[5]
GLYCEROPHOSPHATE
ACYLTRANSFERASE
FROM
E. coli
55
[5] G l y c e r o p h o s p h a t e A c y l t r a n s f e r a s e f r o m Escherichia coli B y MARK A. SCHEIDELER a n d ROBERT M. BELL
Introduction
The initial step in the assembly of membrane phospholipids in Escherichia coli is controlled by the membrane-associated sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15). Two water-soluble substrates, snglycerol 3-phosphate and an acyl-coenzyme A (CoA) or acyl carrier protein (ACP) thioester, are catalytically utilized to form a 1-acylglycerol 3-phosphate (lysophosphatidic acid, LPA) lipid product.~ The further acylation of LPA to phosphatidic acid (PA) is promoted by a distinct monoacylglycerol-3-phosphate acyltransferase. 2 Since both enzyme activities are tightly associated with the cytoplasmic side of the inner membrane, 3 it has been difficult to determine clearly the positional specificity and acyl thioester preference of the glycerophosphate acyltransferase or to establish directly its role in the thermal regulation of phospholipid acyl chain composition and membrane fluidity. For this reason, it has been necessary to first devise purification conditions which yield stable and homogeneous enzyme preparations which are devoid of the monoacylglycerophosphate acyltransferase activity. Several key strategies have been implemented in order to purify and characterize the glycerophosphate acyltransferase. These are (1) cloning of the plsB structural gene encoding the glycerophosphate acyltransferase and construction of strains which overproduce the enzyme; (2) the effective use of nonionic detergents to solubilize the glycerophosphate acyltransferase from membranes and the development of chromatographic steps which resolve the solubilized, hydrophobic proteins; and (3) the design of a quantitatively efficient reconstitution methodology to restore activity to enzyme preparations which are stable, but inactive, in the presence of detergent. B a c t e r i a l S t rai n s
The plsB structural gene encoding the 83,000 M r glycerophosphate acyltransferase polypeptide4 was cloned by monitoring the ability of DNA I C. R. H. Raetz and W. Dowhan, J. Biol. Chem. 265, 1235 (1990). 2 j. Coleman, J. Biol. Chem. 265, 17215 0990). 3 C. O. Rock, S. E. Goeltz, and J. E. Cronan, Jr., J. BioL Chem. 256, 737 (1981). 4 Apparent molecular weight estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Alignment of the open reading frame of the sequenced
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
ACYLTRANSFERASES
[5]
from a total E. coli library to overcome the glycerol 3-phosphate growth requirement of a plsB- mutant strain) A strain (VL3/pVL1) containing hybrid plasmids constructed from this DNA expresses a level of enzyme activity that is 10-fold higher than that present in wild-type E. coli6'7; this strain was used in the initial purification experiments. In later studies, the enzyme was purified from a bacterial strain (VL3/pLB3-4) with a higher plasmid copy number and 30-fold higher level of expression. 8 Bacterial strains containing hybrid plasmids in which the plsB gene was under the control of a heat- (hPL) or isopropyl-/3-o-thiogalactopyranoside(tac) inducible promoter have permitted structural studies of the enzyme. 9,10
Assay of Glycerophosphate Acyltransferase Glycerophosphate acyltransferase is routinely estimated at 25° by monitoring the incorporation of [3H]glycerophosphate into chloroform-soluble material) I The sn-[3H]glycerol 3-phosphate used in the assay is prepared enzymatically with glycerol kinase from [2-3H]glycerol (200 mCi/mmol, New England Nuclear, Boston, MA).~2 The specific activity of the product resolved on Dowex AG-1X8 is then diluted with sn-glycerol 3-phosphate. Alternatively, the [2-3H]glycerol can be diluted with glycerol prior to phosphorylation. The palmitoyl- and oleoyl-CoA acyl donors used in the assay can be purchased from Pharmacia P-L Biochemicals (Piscataway, N J). Palmitoyl- and cis-vaccenoyl-ACP are enzymatically synthesized by incubating ACP with free fatty acid and acyl-ACP synthetase.~3 The efficiency of this reaction is enhanced by using the reduced ACP species resolved from total E. coli ACP. Briefly, I0 ml of ACP (7.5 mg/ml) is incubated overnight at 4° with 100 rnM dithiothreitol (DTT) to maximize formation of the reduced species. Hydroxylamine is added to a concentration of 0.2 M, pH 8.0, and the solution is passed over a 1.5 x 40 cm
DNA fragment beating plsB with partial peptide sequence information obtained for the purified enzyme indicates a molecular weight of 91,260. 5 V. A. Lightner, T. J. Larson, P. Tailleur, G. D. Kantor, C. R. H. Raetz, R. M. Bell, and P. Modrich, J. Biol. Chem. 255, 9413 (1980). 6 p. R. Green, A. H. Merrill, Jr., and R. M. Bell, J. Biol. Chem. 256, 11151 (1981). 7 M. A. Scheideler and R. M. Bell, J. Biol. Chem. 261, 10990 (1986). 8 M. A. Scheideler and R. M. Bell, J. Biol. Chem. 264, 12455 (1989). 9 W. O. Wilkison, J. P. Walsh, J. M. Corless, and R. M. Bell, J. Biol. Chem. 261, 9951 (1986). 10 W. O. Wilkison and R. M. Bell, J. Biol. Chem. 263, 14505 (1988). II M. D. Snider and E. P. Kennedy, J. Bacteriol. 130, 1072 (1977). 12 y . Chang and E. P. Kennedy, J. Lipid Res. 8, 447 (1967). 13 C. O. Rock and J. L. Garwin, J. Biol. Chem. 254, 7123 (1979).
