ANALYTICAL
BIOCHEMISTRY
204,
34-39
(19%)
Preparation of Fatty-Acylated Derivatives of Acyl Carrier Protein Using Vibrio harveyi Acyl-ACP Synthetase’ Zhiwei
Shen, Debra
Fice, and David
M. Byers2
Departments of Pediatrics and Bi0chemistr.y and The Atlantic Daihousie Unibersity, Halifax, Nova Scotia, Canada
Received
September
Centre,
5,199l
A simple two-step purification of Vibn’o harveyi fatty acyl-acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. AcylACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at -20°C in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acylACP preparation and analysis. o 1992 Academic PPSSS, hc.
Acyl carrier protein (ACP)3 is a small multifunctional protein that plays a key role in fatty acid metabolism in bacteria, plants, and other organisms [reviewed in (l)]. Although studied principally as an intermediate in fatty acid biosynthesis, ACP is also involved in the synthesis ’ This work was supported by a Scholarship (D.M.B.) and a grant from the Medical Research Council of Canada. 2 To whom correspondence should be addressed at Dalhousie University, Atlantic Research Centre, Room C-202, Clinical Research Centre, 5849 University Avenue, Halifax, Nova Scotia, Canada B3H 4H7, Fax: (902) 494-1394. a Abbreviations used: ACP, acyl carrier protein; SDS-PAGE, SOdium dodecyl sulfate-polyacrylamide gel electrophoresis; BCA, bicinchoninic acid, Mes, 4-morpholineethanesulfonic acid, DTT, dithiothreitol.
34
Research
of phospholipids (2), lipopolysaccharides (3), oligosaccharides (4), and proteins (5). Most of these functions involve fatty acyl moieties linked to a phosphopantetheine prosthetic group of ACP through a thioester linkage to form acyl-ACP, which participates in transfer, reduction, dehydration, and elongation reactions. AcylACP derivatives used to characterize these reactions can be synthesized enzymatically using Escherichia coli acyl-ACP synthetaseBacylglycerolphosphatidylethanolamine acyltransferase (6-9) or plant extracts (10). Although E. coli acyl-ACP synthetase is widely used in long chain acyl-ACP preparations with chain lengths C,, to Cls, Triton X-100 is required for the reaction and high concentrations of LiCl are essential for release of acyl-ACP in the synthetase partial reaction of this enzyme (9). Moreover, acyl-ACP with chain lengths less than C,, must normally be synthesized separately by chemical methods (11). We have recently reported the discovery of a novel acyl-ACP synthetase from the luminescent marine bacterium Vibrio harueyi (12) and have suggested that this enzyme may be involved in the activation of endogenous myristic acid produced during bioluminescence (13) or in the elongation of exogenous fatty acids observed in this organism (14). Although the function of this enzyme remains unknown, it exhibits several properties different from E. coli acyl-ACP synthetase including its soluble nature, inhibition by Triton X-100 and salt, and different fatty acid chain length specificity (12). In the present investigation, V. harveyi acyl-ACP synthetase has been partially purified and its potential for the preparation of acyl-ACP derivatives investigated.
MATERIALS
AND
METHODS
Materials. [I-l*C]Octanoic acid (53.5 Ci/mol) and [l-l*C]palmitic acid (58 Ci/mol) were obtained from DuPont Canada Ltd. (New England Nuclear Products, 0003-2697192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENZYMATIC
PREPARATION
Montreal, PQ, Canada), while [l-14C]lauric acid (57 Ci/ mol) and [l-‘4C]myristic acid (54 Ci/mol) were from Amersham Canada Ltd. (Oakville, ON, Canada). [li4C]Butyric (1.6 Ci/mol), [l-‘4C]hexanoic (8 Ci/mol), and [ l-14C]decanoic (10.6 Ci/mol) acids were purchased from Sigma Chemical Co. (St. Louis, MO.). [9,10-3H]Myristic acid (25 Ci/mmol) was obtained by thin-layer chromatographic purification of the products of tritiation of myristoleic acid prepared by Amersham. P-Mercaptoethanol, ATP, and myristic acid were purchased from Sigma, while dithiothreitol was from BoehringerMannheim Canada Ltd. (Laval, PQ, Canada). SDSpolyacrylamide gel electrophoresis was performed using a Mini-Protean II slab cell from Bio-Rad (Canada) Ltd. (Missisauga, ON); molecular weight standards for SDS-PAGE were purchased from Pharmacia (Canada) Ltd. (Montreal, PQ). En3Hance was obtained from DuPont Canada Ltd. E. coli ACP (Sigma) exhibited a single major band on SDS-PAGE and was used without further purification. Purification of V. harveyi acyl-ACP synthetase. V. harveyi B392 strains were grown in complex medium containing 1% NaCl (13) to an absorbance at 660 nm of 2.0 (corresponding to about 10’ cells/ml). Bacteria were harvested by centrifugation (9OOOg, 20 min) and stored at -20°C or used immediately for purification of acylACP synthetase. The dark mutant strain Ml7 (15) was chosen as an enzyme source for most initial preparations as it lacks a luminescence-specific acyl-ACP esterase that might be expected to interfere with the acylACP synthetase assay (16), although the wild-type strain was subsequently shown to produce similar results. Acyl-ACP synthetase activity is constitutive in these cells (12) and can be purified from either logarithmic or early stationary phase bacteria. All preparative procedures were carried out at 4”C, except where noted. An extract, was prepared from the paste of 5 liters of V. harveyi cells by sonication (six 30-s bursts) in 50 ml of buffer A (20 mM Tris-HCl, 10% glycerol, 1 mM EDTA, 10 pM dithiothreitol at pH 7.5), followed by centrifugation (15,OOOg, 20 min). The resulting pellet was resuspended and sonicated (four 45-s bursts) in 25 ml of buffer A. The combined supernatants were adjusted to 10 mM MgSO, and applied directly to a column (10 X 2.5 cm) containing Cibacron blue F3GAagarose (Pierce, Rockford, 11) equilibrated with buffer A containing 10 mM MgSO,. The column was eluted with this buffer followed by buffer A containing 0.5 M NaCl. Fractions containing acyl-ACP synthetase activity were pooled and concentrated by adding solid ammonium sulfate to 75% saturation. The ammonium sulfate pellet was resuspended in 2-4 ml of buffer A, clarified by centrifugation, and applied to a Sephacryl S-300 high resolution column (95 X 1.5 cm) equilibrated with buffer A. Fractions containing enzyme activity were pooled and
OF
ACYL
CARRIER
PROTEIN
35
