Physical and Chemical Characterization of the Isomerase of ...

Tel.

THE JOURNAL OF RIOLOG~~L CHENISTRY Issue of January 25, pp. 256-264, 196i

242, No. 2,

Printed

Physical Histidine

in

U.S.A.

and Chemical Biosynthesis

Characterization in Salmonella

of the Isomerase typhimurium

of

(Received for publication, ~\IICIIAEL

IV.

~IARGOLIES*

From the Laboratory Institutes of Health,

AND

ROBERT

July 28, 1966)

F. GOLDBERGER

of Chemical Biology, National Bethesda, Maryland 20014

Institute

SUMMARY

and

Metabolic

Diseases,

National

which is encoded in the A gene of the histidine operon. Hartman has shown that mutants of this gene show no intragenic complementation in over 150 tests by abortive transduction (2). The amino acid composition and chemical and immunological properties of the enzyme are reported here, together with evidence that it consists of a single polypeptide chain corresponding to the single complementation group. MATERIALS

AND

Preparation

METHODS

of Enzyme

Growth of bacteria, preparation of cell-free extracts, and purification of the isomerase were performed as described previously (6). Polyacrylamide disc gel electrophoresis was performed according to Davis (7), and gels were stained for protein or isomerase activity as previously reported (6). Assays Protein was estimated in crude preparations by the method of Lowry et al. (8), with the use of bovine insulin (Lilly) as standard. Amounts of pure isomerase were calculated on the basis of the optical density at 280 rnp of enzyme in 0.05 M Tris-HCl, pH 8.0 (see “Results”). Assays for isomerase activity were done as previously described (6). Carboxymethylation

The structural genes for the 10 enzymes for histidine biosynthesis in Salmonella typhimurium are located on the chromosome in a cluster known as the histidine operon. Because of the large body of information now available on the biochemical and genetic aspects of this system (l-3), it was thought especially desirable to obtain data on the physical and chemical characteristics of the enzymes themselves. The isolation and characterization of the enzymes for histidine biosynthesis have therefore been undertaken. The results for several of these enzymes have been reported (4-6). We previously described a method (6) for the isolation of the enzyme catalyzing the fourth step in the pathway for histidine biosynthesis in S. typhimurium, an isomerase, the structure of * Present address, General Surgical Services, General Hospital, Boston, Massachusetts 02114.

Massachusetts

Reduction and alkylation of enzyme were performed according to the method of Anfinsen and Haber (9) as modified by Craven, Steers, and Anfinsen (10). To 10 mg of purified isomerase in 0.05 M Tris-HCl, pH 8.0, was added sufficient recrystallized urea to bring the solution to 8 M in urea (1 ml total volume). After addition of P-mercaptoethanol (Eastman) to a concentration of 0.1 M, the tube was stoppered and incubated for 4 hours at 40”. Following reduction, the solution was diluted 4-fold with 0.1 M Tris-HCl, pH 8.5. A 4-fold molar excess of twice recrystallized iodoacetic acid (Eastman) was dissolved in sufficient Tris base to bring the pH to 8.5. The iodoacetic acid was then added to the reduced enzyme and incubated for 15 min at room temperature without change in pH. An amount of fl-mercaptoethanol equimolar to the iodoacetic acid used was then added to stop the reaction, and the mixture was dialyzed against buffer appropriate for further use (see below). 256

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The amino acid composition and chemical and immunological properties of the isomerase catalyzing the fourth step in histidine biosynthesis in Salmonella typhimwium are reported. The molecular weight of the native enzyme is 29,000, as determined by high speed equilibrium centrifugation; it is unchanged after complete reduction and alkylation, indicating the presence of a single polypeptide chain. This conclusion is supported by the finding of a single carboxyl-terminal amino acid, valine, both by digestion with carboxypeptidase A and by hydrazinolysis. Furthermore, the number of spots seen on peptide maps of tryptic digests of carboxymethylated isomerase is approximately the same as the number of arginine plus lysine residues found by amino acid analyses. These results agree with results of genetic studies (1) which show that the structural gene for this enzyme consists of a single complementation group. The minor active isomers of the enzyme, previously described, appear to result from modifications in vitro of a single protein. Antibody specific to the isomerase has been prepared. Carboxymethylated isomerase and isomerase derived from extracts of repressed and derepressed histidine auxotrophs with mutations in all regions of the histidine operon, of constitutive mutants, and of the wild type strains LT-2 and LT-7 are antigenically identical.

