- Email: [email protected]
[67] Adenosine deaminase from human erythrocytes
502
(67]
PURINE METABOLIZING ENZYMES
are included in the assay. The apparent Km for AMP is ~ 1 mM under standard assay conditions. Inorganic phosphate, inorganic pyrophosphate, 2,3-DPG, and GTP are inhibitors of catalytic activity. Similar results have been reported by Lian and Harkness r and by Yung. s
Relationship to the Erythrocyte Membrane. Partially purified AMP deaminase binds readily and reversibly to the inner (cytoplasmic) surface of isolated human erythrocyte membranes. The binding to the erythrocyte membrane is a hyperbolic function of the amount of enzyme added. Little if any binding to the outer membrane surface occurs. 2,3DPG, 5'-AMP, and 5'-ATP markedly reduce the amount of enzyme that binds to the membrane. Membrane-bound enzyme is less than 20% as active as enzyme fully dissociated from the membrane. These relationships have not been investigated with the fully purified enzyme preparations. 7 C. Y. Lian and D. Harkness, Biochim. Biophys. Acta 341, 27 (1974). s S. L. Yung, Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 1604 (1976).
[67] A d e n o s i n e
Deaminase
from Human
By R. P. AGARWAL and R. E.
PARKS,
Erythrocytes JR.
Adenosine + H~O ~ Inosine + NH3
Assay Method Among various methods available, the direct spectrophotometric method is most commonly employed for the assay of adenosine deaminase.
Principle. The activity is determined by measuring the rate of decrease in absorbancy at 265 nm resulting from the conversion of adenosine to inosine. 1 Molar absorption changes and optimal wavelengths of various adenosine analogs that may be employed as substrates for the enzymic assay have been described elsewhere. ~'3 I H. M. Kalckar, J. Biol. Chem. 167, 461 (1947). 2 R. P. Agarwal, S. M. Sagar, and R. E. Parks, Jr., Biochem. Pharmacol. 24, 693 (1975). 3 C. L. Zielke and C. H. Suelter, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 4, p. 47. Academic Press, New York, 1971.
METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress,Inc. At! fightsof reproductionin arty formreserved. ISBN 0-12-181951-5
[67]
ADENOSINE DEAMINASE FROM HUMAN ERYTHROCYTES
503
Reagents
P o t a s s i u m p h o s p h a t e buffer, 50 raM, p H 7.4 Adenosine, 10 m M Procedure. ~ The reaction mixture (in a 1-ml cuvette; diam = 1.0 cm) contains 0.9 ml buffer, 10 t~l of adenosine (0.1 tzmole), the e n z y m e , and w a t e r to m a k e 1 ml. The d e c r e a s e in a b s o r b a n c y is followed at 265 nm at 30°. 2 One unit of adenosine deaminase is the a m o u n t of e n z y m e that catalyzes the deamination of 1 /~mole o f adenosine per minute ( - A A = 8.6 min -1 m1-1) under the conditions of the assay. Specific activity is e x p r e s s e d as units p e r milligram of protein. Protein concentrations m a y be determined either by m e t h o d s such as that of L o w r y et al. 4 or b y U V absorption at 280 nm. 5 Other assay methods include: (1) m e a s u r e m e n t of inosine production by coupling the adenosine deaminase reaction to the purine nucleoside p h o s p h o r y l a s e and xanthine oxidase reactions; 6 (2) m e a s u r e m e n t of a m m o n i a production by reaction with a-ketoglutarate in the p r e s e n c e of glutamic acid d e h y d r o g e n a s e and N A D H 2 or by a microdiffusion procedure. 7-9 The latter method is useful for studies of the e n z y m e in crude suspensions, intact cells, or with adenosine analogs where spectrophotometric assays are inconvenient or inaccurate, a-l~
Purification Pro ce dur e 2 Step 1. Preparation o f H e m o l y s a t e . T h e preparation of h e m o l y s a t e s is described by Agarwal et al.la Step 2. Calcium Phosphate Gel Negative Adsorption. z,13 This procedure is identical to that described by Agarwal et al. ~s Although this 4 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). O. Warburg and W. Christian, Biochem. Z. 310, 384 O941); also see Vol. 3 [73]. 6 D. A. Hopkinson, P. J. L. Cook, and H. Harris, Ann. Hum. Genet. 32, 361 (1969). r D. Seligson and H. Seligson, J, Lab. Clin. Med. 38, 324 (1951). A. L. Chaney and E. P, Marbach, Clin. Chem. 8, 130 (1962).