[5]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM E.
coli
57
Sephadex G-25 (fine) column to separate ACP and DTT. ACP elution is monitored by protein analysis using a modification of the Lowry method t4 and DTT by absorbance at A410. Reduced ACP binds to Affi-Gel 501 (BioRad, Richmond, CA) and is eluted with 5 mM 2-mercaptoethanol; this accounts for 25% of the total ACP. The final 100 /~1 glycerophosphate acyltransferase reaction mixture contains 150 mM Tris, pH 8.2, 0.2 M NaCI, 5 mM 2-mercaptoethanol, 1 mg/ml bovine serum albumin (BSA), 0-0.3 milliunits (mU) of the enzyme activity present in membranes or reconstituted samples (see below), and an acyl donor such as palmitoyl-CoA. After these additions arqmade at 4° the samples are equilibrated for 10 min at 25°. After the reaction is initiated by the addition of [3H]glycerophosphate, the incorporation of radiolabel into LPA is linear for at least 7 min at 25° and is terminated by the addition of 0.6 ml of 1% HCIO 4 (v/v). The labeled lipid products are extracted as follows: 3 ml of CHCI3/MeOH (1 : 2, v/v) is mixed with each sample. Two separate phases result after the further addition of 1 ml of CHC13 and 1 ml of 1% HCIO 4 (v/v). After brief centrifugation in a clinical centrifuge, the upper phase is aspirated and the remaining lower phase washed twice with 3 ml of I% H C 1 0 4 (v/v). A 1-ml sample is then added to a scintillation vial, evaporated to dryness, and mixed with 5 ml of Aquasol-2 (New England Nuclear) prior to counting. A unit of enzyme activity is defined as the incorporation of 1/xmol of glycerophosphate per minute under these conditions. The routine measurement of maximal enzyme velocity is performed in the presence of saturating palmitoyl-CoA and [3H]glycerophosphate; final concentrations of these substrates are 25 /~M and 5 raM, respectively. Adding BSA to the assay alleviates the detergent effect of palmitoyl- and oleoyl-CoA, but it is not required when using acyl-ACP derivatives or mixed micellar samples (see below). A total ionic strength of 0.2-0.4 M is required for optimal utilization of both acyl-CoA and acyl-ACP substrates; however, the presence of divalent cations in the assay has proved nonessential. 6 The glycerophosphate acyltransferase has a broad pH optimum which in homogeneous preparations is dictated by the lipids used to reconstitute activity. 8 Purification of Glycerophosphate Acyltransferase Membrane Preparation and Solubilization
Cells from E. coli strain VL3/pVLI are harvested by centrifugation at 5000 g for 15 min at 4°, washed once in 50 mM Tris, pH 8.2, containing 10 mM MgCI2, and resuspended (10 g wet weight/100 ml) in this same 14 G. L. Petersen, Anal. Biochem. 83, 346 (1977).
58
ACYLTRANSFERASES
[5]
buffer. The cell suspension is mixed with DNase I (0.5 mg/100 ml, Sigma, St. Louis, MO) at 4 ° for 30 min; this greatly reduces the viscosity of the preparation, thereby facilitating cell breakage. The cell digest is disrupted by two passes through an ice-cold French pressure cell at 16,000 psi. The flow-through changes color from light to dark brown as cells are broken; stabilization of the color signals that maximal disruption has been achieved. Membranes are collected by centrifugation at 200,000 g for 1 hr at 4°, homogenized (5 mg/ml of protein) in 50 mM Tris, pH 8.2, containing 0.5 M NaCl, and stirred at 4 ° for 45 min. Although salt extraction leads to a loss in enzyme activity (Table I) several proteins are removed in this step which are not resolved by subsequent chromatography steps. The salt-extracted membranes are recovered by centrifugation at 200,000 g for 1 hr at 4° and homogenized (10 mg/ml) in elution buffer [25 mM Tris, pH 8.2, containing 20% glycerol (w/v) and 5 mM 2-mercaptoethanol]. The glycerophosphate acyltransferase is extracted by adding 20% Triton X-100 (w/v) to achieve a final detergent concentration of 0.2% (w/v). After stirring for 45 min at 4°, the 83,000 Mr glycerophosphate acyltransferase polypeptide 4 is recovered in the supernatant following high-speed centrifugation. The ratio of detergent to protein is critical in this step; higher Triton X-100 concentrations than TABLE I RECOVERY OF GLYCEROPHOSPHATE ACYLTRANSFERASEACTIVITY AND 83,000 M r POLYPEPTIDE DURING PURIFICATIONa
Purification step Membranes Salt-extracted membranes Triton extract Matrex Gel Green A Octyl-Sepharose CL-4B Hydroxylapatite-HTP
Total activity b (units) 58 (14) 40 (10) 32 3.2 3.2 1.0
Total protein (mg)
Yield of 83,000 M r polypeptide (% of [3H]leucinelabeled protein) c
Purification (-fold) 83,000 Mr polypeptide
Specific activity
650
2.6
1
1
375
3.9
1.5
1.2
5.6 22.8 81.0 100.0
2.2 8.8 31.2 38.0
2.4 4.8 18.0 38.0
150 7.5 2.0 0.3
a Chromatography steps were performed in the presence of 0.5% Triton X-100 (w/v). Reprinted from Ref. 7 with permission of the Journal of Biological Chemistry. b Activities listed are for samples that were solubilized and reconstituted. Values in parentheses were obtained with membranes which were not solubilized and reconstituted prior to assay of glycerophosphate acyltransferase activity. c Procedures used to label VL3/pVL1 cell protein with [3H]leucine and quantitate the recovery of 83,000 M r polypeptide are described in Ref. 7.
