Modified assay procedures for the phosphotransferase system in enteric bacteria

9NALYTICAL

BIOCHEMISTRY

Modified

95, 293-304 (1979)

Assay Procedures for the Phosphotransferase System in Enteric Bacteria1’2

E. B. WAYGOOD,~ NORMAN D. MEADOW, AND SAUL ROSEMAN Department of Biology and The McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218 Received December 18, 1978 Conditions for the assay of individual components of the bacterial phosphotransferase system (PTS) are presented which offer two important improvements over earlier methods. First, a lactate dehydrogenase-coupled assay for phosphocarrier proteins (HPr, FPr, and Factor IIIGiC) which permits their measurement in either pure or partially pure form was developed. Quantitation by this assay does not rely on the level of activity of the enzymes used. Second, conditions under which Enzyme I activity was proportional to enzyme concentration are given. With these methods levels of PIS components have been measured that are 2to 20-fold higher than those previously reported. These levels can now account for various PTS functions measured in Go. Further, we have shown that the phosphocarrier proteins HPr and Factor IIIclc are substrates for their respective enzymes which show typical Michaelis-Menten kinetics. In addition, a method for the partial purification of Enzyme II-BGIC essentially free of Enzyme IIMan activity is presented.

The phosphotransferase system in bacteria is responsible for the phosphorylation and concomitant transport of various carbohydrates across bacterial membranes, resulting in the intracellular accumulation of the corresponding sugar phosphates. In the overall reaction, the phosphoryl group is transferred from phosphoenolpyruvate to the sugar; this reaction requires three or four proteins as indicated in Fig. 1. The phosphoryl group is transferred from PEP4 to Enzyme I and then to a histidine-containing carrier protein, HPr (l-5); these two proteins are ’ This paper is dedicated to the memory of Dr. Alvin Nason. 2 Contribution 994 from the McCollum-Pratt Institute. 3 Present address, Department of Biochemistry. University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO, Canada. 4 Abbreviations used: PEP, phosphoenolpyruvate; HPr, histidine-containing carrier protein; PTS, phosphotransferase system (designations of PTS components are given in the legend to Fig. 1); FPr, phosphocarrier protein.

soluble and are required for phosphorylation of all sugars that are substrates of the PTS. The phosphoryl group is then transferred to the sugar via two sugar-specific proteins; one of these, designated II-B, is always membrane-bound, whereas the other may be membrane-bound (II-A) or soluble (III) (6-8). Several sugar-specific Enzymes II-B have been identified (9); these are designated as shown in Fig. 1. Moreover, when certain organisms are grown on fructose, a phosphocarrier protein designated FPr (lo- 14) is induced which can perform many of the functions of HPr. All of these proteins and enzymes are found in crude extracts of enteric bacteria such as Salmonella typhimurium and Escherichia coli. The obvious difficulty in assaying such extracts for individual components of the PTS is compounded by the complex kinetics of the system (9), particularly when the net enzymatic reaction rate is measured, i.e., transfer of the phosphoryl group from P-enolpyruvate to sugar. 293

0003-2697/79/070293-12$02.00/O Copyright All rights

0 I979 by Academic Press. Inc. of reproductton m any form reserved.

302

WAYGOOD,

MEADOW, TABLE

AND ROSEMAN 2

ASSAM OF HPr IN CRUDE EXTRACTS OF GLYCEROL-GROWN WITH SEVERAL SUGAR SUBSTRATES AND ENZYME

Concentrations Concentrations of sugar in assay (mh@ Methyl cu-glucoside, 10 Glucose, 10 Mannose, 10 Glucose, 1 Mannose, 1

zsc103 0.33 0.37 0.33 0.33 0.33 (0.34 t 0.02)C

S. typhimurium II PREPARATIONS”

SB3507

of HPr (nmol/mg protein) with membranes from SB2950

SB4004

0.29 0.29 0.28 NW ND (0.29 _’ 0.01)

0.34 0.42 0.37 0.31 0.27 (0.34 c 0.06)

a All membrane preparations were obtained from glycerol-grown cells. E. coli strain ZSC103 (18) was a strain obtained from Dr. W. Epstein, University of Chicago. Strain SB4004 was trpE22.3 crr24 (i.e., contained no Factor IIIG1c) obtained from Dr. J. Stock. The assays were performed as described in the text. b Not determined. e Average values.

ptsG

measure absolute concentrations of the protein in partially purified preparations. In crude extracts, the sugar phosphorylation assay was used either with membranes as the source of Enzyme II-BGIC or with partially purified II-B G1c.The response of Enzyme II-BGIC, whether partially purified or in crude membranes, to Factor IIIG’c concentration was that of an enzyme with MichaelisMenten kinetics. The K, value for Factor IIIGIC was 4 FM for both sources of Enzyme II-BGIC. Measurement of Enzymes II Any enzyme is defined by its kinetic constants, and Enzymes II, as mentioned above, show simple Michaelis-Menten behavior (in vitro). They can be defined by their sugar and phosphocarrier protein interactions. “saturating”) concentrations Optimal (i.e., by the sugar substrates of the Enzymes II are easily attained. However, saturation by the phosphocarrier proteins is not easily obtained on a routine basis because of the quantity of the phosphocarrier protein required. Using optimal sugar concentrations, the K, values with Enzyme IIMan, II-BG’c, or II-Bm are: HPr, 20 PM; Factor IIIG’c, 4 PM; FPr, 4 PM. While it is impractical to use saturating concentrations of these

