- Email: [email protected]
[15] Bioluminescence-related acyltransferases from Photobacterium phosphoreum and Vibrio harveyi
[15]
BIOLUMINESCENCE-RELATED ACYLTRANSFERASES
[15] B i o l u m i n e s c e n c e - R e l a t e d Photobacterium By
DAVID DYERS,
phosphoreum
Acyltransferases from and Vibrio harveyi
ANGEL RODRIGUEZ, L u c EDWARD
183
CAREY,
and
MEIGHEN
The long-chain aldehyde substrate of bacterial luciferase is synthesized via the reduction of fatty acids. ~,2 The enzymes responsible for supplying endogenous fatty acids for this process have recently been isolated from P h o t o b a c t e r i u m p h o s p h o r e u m 3 and Vibrio harvey? (Fig. 1, reaction A). These e n z y m e s have subunit molecular weights of 34 and 32 kDa, respectively, and possess acyl-CoA acyltransferase activity in v i t r o ) Recent evidence has indicated that acyl-acyl-carrier protein (acyl-ACP) might be the precursor involved in generating fatty acyl groups for bioluminescence. 6 Both acyltransferases appear to be induced during the midexponential growth phase characteristic of bacterial luminescence in vioo. 7,8 Assay R e a g e n t s . [3H]Tetradecanoyl-CoA is synthesized from [3H]tetradecanoic acid (Amersham) 9,1° and stored frozen under N2 at - 2 0 ° in 50 m M phosphate (pH 6). Silica gel N-HR/UV254 plates and En3Hance spray are obtained from Fisher and N e w England Nuclear, respectively. Organic solvents are reagent grade and are used without further purification. Phosphate buffers are prepared by mixing NaH2PO4 and K2HPO4 in the appropriate ratio. Method. The assay is based on the cleavage of [3H]tetradecanoyl-CoA to form a labeled hexane-soluble product. 10For the P. p h o s p h o r e u m acyl1 S. U l i t z u r and J. W. H a s t i n g s , Proc. Natl. Acad. Sci. U.S.A. 75, 266 (1978).
2 D. Riendeau and E. Meighen, J. Biol. Chem. 254, 7488 (1979). 3 L. M. Carey, A. Rodriguez, and E. Meighen, J. Biol. Chem. 259, 10216(1984). 4 D. Byers and E. Meighen, J. Biol. Chem. 260, 6938 (1985). 5 The term "acyltransferase" has been used for these enzymes, although they exhibit acylhydrolase (transfer to water) activity under certain conditions. "Acyl-CoA cleavage" refers to the cleavage of acyl derivatives to form hexane-soluble products, without regard to the final acceptor. 6 D. Byers and E. Meighen, Proc. Natl. Acad. Sci. U.S.A. 82, 6085 (1985). 7 L. Wall, A. Rodriguez, and E. Meighen, J. Biol. Chem. 259, 1409 (1984). 8 L. Wall, D. M. Byers, and E. A. Meighen, J. Bacteriol. 159, 720 (1984). 9 j. E. Bishop and A. K. Hajra, Anal. Biochem. 106, 344 (1980). ~0A. Rodriguez, D. Riendeau, and E. Meighen, J. Biol. Chem. 258, 5233 (1983). METHODS IN ENZYMOLOGY, VOL. 133
Copyright © 1986by AcademicPress, Inc. All rights of reproductionin any form reserved.
184
BIOLUMINESCENCE A
Fatty acid Precursor
B ~
Fatty acid
[15] C
~
Aldehyde
~
Fatty acid + Light
FIG. 1. A scheme outlining the acyltransferase (A), reductase (B), and luciferase (C) reactions of bacterial bioluminescence.
