J. Biochem. 82, 1585-1590 (1977)
Two Forms of Famesyl Pyrophosphate Synthetase from Hog Liver1 Tanetoshi KOYAMA, Yukiko SAITO, Kyozo OGURA, and Shuichi SETO Chemical Research Institute of Non-Aqueous Solutions, Tohoku University, Sendai, Miyagi 980 Received for publication, June 22, 1977
Two forms of farnesyl pyrophosphate synthetase were separated from hog liver extracts by DEAE-Sephadex chromatography. They were designated as farnesyl pyrophosphate synthetase A and B, in order of elution. Both enzymes catalyzed the exclusive formation of E,E-farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl pyrophosphate or geranyl pyrophosphate. They also showed no detectable differences in pH optima, molecular weights, and susceptibilities to metal ions. However, the catalytic activity of the synthetase B was greatly stimulated by the addition of common sulfhydryl reagents. This stimulation was the result of conversion of the synthetase B into the synthetase A. Conversely the synthetase A was converted into form B when it was dialyzed against a buffer solution containing cupric ions. It is suggested that the formation and cleavage of disulfide bond(s) is involved in the interconversion between the two forms.
Farnesyl pyrophosphate synthetase [dimethylallyl pyrophosphate: isopentenyl pyrophosphate dimethylallyltransferase, EC 2.5.1.1] has been purified from various organisms {1-8). Although the catalytic function of these enzymes from various sources is essentially the same, in that they catalyze the condensation of isopentenyl pyrophosphate with either dimethylallyl pyrophosphate or geranyl pyrophosphate to give farnesyl pyrophosphate, there are apparent differences in properties between animal and plant enzymes.(P). Green and West have demonstrated the existence of two forms of farnesyl pyrophosphate synthetase in soluble 1
This work was supported by a Grant-in-Aid for Scientific Research, No. 174179, from the Ministry of Education, Science and Culture of Japan. Vol. 82, No. 6, 1977
1585
extracts from castor bean seedlings (6), but no report has appeared on such multiplicity of any animal enzyme.We now report that two forms of farnesyl pyrophosphate synthetase were also separated from hog liver, and that they showed different properties from those of higher plant origin. EXPERIMENTAL PROCEDURE Enzyme Assay—The incubation mixture contained, in a final volume of 1.0 ml, 20 fxmo\ of Tris-HCI buffer, pH 7.7, 5 ^mol of MgCI,, 25 nmol of either dimethylallyl or geranyl pyrophosphate, 25 nmol of [l-14C]isopentenyl pyrophosphate (30 nCi) and a suitable amount of enzyme protein. After the addition of enzyme, the reaction mix-
1586 ture was incubated for 15 min at 37°C. The reaction was terminated by the addition of 0.3 ml of 1 N H O and the mixture was incubated again for 15 min to complete the hydrolysis of acidlabile allylic pyrophosphate. The mixture was then made alkaline by the addition of 0.5 ml of 1 N NaOH, and the free alcohols liberated were extracted with 4.0 ml of hexane. The hexane extract was washed with water and the radioactivity in a 2 ml aliquot of the extract was measured by liquid scintillation counting. One unit of enzyme activity was defined as the activity required to incorporate 1 nmol of [l-"C]isopentenyl pyrophosphate into allylic pyrophosphate per min. Specific activity is expressed in units of enzyme activity per mg of protein. Protein concentration was measured in terms of the absorbance at 280 nm using an A\£m value of 10. Enzyme Purification—All procedures were performed at 4°C. Fresh hog liver (800 g) was homogenized in 1,600 ml of 50mM Tris-HCl buffer, pH 6.8, for 75 s in a Waring blender. The supernatant obtained by centrifugation at 26,000 x g for 30 min was filtered through gauze. Ammonium sulfate (443 g) was added to 1,570 ml of the filtrate (45% saturation), and the mixture was centrifuged at 26,000 x g for 30 min. To 1,390 ml of the resulting supernatant, 91 g of ammonium sulfate was added (55% saturation), and the mixture was centrifuged at 26,000 xg for 30 min. The pellet was dissolved in 150 ml of 50 ITIM TrisHCl buffer, pH6.8. This solution was filtered through a Sephadex G-25 column (2.7x90cm) equilibrated with 50 ITIM Tris-HCl buffer, pH 6.8, containing 50 ITIM NaCl. The