8
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 200, No. 2, April 1, pp. 609-616, 1980 Prenyltransferase from Gossypium hirsutum’ ROBERT WIDMAIER, JEAN HOWE, AND PETER HEINSTEIN2 Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907 Received December 18, 1978; revised November 15, 1979 A protein fraction has been purified from Gossypium hirsutum var. Coker 413 which synthesized all four geometrical isomers of farnesyl pyrophosphate from isopentenyl pyrophosphate alone, from isopentenyl pyrophosphate and geranyl or neryl pyrophos- phate. Electrophoretic analysis showed that this protein fraction consisted of three proteins. One of these proteins contained isopentenyl pyrophosphate e dimethylallyl pyrophos- phate isomerase activity. The other two proteins were insufficiently pure to characterize. Estimation of molecular weights by electrophoresis of the three proteins revealed values in the order of 3 x 104 to 1.3 x 105. However the same protein fraction eluted as one peak from Sepharose 6B molecular sieve columns, indicative of a larger protein component as could be accounted for by the electrophoretic molecular weight estimation. From these results and from the different products synthesized it is proposed that isopentenyl pyrophosphate e dimethylallyl pyrophosphate isomerase and prenyltransferase (farnesyl pyrophosphate synthetase) exists as a multiprotein complex in G. hirsutum. Prenyltransferase (EC 2.5.1.1) catalyzes the chain elongation reaction in polyiso- prenoid synthesis through the nucleophilic addition of isopentenyl pyrophosphate (IPP)3 to an allylic pyrophosphate with the con- comitant formation of next higher homol- ogous allylic pyrophosphate. The enzyme has been isolated from animal tissues (l-3), plants (4-Q, and microorganisms (7, 8). The products arising from these condensations are usually cis -trans- and trans -trans-farnesyl pyrophosphate (FPP) (1, 5). However, an analysis of the products synthesized by a partially purified protein fraction from cotton root homogenates (9) showed that all four isomers of FPP can be obtained, if the substrates were IPP and geranyl pyrophosphate (GPP) or neryl ’ This investigation was supported by Grant GM 23249, awarded by the National Institute of General Medical Sciences, DHE W. ’ To whom reprint requests should be addressed. 3 Abbreviations used: IPP, isopentenyl pyrophos- phate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; NPP, neryl pyrophosphate; FPP, farnesyl pyrophosphate; tic, thin-layer chro- matography; glc, gas-liquid chromatography; SDS, sodium dodecyl sulfate. pyrophosphate (NPP) (9). It was not pos- sible to deduce from the data if one en- zyme catalyzed both cis-cis- and trans- trans-FPP synthesis or whether two enzymes existed, one synthesizing cis- trans- and trans-trans-FPP from IPP + GPP and the other synthesizing trans- cis- and cis-cis-FPP from IPP and NPP, respectively. Additional experiments are described in this paper which indicate that IPP-dimethylallylpyrophosphate (DMAPP) isomerase activity, as well as cis-prenyl- transferase and trans-prenyltransferase are present in cotton root homogenates. These enzymes, however, are not readily separated and perhaps exist as a multi- protein complex. MATERIALS AND METHODS Preparation of Substrates [1-‘*C]Isopentenenol was synthesized as previously described (9). Geraniol and nerol were purchased from Tridom Chemical Inc. and were found to be 98 and 97% pure, respectively, by gas chromatography. [lJH]Geraniol and [lJH]nerol were prepared from the respective aldehyde through reduction with sodium bororH]hydride (Amersham) in isopropanol. Geranial and neral were obtained from the respective alcohol 609 0003-9861/80/040609-08$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

Prenyltransferase from Gossypium hirsutum

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 200, No. 2, April 1, pp. 609-616, 1980

Prenyltransferase from Gossypium hirsutum’

