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[16] Rubber transferase from Hevea brasiliensis latex
476
ENZYME SYSTEMS: TERPENOID AND STEROL BIOSYNTHESIS
[16] Rubber
[16]
T r a n s f e r a s e f r o m H e v e a brasiliensis L a t e x By B. L. A~CHER and E. G. C0CKBAIN
CH3
CH3
[
I
- - [ C H 2 - - C ~ C H - - C H 2 ] . - - P P q- CH2~C--CH2--CH2--PP--~ Rubber pyrophosphate isopentenyl pyrophosphate CH3 [CH~--C~---CH--CH2].+I--PP -}- PP Rubber transferase catalyzes the incorporation of isopentenyl-PP into rubber (cis-l,4-polyisoprene). By analogy with the reaction between geranyl-PP and isopentenyl-PP to form farnesyl-PP, ~,2 it is believed that rubber transferase catalyzes the transfer of cis-l,4-polyisoprenyl-PP to isopentenyl-PP with the elimination of inorganic pyrophosphate, the reaction occurring at the surface of the rubber particles in Hevea latex. ~ The enzyme, which is distributed between the aqueous phase of the latex and the surface of the rubber particles,* has been partially purified. Assay Method
Principle. The activities of rubber transferase preparations are compared by measuring the rates of incorporation of isopentenyl-PP-l-l~C into rubber. A suspension of washed rubber particles prepared from fresh Hevea latex serves as the acceptor for isopentenyl-PP in the assay. Essential cofactors for the incorporation are magnesium and glutathione (or cysteine). Different preparations of washed rubber particles, prepared by the same procedure, usually show different rates of incorporation of isopentenyl-PP into rubber in the presence of the same amounts of rubber transferase and cofactors. This variability is most likely due to variations in the size distribution of the rubber particles and/or the number of rubber molecules with terminal pyrophosphate groups at the surface of the particles. No method of determining the latter figure is available, nor has it been possible so far to establish assay conditions in which the rate of incorporation of isopentenyl-PP is zero order with respect to the rubber content of the suspension. Consequently, the assay method described
1G. Popjhk, Tetrahedron Letters 19, 19 (1959). 2F. Lynen, H. Eggerer, U. Henning, and I. Kessel, Angew. Chem. 70, 738 (1958). aB. L. Archer, B. G. Audley, E. G. Cockbain, and G. P. McSweeney,Biochem. J. 89, 565 (1963). 'A. I. McMullen and G. P. McSweeney, Biochem. J. 101, 42 (1966).
[16]
RUBBER BIOSYNTHESIS
IN Hevea LATEX
477
below is suitable only for comparing the relative transferase contents of different preparations, using the same suspension of washed rubber particles. The rate of incorporation of isopentenyl-PP into the washed rubber does not increase linearly with transferase concentration but obeys the relationship 5 1
A
= ~
+ B
(1)
where R is the rate of incorporation of isopentenyl-PP, [P] is the bulk concentration of the protein expressed on the volume of aqueous phase in the rubber suspension, and A and B are constants. Plots of 1/R against 1/[P] for different preparations of rubber transferase give straight lines from which the corresponding values of A are obtained. It can readily be shown from Eq. (1) that the transferase activity per unit weight of a preparation is inversely proportional to the value of A for that preparation. Reagents
Tris-HC1 buffer: The solution is 0.05M with respect to tris(hydroxymethyl)aminomethane and 0.0268M with respect to HCI; pH=8.2at20 ° Cofaetor solution: 19.2 mg of glutathione is dissolved in 0.5 ml of 2.8% (w/v) Tris in water and 0.5 ml of 0.0625 M MgS04 added Isopentenyl-PP-l-14C: 2.2 mg of the lithium salt is dissolved in water and diluted to 1 ml; 30 /~l of the solution contains 0.25 micromole of isopentenyl-PP (approximately 1400 dps) EDTA: 0 .2M ethylenediaminetetraacetic acid (sodium salt) adjusted to pH 8.0 with NaOH Trichloroacetic acid: 1% (w/v) solution in pure toluene Acetic acid-ethanol: 5% v / v acetic acid in ethanol Preparation o] Washed Rubber Particles. All operations are carried out at 0-5 °. Latex is obtained from Hevea brasiliensis trees in commercia1 production, by tapping into clean vessels cooled in ice. The latex is centrifuged at 2000 g for I0 minutes to remove the sedimentable nonrubber bottom fraction particles2 Then 60 ml of the upper fraction is diluted with 30 ml of Tris-HC1 buffer and centrifuged at 7000 g for 1
°B. L. Archer, Proc. Nat. Rubber Producers" Res. Assoc. Jubilee ConS. Cambridge, 1964 p. 101. Maclaren, London, 1965. ' L. N. S. Homans, J. W. van Dalfsen, and G. E. van Gils, Nature 161, 177 (1948).