protein fraction was applied to a 4 x 55 cm column of DEAE-Sephadex A-50 previously equilibrated with 50 ITIM Tris-HCl buffer, pH6.8, containing 50 mM NaCl. The column was then developed with a linear gradient of 2 liters of 50 to 300 mM NaCl in 50 mM TrisHCl buffer, pH 6.8 (Fig. 1). Interconversion between Two Enzyme Forms— To 550 ml of the enzyme fraction which had a larger elution volume in the DEAE-Sephadex chromatography described above (fraction numbers 223-262), 256 g of ammonium sulfate was added (70% saturation), and the mixture was centrifuged at 26,000 x g for 60 min. The pellet was dissolved in 50 ml of 50 mM Tris-HCl buffer, pH 6.8, and the solution was dialyzed against 50 mM Tris-HCl
T. KOYAMA, Y. SAITO, K. OGURA, and S. SETO buffer, pH 6.8, containing 10 mM 2-mercaptoethanol and 50 mM NaCl for 18 h. Then 10 ml of the dialyzed solution (specific activity, 15.6 units/mg) was loaded onto a 1.7x20 cm column of DEAE-Sephadex A-50 which had been washed with 50 mM Tris-HCl buffer, pH 6.8, containing 10 mM 2-mercaptoethanol and 50 mM NaCl. The column was developed with a linear gradient of NaCl (total volume, 400 ml) similar to that used in the purification procedure, except that 10 mM 2-mercaptoethanol was contained in the eluent (Fig. 2). The enzyme which emerged in the earlier fraction (fraction numbers 25-40) was combined and concentrated with a Diafilter apparatus fitted with a G-10 T membrane. After dialysis against 50 mM Tris-HCl buffer, pH 6.8, containing 50 mM NaCl for 17 h, the solution (10 ml; specific activity, 181.8 units/mg) was applied to a 1.7x20 cm column of DEAE-Sephadex A-50 and eluted with a linear gradient similar to that described above except that no 2-mercaptoethanol was contained (Fig. 3). The enzyme protein which emerged in the earlier fraction in the chromatography of Fig. 1 (fraction numbers 163-203) was collected and concentrated with a Diafilter apparatus fitted with a G-10 T membrane. Then one-fifth of the resulting solution (5 ml, specific activity, 3.3 units/ mg) was dialyzed for 16 h against 50 ITIM TrisHCl buffer, pH 6.8, in the presence of 50 fiM CuSO4. The solution was applied to a 1 x 18 cm column of DEAE-Sephadex A-50 equilibrated with 50 mM Tris-HCl buffer, pH 6.8, containing 50 mM NaCl. The column was developed with a linear gradient of 400 ml of 50 to 300 mM NaCl in 50 mM Tris-HCl buffer, pH 6.8 (Fig. 4). RESULTS AND DISCUSSION In DEAE-Sephadex chromatography of the protein fraction precipitating between 45 and 55% saturation of ammonium sulfate, farnesyl pyrophosphate synthetase activity was eluted in two distinct peaks, hereafter designated as farnesyl pyrophosphate synthetase A and B, as shown in Fig. 1. The fractions of synthetase A and B were completely free of isopentenyl pyrophosphate isomerase and phosphatase activities, both of which would interfere with the farnesyl pyrophosphate synthetase assay. /. Biochem.
TWO FORMS OF FARNESYL PYROPHOSPHATE SYNTHETASE
50
100
150
200
1587
250
FRACTION NUMBER Umi/fnc. Fig. 1. DEAE-Sephadex A-50 chromatography of the 45-55% ammonium sulfate fraction. Details of chromatography are described in " EXPERIMENTAL PROC E D U R E . " The start of the gradient is indicated by an arrow. Aliquots (0.1 ml) were assayed for farnesyl pyrophosphate synthetase activity ( O ) ; -4i«o. ( A ) ; conductivity, ( x ) .
TABLE I. Comparison of properties of the two fractions. Dimethylallyl pyrophosphate was generally used as the allylic substrate; figures in parentheses represent data obtained when geranyl pyrophosphate was employed. Synthetase A and B fractions obtained in the first DEAE-Sephadex chromatography were used. Each incubation mixture contained 0.4 unit of enzyme. Details of the assay are described in " EXPERIMENTAL PROCEDURE." Synthetase A pH optima:
ca.
Susceptibilities to metal ions: Mg*+
5 mM
0.5 Mn'*
5 mM
0.5 None Effects of sulfhydryl reagents:t> 2-Mercaptoethanol
10 mM
1 Glutathione
10 mM
5 1 1,4-Dithiothreitol
10 mM
5 1 Effects of sulfhydryl inhibitors; 6 p-Chloromercuribenzoic acid W-Ethylmaleimide Iodoacetamide
8.0 ca.
Molecular weights:*
5 flM
0.5 1 mM 1 mM
100% 93 71 72 0.02 140% 107 18 87 108 109 115 113 5% 75 7 38
Synthetase B
(8.0-9.0)
ca.
000%) (94 ( 76 (62 (0.04
8.0 ca.
90,000
) ) ) )
(104%)
(110 )
.
