- Email: [email protected]
[4] Continuous fluorescence assay for protein prenyltransferases
30
PRENYLATION
[41
when low concentrations of a CaaX-containing peptide substrate (TKCVIM) is present with Ras during incubation but is unaffected when a control peptide (TKVCIM) is present (Fig. 3). Metal Ion Requirements. Binding of Ras or peptide acceptors to FTase requires Zn e÷. This metal atom is tightly bound to the enzyme and can be removed only by extensive dialysis in the presence of EDTA. 7 Therefore, unless Zn 2÷ has been removed, there is no need for exogenous metal to be added to the binding reaction.
Acknowledgments I thank Professor Yoel Kloog and Professor Michael Gelb for critical reading of the manuscript and for helpful suggestions. Y. R. is a recipient of the Aflon Fellowship Award by the Council for Higher Education of Israel.
[41 C o n t i n u o u s F l u o r e s c e n c e A s s a y f o r Protein Prenyltransferases
By P A M E L A
B . CASSIDY, J U L I A M . D O L E N C E , a n d C . D A L E P O U L T E R
Introduction In the reaction catalyzed by protein farnesyltransferase (PFTase), the farnesyl moiety of farnesyl diphosphate (FPP) 1is linked through a thioether bond 2 to a cysteine four amino acid residues from the C terminus of a number of physiologically important proteins. The PFTase enzyme is a heterodimer and has been purified from rat brain, 3 bovine brain, 4 and yeastJ Modified recombinant human 6 and yeast 7 PFTases containing a 1 2-ME, 2-Mercaptoethanol; DM, n-dodecyl-/3-D-maltoside; FPP, farnesyl diphosphate; IPTG, isopropyl-/3-D-thiogalactopyranoside; LB, Luria-Bertani broth; OGP, octyl-/3-D-glucopyranoside; PMSF, phenylmethylsulfonyl fluoride; SB, super broth. 2 W. A. Maltese, FASEB J. 4, 3319 (1990). 3 y. Reiss, J. L. Goldstein, M. C. Seabra, P. J. Casey, and M. S. Brown, Cell (Cambridge, Mass.) 62, 81 (1992). 4 M. D. Schaber, M. B. O'Hara, V. M. Garsky, S. D. Mosser, J. D. Bergstrom, S. L. Moores, M. S. Marshall, P. A. Friedman, R. A. F. Dixon, and J. B. Gibbs, J. Biol. Chem. 265, 14701 (1990). 5 R. Gomez, L. E. Goodman, S. K. Tripathy, E. O'Rourke, V. Manne, and F. Tamanoi, Biochem. J. 289, 25 (1993). 6 C. A. Omer, A. M. Kral, R. E. Diehl, G. C. Prendergast, S. Powers, C. A. Allen, J. B. Gibbs, and N. E. Kohl, Biochemistry 32, 5167 (1993). 7 M. P. Mayer, G. Prestwich, J. M. Dolence, P. D. Milano, H.-Y. Wu, and C. D. Poulter, Gene 132, 41 (1993).
METHODS IN ENZYMOLOGY, VOL. 250
Copyright © 1995 by Academic Press, lnc. All rights of reproduction in any form reserved.
[4]
31
FLUORESCENCE ASSAY FOR PRENYLTRANSFERASES
O ""Y/ N
° (CH3) 2N
0 N
_OH OH
"s"
~
FIG. 1. Dansyl-GCVLS.
Glu-Glu-Phe (EEF) C-terminal epitope in the/3 subunit have been overproduced in Escherichia coli (see also [6] this volume). The EEF epitope permits purification of the enzyme by immunoaffinity chromatography. Detailed kinetic analyses of the PFTase reaction have been carried out for the purified bovine brain s and human 9 enzymes by a single point filter assay using radioactive FPP and modified human Ras protein as substrates. Radioactive farnesylated Ras was precipitated with acidic ethanol, isolated by filtration, and quantified by liquid scintillation spectrometry. During early stages of kinetic experiments with yeast PFTase, we encountered several difficulties with the filter assay. Most notable were the insolubility of modified Ras protein containing CVIA (the CaaX sequence for the yeast a mating factor) above the Km (15 /zm) and a level of reproducibility unsuitable for precise kinetic measurements. These difficulties prompted us to examine the continuous assay initially reported by Pompliano and co-workers 1° based on the change in the fluorescence of the dansylated CaaX peptide, dansyl-GCVLS (Fig. 1), on farnesylation. Dansyl-GCVLS has a fluorescence maximum at 550 nm, which is shifted to 505 nm and enhanced 13-fold on farnesylation. Thus, monitoring the change in fluorescence at 505 nm provides a method for observing the PFTase reaction continuously. In our hands, the fluorescence assay was very sensitive to the type of detergent used, and a significant optimization of the published procedure was required before suitable data were obtained for dansyl-GCVLS or dansyl-GCVIA, a more appropriate substrate for yeast PFTase. The optimized assay is suitable for kinetic measurements and circumvents problems associated with use of radioisotopes. 8 D. L. Pompliano, E. Rands, M. D. Schaber, S. D. Mosser, N. J. Anthony, and J. B. Gibbs, Biochemistry 31, 3800 (1992). 9 D. L. Pompliano, M. D. Schaber, S. D. Mosser, C. M. Omer, J. A. Sharer, and J. B. Gibbs, Biochemistry 32, 8341 (1993). l0 D. L. Pompliano, R. P. Gomez, and N. J. Anthony, J. Am. Chem. Soc. 114, 7945 (1992).
32
PRENYLATION
[41
EcoRV 7.60 !
:!i~
(~ ql
kxy''c
pGP114 7.80 kb
II
/
./"~k~ raml Ndel 5.00 "~~..~'~ EcoRI 4.40
lacZ
/~J" ~
Kpnl 2.64 EcoRl2.80
QEEF,TAATG 3,50
FIG.2. PlasmidpGPll4 used for the production of recombinantyeast PFTase. Materials n-Dodecyl-fl-D-maltoside (DM) is purchased from Calbiochem (La Jolla, CA). Asp-Phe, dansylglycine, and CHAPS are purchased from Sigma (St. Louis, MO). Dansylated peptides are prepared by standard solid-phase synthesis methods on an ABI (Foster City, CA) peptide synthesizer Model 431A using FMOC (fluorenylmethoxycarbonyl) chemistry. Farnesyl diphosphate is synthesized from farnesyl bromide (Aldrich, Milwaukee, WI) and tris-tetrabutylammonium pyrophosphate according to the procedure of Davisson et aL u Quartz cuvettes are purchased from NSG Precision Cells, Inc., Farmingdale, NY. Dimethylformamide is dried over 3 • molecular sieves. Methods Purification o f Yeast Protein Farnesyltransferase
A single colony of E. coli DH5odpGP114 (Fig. 2) is incubated overnight at 37 ° in 3 ml of LB (Luria-Bertani broth: 10 g of Bacto-tryptone, 5 g of yeast extract, 10 g of NaC1, and 0.2 ml of 5 N NaOH, all per liter) containing 300 /~g of ampicillin. A 2-ml portion is used to inoculate 250 ml of SB (super broth: 32 g of Bacto-tryptone, 20 g of yeast extract, 5 g of NaCI, and 5 ml of 1 N NaOH, all per liter) containing 25 mg of ampicillin. 11v. J. Davisson, A. B. Woodside,T. R. Neal, K. E. Stremler, M. Muehlbacher,and C. D. Poulter, J. Org. Chem. 51, 4768 (1986).