ROBERT WIDMAIER, JEAN HOWE, AND PETER HEINSTEIN2

Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907

Received December 18, 1978; revised November 15, 1979

A protein fraction has been purified from Gossypium hirsutum var. Coker 413 which synthesized all four geometrical isomers of farnesyl pyrophosphate from isopentenyl pyrophosphate alone, from isopentenyl pyrophosphate and geranyl or neryl pyrophos- phate. Electrophoretic analysis showed that this protein fraction consisted of three proteins. One of these proteins contained isopentenyl pyrophosphate e dimethylallyl pyrophos- phate isomerase activity. The other two proteins were insufficiently pure to characterize. Estimation of molecular weights by electrophoresis of the three proteins revealed values in the order of 3 x 104 to 1.3 x 105. However the same protein fraction eluted as one peak from Sepharose 6B molecular sieve columns, indicative of a larger protein component as could be accounted for by the electrophoretic molecular weight estimation. From these results and from the different products synthesized it is proposed that isopentenyl pyrophosphate e dimethylallyl pyrophosphate isomerase and prenyltransferase (farnesyl pyrophosphate synthetase) exists as a multiprotein complex in G. hirsutum.

Prenyltransferase (EC 2.5.1.1) catalyzes the chain elongation reaction in polyiso- prenoid synthesis through the nucleophilic addition of isopentenyl pyrophosphate (IPP)3 to an allylic pyrophosphate with the con- comitant formation of next higher homol- ogous allylic pyrophosphate. The enzyme has been isolated from animal tissues (l-3), plants (4-Q, and microorganisms (7, 8). The products arising from these condensations are usually cis -trans- and trans -trans-farnesyl pyrophosphate (FPP) (1, 5). However, an analysis of the products synthesized by a partially purified protein fraction from cotton root homogenates (9) showed that all four isomers of FPP can be obtained, if the substrates were IPP and geranyl pyrophosphate (GPP) or neryl

’ This investigation was supported by Grant GM 23249, awarded by the National Institute of General Medical Sciences, DHE W.

’ To whom reprint requests should be addressed. 3 Abbreviations used: IPP, isopentenyl pyrophos-

phate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; NPP, neryl pyrophosphate; FPP, farnesyl pyrophosphate; tic, thin-layer chro- matography; glc, gas-liquid chromatography; SDS, sodium dodecyl sulfate.

pyrophosphate (NPP) (9). It was not pos- sible to deduce from the data if one en- zyme catalyzed both cis-cis- and trans- trans-FPP synthesis or whether two enzymes existed, one synthesizing cis- trans- and trans-trans-FPP from IPP + GPP and the other synthesizing trans- cis- and cis-cis-FPP from IPP and NPP, respectively. Additional experiments are described in this paper which indicate that IPP-dimethylallylpyrophosphate (DMAPP) isomerase activity, as well as cis-prenyl- transferase and trans-prenyltransferase are present in cotton root homogenates. These enzymes, however, are not readily separated and perhaps exist as a multi- protein complex.

MATERIALS AND METHODS

Preparation of Substrates

[1-‘*C]Isopentenenol was synthesized as previously described (9). Geraniol and nerol were purchased from Tridom Chemical Inc. and were found to be 98 and 97% pure, respectively, by gas chromatography. [lJH]Geraniol and [lJH]nerol were prepared from the respective aldehyde through reduction with sodium bororH]hydride (Amersham) in isopropanol. Geranial and neral were obtained from the respective alcohol

609 0003-9861/80/040609-08$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

610 WIDMAIER, HOWE, AND HEINSTEIN

by MnO, oxidation (10, 11) and purified before re- duction through a silica gel H column with hexane as the eluting solvent. The 3H-alcohols were purified on 10% AgNO,-coated silica gel H thin-layer plates, solvent: cyclohexane-ethyl acetate, 50~50. Pyrophosphorylation of the alcohols was accomplished as previously reported (12) with the following modifications: to a stirred solution of ditriethyl- aminephosphate, dried over P,O,, and trichloro- acetonitrile dissolved in acetonitrile, was added drop- wise a solution of the alcohol in acetonitrile (con- centrations are the same as in Ref. (12)). The solution was kept at room temperature and after stirring overnight, cooled to -60°C. Concentrated NH,OH was then added (equal volume) and the solution brought slowly to room temperature. After repeated extraction with ether, the aqueous phase was con- centrated and the pyrophosphate esters purified through column chromatography on Silica gel H, n-propanol-NH,OH-H,O (6:3:1), 40 psi. The purity of the respective pyrophosphate esters were checked by 31P and proton NMR. GPP, NPP, and tritiated pyrophosphate ester were stored at -20°C and routinely repurified every 3 months by column chromatography as above. Solutions of the pyrophos- phate esters were obtained by dissolving lyophylized samples in 0.2% NH,OH and their concentration was tested by total phosphate and pyrophosphate-phos- phate determination (13).

