the generation of energy by the arginine dihydrolase ... · this pathway was first named in 1940 by...

THE JOURNAL OP BIOLOGICAL CHEMISTRY Vol. 241, No. 10, Iswe of May 25, pp. 222&2236, 1966

Printed in U.S. A.

The Generation of Energy by the Arginine Dihydrolase Pathway in Mycoplasma hominis 07

(Received for publication, November 12, 1965)

ROBERT T. SCHIMKE, C. M. BERLIN, E. W. SWEENEY, AND WILLIAM R. CARROLL

From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service, Bethesda, Maryland SOOl.4

SUMMARY

The nature of the energy source for a representative nonfermenting Myco&~sma, Mycoplasma hominis type II, strain 07, has been studied. The results indicate that this microorganism can obtain energy from the metabolism of arginine by the arginine dihydrolase pathway. The conversion of arginine to ornithine, with the concomitant formation of an equimolar amount of adenosine triphosphate, is quanti- tatively sufficient to account for the estimated energy required for synthesis of macromolecules in growing cells. Thus the results indicate that arginine may be the major source of energy, but they cannot rule out contributions to energy generation from other unknown sources.

Extracts of M. hominis 07 are exceedingly rich in the three enzymes of the arginine dihydrolase pathway. Purifications and characterizations of the three enzymes have been under- taken. Arginine deiminase and omithine transcarbamylase have been purified to an essentially homogeneous state, and have been found to constitute approximately 10 and 4% respectively, of the soluble protein of cell extracts. The physical and kinetic properties are similar to those reported for the enzymes obtained from Strepiococcus.

This pathway was first named in 1940 by Hills (ll), who described the conversion of arginine to citrulline in Streptococcus. It has been described in certain bacteria (12) as well as yeast (13).

MycopZasma (pleuropneumonia-like organisms) constitute a In Streptoccus jaecalis Bauchop and Elsden (14) have shown heterogeneous group of microorganisms characterized by the that the arginine dihydrolase pathway is involved in generating absence of a rigid cell wall, and are distinguished from L forms of adenosine triphosphate for cell growth. There has been some bacteria by the lack of any known relationship to a parent bac- question as to whether this pathway also supplied ATP in M. terium (1). MycopZasma are of particular interest in being the hominis 07, since Smith (10) failed to find sufficient ornithine smallest organisms capable of free life. For instance, one Myco- transcarbamylase activity in cell extracts. In the studies of plasma strain studied extensively, Mycoplasma gallisipticum, Schimke and Barile (8), however, the suggestion was made that has been estimated to have approximately 0.5% of the deoxyribo- arginine did constitute a major energy source for Mycoplasma nucleic acid per cell of that of Escherichia coli (2). The metabolic isolated from contaminated cell cultures, because of their finding properties of Mycoplasma have not been studied extensively, of ornithine as the major metabolic product in growing cells and but in general are characterized by limited synthetic abilities, their demonstration of high activities of all three enzymes of the and extensive requirements for amino acids, purines, pyrimidines, arginine dihydrolase pathway. The present paper presents data and steroids (3-5). Energy requirements for Mycoplasrna are on this enzyme system in M. hominis 07 in support of the con- heterogeneous: certain strains can ferment a variety of carbo- clusion that this enzyme system is used for the formation of ATP. hydrates, whereas another large group of strains, containing many The results indicate that (a) extensive breakdown of arginine to Mycoplasma of human origin, does not (3, 4). The nature of ornithine occurs in growing M. hominis 07 and is compatible with the energy source of one such nonfermenting Mycoplasma strain the idea that the ATP is sufficient to support requirements for

originally isolated from human urethra (6), M. hominis type II, strain 07, constitutes the subject this paper.

Among the metabolic properties of M. hominis 07 is the conversion of arginine to citrulline in intact, resting cells as shown by Smith (7). More recently studies of Schimke and Barile (8), and Barile, Schimke, and Riggs (9) have indicated that the ability to degrade arginine is widespread among Mycoplasma, being present in 10 of 18 Mycoplasma species studied. These workers found that all of four strains of Mycoplasma isolated from contaminated cell cultures contained an active system for the degradation of arginine to citrulline and ornithine (8). The conversion of arginine to ornithine by Mycoplasma occurs by way of the so-called arginine dihydrolase pathway involving the three enzymes arginine deiminase, ornithine transcarbamylase, and carbamate kinase (8, 10) in the reactions shown below.

Arginine + Hz0 arginine deiminase

+ citrulline + NHB (1) ornithine transcarbamylase

Citrulline + Pi -p (2) . , ornithine + carbamyl phosphate

Carbamyl phosphate + ADP < carbamate kinase

, (3)

ATP + CO* + NH3

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growth; and (b) Al. hominis 07 is exceedingly rich in all three enzymes of the arginine dihydrolase pathway. Based on studies of the purification of these enzymes, it is estimated that arginine deiminase and ornithine transcarbamylase constitute 10 and 4%, respectively, of the soluble cell protein.

EXPERIMENTAL PROCEDURE

Growth and Preparation of Cells

M. hominis type II, strain 07 and Mycoplasma laidlauii A were obtained from Dr. M. Barile, Division of Biologics Stand- ards, National Institutes of Health. The cells were grown at 37” without aeration in a brain-heart infusion broth (Difco) containing 1 y0 yeast extract (Difco) and 10% horse serum. The

medium was supplemented with L-arginine (2 mg per ml) unless otherwise stated. Growth was estimated by determining turbidity in a Klett-Summerson calorimeter. Generally 10 ml of cells from a late log phase culture (1 to 2 X log clone-forming units per ml) were inoculated into 500 ml of the broth media. Under these conditions the cells attain maximal growth in 12 to 18 hours.

For the preparation of cell extracts the cells were collected by centrifugation at 15,000 x g for 10 min at 4”. The sedimented cells were suspended in 0.85% NaCl in a volume equivalent to 1% of that of the media from which the cells were obtained. Following centrifugation at 15,000 X g for 15 min at 4”, the cells were resuspended in 0.01 M potassium phosphate, pH 7.0 (1 to 2 ml of buffer for cells from each liter of medium), and exposed to sonic disruption for 10 min in a Raytheon lo-kc sonic oscillator. Each liter of medium yielded approximately 30 mg of cell protein.