9 R. P. Agarwal, G. W. Crabtree, R. E. Parks, Jr., J. A. Nelson, R. Keightley, R, Parkman, F. S. Rosen, R. C. Stern, and S. H. Polmar, J. Clin. Invest. 57, 1025 0976). l0 R. P. Agarwal and R. E. Parks, Jr., Biochem. Pharmacol, 24, 547 (1975). 11R. E. Parks, Jr., G. W. Crabtree, C, M. Kong, R. P. Agarwa|, K. C. Agarwal, and E. M. Scholar, Ann. N. Y. Acad. Sci. 255, 412 (1975). ~2R. P. Agarwal, G. W. Crabtree, K. C. Agarwal, R. E. Parks, Jr., and L. B. Townsend, Proc. Am. Assoc. Cancer Res. 17, 214 (1976). ~3R. P. Agarwal, K. C. Agarwal, and R. E. Parks, Jr., this volume [79].
504
PURINE METABOLIZING ENZYMES
[67]
step does not significantly change the specific activity of the enzyme, it is included primarily to remove most of the purine nucleoside phosphorylase, the activity of which, in human erythrocytes, is at least 50 times greater than the adenosine deaminase activity. Since purine nucleoside phosphorylase reacts with certain products of the adenosine deaminase reaction, e.g., inosine and deoxyinosine, it is desirable to remove this contaminant early during purification. Step 3. DEAE-Cellulose Column Chromatography. 2.13 The supernatant fluid from step 2 is poured onto a column (4.5 × 36 cm) of DEAEcellulose (phosphate form) which is prepared by equilibration with 0.01 M potassium phosphate buffer containing 0.05 M NaC1, pH 7.4. The enzyme is eluted with a linear gradient of 0.05-0.2 M NaCI solution (1 liter each) in 0.01 M potassium phosphate buffer, pH 7.4. The enzyme emerges at approximately 0.12-0.16 M NaC1. Successive fractions are collected and those containing most of the enzymic activity are pooled. When performing purification of adenosine deaminase from large quantities of human blood by the "General Method" described by Agarwal et al., xa this enzyme is found in the ammonium sulfate precipitate of fraction 8.13 This precipitate may be dissolved in a small volume of 0.01 M potassium phosphate, pH 7.4, containing 0.05 M NaCI and dialyzed thoroughly against the same solution. This material may then be subjected to DEAE-cellulose chromatography as described above. Step 4. Ammonium Sulfate Fractionation. To the pooled fractions from step 3, finely dispersed solid ammonium sulfate (231 g/liter) is added slowly by sifting and gentle stirring to final concentration of 40% saturation. The mixture is stirred gently for 10-12 hr at 4 °, and the precipitate is removed by centrifugation at 16,000 g for 60 min. More solid ammonium sulfate (186 g/liter) is added to the supernatant fluid to bring the saturation to 70%. After stirring for 6 hr at 4 °, the precipitate is collected by centrifugation at 16,000 g for 60 min, and the pellet is dissolved in a small volume of 0.1 M Tris'HC1 buffer, pH 7.5. Step 5. Molecular Sieving. The enzyme solution from step 4 is added to a Bio-Gel P-60 column (2 × 60 cm) equilibrated with 0.1 M potassium phosphate buffer, pH 7.4. The enzyme is eluted with the same buffer, and the fractions containing the enzyme are pooled. The pooled enzyme is concentrated by ultrafiltration in an Amicon cell by use of an XM50 membrane under nitrogen pressure (50 psi). Table I summarizes a typical purification procedure of adenosine deaminase from about 100 ml of packed human erythrocytes. Several other methods of purification of erythrocytic adenosine deaminase have been described recently that employ affinity chromatog-
[67]
ADENOSINE DEAMINASE FROM HUMAN ERYTHROCYTES
505
TABLE I PURIFICATION OF ADENOSINE DEAMINASE FROM HUMAN ERYTHROCYTESa
Purification steps 1. 2. 3. 4.