transferase, the enzyme preparation is incubated with 8/zM [3H]tetradecanoyl-CoA (100 Ci/mol) in 1 M phosphate, 50 mM 2-mercaptoethanol, pH 7.0, in a total volume of 100 /zl at room temperature (22o).3 The reaction is stopped by adding 10 ~l of glacial acetic acid and the solution is extracted twice with 1 ml hexane. The hexane washes are combined and counted directly in 10 ml Econofluor (New England Nuclear). The assay for the V. harveyi enzyme is essentially identical, except that the solvent used is 50% ethylene glycol, 50 mM phosphate (pH 7). 4 For both acyltransferases, enzyme concentration and incubation time are chosen such that less than 60% of the substrate is cleaved. The hexane-extractable product of [3H]acyl-CoA cleavage can be analyzed by thin-layer chromatography and fluorography.l° Hexane extracts are concentrated under nitrogen to a volume of -10/~l and spotted on Silica gel plates. The chromatogram is developed in benzene/diethyl ether/acetic acid (90/10/2), dried, sprayed with EnaHance, and exposed to Kodak XAR-5 film at - 7 0 °. Comments. The relatively low specific activities of the P. phosphoreum and V. harveyi acyltransferases in aqueous buffers and the presence of interfering acyl-CoA thioesterases in crude extracts are the major problems faced in the assay for these bioluminescence-related enzymes. The activity of the P. phosphoreum enzyme in 1 M phosphate, 50 mM 2mercaptoethanol (120 nmol/min/mg) is sufficient to allow its resolution at all preparative stages. However, the V. harveyi acyltransferase represents less than 1% of the total acyl-CoA cleavage activity in V. harveyi extracts under similar conditions and high concentrations of ethylene glycol or glycerol must be included in the assay mixture, at least during the initial purification steps. 4 For example, the acyl-CoA cleavage rate of the 32 kDa enzyme in 50 m M phosphate is stimulated 100-fold (to 1500 nmol/min/mg) when 50% ethylene glycol is present; consequently, over 50% of the activity in V. harveyi lysates is attributable to this enzyme. If [3H]tetradecanoyl-CoA is not available, labeled acyl-CoAs of similar chain length can also be used as substrates, although their effects on the activities of interfering enzymes are not known. Another potential substrate is acyl-ACP: it has been found that the cleavage of [3H]tetradecanoyl-ACP to a hexane-soluble product is dependent upon the presence of these acyltransferases in bacterial extracts. 6 Thus, acyl-ACP may in fact be the substrate of choice in studies of enzyme expression under
[15]
BIOLUMINESCENCE-RELATED ACYLTRANSFERASES
185
various conditions. If no labeled substrates are available, acyltransferase activities can be monitored with spectrophotometric assays based on the release of thiol groups or on the hydrolysis of tetradecanoyl-p-nitrophenol. Measurement of fatty acid production by a coupled P. phosphoreum fatty acid reductase-luciferase assay has also been used to demonstrate the hydrolysis of tetradecanoyl-S-mercaptoethanol and tetradecanoyl-lglycerol by the 34 kDa e n z y m e ) Purification
Reagents. DEAE-Sepharose CL-6B and Blue Sepharose CL-6B are purchased from Pharmacia. BioGel HT and Ultrogel AcA 44 are obtained from Bio-Rad and LKB Instruments, Inc., respectively. P. phosphoreum acyltransferase. The 34 kDa acyltransferase can be isolated from the partially purified P. phosphoreum fatty acid reductase complex, with which it copurifies during ion-exchange, gel filtration, and aminohexyl Sepharose chromatography. 1~ No change in the acyl-CoA cleavage/fatty acid reductase activity ratio is observed through these steps, starting from the initial Cellex D pool. 3 The acyltransferase is separated from the 50 and 58 kDa fatty acid reductase subunits on a Blue Sepharose column, using batch elution with 0.2 M NaSCN. H The fractions containing 34 kDa acyl-CoA cleavage activity are pooled and dialyzed vs 50 m M phosphate, 20 mM 2-mercaptoethanol (pH 7) containing 15% glycerol. If desired, the enzyme can be stored at this stage at - 2 0 ° without significant loss of activity after 1 month. Further purification to remove minor contaminants is carried out on an Ultrogel AcA 44 gel filtration column (80 x 1.5 cm) in 0.1 M NaCI, 50 mM phosphate, 20 mM 2-mercaptoethanol (pH 7). The activity is pooled, dialyzed vs 15% glycerol, 50 m M phosphate, 20 m M 2-mercaptoethanol, and stored at -20 °. Cellex D chromatography of P. phosphoreum cell-free extracts separates three peaks of acyl-CoA cleavage activity (Fig. 2, 1-111)3; Peak III corresponds to the activity associated with the aforementioned fatty acid reductase complex. However, Peak II activity is also characteristically stimulated by high concentrations of phosphate (Fig. 2) and is attributed to free 34 kDa acyltransferase, which has been resolved from the other fatty acid reductase subunits. The relative amount of activity in Peak II is variable with different preparations, but the acyltransferase can be purified from this source using Blue Sepharose and gel filtration. A typical yield of the 34 kDa enzyme from Peak III alone is 2-4 mg per 40 g cells. V. harveyi acyltransferase. Although a corresponding fatty acid reductase complex has not been isolated from extracts of V. harveyi, the 32 u The initial preparative stages for purifying the fatty acid reductase complex are described by A. Rodriguez, L. Wall, D. Riendeau, and E. Meighen, this volume [14].
