THE Jomsar. OF RIOLOGIC~I, CHEMISTRY Tol. 218, So. 20, Issue of October 2.5, pp. 6136-6143, 1971

Printed in U.S.A.

D-Alanine : Membrane Acceptor Ligase from Lactobacihs casei*

(Received for publication, May 24, 1971)

VICTOR 11. I~EUSCH, JR.,$ AND FRANCIS C. NEUHAUS$

From the Biochemistry Division, Department of Chemistry, Northwestern University, Evanston, Illinois 60~01

SUMMARY

The enzymatic incorporation of D-alanine into membranes of Lacfobacillus casei (ATCC 7469) is described. The activity is dependent on ATP, supernatant fraction and membrane fragments; it is enhanced by the addition of Mg2+. The incorporation is insensitive to ribonuclease. The product of this reaction is characterized as a hydroxylamine (1 M, pH 7.0, 37”) labile ester that is not extracted into lipophilic solvents. The lability of the incorporated D-alanine in hydroxylamine is similar to that observed for the D-alanine ester residues in the glycerol teichoic acid from this organism. The chromatographic properties of the product are not con- sistent with alanyl phosphatidylglycerol. The K, for D-alanine and D-ol-NH2-n-butyric acid is 18 pM and 850 pM,

respectively. L-[r*C]Alanine is not incorporated. The fol- lowing analogues are effective inhibitors of D-alanine in- corporation: (a) D-(r-NH2-n-butyric acid; (b) DL-alanine hydroxamate; (c) DL-oc-amino-n-butyric acid hydroxamate; (d) DL-alanine amide; and (e) D-serine. Although the speci- ficity profile is similar to that described for the D-alanine- activating enzyme (BADDILEY, J., AND NEUHAUS, F. C., Bio-

them. J., 75, 579 (1960)), the supernatant factor was par- tially separated from this enzyme and alanine racemase by gel filtration on Sephadex G-150. It is proposed that the supernatant fraction contains an enzyme, n-alanine :mem- brane acceptor ligase, that is involved in the introduction of o-alanine ester residues into membranes of this organism.

Teichoic acids are polymers of polyolphosphate found in Grarn-positive bacteria (1). They may be associated either xvi-it11 the cell wall or with the plasma membrane. The most

* This work was supported in part by Grant AI-04615 from the National Institute of Allergy and Infectious Diseases, by Public Health Service Training Grant 5Tl-GM-626, and by Grant HE- 11119 from the National Heart Institute. It is taken in part from a thesis submitted bv V. M. R. in martial fulfillment of the re- ouirements for the degree of Doctor of Philosophy from North- \i-estern University. -

t Sunaorted in Dart bv Public IIealth Service Predoctoral Fellowship FM-44945 from the Institute of General Medical Sci- ences.

5 Supported in part by United States Public Health Service Research Career Development Program Award I-K3-AI-6950 from the National Institute of &4llergy and Infectious Diseases. To whom reprint requests should be sent.

common teichoic acids are polyribitol phosphate and poly- glycerol phosphate, which are often modified by carbohydrates and residues of ester-linked n-alanine.

The biosynthesis of polyglycerol phosphate and polyribitol phospha,te by membrane-associated enzymes has been described (2-4). The incorporation of carbohydrate side chains into these polyolphosphates in vitro has been achieved (5-7). In addition, the biosynthesis of related polymers has also been observed (8-10). However, the enzymatic introduction in vitro of D-

alanine ester residues into polyolphosphate polymers has not been reported. All attempts in our laboratory to demonstrate the incorporation of n-alanine into isolated teichoic acid have been unsuccessful. It was therefore proposed that n-alanine might be incorporated into teichoic acid only when the polymer is intimately associated with the membrane in a particular conformation or environment.

In 1960, Baddiley and Neuhaus (11) described an activating enzyme for n-alanine and proposed that this enzyme might function in the biosynthesis of wall components. It was suh- sequently shown that the n-alanine-activating enzyme is not involved in peptidoglycan synthesis. Since this enzyme cata- lyzes the formation of an enzyme-bound activated n-alanine, it was suggested that it. might function in the biosynthesis of teichoic acid (12).

It is the purpose of this communication to define the reaction that is responsible for the incorporation of n-alanine into mem- branes of Lactobacillus casei. L. casei (ATCC 7469) was chosen for this investigation because it contains a high concentration of the n-alanine-activating enzyme (11). In addition, the plasrna membranes from this organism contain a well characterized teichoic acid that is a linear polyglycerol phosphate with D-

alanine esterified at position 2 of the glycerol moiety (13). The incorporation of n-alanine into membranes from this organism requires a supernatant fraction and ATP. The name n-alanine: membrane acceptor ligase is proposed for the enzyme in the supernatant fraction. 9 preliminary report of this work has been presented (14).

