558
[76]
PURINE METABOLIZING ENZYMES
effectors when present at the 100-200/13,/range. ppGpp is an especially potent inhibitor and at physiological concentrations can account for the drop in guanine nucleotides in the pool during the stringent response to amino acid control of nucleic acid synthesis, lr Physiological Function. In enteric bacteria the 6-OH purine phosphoribosyltransferases reside in the periplasm at the membrane r'a and there mediate the uptake of 6-OH purines. The specificity of the enzymes on the isolated membrane vesicles reflects the metabolic capacities of the cells even though broader substrate specificity is observed upon solubilization. 8 Alternative Purification Scheme. An alternative method of enzyme purification is available which reduces the preparation to two steps. It is not as useful, however, for preparing very large amounts of enzyme. Based on the observation that much of the enzyme is released from the periplasm upon osmotic shock, 7 it can be obtained in the following manner. Cells are grown as described, but must be harvested at midexponential growth. Osmotic shock fluid is prepared by dilution of cells in 20% sucrose solution, 1"100 into 20 ~ MgSO4. The cells are removed by centrifugation. The supernatant "shock fluid" containing the enzyme is filtered through a 0.45 ~m nitrocellulose filter and concentrated by ultrafiltration to a protein concentration of 2-4 mg/ml. An (NH4)2SO4 fraction, 35-50% saturation, is prepared (see step 4 of the purification procedure above) and applied directly to the Ecteolacellulose column and eluted as described in step 9 of the purification procedure. Though the peak fractions from the column are homogeneous after only these two steps, in addition to the preparation of osmotic shock fluid, cells, subjected to osmotic shock immediately upon harvest from mid-exponential growth and never frozen, must be used. ~r j. H o c h s t a d t - O z e r a n d M. Cashel, J. Biol. Chem. 247, 1067 (1972).
[76] A d e n i n e
Phosphoribosyltransferase Escherichia cell
from
By JoY HOCHSTADT A d e n i n e + 5' phosphoribosyl-c~-I pyrophosphate---> 5' A M P + inorganic p y r o p h o s p h a t e
Adenine phosphoribosyltransferase is an inducible enzyme in Escherichia coli I that though readily solubilized into aqueous extracts upon 1 j. H o c h s t a d t - O z e r a n d E. R. S t a d t m a n , J. Biol. Chem. 246, 5294 (1971). M E T H O D S IN E N Z Y M O L O G Y , V O L . LI
Copyright © 1978by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5
[76]
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cell rupture appears to be localized on the cell membrane in situ 2 where it mediates the translocation of adenine into the cell as AMP. z Its location at the cell surface is further indicated by its release from the periplasm upon osmotic shock treatment. 3 Enzyme activity is regulated by nucleotide pool constituents in situ; a experimentally it is quite sensitive to competitive inhibition by a very wide variety of 5' nucleotides. 1
Assay Method
Principle. The assay depends on the chromatographic separation of the radioactivelylabeled product, A M P , from the radioactivelylabeled substrate adenine. Reagents
MgC12, 0.1 M Tris.HC1, pH 7.8, 1 M [8-14C]Adenine, 20 mM (specific radioactivity adjusted to 5-10 mCi/mmole) K+MgZ+EDTA Titriplex, 1 M Mg2PRPP, 20 mM Adenine, 20 mM 5' AMP, 20 mM [K][EDTA], pH 7.0, 1 M Eastman Chromagram with fluorescent indicator #6065 Ammonium acetate, 1 M P r o c e d u r e . The following are added to 12 × 75 mm glass test tubes using Hamilton syringes and Hamilton PB600-1 repeating dispensers: 5 /zl [14C]adenine, 5/xl Tris buffer, 0.5/~1 MgC12, 0.5/zl K+Mg 2+ Titriplex, and enzyme sample in a volume 0.5-10/~1 (containing between 5 ng to 5 /zg of protein according to relative purity). Distilled water is added to make a total volume of 45/~1. Reactions are initiated with the addition of 5 /zl of Mg2PRPP. A control tube is prepared to which 5/zl of distilled water is added instead of MgzPRPP. Reaction mixtures are incubated at 37 ° with shaking usually for 10 min. The reaction is terminated by addition of 5 /zl [K][EDTA]. Cellulose thin layers (plastic-backed) are cut to strips 7-cm high and 1.5 cm times the number of samples wide to a maximum of 20 cm wide. Channels, 6 × 1.5 cm, are ruled on the thin layer with a ruler and pencil. At a point 1 cm from the edge, 0.5 Izl of AMP carrier solution and 0.5/zl of adenine carrier solution are spotted in
2j. Hochstadt-Ozer and E. R. Stadtman, J. Biol. Chem. 246, 5304(1971). aj. Hochstadt-Ozerand E. R. Stadtman, J. Biol. Chem. 246, 5312 (1971).
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PURINE METABOLIZING ENZYMES
[76]
the middle of the 1.5-cm channel. Then 3/xl of the reaction mixture are spotted on top of the carrier AMP/adenine spot. No effort is made to dry the spots prior to placement of the thin layer in the chromatography tank. The chromatograms are then developed in 1 mM ammonium acetate in a glass chromatography tank until the ascending solvent front has reached the upper edge of the 7-cm-high thin layer. The thin layers are removed and dried with a hair dryer. The AMP (Rf = 0.7) and adenine (Re = 0.4) spots are localized under UV light and marked with a pencil; each AMP spot is cut out and placed in a scintillation vial. Toluene-based fluor solution is added to the vial; a few ml are sufficient for the entire thin-layer strip need only be saturated with the fluor solution it need not be totally submerged in the liquid. The samples are counted in a liquid scintillation counter; with an appropriate fluor solution the counting efficiency for 14C on the cellulose-backed thin layers is 75-80%.