Hemolysate Calcium phosphate gel treatment DEAE-cellulosecolumn Ammoniumsulfate fractionation (40-70% saturation) 5. Molecular sieving
Total activity (units)
Specific activity (units/mg Recovery Purification protein) (%) (fold)
21.25 17.0 14.0
2.3 × 10-4 1.4 × 10-4 3.7 × 10-z
100 80 66
1 -161
7.4 3.2
2.7 × 10-x 7.8 × 10-~
35 15
1174 3087
Agarwal et al. z raphy, e.g., adenosine attached to Sepharose, 14 9-(p-aminobenzyl)adenine attached to agarose, 15 and adenosine deaminase specific rabbit antibody attached affinity columns. ~6 The latter method has yielded e n z y m e that appears h o m o g e n e o u s . Properties Stability. The partially purified adenosine deaminase preparation from step 5 above is stable for several weeks at 4 ° and for several years if frozen. 17 This preparation retained 100~ and 36% of activity after heating for 5 rain at 65 ° and 70 °, respectively. 17 Treatment of the e n z y m e with l mM p - c h l o r o m e r c u r i b e n z o a t e at 25 ° for 30 min resulted only in 30% loss of activity. 17 The e n z y m e has a broad pH optimum from p H 6 8. 2 P h y s i c a l P r o p e r t i e s . Molecular weight values o f 30,000-38,000 have been reported for h u m a n erythrocytic adenosine deaminase. 2'14'16Studies with an apparently h o m o g e n e o u s preparation have s h o w n that the e n z y m e has a Stokes radius o f 2 4 / ~ , and s~0,w o f 3.8 × l0 -13, a partial specific volume of 0.729 cm3/g, and a frictional ratio of 1.077.16 Evidence to date indicates that the e n z y m e consists of a single polypeptide chain. Specificity a n d K i n e t i c s . ~ Although the e n z y m e has a broad specificity for adenosine analogs, alterations in the purine ring or sugar moiety
14w. P. Schrader, A. R. Stacy, and B. Pollara, J. Biol. Chem. 251, 4026 (1976). ~ C. A. Rossi, A. Lucacchini, U. Montali, and G. Ronca, Int. J. Pept. Protein Res. 7, 81 0975). 16p. E. Daddona and W. N. Kelley, J. Biol. Chem. 252, 110 (1977). 17Our unpublished results.
506
PURINE METABOLIZING ENZYMES
[67]
result in striking changes in substrate activity. 2 Adenosine analogs modified in the imidazole ring, e.g., formycin A and 8-azaadenosine, have both higher Km and Vmaxvalues, whereas 7-deaza compounds such as toyocamycin and tubercidin are totally devoid of activity either as substrates or inhibitors. 2 The enzyme is inhibited by many compounds, including the products of the reaction and various adenosine analogs. 2'1s-2° Among the most potent inhibitors of this enzyme discovered to date are coformycin (Ki, 10-~ M) and deoxycoformycin (Ki, 2.5 × 10-12 M)) s-2° In fact, these compounds are tight-binding inhibitors and might be regarded as "transition-state" analogs, zx Kinetic parameters of various analogs are provided in Table II. Compounds s u c h as AMP, dAMP, cAMP, dADP, ATP, cytosine, cytidine, CMP, dCMP, CDP, dCDP, and CTP did not show any inhibition, whereas 4-amino-5-imidazole carboxamide ribonucleoside, 4amino-5-imidazole carboxamide-HC1, 2,6-diaminopurine sulfate, 6-chloropurine, iodopurine,, and adenine gave 15-60% inhibition. 