TECSON, J., Biochim. Bio20. GRISOLI.~, phys. Acta 132, 56 (1967). 21. CHANUTIN, A., .~ND CUI~UISH, R. P., Arch. Biochem. Biophys. 121, 96 (1967). 22, BENESCH, R., AND BENESCH: R. E., Biochem. Biophys. Res. Commun. 26, 162 (1967). 23. RAPOPORT, S., AND LUERERING, J., J. Biol. Chem. 196, 583 (1952). 24. ROSE, Z. B., J. Biol. Chem. 243, 4810 (1968). R., AND BENESCH, R. E., Nature 25. BENESCH, London 221, 618 (1969). 26. LENFANT, C., TORRANCE, J., ENGLISH, E., FINCH, C. A., REYNBFARJE, C., RAMOS, J., AND FAURA, J., J. Clin. Invest. 47, 2652 (1968). 27. OSKI, F. A., GOTTLIEB, A. J., DELIVORLP~PADOPOULOS, M., AND MILLER, M., ,V. Engl. J. Med. 280, 1165 (1969). 28. EATON, J. W., AND BREWER, G. J., Proc. Nat. .4cad. Sci. U.S. 61, 756 (1968). 29. ASAKURA, T., SATO, Y., MINAKaMI, S., AND YOSHIKAWA, H., J. Biochem. Tokyo 6, 524 (1966). J., TROTMAN, B., GOTTLIEB, A., 30. WEISSHERG, AND OSKI, F., C&n. Res. 16, 543 (1968). 31. OSKI, F., MILLER, W., DELIVORIA-PAPADOPOULOS, M., AND GOTTLIER, A., Pediat. Res. 3, 376 (1969). 32. BAUER, CH., LUDWIG, I., AND LUDWIG, M., Life Sci. 7, 1339 (1968). I., AND SHIMIZU, K., ilrch. Biochem. 33. TYUMA, 129, 404 (1969). S., AND LUEHERING, J., J. Biol. 34. RAPOPORT, Chem. 189, 683 (1951). 35. PYNES, G. B., AND YOUNATHAN, E. S., J. Biol. Chem. 242, 2119 (1967). S., AND VhRADY, Zs., Acta Physiol. 36. M~NYAI, 14, 103 (1957). 37. DEVERDIER, C. H., Folia Haematol. Leipzig 90, 215 (1968). 38. HARKNESS, D. R., AND ROTH, S., Biochem. Biophys. Res. Commun. 34, 849 (1969). 39. ROSE, Z. B., Fed. Proc. Fed. Amer. Sot. Exp. Biol. 28, 727 (1969). 40. RIEGEL, K., HILPERT, P., AND BARTELS, H., Acta Haematol. 26, 164 (1961).