- Email: [email protected]
[70] Assays for the phosphotransferase system from Salmonella typhimurium
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ASSAYSFOR THE S. typhimurium PTS
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Convenient, albeit semiquantitative, complementation assays can be used. These are performed with extracts from mutants defective in one of the PTS proteins. Such extracts contain all the other PTS proteins and need only be supplemented with the fractions being assayed to reconstitute the complete PTS, capable of phosphorylating sugar. This assay is particularly useful as a "scanning" assay, for example for the detection of activity in fractions obtained by column chromatography. This complementation method is semiquantitative because in a given extract of the mutant cells it is not known whether Enzyme I or Enzyme II-B is in excess (although usually it is Enzyme I), and whether the other PTS proteins in the extract are present at optimal levels for assay of the missing protein. The method is especially subject to error when it is used to assay crude extracts or fractions for a particular PTS protein, since in this case, more than one PTS protein is being added.
[70] A s s a y s for t h e P h o s p h o t r a n s f e r a s e S y s t e m f r o m S a l m o n e l l a t y p h i m u r i u m a- :~ By E. BRUCE WAYGOOD and NORMAN D. MEADOW Included here are procedures for the preparation of membranes and for the partial purification of Enzyme II-B C~c, which, as sources of Enzyme II, are required for the sugar phosphorylation assays described below. Preparations of the other PTS proteins required for these assays (Enzyme I, HPr, and IIl ~') are described in subsequent articles. Sugar Phosphorylation Assay Principle. This assay is based on the separation, by ion-exchange chromatography, of radioactively labeled unreacted sugar from radioactive sugar phosphate formed by the complete phosphotransferase system. The lower limit of the range of the assays depends on the background activity from the particular batches of the PTS proteins being used. The This w o r k was supported by Grant CA 21901 from the National Institutes of Health. N. D. M. was supported in part by a National Institutes of Health Special Fellowship H D 57023 and AG 05029, 2 E. B. Waygood, N. D. M e a d o w , and S. R o s e m a n , Anal. Biochem. 95, 293 (1979). :3 j. B. Stock, E. B. Waygood, N. D. M e a d o w , P. W. Postma, and S. R o s e m a n , J. Biol. Chem. in press (1982).
METHODS IN ENZYMOLOGY, VOL. 90
Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6
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P E P : GLYCOSE PHOSPHOTRANSFERASE SYSTEM
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upper limits are designed so that no more than 25% of the sugar substrate is phosphorylated during the assay period. In the following sections typical values are given.
Reagents Mixture A: 2 ml of 1 M potassium phosphate buffer, pH 7.5; I ml of 0.5 M KF; 0.5 ml of 0.2 M dithioerythritol (DTE) or dithiothreitol (DTT); 0.5 ml of water Mixture B: 1 ml of 0.2 M PEP, potassium or cyclohexylammonium salt; 1 ml of 0.1 M MgCI2 Mixture C: Same as mixture A except that Tris-HC1 buffer, pH 8.0, is used in place of the phosphate buffer Mixture D: Same as mixture A except that pH 6.5 phosphate buffer is used [14C]Methyl a-D-glucopyranoside or 2-deoxyglucose, 0.1 M, specific activity (1-2) × 105 cpm//zmol Assay proteins: These depend on the requirements of the assay. For assaying the soluble PTS proteins (Enzyme I, HPr, IIIClc), either partially purified II-B ~;~c(described below) or washed membranes from S. typhim,ri, m strain SB2950 (deleted for Enzyme I, HPr, III G~) or SB 1687 (II-B ma"-, for the assay of III c~c) are used and are supplemented with the appropriate soluble proteins; it is essential that each of the assay proteins be free of other PTS proteins. Crude extracts: (approximately 20-30 mg protein/ml) from mutant strains of S. typhirnttrium defective in Enzyme I (SB1690, ptsI139 trpB223) 4 or HPr (SB2226,ptsH38 trpB223) 4 are used for semiquantitative assays of the respective proteins in fractions obtained from wild-type strains. The preparation of crude extracts is described in the last section of this article. Enzyme I. All incubation mixtures have 0.1 ml final volumes and contain the following components: 10/xl of mixture D; 10/xl of mixture B; 25 /xg of HPr purified at least through step 4 (see this volume [72]); 5-10 units (0.3-0.6 mg) of washed SB2950 membranes; 10 /xl of [14C]methyl c~-glucoside solution; and 0.03-0. I unit of Enzyme I to be assayed. Prior to assay, it is essential that samples of Enzyme I be dialyzed in the cold against 0.05 M phosphate buffer, pH 6.5, 1 mM DTT, 1 mM EDTA, and that the dialyzate be warmed to 37° before being diluted for assay in the same buffer at 37°. It is then incubated for 5-15 min at 37° before being added to the components of the assay mixture. 5 The assays are conducted 4 j. C. Cordaro and S. R o s e m a n , J. Bacteriol. 112, 17 (1972). N. Weigel, E. B. Waygood, M. A. K u k u r u z i n s k a , A. N a k a z a w a , and S. R o s e m a n , J. Biol. Chem. in press (1982).
[70]
ASSAYS FOR THE S.
