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[33] A d e n o s i n e M o n o p h o s p h a t e Nucleosidase from A z o t o b a c t e r v i n e l a n d i i a n d E s c h e r i c h i a coli
By VERN L. SCHRAMM and HAZEL B. LEUNG MgATP2-
AMP + H20
) Adenine + Ribose 5-PO4
Adenosine monophosphate nucleosidase (AMP nucleosidase) was first described by Hurwitz et al. 1 during studies of the polynucleotide metabolizing enzymes of Azotobacter vinelandii. The enzyme differs from enzymes which are usually classified as nucleotide-degrading enzymes in that it exhibits a high degree of specificity for AMP as substrate and is essentially inactive unless MgATP z-, the allosteric activator, is present. 2 Allosteric inhibition of activity occurs in the presence of Pi. These controls regulate the enzyme activity which has been postulated to control intracellular AMP levels? '4 The resulting adenine is deaminated by adenine deaminase to form hypoxanthine, which is the end-product of AMP catabolism in Azotobacter. AMP nucleosidase is also present in E. coli and has similar kinetic properties. The end-product of AMP catabolism in E. coli appears to be adenine, since adenine deaminase is missing in this organism?
Assay Method
Principle. Hydrolysis of AMP to ribose 5-PO4 and adenine releases the anomeric carbon and therefore causes it to become a reducing sugar. Recent improvements in the reducing sugar assay s allow accurate and sensitive quantitation of the ribose 5-PO4. Previous investigators have used n-butanol extraction of adenine as an assay method for AMP nucleosidase. 7 This method suffers from a lower sensitivity and a lack of specificity, since adenosine is also partially extracted into n-butanol. Conversely, few enzyme activities interfere with the reducing sugar assay. 1j. Hurwitz, L. A. Heppel, and B. L. Horecker, J. Biol. Chem. 226, 525 (1957). 2 V. L. Schramm, J. Biol. Chem. 249, 1729 (1974). 3 V. L. Schramm and H. Leung, J. Biol. Chem. 248, 8313 (1973). aV. L. Schramm and F. C. Lazorik, J. Biol. Chem. 2S0, 1801 (1975). 5 H. Leung and V. L. Schramm, unpublished observations (1976). 6 S. Dygert, L. H. Li, D. Florida, and J. A. Thoma, Anal. Biochem. 13, 367 (1965). 7 M. Yoshino, J. Biochem. (Tokyo) 68, 321 (1970). METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress, [nc. All rightsof reproductionin any form reserved. ISBN 0q2-181951-5
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Reagents
Assay mixture: 0.1 M triethanolamine-HCl, pH 8.0, 4 mM AMP, 7 mM ATP, and 7 mM MgCI2 Alkaline Cu reagent: 16 g Na~CO3, 6.4 g glycine, and 0.18 g CUSO4"5 1-120 in a final volume of 400 ml Indicator reagent: 0.48 g 2,9-dimethyl-l,10-phenanthroline in a final volume of 400 ml; pH adjusted to 3.0 with HC1 Enzyme Dilution. Enzyme is diluted in 0.1 M Tris.HC1, pH 8.0, containing 2 mM AMP, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 3 phenylmethyl sulfonyl fluoride. Diluted enzyme is >90% stable for 8 hr at 0 °. Procedure. Diluted enzyme (0.001-0.01 unit) is added to 0.25 ml of the assay mixture which has been equilibrated to 30 °. The amount of protein added should be kept below 30/zg, since higher concentrations will cause interference in the reducing sugar assay. Blank values must always be run by adding an enzyme aliquot to assay mixture containing 0.3 ml of the alkaline Cu reagent. The reducing sugar formed in an appropriate incubation period (20-2 min for 0.001--0.01 unit, respectively) is estimated by adding 0.3 ml of alkaline Cu reagent, 0.3 ml of 2,9-dimethyl-l,10-phenanthroline reagent, and sufficient HzO to give a final volume of 1.6 ml. The alkaline Cu reagent stops catalysis, and the mixture is stable for several hours at room temperature. Color development occurs when the covered tubes are placed in a boiling HzO bath (95-100 ~) for 8 min. After cooling to 30 ° in a circulating H20 bath, the Cu-2,9-dimethyl-l,10-phenanthroline complex is read at 450 nm. A standard curve with ribose 5-PO4 should be run. The normal sensitivity under these conditions is 0.0025 t~mole of ribose 5-PO4 which will give an A450 of approximately 0.045. Definition o f Unit. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 /xmole of product per minute. Protein is determined by the Folin method. 8
Growth of Calls Azotobacter vinelandii OP a
Cultures of A. vinelandii OP were maintained and grown at 30 ° in nitrogen-free medium 1° containing the following ingredients per liter of SE. Layne, this series, Vol. 3 [73]. aj. A. Bush and P. W. Wilson, Nature (London) 184, 381 (1959). loj. W. Newton, P. W. Wilson, and R. H. Barris, J. Biol. Chem. 204, 445 (1953).
[33]
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deionized water: K2HPO4, 1.4 g; KH2PO4, 0.35 g; MgSO4"7 H~O, 0.2 g; NaC1, 0.1 g; CAC12"2 H20, 0.1 g; Fes(SO4)z'3 H20, 0.01 g; FeClz'4 H20, 3.5 rag; Na2MoO4"2 H20, 0.25 rag; and sucrose, 20 g. The phosphate buffer was autoclaved separately. For growth of large-scale cultures, 50 ml of the media in a 500-ml Erlenmeyer flask were inoculated from an agar slant and grown to mid-log phase (200-400 Klett units using a No. 42 blue filter) on a New Brunswick model VS rotory shaker. Fivemilliliter portions were transferred to six 500-ml portions of media in 2liter Erlenmeyer flasks. These were grown to mid-log phase as before and transferred to 200 liters of media contained in a growth chamber, u Cells were harvested at late-log or early-stationary phase to give approximately 2 kg of cell paste.