186
BIOLUMINESCENCE ~4
9 radlent
~'
I
TT
Wl
n~
[15]
I
o
"o
A
A
I
•
e[o
"~
i
/II
"-O
II I
O" [ 6O
/I 80
I00
o-O'o-~ 120
i I.O. • ~
lO
xo %._
140
Oa,~ ~
I I
~_.
~"
160
Fraction FIG. 2. Elution profile of [3H]tetradecanoyl-CoA cleavage activity of a P. phosphoreum extract after Cellex D chromatography) The column was eluted with a linear 0.05-0.5 M phosphate gradient, pH 7, and assays were conducted in 50 m M (O) or 1 M (O) phosphate containing 50 m M 2-mercaptoethano] as described in the text.
kDa acyltransferase can be separated from luciferase and the majority of the soluble protein by (NH4)2SO4 fractionation. 4 Proteins associated with aldehyde metabolism in V. harveyi, including the acyltransferase, aldehyde dehydrogenase, as well as the 57 and 42 kDa fatty acid-labeled polypeptides thought to be involved in fatty acid reduction, 8 are coprecipitated in 30-50% saturated (NH4)2SO4. This is a possible indication of a complex similar to that found in P. phosphoreum. V. harveyi B392 cells are grown to maximum luminescence (A660 = 1.5-2) at 27 ° in 10 liters of 1% complete medium. 2 The cells are harvested by centrifugation and lysed in 700 ml of 50 mM phosphate, 10 mM 2-mercaptoethanol (pH 7) by sonication (3 × 45 sec) in small batches. A cell-free supernatant is obtained by centrifugation (17,000 g, 25 min). Lysis and subsequent steps are carried out at 4 ° (see Table I). The V. harveyi lysate is made 30% saturated with solid (NH4)2SO4, stirred for 25 min, and centrifuged to remove the precipitate. The resulting supernatant is made 50% saturated with ammonium sulfate, stirred for 30 min, and the precipitate is collected by centrifugation (17,000 g, 30 min). The 30-50% precipitate is dissolved in 100 ml of 50 mM phosphate, I0 m M 2-mercaptoethanol (pH 7) and dialyzed overnight. The dialyzed fraction is applied to a DEAE-Sepharose CL-6B column (2.5 x 20 cm), which is washed with 50 m M phosphate, l0 mM 2-mercaptoethanol at pH 7 (150 ml) followed by a linear 0-0.5 M NaCI gradient (1 liter total) in the same buffer. Fractions with acyl-CoA cleavage activity in 50% ethylene glycol are pooled and concentrated by precipitation in 75% saturated
[15]
BIOLUMINESCENCE-RELATED ACYLTRANSFERASES
PURIFICATION OF THE V.