100% 57 55 52 0.02 1,023% 1,017 188 1,063 1,099 1,118 1,057 1,019
(7. 8-8. 8) 90,000 (100%)
(71 ) (41 ) ( 36 ) (0.02)
(650%)
(1,367)
0.5% 4 4 1
» Estimated by Sephadex G-100 gel filtration (column size; 1.7x58 cm). b Preincubation with an appropriate additive at 37°C for 5 min was carried out prior to the addition of [l-l*C]isopentenyl pyrophosphate.
Vol. 82, No. 6, 1977
1588
T. KOYAMA, Y. SAITO, K. OGURA, and S. SETO
The exclusive formation of iT,E-farnesyl pyrophosphate by both of these two fractions was confirmed by radio-TLC and radio-gas chromatographic identification of [14C]£,.E-farnesol formed by treatment of the product with alkaline phosphatase, as usual (70). As listed in Table I, farnesyl pyrophosphate synthetases A and B showed little difference in pH optima, molecular weights and susceptibilities to metal ions. However, significant differences were observed in the effects of sulfhydryl reagents. The activity of farnesyl pyrophosphate synthetase B was markedly enhanced by the addition of sulfhydryl reagents such as 2-mercaptoethanol, glutathione, and 1,4-dithiothreitol, whereas the synthetase A was hardly activated by these reagents. The effect of glutathione on the synthetase A at 5 IHM or higher concentration was inhibitory rather than stimulative. The synthetase B fraction obtained by the first DEAE-Sephadex chromatography (specific activity, ca. 15) showed about 10-fold stimulation of activity in the presence of sulfhydryl reagents, as shown in Table I. This enzyme, when recycled on DEAE-Sephadex, was recovered in a single peak in the same conductivity region, though the activity decreased so that it was hardly detectable in the absence of sulfhydryl reagents. The synthetase B was more sensitive to inhibition by sulfhydryl-blocking reagents than synthetase A.
To investigate further the effect of sulfhydryl reagents, farnesyl pyrophosphate synthetase B was dialyzed against a buffer solution containing lOmM 2-mercaptoethanol. Then the enzyme solution was chromatographed on DEAE-Sephadex under the same conditions, except that 10 mM 2-mercaptoethanol was contained in the eluent. More than 80 % of the total enzyme activity recovered was located in the earlier emerging fraction corresponding to synthetase A (Fig. 2). This fraction was dialyzed to remove the sulfhydryl reagent and then subjected to the same chromatography except that the eluent contained no sulfhydryl reagent. Although only a single peak corresponding to synthetase A was observed when assayed in the absence of a sulfhydryl reagent, about 43% of the recovered activity could be found at the conductivity region corresponding to the synthetase B when assayed in the presence of 10 mM 1,4 dithiothreitol, as shown in Fig. 3. Direct conversion of the synthetase A into B was observed when synthetase A was dialyzed against a buffer solution containing 50 fiM CuSO4. Almost all the enzyme activity recovered was located at the elution volume corresponding to the synthetase B in DEAE-Sephadex chromatography. Moreover, the stimulation by sulfhydryl reagents, which is characteristic of the synthetase B, was so marked that the enzyme activity of this fraction could be
60 FRACTION NUMBER
70
5ml / f r « .
Fig. 2. DEAE-Sephadex A-50 chromatography of farnesyl pyrophosphate synthetase B fraction in Fig. 1 in the presence of 10 mM 2-mercaptoethanol. Details of the chromatography are described in " EXPERIMENTAL PROCEDURE." The gradient was initiated at the beginning of chromatography. Aliquots (0.1 ml) were assayed for farnesyl pyrophosphate synthetase activity (O); /•*»», ( A ) ; conductivity, ( x ) . / . Biochem.
TWO FORMS OF FARNESYL PYROPHOSPHATE SYNTHETASE
1589
0-4
06 03
c — 04 0-2
02
01
FRACTION NUMBER 5ml / frac. Fig. 3. Rechromatography of famesyl pyrophosphate synthetase A fraction in Fig. 2 in the absence of 2-mercaptoethanol. Details of chromatography are described in " EXPERIMENTAL PROCEDURE." The gradient was initiated at the beginning of the chromatography. Aliquots (0.5 ml) were assayed for famesyl pyrophosphate synthetase activity in the presence of 10 mM 1,4-dithiothreitol (O) or in the absence of sulfhydryl reagents ( • ) . Aat ( A ) , conductivity ( x ) . 015 X
-
^
*
"
—
^
^
12- o
\
K
10"
cO10
6-
DUCT IVITY
^
14'
z=-02 . /
"
/
\
8
o
10
20
30
4 0 5 0 6 0 7 0 8 0 9 0
FRACTION NUMBER
100
5ml / fr«c.