proteins on a routine basis, the ease of the lactate dehydrogenase coupled assay permits the estimation of the concentration of these proteins, and it is therefore practical to use them at a fixed concentration so that the results of all assays may be readily compared. The concentrations of these proteins given under Methods represent a balance between the concentrations required to give high activity and the amount that could be obtained from routine purification. Some examples of measured Enzyme II levels are given in Table 3. Measurement

of Enzyme I

Enzyme I has several properties that affect the enzyme assay, the dominant one being that Enzyme I reversibly dissociates at low temperature from an active dimeric to an inactive monomeric form. This dissociation is known to depend on pH and protein concentration (Fig. 4) as well as temperature. The activation of the enzyme that is induced by increasing the temperature occurs too rapidly to be measured by the sugar phosphorylation assay. Thus the lactate dehydrogenase-coupled assay was modified by greatly reducing the amount of Enzyme I, so that the rate of HPr phos-

PHOSPHOTRANSFERASE TABLE PROPERTIES

stram LT.2.

glucose

295

IN BACTERIA

OF BACTERIAL

1

STRAINS

Genotype

USED

IN THIS

STUDY Use

Propenles

grown”~”

Wild

grown

pt.44

Very low Enzyme

II”,“’

Assay of Factor

pr.sH rrpB223 (HPr pseudorevertant)

Elevated

ll-Bb”’

Away

KY-prsHkrr49

No HPT, Factor Ill’,” Enzyme I. Elevated ,,““”

SB 1687. glucose SB2676.

fructose

SB2950.

lactate

grown

grown

SB3507

vpB223

SB4004

~24

ZSCIO?”

prsG

type

Wild

trpB223

Wild rrpB223

‘I Obtamed from Dr. P. E. Hartman. Department (/ All Strains are S. r\,phimurium except ZSCIO3.

type

Partial purification Enzyme 11-B’:”

Enzyme

type

Negligible

Factor

Very low Enzyme

Ill’,” II-B””

of

Ill’,”

1131

of FPr

II?1

Assay ot HPr and Enzyme

Preparation

of Biology, The Johns Hopkun which is E. cd.

phan where appropriate. Cells used for assay of levels of components were grown to an absorbance of 0.5 to 1.O at 550 nm, harvested by centrifugation, washed with 0.9% NaCl, and immediately resuspended in buffer and disrupted as described below. Cells for large scale preparation of proteins for use as assay components were grown to early stationary phase, harvested by centrifugation, and stored frozen at -20°C until used. Buffers. Optimal conditions for assay of PTS components in crude extracts depended on the use of different buffers. Trischloride buffer was used for Factor IIIG’C, potassium phosphate for Enzyme I or FPr, and either one for HPr. The molarity and pH for each buffer are given in the text. Unless otherwise indicated, all buffers contained 1 mM EDTA and 0.2 mM dithiothreitol or dithioerythritol. Assay procedures. In the complete PTS, two of the four proteins are “enzymes” in the usual sense, i.e., Enzyme I and the II-Bsugar component of the Enzyme II complex. The other two proteins, HPr (or FPr) and IIIsu~ar (or the II-ASUgar component of the Enzyme II complex) are phosphocarrier proteins and act as substrates for the two enzymes. Assays of sugar phosphorylation are therefore designed so that: (a) One of the enzymes is in large excess while the

or Enzyme

RefereWe

Awy

of PTS protens

1

(171

1171

of HPr

Assay of HPr

,IXl

Umverwy.

other is rate determining; (b) the component being measured, such as HPr, is added at levels where it is rate limiting while all other substrates are used at “saturating” concentrations. Conditions for assaying each of the components are given below. The Sugar Phosphorylation

Assay

These assays were performed basically as described by Kundig and Roseman (15). The incubation mixture for assay of each component is given below. Assays were generally performed in O.l-ml volumes and the mixtures were incubated at 37°C for 30 min. The reaction was stopped with 0.5 ml of cold (OOC) water and the mixtures were immediately placed on the ion-exchange columns (15). The columns were eluted with two 3-ml portions of 1 M LiCl, the eluates were collected directly in scintillation vials, 6 ml of scintillation fluid (3a70B, Research Products International) was added, and the samples were counted. HPr assay. For assaying HPr the following components were used in a final volume of 0.1 ml: 10 mM methyl cr-[UJ4C]glucoside, 1 to 2 X lo5 cpm/pmol; 0.05 h4 potassium phosphate, pH 7.5; 2.0 mM dithioerythritol; 12.5 mM KF; 5 mM M&l,; 10 mrw phosphoenolpyruvate (cyclohexylammonium