Enzyme Assays

Arginine Deiminase-Arginine deiminase was assayed by measuring the rate of citrulline formation in a system containing potassium phosphate, 0.1 M, pH 6.5, and n-arginine, 0.05 M.

Incubations of suitable aliquots of enzyme in 1.0 ml of the assay medium were performed at 37” for 5 to 10 min. The reaction was stopped by the addition of 1.0 ml of 0.5 M perchloric acid to the assay tubes. Suitable aliquots were assayed for citrulline by the method of Archibald (15) as modified by Ratner (16). The assay was linear with respect to time of incubation or added enzyme from 0.01 to 4 Imoles of citrulline formed.

Ornithine Transcarbamylase-Ornithine transcarbamylase was assayed as described by Jones in the direction of citrulline synthesis (17). To assay the enzyme in the direction of ornithine formation, an arsenolysis reaction was used with ureido-“C-n- citrulline (New England Nuclear) as substrate. The rate of the reaction was measured by collecting and counting the WOZ as described previously (10). The assay medium contained 0.01 M Tris-Cl, pH 7.3; 0.05 M ureido-W-n-citrulline, specific activity, 34,000 cpm per pmole; and 0.05 M potassium arsenate.

Carbamate Kinase-Carbamate kinase was assayed in the direction of carbamyl phosphate synthesis as described by Jones (17), in which the rate of the reaction is estimated by the citrulline formed in a coupled assay containing an excess of ornithine transcarbamylase and ornithine. The assay medium contained 0.1 M Tris-Cl, 0.1 M NHaCO+ 0.005 M MgClz, 0.01 M ATP, 0.05 M n-ornithine, and 50 units of ornithine transcarbamylase, final pH 8.3. The ornithine transcarbamylase used in this assay was obtained from M. hominis 07 and was free of carbamate kinase.

For assay of carbamate kinase in the direction of .\TP synthesis, the rate of the reaction was estimated in a system in which the ATP formed was coupled by means of glucose and hexokinase to the formation of glucose g-phosphate, which in turn was detected by conversion of TPN to TPNH by means of glucose g-phosphate dehydrogenaoe. The assay medium contained 0.01 M Tris-Cl (pH 8.3), 0.01 M lithium carbamyl phosphate (Sigma), 0.005 M ADP (Pabst), 0.01 M n-glucose, 0.001 M

TPN (Sigma), 10 units of yeast hexokinase (Sigma, crystallized), and 10 units of glucose 6-phosphate dehydrogenase (Sigma, type V). The reaction rate was determined by continuous recording of the increment in optical density at 340 111~ with a Gilford recording apparatus. Assays were performed at 37” with an assay volume of 1 ml. The rates in both directions were linear with respect to time of incubation and extract concentration from 0.1 to 1.5 pmoles of product formed.

An enzyme unit is defined as the amount of enzyme that catalyzes the formation of 1 pmole of product per min.

Determination of Arginine, Omithine, and Citrulline during Growth of M. hominis W-The products of arginine metabolism during growth were determined by using tracer amounts of uniformly labeled W-n-arginine (New England Nuclear) added to the growth medium. The metabolic products of arginine metabolism, most specifically ornithine, were characterized by chromatography of protein-free extracts of growth medium on Amberlite CG-50 (NHa+) columns as described previously (8). In this procedure the basic amino acids, arginine and ornithine, are retained on the column and can be separated by gradient elution with ammonium hydroxide. Suitable aliquots from the columns were counted by liquid scintillation techniques in a scintillation spectrometer with a dioxane phosphor system (8). The radioactive peaks eluted from the Amberlite CG-50 columns were confirmed to be ornithine and arginine by descending paper chromatography in two solvent systems, phenol-ethanol-water- concentrated ammonia (75 : 20 : 9 : 1) and phenol-water (80 : 20). Total arginine in the broth medium was determined by the calorimetric method of Sakaguchi (18) as modified by van Pilsum et al. (19).

Other Procedures-Disk acrylamide gel electrophoresis was performed as described by Davis (20) in a buffer containing Tris- glycine (6 g of Tris, 28 g of glycine per liter), pH 8.3. Either the crude extracts or purified enzymes (200 pg) were subjected to electrophoresis for 30 min at 25” (5 ma per tube). Protein bands were identified by staining with 0.557, Buffalo black (napthol blue black, National Aniline Division) in 7.5% acetic acid, followed by destaining in 7.5% acetic acid at 10 ma per tube (20).

Protein was estimated by the method of Lowry et al. (21) with bovine serum albumin as standard. RNA and DNA were estimated as outlined by Schneider (22) with yeast RNA and sperm DNA (Schwarz) as standards.

RESULTS

Effect of Arginine on Growth of M. hominis Or-The importance of arginine for the metabolism of M. hominis 07 is shown in Fig. 1. In a medium consisting of brain-heart infusion, 1 y. yeast extract, and 10% horse serum, cell growth ceases at absorbance increments of 11 and 43 Klett units when the cells are grown at n-arginine concentrations of 2 and 10 mM, respectively. In these cases growth ceases when the medium is totally depleted of arginine. When additional arginine is added to a concentration


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2230 Arginine Dihydrolase Pathway in 1M. hominis 07 Vol. 241, No. 10

of 50 mM, either initially or after growth has stopped (indicated by the arrow in Fig. l), growth continues at the same rate as initially, but reaches a new plateau. In this case further growth is not limited by arginine, but rather by some unknotin factor. This effect of added arginine on the growth of M. hominis 07 is similar to that reported previously for Mycoplasma strain ERKS isolated from mammalian cell culture (8). Thus, in the experi- ment of Fig. 1 the total yields of cell protein were 1.75, 8.2, and

b3 80 50 mM ARGININE

9 E d 60 0

IO mht ARGININE E w 40 y’

! 2 mM ARGININE _

ARGININE ADDED I I, t I I I I I I Ia I I

‘0 4 8 12 16 20 24

HOURS OF INCUBATION

FIG. 1. Effect of L-arginine on growth of M. hominis 07. M. hominis 07 (5 ml, 5 to 10 X 104 clone-forming units) from a logarithmic phase culture were inoculated into 100 ml of brain-heart infusion broth containing 1% yeast extract and 10% horse serum. Growth was followed by measuring turbidity with a Klett color- imeter. The L-arginine concentrations indicated are those of the final medium before growth: O--O, 2 mM arginine; O--O, 10 mM; n----A, 50 mM; A----A, 2 mM; initially, followed by the addition of arginine to a final concentration of 50 mM at the time indicated by the arrow.