of Arthritis

Issue

of January

M. N. Margolies and R. F. Goldberger

25, 1967 Molecular

Weight Determinations

Molecular weight and homogeneity were estimated in the Spinco model E analytic ultracentrifuge by the meniscus depletion method of Yphantis (ll), with the use of a standard doublesector interference cell. The ultracentrifuge was equipped with Rayleigh interference optics and the temperature for all runs was controlled at 20”. Photographs were taken at zero time and after equilibrium was reached. Attainment of equilibrium required 20 hours of centrifugation except for samples run in 8 M urea; in the latter case photographs were taken after 48 hours of centrifugation. To prepare these samples, enzyme solutions (0.2 to 0.4 mg per ml) were dialyzed for 7 to 10 days against 8 M urea in 0.05 M Tris-HCl, pH 8.0. We assumed v to be 0.725 for the calculation of molecular weights. Acid Hydrolyses and Amino

Acid Analyses

Digestion and Peptide Jdapping

Samples of carboxymethylated isomerase were dialyzed against 0.2 M NH,HCOs and divided into 2-mg (O.O7+mole) aliquots. Two per cent by weight of trypsin (Worthington), previously treated with diisopropyl fluorophosphate (14), was added, and the solution was incubated for 4 hours at 37”. Following lyophilization, peptide maps were prepared by the method of Katz, Dreyer, and Anfinsen (15) on Whatman No. 3MM chromatography paper and stained with 0.25a/, ethanolic ninhydrin (Eastman). Carboxypeptidase

Digestion

diisopropyl

and B for 16 to 20 hours in 0.1 M NH4HC03. In later experiments, 0.2 pmole of carboxymethylated enzyme was incubated with 2 To by weight of carboxypeptidase A alone. Equal aliquots (0.04 pmole) were withdrawn at 0, 2, 4, 6, and 16 hours. Digestions were stopped by freezing and lyophilization. All samples were subjected to amino acid analyses (see above). In addition, control samples, containing amounts of the carboxypeptidase preparation identical with the experimental samples, were incubated in parallel and subjected to amino acid analyses. The amount of each contaminating amino acid in these latter samples was always less than 0.002 pmole. Hydrazinolysis Hydrazinolysis was performed according to the method of Korenman (17). Anhydrous hydrasine (Matheson, Coleman, and Bell) was used without further treatment, and oxidized bovine pancreatic ribonuclease A (Worthington) was employed as a control. Aliquots of 0.05 pmole were withdrawn at zero time and after 10 hours of hydrazinolysis at 110”. Amino acid analyses were performed as described above. Amino-terminal

The carboxypeptidases A and B were obtained from Dr. J. T. Potts. These proteolytic enzymes had been treated with DFPl to remove all endopeptidase activity (16). Following such treatment, they exhibited only exopeptidase activity when tested with the following substrates: bovine pancreatic ribonuclease A, ribonuclease S, ribonuclease S-protein, ribonuclease S-peptide, parathyroid hormone, and an extracellular nuclease from Staphylococcus uureus. In early experiments, 3-mg (O.lpmole) samples of reduced, alkylated isomerase were incubated (5 mg per ml) with 2% by weight each of carboxypeptidases A 1 The abbreviations used are: DFP, phate; DNP-, 2,4-dinitrophenyl-.

I I I I 480 490 500 505 470 x2x2FIG. 1. Molecular weight determinations by the meniscus depletion method of Yphantis (11). Average molecular weight values are indicated for each set of runs. The constant slopes of the plots for log C with respect to X2 indicate homogeneity. A, native isomerase in 0.05 M Tris-HCl, pH 8.0; B, carboxymethylated enzyme in the same buffer plus 8 M urea; C, carboxymethylated isomerase in buffer; D, native enzyme in buffer plus 8 M urea. The agreement in both calculated molecular weight and degree of homogeneity among Samples A, B, and D suggests that the isomerase consists of a single polypeptide chain. Aggregation and precipitation of carboxymethylated enzyme, as seen in C, may be prevented by dialysis against 8 M urea (see B).

fluorophos-

End Group Analyses

Amino-terminal end group analyses of 0.1 pmole of enzyme were done by the fluoroclmitrobenzene method of Sanger (18), with the use of paper chromatographic separation of the ethersoluble 2,4-dinitrophenyl-amino acids, as described by Redfield and Anfinsen (19). The water phase was examined by high voltage electrophoresis (20). The yields of DNP-amino acids were estimated photometrically at 360 rnp after elution of spots from paper into distilled water at 60” for 15 min. Solutions of authentic DNP-amino acids (Calbiochem) were used as standards. Determination of the amino-terminal end group was also attempted by digestion of carboxymethylated isomerase with leucine aminopeptidase (21). Before use, the leucine amino-