Linearity. The reaction goes to completion and often is linear until a majority of the substrate (60-70%) has been utilized. For kinetic studies, concentration of enzyme and incubation times are adjusted, however, to utilize no more than 15-25% of the substrate. Standardization of Substrates. [14C]adenine is standardized by spectral assay using a molar extinction coefficient of 13,400 at 260.5 nm at pH 7. One to three microliters pipetted directly into a scintillation vial, dried completely, and covered with 10 ml of fluor solution gave a counting efficiency of approximately 97%; this was used to confirm radiospecific activity assigned by the manufacturer. Mg2PRPP solutions are standardized by allowing the enzyme reaction to proceed to completion using a limiting amount of Mg2PRPP and an excess of [14C]adenine standardized as above. The amount of AMP formed under such conditions is taken as a measure of the Mg~PRPP present since the reaction does go to virtual completion. Commercially purchased PRPP salts are often found to be 50-60% impure. Purification of substrate PRPP. In order to assay a variety of PRPP salts in addition to those commercially available, as well as to remove contaminants, the following procedure I is employed: 100 /zmole of Mg~PRPP (in a 1.5 ml volume) are passed over a 7.5 × 0.5 cm bed of Dowex 50 which has been well washed with water after preliminary treatment with LiC1 or KC1 (for preparing Li4PRPP or K4PRPP, respectively, for example). Water (3.5 ml) is added to wash through the PRPP in 0.5-ml portions, and the effluent fractions are pooled and stored at - 7 9 ° in 0.5-ml portions. All PRPP activity is standardly recovered from
[76]
ADENINE PHOSPHORIBOSYLTRANSFERASE
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the Dowex 50. The quantity and concentration of the PRPP are then determined by the endpoint assay described above. Definition o f Unit and Specific Activity. One unit is defined as the amount of enzyme that catalyses the phosphoribosylation of 1/xmole of adenine to form I ~mole of AMP per minute. Specific activity is expressed in terms of units per milligram of protein. Protein is determined by the Biuret 4 procedure during initial steps in the purification and thereafter by the method of Lowry et al. ~ Alternate Assay Procedure. When even greater sensitivity is required, assay of enzyme activity is performed in 20 /zl of reaction volume in glass tubes, 6 × 50 mm, containing 0.2 mM [14C]adenine (specific radioactivity >50.0 mCi/mmole), 2 mM Mg2PRPP, 100 mM Tris.HC1, and 1-10 ng of purified enzyme. The surface tension properties of the reaction mixtures in tubes of this geometry required vortexing each tube for a second, once each minute during the incubation, to ensure adequate mixing during the reaction. Other aspects of the assay are identical. Application to Measurements in Crude Extracts. Because nucleotide pool constituents are potent inhibitors of enzyme activity, all crude extracts should be dialyzed or otherwise separated from cellular metabolites prior to assay of the protein fraction for activity. Though the nucleotides are competitive with PRPP, simple increase in PRPP concentrations is not an adequate means of dealing with the situation since, as purchased, the PRPP is contaminated with inorganic pyrophosphate and possibly other inhibitors of the enzyme. Other interfering contaminants (e.g., proteases) of the crude extract can be assessed after enzyme purification by reconstructive mixing of a sample of purified enzyme with a sample of crude extract, each of known activity alone, and determination of the combined activity in the mixture. Purification
Procedure
Growth and Harvest of Organism. As much as a 100-fold enrichment of adenine phosphoribosyltransferase can be obtained by growth under conditions which inhibit de novo purine synthesis and render the organism dependent on exogenous purine and phosphoribosyltransferase 4 M. Dittebrandt, Am. J. Clin. Pathol. 18, 439 (1948). 5 O. H. Lowry, N. J. Rosebough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
562
PURINE METABOLIZINGENZYMES
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activity. 1 The medium designed for this purpose, PAT medium, 1 contains a mixture of "P"urines each at 10-4 M, "A"minopterin or amethopterin (to inhibit de novo purine biosynthesis; it also inhibits thymidine synthesis) 3 × 10 - r M , and compensatory "T"hymine or thymidine 10-s M. These " P A T " additions are made to the basic VogelBonner-Citrate medium as described by Korn and Weisbach. 6 Escherichia coli K12 is grown to the end of exponential growth (turbidity monitored) in either 20-liter glass bottles with aeration or in large-scale fermentation apparatus (e.g., 60-300 liters) at 37 °, harvested by centrifugation, frozen in liquid N2, and stored at - 7 9 °.
Step 1. Preparation o f Cell Extracts. Frozen cells are thawed at room temperature, suspended in 5-10 volumes of 50 mM potassium phosphate buffer (pH 7.5), and sonically disrupted at 0 ° by six 30-sec bursts (at maximum output with Heat Systems-Ultrasonic, Inc., model 185W Sonifier Cell Disruptor) with intervening cooling at 0 °. Debris is removed from this homogenate by centrifugation at 10,000 rpm for 20 min. Step 2. Streptomycin Precipitation. Streptomycin sulfate, 10% by volume, of 10% solution, is added to the supernatant solution (20--40 mg of protein per ml) obtained in step 1. After stirring for 10 min in the cold the mixture is centrifuged. Step 3. Ammonium Sulfate Precipitation P5a. The supernatant solution from step 2 is adjusted to a protein concentration of 10 mg/ml and pH 7.8 with Tris.HC1 (50 mM, final concentration) and a saturated solution of (NH4)2SO4 is added, to a final concentration of 35% saturation by volume. The treatment is at room temperature and equilibration is for 15 min with stirring. The precipitate is collected by centrifugation at 4 ° for 10 min at 10,000 rpm (Sorvall SS-34 rotor). The pellet is discarded. Saturated (NH4)2SO4 solution is added to that supernatant solution to make it 42% saturation by volume. Equilibration and collection of the precipitate are as before; the precipitate is resuspended in 20 mM Tris.HCl (pH 8.0) and saved for the preparation of 6-OH purine PRT. The supernatant fraction is adjusted to 49% saturated ammonium sulfate as above, and the precipitate is collected and resuspended as above. After testing this fraction for adenine and other purine PRT enzyme activities, it can be pooled with either the 35-42% fraction if rich in GPRT/HPRT, pooled with 49-56% fraction if rich in APRT, or discarded according to activities present. The supernatant fluid from this fraction is then made 56% with respect to (NH4)2SO4, and the 56% 6D. Korn and A. Weissbach, Biochim. Biophys. Acta 61, 775 (1962).