187
TABLE I harveyi32 kDa ACYLTRANSFERASEa Acyl-CoA cleavage activity b
Purification step
Volume (ml)
Total protein (mg)
Lysate supernatant (NH4)SO4 30-50% fraction DEAE-Sepharose pool Gel filtrationpool Hydroxylapatitepool
700 100 32 10 2.7
970 125 12.1 4.5 2.6
Total Specific (nmol/min) (nmol/min/mg) 43,000c 12,000c 8,000 5,300 3,800
45 100 660 1,170 1,460
a From Byers and Meighen.4 b [3H]Tetradecanoyl_CoA cleavageassay was performedin 50% ethyleneglycol, 50 mM phosphate, pH 7. c Not all of the activityat these stages is due to the 32 kDa acyltransferase. (NH4)2SO4. This precipitate is dissolved in 50 mM phosphate, 10 mM 2mercaptoethanol, pH 7 (2 ml) and applied to an Ultrogel AcA 44 column (1.5 x 46 cm). Following elution of the column with the same buffer, the appropriate fractions are pooled and concentrated on a small (1 ml) DEAE-Sepharose CL-6B column. The concentrated protein is eluted with 0.5 M NaCI and dialyzed vs 10 mM phosphate, 20 mM 2-mercaptoethanol. The dialyzed sample is applied to a hydroxylapatite column (BioGel HT, 1 ml) and the 32 kDa acyltransferase is eluted in the void volume with 10 mM phosphate, 20 mM 2-mercaptoethanol (pH 7). The purified enzyme is made 10% in glycerol and stored at - 2 0 ° with no appreciable loss of activity after 1 month. Properties The 32 kDa acyltransferase from V. harveyi appears to be monomeric in the phosphate buffers used for its purification, whereas the 34 kDa P. phosphoreum enzyme tends to aggregate unless NaCI (at least 0.1 M) is included in the gel filtration eluant. With both acyltransferases, the acylCoA cleavage activity in phosphate buffer is maximal at pH values above pH 7 and the apparent Km for [3H]tetradecanoyl-CoA is - 1 / ~ M . 3,4 The P. phosphoreum enzyme exhibits substrate inhibition at tetradecanoyl-CoA concentrations above 10/zM, but no such effect is observed for the V. harveyi enzyme (up to 40/zM). Both acyltransferases are inhibited by the sulfhydryl reagent N-ethylmaleimide, although the V. harveyi enzyme appears to be protected from NEM inactivation by high concentrations of phosphate buffer. 4 This observation, together with the phosphate-induced
188
BIOLUMINESCENCE
[ 15]
TABLE II [3H]TETRADECANOYL-CoA CLEAVAGE CATALYZED BY P. phosphoreum AND V. harveyi ACYLTRANSFERASES: EFFECT OF SOLVENT COMPOSITION
Enzyme activity (nmol/min/mg) Solvent composition
P. phosphoreum
V. harveyi
50 mM phosphate, pH 7 +50 mM 2-mercaptoethanol +40% ethyleneglycol +50% glycerol 1 M phosphate, pH 7 +50 mM 2-mercaptoethanol
0 40 1000 2000 0 120
15 40 1500 800 20 --
stimulation of the activities of the 32 and 34 kDa enzymes (Table II), suggests that the effect of this anion could have physiological relevance. Neither enzyme is appreciably affected by similar concentrations of other salts (i.e., NaCI) or buffers. One of the more interesting properties of the bioluminescence-related acyltransferases is the dramatic stimulation of the acyl-CoA cleavage activity observed in the presence of low molecular weight thiol and alcohol acceptors (Table II). 4 In fact the P. phosphoreum enzyme appears to be essentially inactive in phosphate buffer alone, while the V. harveyi enzyme exhibits a low acylhydrolase activity. The large increase in acylCoA cleavage rate by such compounds as glycerol, ethylene glycol, or 2mercaptoethanol is accompanied by the transfer of the fatty acyl moiety to form the O- or S-acyl ester derivative, as observed by TLC. 3,4 Other organic compounds, either without or with poor acceptor capabilities, still produce a significant increase in the acyl-CoA cleavage rate, probably due to effects on the enzyme environment or on the effective substrate concentration. Thus, these enzymes do not function optimally in a purely aqueous environment, with water as an acceptor, suggesting perhaps that their true role is to channel fatty acyl groups to and from specific acceptors (i.e., lipids, other enzymes, etc.). Indeed, it has been demonstrated that the P. phosphoreum 50 kDa acyl-protein synthetase subunit can modify both the rate and final product of acyl-CoA cleavage catalyzed by the 34 kDa acyltransferase. 3 Acknowledgments This work was supported by a PostdoctoralFellowship(D.B.), a Graduate Studentship (L.C.), and a Research Grant (MT-4314)from the MedicalResearch Councilof Canada.