Amino acid analyses were performed according to the method of Spackman, Stein, and Moore (12), with the use of a Beckman model 120B automatic amino acid analyzer coupled with an Infotronics model CRS-1OA digital readout system. Purified native enzyme and the carboxymethylated enzyme were each dialyzed against several changes of 0.2 M NH4HC0a, divided into 1-mg aliquots, and lyophilized to dryness. Duplicate samples of both native and carboxymethylated enzyme were hydrolyzed with constant boiling HCl in sealed evacuated tubes for 19, 45, and 69 hours at 110”. The acid was removed under reduced pressure and the timed hydrolysates were analyzed consecutively. Tryptophan was estimated by the method of Beaven and Holiday (13) with the use of solutions of carboxymethylated enzyme in 0.1 M NaOH. Spectra were recorded with the model 15 Cary spectrophotometer. In addition, peptide maps prepared from tryptic digests of the carboxymethylated enzyme were stained with Ehrlich’s reagent (14) to determine the minimum number of tryptophan residues. The reagent, p-dimethylaminobenzaldehyde, was obtained from Eastman. Trypsin

257

258

Isomerase of Histidine Biosynthesis in S. typhimurium

Vol. 242, No. 2

peptidase(Worthington) wasdialyzed against 0.05 MNH.J?ICOa with 0.01 M MgC& and incubated at 37” for 3 hours. Three milligramsof carboxymethylated isomerase dissolvedin the same buffer were incubated with 0.3 mg of leucineaminopeptidaseat 37” for 16 hours. Aliquots 10.05pmole) were removedinitially and at 16 hours, and lyophilized for amino acid analyses. In addition, parallel incubations of identical amounts of leucine aminopeptidasein the samebuffer wereanalyzed.

Removal of contaminatingantibodiesfrom the immuneserum (see“Results”) wasaccomplishedby incubation of antiserumat 37” for 1 hour with one-tenth volume of a derepressed extract (25 mg of protein per ml) of an isomerase deletion mutant, his61% The reaction mixture was stored overnight at 4” and the supernatantwasseparatedby centrifugation. Two consecutive adsorptionswerenecessaryto removeall detectablecontaminants (see“Results”). RESULTS

Immunizations

New Zealand albino male rabbits, weighing 2500 g, were immunizedagainstvarious samplesof isomerase,with the useof 1 ml of enzyme solution emulsifiedin 1 ml of Freund’s complete adjuvant (Difco) injected subcutaneously,on the 1st and again on the 7th day of immunization. A boosterdosewas given at 30 days. For crude enzyme preparations,20 mg were given at each injection; for purified isomer&se,1 mg was usedfor each injection. Prior to use, enzyme solutionswere renderedsterile by passagethrough a Millipore filter with a pore size of 0.22 p. Blood was collected prior to immunization, at the time of the booster injection, and at 4-day intervals thereafter. Sera obtained by centrifugation were storedat -20”. Examination of Antibody Preer Technique--The minimum number of precipitating antigen-antibody systemspresent was examinedquantitatively by double diffusion in agar by the method of Preer (22). Noble agar (Difco), 0.8%, was employed as the diffusing medium. All tubeswere run in duplicate, with the useof serialdilutions of

Molecular Weight DeterwGnations-We have previously determined the average molecularweight of the native isomerase by the meniscusdepletion method of Yphantis (11) in 0.05 M Tris, pH 8.0, to be 29,000(6). Plots of log C against X2 for all five experimentsconformedto a straight line, indicating homogeneity with respectto size (seeFig. 1A). The data were also subjected to analysis on a Honeywell 800 computer with the programof Dr. Parker Small and Dr. Robert Resnick. For this run, at 37,020 rpm, the whole cell weight average molecular weight (J&J was29,700,the wholecell numberaveragemolecular weight (M,J was27,700,and the whole cell z averagemolecular weight (ME) was32,500. The carboxymethylated isomerasewas subjected to equilibrium centrifugation in order to determine whether the enzyme is composedof subunits. Solutions of reduced and alkylated isomerasein 0.05 M Tris-HCl, pH 8.0, at 0.4, 0.2, and 0.15 mg per ml were examined at 35,600, 31,140, and 35,600rpm, respectively, after 24 hours of centrifugation. The results of a typical experimentare shownin Fig. 1C. The rangeof molecular weights calculatedfrom the slopesof linestangent to the curve


