Proc. Nati. Acad. Sci. USA Vol. 74, No. 9, pp. 3701-3704, September 1977
Biochemistry
Purification and properties of human erythrocyte pyrimidine 5'-nucleotidase (chromatography/stability/kinetics/hemolytic anemia)
J. D. TORRANCE*, D. WHITTAKER*, AND E. BEUTLERtt * Medical Research Council Iron and Red Cell Metabolism Unit, Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa; and t City of Hope National Medical Center, Duarte, California 91010
Contributed by Ernest Beutler, June 13, 1977
ABSTRACT A 250,000-fold purification of pyrimidine 5'nucleotidase from human erythrocytes has been achieved using a combination of DEAE-ceflulose chromatography, ammonium sulfate fractionation, gel filtration, and isoelectric focusing. Polyacrylamide disc and starch gel electrophoreses of the purified material show two strong protein bands. On starch gel these bands exhibited pyrimidine 5'-nucleotidase activity. Two faint protein bands devoid of enzyme activity were also found in the case of polyacrylamide electrophoresis. The enzyme has a H optimum at 7.5 and is most stable between pH 6 and 7.5. The enzyme has a pI of 5.0 and a molecular weight of 28,000 by gel filtration. The Km of the purified enzyme was 10 #M, compared to 40 ,uM when measured in hemolysate. The higher Km in the hemolysate is due to the presence of an inhibitor. Inorganic phosphate was shown to be a competitive inhibitor of pyrimidine 5'-nucleotidase and inorganic phosphate in the hemolysate may be responsible for increasing the Km of the enzyme for the substrate cytidine monophosphate. In 1974 Valentine et al. (1) reported the presence of a pyrimidine-specific 5'-nucleotidase in the soluble fraction of normal human erythrocytes. The enzyme catalyzes the hydrolytic dephosphorylation of pyrimidine 5'-ribose monophosphates but is ineffective with purine nucleotides. A deficiency of the enzyme results in a hereditary nonspherocytic hemolytic anemia in which the erythrocytes contain extremely high levels of pyrimidine nucleotides and show marked basophilic stippling. It was the investigation of this hereditary condition that led Valentine et al. (1) to identify the enzyme. Several additional kindreds having anemia associated with erythrocyte pyrimidine 5'-nucleotidase deficiency have been discovered (2-4) since its description in 1974. It seems likely that this enzyme deficiency will prove to be a fairly common cause of nonspherocytic hemolytic anemia. The characteristics of the enzyme in hemolysates have subsequently been examined by Paglia and Valentine (5). This paper presents a scheme for the partial purification of the enzyme pyrimidine 5'-nucleotidase from human erythrocytes and describes some properties of the partially purified enzyme. METHODS Enzyme Assay. Pyrimidine 5'-nucleotidase activity was measured by a radiometric assay (4). Samples were incubated at 370 with 0.3 mM [2-14C]CMP substrate, 5 mM MgCI2, and 20 mM Tris-HCl buffer at pH 7.6 in a final volume of 100 ul. The [2-14C]CMP substrate (Schwarz/Mann) was purified on Dowex-1 before use (4). The reaction was stopped and unreacted substrate was precipitated by addition of 200 Al each of 0.15 M Ba(OH)2 and ZnSO4. Radioactivity present in [214C]cytidine formed was estimated by counting an aliquot of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
supernatant in 5 ml of Instagel (Packard). Because salt concentrations above 200 mM prevent quantitative precipitation of CMP it is necessary to run a zero time blank to verify that removal of unreacted substrate is complete. Preparation of Hemolysates of Young and Old Erythrocytes. Cells were separated according to their densities by the method of Danon and Marikovsky (6). The blood was first filtered through a column of microcrystalline cellulose and acellulose (Sigma) to remove leukocytes and platelets (7), and then washed three times with saline, and the washed cells were suspended in an equal volume of physiological saline. The cell suspension was layered above phthalate ester in a microhematocrit tube. A set of tubes having phthalate ester densities ranging from 1.122 to 1.070 g/cm3 was prepared by mixing methyl phthalate and di-n-butyl phthalate in the appropriate proportions. The tubes were centrifuged at 40 for 15 min at 12,000 X g using a Hawksley microhematocrit centrifuge. Hemolysates of heavy and light packed cells were made by addition of 20 volumes of a solution containing 0.05 ml of 2mercaptoethanol and 10 ml of neutralized 10% EDTA per liter
(8).
Protein Determinations. Because [bis(2-hydroxyethyl) amino]tris(hydroxymethyl)methane (Bis-Tris) buffers interfere with the Lowry method (9), this technique could be used for protein determinations only after dialysis. Alternatively, measurements of absorbance at 260 and 280 nm (10) were employed.
RESULTS Purification of pyrimidine 5'-nucleotidase All steps in the purification procedure were carried out at a temperature between 0 and 60, and all buffers used contained 0.7 mM 2-mercaptoethanol and 2.7 mM EDTA unless otherwise
specified. Preparation of Hemolysate. A unit of ACD (acid-citratedextrose) blood (either fresh or outdated bank blood) was centrifuged, and the plasma and buffy layer were aspirated. The packed cells were then resuspended in physiological saline, and white cells and platelets were removed by filtering through a 5-cm bed prepared by mixing equal weights of a-cellulose and microcrystalline cellulose (Sigma). The erythrocytes were washed through the cellulose with additional saline (7). After it had been verified that no white cells were present, the erythrocytes were washed three times with saline, and then hemolyzed by adding four volumes of 10 mM Bis-Tris buffer, pH 6.5. Batchwise Purification on DEAE-Cellulose. The pH of the Abbreviation: Bis-Tris, [bis(2-hydroxyethyl)aminojtris(hydroxymethyl)methane. t To whom reprint requests should be addressed.
