305 Biochimica et Biophysics Acta, 575 (1979) 305-308 Q Elsevier/North-Holland Biomedical Press
BBA Report BBA 51262
HYDROPHOBIC C~RO~ATO~RAP~Y
JANINE D. MUTH, TSUNEO
OF PRENYL TRANSFERASES
BABA and CHARLES
M. ALLEN*
Department of Biochemistry, Uniuersity of Florida, J. Hiltis Miller Health Center, Gainesville, FL 32610 (U.S.A.) (Received
June 22nd, 1979)
Key words: Prenyl transferase; Hydrophobic
chromatography
Several prenyl transferases were examined with respect to their affinity for CO--C10 alkyl-Agarose columns. Clo-alkyl Agarose was effective in adsorbing each of the prenyl transferases tested but only undecaprenyl pyrophosphate and octaprenyl pyrophosphate synthetases could be effectively eluted with Triton X-100.
Enzymes which are found associated with lipids because of their location in membranes or because of the hydrophobic nature of their substrates, products or cofactors are attractive candidates for hydrophobic -affinity chromatography. Adsorption of these enzymes to hydrophobic columns and desorption with detergents or specific ligands offer a potentially valuable method for separation and characterization of this class of proteins. The detergent and phospholipid requirements of the prenyl transferase, undecaprenylpyrophosphate (C&,-PI”)synthetase [l, 21 suggested some kind of membrane association for the synthetase. We have examined this potential association to a hydrophobic surface as a means of separating the synthetase from some soluble proteins. The ability of alkyl-Agarose columns to adsorb this and other prenyl transferases is reported here, Lactobacillus plantarum (ATCC 8014) C&-PP synthetase was partially purified through a hydroxyapatite step as previously described except that no Triton X-100 was used during the purification [l].Crude L. ~~a~turu~ C&P synthetase was a 100 000 X g supernatant obtained from the freshly lysed cells. Micrococcus Euteus (ATCC 4698) C&-PP synthetase, all trunsoctaprenyl pyrophosphate (C&P) synthetase and t,t,t-geranylgeranyl *To whom correspondence
should be addressed.
306
pyrophosphate (C,,-PP) synthetase were prepared by DEAE-cellulose chromatography of a 31 300 X g supernatant from lysozyme treated cells. The C,,-PP synthetase was further purified by hydroxylapatite chromatography (Baba, T., unpublished data). Prenyl transferase activity was measured using farnesyl pyrophosphate and A3-[ l-14C]isopentenyl pyrophosphate according to the previously described method [l] . Farnesyl pyrophosphate synthetase was analyzed using geranyl pyrophosphate as a substrate. Protein was determined by the method of Lowry et al. [3] as modified by Wang and Smith [4]. C,,-C,, alkyl-Agarose were obtained from Miles Chemical Corp. The concentration of alkyl groups on the Agarose was 15--20 pmol/ml of swollen gel. The crude and partially purified enzymes were applied to 1 ml columns of C,--C,, alkyl-Agarose equilibrated with 0.05 M potassium phosphate buffer pH 7.5. The columns were washed with 6 ml of equilibration buffer followed by sequential additions of 6 ml aliquots of equilibration buffer containing the eluting components (e.g. salt, Triton X-100, substrate). Two ml fractions were collected and aliquots of each fraction were analyzed for protein and prenyl transferase activity. The adsorption and elution of prenyl transferases from L. plantarum and M. luteus from columns of Agarose containing no alkyl substituent (C,), ethyl (C,), butyl (C,), hexyl (C,), octyl (C,) and decyl (C,,) substituents were studied. Fig. 1 shows the elution profile of enzymic activity and protein of the crude synthetase from L. plantarum on Co-, C6-, Cs- and C,O-Agarose columns using increasing amounts of Triton X-100 (up to 0.5%) in the eluting buffer. The profiles observed using C,- and C,-Agarose were the same as seen with Co-Agarose (data not shown). It was apparent that C,-Agarose (Fig. lA), CZ- and C,-Agarose did not bind the L. plantarum Cs5 -PP synthetase
4
.
co-
o
C6-AGAROSE
.