16No cofactor requirement has been established for the enzyme. Comments Several recent developments have greatly increased the interest in adenosine deaminase: (I) a hereditary deficiency in this enzyme has been associated with a severe combined immunodeficiency syndrome in which the children so affected lack both T- and B-lymphocyte function and often have bone abnormalities; ~'~2"23(2) many analogs of adenosine with chemotherapeutic potential are degraded to inactive compounds by this enzyme, e,g., arabinosyl adenine, formycin, cordycepin, etc. ~ Administration of these analogs with a potent adenosine deaminase inhibitor such as deoxycoformycin has resulted in marked synergy in antitumor activity in experimental animals. 24These observations point to an important role played by adenosine deaminase, both in immunosuppression and chemotherapy. Furthermore, cells in which the adenosine deaminase is absent or inhibited may accumulate remarkably high 18s. Cha, R. P. Agarwal,and R. E. Parks, Jr., Biochem. Pharrnacol. 24, 2187 (1975). 19R. P. Agarwal,T. Spector, and R. E. Parks, Jr., Biochem. Pharmacol. 26, 359 (1977). 20R. P. Agarwal, S. Cha, G. W. Crabtree, and R. E. Parks, Jr., Adv. Chem. Ser. (in press). ~1L. Pauling,Am. Sci. 36, 58 (1948). zz E. R. Giblett, J. E. Anderson, F. Cohen, B. PoUara, and H. J. Meuwissen,Lancet 2, 1067 (1972). ~aH. J. Meuwissen, R. J. Picketing, B. Pollara and I. H. Porter, eds., "Combined ImmunodeficiencyDisease and AdenosineDeaminaseDeficiency:A MolecularDefect." Academic Press, New York, 1975. 2aD. G. Johns and R. H. Adamson,Biochem. PharmacoL 25, 1441 (1976).
[67]
507
ADENOSINE DEAMINASE FROM HUMAN ERYTHROCYTES T A B L E II KINETIC PARAMETERS OF HUMAN ERYTHROCYTIC ADENOSINE DEAMINASEa'b'c
Substmtes or inhibitors
Relative Vmax
Adenosine Forrnycin A 8-A_zaadenosine 6-Chloropurine ribonueleoside 2,6-Diaminopurine ribonucleoside 6-Methylselenopurine ribonucleoside 2'-Deoxyadenosine Xylosyl adenine Arabinosyl adenine Y-Amino-3'-deoxyadenosine 4'-Thioadenosine
100 750--800 310 91 91 88 60 60 47 89 43
Inosine 2'-Deoxyinosine Guanosine 2-Fluoroadenosine 2-Fluorodeoxyadenosine N6-rnethyladenosine N l-methyladenosine 6-Thioguanosine 6-Thioinosine 6-Methylthioinosine Arabinosyl-6-thiopurine 1,6-Dihydro-6-hydroxymethylpurine ribonucleoside Erythro-9-(2-hydroxy-3-nonyl)adenine Coformycin Deoxycoformycin
Km (M) 2.5 1.0 1.3 1.0 7.4 2.7 7.0 3.3 1.0 1.3 1.3
× × × × x × × × × × ×
K~ (M)
10 _5 10-3 10-4 10-z 10 -~ 10 -~ 10-6 10-5 10 -4 10-4 10-5 1.2 6.0 1,4 6.0 1.9 1.7 2.8 9.2 3.3 2.7 3.6
M
w
w
× x x × × × x × × × ×
10 -4 10-~ 10-4 10-~ 10-5 10-5 10-4 10-5 10-4 10-4 10-4
1.3 x 1.6 × 1.0 × 2.5 ×
10-6 10-~ 10 - u 10-12
Agarwal et al. 2 b Cha et al. 18 c Agarwal e t a [ . la
concentrations of adenine nuc|eotides, e.g., ATP, when incubated with adenosine. 9'11'2°'~5 It is possible that the cytotoxicity of adenosine with certain tissues may result from these abnormally elevated levels of adenine nucleotides. 9,~6
z5 L. M. Rose and R. W. Brockman, J. Chromatogr. 133, 335 (1977). 2a S. H. Polmar, R. C. Stern, A. L. Schwartz, E. M. Wetzler, P. A. Chase, and R. Hirschhorn, N . Engl. J. Med. 295, 1337 (1976).