[5]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM E. coli
59
used here do not further enhance the extraction. Although activity in the membrane preparations deteriorates during storage at - 7 0 °, solubilized preparations of the enzyme are indefinitely stable at this temperature.
Chromatographic Fractionation Purification is accomplished following three successive steps of fractionation based on hydrophobic dye interaction, combined sizing and hydrophobic interaction, and ion exchange. The efficiency of 83,000 Mr polypeptide recovery and reconstitution of activity under conditions which are quantitatively efficient is described for each step in Table I. All steps are performed at 4°. Matrex Gel Green A. The Triton X-100 extract (150 mg of protein) is diluted l : l (v/v) with elution buffer containing 0.2% Triton X-100 (w/v) and 0.2% deoxycholate (w/v); this is mixed batchwise with 4 ml of Matrex Gel Green A (Amicon, Danvers, MA) that had been equilibrated in elution buffer containing 0.2% Triton X-100 (w/v) and 0.1% deoxycholate (w/v). After gentle mixing for 1 hr the dye resin is pelleted by centrifugation at 3000 g for 5 min, washed twice with 90 ml of elution buffer containing 0.2% Triton X-100 (w/v) and 0.1% deoxycholate (w/v), and then washed twice with this same buffer containing 0.5 M NaCl. The glycerophosphate acyltransferase is then eluted from the resin by washing twice with 90 ml of this same buffer containing 3 M NaC1. The inclusion of deoxycholate reduces irreversible, nonspecific interactions between enzyme and the dye resin by adding a negative charge to the Triton X-100 micelles. 15 Binding of the glycerophosphate acyltransferase to Matrex Gel Red A is also high, but recoveries are lower. The enzyme does not bind strongly to either Blue or Orange Matrex Gel A resins. Octyl-Sepharose CL-4B. The pooled elution fractions from the previous step are directly loaded onto a 1.5 x 70 cm octyl-Sepharose CL-4B column (Pharmacia) that had been equilibrated in elution buffer containing 0.2% Triton X-100 (w/v). The column is eluted with a 900-ml linear gradient of 3 to 0.5 M NaCl, in elution buffer containing 0.2% Triton X-100 (w/v). Resolution of the glycerophosphate acyltransferase at this step is highly dependent on both the column geometry, indicating a partial fractionation based on size, and ionic strength of the eluting buffer, as predicted for the disruption of a hydrophobic interaction. Hydroxylapatite. The hydroxylapatite step serves to resolve inactive t5 j. B. Robinson, Jr., J. M. Strottman, D. G. Wick, and E. Stellwagen, Proc. Natl. Acad. Sci. U.S.A. 77, 5874 (1980).
60
ACYLTRANSFERASES
[5]
83,000 M r polypeptide generated during the Matrex Gel Green A step and concentrate homogeneous enzyme7; this step can be additionally employed to exchange Triton X-100 with another detergent. 8 The pooled fractions from the previous step are gently mixed for 1 hr with 4 g of hydroxylapatite-HTP (Bio-Rad) that had been equilibrated with elution buffer. The slurry is then loaded onto a 1.5-cm column, washed with 60 ml of elution buffer containing 0.2% Triton X-100 (w/v), and eluted with a 100-ml gradient of 0.05 to 0.5 M potassium phosphate, pH 7.0, in elution buffer containing 0.2% Triton X-100 (w/v). Preparations are homogeneous after this step; only a single 83,000 Mr band is apparent when eluting fractions are resolved electrophoretically on polyacrylamide gels run in the presence of sodium dodecyl sulfate. However, most of the protein eluting in the early (low ionic strength) fractions is inactive. Fractions eluting at high ionic strength contain high levels of glycerophosphate acyltransferase activity. These are pooled and stored at - 70°. Comment. Use of the E. coli strain VL3/pLB3-4 shortens the purification procedure considerably by obviating the need for an octyl-Sepharose CL-4B step) Elution fractions from the Matrex Gel Green A step are diluted 3-fold in elution buffer containing 0.2% Triton X-100 to lower the salt concentration and then directly mixed with hydroxylapatite. In addition, enzyme inactivation during the Matrex Gel Green A step is mostly avoided; only active enzyme is subsequently observed eluting from hydroxylapatite. The large proportion of overproduced enzyme present in membranes from VL3/pLB3-4 is a dimeric species 9 which may be resistant to inactivation. Reconstitution of Glycerophosphate Acyltransferase Incorporation into Vesicles
Glycerophosphate acyltransferse activities of solubilized membrane samples, and of samples taken at each step of the purification, are reconstituted by addition to phospholipid. 7'~1 In this procedure, a 5-/.,1 sample (0-0.3 mU of enzyme activity) in elution buffer containing 0.2% Triton X-100 (w/v) is diluted below the critical micellar concentration ( C M C ) 16 of the detergent, namely, 0.05% (w/v), by addition of 30 p,l of a concentrated solution of preformed phospholipid vesicles. The reconstituted sample is then added to the assay reaction mixture previously described. The recovery of activity is strictly dependent on 16A. Helenius, D. R. McCaslin, E. Fries, and C. Tanford, this series, Vol. 56, p. 734.