antigen against a constant amount of undiluted antiserum. Tubes were examinedfor precipitin bandsat 4-hour intervals in a dissectingmicroscopeequippedwith a calibrated ocular. The equivalencepoint for each antigen-antibody system was determinedfrom plots of p (position of band relative to antigen-agar interface) with respectto concentration of antigen for eachtime of observation,aswell asfrom plots of p with respectto time for each concentrationof antigen. Ouchterlony Technique--Antibody was also examined by double diffusion in agar accordingto the method of Ouchterlony (23), in prepared plates (Hyland Laboratories)with 4-, 5-, and 7-n-m well distances. Plates were incubated at room temperature for 24 hours. ImmunoeZectrophore~Immunoelectrophoresis wasperformed on microscopeslides(24) in 0.1 M Verona1buffer, pH 8.6, for 50 min at 250 volts. The slideswere incubated with antiserum at room temperaturefor 16hours. Inactivation-Precipitation-Antibody was also tested for its ability to neutraliie the enzymic activity of the isomerase. To 0.1 ml of immune serumin each of several centrifuge tubes was added 0.1 ml of serial dilutions of purified isomerase. Control tubes contained preimmunization serum with identical serial dilutions of enzyme. All tubes were incubated at 25” for 90 min and stored overnight at 4”. Following centrifugation, each supernatant was quantitatively assayedin duplicate for isomerase activity. Equivalence points for antisera were FIG. 2. Patterns obtained by electrophoresis in polyacrylamide gels of (1) purified native isomeraseand (3) carboxymethylated determinedfrom plots of concentration of activity in the superisomerase. The pattern noted in Rappearsto be due to aggrega- natant with respectto concentrationof antigen added(seeFig. 8). tion of the carboxymethylated enzyme (cf. Fig. 1). Adsorption of Antibody

Issue of January

25, 1967

M. N. Margolies

and R. F. Goldberger TABLE

I

composition of purified isomerase from hisEll Duplicate samples of 1 mg each were hydrolyzed in acid for 19, from the averages of the 45 and 69.hour values, after complete release of these residues had been effected. Cysteine was deter45, and 69 hours. Numbers of residues of each amino acid are based upon a molecular weight of 29,000 for the native isomerase as mined as S-carboxymethylcysteine from a separate set of analyses determined by equilibrium centrifugation. The amounts of on carboxymethylated protein. Tryptophan was estimated by serine, threonine, and tyrosine decreased linearly with time and the method of Beaven and Holiday (13). The amounts of all other were therefore calculated by extrapolation to zero time. amino acids were calculated by averaging the results obtained Amounts of methionine, valine, and isoleucine were calculated from the paired samples at all hydrolysis times. Amino

acid

Duration

of hydrolysis

Amino acid 19 hrs

45 hrs

I

69 hrs

-I-

wnole

0.186 ...

0.077 0.234 0.065 0.406 0.192 0.178 0.497 0.172

0.432 0.414 0.413 0.037 0.232 0.419 0.103 0.090

0.188 0.081

0.233 0.063 0.400 0.190 0.177

0.488 0.179

0.428 0.414 0.425 0.033 0.221 0.421 0.105 0.093

0.180 0.074 0.232 0.061 0.406 0.181 0.156 0.518 0.169 0.429

0.420 0.445 0.040 0.233 0.422 0.097 0.095

shown varied up to values above 50,000. Comparison of the pattern obtained upon electrophoresis of the carboxymethylated protein in polyacrylamide gels to that obtained with the use of native enzyme (see Fig. 2) also indicated size heterogeneity due Therefore, to aggregation of the reduced and alkylated protein. carboxymethylated protein was first dialyzed against 8 M urea in the same buffer for 7 to 10 days and then examined in the ultracentrifuge. Under these conditions, the protein no longer aggregated or precipitated, and a pattern identical with that for native enzyme was seen (see Fig. 1B). For these experiments, solutions of carboxymethylated isomerase, 0.3 mg per ml, were centrifuged at 33,450 rpm for 48 and 60 hours, and at 37,020 rpm for 48 hours; the molecular weights were calculated to be 25,900, 25,000, and 26,100, respectively. These calculations were made with the use of the same v for runs in both buffer and in buffered Control samples of native enzyme (0.2 to 0.4 urea solutions. pH 8.0, produced mg per ml) in 8 M urea, 0.05 M Tris-HCl, patterns identical with those for the carboxymethylated enzyme in urea when examined by the same technique (see Fig. 1D). Data obtained by centrifugation at 40,000 and 36,000 rpm gave calculated molecular weights of 28,500 and 24,200, respectively. The average molecular weight for the reduced and alkylated enzyme in urea (25,700) compares favorably with that for the native enzyme in urea (26,400). Amino Acid AnalysesThe amino acid compositions of paired samples of native enzyme hydrolyzed in acid for 19, 45, and 69 hours are shown in Table I. The amounts of threonine, serine, and tyrosine decreased linearly with the duration of hydrolysis