[4]
33
FLUORESCENCE ASSAY FOR PRENYLTRANSFERASES
Cultures are incubated at 37° and induced with 0.2 mM isopropyl-/3-Dthiogalactopyranoside (IPTG) (final concentration) when the A600 is approximately 0.6. Cells are harvested after an additional 5 hr of incubation. A 1.5-g portion of wet cells is washed with 200 ml of 50 mM Tris-HCl, pH 7.0, 5 mM 2-mercaptoethanol (2-ME), 1 mM phenylmethylsulfonyl fluoride (PMSF), and disrupted by sonication in 40 ml of the wash buffer. The cell-free extract is clarified by centrifugation at 24,000 g for 30 min at 4°, and the supernatant is dialyzed against two 2-liter changes of wash buffer without PMSF at 4°. The extract is chromatographed on 40 g of DE52, equilibrated with 50 mM Tris-HCl, pH 7.0, 50/xM ZnCI2, 5 mM MgC12, 10 mM 2-ME. The protein is eluted with a stepwise gradient of 0 mM, 100 mM, 200 mM, and 1 M NaCI in the same buffer. The PFTase activity elutes in the 200 mM step. The enzyme is then purified to greater than 90% homogeneity on a 1 ml o~-tubulin immunoaffinity column at 40.7 The DE52-purified material (10 ml) is loaded at 0.1 ml/min directly onto the column, which has been equilibrated with 10 mM sodium phosphate, pH 7.0, 10 mM EDTA, 150 mM NaC1, 10 mM 2-ME. The column is washed with the equilibration buffer until the UV absorbance of the eluant returns to baseline (4-6 ml), and the PFTase is eluted with equilibration buffer containing 5 mM Asp-Phe. The column is then washed with equilibration buffer containing 600 mM NaC1 to remove remaining nonspecifically bound proteins and stored at 4° in equilibration buffer containing 0.05% (w/v) thimerosal. Purified PFTase is stored at -70 ° in elution buffer containing 40% glycerol. Table I summarizes the results of the purification of recombinant yeast PFTase. Peptide affinity columns used by others to purify PFTase from mammalian sources 3,4were not successful with the yeast enzyme, apparently because of weaker binding interactions with the peptide substrates.
Synthesis of Farnesylated Dansyl-Pentapeptide In a 1.5-ml Eppendorf tube, 5.0 mg of dansyl-GCVIA is dissolved in 200 /zl of anhydrous dimethylformamide.1° Diisopropylethylamine (1.1
TABLE I PURIFICATION OF RECOMBINANTYEASTPROTEIN FARNESYLTRANSFERASE
Step Supernatant DE-52 chromatography Immunoaffinity chromatography
Protein (mg) 67 12 0.85
Units (/xmol/min)
Yield (%)
Specific activity Ozmol/min/mg)
Purification (-fold)
1.4 0.75
100 54
0.021 0.068
1 3.2
0.35
25
0.53
25
34
PRENYLATION
[41
equivalents) and farnesyl bromide (1.1 equivalents) are added. The solution is mixed by vortexing and is allowed to stand for 1 hr at room temperature. Water (1 ml) is added, and the solution is frozen and lyophilized. The residue is dissolved in 50% acetic acid in acetonitrile (CH3CN) and purified by preparative high-performance liquid chromatography (HPLC) on a Vydac protein and peptide Cls reversed-phase column (Phenomenex, Torrence, CA) by elution with a linear gradient of 40-100% CH3CN/0.1% trifluoroacetic acid (TFA) in water/0.1% (v/v) TFA. The farnesylated product is obtained in 34% yield.
Preparation of Substrate Stock Solutions Peptide Stock Solution. Because it is difficult to weigh milligram quantities of dansylated peptides accurately, the concentration of the dansylated peptide is determined by UV measurements of absorbance at 340 nm. Because Beer's law is not obeyed over a wide range of concentrations, a calibration curve is generated using dansylglycine as the standard. Dansylglycine (6.2 mg) is dissolved in peptide buffer (20 mM Tris-HC1, pH 7.0, 10 mM EDTA) in a 5-ml volumetric flask, and peptide buffer is added to a final volume of 5 ml. Solutions of 0.02, 0.04, 0.08, 0.12, and 0.16 mM are prepared from the stock solution (4.02 mM) by dilution with peptide buffer. Absorbance is measured at 340 nm using 0.5-ml quartz cuvettes. A linear regression analysis of a plot of absorbance versus concentration gives a correlation coefficient of r e greater than 0.99 over the range of concentrations measured. A stock solution of dansylated peptide ( - 1 raM) is prepared by dissolving the appropriate amount of the peptide in peptide buffer. The solution is then diluted to the appropriate concentration within the absorbances of the calibration curve with peptide buffer. The peptide stock solution is stored in 100-/xl portions. Samples are stable at - 2 0 ° for several months. Farnesyl Diphosphate Stock Solution. A stock solution of FPP (--1015 mM) is prepared by dissolving the compound in 25 mM ammonium bicarbonate. The precise concentration is determined by phosphate analysis essentially as described by Reed and Rilling. a2 This procedure is extremely sensitive to contaminating sources of phosphate such as detergents, and it is recommended that all glassware be soaked in 5% (w/v) KOH in 2-propanol overnight and then rinsed with deionized distilled water before use. Reagents Isobutanol/toluene (1 : 1, v/v) 1.9 M HC104:16.8 ml of 70% HC104 and 83.2 ml of water 12B. C. Reed and H. C. Rilling,Biochemistry 15, 3739 (1976).
[4-]
FLUORESCENCE ASSAY FOR PRENYLTRANSFERASES
35
Molybdate • H 2 8 0 4 " 4.57 g of ( N H 4 ) 6 M o 7 0 2 4 • 4H20, 97 ml of water, and 2.1 ml of H 2 8 0 4 0.10 m M KHzPO4 Diphosphate solution, diluted to approximately 1 m M In duplicate sets of disposable glass test tubes, 0, 0.10, 0.25, 0.50, 0.75, and 1.0 ml of 0.10 mM KHePO4 solution or 10-30/xl of diphosphate solution are added to 0.5 ml of HC104 solution. The tubes are heated in a boiling water bath for 30 min and cooled to room temperature. Water is added to bring the volume of each tube to 3.5 ml, followed by addition of 1.0 ml of the molybdate solution and 2.0 ml of the isobutanol/toluene solution. Each tube is vortexed, and the absorbance of the organic layer is measured at 310 nm in a quartz cuvette. A standard curve is constructed for the phosphate standards using a linear regression analysis (r e > 0.99), and the concentration of FPP is determined by Eq. (1): [FPP](M) =
[ moles Pi determined ] (1 mol FPP Lvol--i-~me o(F--PP~ ) ] \-2~ool ~ - i ]
(1)
Stock solutions of FPP can be stored at - 2 0 ° for several months without noticeable decomposition.
Assay Components Assay buffer: 52 mM Tris-HC1, pH 7.0, 5.8 mM dithiothreitol, 12 mM MgC12, 12/zM ZnC12. The assay buffer is prepared fresh daily n-Dodecyl-/3-D-maltoside (0.4%): DM (40 mg) is dissolved in 9.96 ml of 52 mM Tris-HC1, pH 7.0. The detergent solution is stored in 1ml aliquots at - 2 0 °. Thawed solutions used for kinetic measurements are not returned to storage Dansylated peptide: Peptide solutions are prepared by diluting the peptide stock solution into assay buffer. The dilute solutions can be stored for 2 to 3 days on ice but are not refrozen FPP: The FPP stock solution is diluted into 25 mM ammonium bicarbonate to prepare small quantities of samples. The dilute solutions are stored on ice for up to a week and then discarded. The samples are not refrozen PFTase: A 15-/zl portion of enzyme ( - 2 - 3 /zg, specific activity 0.53 /zmol/mg/ml) is diluted with assay buffer containing 1 mg/ml of bovine serum albumin (BSA) to give concentrations between 25 and 150 nM
Quartz Cuvettes Cleaning. Three-millimeter square quartz cuvettes are used for the assay. Before using for the first time, the cuvettes are cleaned as follows. (1)
36
PRENYLATION
[41
Soak overnight in Chromerge (Baxter, San Francisco, CA), then rinse with deionized distilled H20. (2) Soak overnight in a solution of K O H in methanol (20 pellets/100 ml), then rinse with deionized distilled H20. (3) Soak overnight in concentrated nitric acid, then rinse with deionized distilled H20. The cuvettes are stored in concentrated nitric acid, rinsed several times with deionized distilled H 2 0 just before use, and allowed to air dry. Between assays, they are rinsed several times with deionized distilled H 2 0 and dried with a stream of nitrogen. Matching. The top of one side of one of the cuvettes is lightly etched with a diamond pen, and the fluorescence of a solution containing assay buffer, peptide, and detergent is measured. The other cuvettes are filled with the same solution and matched by determining the orientation which gives a fluorescence intensity nearest that of the first cuvette. The remaining cuvettes are etched on the appropriate side.