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at 37° for 30 min. Reactions can be terminated by chilling to 0° or by adding 2 txmol of EDTA and immediately transferring to the ion-exchange columns described below. Under these conditions, the rate of sugar phosphorylation remains constant for at least 90 rain provided that the quantity of Enzyme I added for assay is within the range specified. HPr. Incubation mixtures contain the following components in volumes of 0.1 ml: 10/xl of Mixture A; 10 txl of mixture B; 10 units of purified or partially purified Enzyme I (at least through the AcA-44 column, step 4, described below); 10/xl of the labeled methyl o~-glucoside solution; 0.35 to 1 unit of Enzyme II TM (about 50 b~g of washed SB2950 membranes); the HPr fraction to be assayed containing from 0.2 to 2 txg of HPr. For determining the quantity of HPr, a standard curve is constructed using the homogeneous protein. Other reaction conditions are as described above except that preincubation is not required for PTS proteins other than Enzyme I. III Ctc. Incubation mixtures contain the following in 0.1-ml volumes: 10 ~1 of mixture C; 10 txl of mixture B; 5 units of Enzyme I; 3 txg of HPr; 0.2 unit of II-B~:~c-, either partially purified or membranes from S B 1687; the fraction to be assayed containing III c'lc equivalent to 1-10 ~g of the homogeneous protein. Standard curves are obtained with the homogeneous protein. Control incubation mixtures are conducted in the absence of III~l,'. Semiquantitative Cornplementation Assay for Enzyme I or HPr. Incubation mixtures contain the following components in 0.1 ml: 10 ~1 of mixture A (for HPr) or D (for Enzyme I); 10/A of mixture B; 10 ~1 of [~4C]methyl ~-glucoside; 50 ~1 of crude extract from SB 1690 (for Enzyme I) or SB2226 (for HPr). It should be emphasized that complementation assays are, at best, semiquantitative for the reasons described above. Enzyme II. These are assayed under conditions similar to those described for the phosphocarrier proteins. Incubation mixtures contain the following components in volumes of 0.1 ml: for II-B TM, 10/xl of mixture A, 10 tzl of mixture B, 10-20 units of Enzyme I (purified at least through step 5), 25 tzg of HPr (purified at least through the CM-23 column), and 10 ~1 of 2-deoxyglucose; for II-BC1% 10/xl of mixture C, 10/A of mixture B, 10-20 units of Enzyme I (purified at least through step 5), 2 #g of HPr (purified at least through the CM-23 column), 20 /~g of III ~ (purified at least through step 4 in procedure A or step 2 in procedure B, this volume [73]), 1 /xl of [~4C]methyl c~-glucoside. These conditions are suitable for the assay of up to 0.25 unit of II-B M~*, but of only 0.025 unit of ii_Br;LC.2,3 Definition of Unit and Specific Activity. HPr and III ~ are defined in absolute terms: quantity in milligrams or micromoles of pure protein; or
426
P E P : GLYCOSE PHOSPHOTRANSFERASE SYSTEM
[70]
they can be defined in terms of relative activities when comparing fractions. They can also be defined as phosphorylation equivalents based on the spectrophotometric assay. This is particularly useful in other bacteria where pure proteins are not available. E n z y m e I and the E n z y m e II are defined in terms of activity; 1 unit converts 1 gmol of sugar to sugar phosphate under the conditions described above (37 °, 30 min) where the enzyme is rate-limiting. Specific activity is expressed as units per milligram of protein. Ion-Evchange Separation oJ" Sugar-Phosphates and S,gars. An ionexchange method for routine assay of incubation mixtures 6 has been modified to conserve expensive materials, such as liquid scintillation fluids, and to permit the use of small counting vials (7 versus 21 ml). In this procedure, the incubation mixture containing labeled sugar substrate and the product, labeled sugar phosphate, is passed over an ion-exchange column, washed with water to remove excess substrate, and then eluted with LiC1 to release the sugar phosphate. The latter is then counted in a liquid scintillation counter. The columns consist of precision-bore glass tubing, 4 mm i.d. × 10 cm, drawn to a coarse point at one end (containing glass wool as a support for the resin), and sealed to a reservoir (1.5 × 3.5 cm) at the other. The columns are supported by Lucite blocks (19 × 19 × 2 cm) drilled to accommodate 25 columns, each of which is held in the block by its reservoir. Analytical grade anion-exchange resin is used (Bio-Rad AG I-X2, 50-100 mesh) and is converted batchwise to the chloride form after washing extensively with HCI, N a O H , etc., in the usual manner. The resin, in 1 M NaC1, is placed in the column to a height of 3.5-4 cm and washed with water before use. All solutions used to wash and to elute the columns contain 0.01% Triton X-100. The incubation samples described above are quantitatively transferred to the columns, washed with at least two 7-ml portions of water, and eluted with three 0.75-ml portions of 1 M LiC1. During elution, the columns are placed over Lucite blocks containing 25 scintillation vials (7 ml capacity each) so that each eluate is collected in a vial. Liquid scintillation fluor (3 ml, National Diagnostic Hydrofluor) is added to each vial, and the samples of sugar phosphate are then counted. The columns are regenerated for use again with a minimum of 2 × 7 ml of 1 M NaC1 followed by washing with water.
Spectrophotometric Assay"
Principle. The extent (and with limiting E n z y m e I, the rate) of phosphorylation of purified PTS proteins, such as HPr, III Gjc, III Lac, can be 6 W. Kundig and S. Roseman, J. Biol, Chem. 246, 1407 (1971).
[70]
ASSAYSfOR ThE S. typhimurium PTS
427
followed spectrophotometrically in a coupled assay system. In this assay, the pyruvate formed during the phosphorylation of the protein is measured by following the oxidation of NADH by lactate dehydrogenase as a function of time, using a recording spectrophotometer at 340 nm with a temperature-regulated water jacket (30°) surrounding the cuvette holder. Rea ,~ents
Potassium phosphate buffer, 1 M, pH 7.5 (for Enzyme I) Tris-HC1 buffer, 1 M, pH 8.0 (for HPr or III (;lc) PEP, 0.2 M, potassium salt MgClz, 0.1 M NADH, 0.0O6 M Lactate dehydrogenase (LDH) rabbit muscle, 10,000 units/ml (units as defined by supplier), Sigma type II PTS proteins: Enzyme I, protein obtained from step 4 and beyond may be used~; HPr, protein obtained from step 4 and beyond may be usedT; III G~e, protein obtained from step 5 of Procedure A or step 2 of Procedure B (see this volume [73]). The volume of the incubation mixture should be the smallest that can be accommodated in the spectrophotometer used, thus minimizing consumption of the PTS proteins. In the descriptions that follow, the volume of additions are given for 1-ml incubation mixtures. HPr. The incubation mixture contains 50 ~zl of potassium phosphate buffer, 5 ~1 of PEP, 50 ~! of MgC12, 20/xl of NADH, 4 gl of LDH, and 20 units of Enzyme I. Quantities of HPr greater than 10 nmol are used in order to obtain accurately measurable absorbance changes. The reaction is usually started by the addition of Enzyme I, which is contained in as small a volume as is practical. This quantity of Enzyme I should be sufficient to complete the reaction within a few minutes. Phospho-HPr spontaneously hydrolyzes, and thus there is a continual oxidation of NADH following the initial rapid rate, but corrections can be made for this hydrolysis reaction. 2 Controls contain either Enzyme I or the PTS protein fraction being assayed, but in the absence of LDH to ascertain that there is no NADH oxidation by contaminating enzymes. II1 ~c. The incubation mixtures contain 50/xl of Tris-HC1 buffer, 5/zl of PEP, 50/xl of MgC12, 20/~1 ofNADH, 5/zl of LDH, 3-6 units of Enzyme I, HPr, 5% (or less) of the molar quantity of III ca'. Quantities of III ~" greater than l0 nmol are used in order to obtain accurately measurable absorbance changes. The reaction is usually started by the addition of either the Enzyme I or HPr, which is contained in as small a volume as practical. Phospho-III (~c is more stable than phospho-HPr, making correction for 7 D. A. Beneski, A. Nakazawa, N, Weigel, P. E. Hartman, and S, Roseman, J. Biol. Chem. in pres~ (1982).