Escherichia coli K12 For growth of E. coli K12, a similar procedure was used except the cells were maintained and grown at 37 ° in medium containing the following ingredients per liter of deionized water: K2HPO4, 10 g; KH2PO4, 2.5 g; MgSO4"7 H20, 0.2 g; CAC12"2 H20, 0.01 g; Fe2(SO4)3"3 H20, 0.01 g; (NH4)~SO4, 1 g; and glucose 5 g. Medium and glucose were autoclaved separately and mixed before use. Inoculation of the 200-liter growth chamber was with 100 ml of mid-log phase cells. Cells were harvested in log phase (Klett 490) to give approximately 1 kg of cell paste. Cell pastes or cells suspended 1 : 1 (w: w) in 0.4 M phosphate, pH 7.5, can be frozen and stored at - 2 0 ° or -85 °. At - 2 0 °, the enzyme activity is stable for 6 months followed by a slow decline' of activity. Cells frozen and stored at -85 ° show no loss of enzyme activity for up to 3 years. This applies to both Azotobacter and Escherichia cell pastes. Purification Procedures
AMP Nucleosidase from Azotobacter vinelandii OP Unless otherwise mentioned, all buffers used during the purification procedure contained EDTA (10 -4 M), dithiothreitol (10 -4 M), and phenylmethylsulfonyl fluoride (3 × 10-6 M). Temperatures were at 0-5 ° except where noted.
Step 1. Initial Extract. Frozen cells (954 g) were thawed in a 30 ° water bath and suspended by mixing with 1.0 liter of 0.4 M potassium phosphate buffer (pH 7.5) containing EDTA (2 × 10-4 M), dithiothreitol (2 x 10-4 M), and PMSF (6 × 10-6 M). The suspension was passed 11V. L. Schramm, Anal. Biochem. 57, 377 (1974).
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NUCLEOTIDASES AND NUCLEOSIDASES
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through an Aminco French pressure cell at 15,000-25,000 psi and diluted to 3.3 liters with 0.2 M potassium phosphate buffer (pH 7.5). A 5-ml sample of the extract was centrifuged at 12,000 g for 15 min, and the supernatant fluid was used to determine activity and protein.
Step 2. Heat Treatment. The initial extract, placed in a 3.5-liter stainless-steel beaker, was heated to 60 ° while being stirred over a period of 30 rain. The extract was maintained at 60 ° for 10 min and then cooled by placing the beaker in an ice bath. The supernatant fluid (2.5 liters) was recovered after centrifugation at 12,000 g for 8 hr. Heating rates were not critical during this step, and similar results were obtained when the temperature was increased to 60 ° in as little as 2 min. Step 3. Ammonium Sulfate Fractionation. The supernatant fluid from the heat treatment was brought to 0.37 saturation at 2 ° by the addition of solid ammonium sulfate over a period of 13 min followed by an additional 17-min stirring. The precipitate was removed by centrifugation (12,000 g for 40 min) and suspended in 150 ml of 0.1 M Tris.HCl buffer (pH 8.0) containing 0.15 M NaC1 and 2 mM AMP (the Tris-NaC1AMP buffer) to give a total volume of 208 ml. Step 4. Desalt on G-25 Sephadex. The ammonium sulfate fraction was applied to a large (4.6 × 56 cm) G-25 Sephadex column which had been equilibrated with the Tris-NaC1-AMP buffer, and eluted with the same buffer. Those protein fractions free of ammonium sulfate, as determined by Nessler reagent, were pooled to give 250 ml. Step 5. First DEAE A-50 Sephadex Fractionation. The desalted protein solution was applied to a 4.7 × 12 cm DEAE A-50 Sephadex column equilibrated against the Tris-NaCI-AMP buffer. The protein was eluted with a 2-liter linear gradient of NaC1 (0.15-0.30 M) in the same buffer. The enzyme peak appeared at 0.23 M NaCI and was diluted with the Tris-AMP buffer, containing no NaC1, to reduce the NaCI concentration to 0.15 M. The solution was concentrated to 230 ml on an Amicon Diaflow pressure dialysis unit. Step 6. Second DEAE A-50 Sephadex Fractionation. The solution from the previous step was applied to a 2.5 × 50 cm DEAE A-50 Sephadex column, and the column was developed using a 2-liter gradient from 0.15 to 0.28 M NaCI. Fractions of the highest activity were pooled and concentrated to 57 ml in an Amicon Diaflow concentrator. Step 7. Hydroxylapatite Fractionation. Without further adjustment of the NaCI concentration, the fraction from the second DEAE column was applied to a 1.5 × 7.5 cm column of hydroxylapatite:cellulose
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267
mixture (1.4:1.0 dry weight basis) which had been equilibrated by washing with 10 volumes of Tris-NaCI-AMP buffer. The column was eluted sequentially with 25-ml portions of the buffer containing 20, 40, 80, 160, and 320 mM KzHPO4 (pH 8.0). Fractions containing enzymic activity were pooled and concentrated on an Amicon Diaflow concentrator to 7.4 ml.
