3452
BI OCHEM I STRY
CARLIER,
HOLOWKA,
AND HAMMES
Interaction of Photoreactive and Fluorescent Nucleotides with Chloroplast Coupling Factor 1 Marie-France Carher,$ David A. Holowka,s and Gordon G . Hammes*
ABSTRACT: The photoreactive nucleotide 3’-0- [ 3- [N-(4azido-2-nitrophenyl)amino]propionyl] -ATP is a substrate of heat-activated chloroplast coupling factor 1 (CF,) and can be incorporated stoichiometrically (1 :1) into the latent enzyme. Photolysis of the isolated [3H]nucleotide-CF, complex and analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the /3 subunit was covalently labeled. The analogous photoreactive ADP nucleotide binds to different sites which are specific for ADP. Photolysis of a mixture of the tritium-labeled photoreactive ADP and C F , followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both the CY and /3 subunits are covalently labeled, in approximately equal amounts. In both cases, covalent labeling inactivates the enzyme, but a clear-cut correlation between the extent of labeling and loss of activity
could not be established. The fluorescent nucleotide 1 , p etheno-ATP (t-ATP) also can be stoichiometrically (1:l) incorporated into the latent enzyme, with a concomitant quenching in the fluorescence of about 30%. If a tyrosine residue of the /3 subunit in the fluorescent nucleotide-CF, complex is modified with 7-chloro-4-nitrobenz-2-oxa1,3diazole (NBD-Cl), the fluorescence is first quenched and later enhanced. The tyrosine modification increases the rate of dissociation of €-ATP from the C F I , causing the enhancement of fluorescence, a decrease in fluorescence polarization, and an apparent decrease in the resonance energy transfer from t-ATP to tyrosine as the reaction proceeds. Extrapolation of the energy-transfer efficiency to zero time suggests that the t-ATP is bound 35 A from the NBD-tyrosine residue.
T h e coupling factor C F , ’ from spinach chloroplasts has a molecular weight of 325 000 (Farron, 1970) and probably has a subunit structure of ~ ~ ~ / 3(Binder ~ y 8 t et~ al., 1978; Baird & Hammes, 1976), where the Greek letters designate different polypeptide chains. The interaction of nucleotides with C F , has been extensively studied [cf. Cantley & Hammes (1975a,b), Tiefert et al. (1977), VanderMeulen & Govindjee (19771, Girault et al. (1973), and Girault & Galmiche (1977)]. In an accompanying paper, we have shown that at least two types of nucleotide sites exist (Carlier & Hammes, 1979). A single ATP is incorporated into both latent and heat-activated CF, and is hydrolyzed into ADP and Pi. The ADP molecule remains tightly incorporated into the enzyme after extensive Sephadex chromatography. Two additional nucleotide binding sites have been observed which bind ADP with a dissociation constant of about 1 FM. In the work reported here, we have specifically photochemically labeled the two types of nucleotide binding sites with arylazido-ATP and arylazido-ADP. The former specifically labels the /3 subunit, while the latter labels both CY and /3 subunits in approximately equal amounts. The fluorescent nucleotide t-ATP has been incorporated into the very tight nucleotide binding site, and the distance between the C F l bound t-ATP and an NBD-modified tyrosine residue on the /3 subunit has been determined. Changes in the fluorescent properties of the t-ATP during the reaction of NBD-CI with the enzyme indicate that dissociation of t-ATP is associated with the tyrosine modification. Materials and Methods Chemicals. The NBD-CI, ADP, €-ATP, ATP (vanadium-free), DL-dithiothreitol, and /3-mercaptoethanol were