BIOLUMINESCENCE-RELATED ACYLTRANSFERASES
[15] B i o l u m i n e s c e n c e - R e l a t e d Photobacterium By
DAVID DYERS,
phosphoreum
Acyltransferases from and Vibrio harveyi
ANGEL RODRIGUEZ, L u c EDWARD
183
CAREY,
and
MEIGHEN
The long-chain aldehyde substrate of bacterial luciferase is synthesized via the reduction of fatty acids. ~,2 The enzymes responsible for supplying endogenous fatty acids for this process have recently been isolated from P h o t o b a c t e r i u m p h o s p h o r e u m 3 and Vibrio harvey? (Fig. 1, reaction A). These e n z y m e s have subunit molecular weights of 34 and 32 kDa, respectively, and possess acyl-CoA acyltransferase activity in v i t r o ) Recent evidence has indicated that acyl-acyl-carrier protein (acyl-ACP) might be the precursor involved in generating fatty acyl groups for bioluminescence. 6 Both acyltransferases appear to be induced during the midexponential growth phase characteristic of bacterial luminescence in vioo. 7,8 Assay R e a g e n t s . [3H]Tetradecanoyl-CoA is synthesized from [3H]tetradecanoic acid (Amersham) 9,1° and stored frozen under N2 at - 2 0 ° in 50 m M phosphate (pH 6). Silica gel N-HR/UV254 plates and En3Hance spray are obtained from Fisher and N e w England Nuclear, respectively. Organic solvents are reagent grade and are used without further purification. Phosphate buffers are prepared by mixing NaH2PO4 and K2HPO4 in the appropriate ratio. Method. The assay is based on the cleavage of [3H]tetradecanoyl-CoA to form a labeled hexane-soluble product. 10For the P. p h o s p h o r e u m acyl1 S. U l i t z u r and J. W. H a s t i n g s , Proc. Natl. Acad. Sci. U.S.A. 75, 266 (1978).
2 D. Riendeau and E. Meighen, J. Biol. Chem. 254, 7488 (1979). 3 L. M. Carey, A. Rodriguez, and E. Meighen, J. Biol. Chem. 259, 10216(1984). 4 D. Byers and E. Meighen, J. Biol. Chem. 260, 6938 (1985). 5 The term "acyltransferase" has been used for these enzymes, although they exhibit acylhydrolase (transfer to water) activity under certain conditions. "Acyl-CoA cleavage" refers to the cleavage of acyl derivatives to form hexane-soluble products, without regard to the final acceptor. 6 D. Byers and E. Meighen, Proc. Natl. Acad. Sci. U.S.A. 82, 6085 (1985). 7 L. Wall, A. Rodriguez, and E. Meighen, J. Biol. Chem. 259, 1409 (1984). 8 L. Wall, D. M. Byers, and E. A. Meighen, J. Bacteriol. 159, 720 (1984). 9 j. E. Bishop and A. K. Hajra, Anal. Biochem. 106, 344 (1980). ~0A. Rodriguez, D. Riendeau, and E. Meighen, J. Biol. Chem. 258, 5233 (1983). METHODS IN ENZYMOLOGY, VOL. 133
Copyright © 1986by AcademicPress, Inc. All rights of reproductionin any form reserved.
184
BIOLUMINESCENCE A
Fatty acid Precursor
B ~
Fatty acid
[15] C
~
Aldehyde
~
Fatty acid + Light
FIG. 1. A scheme outlining the acyltransferase (A), reductase (B), and luciferase (C) reactions of bacterial bioluminescence.