The four isomers of farnesol used as standards were prepared from cis- and trams-geranylacetone (separated by spinning band distillation) and bromo- methylacetate (15). The resulting esters, cis-cis- and truns-cis-methylfarnesoate from cis-geranylacetone and cis-trans- and truns-truns-methylfarnesoate from truns-geranylacetone were separated by spinning band distillation. The esters were reduced to the alcohols with LiAIH,. The purity of the four isomers was documented by NMR (16) and gas chromatography (17).

Prenyltransferase Assay A typical incubation contained in a total volume of

0.5 ml: [1-14C]IPP, 2 x lo4 M (4.5 x 105 dpmmmol); GPP or NPP, 4.4 x lo4 M; Tris-HCl, 0.1 M, pH 8.0; mercaptoethanol, 0.01 M; MgCl,, 5 X lo9 M. Incuba- tions were placed in a shaking water bath at 32°C for 30 min. The reaction was stopped with 0.1 ml of 2 N HCl in 80% EtOH and after 30 min at 32°C the acid labile isoprene alcohols were extracted as de- scribed previously (9). The assay was linear for protein concentrations up to 150 pg. For the determination of the K, 30-40 pg of protein was used for each incubation. One unit is defined as the amount of enzyme catalyzing the conversion of 1 pmol of [I- laC]IPP into acid labile product per minute under the above assay conditions. In experiments where a de- tailed product analysis was performed, as the analysis of the four geometrical isomers of farnesol synthe-

sized, the incubation contained besides [1J4C]IPP and the ingredients listed above, [1-3H]GPP (1.2 x 10m5 M, 5 x 105 dpmnmol) or [1-3H]NPP (2.04 x 10m5 M, 1.8 x 105 dpmnmol). The incubations were maintained at 32°C for 60 min, stopped by immersion for 1 min in a boiling water bath, and the pH ad- justed to 10.2-10.4 with KOH. Hydrolysis of the pyrophosphate esters was accomplished with alkaline phosphatase (Sigma Chemical Co.) (2 mg protein for each 0.5 ml prenyltransferase incubation). ARer incubation at 32°C for 16 h, the isoprene alcohols were extracted (3 X) with equal volumes of petroleum ether (bp 30-60°C). To the combined petroleum ether extracts isopentenol, dimethylallyl alcohol, nerol, geraniol, and the four farnesol isomers were added as standards, the petroleum ether was evaporated, and the oily residue analyzed by gas chromatog- raphy (1.5% TCEPE on Chrom G, AW-DNCS, 80-100 mesh, 10 ft x 5/16 in. nickel column, carrier gas flow 70 ml/min, temperature programmed 40- 16o”C, G”C/min) or by C-18 phase-bonded silica column chromatography (14) which separated the synthesized farnesols from the substrates. The four geometrical isomers were separated by AgNO, tic (14). To insure that the isomerization of the four isomers did not occur during isolation and separation, authentic cis-cis-, cis-bans-, tmns-cis-, and trans-

trans-farnesol was subjected to the extraction and separation procedures above. Although during glc some isomerization and cyclization of the farnesol isomers did occur (14), no such rearrangements could be detected during C-18 phase-bonded Silica column chromatography followed by AgNO, tic analy- sis (14). Similarly when standard [1-YJ]isopentenol was analyzed by glc considerable radioactivity was recovered in eluates with retention times correspond- ing to dimethylallylalcohol, nerol, geraniol, and all four isomers of farnesol. Therefore glc was used only for routine assays, whereas for critical analyses C-18 phase-bonded silica column chromatography followed by AgNO, tic was used (Table III).