Step 8. Sephadex G-200 Gel Filtration. The solution from the previous step was passed through a (2.5 × 100 cm) column of Sephadex G-200 equilibrated with Tris-HC1 buffer (0.1 M, pH 8.0) containing 2.0 mM AMP. Those fractions containing the highest specific activities were combined and concentrated by pressure dialysis (Amicon Diaflow) to 3.2 ml. Polyacrylamide gel electrophoresis of this fraction gave a single band both in the presence and absence of sodium dodecyl sulfate. Crystallization of AMP Nucleosidase. Purified enzyme (4.6 ml; 1.7 mg/ml, specific activity of 30) in 0.1 M Tris.HC1 (pH 8.0) containing 1 mM AMP was precipitated by the addition of solid ammonium sulfate to 0.75 saturation. The precipitate was collected by centrifugation at 29,000 g for 7 min and extracted sequentially with 1 ml of 0.70, 0.66, 0.62, 0.58, 0.54, 0.50, 0.46, and 0.42 saturated ammonium sulfate in 0.1 M Tris.HC1 and 1 mM AMP (pH 8.0). Upon standing overnight at 4 °, crystals appeared in tubes containing 0.42 and 0.46 saturated ammonium sulfate, both having specific activities of 34.3, and both giving a single band in polyacrylamide gel electrophoresis. Microscopic examination revealed the crystalline structure to be hexagonal plates with a diameter of -0.03 ram. These crystals have been used to induce crystallization using enzyme preparations with specific activities as low as 15. The resulting crystalline material always exhibits specific activities near 34 and appears either as hexagonal plates or clusters of needles. Yield is usually 50-80% for the crystallization step. The purification procedure is summarized in Table I. Stability of the Purified Enzyme. Following Sephadex G-200 the enzyme was stable for periods of at least 1 year after freezing in Dry Ice-ethanol mixtures and storing at - 7 0 °. The crystalline enzyme was also stable for periods of at least 1 year at 4 °, stored in the ammonium sulfate solution from which it was crystallized. AMP Nucleosidase from Escherichia coli In general, the above purification procedure can be used for preparation of the E. coli enzyme. The differences in purification and yield are due primarily to E. coli containing less of the enzyme, and the enzyme
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TABLE I PURIFICATIONOF AMP NUCLEOSIDASEFROMAzotobacter vinelandia
Purification procedure Initial extract Heat treatment Ammonium sulfate fractionation Sephadex G-25 First DEAE-Sephadex column Second DEAE-Sephadex column Hydroxylapatite column Sephadex G-200 fractionation° Crystals from G-200 side fractions c
Volume (ml)
Total protein (rag)
Total units (/zmoles/ rain)
3300 2500 208 250 230 57 7.4 3.2 1.1
108,000 32,000 8100 7500 300 130 60 21 6.7
2700 2100 1500 1500 1400 1300 1100 690 230
Specific activity (/zmoles/ Yield min/mg) (%) 0.025 0.067 0.19 0.21 4.6 9.9 18 34 34
100 78 58 58 51 48 39 34a --
a Starting material was 954 g wet weight of cells. b Fractions containing enzyme of specific activity 34 were pooled, concentrated, and frozen for storage. c In this purification, fractions containing substantial activity, but specific activities less than 34, were pooled, concentrated, brought to near precipitation with ammonium sulfate, and crystallization was induced by the addition of 1/zl of a solution containing seed crystals of AMP nucleosidase. These were collected by centrifugation, dissolved in Tris.HCl (0.1 M, pH 8.0) containing 2 mM AMP, dialyzed to remove ammonium sulfate, and assayed for protein and activity. a Percent yield includes both fractions with specific activity of 34. b e i n g l e s s s t a b l e t h a n t h a t f r o m Azotobacter. T h e p u r i f i c a t i o n s c h e m e o u t l i n e d b e l o w will e m p h a s i z e t h e d i f f e r e n c e s in t h e t w o p r o c e d u r e s .
Step 1. Initial Extract. T h i s is s i m i l a r to t h e A. vinelandii p r o c e d u r e e x c e p t t h a t 4 m M MgCI~ a n d 9 m g c r y s t a l l i n e d e o x y r i b o n u c l e a s e ( W o r t h i n g t o n , c r y s t a l l i n e ) w e r e a d d e d to 1.5 k g o f c e l l s d u r i n g t h e t h a w i n g p r o c e d u r e . T h e p H w a s a d j u s t e d to 7.5 w i t h 10 N K O H b e f o r e the next step.
Step 2. Heat Treatment. T h i s is s i m i l a r to t h e Azotobacter p r e p ~ a t i o n e x c e p t t h a t o n l y 30 m i n o f c e n t r i f u g a t i o n a t 12,000 g w e r e r e q u i r e d to r e m o v e t h e p r e c i p i t a t e . Step 3. Ammonium Sulfate Fractionation. T h e f r a c t i o n p r e c i p i t a t i n g b e t w e e n 0.28 a n d 0.39 s a t u r a t i o n o f a m m o n i u m s u l f a t e w a s d i s s o l v e d in a minimum volume of Tris-NaCI-AMP buffer and was dialyzed against 3.5 liters o f t h e s a m e b u f f e r . Step 4. First DEAE A-50 Sephadex Fractionation. T h e d i a l y z e d
[33]
ADENOSINE MONOPHOSPHATE NUCLEOSIDASE
269
solution was eluted from a 3.8 × 21 cm column of DEAE A-50 Sephadex with a 1.5-liter linear gradient of NaC1 (0.16-0.35 M).
Step 5. Second DEAE A-50 Sephadex Fractionation. The active fraction from the first DEAE column was diluted with an equal volume of Tris-AMP buffer (no NaC1) and was placed on a 1.8 × 21 cm column of DEAE A-50 Sephadex. The enzyme was eluted with a 500-ml PO4 gradient (0.05-0.15 M, pH 8.0) in the Tris-NaC1-AMP buffer. Step 6. 1,6-DiaminohexaneSepharose 4B Fractionation. The AMP nucleosidase peak (140 ml) from step 5 was dialyzed overnight against 2 liters of salt-free Tris-AMP buffer and applied to a 1.6 × 20 cm column of Sepharose 4B coupled to 1,6-diaminohexane TM (coupling was done with 1 M 1,6-diaminohexane). The enzyme was eluted with a 800-ml NaC1 gradient (0-1.0 M) followed by 200 ml of 1.1 M NaC1. The gradient was buffered with Tris-AMP buffer. Active fractions were concentrated to 4.2 ml with Amicon ultrafiltration using a PM 30 membrane. Step 7. Sephadex G-200 Gel Filtration. Enzyme from the previous step was eluted from Sephadex G-200 with Tris-NaC1-AMP buffer in which the NaC1 had been increased to 0.2 M. The active fractions were pooled and concentrated using a Sartorius vacuum ultrafiltration device. A summary of the purification is listed in Table II.