Fig. 4. DEAE-Sephadex A-50 chromatography of famesyl pyrophosphate synthetase A fraction in Fig. 1 after dialysis against a buffer solution containing cupric ions. Details of chromatography are described in " EXPERIMENTAL PROCEDURE." The gradient was initiated at the beginning of the chromatography. AJiquots (0.5 ml) were assayed for famesyl pyrophosphate synthetase activity in the presence of 10 mM 1,4-dithiothreitol (O) or in the absence of sulfhydryl reagents ( # ) . A^ ( A ) , conductivity ( x ) . located only by assay in the presence of a sulfhydryl reagent (Fig. 4). These findings clearly show that the famesyl pyrophosphate synthetases A and B arc interconvertible. Green and West reported the existence of two forms of famesyl pyrophosphate synthetase separable by QAE-Sephadex chromatography in a Vol. 82, No. 6, 1977
higher plant, but offered no evidence of interconversion between them. However, they have found that the molecular weight and kinetic properties of each form of the enzyme vary depending on the protein concentration, because of proteinprotein interaction (6). The present enzyme, however, did not show such changes in properties
1590
even at a high protein concentration. Furthermore, the molecular weights of our farnesyl pyrophosphate synthetases A and B, both ca. 90,000 as estimated by Sephadex G-100 gel filtration (Table I), were nearly identical with that reported for the enzyme from avian liver (8) and different from that of the enzyme from castor bean (6). There are also some remarkable differences between the two forms of farnesyl pyrophosphate synthetase from hog liver and from castor bean seedlings with respect to the pH optima and the susceptibilities to metal ions and sulfhydryl inhibitors. The fact that the conversion of farnesyl pyrophosphate synthetase B to A requires a sulfhydryl reagent, whereas the reverse process is stimulated by cupric ions, suggests that the formation and cleavage of disulfide bond(s) is involved in this interconversion, since the air oxidation of sulfhydryl groups in proteins is strongly catalyzed by some transition metal ions, especially cupric ions (77). Reed and Rilling obtained farnesyl pyrophosphate synthetase in a stable crystalline form from avian liver (8). It seems likely that their enzyme corresponds to the form A in this paper, because they employed 10 mM 2-mercaptoethanol throughout the purification procedure. They have recently reported that the avian liver enzyme contains 13 cysteine residues but no disulfide bond (72). Thus, the properties of the synthetase B might be attributed to an oxidized form of the enzyme containing disulfide bond(s). It has also been suggested that the avian enzyme consists of two identical subunits, each of which carries onecatalytic site (72). If a sulfhydryl group is essential for the catalytic activity, there might be two degrees of reversible inactivation, partial and complete, according as only one or both of the subunits are oxidized. The major portion of the synthetase B obtained by the first DEAE-Sephadex chromatography might be suffering from partial
T. KOYAMA, Y. SAITO, K. OGURA, and S. SETO
inactivation and the synthetase B which has no enzyme activity in the absence of a sulfhydryl reagent, as shown in Fig. 3 and 4, might be completely inactivated. It will be of great interest to investigate the role of the sulfhydryl groups to develop a better understanding of the mechanism of farnesyl pyrophosphate synthetase. Note added in proof: While this paper was in press, we were informed by Dr. H.C. Rilling, University of Utah, that he had also made similar observations with farnesyl pyrophosphate synthetase from pig liver {Arch. Biochem. Biophys., in press). We are grateful to him for this preliminary communication. REFERENCES 1. Lynen, F., Agranoff, B.W., Eggerer, H., Henning, U., & Mdslein, E.M. (1959) Angew. Chem. Int. Ed. 71, 657-667 2. Dorsey, J.K., Dorsey, J.A., & Porter, J.W. (1966) / . Biol. Chem. 1A\, 5353-5360 3. Holloway, P.W. & Popjak, G. (1967) Biochem. J. 104, 57-70 4. Ogura, K., Nishino, T., & Seto, S. (1968) /. Biochem. 64, 197-203 5. Ogura, K., Shinka, T., & Seto, S. (1972) J. Biochem. 72,1101-1108 6. Green, T.R. & West, C.A. (1974) Biochemistry 13, 4720-4729 7. Eberhardt, N.L. & Rilling, H.C. (1975) J. Biol. Chem. 250, 863-866 8. Reed, B.C. & Rilling, H.C. (1975) Biochemistry 14, 50-54 9. Nishino, T., Ogura, K., & Seto, S. (1973) Biochim. Biophys. Ada 302, 33-37 10. Ogura, K., Nishino, T., Koyama, T., & Seto, S. (1970) /. Amer. Chem. Soc. 92, 6036-6041 11. Takagi, T. & Isemura, T. (1964) / . Biochem. 56, 344-350 12. Reed, B.C. & Rilling, H.C. (1976) Biochemistry 15, 3739-3745
/ . Biochem.