[5]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM E. coli
61
both the order of mixing and the phospholipid concentration. Quantitative enzyme reactivation observed by this procedure closely parallels the recovery of 83,000 Mr polypeptide during the purification (Table I). The total lipid extract used to make phospholipid vesicles is prepared from E. coli membranes 17 and stored in chloroform at - 2 0 °. The major lipids present are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and diphosphatidylglycerol (cardiolipin, CL) in the approximate molar ratio of 6 : 1 : 1.18 Following chloroform removal under nitrogen, phospholipids are suspended in 150 mM Tris, pH 8.2, containing 10 mM 2-mercaptoethanol at a concentration of 15 mg/ml. Phospholipid vesicles are formed by direct probe sonication of the suspension at room temperature; this should be continued until clearing of the suspension ceases and an orange-blue tint typical of vesicle formation is evident. Titaninum shed by the probe is pelleted by centrifugation in a clinical centrifuge for 5 min. Mixed Micellar Reconstitution
Mixed micellar reconstitution is a soluble assay system in which the glycerophosphate acyltransferase is activated by supplementing detergent micelles containing the enzyme with limiting amounts of specific cofactors. The goal of assaying the glycerophosphate acyltransferase in mixed micelles is to permit in oitro investigation of the lipid-protein stoichiometry required for the reconstitution of in oioo enzyme kinetics. C~2Eg is a chemically pure, nonionic detergent which forms a stable micelle size of 123 mol/micelle above its l l0/xM CMC value, 19 and it does not inhibit glycerophosphate acyltransferase activity when added to assays at concentrations exceeding its CMC. 8 During the glycerophosphate acyltransferase purification, 0.1% C12E8 (W/V) can be substituted for Triton X-100 in the wash and elution steps of hydroxylapatite chromatography. However, CI2E8 is tOO mild to be used in the initial membrane solubilization and fractionation steps. Maximal levels of activity, equal to those measured following vesicle reconstitution of homogeneous enzyme, are achieved by mixing homogeneous glycerophosphate acyltransferase (0-0.3 mU) in 0.1% CI2E8 (w/v) with 450 /xg of E. coli phospholipid vesicles, or with 150 /xg of vesicles formed from pure E. coli phosphatidylglycerol or cardiolipin
17 E. A. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 18 j. E. Cronan, Jr., and P. R. Vagelos, Biochim. Biophys. Acta 265, 25 (1972). 19 C. Tanford, Y. Nozaki, and M. F. Rhode, J. Phys. Chem. 81, 1555 (1977).
62
ACYLTRANSFERASES
[5]
100
75 {E.¢oli)
>
E E
PO ( E_.col._.ji) 5O
i--
_>
I-U
0 25
0 w" 0
I 0.3
I 0.6
I 0.9
I 1.2
PHOSPHOLIPID/C12E 8 Jrnol/mol ) FIG. 1. Specific phospholipid requirement for enzyme activation. Data are given as the final ratio (mol/mol) in the assay of phospholipid to detergent. (Reprinted from Ref. 8 with
permission of the Journal of Biological Chemistry.)
(Fig. 1). It is important to note that the size of a nonionic detergent micelle increases predictably as phospholipid is added, 2° up to 50 mol%. Thus, the final phospholipid/C12E8 ratio after mixing and addition to the assay reaction mixture should not exceed 2:1 (mol/mol). Unilamellar phospholipid vesicles are employed as the lipid source during mixing with detergent owing to their ease of solubilization. Reconstitution of the glycerophosphate acyltransferase at a low phospholipid/C12E8 ratio in cardiolipin/C~2E 8 mixed micelles eliminates the stringent order of enzyme-phospholipid-detergent addition required when using E. coli 20 R. J. Robson and E. A. Dennis, Biochim. Biophys. Acta 508, 513 (1978).
[5]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM E. coli
63
lipid preparations containing a low percentage of activating cofactor lipid. Characterization of Glycerophosphate Acyltransferase Reconstituted samples of homogeneous glycerophosphate acyltransferase acylate sn-glycerol 3-phosphate at the sn-1 position. Reaction products migrate with authentic 1-acylglyerol phosphate on thin-layer chromatography plates, and with 1-acylglycerol following phosphatase treatment. 6 Further, sn-glycerol 3-phosphate analogs which lack a secondary hydroxyl group (dihydroxyacetone phosphate, ethylene glycol phosphate, and 1,3propanediot phosphate) work in its place as substrates. 2~Taken together, these results suggest that the primary acylation product of the homogeneous/reconstituted enzyme is 1-acylglycerol 3-phosphate. The reconstituted enzyme preparations utilize the palmitoyl and cisvaccenoyl thioester analogs of ACP as acyl donor in the assay, in addition to the palmitoyl and oleoyl analogs of coenzyme A . 6'7 However, a detailed kinetic investigation to establish an in vitro acyl donor preference has not yet been done. Large amounts of E. coli phospholipid are required to activate fully the homogeneous glycerophosphate acyltransferase6-8; this result suggests that minor lipids present in the total lipid extract are responsible for activation. Enzyme reconstituted in mixed phospholipid-C~2E8 micelles is activated by approximately 50 mol of phosphatidylglycerol or cardiolipin per mole of enzyme. 8 Phosphatidylethanolamine, the major lipid component of the E. coli extracts, reconstitutes activity inefficiently and in a pHdependent manner. Much of the membrane-associated glycerophosphate acyltransferase activity present in membranes from E. coli strains which overproduce the enzyme is latent until solubilized and reconstituted into an excess of activating phospholipid. The latent activity is organized as crystalline arrays of dimeric enzyme, 9'~° whereas the active form of the enzyme reconstituted into mixed cardiolipin-C~2E8 micelles is monomeric.8Recent kinetic analyses of monomeric and dimeric preparations of homogeneous enzyme have revealed profound differences in their relative efficiencies of glycerol phosphate and palmitoyl-CoA utilization. 22
21 p. R. Green and R. M. Bell, Biochim. Biophys. Acta 795, 348 0984). 22 M. A. Scheideler and R. M. Bell, J. Biol. Chem. 266, 14321 (1991).
