Biochem. J. (1990) 266, 835-841 (Printed in Great Britain)
835
Interaction of ox heart mitochondrial F1-ATPase with immobilized ADP and ATP Seelochan BEHARRY,* Michael J. GRESSERt and Philip D. BRAGG* *Department of Biochemistry, University of British Columbia, 2146 Health Sciences Mall, Vancouver, B.C., Canada V6T 1W5, and tDepartment of Chemistry, Simon Fraser University, Burnaby, B. C., Canada V5A 1S6
The reaction of mitochondrial F1-ATPase with immobilized substrate was studied by using columns of agarose-hexane-ATP. Mg2+ was required for binding of the enzyme to the column matrix. The columnbound enzyme could be eluted fully by ATP and other nucleoside triphosphates. Nucleoside di- and monophosphates were less effective. At a fixed concentration of nucleotide the effectiveness of elution was proportional to the charge on the eluting molecule. The ATP of the column matrix was hydrolysed by the bound F1-ATPase to release phosphate, probably by a uni-site reaction mechanism. Thus the F1-ATPase was bound to the immobilized ATP by a catalytic site. Treatment of the bound F1-ATPase with 4-chloro7-nitrobenzofurazan prevented complete release of the enzyme by ATP. Only one-third of the bound enzyme was now eluted by the nucleotide. The inhibition of release could be due either to the inhibitor blocking cooperative interactions between sites or to its increasing the tightness of binding of immobilized ADP at the catalytic site.
INTRODUCTION
Mitochondrial FiFo, the proton-translocating ATP synthase, is the terminal enzyme of oxidative phosphorylation in which process it synthesizes ATP from ADP and Pi. F1 may be separated from F, as a soluble enzyme able to hydrolyse ATP to ADP and phosphate. F1 consists of five different subunits (a.
..
:
...
....
Fig. 2. SDS/PAGE of fractions eluted by ATP from the agarose-hexane-ATP (type 2) column of Fig. 1 The various fractions were obtained as described in the legend of Fig. 1. The SDS/polyacrylamide gel consisted of a 4 % (w/v)-polyacrylamide stacking gel on top of a 15 % separating gel; 20 ,l samples of the fractions diluted with an equal volume of SDS sample buffer were used. Lane 1, F1 standard; lane 2; sample application; lanes 3-7, buffer washes; lane 8, AMP wash; lanes 9-12, ATP washes without centrifugation; lanes 13-14, ATP wash with centrifugation; lanes 15-18, urea washes. The positions of migration of the a-e subunits of F1 are indicated.
838
S. Beharry, M. J. Gresser and P. D. Bragg
Table 1. Elution of Fl from agarose-hexane-ATP (Type 2) by various compounds
The activity eluted by ATP was set at 1000%. Percentage of bound F1 eluted by 10 mM compound
Compound ATP ITP GTP UTP CTP ADP with phosphate ADP IDP GDP AMP
100 75 74 73 70 66 52 53 51 33 50 66 34 42 16 12
Tripolyphosphate Pyrophosphate Phosphate Vanadate Adenosine Adenine
were no evident subunit differences between column fractions indicative of different species of F1 undergoing separation on the column. The buffers used in the experiments contained 1.6 mm-
Mg2". Omission of this resulted in complete lack of binding of F1 to agarose-hexane-ATP. ATP, as an elutant of the enzyme, could be replaced by a variety of related compounds (Table 1). Hydrolysis of
the elutant was clearly not required for it to be effective. Thus the non-hydrolysable analogues of ATP, p[NH]ppA (Fig. 3) and p[CHJ]ppA, were effective elutants, as also were the nucleoside diphosphates. At a 10 mM concentration of elutant, their effectiveness was related to the charge carried by the molecule. Thus the effectiveness of elution decreased in the series nucleoside triphosphates > nucleoside diphosphates, pyrophosphate > AMP, orthophosphate, orthovanadate > adenosine, adenine (Table 1). In the experiments shown in Table 1, any uneluted F1 could subsequently be eluted from the column with 10 mM-ATP. Hydrolysis of bound ATP Since mitochondrial Fl can bind nucleotides at noncatalytic as well as at catalytic sites (Ferguson et al., 1975; Senior, 1988) the type of site involved in binding to the immobilized ATP was examined. Ox heart mitochondrial F1 as usually prepared contains three molecules of bound adenine nucleotide per molecule of F1. Two molecules are bound at non-catalytic sites, with the third at a catalytic site (Kironde & Cross, 1986, 1514: 13: 12o 110
EC 10g
12 34 5
6 78910101121213
9.a) 8.2 7 con 6:.0 5 321-
.)in _Im1=
j
- - S-
M
-
Fig. 3. SDS/PAGE of fractions eluted by pINHIppA from an agarose-hexane-ATP (Type 2) column. The various fractions were obtained as described in Fig. 1, except that 4 x 1 ml washes, without centrifugation, preceded the initial ATP washes. The gel and sample preparation were as described in Fig. 2, except that 10 p.1 of each sample was applied to the gel. Lane 1, F, standard; lane 2, first fraction of buffer washes; lane 3, AMP wash; lanes 4-7, p[NH]ppA washes, without centrifugation; lanes 8-11, ATP washes, without centrifugation; lanes 12-13, ATP washes, with centrifugation. The positions of migration of the a subunits of F, are indicated.