stored at -20°C for several months with little loss of activity. Enzymaticsynthesis of acyl-ACP. The acyl-ACP synthetase reaction was monitored using a filter disk assay (6) modified for the V. harueyi enzyme as previously described (12). The standard assay for acyl-ACP synthetase activity was performed at 37°C in a total volume of 50 ~1 containing E. coli ACP (20 PM), 0.1 M Tris-HCI (pH 7.8), 10 mM MgSO,, 10 mM ATP, 5 mM dithiothreitol, and 80 pM [3H]myristic acid (1 Ci/mmol). Twentymicroliter samples were removed at 10 and 20 min and applied to Whatman 3 MM filter paper (2 cm’) that was washed three times with methanol/chloroform/acetic acid (6/3/l, v/v) to remove unbound fatty acid. The [3H]myristoyl-ACP product was counted in a liquid scintillation spectrometer with an efficiency of 16% (a counting efficiency of 75% was determined for [‘“Cldecanoyl-ACP). A similar procedure was used for the quantitation of ACP by its conversion to labeled acylACP, except that the reaction mixture contained 0.02 units of V. harveyi acyl-ACP synthetase (one unit defined as the amount of enzyme required for the formation of 1 nmol of acyl-ACP per minute) instead of E. coli ACP and incubation was performed for 4 h. For preparation of acyl-ACP derivatives, ACP from E. coli (50-70 pM) was incubated as described above with partially purified V. harueyi acyl-ACP synthetase (0.51.5 units/ml) and”H- or l-i4C-labeled fatty acids ofvarying chain length in a total volume of 0.3-l ml. To obtain maximal conversion of ACP to acyl-ACP, the dithiothreitol concentration was reduced to 1 mM and the reaction was allowed to proceed for 4 h or longer at 37°C. The extent of conversion of ACP bo acyl-ACP was monitored using the filter disk assay as above and expressed relative to the initial ACP concentration determined using the micro-BCA protein assay (Pierce) with bovine serum albumin as a standard; this assay was validated using an A,,, (1 mg/ml) of 0.20 for stock solutions of E. coli ACP (17). For SDS-PAGE analysis (18), the reaction mixture was terminated by adding an equal volume of sample buffer (125 mM Tris-HCI, pH 6.8, 25%, v/v, glycerol, and 2.5% SDS). Protein samples were separated on 15% polyacrylamide slab gels in the absence of reducing agents and the radioactive acyl-ACP bands were observed by fluorography. In some experiments, the labeled acyl-ACP product was isolated by modification of published methods (19). The reaction mixture was applied to a small (1 ml) DEAE-Sepharose column equilibrated in 10 mM Mes (pH 6.0). The column was eluted with this buffer containing 80% 2-propanol to remove free fatty acid; acyl-ACP was subsequently eluted with 10 mM Mes containing 0.6 M Lick. This fraction was applied to an octyl-Sepharose column to separate acyl-ACP from ACP; fractions containing the former were reapplied to DEAE-Sepharose to remove 2-propanol (19).
36
SHEN,
Fraction
FICE,
AND
BYERS
Fraction
Number
FIG. 1. Fractionation of V. harveyi acyl-ACP synthetase by dye-ligand chromatography. Cell-free extract from the AFM mutant (440 mg protein in 63 ml) was applied to a Cibacron blue F3GA-agarose column (10 X 2.5 cm) in 20 mM Tris-HCl (pH 7.5), 10% glycerol, 1 mM EDTA, 10 KM DTT, and 10 mM MgSO,. After washing with 80 ml of this buffer, enzyme activity was eluted (see arrow) with 20 mM TrisHCl (pH 7.5), 10% glycerol, 1 mM EDTA, 10 pM DTT, 0.5 M NaCl. The flow rate was 1 ml/min and lo-ml fractions were collected. AcylACP synthetase activity (0) was monitored as pmol of [3H]myristoylACP formed/min/pl of fraction in the filter disk assay (see text). The absorbance at 300 nM (- - -) of the fractions was multiplied by two for display on the y-axis.
RESULTS AND DISCUSSION The soluble acyl-ACP synthetase activity previously described in cell-free extracts of V. harveyi (12) is not suitable for quantitative preparation of acyl-ACP derivatives or for sensitive detection of ACP due to the presence of other acyl-ACP-utilizing enzymes and endogenous V. harveyi ACP, respectively. In the present study, we have partially purified the enzyme using a simple two-step procedure involving dye-ligand and gel filtration chromatography. The first step in the procedure takes advantage of enhanced binding of the enzyme to Cibacron blue FSGA agarose in the presence of Mg’+; although some activity could subsequently be eluted by EDTA alone, maximal recovery of synthetase activity was obtained by elution with buffer containing 0.5 M NaCl and 1 mM EDTA (Fig. 1). After concentration, this material eluted in a single peak of activity on Sephacryl S-300 gel filtration, displaced from the major protein peak (Fig. 2). Calibration of this column with protein standards of known molecular weight indicated that acyl-ACP synthetase might be part of an enzyme complex of about 500 kDa; further purification resulted in increased instability of the enzyme, possibly due to disruption of this complex. The presence of glycerol (lo%, v/v) in all buffers during purification was essential for good recovery of activity. In five representative preparations, the average purification of synthetase activity was 25-fold with a yield of 30% (about 500 units of activity). At this stage of purification, V. harveyi acylACP synthetase was stable for several months at -20°C and was not inactivated by repeated freeze/thaw cycles. To determine the potential of Sephacryl S-300-purified V. harueyi acyl-ACP synthetase for the preparation
Number
FIG. 2. Gel filtration of V. harueyi acyl-ACP synthetase. Active fractions from Cibacron blue chromatography were concentrated and applied to a Sephacryl S-300 HR column (95 X 1.5 cm) in 20 mM Tris-HCl (pH 7.5), 10% glycerol, 1 mM EDTA, and 50 pM DTT. The sample contained 30 mg of protein in 2 ml. The flow rate was 11 ml/h and l&ml fractions were collected. The acyl-ACP synthetase activity (0) was monitored as pmol of [3H]myristoyl-ACP formed/min/pl fraction; the absorbance at 300 nM (---) was multiplied by two for display on the y-axis.
of acyl-ACP derivatives, conditions for optimal acylation of E. coli ACP (200 Kg) with [3H]myristic acid were investigated using a filter disk assay (Fig. 3). The time necessary for maximum (90%) conversion of ACP to [3H]myristoyl-ACP was dependent upon the enzyme
60
I 0
60
120
180
Time (min) FIG. 3. Effects of enzyme and fatty acid concentration on myristoyl-ACP formation by V. harueyi acyl-ACP synthetase. (A) E. coli ACP (190 pg, 70 PM final concentration) was incubated with [3H]myristic acid (440 PM, 1 Ci/mmol) and ATP in the presence of (0) 0.5, (A) 1.0, and (w) 1.5 units per milliliter of acyl-ACP synthetase in a total volume of 0.3 ml as outlined in the text. (B) ACP (190 pg) was incubated with (0) 180 FM, (A) 440 FM, and (m) 710 pM [3H]myristic acid (1 Ci/mmol) and ATP in the presence of 1.0 unit per milliliter of acyl-ACP synthetase. At the times indicated, duplicate lo-p1 samples were withdrawn and [3H]myristoyl-ACP was quantitated using the filter disk assay. Results are expressed as percentage conversion of ACP to acyl-ACP.
