Lipid activation of undecaprenol kinase from Lactobacillus plantarum

76

Biochimica

et Biophysics

@ Elsevier/North-Holland

BBA

Acta,

619 (1980)

Biomedical

76-89

Press

57599

LIPID ACTIVATION OF UNDECAPRENOL LACTOBACILLUS PLANTARUM *

JACK

R. KALIN

Department Gainesville, (Received

** and CHARLES

12th,

FROM

M. ALLEN

of Biochemistry, J. Hillis Miller Health FL 32610 (U.S.A). November

KINASE

Center,

University

of Florida,

1979)

Key words: Undecaprenol kinase; Phospholipid; activation; (Lactobacillusplantarum)

Lysylphosphatidylglycerol;

Lipid

Summary Extraction of membranes of Lactobacillus plantarum with Triton X-100/ glycerol solubilized up to 80% of the undecaprenol kinase activity. Fractionation of the extract by gel chromatography separated endogenous phospholipid from the enzyme but simultaneously inactivated the enzyme. The kinase was reactivated by reconstitution with various synthetic phosphatidylcholines and purified L. plantarum phospholipids. Ditetradecanoylphosphatidylcholine and lysylphosphatidylglycerol were the best activators. Furthermore, the optimal environment for enzyme stimulation was provided by different defined molar ratios of Triton X-lOO/phospholipid. The ratios for the phospholipids tested ranged from 1.25 to 6.3. Similar substrate specificity and kinetic constants were observed for both the solubilized and reconstituted enzymes suggesting that no fundamental changes in the enzyme activity occurred during the delipidation-reconstitution process.

Introduction The role of undecaprenol polysaccharide biosynthesis

as the glycosyl carrier lipid in prokaryotic cell wall is well established [l]. The availability of the

* Taken in part from the Ph.D. dissertation of J.R.K., Department of Biochemistry, University of Florida. ** Present address: Kettering-Meyer Laboratory. Southern Research Institute, Birmingham, AL 35205, U.S.A. Abbreviations: dioctanoyl-, didecanoyl-, didodecanoyl-. ditetradecanoyl-, dihexadecanoyl-. dioctadecanoyl-, and dioleoylphosphatidylcholines are abbreviated as phosphatidylcholine (C8 : 0). (Cl0 : 0). (Cl2 : 0). (Cl4 : O), (Cl6 : 0). (Cl8 : 0). and (Cl8 : 1). respectively.

77

monophosphorylated derivative, undecaprenyl monophosphate, has been suggested to be rate controlling in peptidoglygan biosynthesis in Staphylococcus aureus [Z]. Its availability is dependent on the biosynthesis of undecaprenyl pyrophosphate and its dephosphorylation as well as the rephosphorylation of undecaprenol. Most of the enzymes responsible for undecaprenol metabolism are associated with the membrane. The most thoroughly studied enzymes have been undecaprenol kinase and undecaprenyl pyrophosphate synthetase. Undecaprenol kinase from S. ~~~e~s has been purified from membranes and shown to be activated by a variety of lipids and detergents of different charge and hydrophobic character [ 3-51. The most important determinant of a lipid’s potential to activate the kinase was hydratability [ 61. Lactobacillus plantarum undecaprenyl pyrophosphate synthetase has been isolated as a soluble enzyme. It is, however, apparently associated with the membrane, in vivo, as suggested by its dependence on lipid or detergent for in vitro activity [7]. This enzyme is interesting because of its preference for anionic phospholipid for activation. A comparison of the lipid requirements of the kinase and synthethase from the same microorganism will yield useful information regarding the manner in which the lipid environment may ultimately control polyprenol lipid metabolism in L. plan tarum. The undecaprenol kinase has been solubilized from L. plantarum with Triton X-lOO/glycerol [8]. The present work describes detailed studies of the lipid/ detergent requirements necessary for activation of the delipidated enzyme. Optimal stimulation by the endogenous aminoacyl phospholipid, Iysylphosphatidylglyceroi, is of particular interest. Material and Methods Sorbents for chromatography were silicic acid (Biosil HA, finer than 325 mesh, Bio-Rad Laboratories), silica gel G (type 60, E. Merck} and Sil-G (Macherey-Nagel) coated on plastic sheets. All polyprenols were prepared as previously described [8]. The exogenous phospholipids were obtained from Sigma Chemical Co., except phosphatidylcholine (CS :0 and Cl0 :0),bacterial (Escherichia coli) phosphatidylglycerol and cardiolipin which were obtained from Supelco, Inc. [T-~~P]ATP (3 Ci/mol) was obtained from AmershamSearle Corp. Ultrogel AcA 34 was obtained from LKB Instruments. Chemical determinations. Phospholipid content was determined according to the method of Rouser et al. [9] using either Na&IP04 or phosphatidylcholine (Cl4 :0) asthe standard. Neutral and glycolipid contents were determined by the dichromate oxidation method of Amenta [lo] using phosphatidylcholine (Cl4 :0) as the standard. Triton X-100 was measured by its absorbance at 275 nm (El%= 21.0; [ll]). Protein concentrations were determined by the method of Lowry et al. 1121. Sodium dodecyl sulfate was included in the assay for samples containing Triton X-100 [13]. Lipid purification. Lipids were purified from L. plantarum harvested in the late-log phase of growth by a modification of the procedure described by Vorbeck and Marinetti [ 141. Intact cells (100 g wet weight) were suspended in 300 ml of CH30H and heated for 5 min at 65°C. After cooling, 600 ml of