Two Forms of Famesyl Pyrophosphate Synthetase from Hog Liver1 Tanetoshi KOYAMA, Yukiko SAITO, Kyozo OGURA, and Shuichi SETO Chemical Research Institute of Non-Aqueous Solutions, Tohoku University, Sendai, Miyagi 980 Received for publication, June 22, 1977
Two forms of farnesyl pyrophosphate synthetase were separated from hog liver extracts by DEAE-Sephadex chromatography. They were designated as farnesyl pyrophosphate synthetase A and B, in order of elution. Both enzymes catalyzed the exclusive formation of E,E-farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl pyrophosphate or geranyl pyrophosphate. They also showed no detectable differences in pH optima, molecular weights, and susceptibilities to metal ions. However, the catalytic activity of the synthetase B was greatly stimulated by the addition of common sulfhydryl reagents. This stimulation was the result of conversion of the synthetase B into the synthetase A. Conversely the synthetase A was converted into form B when it was dialyzed against a buffer solution containing cupric ions. It is suggested that the formation and cleavage of disulfide bond(s) is involved in the interconversion between the two forms.
Farnesyl pyrophosphate synthetase [dimethylallyl pyrophosphate: isopentenyl pyrophosphate dimethylallyltransferase, EC 2.5.1.1] has been purified from various organisms {1-8). Although the catalytic function of these enzymes from various sources is essentially the same, in that they catalyze the condensation of isopentenyl pyrophosphate with either dimethylallyl pyrophosphate or geranyl pyrophosphate to give farnesyl pyrophosphate, there are apparent differences in properties between animal and plant enzymes.(P). Green and West have demonstrated the existence of two forms of farnesyl pyrophosphate synthetase in soluble 1
This work was supported by a Grant-in-Aid for Scientific Research, No. 174179, from the Ministry of Education, Science and Culture of Japan. Vol. 82, No. 6, 1977
1585
extracts from castor bean seedlings (6), but no report has appeared on such multiplicity of any animal enzyme.We now report that two forms of farnesyl pyrophosphate synthetase were also separated from hog liver, and that they showed different properties from those of higher plant origin. EXPERIMENTAL PROCEDURE Enzyme Assay—The incubation mixture contained, in a final volume of 1.0 ml, 20 fxmo\ of Tris-HCI buffer, pH 7.7, 5 ^mol of MgCI,, 25 nmol of either dimethylallyl or geranyl pyrophosphate, 25 nmol of [l-14C]isopentenyl pyrophosphate (30 nCi) and a suitable amount of enzyme protein. After the addition of enzyme, the reaction mix-
1586 ture was incubated for 15 min at 37°C. The reaction was terminated by the addition of 0.3 ml of 1 N H O and the mixture was incubated again for 15 min to complete the hydrolysis of acidlabile allylic pyrophosphate. The mixture was then made alkaline by the addition of 0.5 ml of 1 N NaOH, and the free alcohols liberated were extracted with 4.0 ml of hexane. The hexane extract was washed with water and the radioactivity in a 2 ml aliquot of the extract was measured by liquid scintillation counting. One unit of enzyme activity was defined as the activity required to incorporate 1 nmol of [l-"C]isopentenyl pyrophosphate into allylic pyrophosphate per min. Specific activity is expressed in units of enzyme activity per mg of protein. Protein concentration was measured in terms of the absorbance at 280 nm using an A\£m value of 10. Enzyme Purification—All procedures were performed at 4°C. Fresh hog liver (800 g) was homogenized in 1,600 ml of 50mM Tris-HCl buffer, pH 6.8, for 75 s in a Waring blender. The supernatant obtained by centrifugation at 26,000 x g for 30 min was filtered through gauze. Ammonium sulfate (443 g) was added to 1,570 ml of the filtrate (45% saturation), and the mixture was centrifuged at 26,000 x g for 30 min. To 1,390 ml of the resulting supernatant, 91 g of ammonium sulfate was added (55% saturation), and the mixture was centrifuged at 26,000 xg for 30 min. The pellet was dissolved in 150 ml of 50 ITIM TrisHCl buffer, pH6.8. This solution was filtered through a Sephadex G-25 column (2.7x90cm) equilibrated with 50 ITIM Tris-HCl buffer, pH 6.8, containing 50 ITIM NaCl. The