478
ENZYME SYSTEMS: TERPENOID AND STEROL BIOSYNTHESIS
[15]
hour. The lower layer containing the finer particles is separated and reeentrifuged at 150,000 q for 1 hour in a swing-out rotor (e.g., Spineo SW 39L). The layer of rubber particles is carefully redispersed in Tris-buffer and diluted to 60 ml. The centrifugation and redispersion are repeated twice more, any coagulated rubber being removed at each stage. The final volume of rubber dispersion should be about 10 ml, and it should contain about 1 g of rubber. It is stable for up to 2 days at 0% Assay Procedure. A number of 100-rag samples of the suspension of washed rubber are weighed into 2 X ~-ineh rimless tubes; cofactor solution (20 t~l) is added, followed by the enzyme preparation to be assayed. Each preparation is used in a range of concentrations varying by a factor of about fifty. The mixtures are each diluted to 220/~1 with 0.05 M Tris buffer, and 30 t~l of IPP-I~C solution is added. Incubations are carried out for 1 hour at 30 ° and terminated by the addition of 0.25 ml of 0.2M EDTA. The contents of the tubes are dried in a stream of air or oxygen at 70 ° while the tubes are rotated at about 100 rpm with their axes horizontal. When dry, the rubber in the tubes is immersed in acetic acidethanol for 1 hour in order to produce a completely nondispersible film which is then washed several times with water. The rubber films are then digested at 100 ° for 1 hour in N KOH in order to remove hydrolyzable impurities, washed with water and extracted with boiling ethanol continuously for 16 hours to ensure removal of low molecular weight isoprenoid contaminants. After superficial drying, each sample of rubber is dissolved in 2 ml of trichloroacetic acid solution in toluene, scintillator is added to give a total volume of 6 ml, and the solutions are counted by a standard scintillation technique. The above purification procedure has been checked by ozonolysis of the rubber and recrystallization of the resulting levulinie acid as the dinitrophenylhydrazone. No retention of nonrubber isoprenoid contaminants could be detected. After correction for the residual biosynthetic activity of the washed rubber in the absence of added enzyme, the reciprocal of the rate of incorporation of 1'C into the rubber is plotted against the reciprocal of the protein concentration for each of the enzyme preparations and their relative purities are determined from the values of 1/A [Eq. (1)]. Protein concentrations are determined by the Folin-Ciocalteau method or by ultraviolet absorptiometry at 260 and 280 m~. 7 Provided the incorporation of 1'C into rubber does not exceed 30%, the uptake vs time graphs are linear for the first hour. As the observed enzymatic activities are dependent on the sample of washed rubber used, it is not possible to define absolute units of rubber transferase activity. The method appears to be satisfactory for both unpurified and partially purified samples of the transferase enzyme. ' E . Layne, see Vol. I I I [73].