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PEP: GLYCOSEPHOSPHOTRANSFERASESYSTEM
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hydrolysis less important than it is during the measurement of HPr. Controls employed are the same as those for HPr described above. Preparation of the Membrane Components of the PTS for Assays
Preparation of Membranes. Growth of cells and preparation of crude extracts are described in the last section of this article. A mutant strain, SB2950 (trpB223 trzA-ptsHI crrA49) is used to prepare membranes for assays since this strain is deleted in the structural genes for the three soluble proteins, Enzyme I, HPr, and III C~c. The membranes contain two important Enzyme II components: (a) II-B me, which phosphorylates methyl a-glucoside (Kin, 6 ~M) and glucose (Kin, 10/.tM) when supplemented with the three soluble proteins; (b) an Enzyme II complex designated lI TM, comprising a II-A and II-B component, which phosphorylates 2-deoxyglucose (Kin, 0.2 raM), glucose (Kin, 0.015 raM), and mannose (Kin, 0.045 mM). In assays with II TM, the required supplements are Enzyme I and HPr, but not III Glc. Since SB2950 contains relatively low levels of Enzyme II-B me, membranes from another mutant strain, SB 1687 (II-BMan-) 8 are used as a source of II-B me. Membranes are prepared from crude extract by centrifugation for 2 hr at 200,000g. The pellet is washed twice with 0.025 M Tris-HC1 buffer, pH 7.5, containing 1 mM EDTA and 0.2 mM DTE, and finally suspended in this buffer to a concentration of 20-40 mg of protein per milliliter. Since such membrane preparations cannot be frozen, they are stored at 0°; their Enzyme II activity is stable for about l0 days. Although the activity of Enzyme II-B mc in membranes from SB 1687 is relatively high compared to the activity of Enzyme II-B TM, the latter enzyme shows some background activity in the sugar phosphorylation assay. The following section describes preparation of partially purified Enzyme II-B me containing very low activity of Enzyme II TM. Preparation oJ'lI-Ba~e,z This protein is required for assay of III G~c.Most membrane preparations contain both this activity and IIMan; the aim of the purification procedure is to remove the latter as well as the soluble PTS proteins in order to reduce blank values (sugar phosphorylation in the absence of IIIGIe). To purify Enzyme II-B (~c, 140 g (wet weight) of frozen S. typhimurium LT-2 cells are resuspended in 1 liter of 25 mM Tris-HC1, pH 7.5, containing 1 mM EDTA, and 0.2 mM dithioerythritol (buffer A). Crude extracts are prepared as described in the last section. The cell suspension is homogenized by two passages through a Manton-Gaulin (Gaulin Co., Everett, Massachusetts) press operated at 8000 psi. The membranes are s M. H. Saier, Jr., R. D. Simoni,and S. Roseman,J. Biol. Chem. 251, 6584 (1976).
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ASSAYS FOR THE S.
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separated from the soluble components by centrifugation of 410 ml of crude extract at 100,000g for 3 hr in a type 42 rotor in a Beckman L2-65B centrifuge. Centrifugation and resuspension (to the original volume with buffer A) are repeated three times. The final suspension is adjusted to a protein concentration of 25 mg/ml. The washed membraiaes (50 ml) are treated with 5.5 ml of a solution of 10% sodium lauroyl sarcosinate (Sarkosyl NL-97, K and K Fine Chemicals, filtered through a glass-fiber filter, Reeve Angel, Clifton, New Jersey, 985H) and stirred for 1 hr at 0°. The 1% detergent solution is centrifuged at 160,000g in a Ti-50 rotor (Beckman L2-65B centrifuge), and the pellets are discarded. The supernatant fraction (49 ml) is adjusted to 30% of saturation with ammonium sulfate by the addition of 26.3 ml of cold saturated ammonium sulfate previously adjusted to pH 7.0 with ammonium hydroxide. The solution is stirred for 1.5 hr at 0° and then centrifuged at 78,000 g (type 30 rotor) for 30 min. The supernatant is adjusted to 60% of saturation with ammonium sulfate by the addition of 47.2 ml of the saturated ammonium sulfate solution, stirred for 1 hr at 0°, and centrifuged as above. The protein is recovered as a pellicle floating on the clear ammonium sulfate solution. Detergent extracts vary in their response to the first treatment with ammonium sulfate. For this reason, small samples of each preparation are tested at several concentrations of ammonium sulfate varying from 25 to 35%, and the concentration that results in maximum removal of extraneous proteins and minimum loss of Enzyme II-B G~cactivity is used to treat the remaining detergent supernatant. The protein is dissolved in 12 ml of buffer A and dialyzed for 18 hr at 4° with three 500-ml changes of the same buffer. The preparation is diluted with buffer A to a protein concentration of 4 mg/ml, chilled to 0° in an ethylene glycol-Dry Ice bath, and stirred; acetone at - 7 7 ° is added slowly to a final concentration of 60% (v/v) while the temperature of the protein solution is slowly reduced to - 10°. The mixture is stirred for 20 min and centrifuged at 10,000g at - 10° for 15 min (Sorvall RC2B centrifuge, SS-34 rotor). The supernatant solutions are decanted, the tubes are inverted and allowed to drain in a freezer, and the pellets are resuspended in buffer A (10 ml) and dialyzed against two 500-ml changes of buffer over 18 hr at 4°. The preparation is frozen at - 2 0 ° in small aliquots. Under these conditions, the preparation is stable for at least 6 months. Upon thawing, each aliquot is sonically dispersed by immersing the tube intermittently in a 50-W bath-type sonicator for a total of about 3-5 min of sonication. The specific activity of Enzyme II-B G~c from the final stage of this scheme is from 5- to 10-fold higher than that of the washed membranes. The recovery of Enzyme II-B G~ activity is approximately 50%. Of greatest importance, however, is the fact that the activity of Enzyme II T M