protein fraction was applied to a 4 x 55 cm column of DEAE-Sephadex A-50 previously equilibrated with 50 ITIM Tris-HCl buffer, pH6.8, containing 50 mM NaCl. The column was then developed with a linear gradient of 2 liters of 50 to 300 mM NaCl in 50 mM TrisHCl buffer, pH 6.8 (Fig. 1). Interconversion between Two Enzyme Forms— To 550 ml of the enzyme fraction which had a larger elution volume in the DEAE-Sephadex chromatography described above (fraction numbers 223-262), 256 g of ammonium sulfate was added (70% saturation), and the mixture was centrifuged at 26,000 x g for 60 min. The pellet was dissolved in 50 ml of 50 mM Tris-HCl buffer, pH 6.8, and the solution was dialyzed against 50 mM Tris-HCl
T. KOYAMA, Y. SAITO, K. OGURA, and S. SETO buffer, pH 6.8, containing 10 mM 2-mercaptoethanol and 50 mM NaCl for 18 h. Then 10 ml of the dialyzed solution (specific activity, 15.6 units/mg) was loaded onto a 1.7x20 cm column of DEAE-Sephadex A-50 which had been washed with 50 mM Tris-HCl buffer, pH 6.8, containing 10 mM 2-mercaptoethanol and 50 mM NaCl. The column was developed with a linear gradient of NaCl (total volume, 400 ml) similar to that used in the purification procedure, except that 10 mM 2-mercaptoethanol was contained in the eluent (Fig. 2). The enzyme which emerged in the earlier fraction (fraction numbers 25-40) was combined and concentrated with a Diafilter apparatus fitted with a G-10 T membrane. After dialysis against 50 mM Tris-HCl buffer, pH 6.8, containing 50 mM NaCl for 17 h, the solution (10 ml; specific activity, 181.8 units/mg) was applied to a 1.7x20 cm column of DEAE-Sephadex A-50 and eluted with a linear gradient similar to that described above except that no 2-mercaptoethanol was contained (Fig. 3). The enzyme protein which emerged in the earlier fraction in the chromatography of Fig. 1 (fraction numbers 163-203) was collected and concentrated with a Diafilter apparatus fitted with a G-10 T membrane. Then one-fifth of the resulting solution (5 ml, specific activity, 3.3 units/ mg) was dialyzed for 16 h against 50 ITIM TrisHCl buffer, pH 6.8, in the presence of 50 fiM CuSO4. The solution was applied to a 1 x 18 cm column of DEAE-Sephadex A-50 equilibrated with 50 mM Tris-HCl buffer, pH 6.8, containing 50 mM NaCl. The column was developed with a linear gradient of 400 ml of 50 to 300 mM NaCl in 50 mM Tris-HCl buffer, pH 6.8 (Fig. 4). RESULTS AND DISCUSSION In DEAE-Sephadex chromatography of the protein fraction precipitating between 45 and 55% saturation of ammonium sulfate, farnesyl pyrophosphate synthetase activity was eluted in two distinct peaks, hereafter designated as farnesyl pyrophosphate synthetase A and B, as shown in Fig. 1. The fractions of synthetase A and B were completely free of isopentenyl pyrophosphate isomerase and phosphatase activities, both of which would interfere with the farnesyl pyrophosphate synthetase assay. /. Biochem.
TWO FORMS OF FARNESYL PYROPHOSPHATE SYNTHETASE
50
100
150
200
1587
250
FRACTION NUMBER Umi/fnc. Fig. 1. DEAE-Sephadex A-50 chromatography of the 45-55% ammonium sulfate fraction. Details of chromatography are described in " EXPERIMENTAL PROC E D U R E . " The start of the gradient is indicated by an arrow. Aliquots (0.1 ml) were assayed for farnesyl pyrophosphate synthetase activity ( O ) ; -4i«o. ( A ) ; conductivity, ( x ) .
TABLE I. Comparison of properties of the two fractions. Dimethylallyl pyrophosphate was generally used as the allylic substrate; figures in parentheses represent data obtained when geranyl pyrophosphate was employed. Synthetase A and B fractions obtained in the first DEAE-Sephadex chromatography were used. Each incubation mixture contained 0.4 unit of enzyme. Details of the assay are described in " EXPERIMENTAL PROCEDURE." Synthetase A pH optima:
ca.
Susceptibilities to metal ions: Mg*+
5 mM
0.5 Mn'*
5 mM
0.5 None Effects of sulfhydryl reagents:t> 2-Mercaptoethanol
10 mM
1 Glutathione
10 mM
5 1 1,4-Dithiothreitol
10 mM
5 1 Effects of sulfhydryl inhibitors; 6 p-Chloromercuribenzoic acid W-Ethylmaleimide Iodoacetamide
8.0 ca.
Molecular weights:*
5 flM
0.5 1 mM 1 mM
100% 93 71 72 0.02 140% 107 18 87 108 109 115 113 5% 75 7 38
Synthetase B
(8.0-9.0)
ca.
000%) (94 ( 76 (62 (0.04
8.0 ca.