EXPERIMENTAL PROCEDURE

Materials-n-[14C]Alanine (uniformly labeled) of specific ac- tivities ranging between 32.3 and 60 mCi per mmole and tet- rasodium [32P]pyrophosphate were purchased from Amersham- Searle. Q4C]Alanine and n-[14C]alanine of specific activity 13.6 mCi per mmole were the products of New England Nuclear. DL-a-Amino-n-[‘4C]butyric acid of specific activity 8.4 mCi per mmole was the product of Calatomic. The D and L isomers of alanine and nn-cr-amino-n-butyric acid were purchased from

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Calbiochem and Cycle Chemicals. D- and L-Serine, DL-a- fragments (60 to 100 pg), and 50 m&I sodium maleate, pH 6.5, in amino-n-butyric acid hydroxamate, and nL-alanine hydroxamate a total volume of 50 ~1. The reaction mixture was incubated were obtained from Sigma. We are indebted to Mr. Roger at 37” for 30 min in a stoppered tube. The reaction was termi- Harned of Commercial Solvents Corporation for a generous nated by the addition of 20 volumes of ice-cold buffer (5 ml1 sample of n-cycloserine. m-Alanine amide was synthesized by sodium maleate, pH 6.5, and 10 rnM MgCIZ). The content’s of the ammonolysis of m-alanine methyl ester and purified by the reaction mixture were transferred to a 0.45 p membrane paper electrophoresis. In addition, nucleoside mono-, di-, and filter, and the filter was washed with 10 ml of the above buffer triphosphates were purchased from Sigma. Sephadex G-25, at room temperature. The damp membrane filter and labeled G-100, and G-150 were obtained from Pharmacia. Membrane membranes were dissolved in 1.0 ml of ethyl acetate by incubat- filters (MF type) and the appropriate filter holder apparatus ing the filter for 30 min at 37” before the addition of 15 ml of were the products of Millipore. The plastic beads (styrene- Triton-toluene scintillation mixture. divinyl benzene copolymer, 20 to 50 mesh, 8% cross-linked) Precipitation of the labeled membrane fragments with 0.3 31 were the kind gift of Dr. Richard Reitz of the Dow Chemical HC104 prior to filtration on the filter did not increase the amount Company. Antifoam 66 was obtained from General Electric of observed product. Membrane filters of several pore sizes Company (Silicone Products). Amberlite SB-2 anion exchange were tested in order to establish an optimal pore size that was paper was the product of Rohm and Haas. D-Amino acid required to retain a maximal amount of membrane fragments oxidase (EC 1.4.3.3) (4.5 units per mg, electrophoretically labeled with n-[14C]alanine. Portions of a standard reaction purified) and ribonuclease (EC 2.7.7.16) were purchased from mixture were applied to filters of pore sizes ranging from 25 rnp Worthington. to 5 p. The results indicated that the 0.45 p filter used in the

Enzyme Preparation-L. casei (ATCC 7469) was grown in a assay procedure retained approximately 90% of the radio- New Brunswick fermentor (15 liters) without aeration at 37”. activity retained by the smallest pore filter available. The The medium contained: 2% glucose, 2% peptone, 1% yeast choice of this size (0.45 p) represented a compromise between extract, 0.5oj, NaHaPOd, and 1% potassium acetate. Addition the rate of filtration and the amount of product retained on the of the following inorganic salts-0.027, MgSO,.7HgO, 0.00075% filter. MnSOc. H20, 0.001 To FeS04.7H20, and 0.001 y. NaCl-in- Analytical Methods-Ascending and descending chromatog- creased the yield of cells. Cells grown to late log phase were raphy was performed with Whatman No. 3MM paper. The harvested with a Sharples continuous flow centrifuge at 45,000 following systems were used: I, propanol-ammonia-water (6:3: 1) rpm and resuspended in 0.02 M Tris-HCl, pH 7.8. A 30% (15); II, isobutyric acid-ammonia-water (66: 1:33) (16); III, suspension of the bacteria was made in this buffer. Disruption butanol-acetic acid-water (4: 1: 5, organic phase) ; IV, iso- of the cells was accomplished by shaking 40 ml of this suspension propanol-12 N HCl-water (65: 17: 18) (17) ; V, methanol-water-10 with 35 g of plastic beads and 1 drop of antifoam in the Bronwill N HCl-pyridine (80: 17.5:2.5: 10) (18); VI, phenol-glacial acetic cell homogenizer (Braun, model MSK) at 4000 cycles per min acid-absolute ethanol-water (40 : 5: 6 : 10) (19) ; VII, butanol- for 7 min with cooling by liquid COZ. The beads were removed propionic acid-water (142:71: 100) (19). by filtration through fine mesh cloth and the homogenate was Measurements of radioactivity were made in polyethylene centrifuged at 100,000 X g for 60 min. The supernatant fraction vials with the Packard Tri-Carb liquid scintillation spectrometer was recentrifuged at 175,000 x g for 10 min. Samples of this (model 314-EX). Samples were counted in 15 ml of the Triton- fraction were either dialyzed against 100 volumes of 0.01 M toluene (1:2) scintillation fluid described by Patterson and piperazine buffer, ~1-1 6.5, for 8 hours or filtered on Sephadex Greene (20) and evaluated by Benson (21). For analysis of G-25. The supernatant fractions were stored in a liquid nitrogen radioactivity on paper chromatograms, I-inch squares were freezer. counted in a scintillation fluid consisting of 0.3% (w/v) 2,5-