3701
3702
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Torrance et al.
FIG. 2. Polyacrylamide disc electrophoresis of 250,000-fold purified pyrimidine 5'-nucleotidase from human erythrocytes. Migration is toward the anode, at the right, and protein is stained with Coomassie blue.
FIG. 1. Isoelectric focusing of Sephacryl S-200 fractions that had pyrimidine 5'-nucleotidase activity. Focusing was carried out at 500 V for 48 hr at 60 with the cathode at the top in a 1% Ampholine column stabilized by a 0-45% sucrose gradient. Protein was estimated by absorbance at 280 nm and enzyme activity is expressed as % substrate converted by a 10-,vl sample in 6 min. The pH values were determined at 60 immediately after removal of the sample from the column.
hemolysate was measured and if necessary readjusted to pH 6.5. The solution was passed through a 5-cm bed of DEAE-cellulose in a 10-cm sintered glass funnel. The DEAE-cellulose, previously equilibrated with 10 mM Bis-Tris-HCI buffer at pH 6.5, was washed with this buffer until all hemoglobin had been eluted. Gentle suction was used to speed up the procedure. The DEAE-cellulose was then washed with 500 ml of the buffer containing 50 mM NaCl before eluting the absorbed pyrimidine 5'-nucleotidase with 500 ml of the same buffer containing 150 mM NaCl. Ammonium Sulfate Precipitation. Solid ammonium sulfate was added to the eluted enzyme solution to give a final concentration of 50%, and immediately thereafter the pH was readjusted to 6.5. After 20 min at 00 the precipitate was removed by centrifugation for 30 min at 14,000 X g and discarded. Additional ammonium sulfate was added to increase the saturation to 80% and the pH was again immediately adjusted to 6.5. After an hour at 00 the precipitate was removed by centrifugation for 1 hr at 14,000 X g, and was dissolved in a minimal volume of 10 mM Bis-Tris at pH 6.5. DEAE-Cellulose Column Chromatography. The residual ammonium sulfate was removed by buffer exchange into 10 mM Bis-Tris at pH 6.5 using Sephadex G-25. The enzyme was then chromatographed on a 1.6 X 20-cm column of DEAEcellulose equilibrated with 10 mM Bis-Tris at pH 6.5. The column was eluted with 500 ml of a linear sodium chloride gradient, 0-250 mM. Gel Filtration on Sephacryl S-200. The fractions containing
the enzyme were pooled and the enzyme was precipitated with 80% ammonium sulfate. The precipitate was collected by centrifugation at 14,000 X g and dissolved in the minimum volume of 10 mM Bis-Tris at pH 6.5. The concentrated enzyme was chromatographed on a 1.6 X 90-cm column of Sephacryl S-200 in 10 mM Bis-Tris at pH 6.5. Isoelectric Focusing. Fractions from the Sephacryl column
that showed pyrimidine 5'-nucleotidase activity were pooled and electrofocused in a 1 10-ml LKB 8100 electrofocusing column with the cathode at the top, using the method described by the manufacturer (11). The column, which was stabilized by a 0-45% sucrose gradient, contained 1% Ampholine (LKB), pH range 4-6, and 1.4 mM 2-mercaptoethanol. The sample was introduced into the middle of the sucrose gradient during column preparation. Focusing was carried out for 48 hr at 60 and 500 V. Fig. 1 shows that the enzyme focused to a sharp band at pH 5.0. The peak containing the pyrimidine 5'-nucleotidase was finally rechromatographed on a 1.6 X 90-cm Sephacryl S-200 column in 10 mM Bis-Tris at pH 6.5 to remove all sugar and Ampholine contamination. The degree of purification and the yield of each of the purification steps are summarized in Table 1. Two different protein concentrations are shown because of the difficulty encountered in protein determination. Electrophoresis. The purity of the electrofocused preparation was assessed by disc electrophoresis using the method of Davis (12) as modified by the Buchler Co. (13). Fig. 2 shows two dense bands and two faint bands of protein. Because staining of the polyacrylamide gels for the enzyme gave poor results, electrophoresis was also carried out in histidine/citrate buffer at pH 7 on a starch gel, in which the enzyme could be stained (14). The gel was coated for 2 hr with 20 ml of 1% ionagar in 0.05 M Tris-HCl buffer at pH 7.8 containing 20 mg of UMP, 5 mg of reduced glutathione, and 120 mg of MgSO4. This was removed and the starch gel was coated with 1% ionagar in 1 M H2SO4 containing 1 g of ascorbic acid and 250 mg of ammonium molybdate. Two blue zones were found, showing the presence of two bands of pyrimidine 5'-nucleotidase.
Table 1. Purification of human erythrocyte pyrimidine 5'-nucleotidase Enzyme activity Yield, % Milliunitst
Spectrophotometric protein assay Purification, Protein, Specific fold mg activity$ X 103
Protein, mg
Lowry protein assay* Specific Purification, fold activityt X 103
1 0.11 0.11 1 100 45,000 5,000 45,000 Hemolysate Batchwise DEAE367 15.1 34 164 111 cellulose 1,486 3,339 5,559 291 471 15.5 90 9.58 Ammonium sulfate 2,646 4,280 4,511 5.98 466 2.56 55.8 4,241 DEAE-cellulose 9,923 1,092 2,790 0.45 45 0.31 45,111 4,962 6,540 7,200 2,233 Sephacryl S-200 0.032 18 0.023 888 252,272 27,750 Electrofocused 354,067 38,947 * the method. for used were protein assays by Lowry Dialyzed samples t One unit of pyrimidine 5'-nucleotidase is defined as the amount of enzyme required to hydrolyze 1 ,mol of CMP per min under the assay conditions. $ The specific activity is defined as units per mg of protein.