GLYCEROPHOSPHATE
ACYLTRANSFERASE
FROM
E. coli
55
[5] G l y c e r o p h o s p h a t e A c y l t r a n s f e r a s e f r o m Escherichia coli B y MARK A. SCHEIDELER a n d ROBERT M. BELL
Introduction
The initial step in the assembly of membrane phospholipids in Escherichia coli is controlled by the membrane-associated sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15). Two water-soluble substrates, snglycerol 3-phosphate and an acyl-coenzyme A (CoA) or acyl carrier protein (ACP) thioester, are catalytically utilized to form a 1-acylglycerol 3-phosphate (lysophosphatidic acid, LPA) lipid product.~ The further acylation of LPA to phosphatidic acid (PA) is promoted by a distinct monoacylglycerol-3-phosphate acyltransferase. 2 Since both enzyme activities are tightly associated with the cytoplasmic side of the inner membrane, 3 it has been difficult to determine clearly the positional specificity and acyl thioester preference of the glycerophosphate acyltransferase or to establish directly its role in the thermal regulation of phospholipid acyl chain composition and membrane fluidity. For this reason, it has been necessary to first devise purification conditions which yield stable and homogeneous enzyme preparations which are devoid of the monoacylglycerophosphate acyltransferase activity. Several key strategies have been implemented in order to purify and characterize the glycerophosphate acyltransferase. These are (1) cloning of the plsB structural gene encoding the glycerophosphate acyltransferase and construction of strains which overproduce the enzyme; (2) the effective use of nonionic detergents to solubilize the glycerophosphate acyltransferase from membranes and the development of chromatographic steps which resolve the solubilized, hydrophobic proteins; and (3) the design of a quantitatively efficient reconstitution methodology to restore activity to enzyme preparations which are stable, but inactive, in the presence of detergent. B a c t e r i a l S t rai n s
The plsB structural gene encoding the 83,000 M r glycerophosphate acyltransferase polypeptide4 was cloned by monitoring the ability of DNA I C. R. H. Raetz and W. Dowhan, J. Biol. Chem. 265, 1235 (1990). 2 j. Coleman, J. Biol. Chem. 265, 17215 0990). 3 C. O. Rock, S. E. Goeltz, and J. E. Cronan, Jr., J. BioL Chem. 256, 737 (1981). 4 Apparent molecular weight estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Alignment of the open reading frame of the sequenced
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
ACYLTRANSFERASES
[5]
from a total E. coli library to overcome the glycerol 3-phosphate growth requirement of a plsB- mutant strain) A strain (VL3/pVL1) containing hybrid plasmids constructed from this DNA expresses a level of enzyme activity that is 10-fold higher than that present in wild-type E. coli6'7; this strain was used in the initial purification experiments. In later studies, the enzyme was purified from a bacterial strain (VL3/pLB3-4) with a higher plasmid copy number and 30-fold higher level of expression. 8 Bacterial strains containing hybrid plasmids in which the plsB gene was under the control of a heat- (hPL) or isopropyl-/3-o-thiogalactopyranoside(tac) inducible promoter have permitted structural studies of the enzyme. 9,10
Assay of Glycerophosphate Acyltransferase Glycerophosphate acyltransferase is routinely estimated at 25° by monitoring the incorporation of [3H]glycerophosphate into chloroform-soluble material) I The sn-[3H]glycerol 3-phosphate used in the assay is prepared enzymatically with glycerol kinase from [2-3H]glycerol (200 mCi/mmol, New England Nuclear, Boston, MA).~2 The specific activity of the product resolved on Dowex AG-1X8 is then diluted with sn-glycerol 3-phosphate. Alternatively, the [2-3H]glycerol can be diluted with glycerol prior to phosphorylation. The palmitoyl- and oleoyl-CoA acyl donors used in the assay can be purchased from Pharmacia P-L Biochemicals (Piscataway, N J). Palmitoyl- and cis-vaccenoyl-ACP are enzymatically synthesized by incubating ACP with free fatty acid and acyl-ACP synthetase.~3 The efficiency of this reaction is enhanced by using the reduced ACP species resolved from total E. coli ACP. Briefly, I0 ml of ACP (7.5 mg/ml) is incubated overnight at 4° with 100 rnM dithiothreitol (DTT) to maximize formation of the reduced species. Hydroxylamine is added to a concentration of 0.2 M, pH 8.0, and the solution is passed over a 1.5 x 40 cm
DNA fragment beating plsB with partial peptide sequence information obtained for the purified enzyme indicates a molecular weight of 91,260. 5 V. A. Lightner, T. J. Larson, P. Tailleur, G. D. Kantor, C. R. H. Raetz, R. M. Bell, and P. Modrich, J. Biol. Chem. 255, 9413 (1980). 6 p. R. Green, A. H. Merrill, Jr., and R. M. Bell, J. Biol. Chem. 256, 11151 (1981). 7 M. A. Scheideler and R. M. Bell, J. Biol. Chem. 261, 10990 (1986). 8 M. A. Scheideler and R. M. Bell, J. Biol. Chem. 264, 12455 (1989). 9 W. O. Wilkison, J. P. Walsh, J. M. Corless, and R. M. Bell, J. Biol. Chem. 261, 9951 (1986). 10 W. O. Wilkison and R. M. Bell, J. Biol. Chem. 263, 14505 (1988). II M. D. Snider and E. P. Kennedy, J. Bacteriol. 130, 1072 (1977). 12 y . Chang and E. P. Kennedy, J. Lipid Res. 8, 447 (1967). 13 C. O. Rock and J. L. Garwin, J. Biol. Chem. 254, 7123 (1979).