(67]
PURINE METABOLIZING ENZYMES
are included in the assay. The apparent Km for AMP is ~ 1 mM under standard assay conditions. Inorganic phosphate, inorganic pyrophosphate, 2,3-DPG, and GTP are inhibitors of catalytic activity. Similar results have been reported by Lian and Harkness r and by Yung. s
Relationship to the Erythrocyte Membrane. Partially purified AMP deaminase binds readily and reversibly to the inner (cytoplasmic) surface of isolated human erythrocyte membranes. The binding to the erythrocyte membrane is a hyperbolic function of the amount of enzyme added. Little if any binding to the outer membrane surface occurs. 2,3DPG, 5'-AMP, and 5'-ATP markedly reduce the amount of enzyme that binds to the membrane. Membrane-bound enzyme is less than 20% as active as enzyme fully dissociated from the membrane. These relationships have not been investigated with the fully purified enzyme preparations. 7 C. Y. Lian and D. Harkness, Biochim. Biophys. Acta 341, 27 (1974). s S. L. Yung, Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 1604 (1976).
[67] A d e n o s i n e
Deaminase
from Human
By R. P. AGARWAL and R. E.
PARKS,
Erythrocytes JR.
Adenosine + H~O ~ Inosine + NH3
Assay Method Among various methods available, the direct spectrophotometric method is most commonly employed for the assay of adenosine deaminase.
Principle. The activity is determined by measuring the rate of decrease in absorbancy at 265 nm resulting from the conversion of adenosine to inosine. 1 Molar absorption changes and optimal wavelengths of various adenosine analogs that may be employed as substrates for the enzymic assay have been described elsewhere. ~'3 I H. M. Kalckar, J. Biol. Chem. 167, 461 (1947). 2 R. P. Agarwal, S. M. Sagar, and R. E. Parks, Jr., Biochem. Pharmacol. 24, 693 (1975). 3 C. L. Zielke and C. H. Suelter, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 4, p. 47. Academic Press, New York, 1971.
METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress,Inc. At! fightsof reproductionin arty formreserved. ISBN 0-12-181951-5
[67]
ADENOSINE DEAMINASE FROM HUMAN ERYTHROCYTES
503
Reagents
P o t a s s i u m p h o s p h a t e buffer, 50 raM, p H 7.4 Adenosine, 10 m M Procedure. ~ The reaction mixture (in a 1-ml cuvette; diam = 1.0 cm) contains 0.9 ml buffer, 10 t~l of adenosine (0.1 tzmole), the e n z y m e , and w a t e r to m a k e 1 ml. The d e c r e a s e in a b s o r b a n c y is followed at 265 nm at 30°. 2 One unit of adenosine deaminase is the a m o u n t of e n z y m e that catalyzes the deamination of 1 /~mole o f adenosine per minute ( - A A = 8.6 min -1 m1-1) under the conditions of the assay. Specific activity is e x p r e s s e d as units p e r milligram of protein. Protein concentrations m a y be determined either by m e t h o d s such as that of L o w r y et al. 4 or b y U V absorption at 280 nm. 5 Other assay methods include: (1) m e a s u r e m e n t of inosine production by coupling the adenosine deaminase reaction to the purine nucleoside p h o s p h o r y l a s e and xanthine oxidase reactions; 6 (2) m e a s u r e m e n t of a m m o n i a production by reaction with a-ketoglutarate in the p r e s e n c e of glutamic acid d e h y d r o g e n a s e and N A D H 2 or by a microdiffusion procedure. 7-9 The latter method is useful for studies of the e n z y m e in crude suspensions, intact cells, or with adenosine analogs where spectrophotometric assays are inconvenient or inaccurate, a-l~
Purification Pro ce dur e 2 Step 1. Preparation o f H e m o l y s a t e . T h e preparation of h e m o l y s a t e s is described by Agarwal et al.la Step 2. Calcium Phosphate Gel Negative Adsorption. z,13 This procedure is identical to that described by Agarwal et al. ~s Although this 4 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). O. Warburg and W. Christian, Biochem. Z. 310, 384 O941); also see Vol. 3 [73]. 6 D. A. Hopkinson, P. J. L. Cook, and H. Harris, Ann. Hum. Genet. 32, 361 (1969). r D. Seligson and H. Seligson, J, Lab. Clin. Med. 38, 324 (1951). A. L. Chaney and E. P, Marbach, Clin. Chem. 8, 130 (1962).