Purijkation of Prenyltransferase

The procedure through DEAE-cellulose chromatog- raphy was essentially as described previously (9). However the source of the root homogenate was Gossypium hirsutum var. Coker 413. The enzyme was further purified through DEAE-Sephadex A-25 chromatography. The concentrated eluate (23 ml, 97 mg protein obtained from 570 ml crude homogenate) from the DEAE-cellulose column was dialyzed over- night against two changes (20-fold excess each) of 0.01 M K,HPO,-KH,PO, buffer, pH 8.0, containing 0.02 M 2-mercaptoethanol and 10% glycerol (buffer B). The dialyzed solution was applied to a DEAE- Sephadex A-25 column (2.5 x 40 cm) and eluted with a O-O.5 M KC1 gradient in buffer B (flow rate 12

PRENYLTRANSFERASE FROM GOSSYPZUM HZRSUTUM 611

ml/h, s-ml fractions). The enzymatically active frac- tions were combined, concentrated (Amicon Ultra- filtrator), and applied to a Sepharose 6B column (1.2 x 80 cm) and eluted with buffer B containing 0.5 M KC1 (flow rate 0.8 ml/h, 2-ml fractions). The active fractions were combined and concentrated.

Electrophoresis

Analytical acrylamide electrophoresis was carried out according to Davis (18) (running pH approxi- mately 9.5) using 5-B% gels. For molecular weight determinations, the SDS-polyacrylamide gel electro- phoretic system of Weber and Osborne (19) was used. A similar method (20) was utilized to estimate the molecular weight of the native protein (running pH approximately 8.5, 5-8.5% gels). An additional electrophoretic system using N-ethylmorpholine and an approximate running pH of 8.9 (21) was used.

Preparative disc gel electrophoresis was carried out in the buffer system and according to the instructions supplied by the manufacturer of the instrument (Canal Co.). Since considerable enzyme activity was lost during preparative disc gel electro- phoresis large scale analytical separation was utilized. Multiple sets of gels each 6 mm in diameter were loaded with 80 to 100 fig of Sepharose 6B purified protein. The buffer system was according to Davis (18). One of the gels was stained for protein with coomassie blue and used as a guide to cut the re- maining gels. Protein was extracted from the gels by macerating the gel sections in buffer B with a Potter-Elvjehem homogenizer. Gel fragments were removed by centrifugation.

Other Reagents and Methods

Protein was determined by the method of Lowry (22). Other chemicals were reagent grade commercial products.

RESULTS

Enzyme Purification

Prenyltransferase previously purified (9) catalyzed the formation of all four geo- metrical isomers of farnesol from either IPP alone or IPP and GPP or NPP as substrate. Since the possibility existed that two enzymes were present, one a cis- prenyltransferase and one a trans-prenyl- transferase, respectively, we attempted to purify the DEAE-cellulose protein fraction further. However DEAE-Sephadex A-25 column chromatography resulted in a sub- stantial loss of protein (Table I) but with little increase in purification. Similar results

were obtained with subsequent Sepharose 6B molecular sieve chromatography. In this case besides substantial loss of protein (75%) no increase in purification was obtained (Table I). Presumably the enzyme fraction obtained from DEAE-cellulose column chromatography is already fairly pure. Separation of DEAE-cellulose purified prenyltransferase on preparative disc gel electrophoresis resulted in the isolation of three protein components. Two of these protein fractions catalyzed the formation of acid labile products. However, the spe- cific activity of either protein was much lower than the original DEAE-cellulose protein fraction applied to the electro- phoretic gel (Table I). The third protein did not catalyze the formation of acid labile products from [1-14C]IPP alone or together with GPP or NPP as the cosubstrate.