17.5 mg for cells harvested at the end of growth in 2, 10, and 50 mM n-arginine, respectively. The effect of arginine on increasing cell yield has also been a consistent finding with other Myco- plasma strains that contain the arginine dihydrolase pathway (9). On the other hand, Mycoplasma strains that do not contain the arginine dihydrolase pathway, such as M. laidlawii A (9), do not show any effect of added arginine on growth characteristics.

A number of arginine analogues were added to the unsupplemented medium at concentrations of 2 mg per ml in order to determine the specificity of the arginine effect in stimulating growth. These included n-ornithine, n-citrulline, n-lysine, L-

glutamine, n-glutamic acid, agmatine, L-homoarginine, ar- gininic acid, guanidinoproprionic acid, ureidoproprionic acid, benzoyl-n-arginine, and benzoyl-n-arginine ethyl ester. None of these compounds substituted for arginine in increasing cell yield. In view of the fact that arginine utilization may serve to generate energy for M. hominis 07, a group of potential energy sources was also added to unsupplemented medium in order to observe whether they could “spare” the arginine requirement. No compound, including glycerol, succinate, acetate, DL-fl-

hydroxybutyrate, or pyruvate, added at concentrations of 2 mg per ml, was effective in promoting growth beyond that determined by the amount of arginine present. The fact that a compound does not stimulate growth could be due to a lack of the required enzymes or to a lack of permeability. In the case of n-citrulline, the requisite enzymes are present, i.e. ornithine transcarbamylase and carbamate kinase, yet n-citrulline is in- effective in promoting growth because it does not appreciably enter the cells (7, 8).

Relationship between Degradation of Arginine and Growth Yields of M. hominis Or-During growth of M. hominis 07, 90% of the arginine utilized can be accounted for by the formation of ornithine (see Table I). This finding is consistent with the utilization of arginine for energy formation. The question may now be

TABLE 1

Relationship between ATP formation from arginine and formation of cellular constituents in M. hominis 07

M. hominis 07 was grown to the specified increments in absorb- of the trichloracetic acid with ethyl ether. Aliquots of the result- ante units during logarithmic phase growth in 110 ml of medium ing extracts were chromatographed on Amberlite CG-50 (NITI+ supplemented with W-I,-arginine. To medium originally con- form) for determination of products of arginine metabolism as taining 6.0 pmoles per ml of arginine were added either no addi- described under “Experimental Procedure.” The column desig- tional L-arginine (Flasks 1 to 3) or 9.5 pmoles per ml of L-arginine nated “Other radioactivity” refers to radioactive products of (Flasks 4 to 6). In addition, uniformly labeled W-L-arginine arginine not adsorbed on the Amberlite CG-50 column. The esti- (specific activity, 170 mC per mmole) was added to the medium to mate of ATP formed was based on the assumption that 1 mole of a final specific activity of arginine of 2800 cpm per pmole (Flasks 1 ATP was formed for each mole of ornithine found. The estimates to 3) or 950 cpm per pmole (Flasks 4 to 6). The cells were removed of ATP required for synthesis of protein, RNA, and DNA were as by centrifugation and washed twice with chilled 0.85% NaCl, dis- follows: protein, 3 moles of ATP per mole of amino acid residue; rupted by sonic disintegration, and assayed for protein, DNA, and RNA and DNA, 4 moles of ATP per mole of nucleic acid resi- RNA directly on the sonic ext,racts as described under “Experi- due. The average amino acid and nucleic acid base compositions mental Procedure.” To the cell-free media was added solid tri- of protein, RNA, and DNA were taken from the results of Moro- chloracetic acid to a final concentration of 10%. The precipitated wit2 et al. (2). protein was removed by centrifugation followed by the extraction

Flask A A (KM)

1 No cells

2 14

3 10

4 18 5 24 6 33

T-

-

DNA RNA Protein

0.85 1.87 0.97 1.70

1.66 3.60

1.78 3.12 3.11 7.05

O 644 0 202 418 25 207 410 21

303 1080 43 336 1020 61 755 672 70

202 I 58 207 56

303 108 336 108 755 204

3.5 3.9

2.8 3.4 3.7


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asked whether sufficient arginine is degraded to account for the energy requirement for synthesis of cellular constituents. To answer this question, M. hominis 07 was grown in medium containing uniformly labeled ‘%-L-arginine. Cells were harvested at various increments in absorbance during logarithmic phase growth, and the yields of cellular DNA, RNA, and protein were determined, as well as the amount of arginine converted to ornithine. The cells were all harvested during logarithmic phase growth because, as shown previously for another Myco- plasma strain, arginine is converted largely to ornithine (Table I) only during rapid growth. When growth ceases in the presence of excess arginine, on the other hand, arginine degradation continues, but citrulline, rather than ornithine, is the predominant product (8). With these determinations and certain estimates as to the amount of ATP required for the synthesis of cellular constituents, a comparison of the amount of ATP required and the amount of ATP formed was made. These results and specific experimental details are shown in Table I. The major metabolic product of arginine degradation during growth was ornithine. In addition, approximately 10 to 15% of the radioactivity present as ornithine was recoverable in material not adsorbed on the Amberlite CG-50 column. This radioactivity presumably represents more acidic metabolic products. It was assumed that for each mole of ornithine formed, 1 mole of ATP was generated (see Reaction 3). The amount of ATP required for the growth of the cells was estimated on the basis of the following considerations. (a) Because of the extensive nutritional requirements of Mycoplasma (3-5) and the rich growth medium employed, no energy was required for the synthesis of amino acids, purines, pyrimidines, and carbohydrates. (b) Although the exact energy requirements for macromolecule synthesis are unknown, we have set them at 3 ATP molecules for the transport and insertion of each amino acid into peptide linkage and 4 ATP molecules for the transport and insertion of each nucleic acid residue into RNA or DNA. (c) The ATP requirements for other constituents, such as cell wall, have not been included, since we have found that the protein, RNA, and DNA constitute 87 to 95yo of the dry weight of M. hominis 07. We thus assume that the energy necessary for the synthesis of protein, RNA, and DNA constitutes the major ATP requirements for the cells during growth.