TABLE II PURIFICATION OF AMP NUCLEOS1DASE FROM Escherichia coli K1z a
Purification procedure Initial extract Heat treatment Ammonium sulfate fractionation First DEAE-Sephadex column Second DEAE-Sephadex column 1,6-Diaminohexane-Sepharose fractionation Sephadex G-200 fractionation
Volume (ml) 2950 5325 200 298 140
Total protein (mg) 446,00~ 35,000 2,500 410 64
4.2 0.2
5.7 3.9
Total units (/zmoles/ min) 1010 685 580 360 260 85 67
Specific activity (#tmoles/ Yield min/mg) (%) 0.002 0.019 0.19 0.88 4. I 15 17
100 68 48 36 26 8 7
Starting material was 1.5 kg wet weight of cells. b Initial extract protein values may not be accurate. 12p. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Natl. Acad. Sci. U.S.A. 61, 636
(1968).
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NUCLEOTIDASES AND NUCLEOSIDASES
[33]
TABLE III PROPERTIES OF AMP NUCLEOSIDASEFRoMAzotobacter vinelandii OP Molecular weight a.b Subunit molecular weight a,b
320,000 54,000
Number of subunits a'b Km for AMP c
6 100 uM 40 uM
M0.s for MgATP 2- c
150 UM
10.5 for Pi c Binding sites for tubercidin 5'PO4 (an AMP analog) b Ko for tubercidin 5'-PO4 b Binding sites for MgATP 2- b Kd for MgATP 2- b Binding sites for P~ b Kd for Pt~
3 15 6 90 pdk/ 6 170/~/
Equilibrium sedimentation SDS gel electrophoresis and equilibrium sedimentation Apparently identical size and charge Km is independent of [MgATP2-], exhibits Michaelis-Menten kinetics Increases Vmax400-fold, Hill coefficient >3, Mo.5 is independent of [AMP] Competitive with MgATP 2-, Hill coefficient of inhibition >3 Binding follows standard saturation isotherm MgATP 2- has no effect on Ka Binding strongly cooperative Ka is [MgATP z-] for 50% saturation At high [Pi] nonspecific binding occurs, binding strongly cooperative Ko is [El for 50% saturation, P~ and MgATP 2- binding are mutually exclusive
a Copyright by the American Chemical Society. V. L. Schramm and L. I. Hochstein, Biochemistry 11, 2777 (1972). b V. L. Schramm, J. Biol. Chem. 251, 3417 (1976). c V. L. Schramm, J. Biol. Chem. 249, 1729 (1974).
Comments on Purification Procedure. The E. coli AMP nucleosidase is present at lower levels than the A. vinelandii enzyme, and it is also less stable as judged by the lower yields. The reason for this instability, and the methods to stabilize the E. coli enzyme, have not yet been investigated. However, the enzyme retains >80% of the initial activity when stored frozen for 6 months at - 8 0 ° in the buffer used for the G-200 TABLE IV KINETIC PROPERTIESOF E. coli AMP NUCLEOSIDASEa Km for AMP
M0.5 for MgATP 210.5 for P~
120/.d,/
40/zM 200 p.M
Km dependent on MgATP 2- Vm~, unaffected, cooperative kinetics at low [MgATP2-], Hill coefficient >2 M0.5 dependent on [AMP], MgATP 2- activation curves cooperative, Hill coefficient > 1.5 Pi competitive with MgATP z-, cooperativity of MgATP ~- curves retained at high [Pl]
H. Leung and V. L. Schramm, unpublished observations (1976).
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271
Sephadex step. Analysis of purified E. coli AMP nucleosidase on polyacrylamide gel electrophoresis gave two bands which stained for protein with Coomassie blue. Similar unstained gels showed that AMP nucleosidase activity was present in both protein bands. This result suggests that the E. coli enzyme is homogeneous following the Sephadex G-200 step. No attempts have been made to crystallize the E. coil enzyme. Properties of AMP Nucleosidases The kinetic patterns of AMP nucleosidases are complex, with cooperative activation by MgATP 2- and cooperative inhibition by Pi. 2 The kinetic constants and physical properties of the A . vinelandii enzyme are listed in Table III, and the kinetic properties of the E. coil enzyme are listed in Table IV.
[34] A n A c i d N u c l e o t i d a s e
from Rat Liver Lysosomes
B y CHAanLAMPOS ARSENIS and OSCAR TOUSTER
Nucleotide + H~O--~nucleoside + phosphate Rat liver lysosomes are capable of degrading nucleic acid to nucleosides and inorganic phosphate.1 This process requires an acidic pH and the presence of nucleases and nucleotidases. Lysosomes have long been known to contain various enzymes exhibiting phosphatase activity toward a wide variety of substrates, including nucleotides. Resolution of these activities was achieved by DEAE-cellulose chromatography, 2'3 yielding an enzyme which acts on nucleotides but not on simple sugar phosphates. 3 It is apparently the only acid nucleotidase that has been found in mammalian cells, and it is the only nucleotidase reported to be active toward 2'-, Y-, and 5'-nucleotides. The following preparation of the acid nucleotidase from an extract of a crude lysosomal fraction, and the description of its properties, are based on a previous report a in which the preparation of the enzyme from highly purified lysosomes ("tritosomes") was also reported. The use of purified lysosomes offers the advantage of yielding a higher purity c. Arsenis, J. S. Gordon, and O. Touster, J. Biol. Chem. 245, 205 (1970). 2C. Arsenis and O. Touster, J. Biol. Chem. 242, 3400 (1967). :~C. Arsenis and O. Touster,J. Biol. Chem. 243, 5702 (1968). METHODS
IN
ENZYMOLOGY, VOL. LI
Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5
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[33] A d e n o s i n e M o n o p h o s p h a t e Nucleosidase from A z o t o b a c t e r v i n e l a n d i i a n d E s c h e r i c h i a coli
By VERN L. SCHRAMM and HAZEL B. LEUNG MgATP2-
AMP + H20
) Adenine + Ribose 5-PO4
Adenosine monophosphate nucleosidase (AMP nucleosidase) was first described by Hurwitz et al. 1 during studies of the polynucleotide metabolizing enzymes of Azotobacter vinelandii. The enzyme differs from enzymes which are usually classified as nucleotide-degrading enzymes in that it exhibits a high degree of specificity for AMP as substrate and is essentially inactive unless MgATP z-, the allosteric activator, is present. 2 Allosteric inhibition of activity occurs in the presence of Pi. These controls regulate the enzyme activity which has been postulated to control intracellular AMP levels? '4 The resulting adenine is deaminated by adenine deaminase to form hypoxanthine, which is the end-product of AMP catabolism in Azotobacter. AMP nucleosidase is also present in E. coli and has similar kinetic properties. The end-product of AMP catabolism in E. coli appears to be adenine, since adenine deaminase is missing in this organism?