Assay Procedure Data are collected on a Spex FluoroMax spectrofluorimeter (Jobin Yuan Spex, Edison, N J). For dansyl-GCVIA, the excitation wavelength is 340 nm, and the emission wavelength is 486 nm. All spectra are obtained at 30 ° in a themostatted cuvette holder. Empty cuvettes are prewarmed in a themostatted holder for 5 min at 30 °. The assay components are assembled in a 1.5-ml E p p e n d o r f tube on ice as follows: 195/xl of assay buffer 25 txl of detergent (DM) 15/xl of peptide (dansyl-GCVIA) 5/xl of PFTase The mixture is incubated at 30 ° for 5 min in a constant temperature bath before the reaction is initiated by the addition of 10/zl of FPP. The contents are quickly mixed by flicking the tube, and a 200-/zl sample is pipetted into the prewarmed cuvette. The fluorescence intensity is measured at a fixed wavelength for 300 sec. Alternatively, PFTase or the peptide can be used to initiate the reaction.
Quantitation of Assay The rate of change in fluorescence 13 is converted to units of concentration per second (/zM/sec) as shown in Eq. (2)14: 13 The Spex FluoroMax spectrofluorimeter measures the rate of fluorescence increase in counts per second per second. 14 E. D. Matayoshi, G. T. Wang, G. A. Kraft, and J. Erickson, Science 247, 954 (1990).
[4]
FLUORESCENCE
Velocity
ASSAY
(IxM) r \sec/=[Velocity~
FOR
37
PRENYLTRANSFERASES
[counts/sec~ ] tzM e ~ )][~(cou~Ts/sec)l(e_---y--f)(2)
The fluorescence enhancement factor (e) is the ratio of the fluorescence of the farnesylated product to the fluorescence of the starting material (dansyl-GCVIA). The conversion factor m is the slope of the line generated in a plot of concentration of S-farnesyldansyl-GCVIA versus fluorescence intensity. It is important that the factors e and m be measured under the same conditions (bandpass, emission, and excitation wavelengths) as those under which reaction velocity measurements are made. Fluorescence enhancement is determined by first measuring the emission at 486 nm, 340 nm excitation, of a solution of assay buffer, 0.04% (w/v) DM, 0.45 /zM dansyl-GCVIA, 20 ~M FPP, and 5/zg of BSA. An identical solution containing 78 ng of PFTase instead of BSA is incubated at 30° until the reaction is complete. At that point, the emission spectrum of the solution is measured. The ratio of the fluorescence of the product to that of the starting material at 486 nm is the enhancement factor (Fig. 3). The conversion factor m is measured by first preparing a stock solution of S-farnesyldansyl-GCVIA. S-Farnesyldansyt-GCVIA ( - 1 mg) is rapidly stirred in 2 ml of 0.4% DM in 52 mM Tris, pH 7.0, under a blanket of nitrogen for 2 hr, then the solution is clarified by centrifugation. The concentration of S-farnesyldansyl-GCVIA is determined as described for the preparation of the dansyl-GCVIA stock solution except that the dansylglycine concen-
S-FarnesyldansyI-GCVlA ,-. 60,000
j 40,000 .F. Q C O
i 20,000.
•
380
i
430
•
i
480
,
1
530
.
i
580
630
Wavelength(nm) FIG. 3. Emission spectra of 2.1/xM dansyl-GCVIA and 2.1/zM S-farnesyldansyl-GCVIA at a bandpass of 2.975 nm for excitation and 1.4875 nm for emission.
38
PRENYLATION
I41
tration curve is generated using 0.4% D M in 52 m M Tris, p H 7.0, as the solvent. The S-farnesyldansyl-GCVIA stock solution is then diluted with 0.4% DM, 52 m M Tris, p H 7.0, to give a series of five solutions with concentrations of 1.2-3/zM. Reaction mixtures are assembled as described in the assay procedure using the S-farnesyldansyl-GCVIA solutions as the detergent component and substituting 5/zg of BSA for PFTase. The emission of each solution at 486 nm is measured at an excitation wavelength of 340 nm. A plot of concentration of S-farnesyldan'syl-GCVIA versus fluorescence intensity is made. The slope of this line is the conversion factor m. Samples are checked for an "inner filter effect" over the range of peptide concentrations used in kinetic studies. Inner filter effects are observed in samples where the optical density (OD = log Io/I) of a solution at the excitation wavelength is sufficiently high that the intensity of light (I) at the center of the cuvette is significantly less than at the front face of the cuvette. Because fluorescence intensity is proportional to the intensity of exciting light, the inner filter effect causes a deviation from linearity in the relationship between concentration and fluorescence. To determine if an inner filter effect is present, the calibration curves used to determine m are checked for linearity with the lowest and highest peptide concentrations encountered during kinetic experiments (0.42 and 4.2/xM). 15 No inner filter effect is seen for the peptide concentrations used in this study. However, in experiments where longer path lengths (larger cuvettes) or higher peptide concentrations are used, an inner filter effect could be encountered. In these cases, fluorescence intensities must be corrected at every peptide concentration used) 4
Discussion
Detergents The assay is particularly sensitive to the type and amount of detergent used. Although 0.04% (w/v) of D M is satisfactory for the assays described herein, alternative concentrations or detergents may be more appropriate for other peptide substrates or enzymes. For kinetic measurements, the progress curve must be smooth, the curve must increase to a stable maximum, and the detergent should not inhibit the enzyme. Figure 4 shows two progress curves. The curve generated using 0.2% octyl-/3-D-glucopyranoside ( O G P ) is typical of an unsatisfactory curve where the fluorescence signal increases to a maximum and then decreases dramatically. Higher concentrais j. p. Lakowicz,"Principles of Fluorescence Spectroscopy,"p. 44. Plenum, New York, 1983.
[4]
FLUORESCENCE ASSAY FOR PRENYLTRANSFERASES
39
3,000,000.
D.
o
0.2% OG P 2D00,000
C
.c_ 4) I¢ O
m
~
1,000D00
O "1
,"r
-"-~
0.04% DM
0 0
400
800
1200
1600
time (sec)
FIG.4. Progress curves in the presence of different detergents. Reaction mixtures contained 10 /~M FPP, 10 /~M dansyl-GCVIA, 56 ng of PFTase, and 0.2% OGP or 0.04% DM at a bandpass of 5.1 nm for excitation and emission.
tions of O G P inhibit the enzyme. In contrast, the curve measured using 0.04% D M increases smoothly to a maximum and is stable at long incubation times. To evaluate detergents for kinetic measurements, the reactions are performed at saturating concentrations of both substrates at levels of enzyme where the reaction reaches completion in a reasonable period of time. It is important to generate a high concentration of product at the end of the reaction to test the ability of the detergent to stabilize the fluorescence signal over the entire range of concentrations encountered during experiments. The evaluation of a detergent should begin at a concentration near the critical micelle concentration (CMC) of the detergent. 16,17 Because CMC values reported in the literature may not correspond exactly to CMCs in the assay buffer, published values are useful only as a guide for examining detergent concentrations. Progress curves are obtained at increasing detergent concentrations until a smooth curve and a stable maximum are achieved. The results for D M (CMC 0 . 0 0 8 1 % ) 17 a r e shown in Table II. At 0.01%, D M gives an irregular progress curve where the rate of increase in fluorescence fluctuates erratically during the course of the reac-
16j. M. Neugebauer, this series, Vol. 182, p. 239. 17Sigma Catalog, p. 1502 (1991).
