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Allosteric changes in citrate synthase observed by electron microscopy
J. Mol. Bdol. (1969) 43, 346-349
Allosteric Changes in Citrate Synthase observed by Electron Microscopy Considerable interest is at present focused on the nature of allosteric regulatory enzymes and the changes produced in them by their inhibitors and activators. Monod, Wyman t Changeux (1965) h eve proposed a model for such enzymes to account for their observed properties. According to this model, an allosteric enzyme, which is itself a specific association of subunits, can exist reversibly in at least two states differing in protein conformation. One of these states is the active form of the enzyme; another by virtue either of a reduced afllnity for substrate or a reduced catalytic activity, is an inactive form of the enzyme. The equilibrium between these forms may be displaoed by the binding of effecters which exhibit preferential a%nity for one of the forms. In this way, displacement of the equilibrium towards the inactive state is produced by the binding of an inhibitory effector, while an activator would produce a shift to the active state. To our knowledge, the main evidence at present in favour of such a model has been drawn indirectly from kinetic studies or from the results of the application of physicochemical techniques such as optical rotatory dispersion, fluorescence and sedimentation. However, since allosteric enzymes, being aggregates of subunits, possessrelatively high molecular weights, it would seem conceivable that a transition from the active to the inactive state (and vice versa) might be accompanied by conformational changes sufficiently gross to be visible directly in the electron microscope. We present data here which indicate a change of shape of an enzyme produced by its allosteric inhibitor, and the reversal of this change by conditions known to reverse the inhibition. It has been shown by one of us (Weitzman, 1966a,b; Weitzman & Jones, 1968) that oitrate synthase from Gram-negative bacteria is an example of an allosteric regulatory enzyme. This key enzyme of the tricarboxylic acid cycle is specifically and powerfully inhibited by NADH, one of the “products” of the action of the cycle. That this inhibition is of the allosteric type has been shown by the desensitization of the enzyme to NADH without loss of enzymic activity. Such desensitization is produced in the presence of 0.2 M-KC2 or at pH values above 9. Weitzman & Dunmore (manuscript in preparation) have purified the enzyme from Acinetobmter lwofi (> 90% homogeneous in acrylamide gel eleotrophoresis). The purified enzyme is approximately 90% inhibited by 0.6 mM-NADH in buffer of composition 20 mu-Tris, brought to pH 8-O with NasEDTA. In the presence of O-2 M-KC& the inhibition by NADH is completely suppressed. The purified enzyme was prepared for examination in the electron microscope by negative-contrast techniques, using unbuffered 2% uranyl acetate as the contrast medium. Our aim has been to “freeze” the conformation of the enzyme molecules by adsorbing them on to the carbon substrate film. Particular care has therefore been taken to wash the substrate film to remove non-adsorbed molecules, either by dropping over first the appropriate buffer solution, then water, or, more simply by the use of 346
346
A. J.
ROWE
AND
P. D. .I. WEITZMAIC