obtained from Sigma Chemical Co. The AMP-PNP was obtained from PL Biochemicals and purified by chromatography on DEAE-cellulose (Yount et al., 1971). The quinine sulfate dihydrate was from Aldrich Chemical Co. The sodium dodecyl sulfate (electrophoresis grade) and Coomassie Blue R were from Bio-Rad Laboratories. Acrylamide, N,N’methylenebis(acrylamide), ammonium peroxidisulfate, 2,5diphenyloxazole, and 2,2’-p-phenylenebis(4-methyl-5phenyloxazole) were from Eastman Kodak Co. The [3H]ATP and [3H]ADP were obtained from New England Nuclear and purified on ascending paper chromatography with Whatman 3MM paper and isobutyric acid-1 N N H 4 0 H (100:60). All standard chemicals were analytical grade, and deionized distilled water was used for all solutions. Photoaffinity Labels. The arylazidoadenine nucleotides were synthesized with unlabeled and 3H-labeled ATP and ADP (Jeng & Guillory, 1975). In the last step of the synthesis, the water extract of the product of the coupling reaction between arylazido-/3-alanine and the nucleotide was filtered and the compound was chromatographed twice on Whatman 3MM paper in the solvent butanol-water-acetic acid (5:3:2, v/v/v). The ATP derivative had an RJof 0.35 and the ADP derivative an Rj of 0.55. The 3H-labeled compounds had a specific radioactivity of about 50-100 Ci/mol. The purity of the final compound was assessed from comparison of the specific radioactivity of the final product with that of the starting material. An extinction coefficient of 4200 M-’ cm-’ at 480 nm and 35 200 M-’ cm-l at 260 nm was used in calculating the concentration of the product (Jeng & Guillory, 1975). The photolabile nucleotides were found to be >98% pure. The photoreactivity of the nucleotides was checked by illuminating a sample spotted on a cellulose thin-layer chromatography
t From the Department of Chemistry, Cornell University, Ithaca, New York 14853. Received December 27, 1978. This work was supported by a grant from the National Institutes of Health (GM 13292). *Permanent address: Laboratoire D’Enzymologie, C.N.R.S., 9 1 Gif-Sur-Yvette, France. Supported by C.N.R.S. and a fellowship from the International Agency for Research on Cancer. *National Institutes of Health Postdoctoral Fellow (GM 05413).
I Abbreviations used: C F I , chloroplast coupling factor; NBD-CI, 7-chloro-4-nitrobenz-2-oxa1,3-diazole; AMP-PNP, adenylyl imidodiphosphate; e-ATP, 1,PP-ethenoadenosine triphosphate; (-ADP, 1,I@ethenoadenosine diphosphate; arylazido-ATP (-ADP), 3’-0-[3-[N-(4azid~2-nitrophenyl)amino]propionyl]adenosinetriphosphate (diphosphate); EDTA, ethylenediaminetetraacetic acid.
0006-2960/79/0418-3452$01.00/00 1979 American Chemical Society
PHOTOREACTIVE AND FLUORESCENT NUCLEOTIDES
plate in isobutyric acid-1 N ammonia (100:60, v/v) for 1 min with a 200-W Xe-Hg arc (Hanovia). No material was found to migrate in the illuminated sample, while a single spot with an Rfvalue of 0.9 was found with the sample not illuminated. This test also indicated the absence of unmodified nucleotides, which have a low Rfvalue, and azido-@-alanine,which has an Rf value of 1. Enzyme. The CF, was prepared from fresh spinach leaves (Lien & Racker, 1971). Molar concentrations of C F I were determined spectrophotometrically as previously described (Carlier & Hammes, 1979). The CF1 was stored at 4 OC as a precipitate in 2 M ammonium sulfate, 10 mM Tris-HC1 (pH 7.2), 2 mM EDTA, and 0.5 mM ATP. The latent enzyme was heat-activated when necessary (Lien & Racker, 1971). The ATPase activity was monitored at 23 "C, pH 8.0, in the presence of 5 mM CaClz either by phosphate analysis with ammonium molybdate (Taussky & Schorr, 1953) or by the pH-stat method (Cantley & Hammes, 1975a). Photoafjnity Labeling of CF,. The tight nucleotide binding site on C F I , which incorporates ATP rapidly in the presence of Mg2+ and Ca2+ (Carlier & Hammes, 1979), was labeled in the following manner. The enzyme first was freed from dissociable nucleotides by column chromatography on Sephadex G25 (1.2 cm i.d. X 50 cm) in 10 mM Tris-HC1 (pH 8.0), 2 mM EDTA, and 25 mM sucrose. The eluted enzyme was concentrated and dialyzed against 40 mM Tris-HC1 (pH 8.0) and 5 mM MgC12using a collodion bag (Schleicher and Schuell) and then incubated in the dark in the presence of 100 p M arylazido- [3H]ATP. For convenience, this incubation normally was done overnight, but the same final result was obtained with a 15-min incubation. The enzyme solution then was precipitated by addition of an equal volume of saturated ammonium sulfate in 10 mM Tris-HC1 (pH 7 . 