Determination of the Molecular Weights of Protein Components by Electrophoresis

Since preparative disc gel electrophoresis apparently caused considerable loss in enzyme activity (Table I) a large scale analytical separation was utilized. Al- though this procedure resulted in a protein recovery of only 35%, the specific activities of the two enzymatic active protein com- ponents were 10 times higher as the spe- cific activities of the proteins separated by preparative disc gel electrophoresis. For identification purposes the three proteins were numbered I, II, and III from the origin (top) of the gel. Molecular weights of protein I, II, and III were estimated by the electrophoretic method of Hedrick and Smith (20). As indicated in Table II, protein I was found to have an approxi- mate molecular weight of 52,000. The approximate molecular weights of protein II and III were 126,000 and 92,000, re- spectively. Electrophoresis of the three proteins in SDS-gels showed that protein I and protein III could not be separated into smaller protein components. The ap- parent weights of protein I and III in SDS-gels were 51,500 and 98,000, respec- tively. Protein II separated into three

612 WIDMAIER, HOWE, AND HEINSTEIN

TABLE I

SUMMARYOFPURIFICATION OF~RENYLTRANSFERASE FROM G. hirsutum

Purification step Total protein

(mg)

Crude homogenate 3723 1.24 105,OOOg Supernatant 3055 1.04 Ammonium sulfate, FE 516 110.69 DEAE-Cellulose 97 537.59 DEAE-Sephadex A-25 39 680.21 Sepharose 6B 13 623.34 Electrophoresis, protein II0 0.53 4.5 Electrophoresis, protein IIP 0.59 6.2

Specific activity” (mu/m&

Total units

4.617 3.177

57.1 52.1 26.5 8.1

Recovery Purification (So) (n-fold)

(100) (1) 68.8 0.8

1241.3 89.3 1132.6 433.5 576.1 548.6 176.1 502.7

a One unit = 1 pm of [1J4C]IPP converted/min. All assays contained [1-‘4C]IPP and GPP as substrates, except protein III, which was assayed with [1-14C]IPP alone.

* The DEAE-Sephadex A-25purified prenyltransferase fraction was used as a protein source for the electro- phoretic separation.

components upon treatment with SDS and electrophoresis in SDS gels. The molecular weights for these three proteins were estimated to be 32,400, 38,800, and 57,700, respectively.

When the Sepharose GB-purified protein was subjected to electrophoresis in the N-ethylmorpholine system (21) only one broad protein peak covering approximately 15% of the upper most part of the gel was obtained.

in the original Davis system (18) showed three protein bands. The separation was not sharp (Fig. 1). Considerable tailing oc- curred between the three protein bands in this electrophoretic system indicating pos- sible interaction between the three proteins.

Substrate Utilization and Reaction Products

The Sepharose GB-purified fraction showed only one band in an electro- phoretic system using asparigin as the trailing ion (20). However, electrophoresis

TABLE II

PROPERTIES OF PRENYLTRANSFERASE PROTEINS

Electrophoresis proteins Sepharose

I II III GB-protein

Molecular weight” 52,000 126,000 92,000

Subunit@ 51,500 32,400 98,000 38,800 57,706

258,000

The Sepharose GB-purified enzyme was capable of synthesizing all four isomers of farnesol from either IPP and GPP, IPP and NPP, or IPP alone (Table III). From the molecular (T/C) ratios of the products it appeared that the utilization of IPP for the synthesis of the cis-cis isomer depended on the cosubstrate. Thus if NPP was the cosubstrate 1.4 molecules of IPP were utilized for each molecule of NPP used. However, when GPP was the cosubstrate 20 molecules of IPP were utilized for each molecule of GPP for the synthesis of cis -cis-farnesol. Six and one-half molecules of IPP were used for each molecule of GPP, but 14 molecules of IPP were utilized for each molecule of NPP for the synthesis of tram -trans-farnesol. The incorporation of IPP into trans-trans-farnesol in the presence of GPP was much more efficient than incorporation of IPP into cis-cis- farnesol in the presence of NPP. The in- corporation of IPP as the only substrate

a Determined by electrophoresis; values are klO% (20); tailing ion was asparagine; approximate running pH 8.5.

b Determined by SDS electrophoresis.

PRENYLTRANSFERASE FROM GOSSYPIUM HIRSUTUM 613

into cis -cis- and tram -trans-farnesol ap- peared to be equally efficient (Table III). This implied isomerization of IPP to DMAPP, however, only traces of dimethylallyl al- cohol were found in these incubations.

Although the substrate utilization and products formed by protein II were carried out, the results were somewhat confusing and since the protein was not homogeneous further characterization is required.