[76]
ADENINEPHOSPHORIBOSYLTRANSFERASE
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precipitate is collected as before to provide the peak APRT fraction for subsequent steps. If new growth conditions are being employed it may be possible that a shift in salting out may occur--an optional additional step is the preparation and APRT assay of a protein fraction salting out between 56-59% saturation. In about 35% of the fractionations, that fraction had the greatest APRT specific activity; however, it never contained more than 10-15% of the total activity. Step 4. Precipitation with Acetone. The protein fraction salting out between 49-56% saturated (NH4)2SO4 (or 42-56%, see above) is adjusted to 10 mg/ml of protein in 20 mM Tris.HCl (pH 8.0). One volume of acetone at - 7 ° is added to 4 volumes of protein solution and stirred in a NaCl-ice bath at - 1 0 °, and then 4 more volumes of acetone at - 1 0 ° are added with stirring. The mixture is centrifuged at - 1 5 ° after 1 more volume of acetone ( - 15~) has been added. The pellet is then collected as before, drained, and resuspended in 20 mM Tris.HCl (pH 8.0) at 0 °. The final volume it is resuspended in is - one-half that of the protein solution originally treated with acetone. This solution is then dialyzed against 1 liter of 20 mM Tris'HC1 at 0 ° for 2 hr. Step 5. Treatment with C~ Gel. The solution from step 4 is adjusted to pH 6.0 with 50 mM potassium phosphate buffer and mixed with a suspension of aged Cv gel in 50 mM potassium phosphate buffer. The ratio of gel to protein is 34.5 mg of gel to 1 mg of protein. After stirring for 15 rain in the cold, the supernatant solution is collected by centrifugation. Step 6. Treatment with Calcium Phosphate Gel. The supernatant solution from step 5 is treated with a suspension of calcium phosphate gel in 50 mM potassium phosphate buffer (pH 6.0). The ratio of calcium phosphate gel to protein is 17.6 mg of gel per milligram of protein. After stirring for 15 min in the cold, the supernatant fluid is collected and dialyzed twice in the cold, against 3 liters of 50 mM potassium phosphate buffer (pH 7.5) for 1 hr each and once against 3 liters of l0 mM potassium phosphate buffer (pH 7.5), and lyophilized. Step 7. Chromatography on Bio-Gel P-150. The lyophilized powder is resuspended to one-tenth its original volume in distilled water and divided into l-ml portions and stored at - 7 9 °. One- or 2-ml portions at a time are applied to a column (1.5 × 30 cm, resin bed) of Bio-Gel P-150 equilibrated with 75 mM potassium phosphate buffer (pH 7.5). Fractions (1-2 ml) are collected in the cold, automatically. The void volume containing contaminating protein is discarded. The enzyme fractions with activity greater than half-peak activity are pooled unless otherwise
564
[76]
PURINE METABOLIZING ENZYMES
PURIFICATION OF ADENINE PHOSPHORIBOSYLTRANSFERASEFROM PAT-MEDIUM-GROWN CELLS a,b
Protein fractionc 1. Untreated extract 2. 3. 4. 5. 6. 7.
Streptomycin supernatant solution Ammonium sulfate precipitate Acetone precipitate C_~gel supernatant Calcium phosphate gel supernatant Bio-Gel P 150 column peak
Specific activity (bemoles/min/ nag protein) 0.010 0.015 0.08 0.70 3.10 7.20 14.00
Percentage recovery 100.0 (54 observed) 73.0 23.2 22.2 21.0 14.0 12.6
a Reprinted from Hochstadt-Ozer and Stadtman, 1p. 5294. b PAT medium is VBC medium plus purines (adenine, guanine, hypoxanthine, xanthine, 0.1 mM each); amethopterin, 0.3/.d,/; thymine, 0.1 raM. c The numbers refer to steps described in the text.
noted. Fractions are stored at - 2 0 ° for 2 weeks or less and at - 7 9 ° for longer periods. The table shows a summary of the purification with respect to specific activity and yields at each step. R e c o v e r y from the streptomycin step is higher than the total activity observed in the crude extract. The extent to which inhibitory conditions or inhibitors present in the crude extract assay contributes to the decreased activity observed is determined by adding a known a m o u n t of e n z y m e activity from step 7 to a sample o f step 1 and noting the activity increment actually observed. It is from this latter figure that the total activity o f the extract and subsequent yields are calculated.
Properties
Stability. The adenine phosphoribosyltransfcrase is reasonably stable to heat treatment o f 5 rain at 60 ° [crude extracts, however, require substrate to stabilize during heat treatment, presumably due to protease which is r e m o v e d during (NH4)2SO4 fractionation]. Activity is unaffected by l0 mM mercaptoethanol. After treatment with calcium phosphate gel (step 6 of purification), instability to storage at 4 ° (or - 2 0 °) is noted. Storage in 0.1 M potassium phosphate buffer (pH 7), however, was found to be a satisfactory means of preventing e n z y m e inactivation. The
[76]
ADENINE PHOSPHORIBOSYLTRANSF ERASE
565
completely purified enzyme may be stored in a very dilute solution (i.e., 0.1 mg/ml at - 7 9 ° for several months), but considerable loss of activity was noted after subsequent storage of such solutions at - 2 0 ° for 2-3 weeks.
