TIIE JOURNAL OF B~LOGICAL CHEMISTRY Vol 249, No. 9, Issue of May 10, PP. 26&2689, 1974

Printed in U.S.A.

The Synthesis of Polyribitol Phosphate

I. I’URIPICATIOK OF POLYRIUITOL PHOSPHATE POLYbIERASE Ai\D LIPOTEICHOIC ACID CARRIER*

(Received for publication, October 4, 1973)

FRASZ FIEDLER~ ASD LUIS GLASER

From the Depadmen t of Biological Chemistry, Washington University School of dfedicil?e, Saint Louis, Mis- souri 63110

SUMMARY

The polyribitol phosphate polymerase from Sta@ylococcus aureus H has been purified 400-fold by extraction with Triton X-100 and conventional fractionation procedures. The en- zyme shows a requirement for phospholipid, and uses as an acceptor lipoteichoic acid carrier (LTC). LTC has been purified to homogeneity, it contains glycerol-phosphate-glu- cose-fatty acids in ratio of 1: 1: 0.1 :O.l and contains traces of glucosamine. Both W- and 32P-labeled LTC have been prepared and they are totally converted to LTC-polyribitol phosphate when incubated with the purified enzyme, CDP- ribitol, and phospholipid.

Tcichoic acids are one of the major components of the cell wall of gram-positive bacteria. Until recently the synthesis of tcichoic acid was poorly understood because all preparations that catalyzed the in vitro synthesis of tcichoic acids were membrane preparations which contained both the cnzymcs catalyzing poly- mer synthesis as well as endogenous acceptor (l-3). Mauck and Glaser recently reported the preparation of a detergcnt-solu- bilizcd enzyme from Bacillus subtilis which catalyzed the syw thesis of polyglycerolphosphate and was dependent on the addi- tion of an acceptor which was similar to or identical with the “membrane teichoic acid” (4) from the same organism, and which we designate as lipoteichoic acid carrier. Because iu B. subtilis both the membrane teichoic acid as well as the cell wall teichoic acids are glyccrolphosphate polymers, it was not clear whether this enzyme system was involved in cell wall synthesis or in cell membrane synthesis. In addition, under the conditions used (4), only a portion of the membrane tcichoic acid could be shown to function as LTC,’ and the precise structural requirements that allowed lipoteichoic acid to function as LTC were not obvious.

In this communication we describe the purification of poly- ribitol phosphate polymerase from Sfaph~2ococcus aweus and of homogeneous LTC from the same organism. Since polyribitol

* This work was supported by Grant GM 18405 from the Na- tional Institutes of Health. A preliminary report of some of these observations was presented at the New York Academy of Science in June 1973.

$ Supported in part by the Deutsche Forschungsgemeinschaft. 1 The abbreviation used is: LTC, lipoteichoic acid carrier.

phosphate is a constituent of the cell wall but not of the cell mem- brane, the system is clearly involved in cell wall biosynthesis.

MATERIALS ASD hlETIIOlX5

[‘4C]Ribitol-5-P was prepared from [U-14C]AMP (Amersham- Searle) by hydrolysis in 1 N IICl for 30 min at 100” to release D- ribose-5-P followed by borohydride reduction. The ribitol-5-P was isolated by chromatography on ljowex 1-formate and con- verted to Cl)P-ribitol bv the mornholvdate method and ourified 1 as described previously (5). CDP-[“Clglycerol was prepared as described previously (1, 4).

Protein was determined by a modified Biuret procedure which can be used in the presence of Triton X-100 (6). Glucose was de- termined with hexokinase and glucose-6-P dehydrogenase (7) ; glycerol and L-a-glycerol-P were determined with glycerol kinase and glycerol-P dehydrogenase (7). Amino sugar was determined by the method of Levvy and McAllan (8) or with an amino acid analyzer. Ribitol was measured with ribitol dehydrogenase (5). Fatty acids were quantitated as the methyl esters by gas-liquid chromatography on a B-foot 3y0 SE-30 column on Gas-chrom Q (9). Phosphate was determined by the method of Ames (10).