40
PRENYLATION
[4]
T A B L E II EFFECTS OF DIFFERENT CONCENTRATIONSOF n-DODECYL4~-D-MALTOS1DEPROGRESS CURVE Concentration (%, w/v)
Fluorescence enhancement (486 nm)
Observed rate (counts/sec/sec)
Adjusted rate a (/zM/sec)
0.010 0.014 0.020 0.040 0.094
ND ND 14x 9x 5x
ND a ND b 1750 c 1650 c 1700 c
ND ND 5.6 x 10 -4 5.5 x 10 -4 6.3 x 10 4
"Progress curve was irregular. ND, Not determined. b Progress curve was smooth, but the fluorescence signal decreased sharply after reaching a maximum. c Progress curve increased smoothly to a stable maximum. d Reaction mixtures contained 3.6/zM dansyl-GCVIA, 50/xM FPP, and 83 ng P F r a s e .
tion. At 0.014%, approximately 1.7 times the published CMC, DM did not give a stable fluorescence signal over long incubation times. However, a satisfactory progress curve is obtained as the detergent concentration is increased to 0.02%. Further increases in detergent concentration have little effect on the rate of the reaction but result in a decrease in the fluorescence enhancement factor. To detect inhibition by detergents, rates from experiments at different detergent concentrations must be adjusted for fluorescence enhancement and for differences in fluorescence per mole of product. The adjusted rate is calculated as shown by Eq. (2). The fluorescence enhancement factor (e) is measured as described above. An estimate of 1/m (fluorescence per mole of product) is made by dividing the magnitude of the fluorescence of a reaction run to completion by the number of moles of product formed. A decrease in the adjusted rate at increasing detergent concentration indicates inhibition of the enzyme by the detergent. Rates from experiments using different detergents also can be compared by this method. Detergent concentrations far above the CMC give a reduced fluorescence enhancement and should be avoided. For example, 0.45% (w/v) Tween 80 (350 times CMC) gives good activity in the farnesylation of modified Ras. 7 However, the shift in the fluorescence maxima between substrate and product observed for moderate detergent concentrations (Fig. 3) disappears at the high concentration of Tween 80 (data not shown). In addition, the fluorescence enhancement decreases to only threefold, and the observed rate of increase in fluorescence is low. Table III shows three detergents and concentrations that are acceptable for use with yeast PFTase and dansyl-GCVIA. The enzyme is not inhibited
[4]
FLUORESCENCE ASSAY FOR PRENYLTRANSFERASES
41
TABLE III DETERGENTS USEFUL IN PROTEIN FARNESYLTRANSFERASE FLUORESCENCE ASSAY
Detergent
Concentration (%, w/v)
DM Triton X-100 CHAPS
0.04 0.028 0.18
by higher concentrations of either D M or Triton X-100, but the fluorescence enhancement decreases. Concentrations of C H A P S higher than 0.18% tend to inhibit the enzyme. Applications The continuous fluorescence assay has proved to be very useful and convenient for a variety of applications. The assay is suitable for analysis of crude and partially purified samples of PFTase as well as purified enzyme. If the activity of an impure sample is not known, it may be necessary to dilute the enzyme 10- to 1000-fold in order to stay in the linear range. Small quantities (2% v/v) of organic solvents such as dimethyl sulfoxide ( D M S O ) and dimethylformamide (DMF) do not interfere with the fluorescence measurements. However, a slight inhibition ( ~ 5 % ) of PFYase activity is observed in the presence of these solvents. The assay is particularly suited to kinetic measurements. Each run takes approximately 6 - 8 min. Because the formation of product is continuously monitored, one can easily be certain that rate m e a s u r e m e n t s are m a d e in the linear range of the reaction. In our experience, triplicate m e a s u r e m e n t s do not vary by more than 10%. Substrate inhibition TM is observed with d a n s y l - G C V I A (Fig. 5) and dansyl-GCVLS. Substrate inhibition is also reported for h u m a n PFTase with peptide substrates. 9 To fit data 19 when d a n s y l - G C V I A is the varied substrate (S), it is necessary to include in the Michaelis-Menten equation a term for uncompetitive substrate inhibition [Eq. (3)]:
Vma×S S/Ki)
V = Km + S(1 +
(3)
where S is the concentration of substrate, Km is the Michaelis constant, and Ki is the substrate inhibition constant. 18W. W. Cleland, this series, Vol. 63, p. 500. 19R. J. Leatherbarrow, "GraFit Version 3.0." Erithacus Software Limited, Staines, U.K., 1992.
42
PRENYLATION
[4]
7.5.
6.5X
5.5-
f.
4.5_ n,3.52.5
2
0
4
6
[dansyI-GCVlA] (pM)
FIG. 5. Substrate inhibition of PFTase by dansyl-GCVIA.Reaction mixtures contained 5 /zM FPP, 0.04% DM, dansyl-GCVIA (0.3-7.25/~M), and PFFase (0.64-2.57 nM). A p p a r e n t Michaelis constants at a fixed concentration of the nonvaried substrate were obtained for FPP and dansyl-GCVIA. The value obtained for FPP (Kin 3 . 0 / z M ) is similar to one we estimated using a filter assay. Using Eq. (3), we calculate an apparent Km for d a n s y l - G C V I A of 2 / z M and a Ki of 2.0/xM. Although there is no published Km for dansyl-GCVIA, the value we obtain is similar to the Km published by Pompliano et al. for 5. ÷
4
% v-
x
3
A
2
A
1 0 0.0
•
i
0.1
•
i
0.2
,
+
0.3
.
i
0.4
•
J
0.5
-
i
0.6
1/[FPP] (pM "I l
FIG. 6. Inhibition of PFTase by KTSCVFM. Reaction mixtures contained 2.4/zM dansylGCVIA, 2-20/zM FPP, KTSCVFM (0, 0/zM; O, 1 /zM; D, 6/zM; •, 11 txM; +, 16 IzM), and 0.9-1.3 nM PFTase.
[5]
INHIBITOR ASSAY USING YEAST
43
dansyl-GCVLS. 1° The maximum observable velocity is 2.7/xmol/min/mg (kca t 3.4/sec). This value is higher than that we reported for the filter bench assay using modified Ras as the substrate. However, the same maximum observable velocity is obtained using an HPLC assay TM where the reaction between [3H]FPP and dansyl-GCVIA is performed under the same conditions used in the fluorescence assay (data not shown). Figure 6 shows reciprocal plots for the inhibition of PFTase in the presence of the heptapeptide KTSCVFM with FPP as the varied substrate. The data were fit 19using the noncompetitive inhibition equation [Eq. (4)]2°: v-
VmaxS
Kin(1 + I/K~s) + S(1 + I/Kii)
(4)
where Kis is the competitive inhibition constant and Kii is the uncompetitive inhibition constant:
Acknowledgments Dansylated peptides were synthesized and purified by Dr. R. Schackmann, Utah Regional Cancer Center Protein/DNA Core Facility (Salt Lake City, UT). J.M.D. is a National Institutes of Health Postdoctoral Fellow, National Institute of General Medical Sciences, GM-15286. This work was supported by NIH Grant GM-21328.
20 A. Cornish-Bowden and C. W. Wharton, "Enzyme Kinetics," p. 37. IRL Press, Washington, D.C., 1988.
[51 In Vivo A s s a y s f o r F a r n e s y l t r a n s f e r a s e Inhibitors w i t h S a c c h a r o m y c e s cerevisiae By HIROSHI MITSUZAWA and FUYUHIKO TAMANOI
Introduction Protein farnesylation has been shown to be important for protein function.l-3 The modification is catalyzed by farnesyltransferase, which transfers a farnesyl group from farnesyl diphosphate to a cysteine residue located in a carboxyl-terminal tetrapeptide sequence (the CaaX motif) of an acceptor 1 A. D. Cox and C. J. Der, Curr. Opin. Cell Biol. 4, 1008 (1992). 2 W. R. Schafer and J. Rine, Annu. Rev. Genet. 30, 209 (1992). 3 S. Clarke, Annu. Rev. Biochem. 61, 355 (1992).