W
I
.I
1 2 3 4 5 6 7 8 9 101112131415161718192021 Fraction no.
Fig. 4. Elution of phosphate released on hydrolysis ofthe agarosehexane-ATP (Type 2) column matrix by F1 A 1.0 ml portion of F1 (0.96 mg) was applied to a 1 ml agarose-hexane-ATP column. Fractions were eluted as follows (see the Materials and methods section): fraction 1-5, buffer washes with centrifugation; 6-9, buffer washes, without centrifugation; 10-1 1, buffer washes with centrifugation; 12-15, 3 M-H2SO4 washes without centrifugation; 16-17, 3 M-H2SO4 washes with centrifugation; 18-21, 2 M-guanidinium chloride washes without centrifugation. An identical control column was eluted similarly, except that 1.0 ml of buffer was applied to the column instead of F1. Phosphate assays were carried out as described in the Materials and methods section. Samples (0.1 ml) of each fraction were assayed and, for plotting, this value was multiplied by the total volume of each fraction. The values given have been corrected for the phosphate content of the same fraction eluted from the control column. The phosphate contents of the fractions from the control column expressed as a percentage of the phosphate contents of equivalent fractions from the column with F1 were: fractions 1-1 1, 0 %; fractions 12-17, 22 %; fractions 18-21, 30 %.
1990
839
Interaction of mitochondrial F1-ATPase with immobilized ADP and ATP Table 2. Effect of Nbf-CI treatment of F1 on binding to Agarose-hexane-ATP (Type 2)
Fl was either treated with Nbf-Cl before application to the column of agarose-hexane-ATP or treated with this reagent after binding to the column. Fl was then eluted with ATP before or after washing the column with DTT to release the reagent from the enzyme (see the Materials and methods section). DTT wash before ATPt
Protein eluted by ATP (mg)
ATPase activity eluted by ATPt (% of control)
+
0.130 0.088 0.057 0.112
100 74 33 66
Nbf-Cl treatment
Precolumn*
On column*
+
+
-, No treatment; +, treated. t , No DTT wash; +, DTT wash. t ATPase activity was assayed in the presence of 0.5 mM-DTT to reverse inhibition by Nbf-Cl. *
1987). Kironde & Cross (1986) have described a procedure whereby F1 can be converted into a form in which all three non-catalytic sites are occupied by adenine nucleotide and all catalytic sites are vacant. We found that F1 prepared in this way had binding and elution behaviour identical with that of the usual enzyme preparation. Therefore it is likely that F1 binds to immobilized ATP through a catalytic site. The possibility that F1 hydrolysed the ATP of the column matrix was examined as follows. F1 was absorbed on the column in phosphate-free equilibration buffer. Unbound Fl was removed by extensive washing with a phosphate-free buffer. Liberated, but unreleased, phosphate bound to the enzyme was eluted from the column with 3 M-H2SO4. The enzyme was subsequently eluted from the column by washing with 2 M-guanidinium chloride. Both treatments released Pi bound to the F1. This was corrected for phosphate released from the column by these treatments in the absence of application of F1 (Fig. 4). A total of 46 nmol of phosphate was released per nmol of F1 bound to the column. This is far in excess of the maximum amount of phosphate (3 nmol) which might possibly originate from phosphate bound to the enzyme or be released by hydrolysis of bound adenine nucleotides. A rate could not be calculated from these data, but the enzyme was bound to the column matrix for about 30 min before the elution procedure was finished. Binding of F1 to columns of agarose-hexane-ADP F1 applied to a column of agarose-hexane-ADP bound to the column matrix, but only to the extent of about one-half of that bound by agarose-hexane-ATP. The bound enzyme was fully eluted by 10 mM-ATP, -ADP, -IDP and -GDP. Effect of Nbf-Cl treatment of F1 on binding to agarose-hexane-ATP Treatment of F1 with Nbf-Cl- results in inhibition of ATPase activity concomitant with modification of tyrosine-3 11 of a f-subunit (Andrews et al., 1984; Sutton & Ferguson, 1985). Almost complete inhibition occurs with the modification of a single fl-subunit per F1 molecule, although the treated enzyme can still bind three molecules of p[NH]ppA (Cross & Nalin, 1982). This suggests that NbF-Cl modification blocks Vol. 266
co-operative interactions between the catalytic sites. The effect of Nbf-Cl treatment of F1 on its interaction with agarose-hexane-ATP was studied in two ways. Pretreatment of the enzyme before application to the column did not affect the ability of the F1 to bind to the column matrix. An amount of enzyme 74 % of that released when untreated F1 was used was eluted from the column by ATP (Table 2). A further 24 % was released when the column was subsequently eluted with ATP in the presence of DTT. If F1 was first bound to the column and then treated with Nbf-Cl, only about one-third of the enzyme could be eluted with ATP alone. Pretreatment with DTT to remove the inhibitor then permitted ATP to release double the amount eluted in the absence of DTT. These results suggest that Nbf-Cl-treated F1 has two modes of binding to agarose-hexane-ATP.
