Dec 25, 2015 - (c: 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in ... the F1-ATPase from the thermophilic bacterium PS3 ... of the complex itself at high temperature and not due .... Single Site Catalysis-To measure single site catalysis, TF1 or ... is 1.0:1.1:0.28:0.16:0.17 (a:p:y:6:c).
THEJOURNALOF BIOLOGICAL CHEMISTRY (c: 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 264, No. 36, Issue of December 25, pp. 21837-21841,1989 Printed in U.S.A.
The Reconstituted CY&^ Complex of the Thermostable F1-ATPase* (Received for publication, December 28, 1988)
Ken Yokoyama, Toru HisaboriS, and Masasuke Yoshida From the Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midori-ku Yokohama 227, Japan
Previously we reported that ATPase activity was recovered when the subunit a + @ + y or a + j3 + 6 of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions. Unlike that of holoenzyme (TF,) and the a + 8 + y mixture, ATPase activity of the a + j3 6 mixture washeat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252,3480-3485). Here, the properties of purified subunit complexes were compared in detail with those of native TFI. The subunit stoichiometries of the complexeswere determined to be a383y1 and ( ~ 3 8 3 6 1 . In general, the properties of the a3D3y complex are very similar to those of TFI, whereas those of the a3j336 complex are significantly different.ATPase activity of the a3P36 complex is cold labile. The a3836 complex showed a less stringent specificity for substrate and divalent cation than TF1 and the a383y complex. Two K,,,values for ATP were exhibitedby the a3836 complex with the lower one being in the rangeof 0.1 WM. Equilibrium dialysis experiments revealed that the a3P36 complex cannot specifically bind ADP in the absence of Mg2+,while TFl and the a3P3y complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively. ADPdependent inactivation of the a3D36complex by dicyclohexylcarbodiimide was not observed. The a3D3y6 complex was readily formed when the y subunit was added to thea3&6 complex, suggesting that the 4 3 6 complex is not a “dead-end”complex. The cause of thermolability of the a3P36 complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the 6 subunit.
+
of the complex of the CY,p, and 6 subunits of TF1has different properties from those of native TF,; thecomplex is insensitive to azide inhibition, unstable at high temperature, and has a different pH dependence (4, 8). Here we describe the subunit stoichiometry, ADP binding,single site catalysis, steady state kinetics, and sensitivity to inactivation by dicyclohexylcarbodiimide (DCCD)’ of both complexes and compare these results with those of TF,. EXPERIMENTAL PROCEDURES
Materials-[y-”PjATP and [14C]ADP were purchased from Du Pont-New England Nuclear and Amersham, respectively. ATP and ADP (sodium salts) were obtained from Kyowa Hakko. GTP, UTP, CTP,rabbit muscle pyruvate kinase, and lactatedehydrogenase were purchased from Sigma. Other chemical reagents were obtained from Wako Pure Chemical Corp. Purification of TF, and its individual subunits was carried out as described in Ref. 4 . The purified preparations were stored in 50% ammonium sulfate solution at 4 “C. Reconstitution of the Complexes-The 01, 0,and y or 6 subunits dissolved in 30 mM Tris sulfate, pH 7.5, were mixed at molar ratios of 3(a):3(P):l(y or 6). The final protein concentrationsof each mixture was approximately 5 mg/ml. The mixtures were dialyzed overnight at room temperature against 30 mM Tris sulfate, pH 7.5. The dialyzed solutions were concentrated to about one-fifth of the original volume by a Centricon 30 microconcentrator (Amicon Corp.). The concentrated solutions were subjected to high performance liquid chromatography on a gel permeation column (Toyo Soda, G3000SW), equilibrated, and eluted with buffer (50 mM Tris sulfate, pH 7.2, 200 mM Na2S04) atroom temperature. Under these conditions, each complex was eluted as a well defined peak and was well resolved from free subunits. The degree of formation of complex from subunits ranged from 10 to 80% of the theoretical yield depending on the batches of subunit used. Addition of solid urea to the subunit mixtures (final concentration, 8 M ) prior to dialysis did not improve the yield of the complexes. The complexes were routinely used for experiments within 1 day of their purification. The complex formed from a, 0,and 6 subunits is cold labile, dissociating into subunits during overnight F1-ATPase is the water-soluble, catalytic moiety of ATP storage at 4 “C; however, addition of 1 M ammonium sulfate or 30% synthase complex which couples proton flow to ATP synthesis glycerol protected the complex from cold inactivation. The complex in energy transducingmembranes (1-3). Ithas a M, of of a, 8, and y subunits is stable at 4 “ C . ATPase Assay-Unless otherwise specified, standard reaction mix380,000, and the complex subunit structure a3P3~161tl. The tures were prepared in a final volume of 0.25 ml, containing 5 mM F1-ATPase from the thermophilic bacterium PS3 (TFl) can ATP, 5 mM MgS04, and 50 mM Tris sulfate, pH 8.0. They were be reconstituted from either native or denatured individual preincubated for 3 min at 23 or 65 “C before initiating the reaction subunits ( 4 , 5 ) . The Escherichia coli