Biochemistry:
Torrance et al.
Proc. Natl. Acad. Sci.
USA
74 (1977)
3703
loo1
(WU L
90
0
4.a 30
Ia
80*
(U ._
C
E
*gC670
.C/ 20-
E N
0
co
C
._) L'
50-
o
10-.
I 5.5
An
5.5
6.0
6.5
7.0
pH at 370
7.5
8.0
8.5
FIG. 3. The effect of pH on erythrocyte pyrimidine 5'-nucleotidase activity. The buffer was an equimolar mixture of Tris and Bis-Tris adjusted to the required pH with HCl. The pH values were measured at 37° in the final incubation medium.
Properties of pyrimidine 5'-nucleotidase Effect of pH on Activity. The pH optimum of purified pyrimidine 5'-nucleotidase was studied using a buffer containing equimolar amounts of Tris and Bis-Tris and adjusted to the required pH with HCI. The concentration of each buffer in the assay was 50 mM and the pH ranged from 5.5 to 8.5. The pH values were determined in the final assay mixture at 37° using a Radiometer thermostated K 497 blood electrode. Fig. 3 shows that the enzyme has a fairly sharp pH optimum at 7.5. Stability of the Enzyme. The effect of pH on the stability of the enzyme was determined by incubating the purified enzyme at 370 with buffer containing equimolar (25 mM) quantities of Tris and Bis-Tris adjusted to the required pH with HCI. After incubation the pH was readjusted towards the pH optimum by addition of Tris buffer at pH 7.6 to a final concentration of 50 mM and the activity was measured. The pH values, measured at 370, ranged from 5.6 to 8.1 during the incubation period and from 7.1 to 7.3 after addition of the pH 7.6 Tris buffer. Fig. 4 shows that the enzyme is most stable at a pH of 6.5 at 370. The error introduced into the measurement of the activity by differences in final pH was estimated from Fig. 3. At the lowest pH the activity would be underestimated by 3%, and at the highest pH overestimated by 5%. These differences would not materially alter the shape of the stability curve. Determination of Molecular Weight. The molecular weight of pyrimidine 5'-nucleotidase was estimated by descending chromatography on a 1.6 X 90-cm Sephadex G-150 column. Cytochrome c, ovalbumin, and bovine serum albumin served as reference markers. The reference proteins were chromatographed in 0.9% sodium chloride buffered by 10 mM Tris at pH 8 without EDTA or 2-mercaptoethanol. The pyrimidine 5'-nucleotidase, chromatographed in 10 mM Bis-Tris at pH 6.5, appeared to have a molecular weight of 28,000. Pyrimidine 5'-Nucleotidase Activity in Young and Old
Erythrocytes. The activity of pyrimidine 5'-nucleotidase decreases markedly with the age of the red cell. Table 2 shows that
6.0
6.5
7.0 pH at 370
7.5
8.0
8.5
FIG. 4. The stability of erythrocyte pyrimidine 5'-nucleotidase at 370 as a function of pH. The buffers were prepared using HCl to adjust the pH of an equimolar mixture of Tris and Bis-Tris. The pH was adjusted back towards the pH optimum with Tris at pH 7.6 before the enzyme activities were measured.
the activity of the enzyme in the lightest cells is 3 to 6 times that in the heaviest cells. The enzyme activity in the 60% of cells having intermediate density approximates the values obtained in the cells before density separation. Kinetics and Magnesium Requirements. The kinetic behavior of pyrimidine 5'-nucleotidase was examined in both crude hemolysate and in the purified state. A high-specificactivity [2-14C]CMP substrate was prepared by purification of the commercial substrate on a Dowex-1 column without adding carrier. A range of substrate concentrations was produced by addition of the appropriate amount of pure nonradioactive CMP. The double reciprocal plot obtained using the purified enzyme is shown in Fig. 5. The Km values obtained in this way had a range of 8-15 ,gM, with a mean of 10 ,uM. In the crude hemolysate the Km of the enzyme was 40juM (Fig. 5). As little as 0.05 mM Mg2+ was sufficient to give maximum enzymatic activity. Inorganic phosphate was a competitive inhibitor of the enzyme. DISCUSSION Enzymes with 5'-nucleotidase activity have been demonstrated to occur in a wide variety of organisms (15-17). Prior to the discovery by Valentine et al. (1) of a pyrimidine-specific 5'nucleotidase in human erythrocytes, all known 5'-nucleotidases were able to dephosphosphorylate both purine and pyrimidine nucleotides. The erythrocyte enzyme also differed markedly Table 2. Pyrimidine 5'-nucleotidase activity in erythrocytes of different density % of
Supranormal 5'-nucleotidase,
cells
5'-nucleotidase, milliunits/g Hb
cells
milliunits/g Hb
100
140
100
350
Normal % of
Whole blood
21 600 27 210 Light Medium 61.5 122 57 353 22 104 Dense 11.5 69 Values are shown for two subjects, one having normal and the other supranormal levels of the enzyme.
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Torrance et al.
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0
0
1/v
This work was supported, in part, by Grant HL 07449 from the National Institutes of Health, and by U.S. Public Health Service International Research Fellowship IF05 TW02190-01 (J.D.T.).
25-
100
0
tween pH 6.0 and 7.5. Activity of the purified enzyme was maximal at a Mg2+ concentration of 0.05 mM, a level much lower than that observed by Paglia and Valentine (5) in hemolysates, where activity was nearly maximal at 1.0 mM but increased slightly in the presense of 10 mM Mg2+. Kinetic studies with the purified enzyme showed that the Km of pyrimidine 5'-nucleotidase for CMP is 10 ,AM (Fig. 5). This is less than that found in hemolysates, 40,uM (Fig. 5) (5), indicating the presence of an inhibitor. Inorganic phosphate at concentrations of 1.3-5.3 mM is an inhibitor of the reaction and the Km for CMP at all concentrations of phosphate tested appeared to be 50 ,M. The exact nature of this inhibition is not yet understood.