Homogeneity and Molecular Weight Estimations. Rechromatography of peak fractions from Bio-Gel P 150 leads to the isolation of a single protein peak, all portions of which exhibit the same specific enzyme activity. The homogeneity of this fraction is further demonstrated by the observation of a single band of protein coincident with enzyme activity after acrylamide gel electrophoresis at pH 7.2 in the presence of 5 mM MgSO4. Under standard conditions of electrophoresis, however (i.e., pH 9.5 and without MgSO4Z), the enzyme dissociates into several catalytically active bands. The smallest active fraction (fastest mobility) on the gel is least stable, but activity can be partially and temporarily enhanced by reaggregation at 4 ° in buffer solutions or in the presence of magnesium or PRPP. Similar dissociation behavior is observed when the enzyme is subjected to gel filtration chromatography at alkaline pH and in the absence of divalent cation. Calibration of the column with protein standards of known molecular weight led to estimation of the active polypeptide moieties of the enzyme at about 20,000, 30,000, and 40,000. z Stoichiornetry. For each mole of adenine and PRPP utilized, 1 mole each of PPi and AMP are generated. Specific Activity and Turnover Number. Based on an estimated molecular weight of 40,0001 for the native enzyme and a specific activity of 14.001 /xmoles of AMP generated per minute per milligram enzyme protein, an enzyme turnover number was calculated to be 5.6 × 10z reactions/rain/40,000 MW moiety. Substrate Specificity. The E. coli enzyme is specific for adenine or 2,6-diamino-purine. Numerous other tested purines, 1 including aminoimidazolicarboximideriboside previously reported to serve as substrate for partially purified bovine adenine phosphoribosyltransferase, 8 do not serve as substrates. Kinetic Constants. Michaelis-Menton kinetics are observed with respect to both PRPP and adenine. Km for PRPP in the presence of a 2fold excess of Mg 2÷ is 125/xM (Mg 2+, though required for the reaction, is also competitive with PRPP1); Km for adenine is approximately 20 p3U. 7A. C. Chrambach, Anal. Biochem. 15, 544 (1964). sj. G. Flaks, M. J. Erwin, and J. M. Buchanan, J. Biol. Chem. 228, 201 (1957).
566
PURINE METABOLIZING ENZYMES
[76]
Metal Ion Requirement. An absolute requirement for either Mn 2+ or Mg 2+ was found. The chloride salt of either metal was found satisfactory. A complex relationship between activity, metal ion concentration, and PRPP concentration exists 1"9 evidence suggests that alternate reaction mechanisms may be possible at varying cation concentrations depending on the relative concentration of PRPP as the free acid, MgPRPP or MgzPRPP 1'9, e.g., 2:1 ratio of Mg ~÷ to PRPP was found to be optimal. Other divalent cations such as Zn 2÷, Ba ~+, and Ca ~+ were highly inhibitory to the reaction. Based on this observation, K+Mg~+EDTA Titraplex is included in the assay to bind continuous traces of such metals. pH Optimum. Enzyme activity has a strikingly sharp pH optimum at pH 7.8.1 Reaction Mechanism. Under most experimental conditions a pingpong mechanism is observed. PRPP + enzyme ~ phosphoribosylenzyme + PP~ Phosphoribosylenzyme + adenine --->AMP + enzyme
Effectors o f Enzyme Activity. All nucleotides with free 5' phosphate groups are effectors. 1 Stimulation of activity at low effector concentrations (especially at saturating PRPP concentrations) is noted for some purine nucleotidesL Higher concentrations of all 5' nucleotides are competitive with PRPP. 6-NH2 purine nucleotides are most inhibitory, 6OH purine nucleotides are moderately inhibitory, and pyrimidine nucleotides are least inhibitory; however, all inhibited activity greater than 50% when included in the reaction mixture (standard assay conditions) at 2 mM. The reaction products AMP and PPf are also inhibitors of the reaction. AMP is competitive with PRPP ~ while PPt competition with PRPP may be cooperative (Mg 2÷ varied with PRPP as in Mg2PRPP) or anticooperative (at constant excess magnesium). ~ Physiological Function. In E. coli cells which are actively producing purine nucleotides by the de novo biosynthetic pathway, approximately 50 enzyme molecules are found per bacterial cell; while in those cells wholely dependent on exogenous purines for growth, approximately 5000 enzyme molecules are found per cell. 1'~ The enzyme, though readily solubilized upon cell rupture as described above, appears to largely reside on the membrane in such derepressed cells where it mediates adenine translocation across the cell membrane. 2 Evidence for this comes both from studies with isolated vesicles 2 in which both 9T. A. Krenitsky, R. Papa:io!annou, and G. B. Elion, J. Biol. Chem. 244, 1263 (1969).
[76]
ADENINE PHOSPHORIBOSYLTRAN SFERASE
567
enzyme activity and transport reaction have been studied 2 and from its recovery with other periplasmic constituents upon osmotic shock. 3 In noninduced cells this group translocation mechanism of adenine uptake may account for only a portion of the small amounts of adenine taken up, TM while in the cells grown on the PAT medium it clearly accounts for virtually all of the u p t a k e ) The enzyme also appears necessary for appropriate regulation of purine biosynthesis since its absence is associated with purine excretion. Other Properties. The enzyme therefore exists in situ in an environment very different from the one in which it is purified, aqueous solution, and characterized. Certain properties which it exhibits when in situ in the membrane differ from what is observed in aqueous solution. These differences disappear when it is released from the membrane by detergent, freeze-thawing, or sonic oscillation; among them are greater sensitivity to effectors in aqueous solution and ability to carry out an exchange reaction between AMP and adenine only while membranebound. Both properties suggest that membrane-localized enzyme exists as phosphoribosyl-enzyme and that the phosphoribosyl moiety is discharged in aqueous solution. One approach to study of this enzyme in a homogeneous environment more reflective of the membrane milieu is to incorporate it into artificial phospholipid bilayers. Alternative Purification Scheme. An alternative method of enzyme purification is available which reduces the preparation to two steps. It is not as useful, however, for preparing very large amounts of enzyme. Based on the observation that much of the enzyme is released from the periplasm upon osmotic shock, 3 it can be obtained in the following manner. Cells are grown as described, but must be harvested at midexponential growth. Osmotic shock fluid is prepared by dilution of cells in 20% sucrose solution, 1:100 into 20 ~ MgSO4. The cells are removed by centrifugation. The supernatant "shock fluid" containing the enzyme is filtered through a 0.45/zm pore size nitrocellulose filter and concentrated by ultrafiltration to a protein concentration of 2--4 rag/ ml. An (NH4)2SO4 fraction, 45-55% saturation, is prepared (see step 3 purification procedure above) and applied directly to a Bio-Gel P 150 column and eluted as described in step 7 of the purification procedure. Though the peak fractions from the column are homogeneous after only two steps in addition to osmotic shock fluid preparation, cells--subjected to osmotic shock immediately upon harvest from mid-exponential growth and never frozen--must be used for this method. 1°S. Roy-Berman and D. W. Visser, J. Biol. Chem. 250, 9270 (1975).