[5]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM E.
coli
57
Sephadex G-25 (fine) column to separate ACP and DTT. ACP elution is monitored by protein analysis using a modification of the Lowry method t4 and DTT by absorbance at A410. Reduced ACP binds to Affi-Gel 501 (BioRad, Richmond, CA) and is eluted with 5 mM 2-mercaptoethanol; this accounts for 25% of the total ACP. The final 100 /~1 glycerophosphate acyltransferase reaction mixture contains 150 mM Tris, pH 8.2, 0.2 M NaCI, 5 mM 2-mercaptoethanol, 1 mg/ml bovine serum albumin (BSA), 0-0.3 milliunits (mU) of the enzyme activity present in membranes or reconstituted samples (see below), and an acyl donor such as palmitoyl-CoA. After these additions arqmade at 4° the samples are equilibrated for 10 min at 25°. After the reaction is initiated by the addition of [3H]glycerophosphate, the incorporation of radiolabel into LPA is linear for at least 7 min at 25° and is terminated by the addition of 0.6 ml of 1% HCIO 4 (v/v). The labeled lipid products are extracted as follows: 3 ml of CHCI3/MeOH (1 : 2, v/v) is mixed with each sample. Two separate phases result after the further addition of 1 ml of CHC13 and 1 ml of 1% HCIO 4 (v/v). After brief centrifugation in a clinical centrifuge, the upper phase is aspirated and the remaining lower phase washed twice with 3 ml of I% H C 1 0 4 (v/v). A 1-ml sample is then added to a scintillation vial, evaporated to dryness, and mixed with 5 ml of Aquasol-2 (New England Nuclear) prior to counting. A unit of enzyme activity is defined as the incorporation of 1/xmol of glycerophosphate per minute under these conditions. The routine measurement of maximal enzyme velocity is performed in the presence of saturating palmitoyl-CoA and [3H]glycerophosphate; final concentrations of these substrates are 25 /~M and 5 raM, respectively. Adding BSA to the assay alleviates the detergent effect of palmitoyl- and oleoyl-CoA, but it is not required when using acyl-ACP derivatives or mixed micellar samples (see below). A total ionic strength of 0.2-0.4 M is required for optimal utilization of both acyl-CoA and acyl-ACP substrates; however, the presence of divalent cations in the assay has proved nonessential. 6 The glycerophosphate acyltransferase has a broad pH optimum which in homogeneous preparations is dictated by the lipids used to reconstitute activity. 8 Purification of Glycerophosphate Acyltransferase Membrane Preparation and Solubilization
Cells from E. coli strain VL3/pVLI are harvested by centrifugation at 5000 g for 15 min at 4°, washed once in 50 mM Tris, pH 8.2, containing 10 mM MgCI2, and resuspended (10 g wet weight/100 ml) in this same 14 G. L. Petersen, Anal. Biochem. 83, 346 (1977).
58
ACYLTRANSFERASES
[5]
buffer. The cell suspension is mixed with DNase I (0.5 mg/100 ml, Sigma, St. Louis, MO) at 4 ° for 30 min; this greatly reduces the viscosity of the preparation, thereby facilitating cell breakage. The cell digest is disrupted by two passes through an ice-cold French pressure cell at 16,000 psi. The flow-through changes color from light to dark brown as cells are broken; stabilization of the color signals that maximal disruption has been achieved. Membranes are collected by centrifugation at 200,000 g for 1 hr at 4°, homogenized (5 mg/ml of protein) in 50 mM Tris, pH 8.2, containing 0.5 M NaCl, and stirred at 4 ° for 45 min. Although salt extraction leads to a loss in enzyme activity (Table I) several proteins are removed in this step which are not resolved by subsequent chromatography steps. The salt-extracted membranes are recovered by centrifugation at 200,000 g for 1 hr at 4° and homogenized (10 mg/ml) in elution buffer [25 mM Tris, pH 8.2, containing 20% glycerol (w/v) and 5 mM 2-mercaptoethanol]. The glycerophosphate acyltransferase is extracted by adding 20% Triton X-100 (w/v) to achieve a final detergent concentration of 0.2% (w/v). After stirring for 45 min at 4°, the 83,000 Mr glycerophosphate acyltransferase polypeptide 4 is recovered in the supernatant following high-speed centrifugation. The ratio of detergent to protein is critical in this step; higher Triton X-100 concentrations than TABLE I RECOVERY OF GLYCEROPHOSPHATE ACYLTRANSFERASEACTIVITY AND 83,000 M r POLYPEPTIDE DURING PURIFICATIONa
Purification step Membranes Salt-extracted membranes Triton extract Matrex Gel Green A Octyl-Sepharose CL-4B Hydroxylapatite-HTP
Total activity b (units) 58 (14) 40 (10) 32 3.2 3.2 1.0
Total protein (mg)
Yield of 83,000 M r polypeptide (% of [3H]leucinelabeled protein) c
Purification (-fold) 83,000 Mr polypeptide
Specific activity
650
2.6
1
1
375
3.9
1.5
1.2
5.6 22.8 81.0 100.0
2.2 8.8 31.2 38.0
2.4 4.8 18.0 38.0
150 7.5 2.0 0.3
a Chromatography steps were performed in the presence of 0.5% Triton X-100 (w/v). Reprinted from Ref. 7 with permission of the Journal of Biological Chemistry. b Activities listed are for samples that were solubilized and reconstituted. Values in parentheses were obtained with membranes which were not solubilized and reconstituted prior to assay of glycerophosphate acyltransferase activity. c Procedures used to label VL3/pVL1 cell protein with [3H]leucine and quantitate the recovery of 83,000 M r polypeptide are described in Ref. 7.