200
100
400
IT
FIG. 5. Lineweaver-Burk plots for purified human erythrocyte pyrimidine 5'-nucleotidase (0) and for the enzyme in a 1:20 hemolysate (0). 1/v is the reciprocal of the number of millimoles of cytidine produced from CMP per min and 1/[SJ is the reciprocal of the concentration of CMP (nM). Open and filled symbols represent two separate experiments. The lines are least square fits for the combined result.
from other 5'-nucleotidases in that it was present in the soluble fraction of the cell, while previously described 5'-nucleotidases were primarily associated with the plasma membrane; indeed, the enzyme is used as a membrane "marker" in histological studies. 5'-Nucleotidases have been purified from many tissues (16) yielding a heterogeneous group of enzymes with a wide range of substrate preferences, pH optima, and metal ion requirements, as well as different responses to a variety of inhibitors. To our knowledge a description of a purification procedure for the pyrimidine-specific 5'-nucleotidase described by Valentine et al. (1) has not previously been published. Using classical purification procedures, we have achieved a 250,000-fold purification of pyrimidine 5'-nucleotidase from human erythrocytes in 18% yield. This preparation was not homogeneous; on disc polyacrylamide gel electrophoresis two dense and two faint bands of protein were obtained. On starch gel it was possible to stain the enzyme (14) and show that there were indeed two bands of enzyme with 5'-nucleotidase activity. Two bands of 5'-nucleotidase activity have also been observed on starch-gel electrophoresis by Giblett (14). Further purification was attempted by affinity chromatography using cytidine bound to Sepharose 4B, but this proved unsuccessful. The substituted agarose was able to bind the enzyme but we were unable to recover the enzyme from the affinity column. Indeed, it was surprising to note that while still bound to the cytidine column the enzyme appeared to retain its ability to hydrolyze [2-14C]CMP. The initial stepwise DEAE-cellulose procedure was included because it very rapidly removed most of the hemoglobin and other protein. This allowed the column DEAE-cellulose step to be carried out on a smaller column, which consistently gave better results than those obtained by applying the hemolysate directly to a large DEAE-cellulose column. The enzyme has a pH optimum at 7.5 and is most stable be-
1. Valentine, W. N., Fink, K., Paglia, D. E., Harris, S. R. & Adams, W. S. (1974) "Hereditary hemolytic anemia with human erythrocyte pyrimidine 5'-nucleotidase deficiency," J. Clin. Invest. 54,866-879. 2. Ben-Bassat, I., Brok-Simoni, F., Kende, G., Holtzmann, F. & Ramot, B. (1976) "A family with red cell pyrimidine 5'-nucleotidase deficiency," Blood 47, 919-922. 3. Rochant, H., Dreyfus, B., Rosa, R. & Boiron, M. (1975) First Case of Pyrimidine 5' Nucleotidase Deficiency in a Male. International Society of Haematology, European and African Third Meeting, London. August 24-28 1975, Abstract 1:19. 4. Torrance, J., West, C. & Beutler, E. (1977) "A simple radiometric assay for pyrimidine-5'-nucleotidase," J. Lab. Clin. Med., in press. 5. Paglia, D. E. & Valentine, W. N. (1975) "Characteristics of a pyrimidine-specific 5'-nucleotidase in human erythrocytes," J.
Biol. Chem. 250,7973-7979. 6. Danon, D. 4 Marikovsky, Y. (1964) "Determination of density distribution of red cell population," J. Lab. Clin. Med. 64, 668-674. 7. Beutler, E., West, C. & Blume, K. G. (1976) "The removal of leukocytes and platelets from whole blood," J. Lab. Clin. Med.
88,328-333. 8. Beutler, E. (1975) Red Cell Metabolism. A Manual of Biochemical Methods (Grune and Stratton, New York), 2nd ed., p. 10. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) "Protein measurement with the Folin phenol reagent," J. Biol. Chem. 193,265-275. 10. Fruton, J. S. & Simmonds, S. (1958) General Biochemistry (John Wiley and Sons, New York), 2nd ed., p. 74. 11. LKB-Produkter Instruction Manual for Ampholine 8100 Electrofocusing Equipment- (LKB-Produkter, Bromma, Swe-
den). 12. Davis, B. J. (1964) "Disc electrophoresis II. Method and application to human serum proteins," Ann. N.Y. Acad. Sci. 121, 404-427. 13. Buchler Co. (1971) Instructions for Polyanalyst and Instructions for PolyPrep 100 and 200 (Buchler Instruments, Fort Lee,
NJ).
14. Anderson, J. A., Teng, Y.-S. & Giblett, E. R. (1975) "Stains for six enzymes potentially applicable to chromosomal assignment by
cell hybridization," [Rotterdam Conference (1974): 2nd National Workshop on Human Gene Mapping], Birth Defects: Original Article Series 11, 295-299. 15. Heppel, L. A. (1961) "5' Nucleotidase," in The Enzymes, ed. Boyer, P. D. (Academic Press, New York), 2nd ed., Vol. 5, pp. 49-53. 16. Bodansky, 0. & Schwartz, M. K. (1968) "5' Nucleotidase," Adv. Clin. Chem. 11, 277-328. 17. Drummond, G. I. & Yamamoto, M. (1971) "Nucleotide phosphomonoesterases," in The Enzymes, ed. Boyer, P. D. (Academic Press, New York), 3rd ed., Vol. 4, pp. 337-354.