DISCUSSION Use of immobilized ATP provides a novel method to study the interaction of F1 with its substrate. F, was bound to columns of agarose-hexane-ATP and could be eluted by ATP and other nucleotides in a process that did not require hydrolysis of the nucleoside triphosphate and the effectiveness of which depended on the charge carried by the eluting molecule. Binding of the enzyme to the column required Mg2+. This fact, together with the observation that the immobilized ATP was hydrolysed by the bound enzyme, suggests that F1 binding is specific and involves an ATP-binding site on the enzyme. Hydrolysis of the immobilized ATP was demonstrated by the liberation from the column of a much greater amount of phosphate in the presence of F1 than in its absence. Some of the liberated phosphate appeared in the fraction containing unbound enzyme. The phosphate (12 nmol) was in large stoichiometric excess over the F1 in this fraction (1.3 nmol) and so could not all be bound to the enzyme. Presumably, this phosphate was liberated by the enzyme as it passed through the column. A much greater amount (62 nmol) of phosphate was retained on the column with the bound enzyme (1.3 nmol) and could be eluted by H2S04 and guanidinium chloride. This phosphate is probably retained by interactions with the column matrix. These interactions have made it impossible to carry out a simple kinetic experiment in which F1 is incubated with agarose-hexane-ATP to measure
S.
840
the rate of hydrolysis of the bound ATP. However, the rate of reaction appears to be low. Only 46 nmol ATP out of a possible 1400 nmol were hydrolysed per nmol of F1 during the 30 min of exposure of the immobilized ATP to the enzyme. The apparent low rate of reaction is consistent with a uni-site mechanism of hydrolysis in which product is released slowly from the enzyme (Grubmeyer & Penefsky, 1981a,b; Grubmeyer et al., 1982; Cross et al., 1982). It is unlikely, for steric reasons, that more than a single molecule of ATP reacts with F1 at any given time. In the absence of free ATP (that is, until elution is commenced) there would be no cooperative interactions which would increase the rate of ATP hydrolysis (Grubmeyer & Penefsky, 1981a,b; Grubmeyer et al., 1982; Cross et al., 1982). The extent of hydrolysis of the immobilized ATP suggests that F1 must be released from its binding site on the column after catalysis, rebinding successively at other immobilized ATP molecules. Release would be favoured by the lower affinity of F1 for agarose-hexane-ADP. The hydrolysis of ATP implies that F1 must be bound through a catalytic site and not through an unoccupied non-catalytic adenine-nucleotide-binding site. Kironde & Cross (1986, 1987) have shown that F1, as isolated by the procedure used in the present study, contains one unoccupied non-catalytic site and two unoccupied catalytic sites. Conversion of this type of F1 into that in which all three non-catalytic sites are occupied by adenine nucleotide and all three catalytic sites are empty (Kironde & Cross, 1986) did not alter the interaction of F1 with immobilized ATP. This supports the view that the enzyme is bound through a catalytic site. Furthermore, the ability of ITP and GTP to elute F1 is evidence for the involvement of the catalytic sites in the release process, since these nucleotides do not bind to non-catalytic sites. There are some indications that F1 may bind in two modes. The F1 is not eluted by ATP (or other agents) as a sharp peak in a few fractions. The tailing which occurs might be due to differences in the tightness of binding by different F1 complexes. Moreover, treatment of F1 bound to immobilized ATP with Nbf-Cl gave two types of species on elution with ATP. Part of the F1 could be eluted readily with ATP, but the remainder required pretreatment with DTT to