Protein III, however, appeared to be at dye front --f

least 95% pure as judged from electro- A B C D

phoresis in three different pH systems, at five different gel concentrations (54%)

FIG. 1. Disc gel electrophoresis of cotton root

and electrophoresis in SDS-gels. This pro- prenyltransferase. (A) Prenyltransferase purified

tein synthesized virtually no farnesol through Sepharose 6B. Electrophoresis in asparagine system, protein concentration 38 pglgel. (B) Same as

isomers nor geraniol or nerol but catalyzed (A), protein concentration 105 pg/geI. (c) Prenyl- the isomerization of IPP to DMAPP (Ta- transferase purified through Sepharose 6B. Electro- ble III). phoresis in Tris-glycine system, protein concentration

92 pg/gel. (D) Prenyltransferase purified through

Kinetic Analysis Sepharose 6B. Electrophoresis in SDS.

Kinetic data are only given for the IPP-DMAPP isomerase (protein III) since ments with protein III were carried out at protein I had no apparent enzymatic ac- 32°C for 30 min using the assay which tivity and protein II was not sufficiently measures the formation of acid labile pure to warrant kinetic analysis. Measure- product from [1J4C]IPP, which was linear

TABLE III

PRODUCTSFORMEDBY PRENYLTRANSFERASE PROTEINS

Products formed”

Protein fraction and substrate(s)

Farnesol Dimethyl

ally1 cis -cis tram -cis cis -tram tram -bans

alcohol Nero1 Geraniol VW (‘W (‘“C) T 3H MC 3H 14C 3H l*C 3H

Sepharose 6B* [1J4C]IPP + [lJH]GPP 0.6 25.1 25.0 10.0 0.5 6.3 2.5 11.1 4.2 622.0 95.0 [l-W]IPP + [1-3H]NPP 0.1 23.8 0 63.2 45.7 1.9 1.2 2.5 2.3 57.8 4.2 [l-W]IPP 3.2 2.4 3.9 23.7 0.68 2.0 26.1

Protein III’ [1-‘4C]IPP + GPP 44.0(59.3) 5.6(1.0) 5.6(1.2) 4.9(0.9) 0.2(O) 0.5(O) 4.9(0.2) [1-14C]IPP + NPP 31.5(52.1) 5.1(0.2) 7.1(0.7) 3.0(1.1) 0.7(O) 1.3(O) 1.5(0.1) [l-‘%]IPP 37.8(53.7) 10.5(1.4) 10.5(0.8) 9.7(0.7) 1.3(O) 3.7(O) 8.0(0.3)

(2 Figures are expressed as nmol of [l-‘%]IPP and nmol of [lJH]GPP or [lJH]NPP converted per mg protein into the products indicated.

b Products were separated by reversed chromatography and AgNO, tic (14), except dimethylallyl alcohol which was separated from isopentenol by gas chromatography after reversed phase chromatography.

c Products of one-half of the incubation mixture were separated by gas chromatography and the other half by reversed phase chromatography and AgNO, tic (14) (values in parentheses).

614 WIDMAIER, HOWE, AND HEINSTEIN

during this incubation period. An amount of protein was used that permitted measure- ment of initial reaction velocity for the incubation period indicated above at all levels of substrate used. Protein III showed Michaelis-Menten kinetics. An apparent K, was found to be 73.6 pM

for IPP and V of 27 nm/min/mg protein. Constants for GPP and NPP as the vari- able substrate could not be obtained for protein III, since the formation of acid labile product from [l-14C]IPP was inde- pendent of either GPP or NPP concentration.

Properties of Sepharose 6B Protein

The Sepharose GB-purified enzyme ap- peared to be stable for 4 weeks when stored at -18°C in buffer B (see Mate- rials and Methods). Thereafter activity is slowly lost. In buffer B, where 0.01 M

Tris*HCl was substituted for 0.01 M

K2HP04-KH2P04, 50% of the activity was lost after 4-5 days at -18°C. The op- timum activity of the Sepharose 6B- purified protein, as measured in the assay buffer (TrissHCl, 0.1 M), was obtained be- tween pH 6.8 and 7.2. The enzyme re- quired Mg2+ at a concentration of 5 mM

for maximum synthesis of farnesol. Iodo- acetic acid or iodoacetamide at concentra- tions up to 10 mM did not inhibit the enzyme. However, 10 mM 2-mercapto- ethanol aided in stabilizing the enzyme.