0.185 0.074 0.235 0.059 0.412 0.184 0.157 0.525 0.167 0.436 0.423 0.449 0.041 0.238 0.424 0.096 0.094

0.179

0.074 0.229 0.062 0.401 0.170 0.139 0.510 0.173 0.422 0.418 0.445 0.041 0.236 0.420 0.093 0.094 -

0.191 0.079 0.239

0.063 0.407 0.175 0.141 0.520 0.162 0.430 0.421 0.450 0.043 0.243 0.425 0.094 0.093

/.mole

0.185 0.076 0.234 0.062 0.405 0.198 0.192 0.510 0.170 0.429 0.418

0.447 0.041 0.237 0.422 0.107 0.093

11.9 4.9 15.1

4.2 26.1 12.8 12.4 32.9 10.9 27.7 27.0 28.9 2.7 15.3 27.2 6.9 6.0 2.9

and were therefore calculated by extrapolation to zero time; the amounts of methionine, valine, and isoleucine were calculated from the average of the 45- and 69-hour samples, after these amino acids had been completely released by hydrolysis; the amounts of all other amino acids were calculated by averaging the results from all samples. Cysteine was estimated from a separate set of timed hydrolyses of paired samples of carboxymethylated protein. The quantitative conversion of cysteine to S-carboxymethylcysteine was shown by the absence of cysteine or cysteic acid in the latter analyses. The reduced and alkylated enzyme had no detectable isomerase activity. There were 2.9 residues of tryptophan per mole of protein as determined from the spectrum of the isomerase in 0.1 M NaOH (Fig. 3). Peptide maps prepared from tryptic digests of carboxymethylated isomerase, stained with Ehrlich’s reagent, revealed two spots. On the basis of the absorbance at 280 rnp of a solution of native isomerase in 0.05 M Tris-HCl, pH 8.0, together with determinations of protein concentration by amino acid analyses, the molar extinction coefficient was calculated to be 3.31 x 104. Peptide MapsPeptide maps prepared from tryptic digests of carboxymethylated isomerase revealed a reproducible pattern consisting of 34 ninhydrin-positive spots; the origin was free of ninhydrin-positive material (see Fig. 4). This agrees with the total number of lysine plus arginine residues as determined by amino acid analyses. Carboxyl-terminal End Group AnalysisInitial incubations of 0.1 pmole of carboxymethylated isomerase with carboxypepti-


Lysine ...................... Histidine. .................. Arginine .................... S-Carboxymethylcysteine Aspartic acid. ............. Threonine.................. Serine ...................... Glutamic acid. ............. Proline ..................... Glycine ..................... Alanine .................... Valine ...................... Methionine ................. Isoleucine.................. Leucine .................... Tyrosine ................... Phenylalanine.............. Tryptophan ................

260

Isomerase

Biosynthesis

(mp)

FIG. 3. Ultraviolet absorption spectra of carboxymethylated isomerase, at a concentration of 0.60 mg per ml, in 0.05 M Tris-HCl. pH 8.0 (-), and, at a concentration-of 0.39 mg per ml, in 0.1 & NaOH (-- -). The tryptophan content of the isomerase was calculated from the latter curve, with the use of the method of Beaven and Holiday (13).

dases A and B for 20 hours resulted in 72% yield of valine as well as yields of 26 y0 for aspartic acid, 27% for leucine, and 19% for alanine. Results of timed digestions of carboxymethylated isomerase with carboxypeptidase A, with the use of 0.05 pmole of isomerase for each analysis, are shown in Fig. 5. The calculated final yield of valine in this experiment was 150%. This result suggests that the penultimate residue is also valine. Alanine, aspartic acid, and leucine were again obtained in low yield; it has not been possible to distinguish the order in which they appear. The identity of the carboxyl-terminal residue was confirmed by hydrazinolysis of 0.07 pmole of the isomerase for 10 hours which resulted in 75 y0 yield of valine No other amino acids were released by hydrazinolysis. A 0.1~pmole sample of oxidized ribonuclease, run in parallel, produced a 52y0 yield of valine.2 Amino-terminal End Group AnaZysisAmino-terminal end group analysis of 0.1 pmole of the isomerase, with the use of 2,4-dinitrofluorobenzene, revealed only a trace of threonine (less than 30/,), although the yield of e-DNP-lysine was greater than 70%. In addition, the carboxymethylated enzyme was refractory to tidase.