release the reagent from F1 before ATP could elute the enzyme. In the first type of behaviour modification had not obviously affected the ability of F1 to bind to immobilized ATP. There is no reason to believe that this fraction of the enzyme represents unmodified F1, since the conditions chosen for modification (e.g. concentration and time of reaction) are known to be effective with free F1 (Ferguson et al., 1975). Furthermore, Nbf-Cl-treated F1 binds p[NH]ppA effectively (Cross & Nalin, 1982). We have no evidence that Nbf-Cl modifies the same tyrosine residue in the boun d as in the free enzyme. It is possible that the two species detected by differences in elution by ATP might represent enzyme molecules modified at different tyrosine residues by the reagent. In the second type of behaviour, the lack of ability to elute F1 from the immobilized ATP until the reagent has been removed from the enzyme with DTT might be for one of two reasons. Nbf-Cl treatment of F1 bound to ATP might increase the tightness of binding of the enzyme to the nucleotide. However, it is possible that the effect of Nbf-Cl treatment is to block co-operativity between catalytic sites on the enzyme (Ferguson et al.,
Beharry, M. J. Gresser and P. D. Bragg
1975; Lunardi et al., 1979). This would be significant if the mechanism of elution of F1 from the immobilized ATP was for the eluting ATP to bind at a second catalytic site as opposed to competing at the first binding site. This is a reasonable mechanism on the basis of the catalytic cycle of the enzyme suggesting by the work of Boyer, Cross, Penefsky and their co-workers (Grubmeyer & Penefsky, 1981a,b; Boyer et al., 1982; Cross et al., 1982; Gresser et al., 1982; Grubmeyer et al., 1982; O'Neal & Boyer, 1984; Penefsky, 1988). Indirect support for the hypothesis that F1 is eluted by nucleotide interacting at a second site is given by our experiments with agarose-hexane-ADP. F1 bound to this matrix is eluted fully by ATP and ADP, and also by IDP. In contrast with ADP, IDP does not inhibit the hydrolysis of ATP (or ITP) over the concentration range used in our experiments (Pullman et al., 1960). Thus it is unlikely that there is direct competition between IDP and ATP at the site of hydrolysis. Thus the ability of IDP to elute F1 from agarose-hexane-ADP is likely due to interaction at the second site. The mechanism for the elution of F1 from immobilized ATP needs further clarification. This work was supported by grants from the Medical Research Council of Canada (to P. D. B.) and the Natural Sciences and Engineering Research Council of Canada (to M. J. G.).
REFERENCES Andrews, W. W., Hill, F. C. & Allison, W. S. (1984) J. Biol. Chem. 259, 8219-8225 Beharry, S. & Gresser, M. J. (1987) J. Biol. Chem. 262,
10630-10637 Boyer, P. D., Kohlbrenner, W. E., McIntosh, D. B., Smith, L. T. & O'Neal, C. C. (1982) Ann. N.Y. Acad. Sci. 402,65-73 Cross, R. L. (1981) Annu. Rev. Biochem. 50, 681-714 Cross, R. L. & Nalin, C. M. (1982) J. Biol. Chem. 257, 2874-2881 Cross, R. L., Grubmeyer, C. & Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105 Ernster, L. (1984) Curr. Top. Cell. Regul. 24, 313-334 Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Biochemistry 10, 2606-2617 Ferguson, S. J., Lloyd, W. J., Lyons, M. H. & Radda, G. K. (1975) Eur. J. Biochem. 54, 117-126 Ferguson, S. J., Lloyd, W. J. & Radda, G. K. (1975) Eur. J. Biochem. 54, 127-133 Gresser, M. J., Myers, J. A. & Boyer, P. D. (1982) J. Biol. Chem. 257, 12030-12038 Gresser, M. J., Beharry, S. & Moennich, D. M. C. (1984) Curr. Top. Cell. Regul. 24, 365-378 Grubmeyer, C. & Penefsky, H. S. (1981a) J. Biol. Chem. 256, 3718-3727 Grubmeyer, C. & Penefsky, H. S. (1981b) J. Biol. Chem. 256, 3728-3734 Grubmeyer, C., Cross, R. L. & Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100 Hutton, R. L. & Boyer, P. D. (1979) J. Biol. Chem. 254,