Properties of Protein III

This enzyme lost activity rapidly upon storage at -18°C. After 6 days only 10% of the activity remained (buffer B). Op- timum activity was measured in 0.1 M

TrisaHCl at a pH of 7.4-8.0 with 2 tnM Mg3+.

DISCUSSION

A protein fraction which catalyzed the formation of all four isomers of FPP from IPP alone or from IPP and GPP or NPP as cosubstrates was purified from the roots of G. hirsutum var. Coker 413. One of the steps in the purification profile consisted of passage through a molecular sieve column, Sepharose 6B. The activity catalyzing FPP

formation eluted as one protein peak. The specific activity was constant across this peak. Chromatography of the same protein fraction on Sephadex G-200 showed one sharp protein peak which moved with the void volume of the column and therefore appeared to be excluded from Sephadex G-200. These observations would indicate the presence of a large molecular weight protein. However subsequent electropho- resis in a Tris-glycine system indicated the presence of a rather small molecular weight proteins (Table II), in the order of 3 x 104 to 1.3 x 105, which should have been separated by molecular sieving. The appearance of small molecular weight pro- tein upon electrophoresis depended some- what on the buffer system and the running pH. In a system which utilized asparagine as a tailing ion only one homogeneous protein band was observed. In a third electrophoretic system using a N-ethyl- morpholine buffer only one band was found. However this band was too broad to be considered homogeneous. A possible ex- planation of these observations could be that some interaction exists between the proteins which could not be overcome under the rather mild conditions prevalent in molecular sieving columns. However, in electrophoretic systems where charge sepa- rations come into play besides molecular size separation, interaction of the proteins was partially overcome and protein III could be separated from two other pro- teins (I and II).

At a first glance the Sepharose 6B pro- tein appeared to catalyze only a prenyl- transferase reaction. Incubations of IPP with either of the two C,, pyrophos- phates produced all four FPP isomers. However, the product which theoretically would be expected from the C,,, cosub- strate (i.e., trans -trans-FPP from GPP and cis-cis-FPP from NPP) was preferen- tially produced (Table III). The observation that (i) incubation of IPP with NPP gives significant amounts of tram -trans-FPP and IPP ‘with GPP significant amounts of cis-cis-FPP, and (ii) IPP alone as sub- strate yielded both cis-cis- and trans- trans-FPP could only be explained that double bond isomerization did occur during

PRENYLTRANSFERASEFROM GOSSYPIUMHIRSUTUM 615

the formation of FPP. Two possibilities exist. Either only one of the geometrical isomers of FPP was synthesized followed by isomerization to the other isomers or an IPP -+ DMAPP isomerase was present which produced significant amounts of DMAPP (even in the presence of GPP or NPP) which then could condense with IPP in either a cis or a bans fashion. The first possibility is unlikely since incubation of IPP alone or IPP with either GPP or NPP did not produce any free alcohols without alkaline phosphatase or acid treat- ment. Isomerization of the AZ double bond in both FPP and NPP or GPP has been shown to involve first hydrolysis of the pyrophosphate ester to the free alcohol, followed by oxidation to the aldehyde, isomerization, and reduction of the alde- hyde (NADH) (23-25). However, iso- merization may occur in G. hirsutum di- rectly on the FPP without hydrolysis and therefore isomerization of the A, and/or A, double bond in the synthesized FPP cannot be totally excluded.