Assay Method
Principle. Hydrolysis of AMP to ribose 5-PO4 and adenine releases the anomeric carbon and therefore causes it to become a reducing sugar. Recent improvements in the reducing sugar assay s allow accurate and sensitive quantitation of the ribose 5-PO4. Previous investigators have used n-butanol extraction of adenine as an assay method for AMP nucleosidase. 7 This method suffers from a lower sensitivity and a lack of specificity, since adenosine is also partially extracted into n-butanol. Conversely, few enzyme activities interfere with the reducing sugar assay. 1j. Hurwitz, L. A. Heppel, and B. L. Horecker, J. Biol. Chem. 226, 525 (1957). 2 V. L. Schramm, J. Biol. Chem. 249, 1729 (1974). 3 V. L. Schramm and H. Leung, J. Biol. Chem. 248, 8313 (1973). aV. L. Schramm and F. C. Lazorik, J. Biol. Chem. 2S0, 1801 (1975). 5 H. Leung and V. L. Schramm, unpublished observations (1976). 6 S. Dygert, L. H. Li, D. Florida, and J. A. Thoma, Anal. Biochem. 13, 367 (1965). 7 M. Yoshino, J. Biochem. (Tokyo) 68, 321 (1970). METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress, [nc. All rightsof reproductionin any form reserved. ISBN 0q2-181951-5
264
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[33]
Reagents
Assay mixture: 0.1 M triethanolamine-HCl, pH 8.0, 4 mM AMP, 7 mM ATP, and 7 mM MgCI2 Alkaline Cu reagent: 16 g Na~CO3, 6.4 g glycine, and 0.18 g CUSO4"5 1-120 in a final volume of 400 ml Indicator reagent: 0.48 g 2,9-dimethyl-l,10-phenanthroline in a final volume of 400 ml; pH adjusted to 3.0 with HC1 Enzyme Dilution. Enzyme is diluted in 0.1 M Tris.HC1, pH 8.0, containing 2 mM AMP, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 3 phenylmethyl sulfonyl fluoride. Diluted enzyme is >90% stable for 8 hr at 0 °. Procedure. Diluted enzyme (0.001-0.01 unit) is added to 0.25 ml of the assay mixture which has been equilibrated to 30 °. The amount of protein added should be kept below 30/zg, since higher concentrations will cause interference in the reducing sugar assay. Blank values must always be run by adding an enzyme aliquot to assay mixture containing 0.3 ml of the alkaline Cu reagent. The reducing sugar formed in an appropriate incubation period (20-2 min for 0.001--0.01 unit, respectively) is estimated by adding 0.3 ml of alkaline Cu reagent, 0.3 ml of 2,9-dimethyl-l,10-phenanthroline reagent, and sufficient HzO to give a final volume of 1.6 ml. The alkaline Cu reagent stops catalysis, and the mixture is stable for several hours at room temperature. Color development occurs when the covered tubes are placed in a boiling HzO bath (95-100 ~) for 8 min. After cooling to 30 ° in a circulating H20 bath, the Cu-2,9-dimethyl-l,10-phenanthroline complex is read at 450 nm. A standard curve with ribose 5-PO4 should be run. The normal sensitivity under these conditions is 0.0025 t~mole of ribose 5-PO4 which will give an A450 of approximately 0.045. Definition o f Unit. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 /xmole of product per minute. Protein is determined by the Folin method. 8
Growth of Calls Azotobacter vinelandii OP a
Cultures of A. vinelandii OP were maintained and grown at 30 ° in nitrogen-free medium 1° containing the following ingredients per liter of SE. Layne, this series, Vol. 3 [73]. aj. A. Bush and P. W. Wilson, Nature (London) 184, 381 (1959). loj. W. Newton, P. W. Wilson, and R. H. Barris, J. Biol. Chem. 204, 445 (1953).
[33]
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deionized water: K2HPO4, 1.4 g; KH2PO4, 0.35 g; MgSO4"7 H~O, 0.2 g; NaC1, 0.1 g; CAC12"2 H20, 0.1 g; Fes(SO4)z'3 H20, 0.01 g; FeClz'4 H20, 3.5 rag; Na2MoO4"2 H20, 0.25 rag; and sucrose, 20 g. The phosphate buffer was autoclaved separately. For growth of large-scale cultures, 50 ml of the media in a 500-ml Erlenmeyer flask were inoculated from an agar slant and grown to mid-log phase (200-400 Klett units using a No. 42 blue filter) on a New Brunswick model VS rotory shaker. Fivemilliliter portions were transferred to six 500-ml portions of media in 2liter Erlenmeyer flasks. These were grown to mid-log phase as before and transferred to 200 liters of media contained in a growth chamber, u Cells were harvested at late-log or early-stationary phase to give approximately 2 kg of cell paste.