296

WAYGOOD,

MEADOW,

salt); 10 units of Enzyme I; and 0.35 to 1 unit of Enzyme II Man from SB2950 membranes. A standard curve was prepared with three or four known concentrations of HPr (determined by the lactate dehydrogenase assay described below), and the quantity of HPr in unknowns was measured by comparison with this standard curve. All phosphocarrier protein samples must be free of membranes (a source of Enzyme II) and these are removed by high-speed centrifugation for 2 h at 200,OOOg. During purification and experiments with HPr, it has been necessary to detect much lower quantities of HPr than are found in crude extracts. This was accomplished by either lowering the sugar concentration or increasing the Enzyme II concentration. The assay conditions were adjusted so that not more than 30% of the sugar substrate was utilized. FPr assay. The assay of FPr is conducted along similar lines, although it has not been as extensively explored. The incubation mixtures were similar to that for HPr, except that [l*C]fructose replaced methyl (Y[14C]glucoside, membranes from SB2676 (see Table 1) were used in place of those from SB2950, and the Enzyme I levels were increased to about 50 units. The reason why increased levels of Enzyme I were needed is not understood, but these levels were required so that the rate of the reaction was not limited by the Enzyme I. The SB2676 membranes were treated by passage through DEAE-cellulose to decrease high-background phosphorylation caused by FPr bound to the membranes (see Preparation of Crude Extracts and Membranes). Because FPr appears to be partially associated with membranes, crude extracts were passed through DEAE-cellulose so that FPr bound to the membranes could be recovered (see below). The assay was standardized with FPr measured by the lactate dehydrogenase-coupled assay. Factor ZZZGzc.This factor was assayed by the method described for HPr with the following changes; 0.05 M Tris-chloride, pH

AND

ROSEMAN

7.5, instead of phosphate buffer; 2 PM HPr; 1 mM methyl a-[14C]glucoside and, in place of membranes from SB2950, membranes from SB 1687 (very low background Enzyme IIMan) or partially purified Enzyme IIG1c, which are preferable, although membranes from glucose-grown wild-type cells can be used. The reaction was standardized with Factor IIIG’C measured by the lactate dehydrogenase assay. Enzyme ZZ. The various Enzymes II in membrane preparations were assayed under conditions similar to those described for the phosphocarrier proteins, with the following modifications: for Enzyme IIMan, 10 mM [14C]2-deoxyglucose and 25 PM HPr; for Enzyme IF”“, 10 mM [14C]fructose and 15 PM FPr; for Enzyme IIGIC, 1 mM methyl (Y[14C]glucoside, 10 PM Factor IIIGIC, and 2 PM HPr. Enzyme I. Assay mixtures for Enzyme I contained the following components: 0.05 M potassium phosphate buffer, pH 6.5,2.0 mM dithiothreitol, 12.5 mM KF, 10 mM phosphoenolpyruvate, 5 mM MgCL 25 PM HPr, 5- 10 units of Enzyme IIMa” in SB2950 membranes, and 10 mM [14C]sugar (1 to 2 x lo5 cpm/pmol). Any of the following sugars can be used: 2-deoxyglucose, mannose, glucose, fructose, or methyl cy-glucoside. The two analogs (2-deoxyglucose and methyl a-glucoside) are preferable for assaying crude extracts. Other essential conditions are given under Results. The Lactate Dehydrogenase-Coupled Assay for Phosphocarrier Protein

Assays were conducted in l-ml volumes (l-cm cuvettes) in a Gilford 2400 recording spectrophotometer at 340 nm, with a temperature-regulated water jacket surrounding the cuvette holder. Phosphate buffer was used in the assay since it is required for Enzyme I stability (19). The incubation mixtures contained the following components: Potassium phosphate buffer, 0.05 to 0.2 M, pH 6.5 to 7.5; NADH, 0.15 mM;