Biochemistry
Purification and properties of human erythrocyte pyrimidine 5'-nucleotidase (chromatography/stability/kinetics/hemolytic anemia)
J. D. TORRANCE*, D. WHITTAKER*, AND E. BEUTLERtt * Medical Research Council Iron and Red Cell Metabolism Unit, Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa; and t City of Hope National Medical Center, Duarte, California 91010
Contributed by Ernest Beutler, June 13, 1977
ABSTRACT A 250,000-fold purification of pyrimidine 5'nucleotidase from human erythrocytes has been achieved using a combination of DEAE-ceflulose chromatography, ammonium sulfate fractionation, gel filtration, and isoelectric focusing. Polyacrylamide disc and starch gel electrophoreses of the purified material show two strong protein bands. On starch gel these bands exhibited pyrimidine 5'-nucleotidase activity. Two faint protein bands devoid of enzyme activity were also found in the case of polyacrylamide electrophoresis. The enzyme has a H optimum at 7.5 and is most stable between pH 6 and 7.5. The enzyme has a pI of 5.0 and a molecular weight of 28,000 by gel filtration. The Km of the purified enzyme was 10 #M, compared to 40 ,uM when measured in hemolysate. The higher Km in the hemolysate is due to the presence of an inhibitor. Inorganic phosphate was shown to be a competitive inhibitor of pyrimidine 5'-nucleotidase and inorganic phosphate in the hemolysate may be responsible for increasing the Km of the enzyme for the substrate cytidine monophosphate. In 1974 Valentine et al. (1) reported the presence of a pyrimidine-specific 5'-nucleotidase in the soluble fraction of normal human erythrocytes. The enzyme catalyzes the hydrolytic dephosphorylation of pyrimidine 5'-ribose monophosphates but is ineffective with purine nucleotides. A deficiency of the enzyme results in a hereditary nonspherocytic hemolytic anemia in which the erythrocytes contain extremely high levels of pyrimidine nucleotides and show marked basophilic stippling. It was the investigation of this hereditary condition that led Valentine et al. (1) to identify the enzyme. Several additional kindreds having anemia associated with erythrocyte pyrimidine 5'-nucleotidase deficiency have been discovered (2-4) since its description in 1974. It seems likely that this enzyme deficiency will prove to be a fairly common cause of nonspherocytic hemolytic anemia. The characteristics of the enzyme in hemolysates have subsequently been examined by Paglia and Valentine (5). This paper presents a scheme for the partial purification of the enzyme pyrimidine 5'-nucleotidase from human erythrocytes and describes some properties of the partially purified enzyme. METHODS Enzyme Assay. Pyrimidine 5'-nucleotidase activity was measured by a radiometric assay (4). Samples were incubated at 370 with 0.3 mM [2-14C]CMP substrate, 5 mM MgCI2, and 20 mM Tris-HCl buffer at pH 7.6 in a final volume of 100 ul. The [2-14C]CMP substrate (Schwarz/Mann) was purified on Dowex-1 before use (4). The reaction was stopped and unreacted substrate was precipitated by addition of 200 Al each of 0.15 M Ba(OH)2 and ZnSO4. Radioactivity present in [214C]cytidine formed was estimated by counting an aliquot of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
supernatant in 5 ml of Instagel (Packard). Because salt concentrations above 200 mM prevent quantitative precipitation of CMP it is necessary to run a zero time blank to verify that removal of unreacted substrate is complete. Preparation of Hemolysates of Young and Old Erythrocytes. Cells were separated according to their densities by the method of Danon and Marikovsky (6). The blood was first filtered through a column of microcrystalline cellulose and acellulose (Sigma) to remove leukocytes and platelets (7), and then washed three times with saline, and the washed cells were suspended in an equal volume of physiological saline. The cell suspension was layered above phthalate ester in a microhematocrit tube. A set of tubes having phthalate ester densities ranging from 1.122 to 1.070 g/cm3 was prepared by mixing methyl phthalate and di-n-butyl phthalate in the appropriate proportions. The tubes were centrifuged at 40 for 15 min at 12,000 X g using a Hawksley microhematocrit centrifuge. Hemolysates of heavy and light packed cells were made by addition of 20 volumes of a solution containing 0.05 ml of 2mercaptoethanol and 10 ml of neutralized 10% EDTA per liter
(8).
Protein Determinations. Because [bis(2-hydroxyethyl) amino]tris(hydroxymethyl)methane (Bis-Tris) buffers interfere with the Lowry method (9), this technique could be used for protein determinations only after dialysis. Alternatively, measurements of absorbance at 260 and 280 nm (10) were employed.
RESULTS Purification of pyrimidine 5'-nucleotidase All steps in the purification procedure were carried out at a temperature between 0 and 60, and all buffers used contained 0.7 mM 2-mercaptoethanol and 2.7 mM EDTA unless otherwise
specified. Preparation of Hemolysate. A unit of ACD (acid-citratedextrose) blood (either fresh or outdated bank blood) was centrifuged, and the plasma and buffy layer were aspirated. The packed cells were then resuspended in physiological saline, and white cells and platelets were removed by filtering through a 5-cm bed prepared by mixing equal weights of a-cellulose and microcrystalline cellulose (Sigma). The erythrocytes were washed through the cellulose with additional saline (7). After it had been verified that no white cells were present, the erythrocytes were washed three times with saline, and then hemolyzed by adding four volumes of 10 mM Bis-Tris buffer, pH 6.5. Batchwise Purification on DEAE-Cellulose. The pH of the Abbreviation: Bis-Tris, [bis(2-hydroxyethyl)aminojtris(hydroxymethyl)methane. t To whom reprint requests should be addressed.