Escherichia coli K12 For growth of E. coli K12, a similar procedure was used except the cells were maintained and grown at 37 ° in medium containing the following ingredients per liter of deionized water: K2HPO4, 10 g; KH2PO4, 2.5 g; MgSO4"7 H20, 0.2 g; CAC12"2 H20, 0.01 g; Fe2(SO4)3"3 H20, 0.01 g; (NH4)~SO4, 1 g; and glucose 5 g. Medium and glucose were autoclaved separately and mixed before use. Inoculation of the 200-liter growth chamber was with 100 ml of mid-log phase cells. Cells were harvested in log phase (Klett 490) to give approximately 1 kg of cell paste. Cell pastes or cells suspended 1 : 1 (w: w) in 0.4 M phosphate, pH 7.5, can be frozen and stored at - 2 0 ° or -85 °. At - 2 0 °, the enzyme activity is stable for 6 months followed by a slow decline' of activity. Cells frozen and stored at -85 ° show no loss of enzyme activity for up to 3 years. This applies to both Azotobacter and Escherichia cell pastes. Purification Procedures
AMP Nucleosidase from Azotobacter vinelandii OP Unless otherwise mentioned, all buffers used during the purification procedure contained EDTA (10 -4 M), dithiothreitol (10 -4 M), and phenylmethylsulfonyl fluoride (3 × 10-6 M). Temperatures were at 0-5 ° except where noted.
Step 1. Initial Extract. Frozen cells (954 g) were thawed in a 30 ° water bath and suspended by mixing with 1.0 liter of 0.4 M potassium phosphate buffer (pH 7.5) containing EDTA (2 × 10-4 M), dithiothreitol (2 x 10-4 M), and PMSF (6 × 10-6 M). The suspension was passed 11V. L. Schramm, Anal. Biochem. 57, 377 (1974).
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through an Aminco French pressure cell at 15,000-25,000 psi and diluted to 3.3 liters with 0.2 M potassium phosphate buffer (pH 7.5). A 5-ml sample of the extract was centrifuged at 12,000 g for 15 min, and the supernatant fluid was used to determine activity and protein.
Step 2. Heat Treatment. The initial extract, placed in a 3.5-liter stainless-steel beaker, was heated to 60 ° while being stirred over a period of 30 rain. The extract was maintained at 60 ° for 10 min and then cooled by placing the beaker in an ice bath. The supernatant fluid (2.5 liters) was recovered after centrifugation at 12,000 g for 8 hr. Heating rates were not critical during this step, and similar results were obtained when the temperature was increased to 60 ° in as little as 2 min. Step 3. Ammonium Sulfate Fractionation. The supernatant fluid from the heat treatment was brought to 0.37 saturation at 2 ° by the addition of solid ammonium sulfate over a period of 13 min followed by an additional 17-min stirring. The precipitate was removed by centrifugation (12,000 g for 40 min) and suspended in 150 ml of 0.1 M Tris.HCl buffer (pH 8.0) containing 0.15 M NaC1 and 2 mM AMP (the Tris-NaC1AMP buffer) to give a total volume of 208 ml. Step 4. Desalt on G-25 Sephadex. The ammonium sulfate fraction was applied to a large (4.6 × 56 cm) G-25 Sephadex column which had been equilibrated with the Tris-NaC1-AMP buffer, and eluted with the same buffer. Those protein fractions free of ammonium sulfate, as determined by Nessler reagent, were pooled to give 250 ml. Step 5. First DEAE A-50 Sephadex Fractionation. The desalted protein solution was applied to a 4.7 × 12 cm DEAE A-50 Sephadex column equilibrated against the Tris-NaCI-AMP buffer. The protein was eluted with a 2-liter linear gradient of NaC1 (0.15-0.30 M) in the same buffer. The enzyme peak appeared at 0.23 M NaCI and was diluted with the Tris-AMP buffer, containing no NaC1, to reduce the NaCI concentration to 0.15 M. The solution was concentrated to 230 ml on an Amicon Diaflow pressure dialysis unit. Step 6. Second DEAE A-50 Sephadex Fractionation. The solution from the previous step was applied to a 2.5 × 50 cm DEAE A-50 Sephadex column, and the column was developed using a 2-liter gradient from 0.15 to 0.28 M NaCI. Fractions of the highest activity were pooled and concentrated to 57 ml in an Amicon Diaflow concentrator. Step 7. Hydroxylapatite Fractionation. Without further adjustment of the NaCI concentration, the fraction from the second DEAE column was applied to a 1.5 × 7.5 cm column of hydroxylapatite:cellulose
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mixture (1.4:1.0 dry weight basis) which had been equilibrated by washing with 10 volumes of Tris-NaCI-AMP buffer. The column was eluted sequentially with 25-ml portions of the buffer containing 20, 40, 80, 160, and 320 mM KzHPO4 (pH 8.0). Fractions containing enzymic activity were pooled and concentrated on an Amicon Diaflow concentrator to 7.4 ml.