C8-
I
2
4
6
8
FRACTION
AGAROSE AGAROSE
C,cAGAROSE
10
12
14
NO
Fig. 1. Chromatography of C,,-PP synthetase from L. plontarum WI alkyl-AgaroSe columns. Crude L. plantarum C,,-PP synthetase (0.2 ml) was applied to columns of C,-C,, alkyl-Agarose and eluted as described in the text with buffers containing the indicated amounts of Triton X-100 (TX-100). Fractions were then analyzed for enzymic activity (A) and protein (B).
since the enzyme eluted immediately with the equilibration buffer. CgAgarose slightly retarded the enzyme movement down the column. C8- and ClO-Agarose were both effective in adsorbing the enzyme. The addition of 0.1 to 0.5% Triton X-100 was necessary, in these cases, for the elution of the enzyme. It can be seen by analysis of the eluted protein (Fig. 1B) that some purification of the crude enzyme was effected by this method. Chromatography of a hydroxyapatite purified &-PP synthetase on C,,-Agarose resulted in elution patterns similar to that seen with the crude enzyme and gave a 2- to 3-fold purification of the enzyme. The enzyme was not eluted from C,,-Agarose using eluting buffers containing high salt (0.2 M potassium phosphate pH 7.5 or 2 M NaCl). Failure of the prenyl transferase to be eluted at high ionic strength precluded simple ion exchange chromatography. The hydroxyapatite purified enzyme also remained bound to Cl0 - Agarose when elution was attempted with a substrate, geranyl pyrophosphate (10 PM) or a related terpenyl monophosphate, geranyl monophosphate (10 PM). The K, of the Cs,-PP synthetase for geranyl pyrophosphate is 3 PM [ 51. Under all the conditions described above, the enzyme could be recovered by elution with 0.5---l% (8-16 mM) Triton X-100. The elution behavior of the enzyme in the presence of Triton X-100 would seem to indicate that the enzyme was not binding to the alkyl-Agarose via the catalytic binding site. This conclusion is supported by two points. First, the binding of the substrate to the active site is not inhibited by Triton X-100 since Triton X-100 activates the enzyme. Therefore Triton X-100 and the substrate do not associate with the enzyme at the same site. Second, the concentration of Triton X-100 resulting in one half maximum stimulation of the synthetase is 6 mM [l] and the enzyme was eluted from alkyl-Agarose by B-16 mM Triton X-100. This suggests that the alkyl groups of the column and Triton X-100 were associated with the enzyme in a similar way. C -, C,,,- and C,,-PP synthetase from M. luteus were also examined for their el?ition patterns from Cl,-Agarose columns. The results of studies with the latter two enzymes and C!&‘P synthetase from L. pluntarum are illustrated in Table I. The relative retentions of these enzymes on Co- and CtO-Agarose are compared. It was apparent that each of the enzymes was recovered from TABLE
I
ADSORPTION
OF PRENYL
TRANSFERASES
TO C,,-ALKYL-AGAROSE
Each enzyme preparation (0.24.7 ml) was applied to a 1 ml Agarose as indicated. Six ml of the appropriate eluting buffers in the text. Aliquots were taken from each fraction (6 ml) for activities recovered from Co- and C,,-Agarose were essentially Percentage
of total activity C,,-PP synthetase M. luteus ____~~
C,,-PP synthetase M. luteus
C,,-PP synthetase L. plantarum
Eluting buffer Agarose
column containing either C,- or C,,were subsequently added as described analysis of enzymic activity. The total the same for each enzyme tested.