3701
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Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Torrance et al.
FIG. 2. Polyacrylamide disc electrophoresis of 250,000-fold purified pyrimidine 5'-nucleotidase from human erythrocytes. Migration is toward the anode, at the right, and protein is stained with Coomassie blue.
FIG. 1. Isoelectric focusing of Sephacryl S-200 fractions that had pyrimidine 5'-nucleotidase activity. Focusing was carried out at 500 V for 48 hr at 60 with the cathode at the top in a 1% Ampholine column stabilized by a 0-45% sucrose gradient. Protein was estimated by absorbance at 280 nm and enzyme activity is expressed as % substrate converted by a 10-,vl sample in 6 min. The pH values were determined at 60 immediately after removal of the sample from the column.
hemolysate was measured and if necessary readjusted to pH 6.5. The solution was passed through a 5-cm bed of DEAE-cellulose in a 10-cm sintered glass funnel. The DEAE-cellulose, previously equilibrated with 10 mM Bis-Tris-HCI buffer at pH 6.5, was washed with this buffer until all hemoglobin had been eluted. Gentle suction was used to speed up the procedure. The DEAE-cellulose was then washed with 500 ml of the buffer containing 50 mM NaCl before eluting the absorbed pyrimidine 5'-nucleotidase with 500 ml of the same buffer containing 150 mM NaCl. Ammonium Sulfate Precipitation. Solid ammonium sulfate was added to the eluted enzyme solution to give a final concentration of 50%, and immediately thereafter the pH was readjusted to 6.5. After 20 min at 00 the precipitate was removed by centrifugation for 30 min at 14,000 X g and discarded. Additional ammonium sulfate was added to increase the saturation to 80% and the pH was again immediately adjusted to 6.5. After an hour at 00 the precipitate was removed by centrifugation for 1 hr at 14,000 X g, and was dissolved in a minimal volume of 10 mM Bis-Tris at pH 6.5. DEAE-Cellulose Column Chromatography. The residual ammonium sulfate was removed by buffer exchange into 10 mM Bis-Tris at pH 6.5 using Sephadex G-25. The enzyme was then chromatographed on a 1.6 X 20-cm column of DEAEcellulose equilibrated with 10 mM Bis-Tris at pH 6.5. The column was eluted with 500 ml of a linear sodium chloride gradient, 0-250 mM. Gel Filtration on Sephacryl S-200. The fractions containing
the enzyme were pooled and the enzyme was precipitated with 80% ammonium sulfate. The precipitate was collected by centrifugation at 14,000 X g and dissolved in the minimum volume of 10 mM Bis-Tris at pH 6.5. The concentrated enzyme was chromatographed on a 1.6 X 90-cm column of Sephacryl S-200 in 10 mM Bis-Tris at pH 6.5. Isoelectric Focusing. Fractions from the Sephacryl column
that showed pyrimidine 5'-nucleotidase activity were pooled and electrofocused in a 1 10-ml LKB 8100 electrofocusing column with the cathode at the top, using the method described by the manufacturer (11). The column, which was stabilized by a 0-45% sucrose gradient, contained 1% Ampholine (LKB), pH range 4-6, and 1.4 mM 2-mercaptoethanol. The sample was introduced into the middle of the sucrose gradient during column preparation. Focusing was carried out for 48 hr at 60 and 500 V. Fig. 1 shows that the enzyme focused to a sharp band at pH 5.0. The peak containing the pyrimidine 5'-nucleotidase was finally rechromatographed on a 1.6 X 90-cm Sephacryl S-200 column in 10 mM Bis-Tris at pH 6.5 to remove all sugar and Ampholine contamination. The degree of purification and the yield of each of the purification steps are summarized in Table 1. Two different protein concentrations are shown because of the difficulty encountered in protein determination. Electrophoresis. The purity of the electrofocused preparation was assessed by disc electrophoresis using the method of Davis (12) as modified by the Buchler Co. (13). Fig. 2 shows two dense bands and two faint bands of protein. Because staining of the polyacrylamide gels for the enzyme gave poor results, electrophoresis was also carried out in histidine/citrate buffer at pH 7 on a starch gel, in which the enzyme could be stained (14). The gel was coated for 2 hr with 20 ml of 1% ionagar in 0.05 M Tris-HCl buffer at pH 7.8 containing 20 mg of UMP, 5 mg of reduced glutathione, and 120 mg of MgSO4. This was removed and the starch gel was coated with 1% ionagar in 1 M H2SO4 containing 1 g of ascorbic acid and 250 mg of ammonium molybdate. Two blue zones were found, showing the presence of two bands of pyrimidine 5'-nucleotidase.
Table 1. Purification of human erythrocyte pyrimidine 5'-nucleotidase Enzyme activity Yield, % Milliunitst
Spectrophotometric protein assay Purification, Protein, Specific fold mg activity$ X 103
Protein, mg
Lowry protein assay* Specific Purification, fold activityt X 103
1 0.11 0.11 1 100 45,000 5,000 45,000 Hemolysate Batchwise DEAE367 15.1 34 164 111 cellulose 1,486 3,339 5,559 291 471 15.5 90 9.58 Ammonium sulfate 2,646 4,280 4,511 5.98 466 2.56 55.8 4,241 DEAE-cellulose 9,923 1,092 2,790 0.45 45 0.31 45,111 4,962 6,540 7,200 2,233 Sephacryl S-200 0.032 18 0.023 888 252,272 27,750 Electrofocused 354,067 38,947 * the method. for used were protein assays by Lowry Dialyzed samples t One unit of pyrimidine 5'-nucleotidase is defined as the amount of enzyme required to hydrolyze 1 ,mol of CMP per min under the assay conditions. $ The specific activity is defined as units per mg of protein.