PHOSPHOTRANSFERASE

IN BACTERIA

297

FPr was found partly bound to the membranes, and therefore the crude extract was passed through a DEAE-cellulose column which extracted both free and bound FPr. The gravity-packed column, with volume 10 times greater than the original wet weight of cells, was equilibrated with 0.01 M potassium phosphate buffer, pH 7.5,l mM EDTA, and 0.2 mM dithiothreitol. The crude extract was diluted with the same buffer lofold before being loaded on the column, which was then washed with 6 vol of 0.05 M KC1 in the same buffer, and the FPr was eluted with 6 vol of 0.2 M KC1 in the same buffer. The FPr could usually be assayed directly in this fraction, but if necessary it was concentrated by lyophilization. When membranes of cells grown on fructose are to be used as a source of Enzyme II-BP’“, the membranes must also be passed Preparation of Crude Extracts through DEAE-cellulose to remove FPr. and Membranes The membranes were prepared by centriPellets of either S. typhimurium or E. fugation as described above and were then co/i cells were suspended in 0.01 M buffer diluted with the buffer given below to about (either Tris-chloride or potassium phos- 2 mg of protein/ml. A total of 10 ml were phate as indicated under Buffers), pH 7.5, loaded onto a lo-ml DEAE-cellulose column containing 1 mM EDTA and 0.2 mM dithioequilibrated with 0.01 M potassium phosthreitol, at a density of 200 g of cells/liter phate buffer, pH 7.5, I mM EDTA, 0.2 mM of suspension. dithiothreitol, and eluted with the same bufThe cells were passed twice through a fer. The turbid fractions were pooled, conFrench pressure cell at 20,000 to 25,000 psi. centrated by centrifugation (Beckman Ti-50 Cell debris was removed by centrifugation rotor at 200,0001: for 2 h), and subsequently for 10 min at 12,OOOg in a Sorvall RCZB resuspended to about 10 mg of protein/ml. centrifuge. Enzyme I could be assayed in this crude extract. For measurement of Preparation of Partially PuriJied Factor IIIGLc, HPr, or Enzyme II the crude Enzyme lI-BG’C extracts required further treatment. The extracts were centrifuged at 200,OOOg with a To purify Enzyme II-BG1’, 200 g (wet Ti-50 rotor in a Beckman centrifuge for 2 h. weight) of frozen S. typhimurium LT-2 cells The supematant fraction containing Factor was resuspended in 1 liter of 25 mM TrisIIIG’C and HPr, and the pellet containing HCl, pH 7.4, containing 1 mM EDTA, and Enzyme II were separated. The pellet was 0.2 mM dithioerythritol (Buffer A). Crude retained, and the supematant fraction was extracts were prepared as described above, recentrifuged. The pellet was resuspended except that the cell suspension was homogin a tissue homogenizer with a Teflon pestle enized by two passages through a Mantonand was used as a source of Enzyme IIMan Gaulin press operated at 8000 psi. The memand II-BGIC when the strains appropriate branes were separated from the soluble for these activities were being processed. components by centrifugation of 410 ml of P-enolpyruvate, 1 to 10 mM; MgC&, 5 or 10 mM; and 35 units of lactate dehydrogenase. The reaction is started by adding about 20 units of Enzyme I in 5 ~1 of buffer. The reaction may be followed at room temperature. The Enzyme I need not be highly purified. A crude extract purified by DEAE-cellulose chromatography to separate Enzyme 1 from the phosphocarrier proteins (IO-fold purification) or a more purified form (19,ZO) may be used. The assay may also be modified to measure the activity of Enzyme I by measuring the rate of HPr phosphorylation in the presence of low levels of Enzyme I. Protein determination. The protein content of preparations was determined either by the method of Lowry et al. (21) or by a microbiuret method (26).

298

WAYGOOD,

MEADOW,

crude extract at 100,000g for 3 h in a Type 42 rotor in a Beckman L2-65B centrifuge. Centrifugation and resuspension (to the original volume with Buffer A) were repeated three times. The final suspension was adjusted to a protein concentration of 25 mg/ ml. The washed membranes (50 ml) were treated with 5.5 ml of a 10% sodium lauroyl sarcosinate (Sarkosyl NL-97, K and K fine Chemicals, filtered through a glass fiber filter, Reeve Angel 984H), and stirred for 1 h at 0°C. The 1% detergent solution was centrifuged at 160,OOOg in a Ti-50 rotor (Beckman L2-65B centrifuge) and the pellets were discarded. The supernatant fraction(49 ml) was adjusted to 30% of saturation with ammonium sulfate by the addition of 26.3 ml of cold saturated ammonium sulfate, pH 7.0. The solution was stirred for 1.5 h at 0°C and then centrifuged at 78,000g (Type 30 rotor) for 30 min. The supematant was adjusted to 60% of saturation with ammonium sulfate by the addition of 47.2 ml of the saturated ammonium sulfate solution, stirred for 1 h at 0°C and centrifuged as above. The protein was recovered as a pellicle floating on the clear ammonium sulfate solution. The protein was dissolved in 12 ml of the above buffer and dialyzed for 18 h at 4°C with three 500-ml changes of Buffer A. The preparation was diluted to a protein concentration of 4 mg/ml, chilled to 0°C in an ethylene glycol-dry ice bath, stirred, and acetone at -77°C was added slowly to a final concentration of 60% (v/v) while the temperature of the protein solution was slowly reduced to - 10°C. The mixture was stirred for 20 min and centrifuged at 10,OOOg at - 10°C for 15 min (Sorval RC2B centrifuge, SS-34 rotor). The supernatant solutions were decanted, the tubes were inverted and allowed to drain in a freezer, and the pellets were resuspended in Buffer A (10 ml), and dialyzed against two 500-ml changes of buffer over 18 h at 4°C. The preparation was frozen at -20°C in small aliquots. Under these conditions, the preparation is stable for at least 6 months. Upon thawing,

AND

ROSEMAN

each aliquot was dispersed by sonication by immersing the tube intermittently in a 50 W bath-type sonicator for a total of 3-5 min of sonication. The specific activity of Enzyme II-BGIC from the final stage of this scheme was from 5- to IO-fold higher than that of the washed membranes. The recovery of Enzyme II-BGle activity was approximately 50%. Of greatest importance, however, was the fact that the activity of Enzyme IIMan was almost totally lost during the preparation. In washed membranes the ratio of activity of Enzyme II-BGIC/Enzyme IIMan was approximately 1, whereas in the final stage the ratio was 180 because much less than 1% of the activity of Enzyme IIMan remained. Detergent concentration was followed by using 14C-labeled sodium lauroyl sarcosinate prepared by the method of Jungermann ef al. (23) using [14C]sarcosine (California Bionuclear Corporation, Sun Valley, Calif.). The concentration of Sarkosyl decreased from 1% in the original extract to 0.6% after centrifugation, 0.2% in the ammonium sulfate pellicle, and was not detectable in the acetone pellet. Activity in this pellet was stimulated 3- to lo-fold (depending on the preparation) by phosphatidylglycerol (15). All the activities were determined with the optimal lipid concentration for each particular step of purification. The washed membranes were not stimulated by phospholipid. PTS Protein