CHC13 were added and the resulting suspension was stirred for 20 min at room temperature. The CHC13 phase was recovered and the aqueous phase was reextracted twice with 900 ml, then 300 ml of CHC13/CH30H (2 : 1, v/v). The organic extracts were filtered through Whatman No. 1 filter paper and evaporated by rotary evaporation at 45°C. The resulting brown syrup was dried by successive evaporations with C,H,/abs. CzH50H (4 : 1, v/v) then extracted four times with 15-ml portions of CHC13 for 15 min at 40°C. The combined amber extracts were filtered through a sintered glass funnel then fractionated on a 2 cm X 9 cm column of Bio-$1 HA (15 g) equilibrated with CHC13. The neutral lipids were eluted with 200 ml of CHC13. The glycosyldiacylglycerols were eluted with successive lOO-ml washes with CHC1JCH,COCH3 (1 : 1, v/v), then CH&OCH3. The phospholipids were eluted with successive lOO-ml washes of CHC1&H30H in the following proportions (v/v): (90 : lo), (85 : 15), (75 : 25), (65 : 35), (50 : 50), (0 : 100). Both cardiolipin and phosphatidylglycerol were eluted with 25%, 35%, and 50% CH,OH in CHC13. Analytical thin-layer chromatography (TLC) of phospholipids was carried out on silica gel G (type 60) plates. The sorbent was washed twice with CHC13/ CH30H (2 : l), layered onto glass plates, and activated for 45 min at 80°C immediately before use. Chromatograms were developed with CHC13/CH30H/ Hz0 (65 : 25 : 4, v/v/v; solvent I) and components were visualized with I* vapor. Following evaporation of I *, the plates were stained for phospholipids with a molybdate spray reagent [15] or for primary amines with a ninhydrin spray reagent [ 161. Preparative TLC was carried out on 0.5 mm silica gel G (type 60) plates prepared in a similar manner to the analytical TLC plates. Cardiolipin, phosphatidylglycerol and lysylphosphatidylglycerol were purified using solvent I (RF 0.83, 0.57, and 0.43, respectively) or CHC13/CH30H/CH3COOH/Hz0 (65 : 25 : 8 : 4, v/v/v/v; solvent II) (RF 0.85, 0.65, and 0.32, respectively). Appropriate zones on the plates were scraped and the phospholipids were eluted from the silica gel by two extractions each with 0.5 ml of CzH50H/ CHCIJHzO/HCOOH (100 : 30 : 20 : 2, v/v/v/v) per cm’ of scraped zone. The pooled eluates were then mixed with an equal volume of CHCl, and one half vol. of water. The resulting CHC13 phase was recovered and concentrated by rotary evaporation. Residual water and formic acid were removed by evaporation with C,H,/C2HSOH as previously described. Lipids were finally resuspended in CHC13/CH30H (1 : 1, v/v) and stored at 0” C under Nz. Structural analysis of lysylphosphatidylglycerol. A portion of the lysylphosphatidylglycerol fraction was hydrolysed in 2.0 ml of 50% CH,OH (v/v) containing 0.1 M sodium borate (pH 9.4) for 90 min at room temperature [17]. Lipid products were removed from the hydrolysis mixture by extracting with 2.0 ml of CHCIJ. The aqueous phase, containing the liberated amino acid, was dried by rotary evaporation and analyzed by TLC on Sil-G-coated plastic sheets in n-C3H,0H/NH40H/H,0 (35 : 18 : 12, v/v/v; solvent III). The developed chromatogram was stained with ninhydrin reagent. Another portion of the lysylphosphatidylglycerol fraction was hydrolyzed in 1.0 ml of 6 M HCl in vacua for 48 h at 115°C and the lysine content quantified in a Beckman Model 120C amino acid analyzer. The composition of the fatty acid methyl esters obtained from L. plantarum lysylphosphatidylglycerol was determined as previously described [ 71.