METHODS IN ENZYMOLOGY, VOL. 250
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
PRENYLATION
[41
when low concentrations of a CaaX-containing peptide substrate (TKCVIM) is present with Ras during incubation but is unaffected when a control peptide (TKVCIM) is present (Fig. 3). Metal Ion Requirements. Binding of Ras or peptide acceptors to FTase requires Zn e÷. This metal atom is tightly bound to the enzyme and can be removed only by extensive dialysis in the presence of EDTA. 7 Therefore, unless Zn 2÷ has been removed, there is no need for exogenous metal to be added to the binding reaction.
Acknowledgments I thank Professor Yoel Kloog and Professor Michael Gelb for critical reading of the manuscript and for helpful suggestions. Y. R. is a recipient of the Aflon Fellowship Award by the Council for Higher Education of Israel.
[41 C o n t i n u o u s F l u o r e s c e n c e A s s a y f o r Protein Prenyltransferases
By P A M E L A
B . CASSIDY, J U L I A M . D O L E N C E , a n d C . D A L E P O U L T E R
Introduction In the reaction catalyzed by protein farnesyltransferase (PFTase), the farnesyl moiety of farnesyl diphosphate (FPP) 1is linked through a thioether bond 2 to a cysteine four amino acid residues from the C terminus of a number of physiologically important proteins. The PFTase enzyme is a heterodimer and has been purified from rat brain, 3 bovine brain, 4 and yeastJ Modified recombinant human 6 and yeast 7 PFTases containing a 1 2-ME, 2-Mercaptoethanol; DM, n-dodecyl-/3-D-maltoside; FPP, farnesyl diphosphate; IPTG, isopropyl-/3-D-thiogalactopyranoside; LB, Luria-Bertani broth; OGP, octyl-/3-D-glucopyranoside; PMSF, phenylmethylsulfonyl fluoride; SB, super broth. 2 W. A. Maltese, FASEB J. 4, 3319 (1990). 3 y. Reiss, J. L. Goldstein, M. C. Seabra, P. J. Casey, and M. S. Brown, Cell (Cambridge, Mass.) 62, 81 (1992). 4 M. D. Schaber, M. B. O'Hara, V. M. Garsky, S. D. Mosser, J. D. Bergstrom, S. L. Moores, M. S. Marshall, P. A. Friedman, R. A. F. Dixon, and J. B. Gibbs, J. Biol. Chem. 265, 14701 (1990). 5 R. Gomez, L. E. Goodman, S. K. Tripathy, E. O'Rourke, V. Manne, and F. Tamanoi, Biochem. J. 289, 25 (1993). 6 C. A. Omer, A. M. Kral, R. E. Diehl, G. C. Prendergast, S. Powers, C. A. Allen, J. B. Gibbs, and N. E. Kohl, Biochemistry 32, 5167 (1993). 7 M. P. Mayer, G. Prestwich, J. M. Dolence, P. D. Milano, H.-Y. Wu, and C. D. Poulter, Gene 132, 41 (1993).
METHODS IN ENZYMOLOGY, VOL. 250
Copyright © 1995 by Academic Press, lnc. All rights of reproduction in any form reserved.
[4]
31
FLUORESCENCE ASSAY FOR PRENYLTRANSFERASES
O ""Y/ N
° (CH3) 2N
0 N
_OH OH
"s"
~
FIG. 1. Dansyl-GCVLS.
Glu-Glu-Phe (EEF) C-terminal epitope in the/3 subunit have been overproduced in Escherichia coli (see also [6] this volume). The EEF epitope permits purification of the enzyme by immunoaffinity chromatography. Detailed kinetic analyses of the PFTase reaction have been carried out for the purified bovine brain s and human 9 enzymes by a single point filter assay using radioactive FPP and modified human Ras protein as substrates. Radioactive farnesylated Ras was precipitated with acidic ethanol, isolated by filtration, and quantified by liquid scintillation spectrometry. During early stages of kinetic experiments with yeast PFTase, we encountered several difficulties with the filter assay. Most notable were the insolubility of modified Ras protein containing CVIA (the CaaX sequence for the yeast a mating factor) above the Km (15 /zm) and a level of reproducibility unsuitable for precise kinetic measurements. These difficulties prompted us to examine the continuous assay initially reported by Pompliano and co-workers 1° based on the change in the fluorescence of the dansylated CaaX peptide, dansyl-GCVLS (Fig. 1), on farnesylation. Dansyl-GCVLS has a fluorescence maximum at 550 nm, which is shifted to 505 nm and enhanced 13-fold on farnesylation. Thus, monitoring the change in fluorescence at 505 nm provides a method for observing the PFTase reaction continuously. In our hands, the fluorescence assay was very sensitive to the type of detergent used, and a significant optimization of the published procedure was required before suitable data were obtained for dansyl-GCVLS or dansyl-GCVIA, a more appropriate substrate for yeast PFTase. The optimized assay is suitable for kinetic measurements and circumvents problems associated with use of radioisotopes. 8 D. L. Pompliano, E. Rands, M. D. Schaber, S. D. Mosser, N. J. Anthony, and J. B. Gibbs, Biochemistry 31, 3800 (1992). 9 D. L. Pompliano, M. D. Schaber, S. D. Mosser, C. M. Omer, J. A. Sharer, and J. B. Gibbs, Biochemistry 32, 8341 (1993). l0 D. L. Pompliano, R. P. Gomez, and N. J. Anthony, J. Am. Chem. Soc. 114, 7945 (1992).
32
PRENYLATION
[41
EcoRV 7.60 !
:!i~
(~ ql
kxy''c
pGP114 7.80 kb
II
/
./"~k~ raml Ndel 5.00 "~~..~'~ EcoRI 4.40
lacZ
/~J" ~
Kpnl 2.64 EcoRl2.80
QEEF,TAATG 3,50
FIG.2. PlasmidpGPll4 used for the production of recombinantyeast PFTase. Materials n-Dodecyl-fl-D-maltoside (DM) is purchased from Calbiochem (La Jolla, CA). Asp-Phe, dansylglycine, and CHAPS are purchased from Sigma (St. Louis, MO). Dansylated peptides are prepared by standard solid-phase synthesis methods on an ABI (Foster City, CA) peptide synthesizer Model 431A using FMOC (fluorenylmethoxycarbonyl) chemistry. Farnesyl diphosphate is synthesized from farnesyl bromide (Aldrich, Milwaukee, WI) and tris-tetrabutylammonium pyrophosphate according to the procedure of Davisson et aL u Quartz cuvettes are purchased from NSG Precision Cells, Inc., Farmingdale, NY. Dimethylformamide is dried over 3 • molecular sieves. Methods Purification o f Yeast Protein Farnesyltransferase
A single colony of E. coli DH5odpGP114 (Fig. 2) is incubated overnight at 37 ° in 3 ml of LB (Luria-Bertani broth: 10 g of Bacto-tryptone, 5 g of yeast extract, 10 g of NaC1, and 0.2 ml of 5 N NaOH, all per liter) containing 300 /~g of ampicillin. A 2-ml portion is used to inoculate 250 ml of SB (super broth: 32 g of Bacto-tryptone, 20 g of yeast extract, 5 g of NaCI, and 5 ml of 1 N NaOH, all per liter) containing 25 mg of ampicillin. 11v. J. Davisson, A. B. Woodside,T. R. Neal, K. E. Stremler, M. Muehlbacher,and C. D. Poulter, J. Org. Chem. 51, 4768 (1986).