Step 8. Sephadex G-200 Gel Filtration. The solution from the previous step was passed through a (2.5 × 100 cm) column of Sephadex G-200 equilibrated with Tris-HC1 buffer (0.1 M, pH 8.0) containing 2.0 mM AMP. Those fractions containing the highest specific activities were combined and concentrated by pressure dialysis (Amicon Diaflow) to 3.2 ml. Polyacrylamide gel electrophoresis of this fraction gave a single band both in the presence and absence of sodium dodecyl sulfate. Crystallization of AMP Nucleosidase. Purified enzyme (4.6 ml; 1.7 mg/ml, specific activity of 30) in 0.1 M Tris.HC1 (pH 8.0) containing 1 mM AMP was precipitated by the addition of solid ammonium sulfate to 0.75 saturation. The precipitate was collected by centrifugation at 29,000 g for 7 min and extracted sequentially with 1 ml of 0.70, 0.66, 0.62, 0.58, 0.54, 0.50, 0.46, and 0.42 saturated ammonium sulfate in 0.1 M Tris.HC1 and 1 mM AMP (pH 8.0). Upon standing overnight at 4 °, crystals appeared in tubes containing 0.42 and 0.46 saturated ammonium sulfate, both having specific activities of 34.3, and both giving a single band in polyacrylamide gel electrophoresis. Microscopic examination revealed the crystalline structure to be hexagonal plates with a diameter of -0.03 ram. These crystals have been used to induce crystallization using enzyme preparations with specific activities as low as 15. The resulting crystalline material always exhibits specific activities near 34 and appears either as hexagonal plates or clusters of needles. Yield is usually 50-80% for the crystallization step. The purification procedure is summarized in Table I. Stability of the Purified Enzyme. Following Sephadex G-200 the enzyme was stable for periods of at least 1 year after freezing in Dry Ice-ethanol mixtures and storing at - 7 0 °. The crystalline enzyme was also stable for periods of at least 1 year at 4 °, stored in the ammonium sulfate solution from which it was crystallized. AMP Nucleosidase from Escherichia coli In general, the above purification procedure can be used for preparation of the E. coli enzyme. The differences in purification and yield are due primarily to E. coli containing less of the enzyme, and the enzyme
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TABLE I PURIFICATIONOF AMP NUCLEOSIDASEFROMAzotobacter vinelandia
Purification procedure Initial extract Heat treatment Ammonium sulfate fractionation Sephadex G-25 First DEAE-Sephadex column Second DEAE-Sephadex column Hydroxylapatite column Sephadex G-200 fractionation° Crystals from G-200 side fractions c
Volume (ml)
Total protein (rag)
Total units (/zmoles/ rain)
3300 2500 208 250 230 57 7.4 3.2 1.1
108,000 32,000 8100 7500 300 130 60 21 6.7
2700 2100 1500 1500 1400 1300 1100 690 230
Specific activity (/zmoles/ Yield min/mg) (%) 0.025 0.067 0.19 0.21 4.6 9.9 18 34 34
100 78 58 58 51 48 39 34a --
a Starting material was 954 g wet weight of cells. b Fractions containing enzyme of specific activity 34 were pooled, concentrated, and frozen for storage. c In this purification, fractions containing substantial activity, but specific activities less than 34, were pooled, concentrated, brought to near precipitation with ammonium sulfate, and crystallization was induced by the addition of 1/zl of a solution containing seed crystals of AMP nucleosidase. These were collected by centrifugation, dissolved in Tris.HCl (0.1 M, pH 8.0) containing 2 mM AMP, dialyzed to remove ammonium sulfate, and assayed for protein and activity. a Percent yield includes both fractions with specific activity of 34. b e i n g l e s s s t a b l e t h a n t h a t f r o m Azotobacter. T h e p u r i f i c a t i o n s c h e m e o u t l i n e d b e l o w will e m p h a s i z e t h e d i f f e r e n c e s in t h e t w o p r o c e d u r e s .
Step 1. Initial Extract. T h i s is s i m i l a r to t h e A. vinelandii p r o c e d u r e e x c e p t t h a t 4 m M MgCI~ a n d 9 m g c r y s t a l l i n e d e o x y r i b o n u c l e a s e ( W o r t h i n g t o n , c r y s t a l l i n e ) w e r e a d d e d to 1.5 k g o f c e l l s d u r i n g t h e t h a w i n g p r o c e d u r e . T h e p H w a s a d j u s t e d to 7.5 w i t h 10 N K O H b e f o r e the next step.
Step 2. Heat Treatment. T h i s is s i m i l a r to t h e Azotobacter p r e p ~ a t i o n e x c e p t t h a t o n l y 30 m i n o f c e n t r i f u g a t i o n a t 12,000 g w e r e r e q u i r e d to r e m o v e t h e p r e c i p i t a t e . Step 3. Ammonium Sulfate Fractionation. T h e f r a c t i o n p r e c i p i t a t i n g b e t w e e n 0.28 a n d 0.39 s a t u r a t i o n o f a m m o n i u m s u l f a t e w a s d i s s o l v e d in a minimum volume of Tris-NaCI-AMP buffer and was dialyzed against 3.5 liters o f t h e s a m e b u f f e r . Step 4. First DEAE A-50 Sephadex Fractionation. T h e d i a l y z e d
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ADENOSINE MONOPHOSPHATE NUCLEOSIDASE
269
solution was eluted from a 3.8 × 21 cm column of DEAE A-50 Sephadex with a 1.5-liter linear gradient of NaC1 (0.16-0.35 M).
Step 5. Second DEAE A-50 Sephadex Fractionation. The active fraction from the first DEAE column was diluted with an equal volume of Tris-AMP buffer (no NaC1) and was placed on a 1.8 × 21 cm column of DEAE A-50 Sephadex. The enzyme was eluted with a 500-ml PO4 gradient (0.05-0.15 M, pH 8.0) in the Tris-NaC1-AMP buffer. Step 6. 1,6-DiaminohexaneSepharose 4B Fractionation. The AMP nucleosidase peak (140 ml) from step 5 was dialyzed overnight against 2 liters of salt-free Tris-AMP buffer and applied to a 1.6 × 20 cm column of Sepharose 4B coupled to 1,6-diaminohexane TM (coupling was done with 1 M 1,6-diaminohexane). The enzyme was eluted with a 800-ml NaC1 gradient (0-1.0 M) followed by 200 ml of 1.1 M NaC1. The gradient was buffered with Tris-AMP buffer. Active fractions were concentrated to 4.2 ml with Amicon ultrafiltration using a PM 30 membrane. Step 7. Sephadex G-200 Gel Filtration. Enzyme from the previous step was eluted from Sephadex G-200 with Tris-NaC1-AMP buffer in which the NaC1 had been increased to 0.2 M. The active fractions were pooled and concentrated using a Sartorius vacuum ultrafiltration device. A summary of the purification is listed in Table II.