[16]
RUBBER BIOSYNTHESIS IN Hevea ~TEX
479
Enzyme Purification All operations are carried out at 0-4 ° . Step I. Preparation o] Serum. Latex is collected, and the bottom fraction is removed as outlined above. The upper fraction is then centrifuged at 60,000 g to remove most of the rubber, and the resulting, almost clear, aqueous serum is freeze-dried. Then 100 g of the dried solids (ex 1600 ml of serum) is dissolved in oxygen-free water, diluted to 300 ml, and centrifuged at 105,000 g for 90 minutes to remove most of the remaining rubber. Step 2. Acidification. The pH of the solution is lowered to 5.4 by the gradual addition of approximately 35 ml of 0.83 M H s P Q . The precipitate of inactive protein is centrifuged off, and the superna5ant is rapidly neutralized to pH 7.0 with 0.1 N Tris. Step 3. Ammonium Sulfate Fractionation. Saturated ammonium sulfate containing 0.01 M magnesium sulfate and adjusted to pH 7.0 is then added stepwise to give fractions precipitating at 25, 50, and 60% of saturation. The mixtures are each left to stand for 30 minutes before centrifugation. The first fraction normally retains the last of the very small rubber particles and leaves a crystal clear supernatant. The last fraction precipitated between 50 and 60% of saturation contains the active enzyme, which, after washing with 65% saturated ammonium sulfate solution, is dissolved in 15 ml of 0.01 M magnesium sulfate and dialyzed overnight against a large excess of 0.2 M Tris-HC1 buffer at pH 8.2 containing 0.001 M MgS04. Step ~. Gel Filtration Chromatography on Sephadex G-IO0. The solution containing the enzyme is applied to a 140 X 2 cm diameter Sephadex G-100 column which has been equilibrated at 0-4 ° with 0.2 M TrisHC1 buffer containing 0.001 M MgS04, and the enzyme eluted with the same solvent. The process is followed by ultraviolet spectrophotometry and the first (inactive) protein peak appearing at 135 ml is discarded. The second peak at 185 ml is collected, concentrated by freeze-drying, and then dialyzed against 0.05 M phosphate buffer pH 8.0 containing 0.001 M MgS04 and 0.005 M cysteine. Step 5. Chromatography on DEAE-Sephadex A-50. The resulting solution from the above dialysis is applied to a 20 X 2 cm diameter column of the anion exchanger which has been equilibrated against the above 0.05 M phosphate buffer. After the application of the enzyme the column is washed with 145 ml of the same buffer and then eluted with a solution which is 0.1M in NaC1, 0.05M in phosphate buffer pH 8.0, 0.001 M in MgSQ, and 0.005M in cysteine. The active fraction is obtained between 100 and 260 ml after the change to the sodium chloride solution.
480
ENZYME SYSTEMS: TERPENOID AND STEROL BIOSYNTHESIS
[16]
Properties After the above procedure the enzyme has been purified about 350 times, based on the weight of protein present, but the product is still contaminated with dimethylallyltransferase (EC 2.5.1.1). Stability. Preparations of rubber transferase are generally unstable when purified. The enzyme is partially denatured within 10 minutes at 40 ° and is completely inactivated at 60 ° . Molecular Weight. An approximate value of 60,000 for the molecular weight of rubber transferase has been obtained using the gel-filtration technique of Whitaker. s pH Optimum. The incorporation of isopentenyl-14C-PP proceeds maximally in the pH range 6.8-7.5. Co/actors. Magnesium and sulfhydryl compounds are essential for full activity of the rubber transferase enzyme. Maximum incorporation rates have been observed at 5 mM for both compounds. Excess glutsthione is not inhibitory. Magnesium cannot be replaced by manganese, cobalt, or iron. Aseorbic acid is not a co/actor. Inhibitors. The enzyme is strongly inhibited by EDTA (0.05 M), or by 5 >( 10-~M p-chloromercuribenzoate, iodoacetamide, or N-ethylmaleimide provided endogenous sulfhydryl compounds are first removed. Farnesyl pyrophosphate also inhibits, but neryl pyrophosphate is acceleratory. Stereochemical Specificity. Rubber transferase is strictly stereospecific and yields exclusively cis-polyisoprene from isopentenyl-PP2
sj. R. Whitaker, Anal. Chem. 35, 1950 (1963). oB. L. Archer, D. Barnard, E. G. Cockbain, J. W. Cornforth, Rita H. Cornforth, and G. Pop]~k, Proc. Roy. Soc. B163, 519 (1966).