Based on the above considerations, it was found that there was a consistent a-fold excess of ATP formed (2.8 to 3.9) over that estimated as necessary for macromolecule synthesis (Table I). Obviously the estimated requirements for ATP do not take into account many unknown requirements. Nevertheless these results indicate that in all likelihood a sufficient amount of arginine is degraded to account for the energy requirements for cell growth.

In addition to the metabolism of arginine, glutamine has been shown to be rapidly metabolized to glutamic acid by extracts of human Mycoplusma (10). Smith has demonstrated a coupling of this reaction to the formation of ATP by a reversal of a glutamine synthetase reaction (23). However, glutamine would not appear to be a likely source of energy in M. hominis 07 for the following reasons. (a) Glutamine is not converted to glutamic acid by growing cells. Thus when the medium is supplemented with 2 mg per ml of L-glutamine with or without supplemental arginine, virtually no conversion of glutamine to glutamic acid can be demonstrated by paper chromatography in the presence of growing cells (solvent system, phenol-ethanol-water-concen-

trated ammonia, 75 :20 :9 : 1). (b) Glutamine does not affect the growth properties as does arginine. (c) Supplementation of medium with glutamine does not affect the conversion of arginine to ornithine as determined in experiments similar to those described in Table I.

Studies on Properties of Arginine Deiminase, Ornithine Transcarbabamylase, and Carbamate Kinase of

M. hominis 07

Extracts of M. hominis 07 contain extremely high activities of the three enzymes comprising the arginine dihydrolase pathway. The specific activities of the three enzymes in the crude extracts were as follows: arginine deiminase, 5.2 to 6.8 units per mg of protein; ornithine transcarbamylase, 142 to 192 units per mg of protein (measured in direction of citrulline formation); and carbamate kinase, 3.0 to 4.0 units per mg of protein (measured in direction of carbamyl phosphate formation).

Growth of the cells in the presence of 2 mg per ml of DL-P-

hydroxybutyrate, glycerol, succinate, pyruvate, or acetate had no effect on the specific activities of the three enzymes, whether growth occurred under aerobic or anaerobic conditions. The specific activities of the three enzymes were also constant throughout the logarithmic phase of growth. However, cells maintained in stationary phase for 4 or more hours rapidly lost carbamate kinase and ornithine transcarbamylase activities, and, to a much lesser extent, arginine deiminase activity.

When crude extracts of Al. hominis 07 were subjected to disk electrophoresis in acrylamide gel, a striking finding was the

presence of an extremely thick protein band or bands at approximately 2 cm, and the presence of few other discrete bands (Fig. 2). This pattern is to be contrasted with the multiple, discrete protein bands found with extracts of M. laidlawii A, a Myco- plasma strain that does not degrade arginine and does not contain the arginine dihydrolase pathway (9). When a companion gel of an extract of strain 07 was cut into 0.5-cm sections, homogenized, and assayed for arginine deiminase, ornithine transcarbamylase, and carbamate kinase, it was evident (Fig. 2) that the thick protein band or bands contained both the arginine deiminase and carbamate kinase activities. Ornithine transcarbamylase did not appreciatively enter the gel. These findings, as will be documented further below, suggested that these enzymes constitute major components of the cell protein of J4. hominis 07.

PuriJication Procedures

Arginine deiminase, ornithine transcarbamylase, and carbamate kinase were purified from extracts of M. hominis 07 by the following procedures. All procedures were performed at 24” unless stated otherwise. The cells were harvested, washed, and sonically disrupted as described in “Experimental Procedure.” The crude sonic extract was centrifuged at 35,000 X g for 20 min. To the resulting extract, containing 15 to 20 mg of protein per ml, was added protamine sulfate (Nutritional Biochemicals) in a proportion of 1 mg of protamine sulfate per 15 mg of protein. The resulting precipitate was removed by centrifugation at 15,000 x g for 10 min. The supernatant fluid contained all three enzyme activities.

Ammonium Sulfate Precipitation

The extract resulting from the protamine precipitation was subjected to ammonium sulfate fractionation. Solid ammonium


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1

I All% AW

FIG. 2. Disk electrophoresis of M. hominis 07 extract. Ex- tracts of M. hominis 07 and M. laidawii A produced by sonic disruption were subjected to disk electrophoresis on acrylamide gel as described under “Experimental Procedure.” Protein (200 pg) was applied to each gel. One gel with an extract of M. hominis 07 was stained for protein. Another gel was cut into 0.5cm seg- ments, each of which was homogenized in 1.0 ml of 0.1 M potassium phosphate, pH 7.0, and aliquots were assayed for arginine deiminase, ornithine transcsrbamylase, and carbamate kinase as described under “Experimental Procedure.”

TABLE II Purification of carbamate kinase from M. hominis 07

The cells from 20 liters of medium were used for this preparation. Assays were performed in the direction of carbamyl phosphate synthesis.

step

Extract................... Protamine................ Ammonium sulfate (50-

6.5%) . Heat..................... DEAE-cellulose chroma-

tography . Calcium hydroxylapatite. Ammonium sulfate..