Preparations

HPr was obtained from S. typhimurium and E. coli by modification (5) of the procedure described by Anderson et al. (1). Enzyme I was obtained from S. ryphimurium as has been reported (16). Factor IIIGIC purification will be described elsewhere. FPr was obtained as has been reported (12). Definition

ofEnzyme

Units

A unit of either Enzyme I or Enzyme II activity is defined as the amount of enzyme catalyzing the phosphorylation of 1 pmol of

PHOSPHOTRANSFERASE

sugar in 30 min when the assays were conducted as described above. A unit of lactate dehydrogenase activity is the amount of enzyme catalyzing the reduction of 1 pmol of pyruvate/min at pH 7.5 and 37°C. RESULTS Measurement

AND DISCUSSION

of Phosphocarrier

Proteins

Assay by coupling to lactate dehydrogenase . In this assay, the formation of pyru-

vate from P-enolpyruvate is followed by coupling the reaction with lactate dehydrogenase and measuring the oxidation of NADH spectrophotometrically according to the equations described below. Thus, it is possible to measure only the first step in the PTS sequence, the phosphorylation of HPr or FPr. This is especially important because many Enzyme II preparations are essentially crude membranes and have significant background levels of other Enzymes II and phosphocarrier proteins. HPr + phosphoenolpyruvate

-

Enzyme I Mg2+

P - HPr + pyruvate Pyruvate

+ NADH

+ H+

lactate > dehydrogenase

lactate + NAD+

[3]

The concentration of HPr is determined by substrate (NADH) depletion, since the phosphorylation of 1 mol of HPr results in the oxidation of 1 mol of NADH, HPr has only one phosphorylation site (1,5), and phospho-HPr is sufficiently stable during the time required for assay (Fig. 1). This is a stoichiometric assay and is not dependent on the rate of phosphorylation of HPr by Enzyme I. In a given solution of HPr, a series of determinations on eight samples covering a 20-fold range of HPr concentration resulted in a measured concentration of 0.146 ? 0.011 mM; the concentration of HPr determined by this method has been

299

IN BACTERIA

shown to agree with a subsequent value obtained by amino acid analysis.$ Other phosphocarrier proteins of the PTS can be measured by this method. It has been used to determine the concentration of FPr preparations from S. typhimurium (12), since FPr can replace HPr in Reaction 131; and it has also been used to determine the concentration of Factor IIIGrC from this organism. For the determination of Factor IIIGIC concentration, either HPr or FPr was added in small amounts (about 50 nM) so that there was no significant change in optical density due to phosphorylation of HPr or FPr. At this time we do not know whether FPr (M, about 45,000) and Factor IIIGrC (M, about 21,000) have one or more phosphorylation sites and thus their concentrations must be given in terms of equivalents of phosphate transferred per mg of protein. The composition of the incubation mixture is given under Methods. Sufficient Enzyme I should be used so that the reaction is complete in a few minutes. When reaction times are extended, P - HPr, which contains a labile N- 1-phosphohistidine ( 1,2), is hydrolyzed at a significant rate. Figure 2 shows how to make the correction necessitated by the hydrolysis of P - HPr. Both phospho-FPr and phospho-Factor III also showed significant rates of hydrolysis. For several reasons, the spectrophotometric assay system cannot be used with crude extracts. The method cannot distinguish between HPr, FPr, and Factor IIIGiC if more than one of these proteins are present in the extract, and the concentrations of the PTS proteins in crude extracts were usually too low for accurate measurement. In addition, S. typhimurium and E. coli contain “NADH oxidase activity” which interferes with the assay. This “NADH oxidase activity” was present in Factor IIIGrr preparations until they were highly purified. The amount of contamination varies, however, and some partially 5 We thank analysis.

Dr.

Nancy

Weigel

for performing

this

300

WAYGOOD,

MEADOW,

FIG. 2. A representation of the spectrophotometric trace obtained from the lactate dehydrogenasecoupled assay for the phosphocarrier proteins HPr, FPr, or Factor IIIGi~. The dotted (. . . .) portion of the trace represents a region that is not usuaiiy recorded since it occurs during the time between the addition of the Enzyme I and the return of the cuvette to the spectrophotometer. The assay mixture (see Methods) was prepared initially without any Enzyme I and these mixtures show a small but variable decrease in absorbance at 340 nm depending on the type and purity of the phosphocarrier protein being assayed. Enzyme I (20 units in 5 &I) was added at time zero. HPr was phosphorylated rapidly, and depletion occurs in a few minutes (1). Hydrolysis of the labile P - HPr converts it back to HPr, which is rephosphorylated. This rate is given by the tangent as drawn and was constant for at least 30 min. The absorbance value “d” represents the theoretical quantity of P - HPr that is hydrolyzed (and rephosphorylated) during time t ifah of the HPr had been phosphorylated at zero time. The total absorbance change (A absorbance) is corrected for the hydrolysis of phospho - HI? by subtracting d/2. This value is always made less than 5% of A absorbance by using appropriate quantities of the phosphocarrier proteins.