ENZYME SYSTEMS: TERPENOID AND STEROL BIOSYNTHESIS
[16] Rubber
[16]
T r a n s f e r a s e f r o m H e v e a brasiliensis L a t e x By B. L. A~CHER and E. G. C0CKBAIN
CH3
CH3
[
I
- - [ C H 2 - - C ~ C H - - C H 2 ] . - - P P q- CH2~C--CH2--CH2--PP--~ Rubber pyrophosphate isopentenyl pyrophosphate CH3 [CH~--C~---CH--CH2].+I--PP -}- PP Rubber transferase catalyzes the incorporation of isopentenyl-PP into rubber (cis-l,4-polyisoprene). By analogy with the reaction between geranyl-PP and isopentenyl-PP to form farnesyl-PP, ~,2 it is believed that rubber transferase catalyzes the transfer of cis-l,4-polyisoprenyl-PP to isopentenyl-PP with the elimination of inorganic pyrophosphate, the reaction occurring at the surface of the rubber particles in Hevea latex. ~ The enzyme, which is distributed between the aqueous phase of the latex and the surface of the rubber particles,* has been partially purified. Assay Method
Principle. The activities of rubber transferase preparations are compared by measuring the rates of incorporation of isopentenyl-PP-l-l~C into rubber. A suspension of washed rubber particles prepared from fresh Hevea latex serves as the acceptor for isopentenyl-PP in the assay. Essential cofactors for the incorporation are magnesium and glutathione (or cysteine). Different preparations of washed rubber particles, prepared by the same procedure, usually show different rates of incorporation of isopentenyl-PP into rubber in the presence of the same amounts of rubber transferase and cofactors. This variability is most likely due to variations in the size distribution of the rubber particles and/or the number of rubber molecules with terminal pyrophosphate groups at the surface of the particles. No method of determining the latter figure is available, nor has it been possible so far to establish assay conditions in which the rate of incorporation of isopentenyl-PP is zero order with respect to the rubber content of the suspension. Consequently, the assay method described
1G. Popjhk, Tetrahedron Letters 19, 19 (1959). 2F. Lynen, H. Eggerer, U. Henning, and I. Kessel, Angew. Chem. 70, 738 (1958). aB. L. Archer, B. G. Audley, E. G. Cockbain, and G. P. McSweeney,Biochem. J. 89, 565 (1963). 'A. I. McMullen and G. P. McSweeney, Biochem. J. 101, 42 (1966).
[16]
RUBBER BIOSYNTHESIS
IN Hevea LATEX
477
below is suitable only for comparing the relative transferase contents of different preparations, using the same suspension of washed rubber particles. The rate of incorporation of isopentenyl-PP into the washed rubber does not increase linearly with transferase concentration but obeys the relationship 5 1
A
= ~
+ B
(1)
where R is the rate of incorporation of isopentenyl-PP, [P] is the bulk concentration of the protein expressed on the volume of aqueous phase in the rubber suspension, and A and B are constants. Plots of 1/R against 1/[P] for different preparations of rubber transferase give straight lines from which the corresponding values of A are obtained. It can readily be shown from Eq. (1) that the transferase activity per unit weight of a preparation is inversely proportional to the value of A for that preparation. Reagents
Tris-HC1 buffer: The solution is 0.05M with respect to tris(hydroxymethyl)aminomethane and 0.0268M with respect to HCI; pH=8.2at20 ° Cofaetor solution: 19.2 mg of glutathione is dissolved in 0.5 ml of 2.8% (w/v) Tris in water and 0.5 ml of 0.0625 M MgS04 added Isopentenyl-PP-l-14C: 2.2 mg of the lithium salt is dissolved in water and diluted to 1 ml; 30 /~l of the solution contains 0.25 micromole of isopentenyl-PP (approximately 1400 dps) EDTA: 0 .2M ethylenediaminetetraacetic acid (sodium salt) adjusted to pH 8.0 with NaOH Trichloroacetic acid: 1% (w/v) solution in pure toluene Acetic acid-ethanol: 5% v / v acetic acid in ethanol Preparation o] Washed Rubber Particles. All operations are carried out at 0-5 °. Latex is obtained from Hevea brasiliensis trees in commercia1 production, by tapping into clean vessels cooled in ice. The latex is centrifuged at 2000 g for I0 minutes to remove the sedimentable nonrubber bottom fraction particles2 Then 60 ml of the upper fraction is diluted with 30 ml of Tris-HC1 buffer and centrifuged at 7000 g for 1
°B. L. Archer, Proc. Nat. Rubber Producers" Res. Assoc. Jubilee ConS. Cambridge, 1964 p. 101. Maclaren, London, 1965. ' L. N. S. Homans, J. W. van Dalfsen, and G. E. van Gils, Nature 161, 177 (1948).