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P E P : GLYCOSE PHOSPHOTRANSFERASE SYSTEM
[70]
is almost totally lost during the preparation. In washed membranes from S. typhimwqum LT-2, the ratio of activity of Enzyme II-B GJc: Enzyme II TM is approximately l, whereas in the final stage the ratio is about 200 because much less than 1% of the activity of Enzyme II TM remains. Detergent concentration is followed by using ~4C-labeled sodium lauroyl sarcosinate prepared by the method of Jungermann et al. 9 from [~4C]sarcosine (California Bionuclear Corporation, Sun Valley, California). The concentration of Sarkosyl decreases from 1% in the original extract to 0.6% after centrifugation, 0.2% in the ammonium sulfate pellicle, and is not detectable in the acetone pellet. Activity in this pellet is stimulated 3- to 10-fold (depending on the preparation) by phosphatidylglycerol.GAll the activities are determined with the optimal lipid concentration for each particular step of purification. The washed membranes are not stimulated by phospholipid. Growth of Cells and Preparation of Crude Extracts The S. typhimuri,m proteins are purified from the wild-type strain, LT-2, or f-~.m a strain auxotrophic for tryptophan, SB3507 (trpB223). These cells are grown in minimal medium containing glucose to induce high levels of Enzyme I, HPr, and III G~'. The minimal salts medium used for these studies is a modified medium A 4 that contains 93.4 mM potassium phosphate buffer (pH 7.2), 7.5 mM (NH4)2SO4, and 0.83 mM MgSO 4. The medium is supplemented with L-tryptophan, 20 rag/liter, for strains requiring this amino acid. A modified medium 638 lacking iron has also been used and contains the following components: 50 mM KH2PO4, 15.1 mM (NH4)zSO4, and 0.81 mM MgSO4; the pH is adjusted to 7.3 with KOH. The liquid medium is autoclaved, and sterilized glucose solution is added to the hot medium, immediately after autoclaving, to a final concentration of 0.2%. The deletion strain, SB2950, is grown in the same medium containing either 0.2% galactose or 0.4% DL-lactate (adjusted to pH 7.0). Cells are grown in the media in a New Brunswick incubator shaker at 37° with the gyratory shaker speed set at 200-250 rpm and using 5-10% inocula. When the cultures reach the late-exponential phase of growth (As0o = 1.5-1.8), the suspensions are chilled, the cells are harvested by centrifugation at 4° in a Sorvall RC2B centrifuge at 10,000g for 15 min, washed with cold 0.9% KC1 solution, and centrifuged. The pellet is suspended in cold (4°) 0.01 M Tris or 25 mM potassium phosphate buffer, pH 7.5, containing 1 mM EDTA and 0.2 mM dithioerythritol (4 ml of the solution for each gram of cell paste). Homogenates of the cells are prepared by passing the cold suspension twice ~ E. Jungerman, J. F. Gerecht, and I. J. Krems, J. A m . Chem. Soc. 78, 172 (1956).
ENZYME I FROM Salmonella typhimurium
[71]
431
through a French pressure cell (Amicon Corp., Lexington, Massachusetts) using an hydraulic pressure of 13 tons per square inch. Cells are also grown in a 200-liter working capacity fermentor (Chemap AG, Switzerland) using the same media. Growth is continued until the stationary phase, the cells are harvested in a continuous-flow centrifuge (Carl Padberg GMBH, West Germany) at 50,000g, and the cell paste is frozen at - 2 0 °. The usual yield from the fermentor is about 1000 g of cell paste. For large-scale preparations, 500 g of cell paste are suspended in the above buffer to a final volume of 2500 ml, and the cells are disrupted in a Manton-Gaulin press at 8000 psi. Crude extract is prepared from the homogenate by centrifugation at 16,300 g for 15 min at 4° to remove cell debris. Crude extracts from mutant strains SB 1690 or SB2226 are stored frozen in 1-ml aliquots until they are used in the sugar phosphorylation assay described above.
[71] E n z y m e I f r o m S a l m o n e l l a t y p h i m u r i u m 1 By MARIA A. K U K U R U Z I N S K A , N A N C Y and E. BRucE WAY6OOD
WEIGEL,
Purification 2 The purification of Enzyme I is started with 500 g of cell paste. Unless otherwise indicated, all steps are conducted at 0-4 ° , and all columns are packed by gravity. The procedure takes advantage of two properties of the enzyme: first, that Enzyme I undergoes a temperature-dependent change in molecular weight; and second, that Enzyme I can be phosphorylated by incubation with phosphoenolpyruvate (PEP) and MgCI~, which changes its charge and chromatographic properties. Step 1. Precipitation with Protamine Sulfate. A 2% (w/v) solution of protamine sulfate (Sigma, Grade II) is gradually added with stirring to the crude extract (see this volume [70]) to a final concentration of 0.33%. The mixture is stirred for an additional 30 min, and the precipitate is removed by centrifugation at 16,300g for 30 min. Step 2. DEAE-Cellulose Chromatography. The supernatant solution is diluted 1 : 1 with 0.01 M Tris buffer, pH 7.5, containing 1 mM EDTA and 1 This work was supported by Grant CA 21901 from the N a t i ona l Institutes of HeaLth. 2 N. Weigel, E. B. Waygood, M. A. K u k u r u z i n s k a , A. N a k a z a w a , and S. R os e ma n, J. Biol. Chem. in press (1982).