Preparation of Membrane Fragments-The pellet resulting from diphenyloxazole in toluene. the 100,000 x g centrifugation was suspended in 200 ml of 20 For the determination of the stereochemical configuration of mM Tris-HCl, pH 7.8; the suspension was centrifuged at 3,000 X alanine, samples were incubated with n-amino acid oxidase g for 30 min. The pellet was discarded, and the supernatant and the [14C]pyruvate was separated from the unreacted amino suspension was recentrifuged at 5,000 x g for 30 min. This acid fraction by ion exchange chromatography. The samples to procedure was repeated until all material which could be sedi- be tested were taken to dryness and dissolved in 100 ~1 of 20 mM mcnted at 5,000 x g for 30 min was eliminated from the prepara- sodium pyrophosphate buffer, pH 8.3. A mixture (100 ~1) tion. For the isolation of membrane fragments, the turbid containing 137 units of catalase and 0.25 unit of n-amino acid 5,000 x g supernatant fraction was centrifuged at 100,000 X g oxidase in 10 pM FAD and 20 InM sodium pyrophosphate, pH for 60 min. The pellet was homogenized and the fragments were 8.3, was added. The samples were incubated for 1 hour at 3i”, sedimented from buffer two times in order to free the membranes diluted with 0.5 ml of 0.2 N sodium citrate buffer, pH 2.2, and of the supernatant enzyme activity. The membranes were applied to l-ml columns of Dowex 50 (Na+). The columns were suspended in either 10 mM Tris-maleate, pH 6.5, or 10 m&x eluted with an additional 1.5 ml of citrate buffer. The eluate piperazine buffer, pI1 6.5, to a concentration of 20 mg, dry containing the pyruvic acid was collected in a scintillation vial weight, per ml and stored in a liquid nitrogen freezer. and counted as described in the preceding paragraph.

Alanine Incorporation Assay-The assay measured the in- corporation of n+Jalanine into membrane fragments which RESULTS

were retained by a 0.45 p pore size membrane filter. The Requirements for Incorporation-The requirements for the reaction mixture contained the following components: 25 mM incorporation of n-[%]alanine into membrane fragments prc- MgCl,, 5 mM ATP (adjusted to pH 6.5 with NaOH), 0.1 mM pared from L. casei are shown in Table I. The activity is n@Jalanine, supernatant fraction (60 to 100 pg) and membrane dependent on ATP, supernatant fraction, and membrane frag-

Issue of October 25, 1971 V. 111. Reusch, Jr., and F. C. Neuhaus 6137

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6138 D-Alanine : Membrane Acceptor Liyase Vol. 246, No. 20

TABLE I Requirements for o-alanine incorporation into

membrane fragments The complete reaction mixture contained: 25 mM MgC12; 5 mrvr

disodium ATP; 0.1 nap n-[i4C]alanine; 100 pg of supernatant frac- tion and 100 pg of membrane fragments; 50 w sodium maleate buffer, pH 6.5, in a final volume of 50 ~1. The amount incor- porated was established by the method described under “Alanine Incorporation Assay.”

Addition Activity

$nwles/hr

Complete... _.........,._.____............. 96.6 Minus ATP. . . . . . . . . . . 2.1 MinusMgZ+............................... 38.2 Minus supernatant fraction. . . . . 2.0 Minus membrane fragments. . . . . . . . . 8.0

Zero time................................... 0.3 Boiled membrane fragments.. . . . . . . . . 3.7 Boiled supernatant fraction. . . . . . . . . . , . 3.1

SUPERNATANT FRACTION (ro, MEMBRANES (~0)

0 5 IO

WATP]) x IO-*

r FIG. l.‘Effect of supernatant fraction (A) and membrane con- FIG. 4. Incorporation of n-[i*C]alanine as a function of the ATP centration (B) on the incorporation of n-[Wlalanine. The ala- concentration. The alanine incorporation assay was used. The nine incorporation assay was used. In A the assay was performed molar ratio of Mgz+ to ATP was maintained at 3:l. From the with 166 pg of membrane protein and in B the assay was performed Lineweaver-Burk plot, the K, for ATP was determined to be 3.3 with 163 fig of supernatant fraction. rnM.