ENZYMATIC TABLE
PREPARATION
1
Acylation of ACP with Fatty Acids of Different Chain Lengths by V. harueyi Acyl-ACP Synthetase Fatty
acid 6:O 8:O lo:o 12:o 14:o 16:0
Acyl-ACP
(%l” 91 90 84 86 91 sgb
a Each reaction mixture (0.9 ml) contained Tris-HCl (0.1 M, pH 7.8), MgCl, (10 mM), ATP (10 mM), dithiothreitol (1 mM), E. coli ACP (400 fig, 50 PM), acyl-ACP synthetase (0.45 units), and l-*%-fatty acids (250-300 FM). After incubation for 6 h at 37”C, labeled acylACP was quantitated using the filter disk assay (mean of triplicate lo-al samples) and expressed as a percentage of the initial ACP concentration. For each sample, acylation was also verified visually by SDS-PAGE and Auorography. b Incubation time for the 16:0 reaction was 24 h.
concentration (Fig. 3A) and incubation for up to 24 h did not decrease the final extent of conversion regardless of the amount of enzyme used. The rate and extent of acylACP formation was also relatively independent of fatty acid concentration at fatty acid to ACP ratios of five or greater, while some decrease in acylation was noted at lower fatty acid concentrations (Fig. 3B). Acyl-ACP formation was unaffected over the range of ACP concentration tested in the present experiments (13-70 PM); this range spans the K,,, of the enzyme for E. coli ACP [20 PM, (12)]. This is in contrast to the reaction catalyzed by E. coli acyl-ACP synthetase, which requires ACP concentrations less than 15 @M for maximum acylation (7). V. harueyi acyl-ACP synthetase is capable of utilizing a broad range of fatty acid substrates for acyl-ACP formation. As shown in Table 1, conversion of ACP to acylACP in the reaction mixture was greater than 85% for all saturated fatty acids tested between 6 and 16 carbons. The rate of acylation with 16:0 was lower than with shorter fatty acids, as noted earlier (12), although increased yields could be obtained by increasing the reaction time or enzyme concentration. Partial acylation (about 20%) with l&O and no appreciable formation of 4:0-ACP was observed under these conditions. Further purification of acyl-ACP by DEAE-Sepharose and octyl-Sepharose chromatography (19) usually resulted in some loss on this scale of preparation, although the resulting yield of product (>60%) was still greater than that observed previously for the E. coli enzyme [between 5 and 50%, (7)]. SDS-PAGE and fluorography of acyl-ACP prepared using 14C-fatty acids revealed a single labeled band corresponding to the acyl-ACP stained with Coomassie blue (Fig. 4). Acyl-ACP products migrated faster than
OF
ACYL
CARRIER
PROTEIN
37
unacylated ACP on SDS-PAGE; this property has been noted previously and has been attributed to enhanced binding of SDS to the acidic ACP molecule upon fatty acylation (10). The biological activity of the 14:0-ACP product synthesized using acyl-ACP synthetase was confirmed by incubation with the V. harveyi bioluminescence-specific acyl esterase, the enzyme responsible for cleavage of 14:0-ACP to form 14:0 for reduction to myristaldehyde (16). Complete cleavage of [“H]14:0ACP by this enzyme was observed both by SDS-PAGE and fluorography as well as by recovery of hexane-extractable [3H]14:0 (data not shown). As the esterase is fairly specific for myristic acid (20), we conclude that V. harueyi acyl-ACP synthetase forms biologically active thioester derivatives of ACP with no further side reaction. Further experiments were conducted to establish whether fatty acylation by V. harveyi acyl-ACP synthetase reaction can be employed to detect and/or quantitate ACP in solution or in extracts from other bacterial species. Monitored using the filter disk assay, acyl-ACP formation with 0.5 units per milliliter of enzyme was linear over a range of E. coli ACP from 1 to 400 pmol during a 4-h incubation (Fig. 5). This sensitivity and range is similar to that reported previously for E. coli acyl-ACP synthetase under comparable conditions (2.5 to 250 pmol) (17). It should be noted that V. harueyi acyl-ACP synthetase activity is inhibited by monovalent salts and detergents such as Triton X-100 (50% inhibition at 0.1 M and 0.05%, v/v, respectively) (la),
FIG. 4. SDS-PAGE analysis of E. coli acyl-ACP products synthesized using V. harveyi acyl-ACP synthetase. ACP from E. cob (2.4 pg) was incubated for 12 h with acyl-ACP synthetase (0.01 units), ATP, and l-“C-labeled fatty acids (80 pM) in a total volume of 20 ~1 as indicated (no fatty acid was present in the first lane). After addition of SDS sample buffer, half of the reaction mixture was separated by SDS-PAGE. The Coomassie blue staining profile is shown in A while a fluorogram (7.day exposure) is shown in B (labeling intensities reflect different specific radioactivities of fatty acid substrates).
38
SHEN,
FICE,
,021 lo?
IO'
102
103
ACP (pmol)
FIG. 5. Quantitation of E. coli ACP by acylation with V. harueyi acyl-ACP synthetase using the filter disk assay. Reaction mixtures (50 ~1 total volume) were prepared as described in the text and contained [3H]myristic acid (80 FM, 1 Ci/mmol), acyl-ACP synthetase (0.02 units), and varying amounts of ACP. After incubation for 4 h at 37”C, 10.~1 samples containing the amount of ACP indicated were removed and [3H]acyl-ACP formation was determined using the filter disk assay. Each point represents the mean counts per minute of triplicate samples from one reaction mixture; the results from two independent experiments are shown on a double logarithmic scale. The assay was linear down to 1 pmol of ACP, or about 200 cpm above blanks minus ACP (600 cpm).