Polyacrylamide gel electrophoresis in sodium dodecyl sulfate was carried out by the method of Laemmli using 15% gels (11). The gels were sliced in 2-mm slices and extracted for 16 hours in 1 ml of 0.2 N HCl at 37” with shaking and counted in Preblend 3a 70 (Research Products International). LTC is totally degraded under these conditions and quantitative recovery of counts is ob- tained from the gels.

Triton X-100, spermidine, and l)EAE-Sephadex A-50 were ob- tained from Sigma, DEAE-cellulose-52 was-obtained from Reeve Angel, Bio-Gel A-1.5m (200 to 400 mesh) and DI~~AI~~-Bio-Gel A (100 to 200 mesh were obtained from Bio-Rad, 32Pi and IU-14C1- glucose from New England Nuclear. Lipids were obtained from the sources indicated: Cardiolipin from Pierce Chemical, phos- phatidylglycerol from Supelco, phosphatidylethanolamine from Pierce- Chemical, phosphatidylFholine from Applied Science. Dipalmitin was a gift from Dr. David Silbert (Washington Uni- versity School of Medicine).

The assay mixture for polyribitol phosphate polymerase con- tains 0.05 M Tris-HCl, 0.02 hz spermidine, 0.001 hl 2.mercaptoetha- nol, 0.1% Triton X-100, pH 8.0, 20 nmoles of C1)P-[14C]ribitol, 25 nmoles of cardiolipin, and 10 nmoles of LTC (expressed as phos- phate) in total volume of 70 ~1. After incubation at 25” for 1 hour, the reaction was stopped by the addition of 2 volumes of ethanol-l M ammonium acetate, pH 7.5 (7.5:3). The polyribitol phosphate was collected on a 0.2.pm Solvinert Millipore filter and washed with ethanol-l M ammonium acetate. The filter was glued to a planchet and counted in a gas flow counter. The specific activity of various preparations of C11)P-[14C]ribitol varied be- tween 150 to 550 cpm per nmole when counted in the same counter. One unit is defined as the quantity of enzyme catalyzing the syn- thesis of 1 nmole of polyribitol phosphate per hour.

Buffer A used in many experiments contains 0.05 M Tris-HCl, 0.02 M spermidine, 0.001 M 2-mercaptoethanol, pH 8.0.

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Fraction Number

49 61 73 85 97 FRACTION NUMBER

RESULTS

Preparation of Polyribitol Phosphate Polymerase--S. aureus H was obtained from Drs. W. Hammes and F. C. Neuhaus, North- western University. The cells were grown in antibiotic medium 3 (Difco) in a 200-liter fermentor to A600 ,,,,, = 1.0. The fer- mentor was cooled rapidly to 5” (15 min) and cells were harvested by centrifugation in a Sharpless centrifuge. The cells were washed in Buffer A and the final packed cells were stored frozen at -20”.

For preparation of the enzyme the packed cells were thawed at room temperature and were broken by shaking for 2 min with glass beads in a Braunwill homogenizer with CO2 cooling. One- tenth milligram of DNase and RNase was added to each flask. All subsequent operations were carried out at 4”. The glass beads were removed by filtration on coarse sintered glass funnel and washed with Buffer A. A crude membrane fraction was collected from the filtrate by centrifugation at 100,000 X g for 1 hour and washed two times by centrifugation with 10 volumes of Buffer A and finally suspended in a minimal volume of Buffer A (membrane fraction).