19

Enzyme assay. The kinase activity was determined by the column assay method described previously [8] except that the product was eluted from the DEAE-Sephadex LH-20 columns with 1 M HCOOH, 0.5 M NH40H in CHCl,/

CHJOH(1:1,v/v). Lipid activators were added to the ~cub~tion tube first and the solvent was removed under a stream of Nz. Substrate, buffer, and the other components were then added. Triton X-100 concentrations present in the incubation mixture were 0.15% or 0.08% (v/v) as indicated. Enzyme purification. The membranes were prepared from L. plantarum as previously described [ 81. 20 ml of this preparation (200 mg of protein) were diluted to a final volume of 100 ml with 5 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, stirred for 30 min at O”C, and centrifuged at 90 000 x g,,, for 60 min at 5°C. The EDTA-washed and pelleted membranes were than extracted by homogenization in 1% Triton X-100, 10% glycerol (v/v), 20 mM Tris-HCl buffer (pH 8.0) at room temperature. The final suspension (50 ml) was stirred for 60 min at room temperature and the insoluble material was removed by centrifugation at 120 000 X gmax for 60 min at 5°C. The supematant was then concentrated 4-fold by ultrafiltration at 0°C through an XM-50 membrane (Amicon Corp.), prior to gel chromatography. A second extraction was necessary to liberate comparable levels of activity when less than 1% detergent was used. Pre-swollen Ultrogel AcA 34 was equilibrated in 0.1% Triton X-100, 10% glycerol, 20 mM Tris-HCl buffer (pH 8.0) at 5”C, and packed into a 2.5 cm X 35 cm column. The void volume (V,) of the column (68 ml) was determined with Blue Dextran. Chromatography was performed at 5°C. Fractions (4 ml) were collected and each was analyzed for Triton X-100, lipid phosphorus, and kinase activity. Lipid phosphorus was determined on aliquots extracted twice with 2.5 ~01s. of CHC1JCH30H (2 : 1, v/v). Kinase activity was determined as previously described in the presence of 0.36 mM phosphatidylcholine (Cl4 :0). Fractions containing the enzyme were pooled and stored frozen at -20” C'in small aliquots. Under these conditions, enzyme activity was stable for a month. This preparation is referred to as the Ultrogel enzyme. Results Purification of endogenous lipids from L. plantarum The lipids were extracted from L. plantarum and fractionated into neutral, glyco-, and phospholipid fractions by silicic acid chromatography. The phospholipids consisted mainly of cardiolipin, phosphatidylglycerol and lysyiphosphatidylglycerol [17]. These were further purified by preparative TLC in solvents I or II, although, solvent II generally resolved components more successfully. Purified cardiolipin and phosphatidylglycerol corn&rated on analytical TLC with commercially obtained E. coli cardiolipin and phosphatidylglycerol standards. The identity of lysylphosphatidylglycerol was suggested by its mobility in solvent I [l?]. It also stained for both phosphorus and primary amine. Structural analysis of lysylphosphatidylglycerol The identity of lysylphosphatidylglycerol was confirmed

by hydrolysis

of

80

TABLE

I

PHOSPHORUS

AND

LYSINE

CONTENT

OF

LYSYLPHOSPHATIDYLGLYCEROL

PURIFIED

FROM

L. PLANTARUM Phosphorus an amino Experiment

was acid

determined

using

the

standard

phosphorus

determination

and lysine

was determined

with

analyzer. Phosphorus

Lysine

Phosphoruspysine

WmoI)

WmoI)