[5]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM E. coli
59
used here do not further enhance the extraction. Although activity in the membrane preparations deteriorates during storage at - 7 0 °, solubilized preparations of the enzyme are indefinitely stable at this temperature.
Chromatographic Fractionation Purification is accomplished following three successive steps of fractionation based on hydrophobic dye interaction, combined sizing and hydrophobic interaction, and ion exchange. The efficiency of 83,000 Mr polypeptide recovery and reconstitution of activity under conditions which are quantitatively efficient is described for each step in Table I. All steps are performed at 4°. Matrex Gel Green A. The Triton X-100 extract (150 mg of protein) is diluted l : l (v/v) with elution buffer containing 0.2% Triton X-100 (w/v) and 0.2% deoxycholate (w/v); this is mixed batchwise with 4 ml of Matrex Gel Green A (Amicon, Danvers, MA) that had been equilibrated in elution buffer containing 0.2% Triton X-100 (w/v) and 0.1% deoxycholate (w/v). After gentle mixing for 1 hr the dye resin is pelleted by centrifugation at 3000 g for 5 min, washed twice with 90 ml of elution buffer containing 0.2% Triton X-100 (w/v) and 0.1% deoxycholate (w/v), and then washed twice with this same buffer containing 0.5 M NaCl. The glycerophosphate acyltransferase is then eluted from the resin by washing twice with 90 ml of this same buffer containing 3 M NaC1. The inclusion of deoxycholate reduces irreversible, nonspecific interactions between enzyme and the dye resin by adding a negative charge to the Triton X-100 micelles. 15 Binding of the glycerophosphate acyltransferase to Matrex Gel Red A is also high, but recoveries are lower. The enzyme does not bind strongly to either Blue or Orange Matrex Gel A resins. Octyl-Sepharose CL-4B. The pooled elution fractions from the previous step are directly loaded onto a 1.5 x 70 cm octyl-Sepharose CL-4B column (Pharmacia) that had been equilibrated in elution buffer containing 0.2% Triton X-100 (w/v). The column is eluted with a 900-ml linear gradient of 3 to 0.5 M NaCl, in elution buffer containing 0.2% Triton X-100 (w/v). Resolution of the glycerophosphate acyltransferase at this step is highly dependent on both the column geometry, indicating a partial fractionation based on size, and ionic strength of the eluting buffer, as predicted for the disruption of a hydrophobic interaction. Hydroxylapatite. The hydroxylapatite step serves to resolve inactive t5 j. B. Robinson, Jr., J. M. Strottman, D. G. Wick, and E. Stellwagen, Proc. Natl. Acad. Sci. U.S.A. 77, 5874 (1980).
60
ACYLTRANSFERASES
[5]
83,000 M r polypeptide generated during the Matrex Gel Green A step and concentrate homogeneous enzyme7; this step can be additionally employed to exchange Triton X-100 with another detergent. 8 The pooled fractions from the previous step are gently mixed for 1 hr with 4 g of hydroxylapatite-HTP (Bio-Rad) that had been equilibrated with elution buffer. The slurry is then loaded onto a 1.5-cm column, washed with 60 ml of elution buffer containing 0.2% Triton X-100 (w/v), and eluted with a 100-ml gradient of 0.05 to 0.5 M potassium phosphate, pH 7.0, in elution buffer containing 0.2% Triton X-100 (w/v). Preparations are homogeneous after this step; only a single 83,000 Mr band is apparent when eluting fractions are resolved electrophoretically on polyacrylamide gels run in the presence of sodium dodecyl sulfate. However, most of the protein eluting in the early (low ionic strength) fractions is inactive. Fractions eluting at high ionic strength contain high levels of glycerophosphate acyltransferase activity. These are pooled and stored at - 70°. Comment. Use of the E. coli strain VL3/pLB3-4 shortens the purification procedure considerably by obviating the need for an octyl-Sepharose CL-4B step) Elution fractions from the Matrex Gel Green A step are diluted 3-fold in elution buffer containing 0.2% Triton X-100 to lower the salt concentration and then directly mixed with hydroxylapatite. In addition, enzyme inactivation during the Matrex Gel Green A step is mostly avoided; only active enzyme is subsequently observed eluting from hydroxylapatite. The large proportion of overproduced enzyme present in membranes from VL3/pLB3-4 is a dimeric species 9 which may be resistant to inactivation. Reconstitution of Glycerophosphate Acyltransferase Incorporation into Vesicles
Glycerophosphate acyltransferse activities of solubilized membrane samples, and of samples taken at each step of the purification, are reconstituted by addition to phospholipid. 7'~1 In this procedure, a 5-/.,1 sample (0-0.3 mU of enzyme activity) in elution buffer containing 0.2% Triton X-100 (w/v) is diluted below the critical micellar concentration ( C M C ) 16 of the detergent, namely, 0.05% (w/v), by addition of 30 p,l of a concentrated solution of preformed phospholipid vesicles. The reconstituted sample is then added to the assay reaction mixture previously described. The recovery of activity is strictly dependent on 16A. Helenius, D. R. McCaslin, E. Fries, and C. Tanford, this series, Vol. 56, p. 734.