AND
BYERS
the major protein component (Fig. 4). Second, V. harveyi acyl-ACP synthetase is a soluble enzyme and exhibits optimal activity at low ionic strength and in the absence of detergents. This is in contrast to E. coli acylACP synthetase, which requires Triton X-100 for solubility and high concentrations of LiCl for release of acyl-ACP (9). Third, the extent of acyl-ACP formation with the V. harveyi enzyme appears to be largely independent of ACP concentration and the scale of the preparation (compare Figs. 3, 4, and Table 1). Finally, V. harveyi acyl-ACP synthetase quantitatively (>85%) acylates ACP using fatty acids from 6 to 16 carbons in length. This range overlaps that of the E. coli enzyme, which provides maximum yields with 14 to 18 carbon fatty acids, although some acylation with 8:0 to 12:0 is also noted (6,19,23). Thus, preparation of medium to long chain fatty acyl-ACP derivatives can be performed using a single procedure, rather than a combination of chemical and enzymatic methods.
ACKNOWLEDGMENTS
although these agents can normally be diluted out or corrected for in the final assay volume. In other experiments (not shown), acylation with the V. harveyi enzyme has been used to monitor the purification of V. harveyi ACP by filter disk assay and to detect endogenous ACP in extracts of Photobacteriumphosphoreum, a luminescent bacteria that appears to lack endogenous acyl-ACP synthetase activity (12), by SDS-PAGE and fluorography. The activity of acyl-ACP synthetase with ACP substrates from three different bacterial species is consistent with the high degree of ACP structural homology among different organisms (21) and suggests that the V. harveyi enzyme may be useful for a wider range of bacterial and plant species. The only other enzyme that has been described with the ability to directly ligate long chain fatty acids to ACP is E. coli acyl-ACP synthetase/2-acylglycerolphosphatidylethanolamine acyltransferase, a 27-kDa inner membrane enzyme that uses a tightly bound molecule of ACP as an intermediate in the activation and transfer of fatty acids to form phosphatidylethanolamine (9). That enzyme has been employed for the preparation of acylACP derivatives to study the reactions of bacterial (2225) and plant (26-28) fatty acid metabolism, phospholipid synthesis (29,30), lipopolysaccharide synthesis (3), and fatty acylation of proteins (5). We believe that V. harveyi acyl-ACP synthetase offers an attractive alternative to the E. coli enzyme for a number of reasons. First, a suitable enzyme preparation that is stable for several months can be obtained after two simple purification steps. This preparation appears to be free of contaminating activities that utilize acyl-ACP (8), allowing long reaction times under conditions in which ACP is
The authors are grateful to Harold W. Cook, Palmer, and Matthew W. Spence for their helpful gestions.
Frederick comments
B. St. C. and sug-
REFERENCES 1. Vanden
Boom,
T., and Cronan,
J. E., Jr. (1989)
Annu.
Reu. Micro-
biol. 43, 317-343. 2. Rock, 3.
C. O., and Jackowski, 10,765. Brozek, K. A., and Raetz, 15,410-15,417.
S. (1982)
J. Biol.
Chem.
C. R. H. (1990)
J. Biol.
4. Therisod,
Proc.
5.
C. (1991)
H., and Kennedy, E. P. (1987) USA 64,8235-8238. Issartel, J.-P., Koronakis, V., and Hughes, 759-761.
6. Ray,
T. K., and 73,4374-4378.
Cronan,
J. E., Jr.
USA
7. Rock,
C. O., and
Garwin,
J. L. (1979)
(1976)
257,10,759-
NC&.
Proc.
J. Biol.
Chem. Ad.
Sci.
Nature
351,
N&l. Chen.
265,
Acad.
Sci.
254,
7123-
J. E., Jr.
(1979)
7128.
8. Spencer, FEBSLett.
9. Cooper, Biol.
A. K.,
Greenspan,
A. D., and
Cronan,
101,253-256.
C. L., Hsu, L., Jackowski, Chem. 264, 7384-7389.
S., and
Rock,
C. 0.
(1989)
J.
10. Jaworski, J. G., and Stumpf, P. K. (1974) Arch. Biochem. Biophys. 162, 166-173. 11. Cronan, J. E., Jr., and Klages, A. L. (1981) Proc. N&l. Acad. Sci. USA
78,
5440-5444.
12. Byers,
D. M., and Holmes, C. G. (1990) Biochem. Cell Biol. 104551051. Byers, D. M. (1988) Biochem. Cell Biol. 66, 741-749. Byers, D. M. (1989) J. Bacterial. 171, 59-64.
13. 14. 15. Wall, L. A., Byers, 159,720-724.
D. M., and Meighen,
E. A. (1984)
68,
J. Bacterial.
ENZYMATIC 16. Byers, D. M., and USA 82,6085%6089.
Meighen,
PREPARATION
E. A. (1985)
Proc.
Natl.
Acad.
OF Sci.
ACYL
23.
CARRIER
39
PROTEIN
Spencer, A. K., Greenspan, Biol. Chem. 253, 5922-5926.
A. D., and Cronan,
24.
19. Rock, C. O., Garwin, J. L.. and Cronan, J. E., Jr. (1981) in Methods in Enzymology (Lowenstein, J. M., Ed.), Vol. 72, pp. 397-403, Academic Press, San Diego, CA.
26. Mattoo, A. Ii., Callahan, F. E., Mehta, R. A., and Ohlrogge, J. B. (1988) Plant Physiol. 89, 7077711. 27. Post-Beittenmiller, D., Jaworski, J. G.! and Ohlrogge, J. B. (1991) J. Biol. Chem. 266, 18581865. 28. Pollard, M. R., Anderson, L., Fan, C., Hawkins, D. J., and Davies, H. M. (1991) Arch. Biochem. Biophys. 284, 306-312.
and
21. Rock, C. O., and 9778-9785. 22.
Guerra, D. J., and 280, 336-345.
Meighen, Cronan, Browse,
E. A. J. E., Jr.
(1991)
J. Biol.
Chem.
266,
J. Biol.
Chem.
254,
(1979)
J. A. (1990)
Arch.
Biochem.
Biophys.
29. Rock, 30. Green, Chem.
C. 0. (1984) J. Biol. Chem. P. R., Merrill, A. H., Jr., 256, 11,151-11,159.
J. Lipid
Res. 25,
J.
17. Rock, C. O., and Cronan, J. E., Jr. (1981) in Methods in Enzymology (Lowenstein, J. M., Ed.), Vol. 71, pp. 341-351, Academic Press, San Diego, CA. 18. Laemmli, U. K. (1970) Nature 227, 680-685.
20. Ferri, S. R., 12,852-12,857.
Cooper, C. L., and Lueking, D. R. (1984) 1232. 25. Garwin, J. L., Klages, A. L., and Cronan, Chem. 255, 11,949-11,956.
J. E., Jr. (1978)
J. E., Jr. (1980)
259, 6188-6194. and Bell, R. M.
(1981)
1222J. Biol.