9 R. P. Agarwal, G. W. Crabtree, R. E. Parks, Jr., J. A. Nelson, R. Keightley, R, Parkman, F. S. Rosen, R. C. Stern, and S. H. Polmar, J. Clin. Invest. 57, 1025 0976). l0 R. P. Agarwal and R. E. Parks, Jr., Biochem. Pharmacol, 24, 547 (1975). 11R. E. Parks, Jr., G. W. Crabtree, C, M. Kong, R. P. Agarwa|, K. C. Agarwal, and E. M. Scholar, Ann. N. Y. Acad. Sci. 255, 412 (1975). ~2R. P. Agarwal, G. W. Crabtree, K. C. Agarwal, R. E. Parks, Jr., and L. B. Townsend, Proc. Am. Assoc. Cancer Res. 17, 214 (1976). ~3R. P. Agarwal, K. C. Agarwal, and R. E. Parks, Jr., this volume [79].
504
PURINE METABOLIZING ENZYMES
[67]
step does not significantly change the specific activity of the enzyme, it is included primarily to remove most of the purine nucleoside phosphorylase, the activity of which, in human erythrocytes, is at least 50 times greater than the adenosine deaminase activity. Since purine nucleoside phosphorylase reacts with certain products of the adenosine deaminase reaction, e.g., inosine and deoxyinosine, it is desirable to remove this contaminant early during purification. Step 3. DEAE-Cellulose Column Chromatography. 2.13 The supernatant fluid from step 2 is poured onto a column (4.5 × 36 cm) of DEAEcellulose (phosphate form) which is prepared by equilibration with 0.01 M potassium phosphate buffer containing 0.05 M NaC1, pH 7.4. The enzyme is eluted with a linear gradient of 0.05-0.2 M NaCI solution (1 liter each) in 0.01 M potassium phosphate buffer, pH 7.4. The enzyme emerges at approximately 0.12-0.16 M NaC1. Successive fractions are collected and those containing most of the enzymic activity are pooled. When performing purification of adenosine deaminase from large quantities of human blood by the "General Method" described by Agarwal et al., xa this enzyme is found in the ammonium sulfate precipitate of fraction 8.13 This precipitate may be dissolved in a small volume of 0.01 M potassium phosphate, pH 7.4, containing 0.05 M NaCI and dialyzed thoroughly against the same solution. This material may then be subjected to DEAE-cellulose chromatography as described above. Step 4. Ammonium Sulfate Fractionation. To the pooled fractions from step 3, finely dispersed solid ammonium sulfate (231 g/liter) is added slowly by sifting and gentle stirring to final concentration of 40% saturation. The mixture is stirred gently for 10-12 hr at 4 °, and the precipitate is removed by centrifugation at 16,000 g for 60 min. More solid ammonium sulfate (186 g/liter) is added to the supernatant fluid to bring the saturation to 70%. After stirring for 6 hr at 4 °, the precipitate is collected by centrifugation at 16,000 g for 60 min, and the pellet is dissolved in a small volume of 0.1 M Tris'HC1 buffer, pH 7.5. Step 5. Molecular Sieving. The enzyme solution from step 4 is added to a Bio-Gel P-60 column (2 × 60 cm) equilibrated with 0.1 M potassium phosphate buffer, pH 7.4. The enzyme is eluted with the same buffer, and the fractions containing the enzyme are pooled. The pooled enzyme is concentrated by ultrafiltration in an Amicon cell by use of an XM50 membrane under nitrogen pressure (50 psi). Table I summarizes a typical purification procedure of adenosine deaminase from about 100 ml of packed human erythrocytes. Several other methods of purification of erythrocytic adenosine deaminase have been described recently that employ affinity chromatog-
[67]
ADENOSINE DEAMINASE FROM HUMAN ERYTHROCYTES
505
TABLE I PURIFICATION OF ADENOSINE DEAMINASE FROM HUMAN ERYTHROCYTESa
Purification steps 1. 2. 3. 4.