90,000
) ) ) )
(104%)
(110 )
.
100% 57 55 52 0.02 1,023% 1,017 188 1,063 1,099 1,118 1,057 1,019
(7. 8-8. 8) 90,000 (100%)
(71 ) (41 ) ( 36 ) (0.02)
(650%)
(1,367)
0.5% 4 4 1
» Estimated by Sephadex G-100 gel filtration (column size; 1.7x58 cm). b Preincubation with an appropriate additive at 37°C for 5 min was carried out prior to the addition of [l-l*C]isopentenyl pyrophosphate.
Vol. 82, No. 6, 1977
1588
T. KOYAMA, Y. SAITO, K. OGURA, and S. SETO
The exclusive formation of iT,E-farnesyl pyrophosphate by both of these two fractions was confirmed by radio-TLC and radio-gas chromatographic identification of [14C]£,.E-farnesol formed by treatment of the product with alkaline phosphatase, as usual (70). As listed in Table I, farnesyl pyrophosphate synthetases A and B showed little difference in pH optima, molecular weights and susceptibilities to metal ions. However, significant differences were observed in the effects of sulfhydryl reagents. The activity of farnesyl pyrophosphate synthetase B was markedly enhanced by the addition of sulfhydryl reagents such as 2-mercaptoethanol, glutathione, and 1,4-dithiothreitol, whereas the synthetase A was hardly activated by these reagents. The effect of glutathione on the synthetase A at 5 IHM or higher concentration was inhibitory rather than stimulative. The synthetase B fraction obtained by the first DEAE-Sephadex chromatography (specific activity, ca. 15) showed about 10-fold stimulation of activity in the presence of sulfhydryl reagents, as shown in Table I. This enzyme, when recycled on DEAE-Sephadex, was recovered in a single peak in the same conductivity region, though the activity decreased so that it was hardly detectable in the absence of sulfhydryl reagents. The synthetase B was more sensitive to inhibition by sulfhydryl-blocking reagents than synthetase A.
To investigate further the effect of sulfhydryl reagents, farnesyl pyrophosphate synthetase B was dialyzed against a buffer solution containing lOmM 2-mercaptoethanol. Then the enzyme solution was chromatographed on DEAE-Sephadex under the same conditions, except that 10 mM 2-mercaptoethanol was contained in the eluent. More than 80 % of the total enzyme activity recovered was located in the earlier emerging fraction corresponding to synthetase A (Fig. 2). This fraction was dialyzed to remove the sulfhydryl reagent and then subjected to the same chromatography except that the eluent contained no sulfhydryl reagent. Although only a single peak corresponding to synthetase A was observed when assayed in the absence of a sulfhydryl reagent, about 43% of the recovered activity could be found at the conductivity region corresponding to the synthetase B when assayed in the presence of 10 mM 1,4 dithiothreitol, as shown in Fig. 3. Direct conversion of the synthetase A into B was observed when synthetase A was dialyzed against a buffer solution containing 50 fiM CuSO4. Almost all the enzyme activity recovered was located at the elution volume corresponding to the synthetase B in DEAE-Sephadex chromatography. Moreover, the stimulation by sulfhydryl reagents, which is characteristic of the synthetase B, was so marked that the enzyme activity of this fraction could be
60 FRACTION NUMBER
70
5ml / f r « .
Fig. 2. DEAE-Sephadex A-50 chromatography of farnesyl pyrophosphate synthetase B fraction in Fig. 1 in the presence of 10 mM 2-mercaptoethanol. Details of the chromatography are described in " EXPERIMENTAL PROCEDURE." The gradient was initiated at the beginning of chromatography. Aliquots (0.1 ml) were assayed for farnesyl pyrophosphate synthetase activity (O); /•*»», ( A ) ; conductivity, ( x ) . / . Biochem.