-

Total protein

m 2038 1839

units

8ooo 7080

units/mg 3.9 3.8

% 100 89

904 5670 6.3 71 303 5120 17 64

193 4310 22 54 72 3750 52 47 29 2106 73 26

Total Specific enzylpe actwty

enzyye actnnty

Yield

sulfate (enzyme grade, Mann) was added to the extract with a maintenance of a neutral pH by addition of ammonium hydroxide (24). Carbamate kinase activity was present largely in the protein fraction precipitated with ammonium sulfate at 50 to 60% of saturation. Arginine deiminase and ornithine trans-

carbamylase activities, on the other hand, were largely present in the protein fraction precipitated with ammonium sulfate at 65 to 80% of saturation.

Carbamate Kinase

The fraction containing carbamate kinase activity was dissolved in 0.01 M potassium phosphate, pH 7.0, to a final protein concentration of 20 mg per ml.

Heat Step-The solution was heated at 60” for 5 min and then rapidly cooled in an ice bath. The large amount of precipitate was removed by centrifugation. The clear supernatant solution was equilibrated with 0.01 M potassium phosphate, pH 7.0, containing 0.005 M 2-mercaptoethanol by passage through a Sephadex G-25 (bead form) column previously equilibrated with this solution. The 2-mercaptoethanol was incorporated to stabilize enzyme activity. When mercaptoethanol wss used in the heat step, however, virtually complete inactivation of enzyme activity occurred. All subsequent steps were performed in the presence of 0.005 M 2-mercaptoethanol.

DEAE-cellulose Chromatography-A column (1.5 X 20 cm) of DEAE-cellulose (25) was equilibrated with 0.01 M potassium phosphate at pH 7.0. After the enzyme solution from the heat step was applied, the column was washed with 50 ml of the equilibrating buffer and then developed with a linear gradient of KC1 from 0 to 0.5 M in the above phosphate buffer (total volume, 200 ml), at a flow rate of 60 ml per hour. Carbamate kinase was eluted in 15 to 20 ml of eluate at a KC1 concentration of approximately 0.3 M. The fractions containing enzyme activity were combined, concentrated by ultrafiltration, and thereafter dialyzed for 24 hours against 0.001 M potassium phosphate, pH 7.0.

Hydroxylapatite Chromatography-A column (0.75 X 10 cm) of calcium hydroxylapatite (Bio-Gel HT, Bio-Bad) was equilibrated with 0.001 M potassium phosphate at pH 7.0. After the dialyzed enzyme solution wss applied to the column, the column was washed with 20 ml of the equilibrating solution, and was developed with a linear gradient of potassium phosphate from 0.001 to 0.25 M at pH 7.0 (total volume, 100 ml). The flow rate was 2.5 ml per hour. Carbamate kinase was eluted in a volume of 15 ml at a potassium phosphate molarity of approximately 0.175 M. The fractions containing the enzyme activity were pooled and concentrated by ultrafiltration to a volume of 4 to 5 ml.

Ammonium Sulfate Precipitation--The concentrated enzyme solution was adjusted to a protein concentration of 5 mg per ml and subjected to ammonium sulfate precipitation with a satu- rated ammonium sulfate solution at pH 7.0. The enzyme was precipitated in the fraction at 60 to 70% of ammonium sulfate saturation.

Table II gives a representative purification of carbamate kinase. Purifications of 25-fold with 20 to 30% yields were readily obtained.

Ornithine Transcarbamylase

Heat-The protamine-treated extract described previously was heated at 60’ for 5 min and then cooled in an ice bath. The resulting precipitate was removed by centrifugation at 15,000 x g for 10 min.

Ammonium Sulfate Fractionation-The heated extract was subjected to fractionation with ammonium sulfate. Both ornithine transcarbamylase and arginine deiminase activities are


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present in the protein fraction precipitating between 65 and 80% of ammonium sulfate saturation. This protein fraction was dissolved in 5 to 10 ml of 0.01 M potassium phosphate at pH 7.0, and the solution was freed of residual ammonium sulfate by passage through a Sephadex G-25 column equilibrated with the above buffer.

DEAE-cellulose Chromatography-A column (1.5 X 20 cm) of DEAE-cellulose was equilibrated with 0.01 M potassium phosphate at pH 7.0. The enzyme solution (10 to 15 ml) was applied to the column, followed by 50 ml of the equilibrating buffer. The ornithine transcarbamylase was not generally retained on the column, and thereby could be separated from the arginine deiminase activity. However, on several occasions ornithine transcarbamylase activity was retained on the DEAE-cellulose, and, when eluted by a KC1 gradient (see “Arginine Deiminase” below), was contaminated with arginine deiminase. This con- tamination was removed during the subsequent chromatography on carboxymethyl cellulose.

Carboxymethyl Cellulose Chromatography-A column (1.5 X 30 cm) of carboxymethyl cellulose was equilibrated with potassium phosphate, 0.01 M, pH 6.4. The enzyme solution obtained as the nonadsorbed fraction from the prior DEAE-cellulose chromatography (see above) was applied to the column. The column was then washed with 50 ml of the equilibrating solution followed by 300 ml of a linear KC1 gradient from 0 to 1.0 M.

The flow rate of the column was 60 ml per hour. Ornithine transcarbamylase was eluted in 20 to 25 ml at approximately 0.4 M KCl. The fractions containing ornithine transcarbamylase activity were combined and the solution was concentrated by ultrafiltration to a volume of 2 to 3 ml.

Sephadex G-200 Chromatography-A column (1.5 X 40 cm) of Sephadex G-200 was equilibrated with 0.1 M potassium phosphate at pH 7.0. The enzyme solution (2 to 3 ml) was applied to the top of the column. The column was developed with the equilibrating buffer at a flow rate of 2 to 3 ml per hour. Omi- thine transcarbamylase was eluted in the void volume.

A representative purification of ornithine transcarbamylase is shown in Table III.

Arginine Deiminase

The initial purification steps involving protamine sulfate, heat, and ammonium sulfate precipitation are similar to those used for ornithine transcarbamylase purification as described above.