purified preparations have been assayed. In S. typhimutium a PEP phosphatase occasionally contaminated partially purified HPr preparations, but careful chromatography on molecular sieve gels removed this activity. With the precautions described above, the concentration of HPr, FPr, or -Factor III can be determined in either partially purified or homogeneous preparations, and the phosphocarrier proteins used in the assay system can also be recovered.

AND ROSEMAN

Assay by sugar phosphorylation. The scheme shown in Fig. 1 describes the components of the PTS in E. coli and S. typhimurium (9) discussed in this paper. The Enzymes II shown in the figure have specificities for both the phosphocarrier protein and for the sugar. We know that a given Enzyme II can react with more than one sugar and more than one phosphocarrier. For example, glucose is a substrate for both Enzyme IIMan and Enzyme II-BGle with a K, of 10 PM; methyl cY-glucoside is also a substrate but K, values are 25 mM for Enzyme IIMan and 6 PM for Enzyme II-BGiC. Enzyme IIMan is also known to interact with both FPr and HPr, but with an apparent K, value for FPr that is about 10 times greater than that for HPr. Thus the choice of the source of Enzyme II in phosphocarrier protein assays is very important. Mutant strains have been isolated, particularly in S. ty~him~rium, which provide the necessary specificity (Table 1). The membranes from SB2950 (deletion in HPr, Enzyme I, UT) were used to eliminate background due to HPr and Factor IIIGiC; SB2676 was used for its elevated levels of Enzyme II-Bfiu; and SB1687 (defective Enzyme IIMan) was used to avoid interference by Enzyme IIMa*. The advantage of the sugar phosphorylation assays involving either Reaction [l] or Reaction [2] (Fig. 1) is that each has a “built-in” phosphocarrier protein regeneration system. Moreover, the ratio of phosphocarrier protein to its phosphorylated form can be altered by manipulating the amounts of Enzyme I and Enzyme II in the reaction. Thus, if Enzyme I is present in great excess over Enzyme II, the ratio of phosphorylated carrier protein to carrier protein is high, whereas an excess of Enzyme II lowers the ratio. We have chosen to use Enzyme I 9 Enzyme II for phosphocarrier protein estimations because Enzyme I can be obtained in considerably purified form, whereas Enzyme II preparations are usually crude membranes which contain many unknown factors including phosphocarrier proteins, phosphatases, and proteases (24,25).

PHOSPHOTRANSFERASE

Thus, given a choice, the concentration of Enzyme II in an assay should be kept low. HPr assay. Apparent K, values for HPr and for phospho-HPr can be determined under conditions where Enzyme I or Enzyme II, respectively, are rate limiting. If the phosphotransfer reactions are sequential as indicated in Fig. 1, one might expect that the K, for HPr (Enzyme I rate limiting) would be independent of the sugar acceptor and of the specific Enzyme II complex used in the assay, whereas apparent K, values for HPr (actually phosphoHPr) under conditions of excess Enzyme I and Iimiting Enzyme II might be expected to vary with the particular Enzyme II, and possibly with different sugar acceptors and a single Enzyme II complex. These results were, in fact, obtained. The apparent K, values for HPr with limiting Enzyme I were about the same using Enzyme ITMa* (Reaction [ 11, Fig. 1) and with partially purified IIIGIC and II-BGiC (Reaction [2], Fig. l), i.e., 2.5 and 5 FM, respectively. By contrast, the apparent K, values for “phospho-HPr” (actually HPr with an excess of Enzyme I and PEP) using washed membranes of SB2950 (pts deletion) as the source of IIMan were as follows (Fig. 3): methyl cY-glucoside, 3 PM; fructose, 4 PM; and mannose, 10 PM. Glucose gave the same K, value as mannose (10 PM) (data not shown). Similar results were obtained using membranes from SB3507: methyl a-glucoside, 2 PM; fructose, 3.5 FM; mannose and glucose 10 FM; and 2-deoxyglucose, 20 PM.‘~ eK, values for HPr given here indicate that methyl a-glucoside and fructose give the best activities with respect to HPr concentration. However, other work (16) has shown that the K, values for these two sugars were 25 and 20 mM, respectively. Thus the 10 mM concentrations were saturating for mannose, glucose, and 2-deoxyglucose, but were not saturating for methyl a-glucoside and fructose. The results in Fig. 3 and a report on Escherichia coli Enzyme II /%glucoside (26) tentatively indicate that the Enzymes II, like the Enzyme I (16). exhibit ping-pang kinetics. Although further work is required, the pingpong mechanism offers an explanation for the apparent K, values obtained at a fixed sugar concentration.