several drops of the contrast medium. Where necessary, specimens were fixed by holding the moist grid over 2% osmic acid solution for 20 seconds. All preparations were examined in a Siemens Elmiskop lA, using a mean magnification of 43,360, an accelerating voltage of 80 kv, a 200-p condenser aperture and a, 50-p objective aperture. Molecular dimensions were estimated by measuring the projected width of a large number of particles, in a direction normal to an arbitrarily defined direction. This method of measurement, which has been applied to shadowed and negatively contrasted macromolecules (Feinstein & Rowe, 1965; Gibbons & Rowe, 1965), minimizes subjectivity in interpretation. In the Tris-EDTA buffer, citrate synthase molecules appear as nearly symmetrical structures (Plate I(a)). Their mean diameter for this preparation was 98.7&0*6 A (standard error of mean) for 100 particles. A spherical, close-packed sphere of protein (@= O-73) of this diameter would have a molecular weight of 398,000. The real molecular weight may be less than this, since the molecule does not always appear to be close-packed and, since one molecular dimension (height) is unknown, could be oblate in shape. Results from shadowing do indeed suggest this (see below). In the presence of NADH (0.5 mM), the molecules have an enlarged mean diameter (Plate I(c) ). The difference is not readily noticed by the naked eye, but is very apparent upon measurement (Plate I(c), histogram). The mean diameter is now 122*7&1*2 A (loo), a highly significant increase (P < 0~0000002 for this and for all differences here reported, by Student’s t test). There is now also a qualita,tive distinction, in that the enzyme can now only be successfully visualized after fixation, whereas enzyme without NADH can readily be visualized with or without fixation. The use of a fixative does not of itself affect the measured diameters. For example, the following diameters were measured on six different specimens of plain enzyme, negatively contrasted with many1 acetate after fixation: 98*3-&1-O A (50); 96.1+0*9 A (50); 96.9f1.3 b (50); 97.9&O-9 A (50); 98.2kO.8 ii (50); 97.3*1-O A (50). As described above, neutral salt relieves the inhibition by NADH. In the electron microscope, the effect of addition of 0.2 ~-Kc1 is to decrease the mean diameter to a value of 109*8*0*5 A (100). Fixation is now no longer a necessary pre-condition for visualization. This reduced value is identical with that found in the presence of O-2 ~-Kc1 alone, 109*3&0*5 A (loo), see Plate I(b) and (d), but is larger than that of enzyme in Tris-EDTA solution. For another preparation of enzyme, the following mean diameters were estimated: enzyme in Tris-EDTA, 97*1&0*4 A (227); enzyme in O-5 m&r-NADH, 12O*ljl*O A (123). The specificity of NADH action previously observed by kinetic studies appears to be borne out by electron microscopy: neither NAD nor NADPH affects the molecular diameter of the enzyme. In very recent work, we have found that the use of ammonium molybdate (pH 6.6) as the negative contrast medium enables the enzyme to be seen without fixation, even in the presence of NADH. For eight specimens from a new preparation of enzyme, mean projected widths measured as 98.4fl.O A (50); 99.5f0.8 A (50); lOO~S&l~O A (50); 99.7fl.l A (50) (plain enzyme); 116*8&-1*4A (50); 117*0*1*2 d (50); 1195f1.5 A (50); 116*2*1*4 A (50) (enzyme+1 m&r-NADH). These values agree adequately with the earlier results. Close examination of the enzyme molecules in ammonium molybdate reveals a minority of molecules (- 10%) showing a definite S-fold symmetry. The over-all
NADH
(iv)
(iii)
5
PLATE III. Pictures obtained by, super-imposition of micrographs of enzyme molecules, ilk ,gwups of eight.. The groups correspond to the rows in Plate II. .A film negative corresponding exart,ly with Plate II was prepared, and t,he individual molecules were cut out and super-imposed to give compound negatives. The apparent over-all diameters seen in the final print depend someirhat on thr printing exposure, but the rhnnges seen in the presence of SADH and referred t,o irl tkw text are not dependent upw~ photographic conditiorls. “: l,OOO,OOO.
(ii)
(i)
I
0)
(ii)
(iii)
PLATE IV. Electron micrographs of shadow-cast molecules. (i) Plain enzyme, x 130,000. (ii) Enzyme + 1 mM-NADH + 0.5 mnr-AMP, x 430,000. (iii) Enz,yme + 1 mn~-NADH. x 430,000.