478
ENZYME SYSTEMS: TERPENOID AND STEROL BIOSYNTHESIS
[15]
hour. The lower layer containing the finer particles is separated and reeentrifuged at 150,000 q for 1 hour in a swing-out rotor (e.g., Spineo SW 39L). The layer of rubber particles is carefully redispersed in Tris-buffer and diluted to 60 ml. The centrifugation and redispersion are repeated twice more, any coagulated rubber being removed at each stage. The final volume of rubber dispersion should be about 10 ml, and it should contain about 1 g of rubber. It is stable for up to 2 days at 0% Assay Procedure. A number of 100-rag samples of the suspension of washed rubber are weighed into 2 X ~-ineh rimless tubes; cofactor solution (20 t~l) is added, followed by the enzyme preparation to be assayed. Each preparation is used in a range of concentrations varying by a factor of about fifty. The mixtures are each diluted to 220/~1 with 0.05 M Tris buffer, and 30 t~l of IPP-I~C solution is added. Incubations are carried out for 1 hour at 30 ° and terminated by the addition of 0.25 ml of 0.2M EDTA. The contents of the tubes are dried in a stream of air or oxygen at 70 ° while the tubes are rotated at about 100 rpm with their axes horizontal. When dry, the rubber in the tubes is immersed in acetic acidethanol for 1 hour in order to produce a completely nondispersible film which is then washed several times with water. The rubber films are then digested at 100 ° for 1 hour in N KOH in order to remove hydrolyzable impurities, washed with water and extracted with boiling ethanol continuously for 16 hours to ensure removal of low molecular weight isoprenoid contaminants. After superficial drying, each sample of rubber is dissolved in 2 ml of trichloroacetic acid solution in toluene, scintillator is added to give a total volume of 6 ml, and the solutions are counted by a standard scintillation technique. The above purification procedure has been checked by ozonolysis of the rubber and recrystallization of the resulting levulinie acid as the dinitrophenylhydrazone. No retention of nonrubber isoprenoid contaminants could be detected. After correction for the residual biosynthetic activity of the washed rubber in the absence of added enzyme, the reciprocal of the rate of incorporation of 1'C into the rubber is plotted against the reciprocal of the protein concentration for each of the enzyme preparations and their relative purities are determined from the values of 1/A [Eq. (1)]. Protein concentrations are determined by the Folin-Ciocalteau method or by ultraviolet absorptiometry at 260 and 280 m~. 7 Provided the incorporation of 1'C into rubber does not exceed 30%, the uptake vs time graphs are linear for the first hour. As the observed enzymatic activities are dependent on the sample of washed rubber used, it is not possible to define absolute units of rubber transferase activity. The method appears to be satisfactory for both unpurified and partially purified samples of the transferase enzyme. ' E . Layne, see Vol. I I I [73].