METHODS IN ENZYMOLOGY. VOL. 90
Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6
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Convenient, albeit semiquantitative, complementation assays can be used. These are performed with extracts from mutants defective in one of the PTS proteins. Such extracts contain all the other PTS proteins and need only be supplemented with the fractions being assayed to reconstitute the complete PTS, capable of phosphorylating sugar. This assay is particularly useful as a "scanning" assay, for example for the detection of activity in fractions obtained by column chromatography. This complementation method is semiquantitative because in a given extract of the mutant cells it is not known whether Enzyme I or Enzyme II-B is in excess (although usually it is Enzyme I), and whether the other PTS proteins in the extract are present at optimal levels for assay of the missing protein. The method is especially subject to error when it is used to assay crude extracts or fractions for a particular PTS protein, since in this case, more than one PTS protein is being added.
[70] A s s a y s for t h e P h o s p h o t r a n s f e r a s e S y s t e m f r o m S a l m o n e l l a t y p h i m u r i u m a- :~ By E. BRUCE WAYGOOD and NORMAN D. MEADOW Included here are procedures for the preparation of membranes and for the partial purification of Enzyme II-B C~c, which, as sources of Enzyme II, are required for the sugar phosphorylation assays described below. Preparations of the other PTS proteins required for these assays (Enzyme I, HPr, and IIl ~') are described in subsequent articles. Sugar Phosphorylation Assay Principle. This assay is based on the separation, by ion-exchange chromatography, of radioactively labeled unreacted sugar from radioactive sugar phosphate formed by the complete phosphotransferase system. The lower limit of the range of the assays depends on the background activity from the particular batches of the PTS proteins being used. The This w o r k was supported by Grant CA 21901 from the National Institutes of Health. N. D. M. was supported in part by a National Institutes of Health Special Fellowship H D 57023 and AG 05029, 2 E. B. Waygood, N. D. M e a d o w , and S. R o s e m a n , Anal. Biochem. 95, 293 (1979). :3 j. B. Stock, E. B. Waygood, N. D. M e a d o w , P. W. Postma, and S. R o s e m a n , J. Biol. Chem. in press (1982).
METHODS IN ENZYMOLOGY, VOL. 90
Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6
424
P E P : GLYCOSE PHOSPHOTRANSFERASE SYSTEM
[70]
upper limits are designed so that no more than 25% of the sugar substrate is phosphorylated during the assay period. In the following sections typical values are given.
Reagents Mixture A: 2 ml of 1 M potassium phosphate buffer, pH 7.5; I ml of 0.5 M KF; 0.5 ml of 0.2 M dithioerythritol (DTE) or dithiothreitol (DTT); 0.5 ml of water Mixture B: 1 ml of 0.2 M PEP, potassium or cyclohexylammonium salt; 1 ml of 0.1 M MgCI2 Mixture C: Same as mixture A except that Tris-HC1 buffer, pH 8.0, is used in place of the phosphate buffer Mixture D: Same as mixture A except that pH 6.5 phosphate buffer is used [14C]Methyl a-D-glucopyranoside or 2-deoxyglucose, 0.1 M, specific activity (1-2) × 105 cpm//zmol Assay proteins: These depend on the requirements of the assay. For assaying the soluble PTS proteins (Enzyme I, HPr, IIIClc), either partially purified II-B ~;~c(described below) or washed membranes from S. typhim,ri, m strain SB2950 (deleted for Enzyme I, HPr, III G~) or SB 1687 (II-B ma"-, for the assay of III c~c) are used and are supplemented with the appropriate soluble proteins; it is essential that each of the assay proteins be free of other PTS proteins. Crude extracts: (approximately 20-30 mg protein/ml) from mutant strains of S. typhirnttrium defective in Enzyme I (SB1690, ptsI139 trpB223) 4 or HPr (SB2226,ptsH38 trpB223) 4 are used for semiquantitative assays of the respective proteins in fractions obtained from wild-type strains. The preparation of crude extracts is described in the last section of this article. Enzyme I. All incubation mixtures have 0.1 ml final volumes and contain the following components: 10/xl of mixture D; 10/xl of mixture B; 25 /xg of HPr purified at least through step 4 (see this volume [72]); 5-10 units (0.3-0.6 mg) of washed SB2950 membranes; 10 /xl of [14C]methyl c~-glucoside solution; and 0.03-0. I unit of Enzyme I to be assayed. Prior to assay, it is essential that samples of Enzyme I be dialyzed in the cold against 0.05 M phosphate buffer, pH 6.5, 1 mM DTT, 1 mM EDTA, and that the dialyzate be warmed to 37° before being diluted for assay in the same buffer at 37°. It is then incubated for 5-15 min at 37° before being added to the components of the assay mixture. 5 The assays are conducted 4 j. C. Cordaro and S. R o s e m a n , J. Bacteriol. 112, 17 (1972). N. Weigel, E. B. Waygood, M. A. K u k u r u z i n s k a , A. N a k a z a w a , and S. R o s e m a n , J. Biol. Chem. in press (1982).