Biochemistry:
Torrance et al.
Proc. Natl. Acad. Sci.
USA
74 (1977)
3703
loo1
(WU L
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0
4.a 30
Ia
80*
(U ._
C
E
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.C/ 20-
E N
0
co
C
._) L'
50-
o
10-.
I 5.5
An
5.5
6.0
6.5
7.0
pH at 370
7.5
8.0
8.5
FIG. 3. The effect of pH on erythrocyte pyrimidine 5'-nucleotidase activity. The buffer was an equimolar mixture of Tris and Bis-Tris adjusted to the required pH with HCl. The pH values were measured at 37° in the final incubation medium.
Properties of pyrimidine 5'-nucleotidase Effect of pH on Activity. The pH optimum of purified pyrimidine 5'-nucleotidase was studied using a buffer containing equimolar amounts of Tris and Bis-Tris and adjusted to the required pH with HCI. The concentration of each buffer in the assay was 50 mM and the pH ranged from 5.5 to 8.5. The pH values were determined in the final assay mixture at 37° using a Radiometer thermostated K 497 blood electrode. Fig. 3 shows that the enzyme has a fairly sharp pH optimum at 7.5. Stability of the Enzyme. The effect of pH on the stability of the enzyme was determined by incubating the purified enzyme at 370 with buffer containing equimolar (25 mM) quantities of Tris and Bis-Tris adjusted to the required pH with HCI. After incubation the pH was readjusted towards the pH optimum by addition of Tris buffer at pH 7.6 to a final concentration of 50 mM and the activity was measured. The pH values, measured at 370, ranged from 5.6 to 8.1 during the incubation period and from 7.1 to 7.3 after addition of the pH 7.6 Tris buffer. Fig. 4 shows that the enzyme is most stable at a pH of 6.5 at 370. The error introduced into the measurement of the activity by differences in final pH was estimated from Fig. 3. At the lowest pH the activity would be underestimated by 3%, and at the highest pH overestimated by 5%. These differences would not materially alter the shape of the stability curve. Determination of Molecular Weight. The molecular weight of pyrimidine 5'-nucleotidase was estimated by descending chromatography on a 1.6 X 90-cm Sephadex G-150 column. Cytochrome c, ovalbumin, and bovine serum albumin served as reference markers. The reference proteins were chromatographed in 0.9% sodium chloride buffered by 10 mM Tris at pH 8 without EDTA or 2-mercaptoethanol. The pyrimidine 5'-nucleotidase, chromatographed in 10 mM Bis-Tris at pH 6.5, appeared to have a molecular weight of 28,000. Pyrimidine 5'-Nucleotidase Activity in Young and Old
Erythrocytes. The activity of pyrimidine 5'-nucleotidase decreases markedly with the age of the red cell. Table 2 shows that
6.0
6.5
7.0 pH at 370
7.5
8.0
8.5
FIG. 4. The stability of erythrocyte pyrimidine 5'-nucleotidase at 370 as a function of pH. The buffers were prepared using HCl to adjust the pH of an equimolar mixture of Tris and Bis-Tris. The pH was adjusted back towards the pH optimum with Tris at pH 7.6 before the enzyme activities were measured.
the activity of the enzyme in the lightest cells is 3 to 6 times that in the heaviest cells. The enzyme activity in the 60% of cells having intermediate density approximates the values obtained in the cells before density separation. Kinetics and Magnesium Requirements. The kinetic behavior of pyrimidine 5'-nucleotidase was examined in both crude hemolysate and in the purified state. A high-specificactivity [2-14C]CMP substrate was prepared by purification of the commercial substrate on a Dowex-1 column without adding carrier. A range of substrate concentrations was produced by addition of the appropriate amount of pure nonradioactive CMP. The double reciprocal plot obtained using the purified enzyme is shown in Fig. 5. The Km values obtained in this way had a range of 8-15 ,gM, with a mean of 10 ,uM. In the crude hemolysate the Km of the enzyme was 40juM (Fig. 5). As little as 0.05 mM Mg2+ was sufficient to give maximum enzymatic activity. Inorganic phosphate was a competitive inhibitor of the enzyme. DISCUSSION Enzymes with 5'-nucleotidase activity have been demonstrated to occur in a wide variety of organisms (15-17). Prior to the discovery by Valentine et al. (1) of a pyrimidine-specific 5'nucleotidase in human erythrocytes, all known 5'-nucleotidases were able to dephosphosphorylate both purine and pyrimidine nucleotides. The erythrocyte enzyme also differed markedly Table 2. Pyrimidine 5'-nucleotidase activity in erythrocytes of different density % of
Supranormal 5'-nucleotidase,
cells
5'-nucleotidase, milliunits/g Hb
cells
milliunits/g Hb
100
140
100
350
Normal % of
Whole blood
21 600 27 210 Light Medium 61.5 122 57 353 22 104 Dense 11.5 69 Values are shown for two subjects, one having normal and the other supranormal levels of the enzyme.
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Torrance et al.
3704
0
0
1/v
This work was supported, in part, by Grant HL 07449 from the National Institutes of Health, and by U.S. Public Health Service International Research Fellowship IF05 TW02190-01 (J.D.T.).
25-
100
0
tween pH 6.0 and 7.5. Activity of the purified enzyme was maximal at a Mg2+ concentration of 0.05 mM, a level much lower than that observed by Paglia and Valentine (5) in hemolysates, where activity was nearly maximal at 1.0 mM but increased slightly in the presense of 10 mM Mg2+. Kinetic studies with the purified enzyme showed that the Km of pyrimidine 5'-nucleotidase for CMP is 10 ,AM (Fig. 5). This is less than that found in hemolysates, 40,uM (Fig. 5) (5), indicating the presence of an inhibitor. Inorganic phosphate at concentrations of 1.3-5.3 mM is an inhibitor of the reaction and the Km for CMP at all concentrations of phosphate tested appeared to be 50 ,M. The exact nature of this inhibition is not yet understood.