(molar

1

0.430

0.386

1.11

2a

0.261

0.233

1.12

2b

0.522

0.533

0.98

ratio)

the lipid and subsequent analysis of the liberated amino acid. The amino acid released by borate hydrolysis corn&rated on TLC in solvent III with a lysine standard (RF 0.17). Alternatively, the amino acid released by hydrolysis in HCI was identified qualitatively as lysine and quantitated with an amino acid analyzer. Lysine and phosphorus were present in stoichiometric amounts (Table I), consistent with the structure of lysylphosphatidylglycerol [17]. The fatty acid composition of lysylphosphatidylglycerol was determined to be 1.3% Cl4 : 0; 31.6% Cl6 : 0; 7.3% Cl6 : 1; 3.4% Cl8 : 0; 46.6% Cl8 : 1; 9.9% ClS-cyclopropane. This composition differed somewhat from those previously determined for L. plan turum phosphatidylglycerol and cardiolipin [ 71. The major difference was in the relative proportions of Cl8 : 1 and ClS-cyclopropane fatty acids. The ratios of saturated to unsaturated plus cyclopropane fatty acids in cardiolipin, phosphatidylglycerol and lysylphosphatidylglycerol were 0.34, 0.47 and 0.57, respectively. Enzyme purification Gel chromatography in the presence of 0.1% Triton X-100 and 10% glycerol was used to fractionate the Triton X-lOO/glycerol-solubilized enzyme and remove phospholipid and excess detergent. Ultrogel AcA 34 was the most useful medium of several tested to give complete resolution of kinase activity and phospholipid. Phospholipid and excess Triton X-100 were included within the gel whereas the kinase activity was eluted near the V,, (Fig. 1). The detergent/ phospholipid peak had an approximate M, of 80-100 000 comparable to the size of a Triton X-100 micelle [ 181. The kinase, presumably highly aggregated, chromatographed with an approximate M, in excess of 300 000. Essentially all of the lipid phosphorus extracted with Triton/glycerol was included within the gel; less than 1% was associated with the peak of activity. Removal of lipid from the enzyme was the apparent reason for poor recovery of activity following gel filtration (lo-20% of the applied activity) since reconstitution of the enzyme with phosphatidylcholine (Cl4 : 0) stimulated activity (Fig. 1). Recovery of activity under these conditions often exceeded that which was applied to the column. Further attempts to purify the kinase were unsuccessful. These included fractionation of the solubilized enzyme preparation by DEAE-cellulose, hydroxypatite, and Blue Sepharose CL-6B (Pharmacia) chromatography. These

TABLE II

PURIFICATION

89.0 30.6 16.45

300.0

mg

Total protein

29.7 10.2 5.5

100.0

90

OF UNDECAPRENOL

KINASE

1.45 15.10 0.10

25.32 5.7 59.6 0.4

100.0 0.016 0.493 0.006

0.084

(Clmollmg)

pm01

%

Lipid Phosphorus/ protein

Total lipid Phosphorus

613 000 32 200 170 800 39 800 210 000 **

CPm

Total kinase activity *

5.3 27.8 6.5 34.2 **

100.0

%

* Kinase activity was measured in the presence of 0.08% Triton X-100. These results are representative of several experiments. ** Kinase activity was measured in the presence of 0.36 mM phosphatidylcholine (Cl4 : 0) in addition to Triton X-100.

EDTA wash TTiton/glycerol extract Ultrogel enzyme

Membrane enzyme

Preparation

PARTIAL

2 044 362 5 577 2 500 12 765 **

@pm/mg)

Specific activity *

1 .oo 0.18 2.73 1.22 6.25 **

Relative PUlification * <-fold)

82

1.20 0.

,400

go.90 0

: !. 300 F:

rm $ .60 0

.30 O-

,m ;T ,200

,100

60

80

100 EIutson

120 Volume

140

160

180

(ml)

Fig. 1. Gel filtration of the solubilized kinase. The Tritoniglycerol-solubilized enzyme preparation was chromatographed using Ultrogel AcA 34. Kinase activity was measured using 0.15 ml of each fraction and 0.15% Triton X-100 in the absence (a) or presence (0) of 0.36 mM phosphatidylcholine (Cl4 : 0). Lipid phosphorus determinations were made using 0.2 ml of each fraction (A) and Triton X-100 was measured by its absorbance at 275 nm (0).

procedures either provided no purification or resulted in substantial loss of enzymatic activity. Table II summarizes the partial purification of the kinase through gel filtration, All enzyme assays were performed in the presence of 0.08% Triton X-100. The phospholipid-reconstituted Ultrogel enzyme represented 34% of the initial membrane-bound kinase activity at a 6-fold purification. Synthetic lipid activation The kinase was activated to varying degrees by a series of synthetic phosphatidylcholines with fatty acyl chains varying from C8 to Cl8 : 1 (Fig. 2A).