Hemolysate Calcium phosphate gel treatment DEAE-cellulosecolumn Ammoniumsulfate fractionation (40-70% saturation) 5. Molecular sieving
Total activity (units)
Specific activity (units/mg Recovery Purification protein) (%) (fold)
21.25 17.0 14.0
2.3 × 10-4 1.4 × 10-4 3.7 × 10-z
100 80 66
1 -161
7.4 3.2
2.7 × 10-x 7.8 × 10-~
35 15
1174 3087
Agarwal et al. z raphy, e.g., adenosine attached to Sepharose, 14 9-(p-aminobenzyl)adenine attached to agarose, 15 and adenosine deaminase specific rabbit antibody attached affinity columns. ~6 The latter method has yielded e n z y m e that appears h o m o g e n e o u s . Properties Stability. The partially purified adenosine deaminase preparation from step 5 above is stable for several weeks at 4 ° and for several years if frozen. 17 This preparation retained 100~ and 36% of activity after heating for 5 rain at 65 ° and 70 °, respectively. 17 Treatment of the e n z y m e with l mM p - c h l o r o m e r c u r i b e n z o a t e at 25 ° for 30 min resulted only in 30% loss of activity. 17 The e n z y m e has a broad pH optimum from p H 6 8. 2 P h y s i c a l P r o p e r t i e s . Molecular weight values o f 30,000-38,000 have been reported for h u m a n erythrocytic adenosine deaminase. 2'14'16Studies with an apparently h o m o g e n e o u s preparation have s h o w n that the e n z y m e has a Stokes radius o f 2 4 / ~ , and s~0,w o f 3.8 × l0 -13, a partial specific volume of 0.729 cm3/g, and a frictional ratio of 1.077.16 Evidence to date indicates that the e n z y m e consists of a single polypeptide chain. Specificity a n d K i n e t i c s . ~ Although the e n z y m e has a broad specificity for adenosine analogs, alterations in the purine ring or sugar moiety
14w. P. Schrader, A. R. Stacy, and B. Pollara, J. Biol. Chem. 251, 4026 (1976). ~ C. A. Rossi, A. Lucacchini, U. Montali, and G. Ronca, Int. J. Pept. Protein Res. 7, 81 0975). 16p. E. Daddona and W. N. Kelley, J. Biol. Chem. 252, 110 (1977). 17Our unpublished results.
506
PURINE METABOLIZING ENZYMES
[67]
result in striking changes in substrate activity. 2 Adenosine analogs modified in the imidazole ring, e.g., formycin A and 8-azaadenosine, have both higher Km and Vmaxvalues, whereas 7-deaza compounds such as toyocamycin and tubercidin are totally devoid of activity either as substrates or inhibitors. 2 The enzyme is inhibited by many compounds, including the products of the reaction and various adenosine analogs. 2'1s-2° Among the most potent inhibitors of this enzyme discovered to date are coformycin (Ki, 10-~ M) and deoxycoformycin (Ki, 2.5 × 10-12 M)) s-2° In fact, these compounds are tight-binding inhibitors and might be regarded as "transition-state" analogs, zx Kinetic parameters of various analogs are provided in Table II. Compounds s u c h as AMP, dAMP, cAMP, dADP, ATP, cytosine, cytidine, CMP, dCMP, CDP, dCDP, and CTP did not show any inhibition, whereas 4-amino-5-imidazole carboxamide ribonucleoside, 4amino-5-imidazole carboxamide-HC1, 2,6-diaminopurine sulfate, 6-chloropurine, iodopurine,, and adenine gave 15-60% inhibition. 