[4]
33
FLUORESCENCE ASSAY FOR PRENYLTRANSFERASES
Cultures are incubated at 37° and induced with 0.2 mM isopropyl-/3-Dthiogalactopyranoside (IPTG) (final concentration) when the A600 is approximately 0.6. Cells are harvested after an additional 5 hr of incubation. A 1.5-g portion of wet cells is washed with 200 ml of 50 mM Tris-HCl, pH 7.0, 5 mM 2-mercaptoethanol (2-ME), 1 mM phenylmethylsulfonyl fluoride (PMSF), and disrupted by sonication in 40 ml of the wash buffer. The cell-free extract is clarified by centrifugation at 24,000 g for 30 min at 4°, and the supernatant is dialyzed against two 2-liter changes of wash buffer without PMSF at 4°. The extract is chromatographed on 40 g of DE52, equilibrated with 50 mM Tris-HCl, pH 7.0, 50/xM ZnCI2, 5 mM MgC12, 10 mM 2-ME. The protein is eluted with a stepwise gradient of 0 mM, 100 mM, 200 mM, and 1 M NaCI in the same buffer. The PFTase activity elutes in the 200 mM step. The enzyme is then purified to greater than 90% homogeneity on a 1 ml o~-tubulin immunoaffinity column at 40.7 The DE52-purified material (10 ml) is loaded at 0.1 ml/min directly onto the column, which has been equilibrated with 10 mM sodium phosphate, pH 7.0, 10 mM EDTA, 150 mM NaC1, 10 mM 2-ME. The column is washed with the equilibration buffer until the UV absorbance of the eluant returns to baseline (4-6 ml), and the PFTase is eluted with equilibration buffer containing 5 mM Asp-Phe. The column is then washed with equilibration buffer containing 600 mM NaC1 to remove remaining nonspecifically bound proteins and stored at 4° in equilibration buffer containing 0.05% (w/v) thimerosal. Purified PFTase is stored at -70 ° in elution buffer containing 40% glycerol. Table I summarizes the results of the purification of recombinant yeast PFTase. Peptide affinity columns used by others to purify PFTase from mammalian sources 3,4were not successful with the yeast enzyme, apparently because of weaker binding interactions with the peptide substrates.
Synthesis of Farnesylated Dansyl-Pentapeptide In a 1.5-ml Eppendorf tube, 5.0 mg of dansyl-GCVIA is dissolved in 200 /zl of anhydrous dimethylformamide.1° Diisopropylethylamine (1.1
TABLE I PURIFICATION OF RECOMBINANTYEASTPROTEIN FARNESYLTRANSFERASE
Step Supernatant DE-52 chromatography Immunoaffinity chromatography
Protein (mg) 67 12 0.85
Units (/xmol/min)
Yield (%)
Specific activity Ozmol/min/mg)
Purification (-fold)
1.4 0.75
100 54
0.021 0.068
1 3.2
0.35
25
0.53
25
34
PRENYLATION
[41
equivalents) and farnesyl bromide (1.1 equivalents) are added. The solution is mixed by vortexing and is allowed to stand for 1 hr at room temperature. Water (1 ml) is added, and the solution is frozen and lyophilized. The residue is dissolved in 50% acetic acid in acetonitrile (CH3CN) and purified by preparative high-performance liquid chromatography (HPLC) on a Vydac protein and peptide Cls reversed-phase column (Phenomenex, Torrence, CA) by elution with a linear gradient of 40-100% CH3CN/0.1% trifluoroacetic acid (TFA) in water/0.1% (v/v) TFA. The farnesylated product is obtained in 34% yield.
Preparation of Substrate Stock Solutions Peptide Stock Solution. Because it is difficult to weigh milligram quantities of dansylated peptides accurately, the concentration of the dansylated peptide is determined by UV measurements of absorbance at 340 nm. Because Beer's law is not obeyed over a wide range of concentrations, a calibration curve is generated using dansylglycine as the standard. Dansylglycine (6.2 mg) is dissolved in peptide buffer (20 mM Tris-HC1, pH 7.0, 10 mM EDTA) in a 5-ml volumetric flask, and peptide buffer is added to a final volume of 5 ml. Solutions of 0.02, 0.04, 0.08, 0.12, and 0.16 mM are prepared from the stock solution (4.02 mM) by dilution with peptide buffer. Absorbance is measured at 340 nm using 0.5-ml quartz cuvettes. A linear regression analysis of a plot of absorbance versus concentration gives a correlation coefficient of r e greater than 0.99 over the range of concentrations measured. A stock solution of dansylated peptide ( - 1 raM) is prepared by dissolving the appropriate amount of the peptide in peptide buffer. The solution is then diluted to the appropriate concentration within the absorbances of the calibration curve with peptide buffer. The peptide stock solution is stored in 100-/xl portions. Samples are stable at - 2 0 ° for several months. Farnesyl Diphosphate Stock Solution. A stock solution of FPP (--1015 mM) is prepared by dissolving the compound in 25 mM ammonium bicarbonate. The precise concentration is determined by phosphate analysis essentially as described by Reed and Rilling. a2 This procedure is extremely sensitive to contaminating sources of phosphate such as detergents, and it is recommended that all glassware be soaked in 5% (w/v) KOH in 2-propanol overnight and then rinsed with deionized distilled water before use. Reagents Isobutanol/toluene (1 : 1, v/v) 1.9 M HC104:16.8 ml of 70% HC104 and 83.2 ml of water 12B. C. Reed and H. C. Rilling,Biochemistry 15, 3739 (1976).
[4-]
FLUORESCENCE ASSAY FOR PRENYLTRANSFERASES
35
Molybdate • H 2 8 0 4 " 4.57 g of ( N H 4 ) 6 M o 7 0 2 4 • 4H20, 97 ml of water, and 2.1 ml of H 2 8 0 4 0.10 m M KHzPO4 Diphosphate solution, diluted to approximately 1 m M In duplicate sets of disposable glass test tubes, 0, 0.10, 0.25, 0.50, 0.75, and 1.0 ml of 0.10 mM KHePO4 solution or 10-30/xl of diphosphate solution are added to 0.5 ml of HC104 solution. The tubes are heated in a boiling water bath for 30 min and cooled to room temperature. Water is added to bring the volume of each tube to 3.5 ml, followed by addition of 1.0 ml of the molybdate solution and 2.0 ml of the isobutanol/toluene solution. Each tube is vortexed, and the absorbance of the organic layer is measured at 310 nm in a quartz cuvette. A standard curve is constructed for the phosphate standards using a linear regression analysis (r e > 0.99), and the concentration of FPP is determined by Eq. (1): [FPP](M) =
[ moles Pi determined ] (1 mol FPP Lvol--i-~me o(F--PP~ ) ] \-2~ool ~ - i ]
(1)
Stock solutions of FPP can be stored at - 2 0 ° for several months without noticeable decomposition.
Assay Components Assay buffer: 52 mM Tris-HC1, pH 7.0, 5.8 mM dithiothreitol, 12 mM MgC12, 12/zM ZnC12. The assay buffer is prepared fresh daily n-Dodecyl-/3-D-maltoside (0.4%): DM (40 mg) is dissolved in 9.96 ml of 52 mM Tris-HC1, pH 7.0. The detergent solution is stored in 1ml aliquots at - 2 0 °. Thawed solutions used for kinetic measurements are not returned to storage Dansylated peptide: Peptide solutions are prepared by diluting the peptide stock solution into assay buffer. The dilute solutions can be stored for 2 to 3 days on ice but are not refrozen FPP: The FPP stock solution is diluted into 25 mM ammonium bicarbonate to prepare small quantities of samples. The dilute solutions are stored on ice for up to a week and then discarded. The samples are not refrozen PFTase: A 15-/zl portion of enzyme ( - 2 - 3 /zg, specific activity 0.53 /zmol/mg/ml) is diluted with assay buffer containing 1 mg/ml of bovine serum albumin (BSA) to give concentrations between 25 and 150 nM
Quartz Cuvettes Cleaning. Three-millimeter square quartz cuvettes are used for the assay. Before using for the first time, the cuvettes are cleaned as follows. (1)
36
PRENYLATION
[41
Soak overnight in Chromerge (Baxter, San Francisco, CA), then rinse with deionized distilled H20. (2) Soak overnight in a solution of K O H in methanol (20 pellets/100 ml), then rinse with deionized distilled H20. (3) Soak overnight in concentrated nitric acid, then rinse with deionized distilled H20. The cuvettes are stored in concentrated nitric acid, rinsed several times with deionized distilled H 2 0 just before use, and allowed to air dry. Between assays, they are rinsed several times with deionized distilled H 2 0 and dried with a stream of nitrogen. Matching. The top of one side of one of the cuvettes is lightly etched with a diamond pen, and the fluorescence of a solution containing assay buffer, peptide, and detergent is measured. The other cuvettes are filled with the same solution and matched by determining the orientation which gives a fluorescence intensity nearest that of the first cuvette. The remaining cuvettes are etched on the appropriate side.