[76]
PURINE METABOLIZING ENZYMES
effectors when present at the 100-200/13,/range. ppGpp is an especially potent inhibitor and at physiological concentrations can account for the drop in guanine nucleotides in the pool during the stringent response to amino acid control of nucleic acid synthesis, lr Physiological Function. In enteric bacteria the 6-OH purine phosphoribosyltransferases reside in the periplasm at the membrane r'a and there mediate the uptake of 6-OH purines. The specificity of the enzymes on the isolated membrane vesicles reflects the metabolic capacities of the cells even though broader substrate specificity is observed upon solubilization. 8 Alternative Purification Scheme. An alternative method of enzyme purification is available which reduces the preparation to two steps. It is not as useful, however, for preparing very large amounts of enzyme. Based on the observation that much of the enzyme is released from the periplasm upon osmotic shock, 7 it can be obtained in the following manner. Cells are grown as described, but must be harvested at midexponential growth. Osmotic shock fluid is prepared by dilution of cells in 20% sucrose solution, 1"100 into 20 ~ MgSO4. The cells are removed by centrifugation. The supernatant "shock fluid" containing the enzyme is filtered through a 0.45 ~m nitrocellulose filter and concentrated by ultrafiltration to a protein concentration of 2-4 mg/ml. An (NH4)2SO4 fraction, 35-50% saturation, is prepared (see step 4 of the purification procedure above) and applied directly to the Ecteolacellulose column and eluted as described in step 9 of the purification procedure. Though the peak fractions from the column are homogeneous after only these two steps, in addition to the preparation of osmotic shock fluid, cells, subjected to osmotic shock immediately upon harvest from mid-exponential growth and never frozen, must be used. ~r j. H o c h s t a d t - O z e r a n d M. Cashel, J. Biol. Chem. 247, 1067 (1972).
[76] A d e n i n e
Phosphoribosyltransferase Escherichia cell
from
By JoY HOCHSTADT A d e n i n e + 5' phosphoribosyl-c~-I pyrophosphate---> 5' A M P + inorganic p y r o p h o s p h a t e
Adenine phosphoribosyltransferase is an inducible enzyme in Escherichia coli I that though readily solubilized into aqueous extracts upon 1 j. H o c h s t a d t - O z e r a n d E. R. S t a d t m a n , J. Biol. Chem. 246, 5294 (1971). M E T H O D S IN E N Z Y M O L O G Y , V O L . LI
Copyright © 1978by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5
[76]
ADENINE PHOSPHORIBOSYLTRANSFERASE
559
cell rupture appears to be localized on the cell membrane in situ 2 where it mediates the translocation of adenine into the cell as AMP. z Its location at the cell surface is further indicated by its release from the periplasm upon osmotic shock treatment. 3 Enzyme activity is regulated by nucleotide pool constituents in situ; a experimentally it is quite sensitive to competitive inhibition by a very wide variety of 5' nucleotides. 1
Assay Method
Principle. The assay depends on the chromatographic separation of the radioactivelylabeled product, A M P , from the radioactivelylabeled substrate adenine. Reagents
MgC12, 0.1 M Tris.HC1, pH 7.8, 1 M [8-14C]Adenine, 20 mM (specific radioactivity adjusted to 5-10 mCi/mmole) K+MgZ+EDTA Titriplex, 1 M Mg2PRPP, 20 mM Adenine, 20 mM 5' AMP, 20 mM [K][EDTA], pH 7.0, 1 M Eastman Chromagram with fluorescent indicator #6065 Ammonium acetate, 1 M P r o c e d u r e . The following are added to 12 × 75 mm glass test tubes using Hamilton syringes and Hamilton PB600-1 repeating dispensers: 5 /zl [14C]adenine, 5/xl Tris buffer, 0.5/~1 MgC12, 0.5/zl K+Mg 2+ Titriplex, and enzyme sample in a volume 0.5-10/~1 (containing between 5 ng to 5 /zg of protein according to relative purity). Distilled water is added to make a total volume of 45/~1. Reactions are initiated with the addition of 5 /zl of Mg2PRPP. A control tube is prepared to which 5/zl of distilled water is added instead of MgzPRPP. Reaction mixtures are incubated at 37 ° with shaking usually for 10 min. The reaction is terminated by addition of 5 /zl [K][EDTA]. Cellulose thin layers (plastic-backed) are cut to strips 7-cm high and 1.5 cm times the number of samples wide to a maximum of 20 cm wide. Channels, 6 × 1.5 cm, are ruled on the thin layer with a ruler and pencil. At a point 1 cm from the edge, 0.5 Izl of AMP carrier solution and 0.5/zl of adenine carrier solution are spotted in
2j. Hochstadt-Ozer and E. R. Stadtman, J. Biol. Chem. 246, 5304(1971). aj. Hochstadt-Ozerand E. R. Stadtman, J. Biol. Chem. 246, 5312 (1971).
560
PURINE METABOLIZING ENZYMES
[76]
the middle of the 1.5-cm channel. Then 3/xl of the reaction mixture are spotted on top of the carrier AMP/adenine spot. No effort is made to dry the spots prior to placement of the thin layer in the chromatography tank. The chromatograms are then developed in 1 mM ammonium acetate in a glass chromatography tank until the ascending solvent front has reached the upper edge of the 7-cm-high thin layer. The thin layers are removed and dried with a hair dryer. The AMP (Rf = 0.7) and adenine (Re = 0.4) spots are localized under UV light and marked with a pencil; each AMP spot is cut out and placed in a scintillation vial. Toluene-based fluor solution is added to the vial; a few ml are sufficient for the entire thin-layer strip need only be saturated with the fluor solution it need not be totally submerged in the liquid. The samples are counted in a liquid scintillation counter; with an appropriate fluor solution the counting efficiency for 14C on the cellulose-backed thin layers is 75-80%.