9990-9993 Kasahara, M. & Penefsky, H. S. (1978) J. Biol. Chem. 253,
4180-4187 Kironde, F. A. S. & Cross, R. L. (1986) J. Biol. Chem. 261,
12544A12549
Kironde, F. A. S. & Cross, R. L. (1987) J. Biol. Chem. 262, 3488-3495
1990
Interaction of mitochondrial F1-ATPase with immobilized ADP and ATP Kodama, T., Fukai, K. & Kometami, K. (1986) J. Biochem. 99, 1465-1472 Laemmli, U. K. (1975) Nature (London) 227, 680-685 Lanzetta, P. A., Alvarez, L. J., Reinach, P. S. & Candia, D. A. (1979) Anal. Biochem. 100, 95-97 Lunardi, J., Satre, M., Bof, M. & Vignais, P. V. (1979) Biochemistry 24, 5310-5316 O'Neal, C. C. & Boyer, P. D. (1984) J. Biol. Chem. 259, 5761-5767 Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899 Penefsky, H. S. (1979) Adv. Enzymol. Relat. Areas Mol. Biol. 49, 223-280 Received 5 September 1989/5 October 1989; accepted 11 October 1989
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Penefsky, H. S. (1988) J. Biol. Chem. 263, 6020-6022 Pullman, M. E., Penefsky, H. S., Datta, A. & Racker, E. (1960) J. Biol. Chem. 3322-3329 Senior, A. E. (1988) Physiol. Rev. 68, 177-231 Stankiewicz, P. J., Gresser, M. J., Tracey, A. S. & Hass, L. F. (1987) Biochemistry 26, 1264-1269 Sutton, R. & Ferguson, S. J. (1985) Eur. J. Biochem. 148, 551-554 Walker, J. E., Fearnley, I. M., Gay, N. J., Gibson, B. W., Northrop, F. D., Powell, S. J., Runswick, M. J., Saraste, M. & Tybulewicz, V. L. J. (1985) J. Mol. Biol. 184, 677701
835
Interaction of ox heart mitochondrial F1-ATPase with immobilized ADP and ATP Seelochan BEHARRY,* Michael J. GRESSERt and Philip D. BRAGG* *Department of Biochemistry, University of British Columbia, 2146 Health Sciences Mall, Vancouver, B.C., Canada V6T 1W5, and tDepartment of Chemistry, Simon Fraser University, Burnaby, B. C., Canada V5A 1S6
The reaction of mitochondrial F1-ATPase with immobilized substrate was studied by using columns of agarose-hexane-ATP. Mg2+ was required for binding of the enzyme to the column matrix. The columnbound enzyme could be eluted fully by ATP and other nucleoside triphosphates. Nucleoside di- and monophosphates were less effective. At a fixed concentration of nucleotide the effectiveness of elution was proportional to the charge on the eluting molecule. The ATP of the column matrix was hydrolysed by the bound F1-ATPase to release phosphate, probably by a uni-site reaction mechanism. Thus the F1-ATPase was bound to the immobilized ATP by a catalytic site. Treatment of the bound F1-ATPase with 4-chloro7-nitrobenzofurazan prevented complete release of the enzyme by ATP. Only one-third of the bound enzyme was now eluted by the nucleotide. The inhibition of release could be due either to the inhibitor blocking cooperative interactions between sites or to its increasing the tightness of binding of immobilized ADP at the catalytic site.
INTRODUCTION
Mitochondrial FiFo, the proton-translocating ATP synthase, is the terminal enzyme of oxidative phosphorylation in which process it synthesizes ATP from ADP and Pi. F1 may be separated from F, as a soluble enzyme able to hydrolyse ATP to ADP and phosphate. F1 consists of five different subunits (a.
..
:
...
....
Fig. 2. SDS/PAGE of fractions eluted by ATP from the agarose-hexane-ATP (type 2) column of Fig. 1 The various fractions were obtained as described in the legend of Fig. 1. The SDS/polyacrylamide gel consisted of a 4 % (w/v)-polyacrylamide stacking gel on top of a 15 % separating gel; 20 ,l samples of the fractions diluted with an equal volume of SDS sample buffer were used. Lane 1, F1 standard; lane 2; sample application; lanes 3-7, buffer washes; lane 8, AMP wash; lanes 9-12, ATP washes without centrifugation; lanes 13-14, ATP wash with centrifugation; lanes 15-18, urea washes. The positions of migration of the a-e subunits of F1 are indicated.