16No cofactor requirement has been established for the enzyme. Comments Several recent developments have greatly increased the interest in adenosine deaminase: (I) a hereditary deficiency in this enzyme has been associated with a severe combined immunodeficiency syndrome in which the children so affected lack both T- and B-lymphocyte function and often have bone abnormalities; ~'~2"23(2) many analogs of adenosine with chemotherapeutic potential are degraded to inactive compounds by this enzyme, e,g., arabinosyl adenine, formycin, cordycepin, etc. ~ Administration of these analogs with a potent adenosine deaminase inhibitor such as deoxycoformycin has resulted in marked synergy in antitumor activity in experimental animals. 24These observations point to an important role played by adenosine deaminase, both in immunosuppression and chemotherapy. Furthermore, cells in which the adenosine deaminase is absent or inhibited may accumulate remarkably high 18s. Cha, R. P. Agarwal,and R. E. Parks, Jr., Biochem. Pharrnacol. 24, 2187 (1975). 19R. P. Agarwal,T. Spector, and R. E. Parks, Jr., Biochem. Pharmacol. 26, 359 (1977). 20R. P. Agarwal, S. Cha, G. W. Crabtree, and R. E. Parks, Jr., Adv. Chem. Ser. (in press). ~1L. Pauling,Am. Sci. 36, 58 (1948). zz E. R. Giblett, J. E. Anderson, F. Cohen, B. PoUara, and H. J. Meuwissen,Lancet 2, 1067 (1972). ~aH. J. Meuwissen, R. J. Picketing, B. Pollara and I. H. Porter, eds., "Combined ImmunodeficiencyDisease and AdenosineDeaminaseDeficiency:A MolecularDefect." Academic Press, New York, 1975. 2aD. G. Johns and R. H. Adamson,Biochem. PharmacoL 25, 1441 (1976).
[67]
507
ADENOSINE DEAMINASE FROM HUMAN ERYTHROCYTES T A B L E II KINETIC PARAMETERS OF HUMAN ERYTHROCYTIC ADENOSINE DEAMINASEa'b'c
Substmtes or inhibitors
Relative Vmax
Adenosine Forrnycin A 8-A_zaadenosine 6-Chloropurine ribonueleoside 2,6-Diaminopurine ribonucleoside 6-Methylselenopurine ribonucleoside 2'-Deoxyadenosine Xylosyl adenine Arabinosyl adenine Y-Amino-3'-deoxyadenosine 4'-Thioadenosine
100 750--800 310 91 91 88 60 60 47 89 43
Inosine 2'-Deoxyinosine Guanosine 2-Fluoroadenosine 2-Fluorodeoxyadenosine N6-rnethyladenosine N l-methyladenosine 6-Thioguanosine 6-Thioinosine 6-Methylthioinosine Arabinosyl-6-thiopurine 1,6-Dihydro-6-hydroxymethylpurine ribonucleoside Erythro-9-(2-hydroxy-3-nonyl)adenine Coformycin Deoxycoformycin
Km (M) 2.5 1.0 1.3 1.0 7.4 2.7 7.0 3.3 1.0 1.3 1.3
× × × × x × × × × × ×
K~ (M)
10 _5 10-3 10-4 10-z 10 -~ 10 -~ 10-6 10-5 10 -4 10-4 10-5 1.2 6.0 1,4 6.0 1.9 1.7 2.8 9.2 3.3 2.7 3.6
M
w
w
× x x × × × x × × × ×
10 -4 10-~ 10-4 10-~ 10-5 10-5 10-4 10-5 10-4 10-4 10-4
1.3 x 1.6 × 1.0 × 2.5 ×
10-6 10-~ 10 - u 10-12
Agarwal et al. 2 b Cha et al. 18 c Agarwal e t a [ . la
concentrations of adenine nuc|eotides, e.g., ATP, when incubated with adenosine. 9'11'2°'~5 It is possible that the cytotoxicity of adenosine with certain tissues may result from these abnormally elevated levels of adenine nucleotides. 9,~6
z5 L. M. Rose and R. W. Brockman, J. Chromatogr. 133, 335 (1977). 2a S. H. Polmar, R. C. Stern, A. L. Schwartz, E. M. Wetzler, P. A. Chase, and R. Hirschhorn, N . Engl. J. Med. 295, 1337 (1976).