TWO FORMS OF FARNESYL PYROPHOSPHATE SYNTHETASE
1589
0-4
06 03
c — 04 0-2
02
01
FRACTION NUMBER 5ml / frac. Fig. 3. Rechromatography of famesyl pyrophosphate synthetase A fraction in Fig. 2 in the absence of 2-mercaptoethanol. Details of chromatography are described in " EXPERIMENTAL PROCEDURE." The gradient was initiated at the beginning of the chromatography. Aliquots (0.5 ml) were assayed for famesyl pyrophosphate synthetase activity in the presence of 10 mM 1,4-dithiothreitol (O) or in the absence of sulfhydryl reagents ( • ) . Aat ( A ) , conductivity ( x ) . 015 X
-
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"
/
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8
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10
20
30
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FRACTION NUMBER
100
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Fig. 4. DEAE-Sephadex A-50 chromatography of famesyl pyrophosphate synthetase A fraction in Fig. 1 after dialysis against a buffer solution containing cupric ions. Details of chromatography are described in " EXPERIMENTAL PROCEDURE." The gradient was initiated at the beginning of the chromatography. AJiquots (0.5 ml) were assayed for famesyl pyrophosphate synthetase activity in the presence of 10 mM 1,4-dithiothreitol (O) or in the absence of sulfhydryl reagents ( # ) . A^ ( A ) , conductivity ( x ) . located only by assay in the presence of a sulfhydryl reagent (Fig. 4). These findings clearly show that the famesyl pyrophosphate synthetases A and B arc interconvertible. Green and West reported the existence of two forms of famesyl pyrophosphate synthetase separable by QAE-Sephadex chromatography in a Vol. 82, No. 6, 1977
higher plant, but offered no evidence of interconversion between them. However, they have found that the molecular weight and kinetic properties of each form of the enzyme vary depending on the protein concentration, because of proteinprotein interaction (6). The present enzyme, however, did not show such changes in properties
1590
even at a high protein concentration. Furthermore, the molecular weights of our farnesyl pyrophosphate synthetases A and B, both ca. 90,000 as estimated by Sephadex G-100 gel filtration (Table I), were nearly identical with that reported for the enzyme from avian liver (8) and different from that of the enzyme from castor bean (6). There are also some remarkable differences between the two forms of farnesyl pyrophosphate synthetase from hog liver and from castor bean seedlings with respect to the pH optima and the susceptibilities to metal ions and sulfhydryl inhibitors. The fact that the conversion of farnesyl pyrophosphate synthetase B to A requires a sulfhydryl reagent, whereas the reverse process is stimulated by cupric ions, suggests that the formation and cleavage of disulfide bond(s) is involved in this interconversion, since the air oxidation of sulfhydryl groups in proteins is strongly catalyzed by some transition metal ions, especially cupric ions (77). Reed and Rilling obtained farnesyl pyrophosphate synthetase in a stable crystalline form from avian liver (8). It seems likely that their enzyme corresponds to the form A in this paper, because they employed 10 mM 2-mercaptoethanol throughout the purification procedure. They have recently reported that the avian liver enzyme contains 13 cysteine residues but no disulfide bond (72). Thus, the properties of the synthetase B might be attributed to an oxidized form of the enzyme containing disulfide bond(s). It has also been suggested that the avian enzyme consists of two identical subunits, each of which carries onecatalytic site (72). If a sulfhydryl group is essential for the catalytic activity, there might be two degrees of reversible inactivation, partial and complete, according as only one or both of the subunits are oxidized. The major portion of the synthetase B obtained by the first DEAE-Sephadex chromatography might be suffering from partial
T. KOYAMA, Y. SAITO, K. OGURA, and S. SETO
inactivation and the synthetase B which has no enzyme activity in the absence of a sulfhydryl reagent, as shown in Fig. 3 and 4, might be completely inactivated. It will be of great interest to investigate the role of the sulfhydryl groups to develop a better understanding of the mechanism of farnesyl pyrophosphate synthetase. Note added in proof: While this paper was in press, we were informed by Dr. H.C. Rilling, University of Utah, that he had also made similar observations with farnesyl pyrophosphate synthetase from pig liver {Arch. Biochem. Biophys., in press). We are grateful to him for this preliminary communication. REFERENCES 1. Lynen, F., Agranoff, B.W., Eggerer, H., Henning, U., & Mdslein, E.M. (1959) Angew. Chem. Int. Ed. 71, 657-667 2. Dorsey, J.K., Dorsey, J.A., & Porter, J.W. (1966) / . Biol. Chem. 1A\, 5353-5360 3. Holloway, P.W. & Popjak, G. (1967) Biochem. J. 104, 57-70 4. Ogura, K., Nishino, T., & Seto, S. (1968) /. Biochem. 64, 197-203 5. Ogura, K., Shinka, T., & Seto, S. (1972) J. Biochem. 72,1101-1108 6. Green, T.R. & West, C.A. (1974) Biochemistry 13, 4720-4729 7. Eberhardt, N.L. & Rilling, H.C. (1975) J. Biol. Chem. 250, 863-866 8. Reed, B.C. & Rilling, H.C. (1975) Biochemistry 14, 50-54 9. Nishino, T., Ogura, K., & Seto, S. (1973) Biochim. Biophys. Ada 302, 33-37 10. Ogura, K., Nishino, T., Koyama, T., & Seto, S. (1970) /. Amer. Chem. Soc. 92, 6036-6041 11. Takagi, T. & Isemura, T. (1964) / . Biochem. 56, 344-350 12. Reed, B.C. & Rilling, H.C. (1976) Biochemistry 15, 3739-3745
/ . Biochem.