J. Biol.
BIOCHEMISTRY
204,
34-39
(19%)
Preparation of Fatty-Acylated Derivatives of Acyl Carrier Protein Using Vibrio harveyi Acyl-ACP Synthetase’ Zhiwei
Shen, Debra
Fice, and David
M. Byers2
Departments of Pediatrics and Bi0chemistr.y and The Atlantic Daihousie Unibersity, Halifax, Nova Scotia, Canada
Received
September
Centre,
5,199l
A simple two-step purification of Vibn’o harveyi fatty acyl-acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. AcylACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at -20°C in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acylACP preparation and analysis. o 1992 Academic PPSSS, hc.
Acyl carrier protein (ACP)3 is a small multifunctional protein that plays a key role in fatty acid metabolism in bacteria, plants, and other organisms [reviewed in (l)]. Although studied principally as an intermediate in fatty acid biosynthesis, ACP is also involved in the synthesis ’ This work was supported by a Scholarship (D.M.B.) and a grant from the Medical Research Council of Canada. 2 To whom correspondence should be addressed at Dalhousie University, Atlantic Research Centre, Room C-202, Clinical Research Centre, 5849 University Avenue, Halifax, Nova Scotia, Canada B3H 4H7, Fax: (902) 494-1394. a Abbreviations used: ACP, acyl carrier protein; SDS-PAGE, SOdium dodecyl sulfate-polyacrylamide gel electrophoresis; BCA, bicinchoninic acid, Mes, 4-morpholineethanesulfonic acid, DTT, dithiothreitol.
34
Research
of phospholipids (2), lipopolysaccharides (3), oligosaccharides (4), and proteins (5). Most of these functions involve fatty acyl moieties linked to a phosphopantetheine prosthetic group of ACP through a thioester linkage to form acyl-ACP, which participates in transfer, reduction, dehydration, and elongation reactions. AcylACP derivatives used to characterize these reactions can be synthesized enzymatically using Escherichia coli acyl-ACP synthetaseBacylglycerolphosphatidylethanolamine acyltransferase (6-9) or plant extracts (10). Although E. coli acyl-ACP synthetase is widely used in long chain acyl-ACP preparations with chain lengths C,, to Cls, Triton X-100 is required for the reaction and high concentrations of LiCl are essential for release of acyl-ACP in the synthetase partial reaction of this enzyme (9). Moreover, acyl-ACP with chain lengths less than C,, must normally be synthesized separately by chemical methods (11). We have recently reported the discovery of a novel acyl-ACP synthetase from the luminescent marine bacterium Vibrio harueyi (12) and have suggested that this enzyme may be involved in the activation of endogenous myristic acid produced during bioluminescence (13) or in the elongation of exogenous fatty acids observed in this organism (14). Although the function of this enzyme remains unknown, it exhibits several properties different from E. coli acyl-ACP synthetase including its soluble nature, inhibition by Triton X-100 and salt, and different fatty acid chain length specificity (12). In the present investigation, V. harveyi acyl-ACP synthetase has been partially purified and its potential for the preparation of acyl-ACP derivatives investigated.
MATERIALS
AND
METHODS
Materials. [I-l*C]Octanoic acid (53.5 Ci/mol) and [l-l*C]palmitic acid (58 Ci/mol) were obtained from DuPont Canada Ltd. (New England Nuclear Products, 0003-2697192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENZYMATIC
PREPARATION
Montreal, PQ, Canada), while [l-14C]lauric acid (57 Ci/ mol) and [l-‘4C]myristic acid (54 Ci/mol) were from Amersham Canada Ltd. (Oakville, ON, Canada). [li4C]Butyric (1.6 Ci/mol), [l-‘4C]hexanoic (8 Ci/mol), and [ l-14C]decanoic (10.6 Ci/mol) acids were purchased from Sigma Chemical Co. (St. Louis, MO.). [9,10-3H]Myristic acid (25 Ci/mmol) was obtained by thin-layer chromatographic purification of the products of tritiation of myristoleic acid prepared by Amersham. P-Mercaptoethanol, ATP, and myristic acid were purchased from Sigma, while dithiothreitol was from BoehringerMannheim Canada Ltd. (Laval, PQ, Canada). SDSpolyacrylamide gel electrophoresis was performed using a Mini-Protean II slab cell from Bio-Rad (Canada) Ltd. (Missisauga, ON); molecular weight standards for SDS-PAGE were purchased from Pharmacia (Canada) Ltd. (Montreal, PQ). En3Hance was obtained from DuPont Canada Ltd. E. coli ACP (Sigma) exhibited a single major band on SDS-PAGE and was used without further purification. Purification of V. harveyi acyl-ACP synthetase. V. harveyi B392 strains were grown in complex medium containing 1% NaCl (13) to an absorbance at 660 nm of 2.0 (corresponding to about 10’ cells/ml). Bacteria were harvested by centrifugation (9OOOg, 20 min) and stored at -20°C or used immediately for purification of acylACP synthetase. The dark mutant strain Ml7 (15) was chosen as an enzyme source for most initial preparations as it lacks a luminescence-specific acyl-ACP esterase that might be expected to interfere with the acylACP synthetase assay (16), although the wild-type strain was subsequently shown to produce similar results. Acyl-ACP synthetase activity is constitutive in these cells (12) and can be purified from either logarithmic or early stationary phase bacteria. All preparative procedures were carried out at 4”C, except where noted. An extract, was prepared from the paste of 5 liters of V. harveyi cells by sonication (six 30-s bursts) in 50 ml of buffer A (20 mM Tris-HCl, 10% glycerol, 1 mM EDTA, 10 pM dithiothreitol at pH 7.5), followed by centrifugation (15,OOOg, 20 min). The resulting pellet was resuspended and sonicated (four 45-s bursts) in 25 ml of buffer A. The combined supernatants were adjusted to 10 mM MgSO, and applied directly to a column (10 X 2.5 cm) containing Cibacron blue F3GAagarose (Pierce, Rockford, 11) equilibrated with buffer A containing 10 mM MgSO,. The column was eluted with this buffer followed by buffer A containing 0.5 M NaCl. Fractions containing acyl-ACP synthetase activity were pooled and concentrated by adding solid ammonium sulfate to 75% saturation. The ammonium sulfate pellet was resuspended in 2-4 ml of buffer A, clarified by centrifugation, and applied to a Sephacryl S-300 high resolution column (95 X 1.5 cm) equilibrated with buffer A. Fractions containing enzyme activity were pooled and
OF
ACYL
CARRIER
PROTEIN
35