LIpId

Concentration

(mM)

i

c

1

3

0.1

0.2 LIpId

‘8”)“’

0.5

’

1.0

Concentration

2.0

“‘L’c” 5.0

10.0

(mM)

Fig. 2. Activation of the kinase activity by synthetic phosphatidylcholines. The specific activity of the Ultrogel enzyme was measured in the presence of 0.08% Triton X-100 (A) or 0.15% Triton X-100 (B), and increasing amounts of phosphatidylcholine C8 : 0 (0). Cl0 : 0 (0). Cl2 : 0 (m), Cl4 : 0 (A), Cl6 : 0 (*), Cl8 : 0 (v), and Cl8 : 1 (0). Protein concentrations were all 0.047 mg/ml in (A) and 0.036 mg/ml for Cl0 : 0, Cl2 : 0, Cl4 : 0, Cl6 : 0. 0.050 mg/ml for Cl8 : 0, and 0.044 mg/ml for 18 : 1 in (B). These data are representative of two or more experiments.

83

The most active phosphatidylcholines, in order, were Cl4 : 0 > Cl8 : 1 > Cl6 : 0 > Cl2 : 0 > Cl0 : 0. Activity was not stimulated by C8 : 0 or Cl8 : 0. There was an inverse relationship between the fatty acyl chain length of a particular phosphatidylcholine and the concentration at which it optimally stimulated activity. This suggested that some type of mixed detergent/phospholipid micelle was necessary for activation of the enzyme. Short-chain length phosphatidylcholines have higher critical micellar concentrations than their longer chain length analogues [ 19,201 and do not insert as readily into a detergent micelle. Consequently, higher phosphatidylcholine concentrations should be necessary to maintain their presence within the micelle at detergent/phospholipid ratios appropriate for activation. Enzyme assays conducted in the presence of 0.15% Triton X-100, confirmed the importance of this ratio (Fig. 2B). The concentrations where optimal stimulation was observed for each of the phosphatidylcholines tested increased when the concentration of detergent was increased (compare Fig. 2A and B). The best stimulation was again observed with Cl4 : 0 followed by Cl2 : 0, Cl0 : 0 and Cl8 : 1. Activation by Cl6 : 0 was good at 0.08% detergent but much poorer at the higher detergent concentration. Phosphatidylcholine (Cl8 : 0), did not stimulate activity at either detergent concentration. The phosphatidylcholine concentrations necessary for optimal enzyme stimulation did not simply double with the 2-fold increase in detergent concentration. The optimal ratio of detergent/phospholipid was determined by measuring kinase activity at different phosphatidylcholine and detergent concentra-

PHOSPHATIDYL (mM)

CHOLINE Triton

X-100

Concentration

(mM)

Fig. 3. Activation of kinase activity by phosphatidylcholine (Cl4 : 0). The specific activity of the Ultrogel preparation was measured in the presence of increasing amounts of phosphatidylcholine (Cl4 : 0) at the indicated detergent concentrations. Protein concentrations were all 0.032 mg/ml. These data are rePre_ sentative of two or more experiments. Fig. 4. Detergent/synthetic phospholipid ratios optimal for kinase activation. The phospholipid and detergent concentrations at which optimal kinase activation was observed have been represented for phosphatidylcholine (Cl2 : 0) (m). Cl4 : 0 (A). and Cl8 : 1 (0). The curves have been fitted to a least-squares plot of the data.