[70]
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typhimurium P T S
425
at 37° for 30 min. Reactions can be terminated by chilling to 0° or by adding 2 txmol of EDTA and immediately transferring to the ion-exchange columns described below. Under these conditions, the rate of sugar phosphorylation remains constant for at least 90 rain provided that the quantity of Enzyme I added for assay is within the range specified. HPr. Incubation mixtures contain the following components in volumes of 0.1 ml: 10/xl of Mixture A; 10 txl of mixture B; 10 units of purified or partially purified Enzyme I (at least through the AcA-44 column, step 4, described below); 10/xl of the labeled methyl o~-glucoside solution; 0.35 to 1 unit of Enzyme II TM (about 50 b~g of washed SB2950 membranes); the HPr fraction to be assayed containing from 0.2 to 2 txg of HPr. For determining the quantity of HPr, a standard curve is constructed using the homogeneous protein. Other reaction conditions are as described above except that preincubation is not required for PTS proteins other than Enzyme I. III Ctc. Incubation mixtures contain the following in 0.1-ml volumes: 10 ~1 of mixture C; 10 txl of mixture B; 5 units of Enzyme I; 3 txg of HPr; 0.2 unit of II-B~:~c-, either partially purified or membranes from S B 1687; the fraction to be assayed containing III c'lc equivalent to 1-10 ~g of the homogeneous protein. Standard curves are obtained with the homogeneous protein. Control incubation mixtures are conducted in the absence of III~l,'. Semiquantitative Cornplementation Assay for Enzyme I or HPr. Incubation mixtures contain the following components in 0.1 ml: 10 ~1 of mixture A (for HPr) or D (for Enzyme I); 10/A of mixture B; 10 ~1 of [~4C]methyl ~-glucoside; 50 ~1 of crude extract from SB 1690 (for Enzyme I) or SB2226 (for HPr). It should be emphasized that complementation assays are, at best, semiquantitative for the reasons described above. Enzyme II. These are assayed under conditions similar to those described for the phosphocarrier proteins. Incubation mixtures contain the following components in volumes of 0.1 ml: for II-B TM, 10/xl of mixture A, 10 tzl of mixture B, 10-20 units of Enzyme I (purified at least through step 5), 25 tzg of HPr (purified at least through the CM-23 column), and 10 ~1 of 2-deoxyglucose; for II-BC1% 10/xl of mixture C, 10/A of mixture B, 10-20 units of Enzyme I (purified at least through step 5), 2 #g of HPr (purified at least through the CM-23 column), 20 /~g of III ~ (purified at least through step 4 in procedure A or step 2 in procedure B, this volume [73]), 1 /xl of [~4C]methyl c~-glucoside. These conditions are suitable for the assay of up to 0.25 unit of II-B M~*, but of only 0.025 unit of ii_Br;LC.2,3 Definition of Unit and Specific Activity. HPr and III ~ are defined in absolute terms: quantity in milligrams or micromoles of pure protein; or
426
P E P : GLYCOSE PHOSPHOTRANSFERASE SYSTEM
[70]
they can be defined in terms of relative activities when comparing fractions. They can also be defined as phosphorylation equivalents based on the spectrophotometric assay. This is particularly useful in other bacteria where pure proteins are not available. E n z y m e I and the E n z y m e II are defined in terms of activity; 1 unit converts 1 gmol of sugar to sugar phosphate under the conditions described above (37 °, 30 min) where the enzyme is rate-limiting. Specific activity is expressed as units per milligram of protein. Ion-Evchange Separation oJ" Sugar-Phosphates and S,gars. An ionexchange method for routine assay of incubation mixtures 6 has been modified to conserve expensive materials, such as liquid scintillation fluids, and to permit the use of small counting vials (7 versus 21 ml). In this procedure, the incubation mixture containing labeled sugar substrate and the product, labeled sugar phosphate, is passed over an ion-exchange column, washed with water to remove excess substrate, and then eluted with LiC1 to release the sugar phosphate. The latter is then counted in a liquid scintillation counter. The columns consist of precision-bore glass tubing, 4 mm i.d. × 10 cm, drawn to a coarse point at one end (containing glass wool as a support for the resin), and sealed to a reservoir (1.5 × 3.5 cm) at the other. The columns are supported by Lucite blocks (19 × 19 × 2 cm) drilled to accommodate 25 columns, each of which is held in the block by its reservoir. Analytical grade anion-exchange resin is used (Bio-Rad AG I-X2, 50-100 mesh) and is converted batchwise to the chloride form after washing extensively with HCI, N a O H , etc., in the usual manner. The resin, in 1 M NaC1, is placed in the column to a height of 3.5-4 cm and washed with water before use. All solutions used to wash and to elute the columns contain 0.01% Triton X-100. The incubation samples described above are quantitatively transferred to the columns, washed with at least two 7-ml portions of water, and eluted with three 0.75-ml portions of 1 M LiC1. During elution, the columns are placed over Lucite blocks containing 25 scintillation vials (7 ml capacity each) so that each eluate is collected in a vial. Liquid scintillation fluor (3 ml, National Diagnostic Hydrofluor) is added to each vial, and the samples of sugar phosphate are then counted. The columns are regenerated for use again with a minimum of 2 × 7 ml of 1 M NaC1 followed by washing with water.
Spectrophotometric Assay"
Principle. The extent (and with limiting E n z y m e I, the rate) of phosphorylation of purified PTS proteins, such as HPr, III Gjc, III Lac, can be 6 W. Kundig and S. Roseman, J. Biol, Chem. 246, 1407 (1971).
[70]
ASSAYSfOR ThE S. typhimurium PTS
427
followed spectrophotometrically in a coupled assay system. In this assay, the pyruvate formed during the phosphorylation of the protein is measured by following the oxidation of NADH by lactate dehydrogenase as a function of time, using a recording spectrophotometer at 340 nm with a temperature-regulated water jacket (30°) surrounding the cuvette holder. Rea ,~ents
Potassium phosphate buffer, 1 M, pH 7.5 (for Enzyme I) Tris-HC1 buffer, 1 M, pH 8.0 (for HPr or III (;lc) PEP, 0.2 M, potassium salt MgClz, 0.1 M NADH, 0.0O6 M Lactate dehydrogenase (LDH) rabbit muscle, 10,000 units/ml (units as defined by supplier), Sigma type II PTS proteins: Enzyme I, protein obtained from step 4 and beyond may be used~; HPr, protein obtained from step 4 and beyond may be usedT; III G~e, protein obtained from step 5 of Procedure A or step 2 of Procedure B (see this volume [73]). The volume of the incubation mixture should be the smallest that can be accommodated in the spectrophotometer used, thus minimizing consumption of the PTS proteins. In the descriptions that follow, the volume of additions are given for 1-ml incubation mixtures. HPr. The incubation mixture contains 50 ~zl of potassium phosphate buffer, 5 ~1 of PEP, 50 ~! of MgC12, 20/xl of NADH, 4 gl of LDH, and 20 units of Enzyme I. Quantities of HPr greater than 10 nmol are used in order to obtain accurately measurable absorbance changes. The reaction is usually started by the addition of Enzyme I, which is contained in as small a volume as is practical. This quantity of Enzyme I should be sufficient to complete the reaction within a few minutes. Phospho-HPr spontaneously hydrolyzes, and thus there is a continual oxidation of NADH following the initial rapid rate, but corrections can be made for this hydrolysis reaction. 2 Controls contain either Enzyme I or the PTS protein fraction being assayed, but in the absence of LDH to ascertain that there is no NADH oxidation by contaminating enzymes. II1 ~c. The incubation mixtures contain 50/xl of Tris-HC1 buffer, 5/zl of PEP, 50/xl of MgC12, 20/~1 ofNADH, 5/zl of LDH, 3-6 units of Enzyme I, HPr, 5% (or less) of the molar quantity of III ca'. Quantities of III ~" greater than l0 nmol are used in order to obtain accurately measurable absorbance changes. The reaction is usually started by the addition of either the Enzyme I or HPr, which is contained in as small a volume as practical. Phospho-III (~c is more stable than phospho-HPr, making correction for 7 D. A. Beneski, A. Nakazawa, N, Weigel, P. E. Hartman, and S, Roseman, J. Biol. Chem. in pres~ (1982).