DEAE-cellulose Chromatography-The dimensions and applica- tion of the solution of this column are those described in the purification of ornithine transcarbamylase (see above). Follow- ing the washing procedure, the column was developed with 300 ml of a linear gradient of KC1 from 0 to 1.0 M in 0.01 M potassium phosphate, pH 7.0, at a flow rate of 60 ml per hour. Arginine deiminase was eluted in 30 to 40 ml at a KC1 concentration of approximately 0.6 M. The fractions containing arginine deiminase were pooled and concentrated by ultrafiltration to a volume of 4 to 8 ml. Any ornithine transcarbamylase present in these fractions was removed on the subsequent Sephadex G-200 chromatography.

Sephadex G-200 Chromatography-A column (1.5 X 40 cm) of Sephadex G-200 was equilibrated with 0.1 M potassium phosphate, pH 7.0. To this column were applied 2 to 5 ml of the concentrated arginine deiminase obtained from the DEAE- cellulose column. The column was washed with the equilibrat-

TABLE III

Purifzcation of ornithine transcarbamylase from M. hominis 07

The cells from 5 liters of media were used for this preparation. Assays were performed in the direction of citrulline synthesis.

step

Extract................... Protamine................ Heat (-60”). Ammonium sulfate (65

80~~) . DEAF-cellulose chroma.

tography Carboxymethyl cellulose

chromatography. Sephadex G-200 chroma-

tography

Total protein

Total enzyme activity

Specific enzpe act1wty

Yield

mg units u?ds/mg % 320 74,500 234 100 265 67,300 256 90 113 55,400 490 75

63 61,500

13.4 54,200

7.5

6.0 38,000

41,060

975

4,050

5,500

6,300

82

73

55

51

TABLE IV

Purification of arginine deiminase from M. hominis 07

The cell yield from 15 liters of medium was used for this prepa-

ration.

step

Extract...................... Protamine................... Heat Ammonium sulfate (6@30yc’,). DEAE-cellulose chromatog-

raphy Sephadex G-200 chromatog-

raphy

Total protein

w unils

604 3240 325 2880

190 2260 90 2520

37.5

30

1820 48 56

1600 53 49

5 8

11 28

Yield

% 100

89 70 78

ing solution. Arginine deiminase was eluted in 20 to 50 ml, starting at approximately 150 ml of eluting solution. The fractions containing arginine deiminase were concentrated by ultrafiltration.

Table IV shows a representative purification of arginine deiminase.

Properties of PuriJied Enzymes

Carbamate Kinase

The best preparations of carbamate kinase were purified 25- fold over starting extracts (Table II). Such preparations were free of arginine deiminase and ornithine transcarbamylase. The absorbance ratio, AY,.w:Aw, of the preparation was 1.65. Enzyme activity was stable at 4” in the presence of mercaptoethanol for up to 2 weeks, even when the concentration of protein was as little as 0.01 mg per ml. Neither a crude extract of M. hominis 07 nor the purified enzyme was capable of using glutamine as the amino donor for carbamyl phosphate synthesis, a reaction first demonstrated in extracts of mushroom by Leven- berg (26). In addition, acetokinase, an enzyme activity capable of carbamyl phosphate synthesis from ATP and carbamate (27), was not demonstrable in either crude extracts or purified enzyme


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2234 Arginine Dihydrolase Pathway in M. hominis 07 Vol. 241, No. 10

when assayed as described by Rose tt al. (28) within a lower limit of 2% of the rate of carbamyl phosphate synthesis.

The pH optimum of the reaction as determined in 0.2 M Tris-Cl buffer was 8.3 to 8.5 in both the direction of carbamyl phosphate synthesis and the direction of ATP synthesis. The K, for ATP, as determined at pH 8.3 in the presence of the concentrations of the other reactants as listed under “Experimental Procedure,” w&s 1.4 x lo-3 M. The apparent K,,, for ADP in the reverse direction was 2.0 x lo-’ M. Kalman and Duffield (29) reported a K,,, for ADP of 1 X lo-3 M for an enzyme purified from S. faecalis. However, their assays were performed at pH 5.0. The effect of varying the NH,HCOa concentration on the reaction velocity was similar to that described by Kalman and Duflield for the Streptococcus enzyme (29). Maximal activity was obtained with concentrations of 0.05 to 0.1 M ammonium bicarbonate. Marked substrate inhibition occurred at higher ammonium bicarbonate concentrations. Carbamyl phosphate, on the other hand, had a high affinity for the enzyme when the reaction was assayed in the direction of ATP synthesis. A K, of 2 x 10m6 M for carbamyl phosphate was found. The maximal reaction velocity obtained in the direction of ATP synthesis was 5 to 10 times that of the velocity in the direction of carbamyl phosphate synthesis, a finding similar to that described by Caravaca and Grisolia (30) for a Streptococcus enzyme.

An estimate of the molecular weight of the Mycoplasma carbamate kinase was made by the sucrose density gradient technique of Martin and Ames (31). A value of 61,000 was found with the use of arginine deiminase with a molecular weight of 78,000 (see below) as the standard.

Disk gel electrophoresis of 25-fold purified preparations of carbamate kinase indicated the presence of two protein bands of essentially equal staining intensity. One of these bands contained carbamate kinase activity and corresponded in migratory

FIG. 3 (left). Ultracentrifugal schlieren diagram of ornithine transcarbamylase isolated from M. hominis 07. Ornithine transcarbamylase (specific activity, 250 units per mg of protein), 5.7 mg per ml in 0.07 M potassium phosphate, pH 7.0, was sedimented in a Spinco model E analytical centrifuge-at 25”. Sedimentation is from left to right. The photograph was made 25 min after at- taining a speed of 59,780 rpm.

FIG. 4 (right). Ultracentrifugal schlieren diagram of arginine deiminase isolated from M. hominis 07. Arginine deiminase (specific activity, 52.8 units per mg of protein), 5.8 mg of protein per ml, in 0.05 M potassium phosphate, pH 7.0, was sedimented in a Sninco model E analvtical centrifuge at 25”. Sedimentation is from left to right. The”photograph W&I made 67 min after attain- ing a speed of 59,780 rpm.

properties to that band containing the enzyme activity as observed in crude extracts (Fig. 2). On the basis of these findings, it is estimated that the maximal limit of purity of the carbamate kinase preparation would be approximately 50%. It is of interest that the carbamate kinase purified from S. jaecalis by Kalman and Duffield (29) had a final specific activity twice that of the enzyme described here. Their enzyme was homogeneous by the criteria of velocity sedimentation in an analytical ultracentrifuge and gel electrophoresis (29).