-----I-“?

0

i- 50 loo 1%

I 1 I I I 1

I I I

5.0 6.0 7.0 8.0

PH FIG. 2. Incorporation of n-[r%]alanine as a function of pH.

The alanine incorporation assay was used with the exception that 10-l M Tris-maleate buffer of the indicated pH was used instead of sodium maleate buffer.

ments; it is enhanced by the addition of Mg2”. Boiled mem- branes and boiled supernatant fraction are not active. The incorporation of n-alanine is linear with time up to 90 min. The effect of membrane fragments and supernatant fraction on the

r

IO 20 30 40

@-ALANINE] x IO‘

FIG. 3. Incorporation of n-[Wlalanine as a function of the o-[Wlalanine concentration. The alanine incorporation assay was used. In A, product formation is presented as a function of n-[i4C]alanine concentration. From the Lineweaver-Burk plot (B), the Michaelis-Menten constant (Km) for n-alanine was deter- mined to be 18 PM.

r

amount of D-alanine incorporation is illustrated in Fig. 1. The reaction is linear with respect to either membrane fragments or supernatant fraction within a restricted concentration range.

Optimal Conditions for Incorporation-The pH optimum for D-alanine incorporation (Fig. 2) is between 6.5 and 7.0. For the experiments reported in this paper, 50 mM sodium maleate, pH 6.5, was used unless otherwise stated. During the latter phases of this work, a 2-fold higher specific activity was observed when 50 mM piperazine buffer, pH 6.5, was used. In Fig. 3A, the effect of n-alanine concentration on the rate of incorporation is illustrated. From the Lineweaver-Burk plot (Fig. 329, a Michaelis-Menten constant (Km) of 18 PM was established. Saturation kinetics was also observed with MgATP. An analysis of the data in Fig. 4 indicates that the &, for ATP is 3.3 rnM.

Significant amounts of n-alanine incorporation may be observed without adding divalent metal ion to the reaction mixture (Table I). Stimulation of incorporation is observed if Ba*f, Mg*+, Ca*f, Mn2+, or Co2+ is added (Table II). The addition of Ni2+ led to a significant inhibition whereas the addi- tion of Cut+ caused complete inhibition of the incorporation reaction. As illustrated in Fig. 5, n-alanine incorporation is inhibited by the addition of KC1 and NaCl. However, in the case of the acetates of K+ and Na+, a significant stimulation is

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Issue of October 25, 1971 V. M. Reusch, Jr., and F. C. Neuhaus 6139

TABLE II Effect of divalent melal ions

The alanine incorporation assay was used with 20 rnivr metal ion. The control contained no added metal ions. With the exception of NiSOd, all salts were chlorides.

Addition I

Incorporation 1 Control

None. Mg2+. Bat+. Ca2+. Mn2+. co2+. X2+. al=+. I

pmoles/hr % 23.6 100 38.0 161 38.3 162 34.5 146 28.9 122 26.6 113 20.2 85.6

0.07 0.4

I I I I

2 4 6 6

[SALT] x IO

I5 B . IO

5 Kl i 0

. 2 4 6 8

[SALT] x IO

FIG. 5. Effect of salts on the incorporation of o-[Wlalanine. The alanine incorporation assay was used. The following salts were added to the reaction mixture: in A, KC1 (A), NaCl (A); in R, KNO, (o), NaN03 (o), potassium acetate (O), sodium acetate (w). The data in B were obtained with a different enzyme prepa- ration.

observed. These data suggest that the observed effects with the Kf and Na+ salts are anion-specific and cation-independent.

In order to harvest cells containing maximum enzyme activity, the n-ala&e-incorporating system was measured as a function of growth. Four parameters were measured during the growth of a culture (Fig. 6): (a) turbidity, (b) specific activity of the D-

alanine-activating enzyme, (c) specific activity of the u-nlanine: membrane acceptor ligase, and (d) specific activity of the mem- brane acceptor. At each time point, an appropriate volume was withdrawn from the culture and chilled on ice, and the bacteria were harvested. The supernatant fraction and the membrane fraction were prepared as described under “Experimental Procedure.” The highest specific activities of the ligase and the membrane acceptor were attained in late log phase. For the results described in this paper, enzyme and membrane fragments were prepared from late log cells.

Amino Acid Xpeci$city-The specificity of the alanine in- corporation system was tested in two types of experiments: (a) direct incorporation; (b) inhibition of n-alanine incorporation. Experiments of the second type were necessary when the ap- propriate labeled substrate w&s not available. However, these experiments do not prove that the competing unlabeled com- pound was incorporated into the membrane in place of D-ala&e.