attempts

Immunological

at prolonged

Studies-When

2 The carboxyl-terminal ribonuclease is known

digestion

by

antiserum

amino acid residue to be valine (25).

leucine

prepared of bovine

aminopep-

by impancreatic

in S. typhimurium

Vol. 242, n-o. 2

munization of rabbits with a crude extract of derepresseds S. typhimurium hisEl1 was examined against purified isomerase by double diffusion in agar, with the Ouchterlony and Preer techniques, only one band was seen. However, the antibody prepared in this way was not sufficiently strong for further As expected, when this antiserum examination of homogeneity. was reacted against the original crude extract, multiple strong bands were seen. However, antisera produced by immunization with purified isomerase were satisfactory for further studies. The results obtained when these antisera were examined in Preer tubes containing serial dilutions of the purified isomerase are shown in Fig. 6. According to plots of p against time, and p against concentration of antigen (and by inspection), the equivalence point for the major system seen was found to be at an antigen concentration of 65 pg per ml. In addition, several minor components were revealed at much higher concentrations of antigen. Corresponding patterns were seen in Ouchterlony plates. The conclusion that the major precipitin system was due to the reaction of the isomerase with its specific antibody is based on three lines of evidence. (a) After the antiserum had been adsorbed with a crude extract of S. typhimurium his-612 (a mutant in which the entire A gene is deleted) and then was reacted against purified isomerase, no contaminating system was observed (see Fig. 6B). Conversely, when the reaction between unadsorbed antibody and serial dilutions of the same his-612 extract was examined in Preer tubes, the major band due to the isomerase was absent. (b) When the unadsorbed antiserum was examined with purified isomerase by immunoelectrophoresis, the major component was due to the isomerase (Fig. 7,1). The contaminating systems were absent when the isomerase was reacted with the adsorbed antiserum (Fig. 7, 2). The minor contaminating bands were also identified by their presence in runs with the use of extracts of the deletion mutant h&61,? against the unadsorbed antibody and their absence in reactions involving the adsorbed antibody (Fig. 7, S). (c) The reaction of isomerase with its antibody could also be studied by the ability of the antiserum to inactivate and precipitate the isomerase. The antigen-antibody precipitate in agar did not stain in the color reaction for isomerase activity (6), nor could resuspended precipitates be shown to contain isomerase activity in the standard assay. A plot of isomerase activity remaining in the supernatants after reaction of antiserum with serial dilutions of purified enzyme, with respect to concentration, is shown in Fig. 8. By comparison with the plot obtained from the same experiment done with control (preimmunization) serum, it is clear that the antiserum removed all enzymic activity from solution up to a concentration of 65 pg per ml. The equivalence point obtained in this way is identical with that obtained from studies of double diffusion in agar. The reaction between crude extracts of derepressed histidine auxotrophs and antibody was also examined by the above techniques. The results obtained with both adsorbed and 3 When S. typhimurium is grown in the presence of excess histidine, the histidine operon is repressed: the enzymes for histidine In histidine biosynthesis are synthesized at a relatively low rate. auxotrophs grown under conditions in which the growth rate is limited by the amount of histidine available, the histidine operon becomes derepressed, and the enzymes for histidine biosynthesis are synthesized at a relatively high rate, producing levels of enzymes up to 30 times those of the wild type (LT-2) organism.


WAVELENGTH

of Histidine

Issue of January

25, 1967

261


/

CHROMATOGRAPHY

-

I 6

I 4

I 2

0

TIME

(hrs.)

FIG. 5. Release of amino acids during timed digestion of carFIG. 4. Peptide maps of tryptic digests of 0.1 pmole of carboxymethylated isomerase with carboxypeptidase A. A total of boxymethylated isomerase prepared by the method of Katz, 0.2 pmole of the reduced and alkylated isomerase was incubated Dreyer, and hnfinsen (15), stained with ninhydrin. The patterns with 2oJc, by weight, carboxypeptidase A at 37”. Valine was obwere reproducible and contained 34 ninhydrin-positive spots. tained early and in high yield, but it has not been possible to distinguish the order in which alanine, aspartic acid, and leucine unadsorbed antibody were similiar to those obtained when were released.