84

tions ranging from 0 to 3 mM and from 0.02% to 0.18%, respectively (Fig. 3). An increase in the detergent concentration required a consistent increase in the phosphatidylcholine concentration necessary for optimal stimulation. In addition, a minimum amount of detergent was necessary for activity regardless of the phospholipid concentration tested. The relationship between the lipid and detergent concentrations optimal for stimulation was linear above 0.05% (0.8 mM) Triton X-100. Consequently, the optimal detergent/phospholipid ratio was calculated from a plot of these two variables (Fig. 4). The reciprocals of the slopes of these lines represent the optimal molar detergent/phospholipid ratios and are 1.25, 1.5, and 3.0 for phosphatidylcholines Cl4 : 0, Cl2 : 0 and Cl8 : 1, respectively. The specific activities measured in the presence of phosphatidylcholines Cl4 : 0, Cl2 : 0 and Cl8 : 1 were greatest at the minimum detergent levels necessary for activation (see Fig. 3 for Cl4 : 0). These values dropped 20-30% as the detergent was raised from 0.06 to 0.18% (l-3 mM). Synthetic lysophosphatidylcholines with fatty acyl chain lengths, C12, C14, and C16, were examined for their ability to activate the kinase, but they proved to be ineffective in the presence of either 0.08% or 0.15% Triton X-100. With 0.02% (0.34 mM) Triton X-100, however, specific activities of 5640 and 4730 cpm/mg protein were observed in the presence of 0.12 mM lysophosphatidylcholine (Cl6 : 0) and 0.17 mM lysophosphatidylcholine (Cl4 : 0), respectively. Activation by L. plantarum lipid. The whole lipid extract and each of the individual lipid fractions were tested for their ability to activate the kinase. Neither the whole lipid extract nor the neutral lipid fraction stimulated activity. The phospholipid fraction was the best and most consistent kinase activator with lysylphosphatidylglycerol proving to be the most active component (Fig. 5). Phosphatidylglycerol activated the kinase about 60% as well as lysylphosphatidylglycerol but cardiolipin did not stimulate at all. The specific activities measured under optimal conditions using lysylphosphatidylglycerol were similar to those measured using phosphatidylcholine (Cl4 : 0). The optimal detergent/phospholipid ratios for activation with the endogenous phospholipids were determined as described for the phosphatidylcholines in Fig. 3. This relationship was also linear above a minimum level of detergent (Fig. 6). The ratios for lysylphosphatidylglycerol and phosphatidylcholine (Cl8 : 1) were similar (3.0), however, the x-intercept for lysylphosphatidylglycerol was 2-fold lower than that for phosphatidylcholine (Cl8 : 1). The ratio for phosphatidylglycerol was 6.3, 2-fold higher than either lysylphosphatidylglycerol or phosphatidylcholine (Cl8 : 1). Its x-intercept more closely approximated that of the phosphatidylcholines (0.04%, 0.7 mM). The specific activities decreased at high detergent concentrations more rapidly in the presence of phosphatidylglycerol and lysylphosphatidylglycerol than in the presence of the phosphatidylcholines. Activity decreased 40% and 50% for lysylphosphatidylcholine and phosphatidylglycerol, respectively, as the detergent level was increased from 0.06% to 0.18%. The glycolipid fraction stimulated kinase activity approx. 25-30% as well as

85

0.75 s ;;; 5 0.50 6 z 0” 0.25 0 .a c

0.02

0.05

0.1

0.2

Lipid Concentration

0.5 (mM)

1. Triton

X-100

Concentration

(mM)

Fig. 5. Activation of the kinase by L. plantarum phospholipids. The specific activity of the Ultrogel enzyme was measured in the presence of 0.08% Triton X-100 and increasing amounts of lysylphosphatidylglycerol (0). phosphatidylglycerol (A), and cardiolipin cm). Protein concentrations were all 0.068 me/ ml. These data are representative of four or more experiments. Fig. 6. Detergent/purified L. plantmum phospholipid ratios optimal for kinase activation. The L.plontarum phospholipid and detergent concentrations at which optimal kinase activation was observed have been represented for lysylphosphatidylglycerol (0) and phosphatidylglycerol (A). The curves have been fitted to a least-squares plot of the data. The curve for phosphatidylcholine (Cl8 : 1) (- - - - - -) has been reproduced from Fig. 4.