2 ) and 2 mM EDTA, centrifuged, and passed through the Sephadex G25 column described above. The eluted enzyme was concentrated and dialyzed against 50 mM NaCl and 0.5 mM Tris-HC1 (pH 8.0). All of these operations were done in the dark. The amount of arylazido-ATP bound to CFI was determined spectrophotometrically and by scintillation counting of an aliquot of the enzyme solution. The enzyme concentration also was determined spectrophotometrically. An extinction coefficient of 4200 M-' cm-l at 475 nm was assumed for the enzyme-bound arylazideATP (Jeng & Guillory, 1975). The arylazido-ATP-CF, complex (5-10 pM) was illuminated with a focused 200-W Hanovia Xe-Hg arc. The ultraviolet radiation below 350 nm was eliminated with a CSO-51 glass filter. The sample was placed 12-14 cm from the arc in a cylindrical cell (light path 0.9 cm, volume 0.7 mL) with a jacket thermostated at 20 OC. Further cooling of the walls of the cuvette was provided by a fan. The sample was irradiated for 15 periods of 1 min separated by periods of 30-s cooling. This procedure ensured complete destruction of the label and prevented further slow unspecific binding. In a control experiment, 200 p M AMP-PNP was added to the isolated CF,-arylazido-ATP complex for 30 min before the irradiation. The nucleotide binding sites on CF, which have been shown to bind ADP with a dissociation constant of 1-2 mM (Cantley & Hammes, 1975a) were labeled as follows. CF1 (-10 pM), free from dissociable nucleotides, was equilibrated with variable concentrations (1 5-1 50 pM) of aryla~ido-[~H]ADP in 40 mM Tris-HC1 (pH 8.0) and 5 mM MgClz or 50 mM NaCl, 0.5 mM Tris-HC1 (pH 8.0), and 5 mM MgC12. The irradiation was performed as described above. In control experiments, ADP (380 pM) or AMP-PNP (2.11 mM) was
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VOL. 18, N O . 16, 1979
3453
added to the CF1 solution with arylazido- [3H]ADP before illumination. After photolysis, a portion of the enzyme was heat-activated at 60 OC for 4 min in the presence of 40 mM ATP and 5 mM dithiothreitol, and the ATPase activity was measured. Gel Electrophoresis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was carried out on the photoaffinity labeled CF1 (Weber & Osborn, 1969). The CF, was denatured in 2% sodium dodecyl sulfate and 100 mM @-mercaptoethanolat 90 "C for a few minutes. Approximately 0.1-0.6 nmol of CF, in 10-60 pL was applied to 10 or 12% [0.27 or 0.33% N,N'-methylenebis(acrylamide)] cylindrical (6 mm i.d. X 100 mm) polyacrylamide gels. The gels were electrophoresed for 15 min at 50 V and then for 3-4 h at 100 V in 70 mM sodium phosphate (pH 8.0) containing 0.1% sodium dodecyl sulfate by using a Gradipore flowcell apparatus (Isolab) thermostated at 20 OC. The 12% gels were electrophoresed for an additional 15 h at 50 V or 6-8 h at 100 V to improve the separation between the CY and @ subunits of CF,. The gels were stained with 0.5% Coomassie Blue R in methanol-water-acetic acid (227:227:46) and destained by diffusion in the same solvent. The gels were spectrophotometrically scanned at 280 or 580 nm by using an ISCO gel scanner (slit width 0.25 nm, scan speed 30 cm/h) and then frozen with dry ice and cut into 1-mm slices with a Mickle gel slicer (Brinkmann Instruments, Inc.). Each slice was incubated for 6-18 h at 50 "C in a scintillation vial containing 0.9 mL of N C S tissue solubilizer (Amersham) and 0.1 mL of H 2 0 (Baird & Hammes, 1976). After that time, 10 mL of a scintillation fluid containing 5 g of 2,5-diphenyloxazole and 0.2 g of 2,2'-p-phenylenebis(4-methyl-5-phenyloxazole) in 1 L of toluene was added to each vial. The radioactivity present in each slice was assayed with a Beckman LS255 liquid scintillation counter. Correction was made for enhancement of the counts (1.9-fold) due to the presence of NCS solubilizer. Fluorescence Measurements. Steady-state fluorescence measurements were made with a Hitachi Perkin-Elmer MPF-3 fluorimeter thermostated at 25 OC using square (3 X 3 mm) microcells. The quantum yield of €-ATP bound to CFI (320-nm excitation) in 40 mM Tris-HC1 (pH 8.0) and 5 mM MgClz was determined by comparison of the areas of the fluorescence emission spectra of free and bound €-ATP at identical concentrations. The