putified enzyme was used as antigen. After adsorption, the antiserum formed only one band with pure and with crude preparations of isomerase,and formed no detectableprecipitate with extracts of the deletion mutant. When examinedby doublediffusion in agarby the Ouchterlony technique, with the useof antiserum prepared against purified isomerase,reactionsof identity were seenbetweenthe following antigen preparations: (a) purified isomerasewith crude extract from the of derepressed h&El 1; (b) partially purified isomerase wild type strain LT-2 (repressed)with crude extract of derepressedhisEll; (c) crude extract of derepressedLT-2* with crude extracts of repressedhi&II (grown on excesshistidine); (d) crude extracts of both repressedand derepressedhistidine auxotrophs (other than A gene mutants) with extracts of various constitutive mutants5; (e) crude extracts of LT-2 with crude extracts of another wild type strain, LT-7; fj) crude extracts of both repressedand derepressedhistidine auxotrophs 4 Derepression of the wild type organism was effected by addition to the growth medium of nn-2-thiazole alanine (Cycle Chemical Company) at a concentration of 0.5 mM. This compound is known to inhibit the first enzyme in the pathway for histidine biosynthesis (26). 5 The enzymes for histidine biosynthesis are present in relatively high concentrations in organisms with certain mutations outside the histidine operon; these are the constitutive mutants

(27).

A Ag

I:1

I:2

Ab

I:1

I:1

Ag

I:1

Ab

PI

I:4

1~8

ItI6

I:32

I:64

I:128

I:1

I:1

Id

I:I

I:I

I:1

I:2

I:4

1:8

I:16

I:32

I:64

I:128

I:1

I:1

Id

I:1

I:I

I:I

I:I

B

FIG. 6. Double diffusion in agar, by the method of Preer (22), of serial dilutions of purified isomerase against constant amounts of undiluted antiserum. A, reaction of isomerase with unadsorbed system with equivaantiserum reveals a major antigen-antibody lence at 65 pg of isomerase per ml. Minor components are seen B, the contaminating sysonly at high antigen concentrations. tems were no longer detected when pure isomerase was reacted against antiserum previously adsorbed with an extract of the deletion mutant !&+6+fd.


0

262

Isomerase of Histidine

Biosynthesis

in S. typhimurium

extract of hisSld against,adsorbedantibody. derived from LT-2 with those derived from LT-7; and (g) purified native isomerasewith carboxymethylated isomerase. Thus, the antigenic site or sites of isomerasefrom all sources listed above are indistinguishable. Examination of Isomers---Asreported previously, the purified isomerasewas homogeneouswith respect to size, but three enzymically active forms couldbe separatedupon electrophoresis in polyacrylamide gels (6). It was concluded that the three forms of isomerasediffered in chargeonly. Several observations suggestthat the variant forms ariseasartifacts of the purification procedure. The ratio observed for the three forms varied in different preparations, depending upon the duration of the isolation procedure. For example, activity stainsof disc gelsof an extract of strain LT-2, which was purified 20-fold over a 16-hourperiod, revealedlessthan 2% of the minor forms. After large scale preparations of purified enzyme, requiring several weeks,the variant forms were observed in higher proportions (10%). Incubation of pure enzyme at pH 2 to 3 for several minutes at 4” resulted in the appearanceof still higher propor-

tions (50%) of the variant forms as well as a new (fourth) variant. Moreover, only one band was seen in immunoelectrophoresis of the purified protein against adsorbed antibody, indicating antigenic identity amongthe isomers. DISCUSSION

We have previously reported that the purified isomerasewas homogeneous asjudgedby electrophoresisin polyacrylamide gels at pH 9.5 and at pH 4.3, electrophoresisin starch gels,and high speed equilibrium centrifugation (6). Further evidence for homogeneitypresentedhereincludesthe observationsthat amino acids other than the carboxyl-terminal valine do not appear following hydrazinolysis, that the number of tryptic peptides predicted from the ammoacid analysesagreeswith the number actually found, and that the purified preparation elicits the production of antibody which, although it contains minor contaminants,is directed predominantly toward the isomerase. The available evidence suggeststhat the product of the A geneof the hiitidine operonin S. typhimurium is a singleprotein.


FIU. 7. Examination of antiseraby slide immunoelectrophoresis.Electrophoresisproceededin 0.1 MVerona1buffer, pH 8.6, for 50 min at 250 volts. Slides were then incubated with antiserum at room temperature for 16 hours. 1, native isomerasereacted against unadaorbed antiserum; 8, native isomerase reacted against antiserum adsorbed with extract of isomerase deletion mutant his4’ld; 3,

Issue of January

ISOMERASE


CONCENTRATION

(mg/ml)

FIG. 8. Neutralization and precipitation of isomerase by antiserum. A plot of isomerase activity remaining in the supernatant with respect to antigen concentrations after reaction of serial dilutions of purified isomerase with control serum (- - -) and antiserum (-) reveals that the equivalence concentration is 65 ag of isomerase per ml. This is identical with the equivalence point found in Preer tubes (upper part of the figure), calculated from plots of p against time and p against concentration.