LETTERS
TO
THE
EDITOR
347
dimensions of these molecules can be shown to be within the normal range of the population, and analysis on gel electrophoresis does not suggest the presence of EO appreciable an impurity. It may be, therefore, that t’hese are indeed typical enzyme molecules, in which either the orientation or the degree of penetration is favourable for visualization of substructure. Plate II illustrates 32 such molecules, chosen at random by an independent party, from fields with and without NADH. The centre-to-centre separation of the three substructures was measured, and averaged (mean of 3) for each molecule. A clear difference is seen in the substructural separation: plain enzyme, 51.7k3.4 d; enzyme+NADH 61&3*5 A (standard deviation given). This difference is consistent with the observed changes in gross conformation. The scatter is only marginally too large to enable individual molecules to be classified as “large” or “small”. Superimposition of images (eight at a time) makes the change readily apparent to the eye (Plate III). In the presence of NADH, the substructures move apart, allowing penetration of electron-dense salt towards the “centre”. It should be said that the visualization of S-fold symmetry does not necessarily imply the presence of three physical subunits. For example, a tetrahedron of four spherical subunits could easily give such an appearance in a minority of cases. If each subunit is taken to have a diameter of 51 8, corresponding to a molecular weigh M = 58,000 (fi = O-73), then the possible number of subunits is limited. We do not yet have sufficient enzyme to determine M for the enzyme exactly, but the presence of four subunits, yielding M = 232,000, would be consistent with the value estimated from the shadowing procedure (see below). These results from the negative-contrast technique led us to consider using metalshadowing as an alternative way of visualizing this change in conformation. Although the presence of relatively large amounts of non-volatile salts in preparations to be metal-shadowed is undesirable in principle, and proved a moderate nuisance in practice, nonetheless very satisfactory pictures were obtained (Plate IV), both with a,nd without NADH. Preparations were also examined in the presence of NADH and AMP, since it has been shown that the inhibition by NADH of the citrate synthase of A. lwofi is specifically overcome by low concentrations of AMP (Weitzman & Jones, 1968; Weitzman, manuscript in preparation). The micrographs were measured and interpreted by a method to be described in detail elsewhere (Rowe & Rowe, manuscript in preparation). Figure 1 summarizes the shadow-width measurements obtained under the three conditions. The correction for metal cap has not been applied to these measurements. The general similarity to the negative-contrast results is obvious. The values inferred for the principal axes of the molecule in the plane of the substrate are as follows: plain enzyme, 107 x 107 A; enzyme+NADH, 96 x 137 A; cnzyme+NADH+AMP, 105 x 105 b. The molecular height, calculated from shadow lengths, showed a considerable degree of scatter in all cases, possibly due to the presence of salt on the substrate. Excluding those data for which salt contamination was grossly apparent, the heights did not significantly differ, and were: plain enzyme, 46 A; enzyme+NADH, 49 A; enzyme+NADH+AMP, 51 A. For an assumed 8 = 0.73, these dimensions correspond to molecular weights of 223,000, 277,000 and 242,000, respectively. These values are consistent with the elution behaviour of the enzyme on Sephadex gels (Weitzman & Dunmore, 1969). 23
348
A. J. ROWE
AND
P. D. J. WEITZMAN
Enzyme
(ii) Enzymet NADH
4
(iii) Enzymet NADH + AMP
50
125 FIG. 1. Histograms showing The widths are not, corrected -46 A (ii).
150
175
200
distribution of shadow widths for metal csp: the correction
with and without effecters present. amounts to -36 A ((i) and (iii)),
It is clear that, within experimental error, these results are in excellent accord with those obtained by the negative contrast technique. Further confirmation of the reality of the change of shape has been obtained from preliminary experiments in the analytical ultracentrifuge. In the presence of 1 ITLMNADH, a retardation of 7% was observed in the sedimentation rate, a,s compared with the control (1 mM-NADPH present). No such rekdation was observed in the additional presence of O-5 mM-AMP. More detailed studies are continuing on this and other aspects of the system. It is useful to consider, at this stage, the exact null hypotheses which our electron microscope data appear to contradict. The$mt null hypothesis is that all the molecules measured, under whatever conditions, constitute a random population. Very adequate data are available to support the rejection of this hypothesis. The second null hypothmis accepts that the molecules in a given preparation may differ in apparent width from those in other preparations, but attributes this to uncontrolled effects occurring during drying down. In other words, there is a random population offields of molecules, from which we may select fields of Yarge” or “small” molecules. The chances that a fortuitous correlation with the presence or absence of NADH would be obtained will be 1 in 2”, where 7~is equal to the number of specimen preparations examined (assuming “large” and “small” to be equally probable). The data quoted in this communication refer to 32 non-selected specimen preparations (23 negative-contrast, 9 shadowed), all of which could unambiguously be classified as “large” or “small”, all in a manner