transferase, the enzyme preparation is incubated with 8/zM [3H]tetradecanoyl-CoA (100 Ci/mol) in 1 M phosphate, 50 mM 2-mercaptoethanol, pH 7.0, in a total volume of 100 /zl at room temperature (22o).3 The reaction is stopped by adding 10 ~l of glacial acetic acid and the solution is extracted twice with 1 ml hexane. The hexane washes are combined and counted directly in 10 ml Econofluor (New England Nuclear). The assay for the V. harveyi enzyme is essentially identical, except that the solvent used is 50% ethylene glycol, 50 mM phosphate (pH 7). 4 For both acyltransferases, enzyme concentration and incubation time are chosen such that less than 60% of the substrate is cleaved. The hexane-extractable product of [3H]acyl-CoA cleavage can be analyzed by thin-layer chromatography and fluorography.l° Hexane extracts are concentrated under nitrogen to a volume of -10/~l and spotted on Silica gel plates. The chromatogram is developed in benzene/diethyl ether/acetic acid (90/10/2), dried, sprayed with EnaHance, and exposed to Kodak XAR-5 film at - 7 0 °. Comments. The relatively low specific activities of the P. phosphoreum and V. harveyi acyltransferases in aqueous buffers and the presence of interfering acyl-CoA thioesterases in crude extracts are the major problems faced in the assay for these bioluminescence-related enzymes. The activity of the P. phosphoreum enzyme in 1 M phosphate, 50 mM 2mercaptoethanol (120 nmol/min/mg) is sufficient to allow its resolution at all preparative stages. However, the V. harveyi acyltransferase represents less than 1% of the total acyl-CoA cleavage activity in V. harveyi extracts under similar conditions and high concentrations of ethylene glycol or glycerol must be included in the assay mixture, at least during the initial purification steps. 4 For example, the acyl-CoA cleavage rate of the 32 kDa enzyme in 50 m M phosphate is stimulated 100-fold (to 1500 nmol/min/mg) when 50% ethylene glycol is present; consequently, over 50% of the activity in V. harveyi lysates is attributable to this enzyme. If [3H]tetradecanoyl-CoA is not available, labeled acyl-CoAs of similar chain length can also be used as substrates, although their effects on the activities of interfering enzymes are not known. Another potential substrate is acyl-ACP: it has been found that the cleavage of [3H]tetradecanoyl-ACP to a hexane-soluble product is dependent upon the presence of these acyltransferases in bacterial extracts. 6 Thus, acyl-ACP may in fact be the substrate of choice in studies of enzyme expression under
[15]
BIOLUMINESCENCE-RELATED ACYLTRANSFERASES
185
various conditions. If no labeled substrates are available, acyltransferase activities can be monitored with spectrophotometric assays based on the release of thiol groups or on the hydrolysis of tetradecanoyl-p-nitrophenol. Measurement of fatty acid production by a coupled P. phosphoreum fatty acid reductase-luciferase assay has also been used to demonstrate the hydrolysis of tetradecanoyl-S-mercaptoethanol and tetradecanoyl-lglycerol by the 34 kDa e n z y m e ) Purification
Reagents. DEAE-Sepharose CL-6B and Blue Sepharose CL-6B are purchased from Pharmacia. BioGel HT and Ultrogel AcA 44 are obtained from Bio-Rad and LKB Instruments, Inc., respectively. P. phosphoreum acyltransferase. The 34 kDa acyltransferase can be isolated from the partially purified P. phosphoreum fatty acid reductase complex, with which it copurifies during ion-exchange, gel filtration, and aminohexyl Sepharose chromatography. 1~ No change in the acyl-CoA cleavage/fatty acid reductase activity ratio is observed through these steps, starting from the initial Cellex D pool. 3 The acyltransferase is separated from the 50 and 58 kDa fatty acid reductase subunits on a Blue Sepharose column, using batch elution with 0.2 M NaSCN. H The fractions containing 34 kDa acyl-CoA cleavage activity are pooled and dialyzed vs 50 m M phosphate, 20 mM 2-mercaptoethanol (pH 7) containing 15% glycerol. If desired, the enzyme can be stored at this stage at - 2 0 ° without significant loss of activity after 1 month. Further purification to remove minor contaminants is carried out on an Ultrogel AcA 44 gel filtration column (80 x 1.5 cm) in 0.1 M NaCI, 50 mM phosphate, 20 mM 2-mercaptoethanol (pH 7). The activity is pooled, dialyzed vs 15% glycerol, 50 m M phosphate, 20 m M 2-mercaptoethanol, and stored at -20 °. Cellex D chromatography of P. phosphoreum cell-free extracts separates three peaks of acyl-CoA cleavage activity (Fig. 2, 1-111)3; Peak III corresponds to the activity associated with the aforementioned fatty acid reductase complex. However, Peak II activity is also characteristically stimulated by high concentrations of phosphate (Fig. 2) and is attributed to free 34 kDa acyltransferase, which has been resolved from the other fatty acid reductase subunits. The relative amount of activity in Peak II is variable with different preparations, but the acyltransferase can be purified from this source using Blue Sepharose and gel filtration. A typical yield of the 34 kDa enzyme from Peak III alone is 2-4 mg per 40 g cells. V. harveyi acyltransferase. Although a corresponding fatty acid reductase complex has not been isolated from extracts of V. harveyi, the 32 u The initial preparative stages for purifying the fatty acid reductase complex are described by A. Rodriguez, L. Wall, D. Riendeau, and E. Meighen, this volume [14].