TABLE II PURIFICATION OF AMP NUCLEOS1DASE FROM Escherichia coli K1z a
Purification procedure Initial extract Heat treatment Ammonium sulfate fractionation First DEAE-Sephadex column Second DEAE-Sephadex column 1,6-Diaminohexane-Sepharose fractionation Sephadex G-200 fractionation
Volume (ml) 2950 5325 200 298 140
Total protein (mg) 446,00~ 35,000 2,500 410 64
4.2 0.2
5.7 3.9
Total units (/zmoles/ min) 1010 685 580 360 260 85 67
Specific activity (#tmoles/ Yield min/mg) (%) 0.002 0.019 0.19 0.88 4. I 15 17
100 68 48 36 26 8 7
Starting material was 1.5 kg wet weight of cells. b Initial extract protein values may not be accurate. 12p. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Natl. Acad. Sci. U.S.A. 61, 636
(1968).
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NUCLEOTIDASES AND NUCLEOSIDASES
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TABLE III PROPERTIES OF AMP NUCLEOSIDASEFRoMAzotobacter vinelandii OP Molecular weight a.b Subunit molecular weight a,b
320,000 54,000
Number of subunits a'b Km for AMP c
6 100 uM 40 uM
M0.s for MgATP 2- c
150 UM
10.5 for Pi c Binding sites for tubercidin 5'PO4 (an AMP analog) b Ko for tubercidin 5'-PO4 b Binding sites for MgATP 2- b Kd for MgATP 2- b Binding sites for P~ b Kd for Pt~
3 15 6 90 pdk/ 6 170/~/
Equilibrium sedimentation SDS gel electrophoresis and equilibrium sedimentation Apparently identical size and charge Km is independent of [MgATP2-], exhibits Michaelis-Menten kinetics Increases Vmax400-fold, Hill coefficient >3, Mo.5 is independent of [AMP] Competitive with MgATP 2-, Hill coefficient of inhibition >3 Binding follows standard saturation isotherm MgATP 2- has no effect on Ka Binding strongly cooperative Ka is [MgATP z-] for 50% saturation At high [Pi] nonspecific binding occurs, binding strongly cooperative Ko is [El for 50% saturation, P~ and MgATP 2- binding are mutually exclusive
a Copyright by the American Chemical Society. V. L. Schramm and L. I. Hochstein, Biochemistry 11, 2777 (1972). b V. L. Schramm, J. Biol. Chem. 251, 3417 (1976). c V. L. Schramm, J. Biol. Chem. 249, 1729 (1974).
Comments on Purification Procedure. The E. coli AMP nucleosidase is present at lower levels than the A. vinelandii enzyme, and it is also less stable as judged by the lower yields. The reason for this instability, and the methods to stabilize the E. coli enzyme, have not yet been investigated. However, the enzyme retains >80% of the initial activity when stored frozen for 6 months at - 8 0 ° in the buffer used for the G-200 TABLE IV KINETIC PROPERTIESOF E. coli AMP NUCLEOSIDASEa Km for AMP
M0.5 for MgATP 210.5 for P~
120/.d,/
40/zM 200 p.M
Km dependent on MgATP 2- Vm~, unaffected, cooperative kinetics at low [MgATP2-], Hill coefficient >2 M0.5 dependent on [AMP], MgATP 2- activation curves cooperative, Hill coefficient > 1.5 Pi competitive with MgATP z-, cooperativity of MgATP ~- curves retained at high [Pl]
H. Leung and V. L. Schramm, unpublished observations (1976).
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271
Sephadex step. Analysis of purified E. coli AMP nucleosidase on polyacrylamide gel electrophoresis gave two bands which stained for protein with Coomassie blue. Similar unstained gels showed that AMP nucleosidase activity was present in both protein bands. This result suggests that the E. coli enzyme is homogeneous following the Sephadex G-200 step. No attempts have been made to crystallize the E. coil enzyme. Properties of AMP Nucleosidases The kinetic patterns of AMP nucleosidases are complex, with cooperative activation by MgATP 2- and cooperative inhibition by Pi. 2 The kinetic constants and physical properties of the A . vinelandii enzyme are listed in Table III, and the kinetic properties of the E. coil enzyme are listed in Table IV.
[34] A n A c i d N u c l e o t i d a s e
from Rat Liver Lysosomes
B y CHAanLAMPOS ARSENIS and OSCAR TOUSTER
Nucleotide + H~O--~nucleoside + phosphate Rat liver lysosomes are capable of degrading nucleic acid to nucleosides and inorganic phosphate.1 This process requires an acidic pH and the presence of nucleases and nucleotidases. Lysosomes have long been known to contain various enzymes exhibiting phosphatase activity toward a wide variety of substrates, including nucleotides. Resolution of these activities was achieved by DEAE-cellulose chromatography, 2'3 yielding an enzyme which acts on nucleotides but not on simple sugar phosphates. 3 It is apparently the only acid nucleotidase that has been found in mammalian cells, and it is the only nucleotidase reported to be active toward 2'-, Y-, and 5'-nucleotides. The following preparation of the acid nucleotidase from an extract of a crude lysosomal fraction, and the description of its properties, are based on a previous report a in which the preparation of the enzyme from highly purified lysosomes ("tritosomes") was also reported. The use of purified lysosomes offers the advantage of yielding a higher purity c. Arsenis, J. S. Gordon, and O. Touster, J. Biol. Chem. 245, 205 (1970). 2C. Arsenis and O. Touster, J. Biol. Chem. 242, 3400 (1967). :~C. Arsenis and O. Touster,J. Biol. Chem. 243, 5702 (1968). METHODS
IN
ENZYMOLOGY, VOL. LI
Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5