F1-ATPase is also recon- with enzyme. The reaction was terminated by addition of 0.25 ml of stitutable from native subunits (6, 7). In both cases, isolated 2% perchloric acid and the amount of P, was assayed as described native a and /3 subunits do not have ATPase activity although previously (4). One unit of activity was defined as the amount of enzyme that liberates 1 pmol of P,/min. A coupled enzyme assay was they can bind 1 mol of adenine nucleotide/mol of subunit. used for experiments in which the dependence of activity on ATP ATPase activity appears when the complexes are reconsti- concentration was measured. The assay mixture (490 p l ) contained tuted from the a , p, and y subunits. Thecomplex reconstituted 10 mM KCI, 2 mM phosphoenolpyruvate, 20 pg/ml of pyruvate kinase, from the a , (I, and 6 subunits of TF1 also shows ATPase 20 pg/ml of lactate dehydrogenase, and 0.32 mM NADH, in addition activity, while the same combinationof the E. coli F1 subunits tothe ingredients of thestandard reactionmixture(seeabove). does not yield an active complex (6, 7 ) .The ATPase activity Reactions were initiated by addition of 10 pl of enzyme solution, and the rate of NADH oxidation was measured at 23 “C spectrophotometrically. The final level of enzyme in the mixture was 5,10, and 20 * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 I The abbreviations used are: DCCD, dicyclohexylcarbodiimide; solely to indicate this fact. MES, 2-(N-morpholino)ethanesulfonicacid Tricine; N-[2-hydroxy$ Present address: Dept. of Biology, Yokohama City University, 1,l-bis(hydroxymethyl)ethyl]glycine;HPLC, high performance liquid Seto, Kanayawa-ku Yokohama, 236 Japan. chromatography; SDS, sodium dodecyl sulfate.
21837
21838
The a3P36Complex of Fl-ATPase
pg for TFI, a3p3y,and a3pn6,respectively. A linear relation between the rate of NADH oxidation and the amount of the enzyme added . was found at the lowest ATP concentrationtested, 0.5 p ~ The were calculated by a non-linear least squares Michaelis constants(K,,,) method after plotting the experimental data in the form of both a Lineweaver-Burk plot and a Hanes-Woolf plot, and the two values thus obtained were average. VmaX values were then calculated so as to give a best fit in an Eadie-Hofstee plotusing the averaged K,,, values calculated as described above. Single Site Catalysis-To measure single sitecatalysis, TF1 or either complex was incubated with a substoichiometric amount of radioactive ATP as described in Ref. 9. Forassays a t 23 "C the reaction mixtures (50 pl) contained 0.25 M sucrose, 40 mM MES, 40 mM Tris, 1 mM KzHP04, 0.5 mM MgSO,, 150 nM [yn2P]ATP, and 500 nM of TF, or either complex. Fortheassays at 65 "C, the concentrations of [y3'P]ATP andTFI or thecomplexes were reduced to 30 and 110 nM, respectively. In acid quench experiments, reactions were quenched by addition of 5 pl of 60% perchloric acid at the times indicated. The precipitated proteins were removed by brief centrifugationafteraddition of K2C03,andtheprecipitated KCIO, was removed by a second centrifugation. The supernatant thus obtained was subjected to ion exchange HPLC (DEAE-ZSW, Toyo Soda) and radioactivity and absorbance a t 260 nm were monitored (9). Forcold chase experiments, 20 p1 of 17 mM ATP was added after the incubation with [y"P]ATP (see above) and the mixture was incubated an additional 20 s;the reaction was then terminatedwith perchloric acid and prepared for analysis as described above. Binding of [14C]ADP-[14CC]ADP binding to the complexes was determined withequilibriumdialysis as described in Ref. 10. One half-cell contained buffer (10 mM Tricine NaOH, 0.1 M NaC1, p H 8.0) plus either 2 mM MgSO, or 1 mM EDTA and different concentrations of [''CIADP, and the other contained a 4 p~ solution of either complex dissolvedin the same buffer. The volume of each halfcell was 120 p1 and a dialysis membrane (cut off molecular weight 7000, Biomed Instruments Inc., CA) separated each cell. After incubation for 6 h a t room temperature to establish equilibrium, 50 pl of solution was taken from each half-cell and subjected to a scintillation counter. Values of triplicate experimentswere averagedand analyzed. Other Methods-Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was performed as described in Ref. 11. Proteins in the gels were visualized either by Coomassie Brilliant Blue R staining or silver staining. Protein concentrations were determined by the method of Bradford (12). Estimation of the relative amounts of stained protein bands in thepolyacrylamide gels was carried out by the formamide elution method (13). TF, and the complexes, 20 pg each, were electrophoresed in a 14% polyacrylamide gel containing SDS, and gels were stained with 0.25% (w/v) Coomassie Brilliant Blue R in 45% (v/v) methanol and 10% (w/v) acetic acid a t 80 "C for 15 min and destained withsuccessive changes of 5% (v/v) methanol and 7.5% (v/v) acetic acid until the background was colorless. The stained bands were cut out of the gels and bound dye was eluted in 1 ml of formamide in a stoppered amber test tube with shaking a t 50 "C for 7 h. The amount of eluted dye was determined by absorbance a t 595 nm. The values obtained from sixmeasurements were averaged and standard mean deviationswere within 15% of the mean values.