Two hundred forty milliliters of the membrane fraction (1 g of protein) were extracted with stirring for 30 min with 800 ml of Buffer A-0.2% Triton. Residual membranes were collected by centrifugation at 100,000 x g for 1 hour and re-extracted with 300 ml of the same buffer and the residue again removed by centrifugation. The combined supernatant fluids were concen- trated to a volume of 100 ml in an Amicon ultrafiltration cell using an XM-50 membrane. The concentrated extract was di- luted to 400 ml with Buffer A and the concentration step repeated in order to decrease the Triton concentration.2

The concentrated enzyme was loaded on a column (30 x 5 cm) of DEAE-Sephadex A-50 in Buffer A-0.1% Triton and eluted stepwise with 1 literof Buffer A-0.1 y0 Triton; 700 ml of Buffer A- 0.5 M NaCl; 700 ml of Buffer A-l.5 1~ NaCI. The enzyme was

2 Trition X-100 passes through an XM-50 Amicon membrane with only slight retention. The second concentration step serves to remove excess Triton X-100.

FIG. 1 (left). Elution of polyribitol phosphate polymerase from Bio-Gel A-1.5m column. For experimental details see text. o-0, activity assayed no LTC; O--O, activity assayed with LTC; o---O, total phosphate.

FIG. 2 (right). Chromatography of [l%]LTC on DEAE-Bio-Gel A. For details see text.

TABLE I

Purijication of polyribitol phosphate polymerase

Membranes............. Triton extract.. DEAE-Sephadex eluate. Bio-Gel eluate..

-

-

I Polymerase activity

Volume Total units

ml

240 4,450 100 13,200 23" 13,800 30* 2,596

-

_- Unitsjmg

protein

4.4 66.5

430

2,080

P

.-

-

urification

1 15 98

475

a Fourteen milliliters of this fraction were used after concentra- tion for chromatography on Bio-Gel column.

* Only fractions dependent on added LTC are shown; the total recovery of enzymes from the Bio-Gel column was essentially quantitative.

then eluted with a linear gradient of 700 ml of Buffer A-0.05% Triton-1.5 M NaCl to the same buffer with 0.2% Triton. Twen- ty-milliliter fractions were collected. The enzyme was eluted in Fractions 8 to 31a and was concentrated in an Amicon cell with an XM-50 membrane to a volume of 20 ml, and dialyzed against Buffer A-0.5’% Triton.

Fourteen milliliters of the dialyzed enzyme were concentrated in an Amicon concentrator to 3 ml, and 2.7 ml were loaded on a column (2.5 x 140 cm) of Bio-Gel A-l.5 in Buffer A-0.1 ‘% Triton and eluted with the same buffer at a flow rate of 12 ml per hour. Three-milliliter fractions were collected.

A typical purification is shown in Table I. The recovery from the Bio-Gel column was essentially quantitative but, as shown in Fig. 1, only the first enzyme fractions eluted from the column are dependent on the addition of LTC and phospholipid (not shown). The remaining enzyme fractions could be concentrated and re-

a The polyribitol phosphate polymerase is strongly inhibited by 1.5 M NaCl, therefore aliquots of each fraction were dialyzed against Buffer A before assay.

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chromatographed on the Bio-Gel column to yield more resolved salt was removed by repeated dilution and concentration of the enzyme. solution (1.1 X lo7 cpm).

The enzyme is stable at all stages of purification at 4” in Buffer A-0.1% Triton, but is inactivated by freezing and thawing. The enzyme was sterilized by filtration through sterile 0.45.pm Millipore filters and stored at 4” for 3 to 4 months without loss of activity. The enzyme is free of the UDP-N-acetyl-n-glucosa- mine polyribitol phosphate glycosyltransferase which is present in the membranes of this strain of S. aureus (12). The great increase in enzyme activity during Triton extraction of the mem- branes may represent the removal of an inhibitor, but more likely represents the increased mobility of lipid, enzyme, and LTC in the extract as compared to the intact membrane, under assay conditions at 25”.