The second possibility, IPP + DMAPP isomerization initially appeared to be un- likely since apparently only traces of di- methylallylalcohol was produced by the Sepharose 6B protein in any of the incuba- tions, as has been reported previously (9). However separation of the Sepharose 6B protein complex through disc gel electro- phoresis allowed the isolation of a protein which catalyzed the formation of DMAPP from IPP but had only traces of prenyl- transferase activity. This observation could explain the formation of significant amounts of bans -trans- and cis -trans-FPP from IPP + NPP and of cis-cis- and trans- cis-FPP from IPP + GPP. These results were in agreement with the postulate that the Sepharose 6B protein contained IPP + DMAPP isomerase as well as prenyl- transferase, that only small amounts of the intermediates, i.,e., C, and C,, allylpyro- phosphates were accumulated and that in incubations where both IPP and GPP or NPP were utilized as substrates for the formation of mainly cis-cis- and trans- trans-FPP a parallel reaction occurred where IPP was converted to DMAPP and subsequently condensed with IPP to form

both A&s and A&runs isomers of FPP. The latter possibility was supported by the molecular (T/C) ratios of synthesized FPP isomers from [1J4C]IPP and [1-3H]GPP or [1-3H]NPP. In each case the molecular (T/C) ratio of the theoretically expected geometrical isomer of FPP, i.e., cis- cis-FPP from IPP + NPP and trans- trans-FPP from IPP + GPP was much closer to 1 as the molecular (T/C) ratio of the theoretically unfavored products. A ratio of 1 would be expected when a molecule of IPP condenses with one molecule of GPP or NPP.

The buffer effect as reported previously (9) for the DEAE-cellulose purified enzyme was not apparent with the Sephadex 6B- protein complex. This apparent discrepancy was attributed to the further purification of the protein in this communication and to the excessive degradation and rearrange- ment during determination of the reaction products (especially farnesol) by glc (14) in the previous publication (9).

Although electrophoretic analysis at best yields only approximate molecular weights, it should be pointed out that the sum of the molecular weights of proteins I, II, and III is within 10% of the molecular weight of the Sepharose GB-protein. Similarly the sum of the molecular weights of the sub- units of protein II agree within 10% with the apparent native molecular weight of protein II. Somewhat puzzling is the pres- ence of protein I. This protein is too small to be carried along as an impurity through molecular sieve columns yet has no appar- ent activity with the substrates utilized. Again separation of proteins I and II were not clean, considerable overlap occurred. This and the fact that the approximate molecular weight of one of the subunits of protein II is quite similar to the molecular weight of protein I could indicate that protein I is a part of protein II and only partially separated in this electrophoretic system. Nevertheless its function in the multiprotein complex remains obscure.

A comparison of the cotton root trans- ferase complex with other prenyltrans- ferases isolated from plant material such as Riciuns communis (6), Pinus radiata (5), Citrus sinensis (5), and pumpkin

616 WIDMAIER, HOWE, AND HEINSTEIN

seed (4) is difficult, since the cotton root transferase apparently exists in a complex with the IPP-DMAPP isomerase. The pH optimum 6.8-7.2, of the cotton root trans- ferase complex is similar to the castor bean transferase, 6.8-7.0 (6). Compared to the mammalian prenyltransferase, which is inhibited 80% upon preincubation with 2 mM iodoacetamide (23), the plant enzymes apparently are much less sensitive to this sulfhydryl inhibitor. Castor bean trans- ferase requires 10 mM iodoacetamide for 40% inhibition (6), whereas cotton root transferase was not inhibited by 10 mM

iodoacetamide. Whereas the cotton root transferase metal requirements appeared to be similar for all plant prenyltrans- ferase namely Mg2+ at a concentration of 2-5 IrIM. Another apparent similarity be- tween the castor bean enzyme and the cotton root transferase is the stabilizing effect of phosphate buffer. Enzyme stored in phosphate buffer was much more stable than enzyme stored in Tris * HCl buffer except for protein III. The IPP-DMAPP isomerase was very unstable even in phos- phate buffer. However activity was higher when the enzyme was assayed in Tris . HCl buffer than in phosphate buffer.

The question whether one protein cata- lyzes the formation of cis-cis-FPP and bans -trans-FPP or whether one subunit of protein II catalyzes the formation of cis-cis-FPP and the other subunit cata- lyzes the formation of bans -trans-FPP has not been resolved. Perhaps this ques- tion is not relevant if one considers the mechanism proposed by Jedlicki et al. (5), where both cis and bans bonds could be formed upon concerted rotation of a transi- tion state substrate-enzyme complex.

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