column
Buffer Wash 0.02-0.05% Triton X-100 0.1% Triton X-100 0.5% Triton X-100 1% Triton X-100
C0
C 10
C0
CM
92 8 0
2 2 10 86 -
97 2 0 0 0
0 0 4 65 29
-
~-
C0
C,,
100 0 0 0 -
0 3 5 92 -
__-
308
ClO-Agarose by elution with 0.5--l% Triton X-100. In other experiments, C&Y’ synthetase from M. luteus was recovered completely in the equilibration buffer wash from C,-Agarose but less than 15% of the activity was recovered from C,,,-Agarose under the elution conditions described for the other enzymes. Similarly, farnesyl pyrophosphate synthetase from Bakers’ yeast (partially purified through the first ammonium sulfate fractionation step [ 61) was adsorbed to ClO-Agarose, but was also recovered in less than 10% yield on elution with 1% Triton X-100. Therefore, the long chain prenyl pyrophosphate synthetases can be quantitatively eluted from C,,,-Agarose whereas the shorter chain prenyl pyrophosphate synthetases were bound tightly and were not eluted effectively under the conditions tested. The behavior of all the enzymes on Co-, CZ-, and CqAgarose was similar. Although the recovery of Cs,-PP synthetase from Cg- and C,-Agarose was good (Fig. l), similar studies with C,,-PP and C,,-PP synthetases showed less than 40% recovery of activity (data not shown). The technique of hydrophobic column chromatography has been used successfully in a number of cases to separate and purify enzymes [ 7-91. The results presented here describe the first application of this technique to the adsorption and desorption of prenyl transferases. This technique might be applied to the separation of several different prenyl transferases which have similar substrates but different chain length products and different in vitro solution requirements (e.g. lipid requirements). They may be selectively eluted with different detergents or ‘water structure’ disrupting agents. This technique might also be applied to the separation of isoprenoid metabolizing enzymes other than these prenyl transferases. This work was supported by a grant from the NIH (GM-23193).
References Allen,
CM.,
Allen,
C.M.
Lowry,
O.H.,
Wang.
C.S.
Baba, Shaltiel,
Z..
Hofstee,
M.V.
Muth.
and
J.D.
Rosebrough,
and
T. and
Eberhardt. Er-el.
Keenan, and
Smith,
Allen,
M.L. S. (1974)
Fur.
(1975)
(1978)
Rilling.
H.C.
(1975)
Shaltiel,
and
Arch.
and
Biochem.
Randall, 17,
J. Biol.
34,
Biophys.
175.
236-248
R.J.
(1951)
J. Biol.
Chem.
193,
265-275
63.414-417 5598-5604 Chem.
250,
863-866
126-140
S. (1972) 52.
Biochem.
16.2908-2915
Biochem.
Biochemistry
Y.
Anal.
A.L.
Anal.
Enzymol.
(1973)
J. (1976)
Biochemistry,
Methods
Zaidenzaig, B.H.J.
N.J..
R.L.
C.M.
and
Sack,
(1977)
Biochem.
430-448
Biophys.
Res.
Commun.
49,
383-390
BBA Report BBA 51262
HYDROPHOBIC C~RO~ATO~RAP~Y
JANINE D. MUTH, TSUNEO
OF PRENYL TRANSFERASES
BABA and CHARLES
M. ALLEN*
Department of Biochemistry, Uniuersity of Florida, J. Hiltis Miller Health Center, Gainesville, FL 32610 (U.S.A.) (Received
June 22nd, 1979)
Key words: Prenyl transferase; Hydrophobic
chromatography
Several prenyl transferases were examined with respect to their affinity for CO--C10 alkyl-Agarose columns. Clo-alkyl Agarose was effective in adsorbing each of the prenyl transferases tested but only undecaprenyl pyrophosphate and octaprenyl pyrophosphate synthetases could be effectively eluted with Triton X-100.
Enzymes which are found associated with lipids because of their location in membranes or because of the hydrophobic nature of their substrates, products or cofactors are attractive candidates for hydrophobic -affinity chromatography. Adsorption of these enzymes to hydrophobic columns and desorption with detergents or specific ligands offer a potentially valuable method for separation and characterization of this class of proteins. The detergent and phospholipid requirements of the prenyl transferase, undecaprenylpyrophosphate (C&,-PI”)synthetase [l, 21 suggested some kind of membrane association for the synthetase. We have examined this potential association to a hydrophobic surface as a means of separating the synthetase from some soluble proteins. The ability of alkyl-Agarose columns to adsorb this and other prenyl transferases is reported here, Lactobacillus plantarum (ATCC 8014) C&-PP synthetase was partially purified through a hydroxyapatite step as previously described except that no Triton X-100 was used during the purification [l].Crude L. ~~a~turu~ C&P synthetase was a 100 000 X g supernatant obtained from the freshly lysed cells. Micrococcus Euteus (ATCC 4698) C&-PP synthetase, all trunsoctaprenyl pyrophosphate (C&P) synthetase and t,t,t-geranylgeranyl *To whom correspondence
should be addressed.