838
S. Beharry, M. J. Gresser and P. D. Bragg
Table 1. Elution of Fl from agarose-hexane-ATP (Type 2) by various compounds
The activity eluted by ATP was set at 1000%. Percentage of bound F1 eluted by 10 mM compound
Compound ATP ITP GTP UTP CTP ADP with phosphate ADP IDP GDP AMP
100 75 74 73 70 66 52 53 51 33 50 66 34 42 16 12
Tripolyphosphate Pyrophosphate Phosphate Vanadate Adenosine Adenine
were no evident subunit differences between column fractions indicative of different species of F1 undergoing separation on the column. The buffers used in the experiments contained 1.6 mm-
Mg2". Omission of this resulted in complete lack of binding of F1 to agarose-hexane-ATP. ATP, as an elutant of the enzyme, could be replaced by a variety of related compounds (Table 1). Hydrolysis of
the elutant was clearly not required for it to be effective. Thus the non-hydrolysable analogues of ATP, p[NH]ppA (Fig. 3) and p[CHJ]ppA, were effective elutants, as also were the nucleoside diphosphates. At a 10 mM concentration of elutant, their effectiveness was related to the charge carried by the molecule. Thus the effectiveness of elution decreased in the series nucleoside triphosphates > nucleoside diphosphates, pyrophosphate > AMP, orthophosphate, orthovanadate > adenosine, adenine (Table 1). In the experiments shown in Table 1, any uneluted F1 could subsequently be eluted from the column with 10 mM-ATP. Hydrolysis of bound ATP Since mitochondrial Fl can bind nucleotides at noncatalytic as well as at catalytic sites (Ferguson et al., 1975; Senior, 1988) the type of site involved in binding to the immobilized ATP was examined. Ox heart mitochondrial F1 as usually prepared contains three molecules of bound adenine nucleotide per molecule of F1. Two molecules are bound at non-catalytic sites, with the third at a catalytic site (Kironde & Cross, 1986, 1514: 13: 12o 110
EC 10g
12 34 5
6 78910101121213
9.a) 8.2 7 con 6:.0 5 321-
.)in _Im1=
j
- - S-
M
-
Fig. 3. SDS/PAGE of fractions eluted by pINHIppA from an agarose-hexane-ATP (Type 2) column. The various fractions were obtained as described in Fig. 1, except that 4 x 1 ml washes, without centrifugation, preceded the initial ATP washes. The gel and sample preparation were as described in Fig. 2, except that 10 p.1 of each sample was applied to the gel. Lane 1, F, standard; lane 2, first fraction of buffer washes; lane 3, AMP wash; lanes 4-7, p[NH]ppA washes, without centrifugation; lanes 8-11, ATP washes, without centrifugation; lanes 12-13, ATP washes, with centrifugation. The positions of migration of the a subunits of F, are indicated.
W
I
.I
1 2 3 4 5 6 7 8 9 101112131415161718192021 Fraction no.
Fig. 4. Elution of phosphate released on hydrolysis ofthe agarosehexane-ATP (Type 2) column matrix by F1 A 1.0 ml portion of F1 (0.96 mg) was applied to a 1 ml agarose-hexane-ATP column. Fractions were eluted as follows (see the Materials and methods section): fraction 1-5, buffer washes with centrifugation; 6-9, buffer washes, without centrifugation; 10-1 1, buffer washes with centrifugation; 12-15, 3 M-H2SO4 washes without centrifugation; 16-17, 3 M-H2SO4 washes with centrifugation; 18-21, 2 M-guanidinium chloride washes without centrifugation. An identical control column was eluted similarly, except that 1.0 ml of buffer was applied to the column instead of F1. Phosphate assays were carried out as described in the Materials and methods section. Samples (0.1 ml) of each fraction were assayed and, for plotting, this value was multiplied by the total volume of each fraction. The values given have been corrected for the phosphate content of the same fraction eluted from the control column. The phosphate contents of the fractions from the control column expressed as a percentage of the phosphate contents of equivalent fractions from the column with F1 were: fractions 1-1 1, 0 %; fractions 12-17, 22 %; fractions 18-21, 30 %.