Ornithine Transcarbamylase

The preparations of ornithine transcarbamylase were purified approximately 25-fold over crude extracts. The specific activity of the final preparations was 3- to B-fold greater than that described by Ravel et al. (32) for an ornithine transcarbamylase purified from Streptococcus la&is grown with arginine as the energy source. The absorbance ratio, A280:A260, of the preparation was 1.75. Ornithine transcarbamylase was stable at 4” for at least 3 weeks without loss of activity.

The pH optimum measured in the direction of citrulline formation was 8.4 in 0.1 M Tris-Cl. The pH optimum measured in the direction of carbamyl phosphate formation was extremely sharp at 7.3 in 0.1 M Tris-Cl. The K,,, for carbamyl phosphate was 2.3 X 10-a M at pH 8.4, and that for L-ornithine was 3.6 X 10-S M at pH 8.4. These K,,, values are similar to those reported by Ravel et al. (32) for the S. Zactis enzyme. The K, for citrulline, as determined in the direction of carbamyl phosphate synthesis at pH 7.3 by an arsenolysis reaction (see “Experimental Proce- dure”), was approximately 4 to 7 X lop4 M. The TI,.,: measured in the direction of citrulline synthesis at pH 8.4 was approximately 10 times that found for the reaction in the direction of carbamyl phosphate synthesis in an arsenolysis reaction performed at pH 7.3.

The purified preparations of ornithine transcarbamylase appeared to be essentially homogeneous as determined by ultracentrifuge analysis. Fig. 3 presents an ultracentrifugal pattern of an ornithine transcarbamylase preparation. A sedimentation constant of 16.6 X lo-l3 at 25” in 0.05 M potassium phosphate, pH 7.0, was obtained. A similar sedimentation constant was calculated for the enzyme activity in a sucrose gradient centrifugation by the method of Martin and Ames (31). The large size of the molecule (estimated molecular weight of 360,000) pre- cluded extensive migration in acrylamide gel (see Fig. 2). How- ever, both anodal and cathodal electrophoresis of purified preparations of ornithine transcarbamylase failed to reveal any protein bands that penetrated into the gel. The large molecular weight of the enzyme is to be contrasted with the estimated value of 60,000 for an ornithine transcarbamylase purified from E. coli by Rogers and Novelli (33).

Assuming that enzyme purified 25-fold results in a homogeneous preparation, it follows that approximately 4% of the protein of M. hominis 07 is ornithine transcarbamylase.

Arginine Deiminase

The preparations of arginine deiminase were purified 9- to lo-fold over crude extracts and contained no carbamate kinase or ornithine transcarbamylase activity. The final specific activity, 53 units per mg of protein, is higher than the specific activity of 41 units per mg obtained by Petrack, Sullivan, and Ratner (34) for an arginine deiminase purified 60-fold from S.


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Issue of May 25, 1966 Schimlce, Berlin, Sweeney, and Carroll 2235

faecalis. The absorbance ratio, Aa0:At6,,, was 1.65 to 1.75. Arginine deiminase could be stored at 4” for at least 4 weeks without loss of activity. The pH optimum was 6.5 to 6.7 in 0.1 M potassium phosphate. The K, for arginine was estimated at 1 to 4 X 10e4 M. No formation of arginine from ammonia and citrulline could be demonstrated over a wide pH range. These properties are similar to those of the arginine deiminase purified by Petrack, Sullivan, and Ratner (34) from S. fuecalis.

Preparations of arginine deiminase purified 9- to lo-fold were essentially homogeneous by the criteria of velocity ultracentrifu- gation (Fig. 4), free diffusion, and gel electrophoresis. The electrophoretic migration of the isolated protein was identical with that of the band of active enzyme observed in crude extracts (Fig. 2). Four velocity ultracentrifuge runs at enzyme concentrations ranging from 1.3 to 9.3 mg per ml showed no significant concentration dependence and gave an average sedimentation constant of 5.65 X lo-l3 at 25” in 0.05 M potassium phosphate buffer, pH 7.0. A single free diffusion measurement under the same conditions was made in the Aminco apparatus and analyzed by the Longsworth procedure (35). There was no appreciable concentration dependence, and the diffusion constant was 6.75 X 10m7 (25”, phosphate buffer). I f a partial specific volume of 0.73 is assumed, a molecular weight of 78,300 is given by these values of s and D.

Assuming that enzyme purified lo-fold results in a homogeneous preparation, arginine deiminase appears to constitute 10% of the cell protein of M. hominis 07.

DISCUSSION

The results presented in this paper indicate that M. hominis 07 obtains energy from the degradation of arginine by the arginine dihydrolase pathway. Both the growth experiments and the studies with the individual enzymes of the pathway support this conclusion. Thus growth in a rich medium was limited by the arginine content (Fig. I), a limitation not overcome by a variety of arginine analogues and other amino acids, as well as other potential energy sources. In addition, the amount of arginine converted to ornithine was sufficient to account for the estimated energy requirements for synthesis of cellular macromolecules (Table I). Finally, the extremely high activities of the arginine dihydrolase pathway enzymes, arginine deiminase, ornithine transcarbamylase, and carbamate kinase, suggest that this pathway plays an important role in the microorganism. In originally describing the reactions of the arginine dihydrolase pathway in 111. hominis 07, Smith (10) concluded that arginine breakdown could not supply sufficient energy for growth on the basis of his finding low activities of ornithine transcarbamylase in cell extracts. The data with growing cells (Table I) indicate that the reaction does occur. It is suggested that Smith’s inability to demonstrate ornithine transcarbamylase may be related to harvesting cells beyond t’he log phase, a time when we have found ornithine transcarbamylase activity to be dimin- ished markedly.