Linearity. The reaction goes to completion and often is linear until a majority of the substrate (60-70%) has been utilized. For kinetic studies, concentration of enzyme and incubation times are adjusted, however, to utilize no more than 15-25% of the substrate. Standardization of Substrates. [14C]adenine is standardized by spectral assay using a molar extinction coefficient of 13,400 at 260.5 nm at pH 7. One to three microliters pipetted directly into a scintillation vial, dried completely, and covered with 10 ml of fluor solution gave a counting efficiency of approximately 97%; this was used to confirm radiospecific activity assigned by the manufacturer. Mg2PRPP solutions are standardized by allowing the enzyme reaction to proceed to completion using a limiting amount of Mg2PRPP and an excess of [14C]adenine standardized as above. The amount of AMP formed under such conditions is taken as a measure of the Mg~PRPP present since the reaction does go to virtual completion. Commercially purchased PRPP salts are often found to be 50-60% impure. Purification of substrate PRPP. In order to assay a variety of PRPP salts in addition to those commercially available, as well as to remove contaminants, the following procedure I is employed: 100 /zmole of Mg~PRPP (in a 1.5 ml volume) are passed over a 7.5 × 0.5 cm bed of Dowex 50 which has been well washed with water after preliminary treatment with LiC1 or KC1 (for preparing Li4PRPP or K4PRPP, respectively, for example). Water (3.5 ml) is added to wash through the PRPP in 0.5-ml portions, and the effluent fractions are pooled and stored at - 7 9 ° in 0.5-ml portions. All PRPP activity is standardly recovered from
[76]
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the Dowex 50. The quantity and concentration of the PRPP are then determined by the endpoint assay described above. Definition o f Unit and Specific Activity. One unit is defined as the amount of enzyme that catalyses the phosphoribosylation of 1/xmole of adenine to form I ~mole of AMP per minute. Specific activity is expressed in terms of units per milligram of protein. Protein is determined by the Biuret 4 procedure during initial steps in the purification and thereafter by the method of Lowry et al. ~ Alternate Assay Procedure. When even greater sensitivity is required, assay of enzyme activity is performed in 20 /zl of reaction volume in glass tubes, 6 × 50 mm, containing 0.2 mM [14C]adenine (specific radioactivity >50.0 mCi/mmole), 2 mM Mg2PRPP, 100 mM Tris.HC1, and 1-10 ng of purified enzyme. The surface tension properties of the reaction mixtures in tubes of this geometry required vortexing each tube for a second, once each minute during the incubation, to ensure adequate mixing during the reaction. Other aspects of the assay are identical. Application to Measurements in Crude Extracts. Because nucleotide pool constituents are potent inhibitors of enzyme activity, all crude extracts should be dialyzed or otherwise separated from cellular metabolites prior to assay of the protein fraction for activity. Though the nucleotides are competitive with PRPP, simple increase in PRPP concentrations is not an adequate means of dealing with the situation since, as purchased, the PRPP is contaminated with inorganic pyrophosphate and possibly other inhibitors of the enzyme. Other interfering contaminants (e.g., proteases) of the crude extract can be assessed after enzyme purification by reconstructive mixing of a sample of purified enzyme with a sample of crude extract, each of known activity alone, and determination of the combined activity in the mixture. Purification
Procedure
Growth and Harvest of Organism. As much as a 100-fold enrichment of adenine phosphoribosyltransferase can be obtained by growth under conditions which inhibit de novo purine synthesis and render the organism dependent on exogenous purine and phosphoribosyltransferase 4 M. Dittebrandt, Am. J. Clin. Pathol. 18, 439 (1948). 5 O. H. Lowry, N. J. Rosebough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
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PURINE METABOLIZINGENZYMES
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activity. 1 The medium designed for this purpose, PAT medium, 1 contains a mixture of "P"urines each at 10-4 M, "A"minopterin or amethopterin (to inhibit de novo purine biosynthesis; it also inhibits thymidine synthesis) 3 × 10 - r M , and compensatory "T"hymine or thymidine 10-s M. These " P A T " additions are made to the basic VogelBonner-Citrate medium as described by Korn and Weisbach. 6 Escherichia coli K12 is grown to the end of exponential growth (turbidity monitored) in either 20-liter glass bottles with aeration or in large-scale fermentation apparatus (e.g., 60-300 liters) at 37 °, harvested by centrifugation, frozen in liquid N2, and stored at - 7 9 °.
Step 1. Preparation o f Cell Extracts. Frozen cells are thawed at room temperature, suspended in 5-10 volumes of 50 mM potassium phosphate buffer (pH 7.5), and sonically disrupted at 0 ° by six 30-sec bursts (at maximum output with Heat Systems-Ultrasonic, Inc., model 185W Sonifier Cell Disruptor) with intervening cooling at 0 °. Debris is removed from this homogenate by centrifugation at 10,000 rpm for 20 min. Step 2. Streptomycin Precipitation. Streptomycin sulfate, 10% by volume, of 10% solution, is added to the supernatant solution (20--40 mg of protein per ml) obtained in step 1. After stirring for 10 min in the cold the mixture is centrifuged. Step 3. Ammonium Sulfate Precipitation P5a. The supernatant solution from step 2 is adjusted to a protein concentration of 10 mg/ml and pH 7.8 with Tris.HC1 (50 mM, final concentration) and a saturated solution of (NH4)2SO4 is added, to a final concentration of 35% saturation by volume. The treatment is at room temperature and equilibration is for 15 min with stirring. The precipitate is collected by centrifugation at 4 ° for 10 min at 10,000 rpm (Sorvall SS-34 rotor). The pellet is discarded. Saturated (NH4)2SO4 solution is added to that supernatant solution to make it 42% saturation by volume. Equilibration and collection of the precipitate are as before; the precipitate is resuspended in 20 mM Tris.HCl (pH 8.0) and saved for the preparation of 6-OH purine PRT. The supernatant fraction is adjusted to 49% saturated ammonium sulfate as above, and the precipitate is collected and resuspended as above. After testing this fraction for adenine and other purine PRT enzyme activities, it can be pooled with either the 35-42% fraction if rich in GPRT/HPRT, pooled with 49-56% fraction if rich in APRT, or discarded according to activities present. The supernatant fluid from this fraction is then made 56% with respect to (NH4)2SO4, and the 56% 6D. Korn and A. Weissbach, Biochim. Biophys. Acta 61, 775 (1962).