1990
839
Interaction of mitochondrial F1-ATPase with immobilized ADP and ATP Table 2. Effect of Nbf-CI treatment of F1 on binding to Agarose-hexane-ATP (Type 2)
Fl was either treated with Nbf-Cl before application to the column of agarose-hexane-ATP or treated with this reagent after binding to the column. Fl was then eluted with ATP before or after washing the column with DTT to release the reagent from the enzyme (see the Materials and methods section). DTT wash before ATPt
Protein eluted by ATP (mg)
ATPase activity eluted by ATPt (% of control)
+
0.130 0.088 0.057 0.112
100 74 33 66
Nbf-Cl treatment
Precolumn*
On column*
+
+
-, No treatment; +, treated. t , No DTT wash; +, DTT wash. t ATPase activity was assayed in the presence of 0.5 mM-DTT to reverse inhibition by Nbf-Cl. *
1987). Kironde & Cross (1986) have described a procedure whereby F1 can be converted into a form in which all three non-catalytic sites are occupied by adenine nucleotide and all catalytic sites are vacant. We found that F1 prepared in this way had binding and elution behaviour identical with that of the usual enzyme preparation. Therefore it is likely that F1 binds to immobilized ATP through a catalytic site. The possibility that F1 hydrolysed the ATP of the column matrix was examined as follows. F1 was absorbed on the column in phosphate-free equilibration buffer. Unbound Fl was removed by extensive washing with a phosphate-free buffer. Liberated, but unreleased, phosphate bound to the enzyme was eluted from the column with 3 M-H2SO4. The enzyme was subsequently eluted from the column by washing with 2 M-guanidinium chloride. Both treatments released Pi bound to the F1. This was corrected for phosphate released from the column by these treatments in the absence of application of F1 (Fig. 4). A total of 46 nmol of phosphate was released per nmol of F1 bound to the column. This is far in excess of the maximum amount of phosphate (3 nmol) which might possibly originate from phosphate bound to the enzyme or be released by hydrolysis of bound adenine nucleotides. A rate could not be calculated from these data, but the enzyme was bound to the column matrix for about 30 min before the elution procedure was finished. Binding of F1 to columns of agarose-hexane-ADP F1 applied to a column of agarose-hexane-ADP bound to the column matrix, but only to the extent of about one-half of that bound by agarose-hexane-ATP. The bound enzyme was fully eluted by 10 mM-ATP, -ADP, -IDP and -GDP. Effect of Nbf-Cl treatment of F1 on binding to agarose-hexane-ATP Treatment of F1 with Nbf-Cl- results in inhibition of ATPase activity concomitant with modification of tyrosine-3 11 of a f-subunit (Andrews et al., 1984; Sutton & Ferguson, 1985). Almost complete inhibition occurs with the modification of a single fl-subunit per F1 molecule, although the treated enzyme can still bind three molecules of p[NH]ppA (Cross & Nalin, 1982). This suggests that NbF-Cl modification blocks Vol. 266
co-operative interactions between the catalytic sites. The effect of Nbf-Cl treatment of F1 on its interaction with agarose-hexane-ATP was studied in two ways. Pretreatment of the enzyme before application to the column did not affect the ability of the F1 to bind to the column matrix. An amount of enzyme 74 % of that released when untreated F1 was used was eluted from the column by ATP (Table 2). A further 24 % was released when the column was subsequently eluted with ATP in the presence of DTT. If F1 was first bound to the column and then treated with Nbf-Cl, only about one-third of the enzyme could be eluted with ATP alone. Pretreatment with DTT to remove the inhibitor then permitted ATP to release double the amount eluted in the absence of DTT. These results suggest that Nbf-Cl-treated F1 has two modes of binding to agarose-hexane-ATP.
DISCUSSION Use of immobilized ATP provides a novel method to study the interaction of F1 with its substrate. F, was bound to columns of agarose-hexane-ATP and could be eluted by ATP and other nucleotides in a process that did not require hydrolysis of the nucleoside triphosphate and the effectiveness of which depended on the charge carried by the eluting molecule. Binding of the enzyme to the column required Mg2+. This fact, together with the observation that the immobilized ATP was hydrolysed by the bound enzyme, suggests that F1 binding is specific and involves an ATP-binding site on the enzyme. Hydrolysis of the immobilized ATP was demonstrated by the liberation from the column of a much greater amount of phosphate in the presence of F1 than in its absence. Some of the liberated phosphate appeared in the fraction containing unbound enzyme. The phosphate (12 nmol) was in large stoichiometric excess over the F1 in this fraction (1.3 nmol) and so could not all be bound to the enzyme. Presumably, this phosphate was liberated by the enzyme as it passed through the column. A much greater amount (62 nmol) of phosphate was retained on the column with the bound enzyme (1.3 nmol) and could be eluted by H2S04 and guanidinium chloride. This phosphate is probably retained by interactions with the column matrix. These interactions have made it impossible to carry out a simple kinetic experiment in which F1 is incubated with agarose-hexane-ATP to measure
S.