306
pyrophosphate (C,,-PP) synthetase were prepared by DEAE-cellulose chromatography of a 31 300 X g supernatant from lysozyme treated cells. The C,,-PP synthetase was further purified by hydroxylapatite chromatography (Baba, T., unpublished data). Prenyl transferase activity was measured using farnesyl pyrophosphate and A3-[ l-14C]isopentenyl pyrophosphate according to the previously described method [l] . Farnesyl pyrophosphate synthetase was analyzed using geranyl pyrophosphate as a substrate. Protein was determined by the method of Lowry et al. [3] as modified by Wang and Smith [4]. C,,-C,, alkyl-Agarose were obtained from Miles Chemical Corp. The concentration of alkyl groups on the Agarose was 15--20 pmol/ml of swollen gel. The crude and partially purified enzymes were applied to 1 ml columns of C,--C,, alkyl-Agarose equilibrated with 0.05 M potassium phosphate buffer pH 7.5. The columns were washed with 6 ml of equilibration buffer followed by sequential additions of 6 ml aliquots of equilibration buffer containing the eluting components (e.g. salt, Triton X-100, substrate). Two ml fractions were collected and aliquots of each fraction were analyzed for protein and prenyl transferase activity. The adsorption and elution of prenyl transferases from L. plantarum and M. luteus from columns of Agarose containing no alkyl substituent (C,), ethyl (C,), butyl (C,), hexyl (C,), octyl (C,) and decyl (C,,) substituents were studied. Fig. 1 shows the elution profile of enzymic activity and protein of the crude synthetase from L. plantarum on Co-, C6-, Cs- and C,O-Agarose columns using increasing amounts of Triton X-100 (up to 0.5%) in the eluting buffer. The profiles observed using C,- and C,-Agarose were the same as seen with Co-Agarose (data not shown). It was apparent that C,-Agarose (Fig. lA), CZ- and C,-Agarose did not bind the L. plantarum Cs5 -PP synthetase
4
.
co-
o
C6-AGAROSE
.
C8-
I
2
4
6
8
FRACTION
AGAROSE AGAROSE
C,cAGAROSE
10
12
14
NO
Fig. 1. Chromatography of C,,-PP synthetase from L. plontarum WI alkyl-AgaroSe columns. Crude L. plantarum C,,-PP synthetase (0.2 ml) was applied to columns of C,-C,, alkyl-Agarose and eluted as described in the text with buffers containing the indicated amounts of Triton X-100 (TX-100). Fractions were then analyzed for enzymic activity (A) and protein (B).
since the enzyme eluted immediately with the equilibration buffer. CgAgarose slightly retarded the enzyme movement down the column. C8- and ClO-Agarose were both effective in adsorbing the enzyme. The addition of 0.1 to 0.5% Triton X-100 was necessary, in these cases, for the elution of the enzyme. It can be seen by analysis of the eluted protein (Fig. 1B) that some purification of the crude enzyme was effected by this method. Chromatography of a hydroxyapatite purified &-PP synthetase on C,,-Agarose resulted in elution patterns similar to that seen with the crude enzyme and gave a 2- to 3-fold purification of the enzyme. The enzyme was not eluted from C,,-Agarose using eluting buffers containing high salt (0.2 M potassium phosphate pH 7.5 or 2 M NaCl). Failure of the prenyl transferase to be eluted at high ionic strength precluded simple ion exchange chromatography. The hydroxyapatite purified enzyme also remained bound to Cl0 - Agarose when elution was attempted with a substrate, geranyl pyrophosphate (10 PM) or a related terpenyl monophosphate, geranyl monophosphate (10 PM). The K, of the Cs,-PP synthetase for geranyl pyrophosphate is 3 PM [ 51. Under all the conditions described above, the enzyme could be recovered by elution with 0.5---l% (8-16 mM) Triton X-100. The elution behavior of the enzyme in the presence of Triton X-100 would seem to indicate that the enzyme was not binding to the alkyl-Agarose via the catalytic binding site. This conclusion is supported by two points. First, the binding of the substrate to the active site is not inhibited by Triton X-100 since Triton X-100 activates the enzyme. Therefore Triton X-100 and the substrate do not associate with the enzyme at the same site. Second, the concentration of Triton X-100 resulting in one half maximum stimulation of the synthetase is 6 mM [l] and the enzyme was eluted from alkyl-Agarose by B-16 mM Triton X-100. This suggests that the alkyl groups of the column and Triton X-100 were associated with the enzyme in a similar way. C -, C,,,- and C,,-PP synthetase from M. luteus were also examined for their el?ition patterns from Cl,-Agarose columns. The results of studies with the latter two enzymes and C!&‘P synthetase from L. pluntarum are illustrated in Table I. The relative retentions of these enzymes on Co- and CtO-Agarose are compared. It was apparent that each of the enzymes was recovered from TABLE
I
ADSORPTION
OF PRENYL
TRANSFERASES
TO C,,-ALKYL-AGAROSE
Each enzyme preparation (0.24.7 ml) was applied to a 1 ml Agarose as indicated. Six ml of the appropriate eluting buffers in the text. Aliquots were taken from each fraction (6 ml) for activities recovered from Co- and C,,-Agarose were essentially Percentage
of total activity C,,-PP synthetase M. luteus ____~~
C,,-PP synthetase M. luteus
C,,-PP synthetase L. plantarum
Eluting buffer Agarose
column containing either C,- or C,,were subsequently added as described analysis of enzymic activity. The total the same for each enzyme tested.