Assay Procedure Data are collected on a Spex FluoroMax spectrofluorimeter (Jobin Yuan Spex, Edison, N J). For dansyl-GCVIA, the excitation wavelength is 340 nm, and the emission wavelength is 486 nm. All spectra are obtained at 30 ° in a themostatted cuvette holder. Empty cuvettes are prewarmed in a themostatted holder for 5 min at 30 °. The assay components are assembled in a 1.5-ml E p p e n d o r f tube on ice as follows: 195/xl of assay buffer 25 txl of detergent (DM) 15/xl of peptide (dansyl-GCVIA) 5/xl of PFTase The mixture is incubated at 30 ° for 5 min in a constant temperature bath before the reaction is initiated by the addition of 10/zl of FPP. The contents are quickly mixed by flicking the tube, and a 200-/zl sample is pipetted into the prewarmed cuvette. The fluorescence intensity is measured at a fixed wavelength for 300 sec. Alternatively, PFTase or the peptide can be used to initiate the reaction.
Quantitation of Assay The rate of change in fluorescence 13 is converted to units of concentration per second (/zM/sec) as shown in Eq. (2)14: 13 The Spex FluoroMax spectrofluorimeter measures the rate of fluorescence increase in counts per second per second. 14 E. D. Matayoshi, G. T. Wang, G. A. Kraft, and J. Erickson, Science 247, 954 (1990).
[4]
FLUORESCENCE
Velocity
ASSAY
(IxM) r \sec/=[Velocity~
FOR
37
PRENYLTRANSFERASES
[counts/sec~ ] tzM e ~ )][~(cou~Ts/sec)l(e_---y--f)(2)
The fluorescence enhancement factor (e) is the ratio of the fluorescence of the farnesylated product to the fluorescence of the starting material (dansyl-GCVIA). The conversion factor m is the slope of the line generated in a plot of concentration of S-farnesyldansyl-GCVIA versus fluorescence intensity. It is important that the factors e and m be measured under the same conditions (bandpass, emission, and excitation wavelengths) as those under which reaction velocity measurements are made. Fluorescence enhancement is determined by first measuring the emission at 486 nm, 340 nm excitation, of a solution of assay buffer, 0.04% (w/v) DM, 0.45 /zM dansyl-GCVIA, 20 ~M FPP, and 5/zg of BSA. An identical solution containing 78 ng of PFTase instead of BSA is incubated at 30° until the reaction is complete. At that point, the emission spectrum of the solution is measured. The ratio of the fluorescence of the product to that of the starting material at 486 nm is the enhancement factor (Fig. 3). The conversion factor m is measured by first preparing a stock solution of S-farnesyldansyl-GCVIA. S-Farnesyldansyt-GCVIA ( - 1 mg) is rapidly stirred in 2 ml of 0.4% DM in 52 mM Tris, pH 7.0, under a blanket of nitrogen for 2 hr, then the solution is clarified by centrifugation. The concentration of S-farnesyldansyl-GCVIA is determined as described for the preparation of the dansyl-GCVIA stock solution except that the dansylglycine concen-
S-FarnesyldansyI-GCVlA ,-. 60,000
j 40,000 .F. Q C O
i 20,000.
•
380
i
430
•
i
480
,
1
530
.
i
580
630
Wavelength(nm) FIG. 3. Emission spectra of 2.1/xM dansyl-GCVIA and 2.1/zM S-farnesyldansyl-GCVIA at a bandpass of 2.975 nm for excitation and 1.4875 nm for emission.
38
PRENYLATION
I41
tration curve is generated using 0.4% D M in 52 m M Tris, p H 7.0, as the solvent. The S-farnesyldansyl-GCVIA stock solution is then diluted with 0.4% DM, 52 m M Tris, p H 7.0, to give a series of five solutions with concentrations of 1.2-3/zM. Reaction mixtures are assembled as described in the assay procedure using the S-farnesyldansyl-GCVIA solutions as the detergent component and substituting 5/zg of BSA for PFTase. The emission of each solution at 486 nm is measured at an excitation wavelength of 340 nm. A plot of concentration of S-farnesyldan'syl-GCVIA versus fluorescence intensity is made. The slope of this line is the conversion factor m. Samples are checked for an "inner filter effect" over the range of peptide concentrations used in kinetic studies. Inner filter effects are observed in samples where the optical density (OD = log Io/I) of a solution at the excitation wavelength is sufficiently high that the intensity of light (I) at the center of the cuvette is significantly less than at the front face of the cuvette. Because fluorescence intensity is proportional to the intensity of exciting light, the inner filter effect causes a deviation from linearity in the relationship between concentration and fluorescence. To determine if an inner filter effect is present, the calibration curves used to determine m are checked for linearity with the lowest and highest peptide concentrations encountered during kinetic experiments (0.42 and 4.2/xM). 15 No inner filter effect is seen for the peptide concentrations used in this study. However, in experiments where longer path lengths (larger cuvettes) or higher peptide concentrations are used, an inner filter effect could be encountered. In these cases, fluorescence intensities must be corrected at every peptide concentration used) 4
Discussion
Detergents The assay is particularly sensitive to the type and amount of detergent used. Although 0.04% (w/v) of D M is satisfactory for the assays described herein, alternative concentrations or detergents may be more appropriate for other peptide substrates or enzymes. For kinetic measurements, the progress curve must be smooth, the curve must increase to a stable maximum, and the detergent should not inhibit the enzyme. Figure 4 shows two progress curves. The curve generated using 0.2% octyl-/3-D-glucopyranoside ( O G P ) is typical of an unsatisfactory curve where the fluorescence signal increases to a maximum and then decreases dramatically. Higher concentrais j. p. Lakowicz,"Principles of Fluorescence Spectroscopy,"p. 44. Plenum, New York, 1983.
[4]
FLUORESCENCE ASSAY FOR PRENYLTRANSFERASES
39
3,000,000.
D.
o
0.2% OG P 2D00,000
C
.c_ 4) I¢ O
m
~
1,000D00
O "1
,"r
-"-~
0.04% DM
0 0
400
800
1200
1600
time (sec)
FIG.4. Progress curves in the presence of different detergents. Reaction mixtures contained 10 /~M FPP, 10 /~M dansyl-GCVIA, 56 ng of PFTase, and 0.2% OGP or 0.04% DM at a bandpass of 5.1 nm for excitation and emission.
tions of O G P inhibit the enzyme. In contrast, the curve measured using 0.04% D M increases smoothly to a maximum and is stable at long incubation times. To evaluate detergents for kinetic measurements, the reactions are performed at saturating concentrations of both substrates at levels of enzyme where the reaction reaches completion in a reasonable period of time. It is important to generate a high concentration of product at the end of the reaction to test the ability of the detergent to stabilize the fluorescence signal over the entire range of concentrations encountered during experiments. The evaluation of a detergent should begin at a concentration near the critical micelle concentration (CMC) of the detergent. 16,17 Because CMC values reported in the literature may not correspond exactly to CMCs in the assay buffer, published values are useful only as a guide for examining detergent concentrations. Progress curves are obtained at increasing detergent concentrations until a smooth curve and a stable maximum are achieved. The results for D M (CMC 0 . 0 0 8 1 % ) 17 a r e shown in Table II. At 0.01%, D M gives an irregular progress curve where the rate of increase in fluorescence fluctuates erratically during the course of the reac-
16j. M. Neugebauer, this series, Vol. 182, p. 239. 17Sigma Catalog, p. 1502 (1991).