840
the rate of hydrolysis of the bound ATP. However, the rate of reaction appears to be low. Only 46 nmol ATP out of a possible 1400 nmol were hydrolysed per nmol of F1 during the 30 min of exposure of the immobilized ATP to the enzyme. The apparent low rate of reaction is consistent with a uni-site mechanism of hydrolysis in which product is released slowly from the enzyme (Grubmeyer & Penefsky, 1981a,b; Grubmeyer et al., 1982; Cross et al., 1982). It is unlikely, for steric reasons, that more than a single molecule of ATP reacts with F1 at any given time. In the absence of free ATP (that is, until elution is commenced) there would be no cooperative interactions which would increase the rate of ATP hydrolysis (Grubmeyer & Penefsky, 1981a,b; Grubmeyer et al., 1982; Cross et al., 1982). The extent of hydrolysis of the immobilized ATP suggests that F1 must be released from its binding site on the column after catalysis, rebinding successively at other immobilized ATP molecules. Release would be favoured by the lower affinity of F1 for agarose-hexane-ADP. The hydrolysis of ATP implies that F1 must be bound through a catalytic site and not through an unoccupied non-catalytic adenine-nucleotide-binding site. Kironde & Cross (1986, 1987) have shown that F1, as isolated by the procedure used in the present study, contains one unoccupied non-catalytic site and two unoccupied catalytic sites. Conversion of this type of F1 into that in which all three non-catalytic sites are occupied by adenine nucleotide and all three catalytic sites are empty (Kironde & Cross, 1986) did not alter the interaction of F1 with immobilized ATP. This supports the view that the enzyme is bound through a catalytic site. Furthermore, the ability of ITP and GTP to elute F1 is evidence for the involvement of the catalytic sites in the release process, since these nucleotides do not bind to non-catalytic sites. There are some indications that F1 may bind in two modes. The F1 is not eluted by ATP (or other agents) as a sharp peak in a few fractions. The tailing which occurs might be due to differences in the tightness of binding by different F1 complexes. Moreover, treatment of F1 bound to immobilized ATP with Nbf-Cl gave two types of species on elution with ATP. Part of the F1 could be eluted readily with ATP, but the remainder required pretreatment with DTT to release the reagent from F1 before ATP could elute the enzyme. In the first type of behaviour modification had not obviously affected the ability of F1 to bind to immobilized ATP. There is no reason to believe that this fraction of the enzyme represents unmodified F1, since the conditions chosen for modification (e.g. concentration and time of reaction) are known to be effective with free F1 (Ferguson et al., 1975). Furthermore, Nbf-Cl-treated F1 binds p[NH]ppA effectively (Cross & Nalin, 1982). We have no evidence that Nbf-Cl modifies the same tyrosine residue in the boun d as in the free enzyme. It is possible that the two species detected by differences in elution by ATP might represent enzyme molecules modified at different tyrosine residues by the reagent. In the second type of behaviour, the lack of ability to elute F1 from the immobilized ATP until the reagent has been removed from the enzyme with DTT might be for one of two reasons. Nbf-Cl treatment of F1 bound to ATP might increase the tightness of binding of the enzyme to the nucleotide. However, it is possible that the effect of Nbf-Cl treatment is to block co-operativity between catalytic sites on the enzyme (Ferguson et al.,
Beharry, M. J. Gresser and P. D. Bragg
1975; Lunardi et al., 1979). This would be significant if the mechanism of elution of F1 from the immobilized ATP was for the eluting ATP to bind at a second catalytic site as opposed to competing at the first binding site. This is a reasonable mechanism on the basis of the catalytic cycle of the enzyme suggesting by the work of Boyer, Cross, Penefsky and their co-workers (Grubmeyer & Penefsky, 1981a,b; Boyer et al., 1982; Cross et al., 1982; Gresser et al., 1982; Grubmeyer et al., 1982; O'Neal & Boyer, 1984; Penefsky, 1988). Indirect support for the hypothesis that F1 is eluted by nucleotide interacting at a second site is given by our experiments with agarose-hexane-ADP. F1 bound to this matrix is eluted fully by ATP and ADP, and also by IDP. In contrast with ADP, IDP does not inhibit the hydrolysis of ATP (or ITP) over the concentration range used in our experiments (Pullman et al., 1960). Thus it is unlikely that there is direct competition between IDP and ATP at the site of hydrolysis. Thus the ability of IDP to elute F1 from agarose-hexane-ADP is likely due to interaction at the second site. The mechanism for the elution of F1 from immobilized ATP needs further clarification. This work was supported by grants from the Medical Research Council of Canada (to P. D. B.) and the Natural Sciences and Engineering Research Council of Canada (to M. J. G.).
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