column
Buffer Wash 0.02-0.05% Triton X-100 0.1% Triton X-100 0.5% Triton X-100 1% Triton X-100
C0
C 10
C0
CM
92 8 0
2 2 10 86 -
97 2 0 0 0
0 0 4 65 29
-
~-
C0
C,,
100 0 0 0 -
0 3 5 92 -
__-
308
ClO-Agarose by elution with 0.5--l% Triton X-100. In other experiments, C&Y’ synthetase from M. luteus was recovered completely in the equilibration buffer wash from C,-Agarose but less than 15% of the activity was recovered from C,,,-Agarose under the elution conditions described for the other enzymes. Similarly, farnesyl pyrophosphate synthetase from Bakers’ yeast (partially purified through the first ammonium sulfate fractionation step [ 61) was adsorbed to ClO-Agarose, but was also recovered in less than 10% yield on elution with 1% Triton X-100. Therefore, the long chain prenyl pyrophosphate synthetases can be quantitatively eluted from C,,,-Agarose whereas the shorter chain prenyl pyrophosphate synthetases were bound tightly and were not eluted effectively under the conditions tested. The behavior of all the enzymes on Co-, CZ-, and CqAgarose was similar. Although the recovery of Cs,-PP synthetase from Cg- and C,-Agarose was good (Fig. l), similar studies with C,,-PP and C,,-PP synthetases showed less than 40% recovery of activity (data not shown). The technique of hydrophobic column chromatography has been used successfully in a number of cases to separate and purify enzymes [ 7-91. The results presented here describe the first application of this technique to the adsorption and desorption of prenyl transferases. This technique might be applied to the separation of several different prenyl transferases which have similar substrates but different chain length products and different in vitro solution requirements (e.g. lipid requirements). They may be selectively eluted with different detergents or ‘water structure’ disrupting agents. This technique might also be applied to the separation of isoprenoid metabolizing enzymes other than these prenyl transferases. This work was supported by a grant from the NIH (GM-23193).
References Allen,
CM.,
Allen,
C.M.
Lowry,
O.H.,
Wang.
C.S.
Baba, Shaltiel,
Z..
Hofstee,
M.V.
Muth.
and
J.D.
Rosebrough,
and
T. and
Eberhardt. Er-el.
Keenan, and
Smith,
Allen,
M.L. S. (1974)
Fur.
(1975)
(1978)
Rilling.
H.C.
(1975)
Shaltiel,
and
Arch.
and
Biochem.
Randall, 17,
J. Biol.
34,
Biophys.
175.
236-248
R.J.
(1951)
J. Biol.
Chem.
193,
265-275
63.414-417 5598-5604 Chem.
250,
863-866
126-140
S. (1972) 52.
Biochem.
16.2908-2915
Biochem.
Biochemistry
Y.
Anal.
A.L.
Anal.
Enzymol.
(1973)
J. (1976)
Biochemistry,
Methods
Zaidenzaig, B.H.J.
N.J..
R.L.
C.M.
and
Sack,
(1977)
Biochem.
430-448
Biophys.
Res.
Commun.
49,
383-390