301

IN BACTERIA ZOT 15.i/V

-0.4

0

04

0.8

I2

16

20

FIG. 3. Effect of concentration of HPr on Enzyme Wan activity. Enzyme II”“” in SB2950 membranes was assayed as described in the text with the following sugars at 10 mM concentration as substrates: mannose (m); fructose (A); and methyl a-glucoside (a). The apparent K, values for P - HPr were: methyl a-glucoside, 3 PM; fructose, 4 PM; and mannose. 10 PM. The initial velocities are expressed as micromoles of sugar-P formed30 min.

These results were obtained with each sugar at 10 mM concentration. The recommendation given under Methods that methyl a-glucoside should be used as the sugar substrate for routine assay of HPr may seem surprising since methyl LXglucoside is a poor substrate for Enzyme IIMan. However it has proved to be useful for HPr determinations for the following reasons: (a) In lactate-grown SB2950 the levels of Enzyme IIMan are about 30 times greater than levels of Enzyme II-BGlc, and thus the Enzyme II-BCIC background is comparatively small. (b) The K, for HPr of Enzyme IIMan with methyl a-glucoside is only 2 to 3 PM, so that relatively high reaction rates are obtained at relatively low concentrations of HPr. The concentrations of HPr in crude extracts of bacterial cells are often about 3 FM. (c) With 10 mM methyl a-glucoside, the background due to phosphorylation of the sugar by systems other than the PTS is very low. The method was applicable to a wide variety of sugars and membranes. The various estimates of HPr in a crude extract of glycerol-grown S. typhimurium are shown in Table 2. Factor ZZZGlcassay. The coupled lactate dehydrogenase assay could be used to

302

WAYGOOD,

MEADOW, TABLE

AND ROSEMAN 2

ASSAM OF HPr IN CRUDE EXTRACTS OF GLYCEROL-GROWN WITH SEVERAL SUGAR SUBSTRATES AND ENZYME

Concentrations Concentrations of sugar in assay (mh@ Methyl cu-glucoside, 10 Glucose, 10 Mannose, 10 Glucose, 1 Mannose, 1

zsc103 0.33 0.37 0.33 0.33 0.33 (0.34 t 0.02)C

S. typhimurium II PREPARATIONS”

SB3507

of HPr (nmol/mg protein) with membranes from SB2950

SB4004

0.29 0.29 0.28 NW ND (0.29 _’ 0.01)

0.34 0.42 0.37 0.31 0.27 (0.34 c 0.06)

a All membrane preparations were obtained from glycerol-grown cells. E. coli strain ZSC103 (18) was a strain obtained from Dr. W. Epstein, University of Chicago. Strain SB4004 was trpE22.3 crr24 (i.e., contained no Factor IIIG1c) obtained from Dr. J. Stock. The assays were performed as described in the text. b Not determined. e Average values.

ptsG

measure absolute concentrations of the protein in partially purified preparations. In crude extracts, the sugar phosphorylation assay was used either with membranes as the source of Enzyme II-BGIC or with partially purified II-B G1c.The response of Enzyme II-BGIC, whether partially purified or in crude membranes, to Factor IIIG’c concentration was that of an enzyme with MichaelisMenten kinetics. The K, value for Factor IIIGIC was 4 FM for both sources of Enzyme II-BGIC. Measurement of Enzymes II Any enzyme is defined by its kinetic constants, and Enzymes II, as mentioned above, show simple Michaelis-Menten behavior (in vitro). They can be defined by their sugar and phosphocarrier protein interactions. “saturating”) concentrations Optimal (i.e., by the sugar substrates of the Enzymes II are easily attained. However, saturation by the phosphocarrier proteins is not easily obtained on a routine basis because of the quantity of the phosphocarrier protein required. Using optimal sugar concentrations, the K, values with Enzyme IIMan, II-BG’c, or II-Bm are: HPr, 20 PM; Factor IIIG’c, 4 PM; FPr, 4 PM. While it is impractical to use saturating concentrations of these

proteins on a routine basis, the ease of the lactate dehydrogenase coupled assay permits the estimation of the concentration of these proteins, and it is therefore practical to use them at a fixed concentration so that the results of all assays may be readily compared. The concentrations of these proteins given under Methods represent a balance between the concentrations required to give high activity and the amount that could be obtained from routine purification. Some examples of measured Enzyme II levels are given in Table 3. Measurement

of Enzyme I

Enzyme I has several properties that affect the enzyme assay, the dominant one being that Enzyme I reversibly dissociates at low temperature from an active dimeric to an inactive monomeric form. This dissociation is known to depend on pH and protein concentration (Fig. 4) as well as temperature. The activation of the enzyme that is induced by increasing the temperature occurs too rapidly to be measured by the sugar phosphorylation assay. Thus the lactate dehydrogenase-coupled assay was modified by greatly reducing the amount of Enzyme I, so that the rate of HPr phos-