Although material at this step appeared pure when labeled with 32P, the 14C-labeled material was heterogeneous (see Fig. 7A) and required further purification. An aliquot, 5 x lo5 cpm, was chromatographed on a column (0.9 x 30 cm) of DEAE-Bio-Gel A in 0.05 M acetate, pH 5.5-0.1% Triton and eluted with a linear gradient of 250 ml of this buffer to 250 ml of 0.6 M acetate, pH 5.5-0.1% Triton. Fractions (3.6 ml) were collected. The elu- tion profile is shown in Fig. 2. Only the last peak, Fractions 81 to 95, had LTC activity.

Preparation of LTC-Radioactive LTC was prepared by grow- ing S. aureus H in 1% Peptone (Difco) (the lot used contained 0.7 pmole of phosphate per ml). To 600 ml of this medium were added 1 mCi of 32Pi or 2 mCi of [ W4C]glucose, when the culture reached Aso0 ,,,,, = 0.2. The cells were harvested at Asoo ,,,,, = 0.8. Membranes were prepared from these cells as described for the preparation of the enzyme. The preparation of W- labeled LTC is described in detail.

Unlabeled and 32P-labeled LTC were prepared by the same method. LTC could also be estracted from membranes by the phenol procedure (I), but it was not routinely used in the purifi- cation of LTC.

The membranes were lyophilized and extracted three times for 30 min at 25” with chloroform-methanol 2 : 1. Two and one- half milliliters of solvent was used for 10 mg of membrane protein. The residue was dried under vacuum and extracted twice at room temperature with Buffer A-0.2% Triton. Three milliliters were used per 10 mg of membrane protein. Residual material was removed by centrifugation at 30,000 x g for 10 min. The su- pernatant fluid was concentrated by ultrafiltration to 4 ml, heated at 100” for 3 min, and after cooling the insoluble residue was removed by centrifugation at 30,000 x g for 10 min (3.5 X 107 cpm). The supernatant fluid was loaded on a DEAE-cellu- lose .column (0.9 x 30 cm) in 0.01 M Tris-HCl, pH 8.0.0.1% Triton and eluted with a linear gradient of 200 ml of 0.01 M

Tris-HCl, pH S.O-0.1% Triton to 200 ml of the same buffer containing 0.5 M NaCl. Fractions of 2.5 ml were collected at a rate of 10 ml per hour. LTC was eluted in Fractions 20 to 24. The fractions were concentrated by ultrafiltration, and excess

Properties of Polyribitol Phosphate Polymerase-The depend- ence of the velocity of the most purified polymerase on LTC concentration is shown in Fig. 3. The enzyme is essentially totally dependent on LTC addition. In Fig. 4 is shown the de- pendence of the enzyme activity on cardiolipin addition at vari- ous Triton X-100 concentrations. As can be seen, Triton X-100 will not substitute for cardiolipin, and at very low Triton con- centrations the enzyme has negligible activity even in the pres- ence of cardiolipin. The detergent appears to be required for the interaction of LTC, enzyme, and phospholipid. The optimal concentration of cardiolipin is 25 nmoles in a 70-~1 reaction mix- ture (data not shown). Table II shows that a variety of phos- pholipids will substitute for cardiolipin, and that the enzyme has a very broad specificity for the phospholipid. Cardiolipin pre- pared from S. aureus H is no more active than cardiolipin from bovine heart.

The enzyme is very stable at 25” under assay conditions and activity continues up to 24 hours (Fig. 5). In contrast, the enzyme loses activity rapidly (2 to 3 min) at 37”. As shown previously by others (2, 13) the enzyme is totally dependent on divalent cation or spermidine and under our assay conditions optimal activity is obtained at 25 mM spermidine (data not shown). The enzyme has no activity with CDP-glycerol, but CDP-glycerol and a variety of other cytidine nucleotides are

600 1

“4 \ \ \ .

.- -. nmOleS of L rc P

‘Or-----l 200 8-

1 50 l-----l+ n 4- 6- a

-CARDIOLIPIN ‘b.