428
PEP: GLYCOSEPHOSPHOTRANSFERASESYSTEM
[70]
hydrolysis less important than it is during the measurement of HPr. Controls employed are the same as those for HPr described above. Preparation of the Membrane Components of the PTS for Assays
Preparation of Membranes. Growth of cells and preparation of crude extracts are described in the last section of this article. A mutant strain, SB2950 (trpB223 trzA-ptsHI crrA49) is used to prepare membranes for assays since this strain is deleted in the structural genes for the three soluble proteins, Enzyme I, HPr, and III C~c. The membranes contain two important Enzyme II components: (a) II-B me, which phosphorylates methyl a-glucoside (Kin, 6 ~M) and glucose (Kin, 10/.tM) when supplemented with the three soluble proteins; (b) an Enzyme II complex designated lI TM, comprising a II-A and II-B component, which phosphorylates 2-deoxyglucose (Kin, 0.2 raM), glucose (Kin, 0.015 raM), and mannose (Kin, 0.045 mM). In assays with II TM, the required supplements are Enzyme I and HPr, but not III Glc. Since SB2950 contains relatively low levels of Enzyme II-B me, membranes from another mutant strain, SB 1687 (II-BMan-) 8 are used as a source of II-B me. Membranes are prepared from crude extract by centrifugation for 2 hr at 200,000g. The pellet is washed twice with 0.025 M Tris-HC1 buffer, pH 7.5, containing 1 mM EDTA and 0.2 mM DTE, and finally suspended in this buffer to a concentration of 20-40 mg of protein per milliliter. Since such membrane preparations cannot be frozen, they are stored at 0°; their Enzyme II activity is stable for about l0 days. Although the activity of Enzyme II-B mc in membranes from SB 1687 is relatively high compared to the activity of Enzyme II-B TM, the latter enzyme shows some background activity in the sugar phosphorylation assay. The following section describes preparation of partially purified Enzyme II-B me containing very low activity of Enzyme II TM. Preparation oJ'lI-Ba~e,z This protein is required for assay of III G~c.Most membrane preparations contain both this activity and IIMan; the aim of the purification procedure is to remove the latter as well as the soluble PTS proteins in order to reduce blank values (sugar phosphorylation in the absence of IIIGIe). To purify Enzyme II-B (~c, 140 g (wet weight) of frozen S. typhimurium LT-2 cells are resuspended in 1 liter of 25 mM Tris-HC1, pH 7.5, containing 1 mM EDTA, and 0.2 mM dithioerythritol (buffer A). Crude extracts are prepared as described in the last section. The cell suspension is homogenized by two passages through a Manton-Gaulin (Gaulin Co., Everett, Massachusetts) press operated at 8000 psi. The membranes are s M. H. Saier, Jr., R. D. Simoni,and S. Roseman,J. Biol. Chem. 251, 6584 (1976).
[70]
ASSAYS FOR THE S.
typhimurium PTS
429
separated from the soluble components by centrifugation of 410 ml of crude extract at 100,000g for 3 hr in a type 42 rotor in a Beckman L2-65B centrifuge. Centrifugation and resuspension (to the original volume with buffer A) are repeated three times. The final suspension is adjusted to a protein concentration of 25 mg/ml. The washed membraiaes (50 ml) are treated with 5.5 ml of a solution of 10% sodium lauroyl sarcosinate (Sarkosyl NL-97, K and K Fine Chemicals, filtered through a glass-fiber filter, Reeve Angel, Clifton, New Jersey, 985H) and stirred for 1 hr at 0°. The 1% detergent solution is centrifuged at 160,000g in a Ti-50 rotor (Beckman L2-65B centrifuge), and the pellets are discarded. The supernatant fraction (49 ml) is adjusted to 30% of saturation with ammonium sulfate by the addition of 26.3 ml of cold saturated ammonium sulfate previously adjusted to pH 7.0 with ammonium hydroxide. The solution is stirred for 1.5 hr at 0° and then centrifuged at 78,000 g (type 30 rotor) for 30 min. The supernatant is adjusted to 60% of saturation with ammonium sulfate by the addition of 47.2 ml of the saturated ammonium sulfate solution, stirred for 1 hr at 0°, and centrifuged as above. The protein is recovered as a pellicle floating on the clear ammonium sulfate solution. Detergent extracts vary in their response to the first treatment with ammonium sulfate. For this reason, small samples of each preparation are tested at several concentrations of ammonium sulfate varying from 25 to 35%, and the concentration that results in maximum removal of extraneous proteins and minimum loss of Enzyme II-B G~cactivity is used to treat the remaining detergent supernatant. The protein is dissolved in 12 ml of buffer A and dialyzed for 18 hr at 4° with three 500-ml changes of the same buffer. The preparation is diluted with buffer A to a protein concentration of 4 mg/ml, chilled to 0° in an ethylene glycol-Dry Ice bath, and stirred; acetone at - 7 7 ° is added slowly to a final concentration of 60% (v/v) while the temperature of the protein solution is slowly reduced to - 10°. The mixture is stirred for 20 min and centrifuged at 10,000g at - 10° for 15 min (Sorvall RC2B centrifuge, SS-34 rotor). The supernatant solutions are decanted, the tubes are inverted and allowed to drain in a freezer, and the pellets are resuspended in buffer A (10 ml) and dialyzed against two 500-ml changes of buffer over 18 hr at 4°. The preparation is frozen at - 2 0 ° in small aliquots. Under these conditions, the preparation is stable for at least 6 months. Upon thawing, each aliquot is sonically dispersed by immersing the tube intermittently in a 50-W bath-type sonicator for a total of about 3-5 min of sonication. The specific activity of Enzyme II-B G~c from the final stage of this scheme is from 5- to 10-fold higher than that of the washed membranes. The recovery of Enzyme II-B G~ activity is approximately 50%. Of greatest importance, however, is the fact that the activity of Enzyme II T M