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precipitate is collected as before to provide the peak APRT fraction for subsequent steps. If new growth conditions are being employed it may be possible that a shift in salting out may occur--an optional additional step is the preparation and APRT assay of a protein fraction salting out between 56-59% saturation. In about 35% of the fractionations, that fraction had the greatest APRT specific activity; however, it never contained more than 10-15% of the total activity. Step 4. Precipitation with Acetone. The protein fraction salting out between 49-56% saturated (NH4)2SO4 (or 42-56%, see above) is adjusted to 10 mg/ml of protein in 20 mM Tris.HCl (pH 8.0). One volume of acetone at - 7 ° is added to 4 volumes of protein solution and stirred in a NaCl-ice bath at - 1 0 °, and then 4 more volumes of acetone at - 1 0 ° are added with stirring. The mixture is centrifuged at - 1 5 ° after 1 more volume of acetone ( - 15~) has been added. The pellet is then collected as before, drained, and resuspended in 20 mM Tris.HCl (pH 8.0) at 0 °. The final volume it is resuspended in is - one-half that of the protein solution originally treated with acetone. This solution is then dialyzed against 1 liter of 20 mM Tris'HC1 at 0 ° for 2 hr. Step 5. Treatment with C~ Gel. The solution from step 4 is adjusted to pH 6.0 with 50 mM potassium phosphate buffer and mixed with a suspension of aged Cv gel in 50 mM potassium phosphate buffer. The ratio of gel to protein is 34.5 mg of gel to 1 mg of protein. After stirring for 15 rain in the cold, the supernatant solution is collected by centrifugation. Step 6. Treatment with Calcium Phosphate Gel. The supernatant solution from step 5 is treated with a suspension of calcium phosphate gel in 50 mM potassium phosphate buffer (pH 6.0). The ratio of calcium phosphate gel to protein is 17.6 mg of gel per milligram of protein. After stirring for 15 min in the cold, the supernatant fluid is collected and dialyzed twice in the cold, against 3 liters of 50 mM potassium phosphate buffer (pH 7.5) for 1 hr each and once against 3 liters of l0 mM potassium phosphate buffer (pH 7.5), and lyophilized. Step 7. Chromatography on Bio-Gel P-150. The lyophilized powder is resuspended to one-tenth its original volume in distilled water and divided into l-ml portions and stored at - 7 9 °. One- or 2-ml portions at a time are applied to a column (1.5 × 30 cm, resin bed) of Bio-Gel P-150 equilibrated with 75 mM potassium phosphate buffer (pH 7.5). Fractions (1-2 ml) are collected in the cold, automatically. The void volume containing contaminating protein is discarded. The enzyme fractions with activity greater than half-peak activity are pooled unless otherwise
564
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PURINE METABOLIZING ENZYMES
PURIFICATION OF ADENINE PHOSPHORIBOSYLTRANSFERASEFROM PAT-MEDIUM-GROWN CELLS a,b
Protein fractionc 1. Untreated extract 2. 3. 4. 5. 6. 7.
Streptomycin supernatant solution Ammonium sulfate precipitate Acetone precipitate C_~gel supernatant Calcium phosphate gel supernatant Bio-Gel P 150 column peak
Specific activity (bemoles/min/ nag protein) 0.010 0.015 0.08 0.70 3.10 7.20 14.00
Percentage recovery 100.0 (54 observed) 73.0 23.2 22.2 21.0 14.0 12.6
a Reprinted from Hochstadt-Ozer and Stadtman, 1p. 5294. b PAT medium is VBC medium plus purines (adenine, guanine, hypoxanthine, xanthine, 0.1 mM each); amethopterin, 0.3/.d,/; thymine, 0.1 raM. c The numbers refer to steps described in the text.
noted. Fractions are stored at - 2 0 ° for 2 weeks or less and at - 7 9 ° for longer periods. The table shows a summary of the purification with respect to specific activity and yields at each step. R e c o v e r y from the streptomycin step is higher than the total activity observed in the crude extract. The extent to which inhibitory conditions or inhibitors present in the crude extract assay contributes to the decreased activity observed is determined by adding a known a m o u n t of e n z y m e activity from step 7 to a sample o f step 1 and noting the activity increment actually observed. It is from this latter figure that the total activity o f the extract and subsequent yields are calculated.
Properties
Stability. The adenine phosphoribosyltransfcrase is reasonably stable to heat treatment o f 5 rain at 60 ° [crude extracts, however, require substrate to stabilize during heat treatment, presumably due to protease which is r e m o v e d during (NH4)2SO4 fractionation]. Activity is unaffected by l0 mM mercaptoethanol. After treatment with calcium phosphate gel (step 6 of purification), instability to storage at 4 ° (or - 2 0 °) is noted. Storage in 0.1 M potassium phosphate buffer (pH 7), however, was found to be a satisfactory means of preventing e n z y m e inactivation. The
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ADENINE PHOSPHORIBOSYLTRANSF ERASE
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completely purified enzyme may be stored in a very dilute solution (i.e., 0.1 mg/ml at - 7 9 ° for several months), but considerable loss of activity was noted after subsequent storage of such solutions at - 2 0 ° for 2-3 weeks.