lysylphosphatidylglycerol at a lipid concentration of 0.3 mg/ml. The stimulation effects by lysylphosphatidylglycerol and the glycolipid fraction together were not additive. In fact, when tested in mixtures, the glycolipid fraction inhibited the stimulation normally supported by lysylphosphatidylglycerol alone. Substrate specificity The substrate specificities and kinetics previously studied using the Triton/ NH&l-solubilized enzyme [8] were reexamined with the Ultrogel enzyme assayed in the presence of 0.08% Triton X-100 and 0.29 mM phosphatidylcholine (Cl4 : 0). Undecaprenol served as the best substrate; solanesol served 87% as well at equimolar concentrations. Dolichol and truns,tram, trans-geranylgeraniol served 26% and 28% as well as undecaprenol, respectively. The Triton/NH,Cl-solubilized enzyme, however, used these latter two substrates only 13% and 6% as well, respectively [8]. All-trans-retinol was a poor substrate. The apparent V values determined with undecaprenol and dolichol were 1.55 and 0.25 nmol/mg protein per min, respectively. These values are 2-fold higher than those determined using the Triton/NH,Cl-solubilized enzyme preparation, i.e. 0.91 and 0.12 nmol/mg protein per min. The apparent Km values determined for undecaprenol and dolichol were 8.0 and 5.5 ,uM, respectively, and are lower than those determined for the Triton/NH&l-solubilized enzyme preparation (12.7 PM and 11.4 PM, respectively).

86

Discussion Undecaprenol kinase has been partially purified and characterized respect to its requirements for endogenous and exogenous phospholipids.

with

Enzyme purification Fractionation of the Triton X-lOO/glycerol extract by gel chromatography in detergent separated 50% of the solubilized protein and 99% of the lipid phosphorus from the enzyme. Recovery of the activity following gel chromatography depended upon reconstituting the enzyme with lipids. The S. aureus kinase, delipidated and deactivated by DEAE-cellulose chromatography, was similarly reactivated by addition of phospholipids [21]. The detergent/phospholipid ratio present in the incubation mixture had a profound effect on kinase activity. Since membrane and solubilized enzyme preparations contained endogenous phospholipid, measurements of activity at various protein concentrations using these preparations fluctuated due to Generally, the specific activities of changing detergent/phospholipid ratios. membrane and solubilized enzyme were greatest when measured at low detergent (0.05%) and protein (and accompanying phospholipid) concentrations (less than 0.2 mg protein/ml). Once the L. plantarum enzyme had been delipidated by gel filtration, however, phospholipid concentrations in the assay could be precisely controlled and more consistent specific activities were obtained. Consequently, the relative purification and yield of the phospholipid-reconstituted Ultrogel enzyme ranged from 5-fold to 12-fold and 25-80%, respectively, depending upon the activity observed with the membrane-associated enzyme. Synthetic lipid activation The Ultrogel enzyme was stimulated by synthetic phosphatidylcholines with fatty acyl chain lengths ranging from Cl0 to Cl8 : 1. Egg lecithin, did not activate the kinase. In contrast the S. aureus kinase was optimally activated by the synthetic phosphatidylcholines (C8 : 0 and Cl0 : 0) [4]. This enzyme was also activated by egg lecithin. The L. plantarum kinase was optimally activated by defined molar ratios of detergent/phospholipid. Furthermore, there was a minimum detergent concentration, below which no activation was observed, suggesting that a micellar environment is required for the activity. Mixed micelles of Triton X-100 and phosphatidylcholine (Cl4 : 0) have been observed at the molar ratios which optimally activated the kinase [22]. Although the critical micellar concentration for Triton X-100 in water is 0.24 mM [18], the minimum concentration of Triton X-100 required in addition to phospholipid for activation was 0.7 mM. This may reflect a change in the critical micellar concentration of Triton X-100, since Me#O, glycerol and ethanol (all present in the assay) are able to elevate an amphiphile’s micellar concentration [18,20]. In contrast to this enzyme, the S. aureus kinase did not necessarily require supplemental micellar concentrations of detergent for activation [4,23]. Neither the L. plan tarum nor S. aureus kinase was stimulated by Triton X-100 alone. The detergentjphospholipid ratio where maximum stimulation was observed

87

for each of the synthetic phosphatidylcholines correlates with the molecular cross-sectional surface areas of the pure phospholipids [24] and hydratability [25]. Both of these characteristics may dictate the relative amount of detergent which can be accomodated within the ordered lipid structure. Consequently, the optimally activating mixed micelles may simply provide a detergentexpanded phospholipid surface. The S. aureus kinase was also activated by hydratable lipids [ 6,231. In fact, this was the best single determinant in predicting the ability of a lipid to activate the enzyme. The lysophosphatidylcholines were the only lipids tested that activated the kinase in the absence of micellar concentrations of detergent. Kinase activity, however, was 3-&fold lower than in the presence of mixed micelles of the respective diacylphosphatidylcholines and Triton X-100. In contrast, the S. aureus kinase was activated maximally by bovine lysophosphatidylserine and lysophosphatidylethanolamine, which stimulated activity 20--50-fold compared to their respective parent diacyl forms and 3-fold compared to egg lecithin [ 41. L. plan tarum lipid activation