quantum yield of unbound €-ATPwas assumed to be 0.59 (Cantley & Hammes, 1975b). The steady-state polarization of fluorescence of CF,-bound €-ATP was measured in 40 mM Tris-HC1 (pH 8.0) and 5 mM MgClz with an excitation slit width of 3 nm at 320 nm and an emission slit width of 5 nm at 400 nm (Cantley & Hammes, 1975b). Fluorescence resonance energy transfer from the €-ATP to a NBD-modified tyrosine on the P subunit of C F I was studied as follows. The CF1-eATP complex (7-9 gM) was equilibrated in 40 mM Tris-HC1 (pH 8.0), 2 mM ADP (to prevent reaction of NBD-CI with sulfhydryl groups), and 5 mM MgC12. A 2.1-2.5-fold excess of NBD-C1 over CF, then was added to the solution, and the change in absorbance at 400 nm and in fluorescence at 400 nm (excitation 320 nm) accompanying formation of the NBD-tyrosine adduct was recorded. Fluorescence measurements were not made continuously to avoid possible photodecomposition reactions. At various times during the course of the labeling reaction, 1.5 pL of a 1.3 M P-mercaptoethanol solution was added to 200 pL of the reaction mixture to displace the NBD from the tyrosine, and the increase in fluorescence was measured. The extent of labeling of CF, with NBD was determined from the absorbance at 400 nm by assuming an extinction coefficient
3454
CARLIER, HOLOWKA, A N D HAMMES
B IOC H E M I S T R Y
0.8,
,
1
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008
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04-
03-
0.2 -
0' Migration
Ol-
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400
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500
Wavelength , n m FIGURE 1: The absorption spectrum of 4.5 fiM CF, in 40 m M Tris-HC1 (pH 8.0) and 5 mM MgClz after incubation with 100 fiM arylazido-ATP (-) or 100 WMarylazida-ADP (- - -) and chromatography on Sephadex G25. of 10 700 M-' cm-I for the Tyr-NBD derivative (Cantley & Hammes, 1975a). The inner filter effects due to the NBD-C1 and P-mercaptcethanol were determined in control experiments with €-ATP in the absence of enzyme, and appropriate corrections to the fluorescence were made. Results Photolabeling of CF1with Arylazido-ATP. Previous studies have shown that CF, rapidly incorporates 1 ATP/CF, in the presence of Mg2+ (Carlier & Hammes, 1979). The ATP hydrolyzes to A D P and P,; however, the A D P cannot be displaced by further addition of ATP for many hours. Similar tight binding does not occur with A D P or AMP-PNP. Arylazido-ATP was found to behave analogously to ATP. When 100 pM a r y l a ~ i d o - [ ~ H ] A Twas P incubated with 10-20 k M CF, in 40 m M Tris-HC1 (pH 8.0) and 5 m M MgClz for 20 min in the dark, 1.0 f 0.15 nucleotide/CF, was found bound by spectrophotometric and radioactivity analyses after chromatography on Sephadex G25, concentration, and dialysis. The absorption spectrum of arylazido-ATP-CF, is presented in Figure 1 , A similar experiment with a r y l a ~ i d o - [ ~ H ] A D P did not result in incorporation of the nucleotide analogue (Figure 1). Thus, arylazido-ATP interacts with CF, similarly to A T P and differently than arylazido-ADP. The arylazido-ATP is a substrate of heat-activated C F , freed from dissociable nucleotides by Sephadex chromatography; the activity with 1 m M arylazido-ATP and 5 m M CaCI,, p H 8.0, a t 20 O C was 10% that of a similar solution with 1 m M A T P substituted for arylazido-ATP. The arylazido-ATP acts as a competitive inhibitor when ATP is used as a substrate, with a competitive inhibition constant of about 20 p M . Under identical conditions, A T P was found to have a Michaelis constant of 60 pM. This value of the Michaelis constant is considerably smaller than that obtained when the enzyme is incubated with nucleotides before assaying (Cantley & Hammes, 1975a). Illumination of the arylazido-ATP-CF, for 15 min, followed by heat activation, resulted in 17-24% inhibition of the ATPase activity. In control experiments with illuminated ATP-CF, or with arylazido-ATP-CF, stored in the dark, less than 3% inhibition occurred. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the photolyzed sample showed almost
-
--.