The finding of ornithine as the major product of arginine metabolism in growing cultures of M. hominis 07 is to be contrasted with the finding of Smith (7) that in resting cultures citrulline is the predominant product of arginine metabolism. This difference in reaction product can be explained by the constant formation of ADP from ATP during cellular growth, which thereby allows for the continual utilization of carbamyl phosphate. Thus, because the equilibrium of the ornithine

transcarbamylase reaction strongly favors citrulline formation (36), the continued formation of ornithine could occur only if the products of the reaction, i.e. carbamyl phosphate or ornithine, or both, are continually removed from the reaction site. The effect of growth conditions on the specific product of arginine metabolism is similar to that observed by Schimke and Barile (8) with a Mycoplasma strain isolated from contaminated tissue culture.

Although the results presented suggest that arginine supplies a major part of the energy required for the growth of M. hominis 07, it cannot be stated that arginine serves as the only energy source. On the basis of demonstrating some oxidation of short chain fatty acids by nonfermenting Mycopksma (37), it has been suggested that short chain fatty acids may be potential energy sources in some instances (4). M. hominis 07 also contain certain enzymes of the tricarboxylic acid cycle (38), although the activities are quite low. In the studies presented herein, however, whether because of lack of requisite enzymes or because of lack of permeability, a series of compounds, including pyruvate, succinate, glycerol, and /3-hydroxybutyrate, did not stimulate growth, as opposed to the effect of the addition of arginine. Thus, at the present time the degradation of arginine stands out as the major metabolic process known to be functioning in the generation of ATP in M. hominis 07.

Extracts of M. hominis 07 are exceedingly rich in all three enzymes of the arginine dihydrolase pathway. Growth of the microorganism in medium containing a variety of potential energy sources did not affect the specific activities of the enzymes, indicating that no catabolic repression phenomenon occurs with these enzymes (39). In view of the fact that the cells do not grow in the absence of arginine, it is not possible to determine whether this pathway is constitutive or inducible. Based on the conclusion that the purified preparations of arginine deiminase and ornithine transcarbamylase are essentially homogeneous, arginine deiminase and ornithine transcarbamylase would appear to constitute 10 and 4%, respectively, of the total soluble protein of M. hominis 07. The kinetic properties of the three enzymes are generally consistent with their functioning in the direction of ATP synthesis. Thus arginine deiminase catalyzes an essentially irreversible reaction (34). The reversible carbamate kinase reaction favors ATP synthesis, just as with the Xtreptococcus enzyme (40). Of particular interest are the findings that the K, values of the reactants in the direction of ATP synthesis are greater by an order of magnitude than those in the opposite direction for both ornithine transcarbamylase and carbamate kinase. This finding is particularly noteworthy for carbamate kinase with respect to the effects of substrate concentrations on the reaction velocity. Thus, the K, for carbamyl phosphate in the direction of ATP synthesis is 2 x 10e5 M,

whereas ammonium bicarbonate concentrations of the order of lo-2 M are required for measurable activity in the opposite direction. This finding, as well as the occurrence of carbamate kinase in association with arginine deiminase and ornithine transcarbamylase in both Mycoplasma (8, 9) and Streptococcus (12, 17), suggests that carbamate kinase functions physiologically in the generation of ATP rather than in the formation of carbamyl phosphate.

The suggested physiological role of carbamate kinase of Myco- plasma and Streptococcus in the generation of ATP from carbamyl phosphate may be further supported by contrasting its properties with those of enzymes from other sources capable of carbamyl


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2236 Arginine Dihydrolase Pathway in M. hominis 07 Vol. 241, ic’o. 10

phosphate synthesis. Thus the carbamyl phosphate synthetase in livers of ureotelic animals catalyzes an irreversible formation of carbamyl phosphate and requires acetylglutamate as a co- factor (12). Another enzyme, first described by Levenberg in mushroom extracts (26), renuires glutamine as the amino donor, and also catalyzes an essemially irreversible reaction. The glutamine-dependent carbamyl phosphate synthetase has now been described in E’. coli (41). Recent genetic evidence would suggest that it is responsible for the carbamyl phosphate utilized in pyrimidine and arginine biosynthesis (42). A third enzyme, acetokinase, also present in E. coli is capable of catalyzing the formation of ATP from carbamyl phosphate and ADP, although at 5% of the rate of formation of ATP from acetyl phosphate

(27). The presence of the arginine dihydrolase pathway is not limited

among Mycoplasma to M. hominis 07. Barile and Schimke (43) have recently surveyed 18 different species of Mycoplasma and have found that 10 contain the enzymes of this pathway. Such a widespread occurrence of a pathway among a specific group of microorganisms is to be contrasted with its lack in animal tissues and its limited distribution in bacteria (12). Such a finding may suggest a common origin for a number of J4ycopZasma species. According to one theory, Mycoplasma originate from parent bacteria by the irreversible loss of certain metabolic properties, including the ability to synthesize cell wall constituents (5). In view of the small amount of DNA per Mycoplasma cell’ (2), this loss presumably also involves loss of DNA from the chromo- some. The marked similarities in properties of the enzymes of the arginine dihydrolase pathway of M. hominis 07 and those of Streptococcus as discussed under “Results” suggest that a Strepto- coccus would seem a likely “parent” of Mycoplasma containing this pathway (8).

The reason for the relatively unique and extensive utilization of arginine by such a large number of Mycoplasma is unknown. A possible suggestion for this finding might be sought in the uniquely small size and limited DNA content of Mycoplasma. Thus, with the arginine dihydrolase pathway only three enzymes are required for the synthesis of ATP, as compared with the generally far greater number of enzymes required for fermenta- tion and oxidative metabolism. Thus, if physiological conditions involved in growth of Mycoplasma in cells and body fluids were such as to impart survival value to an organism with small dimensions and a small DNA content, then an organism with a functioning arginine dihydrolase pathway, with the necessity of coding for only three enzymes for energy metabolism, might have a greater chance of survival.

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Robert T. Schimke, C. M. Berlin, E. W. Sweeney and William R. Carroll 07Mycoplasma hominis

The Generation of Energy by the Arginine Dihydrolase Pathway in

1966, 241:2228-2236.J. Biol. Chem.

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