---n--.-o -________ o 11 'f,,,,

0 02 04 0 5 10 15 20 35 % Triton TIME (hours)

--

FIG. 3 (Zejl). Dependence of velocity of the polyribitol phos- phate polymerase on LTC concentration. Standard assay was

enzyme velocity. Standard assay conditions were used with

used with LTC concentration (expressed as nanomoles of phos- (O--O) and without (O---O) 25 nmoles of cardiolipin per reaction mixture. Triton concentrations were as indicated.

phate per reaction mixture) as indicated. l&” 1 I”” -,,.-\ E.n-..-a -P ^--A:^,:-:- .-2 m-!L-- FIG. 5 (rig/@. Effect of time on polyribitol phosphate synthe- -JJlrl-- -- -L ‘1, ~1 1 1 ..I ‘7n 1 , n,\m .I ., 1

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TABLE II

Stimulation of polyribitol phosphate polymerase by lipids

Standard assay was used with 25 nmoles of each lipid; this level is optimal for cardiolipin.

Lipid Polyribitol formed/hour

nmoles

None......... ._........... 0.23 Cardiolipin .__ ,__._._._.__. ._ ._ 1.35 Phosphatidylglycerol. 0.90 Phosphatidylethanolamine 1.23 Phosphatidylcholine. 0.63 Dipalmitin.. 0.19

TABLE III

Inhibition of polyribitol phosphate synthesis by cytidine nucleotides

Standard assay was used with additions as indicated.

Experiment and addition I ‘olyribitol-P formed Inhibition

/moles nmoles % I None 2.9 0

CDP-glycerol (0.28) 1.41 54 CDP-glucose (0.60) 2.52 12 CDP-ethanolamine (0.45) 2.68 8 Cytidine (0.60) 2.70 7

II None 2.0 0 CMP 0.018 1.86 7

0.027 1.42 29 0.036 1.35 33 0.090 1.1 45 0.270 0.30 85

-

FIG. 6. Effect of addition of polyribitol phosphate on electro- phoretic mobility of [32P]LTC. The top Jigure shows the mobility of [32P]LTC on g-cm polyacrylamide gels. The bottom $gure shows an identical sample which had been incubated for 24 hours with 6.5 units of enzyme and 0.1 pmole of CDP-ribitol under standard assay conditions. The control gel (top) contained all of the assay components, but sodium dodecyl sulfate was added at zero time. In this and subsequent figures Slice 1 is the position of the tracking dye.

SLICE NUMBER

FIG. 7. Effect of addition of polyribitol phosphate on electro- phoretic mobility of [i%]LTC. The experimental conditions are as in Fig. 6. In A [r%]LTC eluted from DEAE-cellulose was used. Ih n [14C]LTC eluted from DEAE-Bio-Gel was used. O---O, LTC before addition of polyribitol phosphate; O--C, LTC after addition of polyribitol phosphate.

TABLE IV

Analysis of LTC from Staphylococcus aureus Glycerol-P and glucose were determined after hydrolysis in 3 N

HCl at 100” for 3 hours. We assume that total glycerol-P is 2.3 times the observed n-Lu-glycerol-P (15, 16). Total glycerol was determined after dephosphorylation of the acid hydrolyzed material with Escherichia coli alkaline phosphatase. Fatty acids were determined as the methyl esters after saponification in 0.1 N NaOH in 50% methanol for 3 hours at 37”. Forty per cent of the fatty acids were iso-C-15, 287, 16:0, and smaller quantities of other fatty acids, similar to the fatty acid composition of the total lipids in this organism (17).

Amount

Total phosphate.. 1.02 Glycerol-P.............................. 1.0 Glycerol................................ 1.1 Glucose................................. 0.07 Fatty acids............................. 0.10

weak inhibitors of the enzyme (Table III). It is of interest that CMP, a product of the reaction, is a relatively poor inhibitor and that CDP-ethanolamine, which is a molecule of approximately the same size as CDP-glycerol, gives no inhibition, presumably because the positive charge on the ethanolamine groups prevents binding to the enzyme.