430
P E P : GLYCOSE PHOSPHOTRANSFERASE SYSTEM
[70]
is almost totally lost during the preparation. In washed membranes from S. typhimwqum LT-2, the ratio of activity of Enzyme II-B GJc: Enzyme II TM is approximately l, whereas in the final stage the ratio is about 200 because much less than 1% of the activity of Enzyme II TM remains. Detergent concentration is followed by using ~4C-labeled sodium lauroyl sarcosinate prepared by the method of Jungermann et al. 9 from [~4C]sarcosine (California Bionuclear Corporation, Sun Valley, California). The concentration of Sarkosyl decreases from 1% in the original extract to 0.6% after centrifugation, 0.2% in the ammonium sulfate pellicle, and is not detectable in the acetone pellet. Activity in this pellet is stimulated 3- to 10-fold (depending on the preparation) by phosphatidylglycerol.GAll the activities are determined with the optimal lipid concentration for each particular step of purification. The washed membranes are not stimulated by phospholipid. Growth of Cells and Preparation of Crude Extracts The S. typhimuri,m proteins are purified from the wild-type strain, LT-2, or f-~.m a strain auxotrophic for tryptophan, SB3507 (trpB223). These cells are grown in minimal medium containing glucose to induce high levels of Enzyme I, HPr, and III G~'. The minimal salts medium used for these studies is a modified medium A 4 that contains 93.4 mM potassium phosphate buffer (pH 7.2), 7.5 mM (NH4)2SO4, and 0.83 mM MgSO 4. The medium is supplemented with L-tryptophan, 20 rag/liter, for strains requiring this amino acid. A modified medium 638 lacking iron has also been used and contains the following components: 50 mM KH2PO4, 15.1 mM (NH4)zSO4, and 0.81 mM MgSO4; the pH is adjusted to 7.3 with KOH. The liquid medium is autoclaved, and sterilized glucose solution is added to the hot medium, immediately after autoclaving, to a final concentration of 0.2%. The deletion strain, SB2950, is grown in the same medium containing either 0.2% galactose or 0.4% DL-lactate (adjusted to pH 7.0). Cells are grown in the media in a New Brunswick incubator shaker at 37° with the gyratory shaker speed set at 200-250 rpm and using 5-10% inocula. When the cultures reach the late-exponential phase of growth (As0o = 1.5-1.8), the suspensions are chilled, the cells are harvested by centrifugation at 4° in a Sorvall RC2B centrifuge at 10,000g for 15 min, washed with cold 0.9% KC1 solution, and centrifuged. The pellet is suspended in cold (4°) 0.01 M Tris or 25 mM potassium phosphate buffer, pH 7.5, containing 1 mM EDTA and 0.2 mM dithioerythritol (4 ml of the solution for each gram of cell paste). Homogenates of the cells are prepared by passing the cold suspension twice ~ E. Jungerman, J. F. Gerecht, and I. J. Krems, J. A m . Chem. Soc. 78, 172 (1956).
ENZYME I FROM Salmonella typhimurium
[71]
431
through a French pressure cell (Amicon Corp., Lexington, Massachusetts) using an hydraulic pressure of 13 tons per square inch. Cells are also grown in a 200-liter working capacity fermentor (Chemap AG, Switzerland) using the same media. Growth is continued until the stationary phase, the cells are harvested in a continuous-flow centrifuge (Carl Padberg GMBH, West Germany) at 50,000g, and the cell paste is frozen at - 2 0 °. The usual yield from the fermentor is about 1000 g of cell paste. For large-scale preparations, 500 g of cell paste are suspended in the above buffer to a final volume of 2500 ml, and the cells are disrupted in a Manton-Gaulin press at 8000 psi. Crude extract is prepared from the homogenate by centrifugation at 16,300 g for 15 min at 4° to remove cell debris. Crude extracts from mutant strains SB 1690 or SB2226 are stored frozen in 1-ml aliquots until they are used in the sugar phosphorylation assay described above.
[71] E n z y m e I f r o m S a l m o n e l l a t y p h i m u r i u m 1 By MARIA A. K U K U R U Z I N S K A , N A N C Y and E. BRucE WAY6OOD
WEIGEL,
Purification 2 The purification of Enzyme I is started with 500 g of cell paste. Unless otherwise indicated, all steps are conducted at 0-4 ° , and all columns are packed by gravity. The procedure takes advantage of two properties of the enzyme: first, that Enzyme I undergoes a temperature-dependent change in molecular weight; and second, that Enzyme I can be phosphorylated by incubation with phosphoenolpyruvate (PEP) and MgCI~, which changes its charge and chromatographic properties. Step 1. Precipitation with Protamine Sulfate. A 2% (w/v) solution of protamine sulfate (Sigma, Grade II) is gradually added with stirring to the crude extract (see this volume [70]) to a final concentration of 0.33%. The mixture is stirred for an additional 30 min, and the precipitate is removed by centrifugation at 16,300g for 30 min. Step 2. DEAE-Cellulose Chromatography. The supernatant solution is diluted 1 : 1 with 0.01 M Tris buffer, pH 7.5, containing 1 mM EDTA and 1 This work was supported by Grant CA 21901 from the N a t i ona l Institutes of HeaLth. 2 N. Weigel, E. B. Waygood, M. A. K u k u r u z i n s k a , A. N a k a z a w a , and S. R os e ma n, J. Biol. Chem. in press (1982).
METHODS IN ENZYMOLOGY. VOL. 90
Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6