stored at -20°C for several months with little loss of activity. Enzymaticsynthesis of acyl-ACP. The acyl-ACP synthetase reaction was monitored using a filter disk assay (6) modified for the V. harueyi enzyme as previously described (12). The standard assay for acyl-ACP synthetase activity was performed at 37°C in a total volume of 50 ~1 containing E. coli ACP (20 PM), 0.1 M Tris-HCI (pH 7.8), 10 mM MgSO,, 10 mM ATP, 5 mM dithiothreitol, and 80 pM [3H]myristic acid (1 Ci/mmol). Twentymicroliter samples were removed at 10 and 20 min and applied to Whatman 3 MM filter paper (2 cm’) that was washed three times with methanol/chloroform/acetic acid (6/3/l, v/v) to remove unbound fatty acid. The [3H]myristoyl-ACP product was counted in a liquid scintillation spectrometer with an efficiency of 16% (a counting efficiency of 75% was determined for [‘“Cldecanoyl-ACP). A similar procedure was used for the quantitation of ACP by its conversion to labeled acylACP, except that the reaction mixture contained 0.02 units of V. harveyi acyl-ACP synthetase (one unit defined as the amount of enzyme required for the formation of 1 nmol of acyl-ACP per minute) instead of E. coli ACP and incubation was performed for 4 h. For preparation of acyl-ACP derivatives, ACP from E. coli (50-70 pM) was incubated as described above with partially purified V. harueyi acyl-ACP synthetase (0.51.5 units/ml) and”H- or l-i4C-labeled fatty acids ofvarying chain length in a total volume of 0.3-l ml. To obtain maximal conversion of ACP to acyl-ACP, the dithiothreitol concentration was reduced to 1 mM and the reaction was allowed to proceed for 4 h or longer at 37°C. The extent of conversion of ACP bo acyl-ACP was monitored using the filter disk assay as above and expressed relative to the initial ACP concentration determined using the micro-BCA protein assay (Pierce) with bovine serum albumin as a standard; this assay was validated using an A,,, (1 mg/ml) of 0.20 for stock solutions of E. coli ACP (17). For SDS-PAGE analysis (18), the reaction mixture was terminated by adding an equal volume of sample buffer (125 mM Tris-HCI, pH 6.8, 25%, v/v, glycerol, and 2.5% SDS). Protein samples were separated on 15% polyacrylamide slab gels in the absence of reducing agents and the radioactive acyl-ACP bands were observed by fluorography. In some experiments, the labeled acyl-ACP product was isolated by modification of published methods (19). The reaction mixture was applied to a small (1 ml) DEAE-Sepharose column equilibrated in 10 mM Mes (pH 6.0). The column was eluted with this buffer containing 80% 2-propanol to remove free fatty acid; acyl-ACP was subsequently eluted with 10 mM Mes containing 0.6 M Lick. This fraction was applied to an octyl-Sepharose column to separate acyl-ACP from ACP; fractions containing the former were reapplied to DEAE-Sepharose to remove 2-propanol (19).
36
SHEN,
Fraction
FICE,
AND
BYERS
Fraction
Number
FIG. 1. Fractionation of V. harveyi acyl-ACP synthetase by dye-ligand chromatography. Cell-free extract from the AFM mutant (440 mg protein in 63 ml) was applied to a Cibacron blue F3GA-agarose column (10 X 2.5 cm) in 20 mM Tris-HCl (pH 7.5), 10% glycerol, 1 mM EDTA, 10 KM DTT, and 10 mM MgSO,. After washing with 80 ml of this buffer, enzyme activity was eluted (see arrow) with 20 mM TrisHCl (pH 7.5), 10% glycerol, 1 mM EDTA, 10 pM DTT, 0.5 M NaCl. The flow rate was 1 ml/min and lo-ml fractions were collected. AcylACP synthetase activity (0) was monitored as pmol of [3H]myristoylACP formed/min/pl of fraction in the filter disk assay (see text). The absorbance at 300 nM (- - -) of the fractions was multiplied by two for display on the y-axis.
RESULTS AND DISCUSSION The soluble acyl-ACP synthetase activity previously described in cell-free extracts of V. harveyi (12) is not suitable for quantitative preparation of acyl-ACP derivatives or for sensitive detection of ACP due to the presence of other acyl-ACP-utilizing enzymes and endogenous V. harveyi ACP, respectively. In the present study, we have partially purified the enzyme using a simple two-step procedure involving dye-ligand and gel filtration chromatography. The first step in the procedure takes advantage of enhanced binding of the enzyme to Cibacron blue FSGA agarose in the presence of Mg’+; although some activity could subsequently be eluted by EDTA alone, maximal recovery of synthetase activity was obtained by elution with buffer containing 0.5 M NaCl and 1 mM EDTA (Fig. 1). After concentration, this material eluted in a single peak of activity on Sephacryl S-300 gel filtration, displaced from the major protein peak (Fig. 2). Calibration of this column with protein standards of known molecular weight indicated that acyl-ACP synthetase might be part of an enzyme complex of about 500 kDa; further purification resulted in increased instability of the enzyme, possibly due to disruption of this complex. The presence of glycerol (lo%, v/v) in all buffers during purification was essential for good recovery of activity. In five representative preparations, the average purification of synthetase activity was 25-fold with a yield of 30% (about 500 units of activity). At this stage of purification, V. harveyi acylACP synthetase was stable for several months at -20°C and was not inactivated by repeated freeze/thaw cycles. To determine the potential of Sephacryl S-300-purified V. harueyi acyl-ACP synthetase for the preparation
Number
FIG. 2. Gel filtration of V. harueyi acyl-ACP synthetase. Active fractions from Cibacron blue chromatography were concentrated and applied to a Sephacryl S-300 HR column (95 X 1.5 cm) in 20 mM Tris-HCl (pH 7.5), 10% glycerol, 1 mM EDTA, and 50 pM DTT. The sample contained 30 mg of protein in 2 ml. The flow rate was 11 ml/h and l&ml fractions were collected. The acyl-ACP synthetase activity (0) was monitored as pmol of [3H]myristoyl-ACP formed/min/pl fraction; the absorbance at 300 nM (---) was multiplied by two for display on the y-axis.
of acyl-ACP derivatives, conditions for optimal acylation of E. coli ACP (200 Kg) with [3H]myristic acid were investigated using a filter disk assay (Fig. 3). The time necessary for maximum (90%) conversion of ACP to [3H]myristoyl-ACP was dependent upon the enzyme
60
I 0
60
120
180
Time (min) FIG. 3. Effects of enzyme and fatty acid concentration on myristoyl-ACP formation by V. harueyi acyl-ACP synthetase. (A) E. coli ACP (190 pg, 70 PM final concentration) was incubated with [3H]myristic acid (440 PM, 1 Ci/mmol) and ATP in the presence of (0) 0.5, (A) 1.0, and (w) 1.5 units per milliliter of acyl-ACP synthetase in a total volume of 0.3 ml as outlined in the text. (B) ACP (190 pg) was incubated with (0) 180 FM, (A) 440 FM, and (m) 710 pM [3H]myristic acid (1 Ci/mmol) and ATP in the presence of 1.0 unit per milliliter of acyl-ACP synthetase. At the times indicated, duplicate lo-p1 samples were withdrawn and [3H]myristoyl-ACP was quantitated using the filter disk assay. Results are expressed as percentage conversion of ACP to acyl-ACP.