Four substrates, n-[14C]a1anine, L-[14C]alanine, nn-a-amino-n- [%]butyric acid, and D-[14c]Ala-D-[14C]Ala, were tested by

I I I I

6 12 18 24

Houf?s FIG. 6. Specific activities of the n-alanine-activating enzyme,

o-alanine:membrane acceptor ligase, and membrane acceptor as a function of the growth phase. Enzyme preparatian and mem- brane fragments were prepared from cells harvested as described in the text. In Al the growth of the culture was monitored by turbidity at 600 nm (0). In AR the variation in specific activity of the n-alanine-activating enzyme (0) was measured (11). In BS the variation in specific activity of the n-alanine:membrane acceptor ligase was measured by assaying each of the enzyme preparations with the membrane fragments prepared from cells harvested at 12 hours (A). In B4 the variation in specific activity of the membrane acceptor was measured by assaying each of the membrane fragment preparations with enzyme preparation from cells harvested at 12 hours (0).

direct incorporation (Table III). L-[14C]Alanine was not in- corporated under conditions in which extensive incorporation of the D isomer occurred. The incorporation of n-alanine was not inhibited by the addition of L-alanine to the reaction mixture. In addition to n-alanine, nL-or-anlino-‘tl-[“C]but’yric acid was incorporated. The results obtained with D- and L-

alanine imply that only the D isomer of cr-amino-n-butyric acid is utilized. From the Lineweaver-Burk plot (Fig. 7), a Michaelis-Menten constant of 1.7 rnhf was est,ablished for DL-

cr-amino-n-[i4C]butyric acid. Assuming that only the D iso- mer is incorporated, the Km is 0.85 mtir. This assumption is supported by the observation that L-or-amino-n-butyric acid does not inhibit the incorporation of n-[i%J]alanine. ~-[i~C]Bls-

n-[i%]Ala was not incorporated. Competition experiments have been carried out with a variety

of D-alanine analogues. The following amino acids were not effective inhibitors: glycine, D- or L-threonine, D- or L-isoleucine, and L-serine. In addition, D- and L-cycloserine, m-alaninol, and either N-acetyl-n- or L-alanine were ineffective inhibitors. The

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6140 D-Alanine : &Jenzbrane Acceptor Ligase

TABLE 111

SpecijZQ profile bzJ direct incorporation

The alanine incorporation assay was used. The concentration of the labeled amino acids was 72 PM and the concentration of di- peptide was 50 PM. The dipeptide was synthesized by the pro- cedure of Xeuhaus and Struve (22).

Substrate Activity

$moles/hr

D-[l%]Alanine. . 23.8 L-[‘%]Alanine. 0.60n uL-ol-Amino-n-[%]butyric acid 0.96 r+[14C]Ala-o-[14C]Ala.. 0.0

a The low activity observed with L-alanine is the result of ala- nine racemase. This activity is inhibited by D-cycloserine.

(I/[~L-U-~-n-AMIN~U~RIC]) x 16~

FIG. 7. Incorporation of uL-a-amino-n-[%]butyric acid as a function of concentration. The alanine incorporation assay was used with nL-a-amino-n-[14C]butyric acid as the substrate. The data are presented as a Lineweaver-Burk plot. The Michaelis- blenten constant (K,,) was determined to be 1.7 mM.

TABLE IV

Inhibitor speci$city projZe

With the exception of D-cycloserine, the data presented in Sec- tion B were obtained with IJL analogues. The constants were evaluated from Lineweaver-Burk plots (23). The values pre- sented are half of those values determined and hence represent the inhibitor constants for the I) isomer.

Analogue Ki

A. 1).Alaninc I)-ol-Amino-n-butyric acid. n-Serine

(1.8 x 10-q 6.0 X 10-4b 3.4 x 10-Z

13. n-Alanine hydroxamate. 1,.ol-ilmiiio-n-brltyric acid hyl

droxamate. I>-Alanine amide.. 1).Alaninol. I)-Cycloserine.

a K, established from Fig. 3B. b I<i determined from Fig. 8d. c Iii established from Fig. 88.