10 15 20 ELUTION TIME(min1 FIG. 1. Purification of the as&? and a&6 complex with gel permeation HPLC (left),and SDS-polyacrylamide gel electrophoresis of the isolated as&,? and as&& complexes (high).The results of TF, are shown as a control. Conditions of gel permeation HPLC and electrophoresis are described under "Experimental Procedures." The electrophoresed gel was stained by the silver staining methods: Lane 1, TF,; Lane 2, a3&y; Lane 3,a&d.
TABLE I Hydrolysis of ATP, GTP, CTP, and UTP by TF, and the a&y and a3p38complexes The incubation mixtures contained 5 mM MgSO, 5 mM of indicated nucleotide triphosphates (sodium salt), 50mM Tris sulfate,p H 8.0, and 5 pg of TF,, nn&y or a3836 in a total volume of 0.25 ml. After a 5-minincubation at 65 or 23 "C, released Pi wasmeasured as described under "ExperimentalProcedures." Values are not presented for a&$ a t 65 "C due to the heatlability of this complex. Substrate
ATP GTP CTP UTP
TF, 23°C
5.1 14 cO.1
co.1
4
3
a3836
7
-
65'C 23°C 23'C65'C activity (unitlmg protein)
8.5 27 ~0.1 2.2
2.2 7.0 cO.1 4.8
5.2 13 4.3 16
1.1
0.94 0.67 1.8
Thus, the ratios of the bound dye to each subunit of the complexes are very close to the ratiofound with TFI, and the calculated molar ratios of subunits area&.9yo.w and a&.d0.94. Therefore, we conclude that the subunitstoichiometry of the complexes is an&yland a&& and will refer to thecomplexes as aaPsyand aa&6 hereafter. Hydrolysis of Other Nucleotides and Specificity for Divalent Cation-At 23 "C, under the conditions described, TF1 does not hydrolyze CTP and UTP, while anpay hydrolyzes U T P but not CTP and an/3aGhydrolyzes both CTP and UTP (Table RESULTS I). At 65 "C, TFI does not hydrolyze CTP but adhy does Purification of Complexes and SubunitStoichiometry-The hydrolyze it. Therefore,substrate specificity is apparently complexes were purified with by gel permeation HPLC from decreased in the order TF, > a&y > an&d(Table I). The mixtures of a, p, and either y or 6 subunits of TF, (Fig. 1, divalentmetalrequirement of the complexesfor ATPase left). The complexes and free subunits eluted at13.5 and 17.0 activity wasmeasured a t 1 mM metal ion and 1 mM ATP min, respectively. Polyacrylamide gel electrophoresis in the (Table 11). Under these conditions, almost all the metal ion presence of SDS of the purified complexes clearly showed in the solution should exist as metal.ATPcomplex because that each complex was free from contaminating proteins(Fig. of the strong metal chelating ability of ATP; therefore, the 1, right). The similar retention time of the complexes and TFl effect of metal ions on the activity ought to reflect the specistrongly suggests that the number of copies of the a and p ficity of the enzyme forthe metal. ATP complexes. The a&y subunits in the reconstituted complexes is the same as in complex shows activity in the presenceof Ca2+,whereas TFI native TF,, that is, three a and three p. This was further is inactive under the sameconditions. The an&dcomplex can confirmed by measuring the amount of dye extracted from also use Hg'+, Ni2+,Cu'+, and Fe", in addition to Ca2+, as the stained subunit bands in the electrophoresed gel of TFl cofactor. Thus thecomplexes, especiallyanP36,appear to have and thepurified complexes. The ratio of bound dye with TF, catalytic site(s) with less stringent specificity for nucleotide is 1.0:1.1:0.28:0.16:0.17 (a:p:y:6:c).The ratios determined for and divalent cation. the complexes are 1.0:1.05:0.27 (a:P:y)and 1.0:1.0:0.15 (a:@:S). Kinetics of A T P Hydrolysis-As has been well documented
21839
The a3p36Complex of Fl-ATPase TABLEI1 Divalent metal cation dependence of ATPase activity of TFI and the a&y and a3&8 complexes The assay mixtures containing 1 mM metal chloride, 1 mM ATP, 50 mM Tris sulfate, pH 8.0, and 5 pg of TF,, a&y, or a&8 were incubated for 5 min at 23 "C, and released Pi was determined as described under "Experimental Procedures." The values are specific activitv. ATPase activity ~~
TFI 4s-Y unitfmg protein
Metal cation
5.3 1.6 10 0.77 3.6 3.3 1.6 (0.1
M e
Mn2+ Zn2+ co2+ Cd2+ Ca2+ cuz+ Fez+ Ni"
1.1
1.4 1.7 2.0 1.1 0.3 0.61 0.67