The enzyme velocity was independent of CDP-ribitol concen- tration at levels as low as 9 x lop5 M, and a precise K, value was not determined. The enzyme was inhibited by incubation with 0.4 mM p-hydroxymercuribenzoate. This inhibition could be reversed with excess 2-mercaptoethanol. N-Ethylmaleimide and iodoacetamide did not inhibit the enzyme.

Role of LTC in Polyribitol Phosphate Polymerase-LTC could function in the polyribitol phosphate-synthesizing system either as an activator or as an acceptor. In Figs. 6 and 7, we show that [32P]LTC and [%]LTC behave as single components on polyacrylamide gel chromatography. With both labeled prep- arations the mobility of LTC is changed to that of a higher molecular weight component after incubation with enzyme, CDP-

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Froct ion Number

FIG. 8. Degradation of [32P]LTC. Panel 1 shows electrophoretic pattern of [32P]LTC. Panel 2 shows the same material after incu- bation in 0.1 N NaOH at 37” for 2 hours. Panel 3 shows the effect of incubation of LTC in 0.1 N HCl at 37” for 2 hours. Polyacrylamide gels (5 cm) were used.

ribitol, and cardiolipin. As is shown in the accompanying paper (14), this higher molecular weight peak is also labeled from CDP-[14C]ribitol. We interpret this observation to indicate that polyribitol phosphate becomes covalently linked to LTC and changes its chromatographic mobility. While [32P]LTC behaves as a single component after DEAE-Sephadex chromatography, it is clearly not pure when 14C-labeled LTC is examined after DEAE-cellulose chromatography (Fig. 7). After chromatog- raphy on DEAE-Bio-Gel, LTC labeled with either 14C or 32P behaves as a single component, and can totally be transformed to material with chromatographic mobility of LTC-polyribitol phosphate.

LTC contains glycerol, phosphate, glucose, and fatty acids. The glycerol is present as glycerol-P and accounts for the total phosphate in LTC (Table IV). Examination of [r4C]LTC after dephosphorylation with 60% HF followed by acid hydrolysis (3 N HCl, 3 hours, 100”) revealed the same components and trace amounts of glucosamine and ribitol, each at approximately >{o the concentration of glucose in the preparation.4

The glucosamine content was too small to be present in every molecule of LTC, nevertheless when [r4C]LTC was converted to LTC-polyribitol phosphate, and the product eluted from poly- acrylamide gels, the product contained all of the glucosamine present in the original LTC. Elution of the same regions of gels on which free LTC had been chromatographed yielded no glucos- amine, thus suggesting that glucosamine is a component of some LTC molecules. Glucosamine has previously been found in B.

subtilis LTC (4) ; whether it is present in a labile linkage in S. uureus LTC and removed during purification is not known.

The small quantities of ribitol may represent a minute quantity of LTC-polyribitol phosphate in the preparation, as is suggested by the small peak of radioactivity in free [r4C]LTC, or [32P]LTC preparation, in the position of LTC-polyribitol phosphate (see Figs. 6 and 7).

4 [r4C]Ribitol was determined by paper chromatography using butanol-pyridine-Hz0 (6:4:3) as the solvent. [‘%]Glucosamine was determined with an amino acid analyzer, equipped with stream splitter.

LTC is very acid and alkali labile. Activity is lost at 37” in 2 hours in either 0.1 N HCl or 0.1 N NaOH and this loss in activity is associated with drastic changes in chromatographic mobility (Fig. 8).

Degradation of S. aureus LTC with 60% HF, which removes phosphate groups, releases 80% of the glycerol as free glycerol showing that it is unsubstituted. The remainder of the glycerol can be released by acid hydrolysis and presumably has linked to it the fatty acid residues and the glucose present in LTC. Only trace amounts of amino acids are found in pure LTC. Based on its composition and properties, LTC is identical with the mem- brane lipoteichoic acid (18-21) and behaves as a polyglycerol- phosphate with a hydrophobic region which contains fatty acids5

The presence of polyglycerolphosphate is per se not adequate for LTC activity; polyglycerolphosphate from cell walls of B.

subtilis obtained by lysozyme digestion, or the same material partially degraded with teichoicase (23), shows no LTC activity, suggesting that the lipid moiety of LTC is essential for LTC activity with the S. aureus enzyme. It should be noted that the same material showed very weak LTC activity with the poly- glycerolphosphate polymerase from B. subtilis (4).