1.3 x lo-3c

7.0 x 10-s 7.5 x 10-z 7.0 x 10-z

>O.l

Vol. 246, No. 20

I I

D-a-NH.-fi-BUTYRIC ACID

I I

B DL-ALANINE HYDROXAMATE

1

0 5 IO

(I/CD-ALANINE]) x lo-’

FIG. 8. Inhibition of incorporation of u-[‘Glalanine by I)-w amino-n-butyric acid (A) and oL-alanine hydroxamate (B). 111 A, the alanine incorporation assay was used with varying conccrl- trations of D-[‘%]alanine and the following concentrations of n-01- amino-n-butyric acid: 0, no inhibitor; 0, 1.0 mM; 0, 2.0 mu; n , 3.0 mM; A, 4.0 rnM. In B, the alanine incorporation assay was used with varying concentrations of o-[%Z]alanine and the following concentrations of nL-alanine hydroxamate: 0, no in- hibitor; 0, 4.5 rnM; 0, 8.9 mM; n , 13.4 mM; A, 17.8 mM.

only analogues that were effective as inhibitors of n-slanine incorporation are: (a) D-cr-amino-n-butyric acid, (b) ur,-alanine

hydroxamate, (c) DL-oc-amino-n-butyric acid hydroxamate, (d) DL-alanine amide, and (e) D-serine. Lineweaver-Burk plots of inhibition data indicated that each of these structural analogues inhibits in a competitive manner. For two of the more effective inhibitors, n-cr-amino-n-butyric acid and nL-alanine hydroxa- mate, the Lineweaver-Burk plots are shown in Fig. 8, d and U. The values of Ki, summarized in Table IV, indicate a high degree of specificity for n-ala&e.

Nucleotide SpeciJicity-The incorporation of u-alanine into membranes is dependent upon ATP (Table I). Additional experiments indicate that the requirement for i\TP may be fulfilled by ADP but not by AMP. The nucleotidc requirement may also be satisfied by GTP, CTP, and UTP (Table V).

Role of o-Alanine-activating Enzyme--The supernatant fraction

prepared from L. casei contains a high concentration of the I)- alanine-activating enzyme. The enzyme (E) catalyzes the activation of n-alanine according to the reaction:

E + o-alanine + ATP F? E.A&IP-I)-alanine + PI’ (1)

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Issue of October 25, 1971 V. M. Reusch, Jr., and F. C. Neuhaus 6141

TABLE V

Nucleotide specijicity of o-alar&e incorporation

In Experiment I, the final concentrations of nucleotide were: ATP, 4.5 mM; ADP, 4.9 mM; AMP, 4.5 mM. In Experiment II, the final concentration of nucleotide was 3.0 mM in all cases. Experiments I and II were performed with different enzyme preparations. The alanine incorporation assay was used.

Nucleotide

I. ATP. ................. ADP .................. AMP .................

II. ATP. ................. GTP .................. UTP. ................. CTP ..................

Incorporation

@noles/hr % 33.4 100 27.8 83

0.0 0 15.3 100 12.8 84 19.3 126 16.4 107

A A

1: A D-ALANINE ACTIVA-

TING ENZYME

I ‘, 0 LIGASE

f 1,

WV0

I I I I

1 B . EXCHANGE ACTIVITY A HYCROXAMATE ACTIVITY

/ 0 LIGASE

I r-1 t; 75 4 t

3 I- Bo * 261

v. WI

FIG. 9. Resolution of alanine racemase, the D-alanine-activat- ing enzyme, and the o-alanine:membrane acceptor ligase. In A, the enzyme preparation (40 mg) was applied to a column of Sepha- dex G-150 (1.1 x 53 cm). The column was eluted with 5 mM piperazine chloride buffer, pH 6.5, and the fractions were assayed with the alanine incorporation assay. The hydroxamate assay (11) was used to assay the n-alanine-activating enzyme. Alanine racemase was assayed by the method of Lynch and Neuhaus (33). In B, the enzyme preparation (60 mg) was applied to a column of Sephadex G-100 (2.8 X 40 cm). The column was eluted with 5 mM piperazine chloride, pH 6.5, that contained millimolar mercapto- ethanol. The hydroxamate and exchange activities catalyzed by the n-alanine-activating enzyme were measured by the methods of Baddiley and Neuhaus (11). For comparison, the peak frac- tion (2 ml) of n-alanine-activating enzyme catalyzed the forma- t,ion of 10,200 nmoles per hour and the peak fraction (2 ml) of D- alanine-membrane acceptor ligase catalyzed the transfer of 0.375 nmoles per hour.

2 4 6 6 2 4 6 6 MINUTES MINUTES

FIG. 10. Loss of D-[Wlalanine ester residues from labeled mem- branes and the release of hydroxamic acids from membranes. In l , 1 M H2NOH, pH 7.0, was added to labeled membranes prepared in the alanine incorporation assay and the mixture was incubated at 37” for the indicated time. Samples were filtered and assayed for radioactivity as described in the alanine incorporation assay. In 0, a suspension of membrane fragments was made 1 M in HzNOH, pH 7.0, and incubated at 37”. Samples were removed at the indicated time and added to 3 M trichloroacetic acid con- taining 1.5 M HCl. The content of hydroxamic acids was assayed by the FeC13 method with authentic alanine hydroxamate as the standard (11). In B, the data are plotted according to first order kinetics, and the values of Z’t,z were determined.

and is assayed by either a [a2P]PP-ATP exchange or by the formation of n-alanine hydroxamate when the reaction mixture contains hydroxylamine (11). It was proposed that this enzyme might catalyze the incorporation of n-alanine into membrane fragments.