[5]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM E. coli
61
both the order of mixing and the phospholipid concentration. Quantitative enzyme reactivation observed by this procedure closely parallels the recovery of 83,000 Mr polypeptide during the purification (Table I). The total lipid extract used to make phospholipid vesicles is prepared from E. coli membranes 17 and stored in chloroform at - 2 0 °. The major lipids present are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and diphosphatidylglycerol (cardiolipin, CL) in the approximate molar ratio of 6 : 1 : 1.18 Following chloroform removal under nitrogen, phospholipids are suspended in 150 mM Tris, pH 8.2, containing 10 mM 2-mercaptoethanol at a concentration of 15 mg/ml. Phospholipid vesicles are formed by direct probe sonication of the suspension at room temperature; this should be continued until clearing of the suspension ceases and an orange-blue tint typical of vesicle formation is evident. Titaninum shed by the probe is pelleted by centrifugation in a clinical centrifuge for 5 min. Mixed Micellar Reconstitution
Mixed micellar reconstitution is a soluble assay system in which the glycerophosphate acyltransferase is activated by supplementing detergent micelles containing the enzyme with limiting amounts of specific cofactors. The goal of assaying the glycerophosphate acyltransferase in mixed micelles is to permit in oitro investigation of the lipid-protein stoichiometry required for the reconstitution of in oioo enzyme kinetics. C~2Eg is a chemically pure, nonionic detergent which forms a stable micelle size of 123 mol/micelle above its l l0/xM CMC value, 19 and it does not inhibit glycerophosphate acyltransferase activity when added to assays at concentrations exceeding its CMC. 8 During the glycerophosphate acyltransferase purification, 0.1% C12E8 (W/V) can be substituted for Triton X-100 in the wash and elution steps of hydroxylapatite chromatography. However, CI2E8 is tOO mild to be used in the initial membrane solubilization and fractionation steps. Maximal levels of activity, equal to those measured following vesicle reconstitution of homogeneous enzyme, are achieved by mixing homogeneous glycerophosphate acyltransferase (0-0.3 mU) in 0.1% CI2E8 (w/v) with 450 /xg of E. coli phospholipid vesicles, or with 150 /xg of vesicles formed from pure E. coli phosphatidylglycerol or cardiolipin
17 E. A. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 18 j. E. Cronan, Jr., and P. R. Vagelos, Biochim. Biophys. Acta 265, 25 (1972). 19 C. Tanford, Y. Nozaki, and M. F. Rhode, J. Phys. Chem. 81, 1555 (1977).
62
ACYLTRANSFERASES
[5]
100
75 {E.¢oli)
>
E E
PO ( E_.col._.ji) 5O
i--
_>
I-U
0 25
0 w" 0
I 0.3
I 0.6
I 0.9
I 1.2
PHOSPHOLIPID/C12E 8 Jrnol/mol ) FIG. 1. Specific phospholipid requirement for enzyme activation. Data are given as the final ratio (mol/mol) in the assay of phospholipid to detergent. (Reprinted from Ref. 8 with
permission of the Journal of Biological Chemistry.)
(Fig. 1). It is important to note that the size of a nonionic detergent micelle increases predictably as phospholipid is added, 2° up to 50 mol%. Thus, the final phospholipid/C12E8 ratio after mixing and addition to the assay reaction mixture should not exceed 2:1 (mol/mol). Unilamellar phospholipid vesicles are employed as the lipid source during mixing with detergent owing to their ease of solubilization. Reconstitution of the glycerophosphate acyltransferase at a low phospholipid/C12E8 ratio in cardiolipin/C~2E 8 mixed micelles eliminates the stringent order of enzyme-phospholipid-detergent addition required when using E. coli 20 R. J. Robson and E. A. Dennis, Biochim. Biophys. Acta 508, 513 (1978).
[5]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM E. coli
63
lipid preparations containing a low percentage of activating cofactor lipid. Characterization of Glycerophosphate Acyltransferase Reconstituted samples of homogeneous glycerophosphate acyltransferase acylate sn-glycerol 3-phosphate at the sn-1 position. Reaction products migrate with authentic 1-acylglyerol phosphate on thin-layer chromatography plates, and with 1-acylglycerol following phosphatase treatment. 6 Further, sn-glycerol 3-phosphate analogs which lack a secondary hydroxyl group (dihydroxyacetone phosphate, ethylene glycol phosphate, and 1,3propanediot phosphate) work in its place as substrates. 2~Taken together, these results suggest that the primary acylation product of the homogeneous/reconstituted enzyme is 1-acylglycerol 3-phosphate. The reconstituted enzyme preparations utilize the palmitoyl and cisvaccenoyl thioester analogs of ACP as acyl donor in the assay, in addition to the palmitoyl and oleoyl analogs of coenzyme A . 6'7 However, a detailed kinetic investigation to establish an in vitro acyl donor preference has not yet been done. Large amounts of E. coli phospholipid are required to activate fully the homogeneous glycerophosphate acyltransferase6-8; this result suggests that minor lipids present in the total lipid extract are responsible for activation. Enzyme reconstituted in mixed phospholipid-C~2E8 micelles is activated by approximately 50 mol of phosphatidylglycerol or cardiolipin per mole of enzyme. 8 Phosphatidylethanolamine, the major lipid component of the E. coli extracts, reconstitutes activity inefficiently and in a pHdependent manner. Much of the membrane-associated glycerophosphate acyltransferase activity present in membranes from E. coli strains which overproduce the enzyme is latent until solubilized and reconstituted into an excess of activating phospholipid. The latent activity is organized as crystalline arrays of dimeric enzyme, 9'~° whereas the active form of the enzyme reconstituted into mixed cardiolipin-C~2E8 micelles is monomeric.8Recent kinetic analyses of monomeric and dimeric preparations of homogeneous enzyme have revealed profound differences in their relative efficiencies of glycerol phosphate and palmitoyl-CoA utilization. 22
21 p. R. Green and R. M. Bell, Biochim. Biophys. Acta 795, 348 0984). 22 M. A. Scheideler and R. M. Bell, J. Biol. Chem. 266, 14321 (1991).