Homogeneity and Molecular Weight Estimations. Rechromatography of peak fractions from Bio-Gel P 150 leads to the isolation of a single protein peak, all portions of which exhibit the same specific enzyme activity. The homogeneity of this fraction is further demonstrated by the observation of a single band of protein coincident with enzyme activity after acrylamide gel electrophoresis at pH 7.2 in the presence of 5 mM MgSO4. Under standard conditions of electrophoresis, however (i.e., pH 9.5 and without MgSO4Z), the enzyme dissociates into several catalytically active bands. The smallest active fraction (fastest mobility) on the gel is least stable, but activity can be partially and temporarily enhanced by reaggregation at 4 ° in buffer solutions or in the presence of magnesium or PRPP. Similar dissociation behavior is observed when the enzyme is subjected to gel filtration chromatography at alkaline pH and in the absence of divalent cation. Calibration of the column with protein standards of known molecular weight led to estimation of the active polypeptide moieties of the enzyme at about 20,000, 30,000, and 40,000. z Stoichiornetry. For each mole of adenine and PRPP utilized, 1 mole each of PPi and AMP are generated. Specific Activity and Turnover Number. Based on an estimated molecular weight of 40,0001 for the native enzyme and a specific activity of 14.001 /xmoles of AMP generated per minute per milligram enzyme protein, an enzyme turnover number was calculated to be 5.6 × 10z reactions/rain/40,000 MW moiety. Substrate Specificity. The E. coli enzyme is specific for adenine or 2,6-diamino-purine. Numerous other tested purines, 1 including aminoimidazolicarboximideriboside previously reported to serve as substrate for partially purified bovine adenine phosphoribosyltransferase, 8 do not serve as substrates. Kinetic Constants. Michaelis-Menton kinetics are observed with respect to both PRPP and adenine. Km for PRPP in the presence of a 2fold excess of Mg 2÷ is 125/xM (Mg 2+, though required for the reaction, is also competitive with PRPP1); Km for adenine is approximately 20 p3U. 7A. C. Chrambach, Anal. Biochem. 15, 544 (1964). sj. G. Flaks, M. J. Erwin, and J. M. Buchanan, J. Biol. Chem. 228, 201 (1957).
566
PURINE METABOLIZING ENZYMES
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Metal Ion Requirement. An absolute requirement for either Mn 2+ or Mg 2+ was found. The chloride salt of either metal was found satisfactory. A complex relationship between activity, metal ion concentration, and PRPP concentration exists 1"9 evidence suggests that alternate reaction mechanisms may be possible at varying cation concentrations depending on the relative concentration of PRPP as the free acid, MgPRPP or MgzPRPP 1'9, e.g., 2:1 ratio of Mg ~÷ to PRPP was found to be optimal. Other divalent cations such as Zn 2÷, Ba ~+, and Ca ~+ were highly inhibitory to the reaction. Based on this observation, K+Mg~+EDTA Titraplex is included in the assay to bind continuous traces of such metals. pH Optimum. Enzyme activity has a strikingly sharp pH optimum at pH 7.8.1 Reaction Mechanism. Under most experimental conditions a pingpong mechanism is observed. PRPP + enzyme ~ phosphoribosylenzyme + PP~ Phosphoribosylenzyme + adenine --->AMP + enzyme
Effectors o f Enzyme Activity. All nucleotides with free 5' phosphate groups are effectors. 1 Stimulation of activity at low effector concentrations (especially at saturating PRPP concentrations) is noted for some purine nucleotidesL Higher concentrations of all 5' nucleotides are competitive with PRPP. 6-NH2 purine nucleotides are most inhibitory, 6OH purine nucleotides are moderately inhibitory, and pyrimidine nucleotides are least inhibitory; however, all inhibited activity greater than 50% when included in the reaction mixture (standard assay conditions) at 2 mM. The reaction products AMP and PPf are also inhibitors of the reaction. AMP is competitive with PRPP ~ while PPt competition with PRPP may be cooperative (Mg 2÷ varied with PRPP as in Mg2PRPP) or anticooperative (at constant excess magnesium). ~ Physiological Function. In E. coli cells which are actively producing purine nucleotides by the de novo biosynthetic pathway, approximately 50 enzyme molecules are found per bacterial cell; while in those cells wholely dependent on exogenous purines for growth, approximately 5000 enzyme molecules are found per cell. 1'~ The enzyme, though readily solubilized upon cell rupture as described above, appears to largely reside on the membrane in such derepressed cells where it mediates adenine translocation across the cell membrane. 2 Evidence for this comes both from studies with isolated vesicles 2 in which both 9T. A. Krenitsky, R. Papa:io!annou, and G. B. Elion, J. Biol. Chem. 244, 1263 (1969).
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ADENINE PHOSPHORIBOSYLTRAN SFERASE
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enzyme activity and transport reaction have been studied 2 and from its recovery with other periplasmic constituents upon osmotic shock. 3 In noninduced cells this group translocation mechanism of adenine uptake may account for only a portion of the small amounts of adenine taken up, TM while in the cells grown on the PAT medium it clearly accounts for virtually all of the u p t a k e ) The enzyme also appears necessary for appropriate regulation of purine biosynthesis since its absence is associated with purine excretion. Other Properties. The enzyme therefore exists in situ in an environment very different from the one in which it is purified, aqueous solution, and characterized. Certain properties which it exhibits when in situ in the membrane differ from what is observed in aqueous solution. These differences disappear when it is released from the membrane by detergent, freeze-thawing, or sonic oscillation; among them are greater sensitivity to effectors in aqueous solution and ability to carry out an exchange reaction between AMP and adenine only while membranebound. Both properties suggest that membrane-localized enzyme exists as phosphoribosyl-enzyme and that the phosphoribosyl moiety is discharged in aqueous solution. One approach to study of this enzyme in a homogeneous environment more reflective of the membrane milieu is to incorporate it into artificial phospholipid bilayers. Alternative Purification Scheme. An alternative method of enzyme purification is available which reduces the preparation to two steps. It is not as useful, however, for preparing very large amounts of enzyme. Based on the observation that much of the enzyme is released from the periplasm upon osmotic shock, 3 it can be obtained in the following manner. Cells are grown as described, but must be harvested at midexponential growth. Osmotic shock fluid is prepared by dilution of cells in 20% sucrose solution, 1:100 into 20 ~ MgSO4. The cells are removed by centrifugation. The supernatant "shock fluid" containing the enzyme is filtered through a 0.45/zm pore size nitrocellulose filter and concentrated by ultrafiltration to a protein concentration of 2--4 rag/ ml. An (NH4)2SO4 fraction, 45-55% saturation, is prepared (see step 3 purification procedure above) and applied directly to a Bio-Gel P 150 column and eluted as described in step 7 of the purification procedure. Though the peak fractions from the column are homogeneous after only two steps in addition to osmotic shock fluid preparation, cells--subjected to osmotic shock immediately upon harvest from mid-exponential growth and never frozen--must be used for this method. 1°S. Roy-Berman and D. W. Visser, J. Biol. Chem. 250, 9270 (1975).