The supernatant fraction described under “Experimental Procedure” was filtered on Sephadex G-150 (Fig. 9A), and the fractions were assayed for alanine racemase, the n-alanine- activating enzyme, and the n-alanine: membrane acceptor ligase. The three enzymes were partially resolved by this procedure. In a separate experiment, it was shown that the [32P]PP-ATP ex- change reaction catalyzed by the n-alanine-activating enzyme has the same IJ’~ as the hydroxamate activity and is separate from the n-alanine : membrane acceptor ligase (Fig. 9B). From calibrated columns of Sephadex G-150, the molecular weights of the n-alanine-activating enzyme and the n-alanine : membrane acceptor ligate were estimated to be 58,000 and 39,000, re- spectively (23).

E$ect of Ribonuclease-Gould et al. (24) have demonstrated the RNase-sensitive incorporation of L-ala&e into phosphatidyl- glycerol by membrane preparations from Clostridium welchii. In the case of the n-alanine incorporation system from L. casei, as much as 38 pug of RNase per ml had no effect on the reaction. Thus, the incorporation of n-alanine into membranes from L. casei does not appear to require the mediation of RNA.

Properties of Membrane-bound Product-Labeled membranes were isolated from a reaction mixture and hydrolyzed in 6 N HCl for 18 hours at 110”. The radioactive product cochromat- ographed with alanine in Systems I, III, IV, and V. The remainder of the hydrolysate was treated with n-amino acid oxidase as described under “Experimental Procedure.” Greater than 95$$ of the [l%]alanine in the sample was in the L, con- figuration.

When the labeled membranes were treated with neutral 1 M

hydroxylamine, a product was formed that cochromatographed

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6142 o-,4 lanine

I I I I I I

0.2 0.4 0.6 0.8

FIG. 11. Analysis of reaction mixture. The reaction mixture described in the alanine incorporation assay was used. Aliquots (50 ~1) were removed and placed in a boiling water bath for 2min. The samples were spotted on Whatman No. 3MM paper and chromatographed in Solvent VI. Koostra and Smith (19) re- ported that the RF of alanyl phosphatidylglycerol is 0.31 in this system. The large peak of radioact,ivity at high RF represents uncombined [Wlalanine from the reaction mixture.

with alanine hydroxamate in four solvent systems (I, III, IV, and V). In order to obtain a time curve for the deacylation by H2NOH, samples of labeled membranes were suspended in neutral 1 rvr H2NOI-I and incubated at 37”. Portions of the mixture were removed at each time point and filtered as de- scribed under “Alanine Incorporation Assay.” The dat,a shown in Fig. 10A illustrate the release of n-[W]alanine hydroxamate from the labeled membranes. For comparison, the release of total hydroxamic acids from membrane fragments is shown. The data for the release of total hydroxamic acids and D-[W-

alanine hydroxamic acid are consistent with first order kinetics (Fig. 10B). The release of total hydroxamic acids had a half- life of 2.9 min and the release of n-[‘4C]alanine hydroxamate had a half-life of 2.1 min.

In order to determine the chromatographic properties of the product of incorporation, samples of a reaction mixture were removed at 0, 30, and 60 min and each was chromatographed in Solvents II, VI, and VII. In each solvent syst,em, the product of the incorporation system accumulated with time at the origin. The results with Solvent VI are shown in Fig. 11. In contrast,

Membrane Acceptor Liyase Vol. 246, No. 20

Koostra and Smith (19) report RF values of 0.31 and O.lT for alanyl phosphatidylglycerol in Solvents VI and VII, respectively. In a separate series of experiments, attempts have been made to extract the product of the n-alnnine incorporation system into chloroform-methanol (2: 1). This extraction is routinely used by Nesbitt and Lennarz (25) in assaying L-lysyl phosphatidyl- glycerol synthetase. No material corresponding to alanyl phosphatidylglycerol was extracted from the product of the n-alanine incorporation system.

DISCUSSION

The results describe the incorporation of n-alaniue into membrane fragments from L. casei according to the following reaction:

ATP + n-alanine + membrane-acceptor Mgz’: (2)

n-alanyl-membrane acceptor + (ADP f Pi)

The reaction requires the presence of a nucleotide triphosphate or diphosphate in addition to membranes and supernatant fraction. The addition of hid 02+ to dialyzed membranes and supernatant fraction enhances the activity. It is proposed that the supernatant fraction contains an enzyme, o-alanine : mem- brane acceptor ligase. This enzyme is heat-labile and has an approximate molecular weight of 39,000. Since the D-

alanine: membrane acceptor ligase is not completely resolved from the n-alanine-activating enzyme and since the absolute activit.y of the activating enzyme is large in comparison to the ligase, it is not possible to conclude unequivocally that the D alanine-activating enzyme is not a component of this system. One feature of this system is not cousistent with the participation of the n-alanine-activating enzyme. The Michaelis-Menten constant for the incorporatiou of n-alanine into membraues is 18 PM whereas the K, for n-alanine in the reaction catalyzed by the activating enzyme is 70 m&f (1 I).