DISCUSSION

The purified polyribitol phosphate polymerase shows an abso- lute requirement for LTC and phospholipid. The requirement for phospholipid is not unsual for a membrane-bound enzyme, and two of the most active phospholipids, cardiolipin and phos- phatidylglycerol, are major constituents of the membrane of S. uureus H (17).

The requirement of the polyribitol phosphate polymerase for LTC establishes a function for membrane teichoic acid in cell wall biosynthesis. We propose that the membrane teichoic acids which are amphipathic molecules containing polyglycerolphos- phate and fatty acids, serve as an assembly point for the synthesis of cell wall teichoic acid from which these polymers are then

6 The LTC from Bacillus subtilis after degradation with 60% HF (22) yields a butanol-soluble fragment containing all of the fatty acids, present in LTC, as well as glycerol and glucose (F. Fiedler and L. Glaser, unpublished observations).

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transferred to nascent peptidoglycan with regeneration of free 7. BERGnmYI’:R, H. U. (ed) (1965) Methods of E/zzy?,zalic Analysis,

LTC. It has been shown previously that teichoic acids can only 1st Ed, Academic Press, New York

be linked to nascent peptidoglycan but not to peptidoglycan al- 8. LWVY, G. II., .\ND McALL.\N, H. (1959) Biochem. J. ‘73, 127

ready present in the cell wall (24). 9. SILBERT, II. F., COHEN, M., AND H.\I<DEIL, M. E. (1972) J.

Biol. Chem. 247, 1699-1707 Two chemically different linkages are synthesized by the poly- 10. AMES, B. N. (1966) Methods Empol. 8, 115-118

ribitol phosphate polymerase; the first is the linkage of ribitol-1’ 11. L~MMLI, U. K. (1970) iValure 227, 68@685

to LTC and the second is the linkage of ribitol-1’ units to each 12. NITHENSON, S. G., ISHIMOTO, N., ANDERSON, J. S., AND

other. Whether, in fact, one or more enzymes arc involved in STHOMINGER, J. I,. (1966) J. Biol. Chem. 241, 651-658

this process is not known at present. It is noteworthy that LTC 13. Smw, 1). R. I)., MIRELM.\N, I>., CII.\TTEIIJEE, A. N., AND

P.IIIK, J. T. (1970) J. l3iol. Chem. 246, 5101-5106

preparations contain very few nascent polyribitol phosphate 14. FII~:DLEII, F., AND GL.\sEI<, L. (1974) J. Biol. Chem. 249,

chains. This suggests that in viva once a chain is completed, it 2G90- 2G95

is rapidly transferred to pcptidoglycan. 15. BURGER, M. hl., AND GL.w:R, L. (1966) J. Biol. Chem. 241,

494-506 16. B.\ILLY, RI. C. (1942) Hull. Sot. Chem. France 9, 314

Acknowledgment-We are grateful to 1Iixs Isarbara Hochenedel 17. W.UCD, J. B., AND PERKINS, II. R. (1968) Biochem. J. 106, 391-

for expert technical assistance. 400

18. WICKISN, A. J., AND KNOX, K. W. (1970) J. Germ. i\ficrobioZ. 60,

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Franz Fiedler and Luis GlaserPHOSPHATE POLYMERASE AND LIPOTEICHOIC ACID CARRIER

The Synthesis of Polyribitol Phosphate: I. PURIFICATION OF POLYRIBITOL

1974, 249:2684-2689.J. Biol. Chem. 

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