263

The variant forms distinguishedby their different electrophoretic mobilitiesappearto ariseas artifacts during large scalepreparations of the enzyme. Such variants might be produced, for example, by deamidat,ionof glutamine or asparagineresidues during the isolation procedure. The conclusionthat the isomeraseis composedof a single polypeptide chain is basedupon the following evidence. (a) The molecular weight and homogeneity, as determined by ultracentrifugation, were not affected by exposureto 8 M urea or by carboxymethylation. (6) There appears to be only one carboxyl-terminal residue, valine, as determined by both hydrazinolysis and digestion with carboxypeptidase A. (c) The possibility that the isomerase is composedof two or moreidentical subunitsis ruled out by the finding that the number of peptides produced by tryptic digestion correspondsclosely to the total numberof lysine plusarginineresiduesdeterminedby aminoacid analyses. The studiespresentedhere show that the isomeraseisolated from a derepressed histidine auxotroph (hisEll) is antigenically identical with that of repressedand derepressed histidine auxotrophs with mutations in other genesof the histidine operon, constitutive mutants, the wild type strains LT-2 and LT-7, and carboxymethylated isomerase. Those A gene mutations which result in the production of isomerasewith amino acid substitutions that alter antigenicity are widely distributed throughout the map of the A gene.6 Thus, the tertiary conformation of the isomerasewhich is required for antigenic specificity may be destroyed by a change in any of a largenumber of amino acidswidely distributed along the polypeptide chain. The conformation required for enzymic activity is probably even more specificsince it is destroyed by carboxymethylation of the native enzyme while antigenicity is preserved and since extracts of many A gene mutants are enzymically inactive but antigenically competent. Acknowbdgmenk-The authors are indebted to Dr. Christian B. Anfinsen for critical suggestionsduring the course of this work. We wish to thank Dr. Edward Steers,Jr., for performanceof the ultracentrifugation and for advice in the immunological studies. Dr. Bruce N. Ames kindly suppliedthe mutants used. We also thank Mrs. Marilyn Meyers for technical assistanceand Mr. Clifford Lee for performing the amino acid REFERENCES 1. LOPER,

J. C., GRABNAR, M., STAHL, R. C., HARTMAN, Z., AND P. E., Brookhaven Symp. Biol., 17, 15 (1964). AND HARTMAN, P. E., ColdSpring Harbor Symp. Quant. Biol., 28, 349 (1963). 3. GOLDBERGER, R. F., AND BERBERICH, M. A., in H. J. VOGEL, J. 0. LAMPEN, AND V. BRYSON (Editors), Organizationalbiosunnthesis, AcademicPress,New York, in press. 4. LOPER, J. C., AND ADAMS, E., J. Biol. Chem.,249,788(1965). 5. MARTIN, R. G.,AND GOLDBERGER, R. F., J. Biol. Chem., in press. 6. MARGOLIES, M. N., AND GOLDBERGER, R. F., J. Biol. Chem., 241,3262(1966). 7. DAVIS, B. J., Ann. N. Y. Acad. Sci., lai, 494(1964). 8. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem.,193,265(1951). 9. ANFINSEN, C. B., AND HABER, E., J. Biol. Chem., 236, 1361 (1961). 6M. N. MargoliesandR. F. Goldberger,manuscriptin preparation. HARTMAN, 2. AYES, B. N.,

FIO. 9. Reactions of identity among isomerase preparations.from various sources, as seen in Ouchterlony plates. Center well contains adsorbed antibody; incubation with antigens proceeded at room temperature for 16 hours. Antigen wells contain: 1, purified native isomerase; %, crude extract of derepressed hisElf; 3, partially purified (repressed)LT-2; 4, crudeextract of constitutive mutant hisTl601; 6, carboxymethylated isomerase.


G;

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Isomerase of Hi&dine

Biosynthesis

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in S. typhimurium

Vol. 242, No. 2

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Physical and Chemical Characterization of the Isomerase of Histidine Biosynthesis in Salmonella typhimurium Michael N. Margolies and Robert F. Goldberger J. Biol. Chem. 1967, 242:256-264.

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