The product, n-[14C]alanyl-menibrane acceptor, has been characterized as a neutral hydrosylamine-labile ester that is uot extracted into lipophilic solvents. u-[r%]Alanyl hydroxamate was rapidly released from membranes at a rate similar to the formation of total hydroxamic acids from the membrane. It was demonstrated that 82y0 of the total hydroxamic acid fraction released with neutral 1 RI H2NOH is alanine hydrosamate and that 96% of the alanine hydrosamate extracted under these conditions is of the n configuration (23). A major constituent of the membranes of L. casei that, contains n-alanine in ester linkage is glycerol teichoic acid. The T+ for the incorporated n-alanine in neutral 1 M HZNOH is similar to the T+ observed f’o~ the n-alanine ester residues in this teichoic acid (13). In a subsequent paper, evideuce will be presented to support the conclusion that the n-alanine that is incorporated in ~ifro is covalently linked to a polymer that has properties similar to the teichoic acid, alanyl polyglycerophosphate.

The possibility that t,he product of Reaction 2 is n-nlanyl phosphatidylglycerol has been considered. This phosphatide would have properties similar to those described by other in- vestigators (19, 24). Three lines of evidence suggest that this phosphatide is not the product, of the system described in this paper: (a) the product is not extractable with chloroform- methanol (2: 1) ; (b) the chromatographic properties of the product are not consistent with alauyl phosphatidylglycerol; (c) Ikawa (26) has reported the absence of lipid-linked alanine in L. casei 7469. These factors, together with a consideration of

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Issue of October 25, 1971 V. M. Reusch, Jr., and F. C. Neuhaus 6143

RNase insensitivity and the stereospecificity, indicate that alanyl phosphatidylglycerol is not a major product of this system.

Direct, incorporation and competition experiments demon- strated that the alanine incorporation system possesses a high degree of structural and stereochemical specificity for the amino acid substrate. n-[X$Manine is incorporated whereas L-[‘~C]- alanine is not. Of the isomers tested for inhibitory activity, only analogues with the D configuration inhibited the incorpora- tion of n-alanine. The only structural analogue of n-alanine with significant activity in this system is D-ar-amino-n-butyric acid. The lack of substrate specificity for nucleotide triphos- phate contrasts with that observed for amino acid. The question of nucleotide specificity will be re-examined after the ligase has been purified.

If the incorporation of n-alanine into the membranes repre- sents the synthesis of alanyl ester residues in teichoic acid, one must consider why incorporation of alanine into the membrane- associated polymer and not into the isolated polymer is observed. We suggest three possible roles for the membrane which might csplain these observations: (a) the membrane establishes a specific conformation of the acceptor teichoic acid; (b) the membrane contairls an enzyme t,hat is required for the over-all reaction; (c) the membrane facilitates the formation of a specific teichoic acid complex with other membrane constituents. The work of Young (27) and Coyrtte and Ghuysen (28) demonstrates that bacteriophage will not adsorb to isolated phage receptor (t,eiehoic acid). These investigators suggested that phage adsorption requires a fixed conformation of teichoic acid or a precise orientation of sugnr substituents that results from the association of the polymer with the cell wall. In addition, Weiser and Rothfield (29) demonstrated that the transfer of galactosyl residues into lipopolysaccharide from Salmonella typhimltrium requires the prior formation of a complex between polymcl~ and phospholipid. Moreover, pure preparations of tcichoic acid do not act ;LS immunogens (30) but can be rendered immunogenic by preparat~ion of a complex of the teichoic acid with either methylated serum albumin or cetyl pyridinium chloride (31). Wicket1 and Knox (32) have isolated the mem- brane teichoic acid from Lactobacillt~s fermenti as a complex wit’h lipid, a form which ret,aius antigenicity. These experiments suggest that the in vilro incorl)orntion of u-alanine into isolated teichoic, acid may require Cnetor~ which the isolated polymer lacks.

ilcX-iioiuledg?nc?lt-m’e thank Miss Rosemary Linzer for many discusiiolls.

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Victor M. Reusch, Jr. and Francis C. NeuhausLactobacillus caseid-Alanine: Membrane Acceptor Ligase from

1971, 246:6136-6143.J. Biol. Chem. 

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