186
BIOLUMINESCENCE ~4
9 radlent
~'
I
TT
Wl
n~
[15]
I
o
"o
A
A
I
•
e[o
"~
i
/II
"-O
II I
O" [ 6O
/I 80
I00
o-O'o-~ 120
i I.O. • ~
lO
xo %._
140
Oa,~ ~
I I
~_.
~"
160
Fraction FIG. 2. Elution profile of [3H]tetradecanoyl-CoA cleavage activity of a P. phosphoreum extract after Cellex D chromatography) The column was eluted with a linear 0.05-0.5 M phosphate gradient, pH 7, and assays were conducted in 50 m M (O) or 1 M (O) phosphate containing 50 m M 2-mercaptoethano] as described in the text.
kDa acyltransferase can be separated from luciferase and the majority of the soluble protein by (NH4)2SO4 fractionation. 4 Proteins associated with aldehyde metabolism in V. harveyi, including the acyltransferase, aldehyde dehydrogenase, as well as the 57 and 42 kDa fatty acid-labeled polypeptides thought to be involved in fatty acid reduction, 8 are coprecipitated in 30-50% saturated (NH4)2SO4. This is a possible indication of a complex similar to that found in P. phosphoreum. V. harveyi B392 cells are grown to maximum luminescence (A660 = 1.5-2) at 27 ° in 10 liters of 1% complete medium. 2 The cells are harvested by centrifugation and lysed in 700 ml of 50 mM phosphate, 10 mM 2-mercaptoethanol (pH 7) by sonication (3 × 45 sec) in small batches. A cell-free supernatant is obtained by centrifugation (17,000 g, 25 min). Lysis and subsequent steps are carried out at 4 ° (see Table I). The V. harveyi lysate is made 30% saturated with solid (NH4)2SO4, stirred for 25 min, and centrifuged to remove the precipitate. The resulting supernatant is made 50% saturated with ammonium sulfate, stirred for 30 min, and the precipitate is collected by centrifugation (17,000 g, 30 min). The 30-50% precipitate is dissolved in 100 ml of 50 mM phosphate, I0 m M 2-mercaptoethanol (pH 7) and dialyzed overnight. The dialyzed fraction is applied to a DEAE-Sepharose CL-6B column (2.5 x 20 cm), which is washed with 50 m M phosphate, l0 mM 2-mercaptoethanol at pH 7 (150 ml) followed by a linear 0-0.5 M NaCI gradient (1 liter total) in the same buffer. Fractions with acyl-CoA cleavage activity in 50% ethylene glycol are pooled and concentrated by precipitation in 75% saturated
[15]
BIOLUMINESCENCE-RELATED ACYLTRANSFERASES
PURIFICATION OF THE V.