Glycosyl

diacylglycerols are the primary constituents of membrane lipids in species [ 14,261. The phospholipid fraction, comprising 24% of the total lipids [27,28], consists primarily of phosphatidylglycerol (68%) lysylphosphatidylglycerol (23%) and cardiolipin (4%) [29]. There are no phosphatidylcholines in L. plan tarum. Endogenous phospholipids also activated the kinase optimally at defined molar detergent/phospholipid ratios. The best activator, lysylphosphatidylglycerol stimulated activity to the same level as phosphatidylcholine (Cl4 : 0). The optimal ratio (3.0), however, more closely resembled that for phosphatidylcholine (Cl8 : 1). The presence of 63% unsaturated fatty acid and a large polar headgroup in lysylphosphatidylglycerol should create a relatively loosely packed hydrocarbon core. This may facilitate mixed micelle formation with detergent, resulting in enzyme activation at a molar detergent/phospholipid ratio comparable to that for phosphatidylcholine (Cl8 : 1). Phosphatidylglycerol activated the kinase about 60% as well as lysylphosphatidylglycerol but at a 2-fold higher molar detergent/phospholipid ratio (6.3). This is unexpected for a lipid which has a fatty acid composition comparable to, but a molecular cross-sectional surface area less than lysylphosphatidylglycerol [ 30,311. This is unexplainable with present data.

Lactobacillus

Substrate

specificity

The phospholipid-reconstituted Ultrogel enzyme, exhibited a substrate specificity similar to that for Triton X-lOO/NH,Cl-solubilized enzyme previously described [8]. There was, however, an increase in the V for each active substrate tested which reflects both the purification of the enzyme and the improved conditions used to assay the reconstituted kinase. The Triton X-lOO/NH,Cl-solubilized enzyme contained endogenous undecaprenol which was phosphorylated in addition to the exogenously added substrates. The phospholipid-reconstituted Ultrogel enzyme, however, contained no endogenous undecaprenol as evidenced by the lack of product formation in

88

the absence of added substrate. Consequently, higher activities with geranylgeraniol and dolichol relative to undecaprenol for the reconstituted Ultrogel enzyme compared to the Triton/NH&l-solubilized enzyme may reflect this lack of contaminating endogenous substrate. However, no dramatic differences were observed in the kinetic constants of these two enzyme preparations. This suggests that the kinase was unaffected by the removal of its endogenous phospholipid. The kinase preferred C,, polyprenols to shorter (C,,) and longer (CloO) substrates. It also phosphorylated solanesol (C,,) essentially as well as undecaprenol. This suggests that the enzyme is more specific with respect to chain length than stereochemistry, since solanesol contains all truns double bonds. Furthermore, the similar apparent K, values for the isoprenologues, undecaprenol and dolichol, suggest that this chain length specificity occurs during the catalytic event rather than the binding of the substrates to the enzyme. It is now apparent that, like undecaprenyl pyrophosphate synthetase in L. plunturum, undecaprenol kinase requires a fluid lipid surface for activity. The synthetase, however, in contrast to the kinase, is optimally stimulated by a negative surface charge. The positively charged endogenous lipid, lysylphosphatidylglycerol was the best endogenous kinase activator but was ineffective as a synthetase activator [ 71. Surface charge requirements for the kinase, however, were not as stringent since the negatively charged lipid, phosphatidylglycerol, also supported activity. The lipid physical state was more important for the kinase than the synthetase. These differences reflects the different reactions catalyzed by the two enzymes. The kinase, which is situated in the membrane along with one of its substrates, undecaprenol, must remain accessible to the aqueous environment in order to bind its other substrate, ATP. Conversely, the synthetase, which is a soluble enzyme catalyzing the polymerization of water-soluble precursors to undecaprenyl pyrophosphate, must recognize a membrane surface which can accept its product. Acknowledgements We would like to thank Dr. L.O. Ingram for the analysis of the phospholipid fatty acids and Dr. Ben Dunn for the amino acid analysis. This work was supported by a grant from the U.S Public Health Service (GM23193). References 1

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