[16]
RUBBER BIOSYNTHESIS IN Hevea ~TEX
479
Enzyme Purification All operations are carried out at 0-4 ° . Step I. Preparation o] Serum. Latex is collected, and the bottom fraction is removed as outlined above. The upper fraction is then centrifuged at 60,000 g to remove most of the rubber, and the resulting, almost clear, aqueous serum is freeze-dried. Then 100 g of the dried solids (ex 1600 ml of serum) is dissolved in oxygen-free water, diluted to 300 ml, and centrifuged at 105,000 g for 90 minutes to remove most of the remaining rubber. Step 2. Acidification. The pH of the solution is lowered to 5.4 by the gradual addition of approximately 35 ml of 0.83 M H s P Q . The precipitate of inactive protein is centrifuged off, and the superna5ant is rapidly neutralized to pH 7.0 with 0.1 N Tris. Step 3. Ammonium Sulfate Fractionation. Saturated ammonium sulfate containing 0.01 M magnesium sulfate and adjusted to pH 7.0 is then added stepwise to give fractions precipitating at 25, 50, and 60% of saturation. The mixtures are each left to stand for 30 minutes before centrifugation. The first fraction normally retains the last of the very small rubber particles and leaves a crystal clear supernatant. The last fraction precipitated between 50 and 60% of saturation contains the active enzyme, which, after washing with 65% saturated ammonium sulfate solution, is dissolved in 15 ml of 0.01 M magnesium sulfate and dialyzed overnight against a large excess of 0.2 M Tris-HC1 buffer at pH 8.2 containing 0.001 M MgS04. Step ~. Gel Filtration Chromatography on Sephadex G-IO0. The solution containing the enzyme is applied to a 140 X 2 cm diameter Sephadex G-100 column which has been equilibrated at 0-4 ° with 0.2 M TrisHC1 buffer containing 0.001 M MgS04, and the enzyme eluted with the same solvent. The process is followed by ultraviolet spectrophotometry and the first (inactive) protein peak appearing at 135 ml is discarded. The second peak at 185 ml is collected, concentrated by freeze-drying, and then dialyzed against 0.05 M phosphate buffer pH 8.0 containing 0.001 M MgS04 and 0.005 M cysteine. Step 5. Chromatography on DEAE-Sephadex A-50. The resulting solution from the above dialysis is applied to a 20 X 2 cm diameter column of the anion exchanger which has been equilibrated against the above 0.05 M phosphate buffer. After the application of the enzyme the column is washed with 145 ml of the same buffer and then eluted with a solution which is 0.1M in NaC1, 0.05M in phosphate buffer pH 8.0, 0.001 M in MgSQ, and 0.005M in cysteine. The active fraction is obtained between 100 and 260 ml after the change to the sodium chloride solution.
480
ENZYME SYSTEMS: TERPENOID AND STEROL BIOSYNTHESIS
[16]
Properties After the above procedure the enzyme has been purified about 350 times, based on the weight of protein present, but the product is still contaminated with dimethylallyltransferase (EC 2.5.1.1). Stability. Preparations of rubber transferase are generally unstable when purified. The enzyme is partially denatured within 10 minutes at 40 ° and is completely inactivated at 60 ° . Molecular Weight. An approximate value of 60,000 for the molecular weight of rubber transferase has been obtained using the gel-filtration technique of Whitaker. s pH Optimum. The incorporation of isopentenyl-14C-PP proceeds maximally in the pH range 6.8-7.5. Co/actors. Magnesium and sulfhydryl compounds are essential for full activity of the rubber transferase enzyme. Maximum incorporation rates have been observed at 5 mM for both compounds. Excess glutsthione is not inhibitory. Magnesium cannot be replaced by manganese, cobalt, or iron. Aseorbic acid is not a co/actor. Inhibitors. The enzyme is strongly inhibited by EDTA (0.05 M), or by 5 >( 10-~M p-chloromercuribenzoate, iodoacetamide, or N-ethylmaleimide provided endogenous sulfhydryl compounds are first removed. Farnesyl pyrophosphate also inhibits, but neryl pyrophosphate is acceleratory. Stereochemical Specificity. Rubber transferase is strictly stereospecific and yields exclusively cis-polyisoprene from isopentenyl-PP2
sj. R. Whitaker, Anal. Chem. 35, 1950 (1963). oB. L. Archer, D. Barnard, E. G. Cockbain, J. W. Cornforth, Rita H. Cornforth, and G. Pop]~k, Proc. Roy. Soc. B163, 519 (1966).