200
100
400
IT
FIG. 5. Lineweaver-Burk plots for purified human erythrocyte pyrimidine 5'-nucleotidase (0) and for the enzyme in a 1:20 hemolysate (0). 1/v is the reciprocal of the number of millimoles of cytidine produced from CMP per min and 1/[SJ is the reciprocal of the concentration of CMP (nM). Open and filled symbols represent two separate experiments. The lines are least square fits for the combined result.
from other 5'-nucleotidases in that it was present in the soluble fraction of the cell, while previously described 5'-nucleotidases were primarily associated with the plasma membrane; indeed, the enzyme is used as a membrane "marker" in histological studies. 5'-Nucleotidases have been purified from many tissues (16) yielding a heterogeneous group of enzymes with a wide range of substrate preferences, pH optima, and metal ion requirements, as well as different responses to a variety of inhibitors. To our knowledge a description of a purification procedure for the pyrimidine-specific 5'-nucleotidase described by Valentine et al. (1) has not previously been published. Using classical purification procedures, we have achieved a 250,000-fold purification of pyrimidine 5'-nucleotidase from human erythrocytes in 18% yield. This preparation was not homogeneous; on disc polyacrylamide gel electrophoresis two dense and two faint bands of protein were obtained. On starch gel it was possible to stain the enzyme (14) and show that there were indeed two bands of enzyme with 5'-nucleotidase activity. Two bands of 5'-nucleotidase activity have also been observed on starch-gel electrophoresis by Giblett (14). Further purification was attempted by affinity chromatography using cytidine bound to Sepharose 4B, but this proved unsuccessful. The substituted agarose was able to bind the enzyme but we were unable to recover the enzyme from the affinity column. Indeed, it was surprising to note that while still bound to the cytidine column the enzyme appeared to retain its ability to hydrolyze [2-14C]CMP. The initial stepwise DEAE-cellulose procedure was included because it very rapidly removed most of the hemoglobin and other protein. This allowed the column DEAE-cellulose step to be carried out on a smaller column, which consistently gave better results than those obtained by applying the hemolysate directly to a large DEAE-cellulose column. The enzyme has a pH optimum at 7.5 and is most stable be-
1. Valentine, W. N., Fink, K., Paglia, D. E., Harris, S. R. & Adams, W. S. (1974) "Hereditary hemolytic anemia with human erythrocyte pyrimidine 5'-nucleotidase deficiency," J. Clin. Invest. 54,866-879. 2. Ben-Bassat, I., Brok-Simoni, F., Kende, G., Holtzmann, F. & Ramot, B. (1976) "A family with red cell pyrimidine 5'-nucleotidase deficiency," Blood 47, 919-922. 3. Rochant, H., Dreyfus, B., Rosa, R. & Boiron, M. (1975) First Case of Pyrimidine 5' Nucleotidase Deficiency in a Male. International Society of Haematology, European and African Third Meeting, London. August 24-28 1975, Abstract 1:19. 4. Torrance, J., West, C. & Beutler, E. (1977) "A simple radiometric assay for pyrimidine-5'-nucleotidase," J. Lab. Clin. Med., in press. 5. Paglia, D. E. & Valentine, W. N. (1975) "Characteristics of a pyrimidine-specific 5'-nucleotidase in human erythrocytes," J.
Biol. Chem. 250,7973-7979. 6. Danon, D. 4 Marikovsky, Y. (1964) "Determination of density distribution of red cell population," J. Lab. Clin. Med. 64, 668-674. 7. Beutler, E., West, C. & Blume, K. G. (1976) "The removal of leukocytes and platelets from whole blood," J. Lab. Clin. Med.
88,328-333. 8. Beutler, E. (1975) Red Cell Metabolism. A Manual of Biochemical Methods (Grune and Stratton, New York), 2nd ed., p. 10. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) "Protein measurement with the Folin phenol reagent," J. Biol. Chem. 193,265-275. 10. Fruton, J. S. & Simmonds, S. (1958) General Biochemistry (John Wiley and Sons, New York), 2nd ed., p. 74. 11. LKB-Produkter Instruction Manual for Ampholine 8100 Electrofocusing Equipment- (LKB-Produkter, Bromma, Swe-
den). 12. Davis, B. J. (1964) "Disc electrophoresis II. Method and application to human serum proteins," Ann. N.Y. Acad. Sci. 121, 404-427. 13. Buchler Co. (1971) Instructions for Polyanalyst and Instructions for PolyPrep 100 and 200 (Buchler Instruments, Fort Lee,
NJ).
14. Anderson, J. A., Teng, Y.-S. & Giblett, E. R. (1975) "Stains for six enzymes potentially applicable to chromosomal assignment by
cell hybridization," [Rotterdam Conference (1974): 2nd National Workshop on Human Gene Mapping], Birth Defects: Original Article Series 11, 295-299. 15. Heppel, L. A. (1961) "5' Nucleotidase," in The Enzymes, ed. Boyer, P. D. (Academic Press, New York), 2nd ed., Vol. 5, pp. 49-53. 16. Bodansky, 0. & Schwartz, M. K. (1968) "5' Nucleotidase," Adv. Clin. Chem. 11, 277-328. 17. Drummond, G. I. & Yamamoto, M. (1971) "Nucleotide phosphomonoesterases," in The Enzymes, ed. Boyer, P. D. (Academic Press, New York), 3rd ed., Vol. 4, pp. 337-354.