ENZYMATIC TABLE
PREPARATION
1
Acylation of ACP with Fatty Acids of Different Chain Lengths by V. harueyi Acyl-ACP Synthetase Fatty
acid 6:O 8:O lo:o 12:o 14:o 16:0
Acyl-ACP
(%l” 91 90 84 86 91 sgb
a Each reaction mixture (0.9 ml) contained Tris-HCl (0.1 M, pH 7.8), MgCl, (10 mM), ATP (10 mM), dithiothreitol (1 mM), E. coli ACP (400 fig, 50 PM), acyl-ACP synthetase (0.45 units), and l-*%-fatty acids (250-300 FM). After incubation for 6 h at 37”C, labeled acylACP was quantitated using the filter disk assay (mean of triplicate lo-al samples) and expressed as a percentage of the initial ACP concentration. For each sample, acylation was also verified visually by SDS-PAGE and Auorography. b Incubation time for the 16:0 reaction was 24 h.
concentration (Fig. 3A) and incubation for up to 24 h did not decrease the final extent of conversion regardless of the amount of enzyme used. The rate and extent of acylACP formation was also relatively independent of fatty acid concentration at fatty acid to ACP ratios of five or greater, while some decrease in acylation was noted at lower fatty acid concentrations (Fig. 3B). Acyl-ACP formation was unaffected over the range of ACP concentration tested in the present experiments (13-70 PM); this range spans the K,,, of the enzyme for E. coli ACP [20 PM, (12)]. This is in contrast to the reaction catalyzed by E. coli acyl-ACP synthetase, which requires ACP concentrations less than 15 @M for maximum acylation (7). V. harueyi acyl-ACP synthetase is capable of utilizing a broad range of fatty acid substrates for acyl-ACP formation. As shown in Table 1, conversion of ACP to acylACP in the reaction mixture was greater than 85% for all saturated fatty acids tested between 6 and 16 carbons. The rate of acylation with 16:0 was lower than with shorter fatty acids, as noted earlier (12), although increased yields could be obtained by increasing the reaction time or enzyme concentration. Partial acylation (about 20%) with l&O and no appreciable formation of 4:0-ACP was observed under these conditions. Further purification of acyl-ACP by DEAE-Sepharose and octyl-Sepharose chromatography (19) usually resulted in some loss on this scale of preparation, although the resulting yield of product (>60%) was still greater than that observed previously for the E. coli enzyme [between 5 and 50%, (7)]. SDS-PAGE and fluorography of acyl-ACP prepared using 14C-fatty acids revealed a single labeled band corresponding to the acyl-ACP stained with Coomassie blue (Fig. 4). Acyl-ACP products migrated faster than
OF
ACYL
CARRIER
PROTEIN
37
unacylated ACP on SDS-PAGE; this property has been noted previously and has been attributed to enhanced binding of SDS to the acidic ACP molecule upon fatty acylation (10). The biological activity of the 14:0-ACP product synthesized using acyl-ACP synthetase was confirmed by incubation with the V. harveyi bioluminescence-specific acyl esterase, the enzyme responsible for cleavage of 14:0-ACP to form 14:0 for reduction to myristaldehyde (16). Complete cleavage of [“H]14:0ACP by this enzyme was observed both by SDS-PAGE and fluorography as well as by recovery of hexane-extractable [3H]14:0 (data not shown). As the esterase is fairly specific for myristic acid (20), we conclude that V. harueyi acyl-ACP synthetase forms biologically active thioester derivatives of ACP with no further side reaction. Further experiments were conducted to establish whether fatty acylation by V. harveyi acyl-ACP synthetase reaction can be employed to detect and/or quantitate ACP in solution or in extracts from other bacterial species. Monitored using the filter disk assay, acyl-ACP formation with 0.5 units per milliliter of enzyme was linear over a range of E. coli ACP from 1 to 400 pmol during a 4-h incubation (Fig. 5). This sensitivity and range is similar to that reported previously for E. coli acyl-ACP synthetase under comparable conditions (2.5 to 250 pmol) (17). It should be noted that V. harueyi acyl-ACP synthetase activity is inhibited by monovalent salts and detergents such as Triton X-100 (50% inhibition at 0.1 M and 0.05%, v/v, respectively) (la),
FIG. 4. SDS-PAGE analysis of E. coli acyl-ACP products synthesized using V. harveyi acyl-ACP synthetase. ACP from E. cob (2.4 pg) was incubated for 12 h with acyl-ACP synthetase (0.01 units), ATP, and l-“C-labeled fatty acids (80 pM) in a total volume of 20 ~1 as indicated (no fatty acid was present in the first lane). After addition of SDS sample buffer, half of the reaction mixture was separated by SDS-PAGE. The Coomassie blue staining profile is shown in A while a fluorogram (7.day exposure) is shown in B (labeling intensities reflect different specific radioactivities of fatty acid substrates).
38
SHEN,
FICE,
,021 lo?
IO'
102
103
ACP (pmol)
FIG. 5. Quantitation of E. coli ACP by acylation with V. harueyi acyl-ACP synthetase using the filter disk assay. Reaction mixtures (50 ~1 total volume) were prepared as described in the text and contained [3H]myristic acid (80 FM, 1 Ci/mmol), acyl-ACP synthetase (0.02 units), and varying amounts of ACP. After incubation for 4 h at 37”C, 10.~1 samples containing the amount of ACP indicated were removed and [3H]acyl-ACP formation was determined using the filter disk assay. Each point represents the mean counts per minute of triplicate samples from one reaction mixture; the results from two independent experiments are shown on a double logarithmic scale. The assay was linear down to 1 pmol of ACP, or about 200 cpm above blanks minus ACP (600 cpm).