187
TABLE I harveyi32 kDa ACYLTRANSFERASEa Acyl-CoA cleavage activity b
Purification step
Volume (ml)
Total protein (mg)
Lysate supernatant (NH4)SO4 30-50% fraction DEAE-Sepharose pool Gel filtrationpool Hydroxylapatitepool
700 100 32 10 2.7
970 125 12.1 4.5 2.6
Total Specific (nmol/min) (nmol/min/mg) 43,000c 12,000c 8,000 5,300 3,800
45 100 660 1,170 1,460
a From Byers and Meighen.4 b [3H]Tetradecanoyl_CoA cleavageassay was performedin 50% ethyleneglycol, 50 mM phosphate, pH 7. c Not all of the activityat these stages is due to the 32 kDa acyltransferase. (NH4)2SO4. This precipitate is dissolved in 50 mM phosphate, 10 mM 2mercaptoethanol, pH 7 (2 ml) and applied to an Ultrogel AcA 44 column (1.5 x 46 cm). Following elution of the column with the same buffer, the appropriate fractions are pooled and concentrated on a small (1 ml) DEAE-Sepharose CL-6B column. The concentrated protein is eluted with 0.5 M NaCI and dialyzed vs 10 mM phosphate, 20 mM 2-mercaptoethanol. The dialyzed sample is applied to a hydroxylapatite column (BioGel HT, 1 ml) and the 32 kDa acyltransferase is eluted in the void volume with 10 mM phosphate, 20 mM 2-mercaptoethanol (pH 7). The purified enzyme is made 10% in glycerol and stored at - 2 0 ° with no appreciable loss of activity after 1 month. Properties The 32 kDa acyltransferase from V. harveyi appears to be monomeric in the phosphate buffers used for its purification, whereas the 34 kDa P. phosphoreum enzyme tends to aggregate unless NaCI (at least 0.1 M) is included in the gel filtration eluant. With both acyltransferases, the acylCoA cleavage activity in phosphate buffer is maximal at pH values above pH 7 and the apparent Km for [3H]tetradecanoyl-CoA is - 1 / ~ M . 3,4 The P. phosphoreum enzyme exhibits substrate inhibition at tetradecanoyl-CoA concentrations above 10/zM, but no such effect is observed for the V. harveyi enzyme (up to 40/zM). Both acyltransferases are inhibited by the sulfhydryl reagent N-ethylmaleimide, although the V. harveyi enzyme appears to be protected from NEM inactivation by high concentrations of phosphate buffer. 4 This observation, together with the phosphate-induced
188
BIOLUMINESCENCE
[ 15]
TABLE II [3H]TETRADECANOYL-CoA CLEAVAGE CATALYZED BY P. phosphoreum AND V. harveyi ACYLTRANSFERASES: EFFECT OF SOLVENT COMPOSITION
Enzyme activity (nmol/min/mg) Solvent composition
P. phosphoreum
V. harveyi
50 mM phosphate, pH 7 +50 mM 2-mercaptoethanol +40% ethyleneglycol +50% glycerol 1 M phosphate, pH 7 +50 mM 2-mercaptoethanol
0 40 1000 2000 0 120
15 40 1500 800 20 --
stimulation of the activities of the 32 and 34 kDa enzymes (Table II), suggests that the effect of this anion could have physiological relevance. Neither enzyme is appreciably affected by similar concentrations of other salts (i.e., NaCI) or buffers. One of the more interesting properties of the bioluminescence-related acyltransferases is the dramatic stimulation of the acyl-CoA cleavage activity observed in the presence of low molecular weight thiol and alcohol acceptors (Table II). 4 In fact the P. phosphoreum enzyme appears to be essentially inactive in phosphate buffer alone, while the V. harveyi enzyme exhibits a low acylhydrolase activity. The large increase in acylCoA cleavage rate by such compounds as glycerol, ethylene glycol, or 2mercaptoethanol is accompanied by the transfer of the fatty acyl moiety to form the O- or S-acyl ester derivative, as observed by TLC. 3,4 Other organic compounds, either without or with poor acceptor capabilities, still produce a significant increase in the acyl-CoA cleavage rate, probably due to effects on the enzyme environment or on the effective substrate concentration. Thus, these enzymes do not function optimally in a purely aqueous environment, with water as an acceptor, suggesting perhaps that their true role is to channel fatty acyl groups to and from specific acceptors (i.e., lipids, other enzymes, etc.). Indeed, it has been demonstrated that the P. phosphoreum 50 kDa acyl-protein synthetase subunit can modify both the rate and final product of acyl-CoA cleavage catalyzed by the 34 kDa acyltransferase. 3 Acknowledgments This work was supported by a PostdoctoralFellowship(D.B.), a Graduate Studentship (L.C.), and a Research Grant (MT-4314)from the MedicalResearch Councilof Canada.