I
1
FIGLRE 2: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CF, labeled by photolysis of the aryla~ido-[~H]ATP-CF, complex. The line is the absorbance at 280 nm,and the bars represent the radioactivity of gel slices. The peaks associated with the CY, /3, 7,6, and subunits are marked. (a) 10% polyacrylamide gel; (b) 12%
polyacrylamidegel with the 6 and c subunits electrophoresed off the gel; (c) 10% polyacrylamide gel where 100 p M AMP-PNP was added to the aryla~ido-[~H]ATP complex 30 min prior to photolysis; (d) 12% polyacrylamide gel after photolysis of a mixture of 51 pM aryla~ido-[~H]ATP and 17 pM CF,.
exclusive labeling of the P subunit (Figure 2a and 2b). About 0.2 aryla~ido-[~H]ATP/CF, was covalently bound. This value was derived from the total radioactivity counted on the gel and the amount of protein applied to the gel. The same extent of labeling was found when the CF,-arylazido-ATP complex was incubated with 100 FM AMP-PNP before illumination (Figure 2c), indicating (as expected) that the arylazido-ATP could not be displaced from the CF,. Photolabeling experiments also were carried out by adding different amounts of arylazido[jH]ATP to the CF1 freed from nucleotides and illuminating the solution as before. When the molar ratio of arylazido[jH]ATP/CF, was 1-2, radioactivity was found exclusively on the 0 polypeptide. When this ratio was increased to 3, 10% of the radioactivity on the gel was associated with the a subunit (Figure 2d) and this percentage increased when the ratio was increased further. Below a ratio of 3, the amount of photoaffinity label covalently bound to C F , remained constant, and the loss in ATPase activity was never greater than 25%. If C F , was incubated with 100 p M AMP-PNP before adding the arylazido- [ 3H]ATP, essentially no radioactivity appeared on the gels. This is similar to the inhibition of A T P incorporation by ADP previously noted (Carlier & Hammes, 1979). Photolabeling of CF, with Arylazido-ADP. Studies of the equilibrium binding of ADP to CF, have shown two apparently identical binding sites per CF1 (Cantley & Hammes, 1975a). Photoaffinity labeling of CF1 by arylazido- [jH]ADP in the presence of 5 m M Mg2+ shows that the label is incorporated into both the a and subunits in approximately equal amounts (Figure 3a and 3b). If 2.1 1 m M AMP-PNP or 380 p M ADP were present with 28 K M a r y l a ~ i d o - [ ~ H ] A Dand P 12.4 g M CF, during the illumination, the amount of radioactivity on the gels was reduced by - 9 W . No loss in ATPase catalytic activity occurred when AMP-PNP or ADP was present during the illumination. However, in their absence a significant loss in activity occurs. An attempt to correlate the amount of covalently bound label with the ATPase activity was made in a series of experiments. Since the nitrene generated on photoillumination reacts significantly with water (Knowles, 1972), additional a r y l a ~ i d o - [ ~ H ] A Dwas P added to the re-
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VOL.
PHOTOREACTIVE AND FLUORESCENT NUCLEOTIDES
r
I
18, NO.
16,
1979
3455
I
20,
I
E 200
-
I50
u
2.
.= ._ e
150
100
100
50
LL
Q
u
lol/"
0
.o
-0
z
0
50
0
Migration
-
FIGURE 3: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CFI labeled with aryla~ido-[~H]ADP. Photolysis was carried out in a mixture of 38.5 pM aryla~ido-[~H]ADP and 16.9 WMCFI. The line is the absorbance at 560 nm, and the bars represent the radioactivity of gel slices. The peaks associated with the a, p, 7,6 , and t subunits are marked. (a) 10% polyacrylamide gel; (b) 12% po-
(