PHOSPHOTRANSFERASE

phorylation could be determined. Samples of pure Enzyme I were incubated at various temperatures and pH values and then transferred to the measuring cuvette at 25°C and pH 6.5 (optimal conditions). The results showed that enzyme that was preincubated at 0°C and at pH 7.5 had an initial activity which was only 5 or 10% of the maximum activity. The maximum rate was approached after 5 min at optimal conditions. Figure 4C shows that nonlinearity with protein concentration also occurs if preincubation conditions are not optimal. Thus the following conditions were determined to be optimal for the assay of Enzyme I: (a) Enzyme I must be preincubated at the temperature of the assay, after removal from the cold, to ensure linearity of activity with respect to enzyme concentration. (Assay temperature should be 2037°C.) (b) Enzyme I should always be in phosphate buffer; Tris-chloride buffers can cause loss of 75% of the activity (19). (c) For diluting the enzyme and performing the assay, 0.05 M potassium phosphate buffer, pH 6.5, containing 1 mM EDTA and 0.2 mM dithiothreitol should be used. TABLE MEASUREMENT OF ACTIVITIES BY METHODS DESCRIBED

PIS component

Strain

Enzyme I

SB3507

Enzyme IIMa”

SB3507

Enzyme II-BGic

SB2950 SB3507

HPr

SB2950 SB3507

303

IN BACTERlA

C

/

,p

lP 1&q!u ENZYME

2

I tpq)

FIG. 4. Effect of pH and temperature on Enzyme 1 activity. (A) Enzyme I (about 100-fold purified) was preincubated at 37°C for 10 min, and then diluted I: 100 into the respective 0.05 M potassium phosphate buffer at the pH values indicated. The buffers contained 1 mM EDTA and 0.2 mM dithioerythritol. and were also warmed to 37°C. The assay conditions were: 50 mM potassium phosphate buffer, 10 mM methyl o-glucoside, 50 pM HPr, and 0.8 mg of membranes from S. typhimurium SB2950. The mixtures containing the four concentrations of Enzyme I were incubated for 15 min at 37°C. (B) The data in A replotted: only at pH 6.5 is there a linear relationship between Enzyme I concentration and activity within the concentration range used. (0) pH 6.5, (0) pH 7.5, (a) pH 8.0. (C) Conditions were the same as in A, except that the Enzyme I was preincubated at o”C, and was diluted with buffer at room temperature. (0) pH 6.5. (A) pH 8.0.

Lebvels of PTS Components

3 OF PTS COMPONENTS IN THIS PAPER

Carbon source for growth

Activityn

Lactate Fructose Glucose Lactate Glucose Lactate Lactate Glucose Lactate Lactate Glucose

8.9 21.3 24.6 2.8 5.4 15.9 0.08 4.0 0.44 0.22” 0.77

‘I Enzyme activities represent specific activities expressed as micromoles of sugar-P produced/30 min/mg protein in the crude extract. ’ HPr is measured as nanomoles/milligram of protein.

The assays described here have now been used extensively in our laboratory and by others (27). Some of the typical enzymatic activities and concentrations of phosphocarrier proteins determined by these assays are given in Table 3. The enzymatic activities are from 2- to 20-fold higher than those reported previously. It is impossible to compare the previous estimates of HPr with our present methods as HPr levels were reported as a “PTS activity” which was dependent on the amount of Enzyme II-B being used. The cytoplasmic HPr concentrations reported here and in other work range from 20 to 100 FM. These values are sufficient to saturate Enzyme IIMa”. The Enzyme I activities are equivalent to about 400 prnol of sugar-P produced/min/g, dry weight, of cells at 37°C. which is similar

304

WAYGOOD,

MEADOW,

to in viva rates of sugar transport.’ Thus, using the present assay, PTS activities that are measured in homogenates are comparable to measurements on whole cells. ACKNOWLEDGMENTS

AND 10.

11. 12. 13.

This work was supported by a grant (CA 21901) from the National Institutes of Health, a fellowship from the Medical Research Council of Canada (to E. B. W.), and a Special Fellowship (5 F03 AG05029) from the National Institute of Aging (to N. D. M.). We thank Dr. N. Weigel for help with protein purification and for useful discussion. The technical assistance of T. Peng, F. Rachman, and D. Aquino has been invaluable.

16.

REFERENCES

19.

1. Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) .I. Biol. Chem. 246,7023-7033. 2. Simoni, R. D., Nakazawa, T., Hays, J. B., and Roseman, S. (1973)5. Biol. Chem. 248,932-940. 3. Jaffor Ullah, A. H., and Cirillo, V. P. (1976) J. Bacterial. 127, 1298- 1306. 4. Beyreuther, K., Raufuss, H., Schrecker, O., and Hengstenberg, W. (1977) Eur. J. Biochem. 75, 275-286. 5. Weigel, N. (1978) Ph.D. Thesis, The Johns Hopkins University, Baltimore. 6. Hays, J. B., Simoni, R. D., and Roseman, S. (1973) J. Biol. Chem. 248, 941-956. 7. Kundig, W. (1974) J. Supramol. Structure 2, 695-714. 8. Saier, M. H., Jr., and Roseman, S. (1976) J. Biol. Chem. 251,6598-6605. 9. Postma, P., and Roseman, S. (1976) Biochim. Biophys. Acta 457, 213-257.

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22.

23. 24. 25. 26. 27.

’ Unpublished

results.

ROSEMAN

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