AND
BYERS
the major protein component (Fig. 4). Second, V. harveyi acyl-ACP synthetase is a soluble enzyme and exhibits optimal activity at low ionic strength and in the absence of detergents. This is in contrast to E. coli acylACP synthetase, which requires Triton X-100 for solubility and high concentrations of LiCl for release of acyl-ACP (9). Third, the extent of acyl-ACP formation with the V. harveyi enzyme appears to be largely independent of ACP concentration and the scale of the preparation (compare Figs. 3, 4, and Table 1). Finally, V. harveyi acyl-ACP synthetase quantitatively (>85%) acylates ACP using fatty acids from 6 to 16 carbons in length. This range overlaps that of the E. coli enzyme, which provides maximum yields with 14 to 18 carbon fatty acids, although some acylation with 8:0 to 12:0 is also noted (6,19,23). Thus, preparation of medium to long chain fatty acyl-ACP derivatives can be performed using a single procedure, rather than a combination of chemical and enzymatic methods.
ACKNOWLEDGMENTS
although these agents can normally be diluted out or corrected for in the final assay volume. In other experiments (not shown), acylation with the V. harveyi enzyme has been used to monitor the purification of V. harveyi ACP by filter disk assay and to detect endogenous ACP in extracts of Photobacteriumphosphoreum, a luminescent bacteria that appears to lack endogenous acyl-ACP synthetase activity (12), by SDS-PAGE and fluorography. The activity of acyl-ACP synthetase with ACP substrates from three different bacterial species is consistent with the high degree of ACP structural homology among different organisms (21) and suggests that the V. harveyi enzyme may be useful for a wider range of bacterial and plant species. The only other enzyme that has been described with the ability to directly ligate long chain fatty acids to ACP is E. coli acyl-ACP synthetase/2-acylglycerolphosphatidylethanolamine acyltransferase, a 27-kDa inner membrane enzyme that uses a tightly bound molecule of ACP as an intermediate in the activation and transfer of fatty acids to form phosphatidylethanolamine (9). That enzyme has been employed for the preparation of acylACP derivatives to study the reactions of bacterial (2225) and plant (26-28) fatty acid metabolism, phospholipid synthesis (29,30), lipopolysaccharide synthesis (3), and fatty acylation of proteins (5). We believe that V. harveyi acyl-ACP synthetase offers an attractive alternative to the E. coli enzyme for a number of reasons. First, a suitable enzyme preparation that is stable for several months can be obtained after two simple purification steps. This preparation appears to be free of contaminating activities that utilize acyl-ACP (8), allowing long reaction times under conditions in which ACP is
The authors are grateful to Harold W. Cook, Palmer, and Matthew W. Spence for their helpful gestions.
Frederick comments
B. St. C. and sug-
REFERENCES 1. Vanden
Boom,
T., and Cronan,
J. E., Jr. (1989)
Annu.
Reu. Micro-
biol. 43, 317-343. 2. Rock, 3.
C. O., and Jackowski, 10,765. Brozek, K. A., and Raetz, 15,410-15,417.
S. (1982)
J. Biol.
Chem.
C. R. H. (1990)
J. Biol.
4. Therisod,
Proc.
5.
C. (1991)
H., and Kennedy, E. P. (1987) USA 64,8235-8238. Issartel, J.-P., Koronakis, V., and Hughes, 759-761.
6. Ray,
T. K., and 73,4374-4378.
Cronan,
J. E., Jr.
USA
7. Rock,
C. O., and
Garwin,
J. L. (1979)
(1976)
257,10,759-
NC&.
Proc.
J. Biol.
Chem. Ad.
Sci.
Nature
351,
N&l. Chen.
265,
Acad.
Sci.
254,
7123-
J. E., Jr.
(1979)
7128.
8. Spencer, FEBSLett.
9. Cooper, Biol.
A. K.,
Greenspan,
A. D., and
Cronan,
101,253-256.
C. L., Hsu, L., Jackowski, Chem. 264, 7384-7389.
S., and
Rock,
C. 0.
(1989)
J.
10. Jaworski, J. G., and Stumpf, P. K. (1974) Arch. Biochem. Biophys. 162, 166-173. 11. Cronan, J. E., Jr., and Klages, A. L. (1981) Proc. N&l. Acad. Sci. USA
78,
5440-5444.
12. Byers,
D. M., and Holmes, C. G. (1990) Biochem. Cell Biol. 104551051. Byers, D. M. (1988) Biochem. Cell Biol. 66, 741-749. Byers, D. M. (1989) J. Bacterial. 171, 59-64.
13. 14. 15. Wall, L. A., Byers, 159,720-724.
D. M., and Meighen,
E. A. (1984)
68,
J. Bacterial.
ENZYMATIC 16. Byers, D. M., and USA 82,6085%6089.
Meighen,
PREPARATION
E. A. (1985)
Proc.
Natl.
Acad.
OF Sci.
ACYL
23.
CARRIER
39
PROTEIN
Spencer, A. K., Greenspan, Biol. Chem. 253, 5922-5926.
A. D., and Cronan,
24.
19. Rock, C. O., Garwin, J. L.. and Cronan, J. E., Jr. (1981) in Methods in Enzymology (Lowenstein, J. M., Ed.), Vol. 72, pp. 397-403, Academic Press, San Diego, CA.
26. Mattoo, A. Ii., Callahan, F. E., Mehta, R. A., and Ohlrogge, J. B. (1988) Plant Physiol. 89, 7077711. 27. Post-Beittenmiller, D., Jaworski, J. G.! and Ohlrogge, J. B. (1991) J. Biol. Chem. 266, 18581865. 28. Pollard, M. R., Anderson, L., Fan, C., Hawkins, D. J., and Davies, H. M. (1991) Arch. Biochem. Biophys. 284, 306-312.
and
21. Rock, C. O., and 9778-9785. 22.
Guerra, D. J., and 280, 336-345.
Meighen, Cronan, Browse,
E. A. J. E., Jr.
(1991)
J. Biol.
Chem.
266,
J. Biol.
Chem.
254,
(1979)
J. A. (1990)
Arch.
Biochem.
Biophys.
29. Rock, 30. Green, Chem.
C. 0. (1984) J. Biol. Chem. P. R., Merrill, A. H., Jr., 256, 11,151-11,159.
J. Lipid
Res. 25,
J.
17. Rock, C. O., and Cronan, J. E., Jr. (1981) in Methods in Enzymology (Lowenstein, J. M., Ed.), Vol. 71, pp. 341-351, Academic Press, San Diego, CA. 18. Laemmli, U. K. (1970) Nature 227, 680-685.
20. Ferri, S. R., 12,852-12,857.
Cooper, C. L., and Lueking, D. R. (1984) 1232. 25. Garwin, J. L., Klages, A. L., and Cronan, Chem. 255, 11,949-11,956.
J. E., Jr. (1978)
J. E., Jr. (1980)
259, 6188-6194. and Bell, R. M.
(1981)
1222J. Biol.
J. Biol.