40
PRENYLATION
[4]
T A B L E II EFFECTS OF DIFFERENT CONCENTRATIONSOF n-DODECYL4~-D-MALTOS1DEPROGRESS CURVE Concentration (%, w/v)
Fluorescence enhancement (486 nm)
Observed rate (counts/sec/sec)
Adjusted rate a (/zM/sec)
0.010 0.014 0.020 0.040 0.094
ND ND 14x 9x 5x
ND a ND b 1750 c 1650 c 1700 c
ND ND 5.6 x 10 -4 5.5 x 10 -4 6.3 x 10 4
"Progress curve was irregular. ND, Not determined. b Progress curve was smooth, but the fluorescence signal decreased sharply after reaching a maximum. c Progress curve increased smoothly to a stable maximum. d Reaction mixtures contained 3.6/zM dansyl-GCVIA, 50/xM FPP, and 83 ng P F r a s e .
tion. At 0.014%, approximately 1.7 times the published CMC, DM did not give a stable fluorescence signal over long incubation times. However, a satisfactory progress curve is obtained as the detergent concentration is increased to 0.02%. Further increases in detergent concentration have little effect on the rate of the reaction but result in a decrease in the fluorescence enhancement factor. To detect inhibition by detergents, rates from experiments at different detergent concentrations must be adjusted for fluorescence enhancement and for differences in fluorescence per mole of product. The adjusted rate is calculated as shown by Eq. (2). The fluorescence enhancement factor (e) is measured as described above. An estimate of 1/m (fluorescence per mole of product) is made by dividing the magnitude of the fluorescence of a reaction run to completion by the number of moles of product formed. A decrease in the adjusted rate at increasing detergent concentration indicates inhibition of the enzyme by the detergent. Rates from experiments using different detergents also can be compared by this method. Detergent concentrations far above the CMC give a reduced fluorescence enhancement and should be avoided. For example, 0.45% (w/v) Tween 80 (350 times CMC) gives good activity in the farnesylation of modified Ras. 7 However, the shift in the fluorescence maxima between substrate and product observed for moderate detergent concentrations (Fig. 3) disappears at the high concentration of Tween 80 (data not shown). In addition, the fluorescence enhancement decreases to only threefold, and the observed rate of increase in fluorescence is low. Table III shows three detergents and concentrations that are acceptable for use with yeast PFTase and dansyl-GCVIA. The enzyme is not inhibited
[4]
FLUORESCENCE ASSAY FOR PRENYLTRANSFERASES
41
TABLE III DETERGENTS USEFUL IN PROTEIN FARNESYLTRANSFERASE FLUORESCENCE ASSAY
Detergent
Concentration (%, w/v)
DM Triton X-100 CHAPS
0.04 0.028 0.18
by higher concentrations of either D M or Triton X-100, but the fluorescence enhancement decreases. Concentrations of C H A P S higher than 0.18% tend to inhibit the enzyme. Applications The continuous fluorescence assay has proved to be very useful and convenient for a variety of applications. The assay is suitable for analysis of crude and partially purified samples of PFTase as well as purified enzyme. If the activity of an impure sample is not known, it may be necessary to dilute the enzyme 10- to 1000-fold in order to stay in the linear range. Small quantities (2% v/v) of organic solvents such as dimethyl sulfoxide ( D M S O ) and dimethylformamide (DMF) do not interfere with the fluorescence measurements. However, a slight inhibition ( ~ 5 % ) of PFYase activity is observed in the presence of these solvents. The assay is particularly suited to kinetic measurements. Each run takes approximately 6 - 8 min. Because the formation of product is continuously monitored, one can easily be certain that rate m e a s u r e m e n t s are m a d e in the linear range of the reaction. In our experience, triplicate m e a s u r e m e n t s do not vary by more than 10%. Substrate inhibition TM is observed with d a n s y l - G C V I A (Fig. 5) and dansyl-GCVLS. Substrate inhibition is also reported for h u m a n PFTase with peptide substrates. 9 To fit data 19 when d a n s y l - G C V I A is the varied substrate (S), it is necessary to include in the Michaelis-Menten equation a term for uncompetitive substrate inhibition [Eq. (3)]:
Vma×S S/Ki)
V = Km + S(1 +
(3)
where S is the concentration of substrate, Km is the Michaelis constant, and Ki is the substrate inhibition constant. 18W. W. Cleland, this series, Vol. 63, p. 500. 19R. J. Leatherbarrow, "GraFit Version 3.0." Erithacus Software Limited, Staines, U.K., 1992.
42
PRENYLATION
[4]
7.5.
6.5X
5.5-
f.
4.5_ n,3.52.5
2
0
4
6
[dansyI-GCVlA] (pM)
FIG. 5. Substrate inhibition of PFTase by dansyl-GCVIA.Reaction mixtures contained 5 /zM FPP, 0.04% DM, dansyl-GCVIA (0.3-7.25/~M), and PFFase (0.64-2.57 nM). A p p a r e n t Michaelis constants at a fixed concentration of the nonvaried substrate were obtained for FPP and dansyl-GCVIA. The value obtained for FPP (Kin 3 . 0 / z M ) is similar to one we estimated using a filter assay. Using Eq. (3), we calculate an apparent Km for d a n s y l - G C V I A of 2 / z M and a Ki of 2.0/xM. Although there is no published Km for dansyl-GCVIA, the value we obtain is similar to the Km published by Pompliano et al. for 5. ÷
4
% v-
x
3
A
2
A
1 0 0.0
•
i
0.1
•
i
0.2
,
+
0.3
.
i
0.4
•
J
0.5
-
i
0.6
1/[FPP] (pM "I l
FIG. 6. Inhibition of PFTase by KTSCVFM. Reaction mixtures contained 2.4/zM dansylGCVIA, 2-20/zM FPP, KTSCVFM (0, 0/zM; O, 1 /zM; D, 6/zM; •, 11 txM; +, 16 IzM), and 0.9-1.3 nM PFTase.
[5]
INHIBITOR ASSAY USING YEAST
43
dansyl-GCVLS. 1° The maximum observable velocity is 2.7/xmol/min/mg (kca t 3.4/sec). This value is higher than that we reported for the filter bench assay using modified Ras as the substrate. However, the same maximum observable velocity is obtained using an HPLC assay TM where the reaction between [3H]FPP and dansyl-GCVIA is performed under the same conditions used in the fluorescence assay (data not shown). Figure 6 shows reciprocal plots for the inhibition of PFTase in the presence of the heptapeptide KTSCVFM with FPP as the varied substrate. The data were fit 19using the noncompetitive inhibition equation [Eq. (4)]2°: v-
VmaxS
Kin(1 + I/K~s) + S(1 + I/Kii)
(4)
where Kis is the competitive inhibition constant and Kii is the uncompetitive inhibition constant:
Acknowledgments Dansylated peptides were synthesized and purified by Dr. R. Schackmann, Utah Regional Cancer Center Protein/DNA Core Facility (Salt Lake City, UT). J.M.D. is a National Institutes of Health Postdoctoral Fellow, National Institute of General Medical Sciences, GM-15286. This work was supported by NIH Grant GM-21328.
20 A. Cornish-Bowden and C. W. Wharton, "Enzyme Kinetics," p. 37. IRL Press, Washington, D.C., 1988.
[51 In Vivo A s s a y s f o r F a r n e s y l t r a n s f e r a s e Inhibitors w i t h S a c c h a r o m y c e s cerevisiae By HIROSHI MITSUZAWA and FUYUHIKO TAMANOI
Introduction Protein farnesylation has been shown to be important for protein function.l-3 The modification is catalyzed by farnesyltransferase, which transfers a farnesyl group from farnesyl diphosphate to a cysteine residue located in a carboxyl-terminal tetrapeptide sequence (the CaaX motif) of an acceptor 1 A. D. Cox and C. J. Der, Curr. Opin. Cell Biol. 4, 1008 (1992). 2 W. R. Schafer and J. Rine, Annu. Rev. Genet. 30, 209 (1992). 3 S. Clarke, Annu. Rev. Biochem. 61, 355 (1992).
METHODS IN ENZYMOLOGY, VOL. 250
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