LETTERS
TO
THE
349
EDITOR
consistent with the non-null hypothesis. Thus for n = 32, the null hypothesis may be rejected. The third null hypothesis is that although the altered conformation is correlated with the presence of NADH during drying down, it is not directly related to an effect of NADH in free solution; i.e. in the presence of NADH, the enzyme becomes deformable, but not actually deformed. Against this we can only urge the sedimentation evidence, which does strongly imply a significant shape change in solution. The degree of confidence which we have in this last case cannot readily, however, be quantified. We can state, however, with a considerable degree of confidence, that our results show that citrate synthase undergoes reversible changes in molecular conformation, in particular in the presence of the allosteric effeotors NADH and AMP. This clearly supports current views of the importance of conformational changes in regulatory proteins. We thank Mr J. Baker, Mm D. Duncan and Mm P. Dunmore for expert technical assistance, and M.S.E. Ltd. for kind assistance with facilities. This work was supported by Science Research Council grants B/SR/3376 and B/SR/652. Department of Biochemistry Adrian School of Biology University of Leicester L&ester, England Received
A. J. ROWE P. D. J. WEITZMAN
13 March 1967, and in revised form 8 May 1969
REFERENCES Feinstein, A. & Rowe, A. J. (1965). Nature, 205, 147. Gibbons, I. R. & Rowe, A. J. (1965). Science, 149, 424. Monod, J., Wyman, J. & Changeux, J. P. (1966). J. Mol. Biol. 12. 88. Weiteman, P. D. J. (1966a). Biochim. biophya. Acta, 128, 213. Weitzman, P. D. J. (1966b). B&hem. J. 101, 44~. Weitzman, P. D. J. BEDunmore, P. (1969). B&him. biophye. Acta, 171, 198. Weitzman, P. D. J. AZJones, D. (1968). Nature, 219, 270.
Allosteric Changes in Citrate Synthase observed by Electron Microscopy Considerable interest is at present focused on the nature of allosteric regulatory enzymes and the changes produced in them by their inhibitors and activators. Monod, Wyman t Changeux (1965) h eve proposed a model for such enzymes to account for their observed properties. According to this model, an allosteric enzyme, which is itself a specific association of subunits, can exist reversibly in at least two states differing in protein conformation. One of these states is the active form of the enzyme; another by virtue either of a reduced afllnity for substrate or a reduced catalytic activity, is an inactive form of the enzyme. The equilibrium between these forms may be displaoed by the binding of effecters which exhibit preferential a%nity for one of the forms. In this way, displacement of the equilibrium towards the inactive state is produced by the binding of an inhibitory effector, while an activator would produce a shift to the active state. To our knowledge, the main evidence at present in favour of such a model has been drawn indirectly from kinetic studies or from the results of the application of physicochemical techniques such as optical rotatory dispersion, fluorescence and sedimentation. However, since allosteric enzymes, being aggregates of subunits, possessrelatively high molecular weights, it would seem conceivable that a transition from the active to the inactive state (and vice versa) might be accompanied by conformational changes sufficiently gross to be visible directly in the electron microscope. We present data here which indicate a change of shape of an enzyme produced by its allosteric inhibitor, and the reversal of this change by conditions known to reverse the inhibition. It has been shown by one of us (Weitzman, 1966a,b; Weitzman & Jones, 1968) that oitrate synthase from Gram-negative bacteria is an example of an allosteric regulatory enzyme. This key enzyme of the tricarboxylic acid cycle is specifically and powerfully inhibited by NADH, one of the “products” of the action of the cycle. That this inhibition is of the allosteric type has been shown by the desensitization of the enzyme to NADH without loss of enzymic activity. Such desensitization is produced in the presence of 0.2 M-KC2 or at pH values above 9. Weitzman & Dunmore (manuscript in preparation) have purified the enzyme from Acinetobmter lwofi (> 90% homogeneous in acrylamide gel eleotrophoresis). The purified enzyme is approximately 90% inhibited by 0.6 mM-NADH in buffer of composition 20 mu-Tris, brought to pH 8-O with NasEDTA. In the presence of O-2 M-KC& the inhibition by NADH is completely suppressed. The purified enzyme was prepared for examination in the electron microscope by negative-contrast techniques, using unbuffered 2% uranyl acetate as the contrast medium. Our aim has been to “freeze” the conformation of the enzyme molecules by adsorbing them on to the carbon substrate film. Particular care has therefore been taken to wash the substrate film to remove non-adsorbed molecules, either by dropping over first the appropriate buffer solution, then water, or, more simply by the use of 346
346
A. J.
ROWE
AND
P. D. .I. WEITZMAIC
several drops of the contrast medium. Where necessary, specimens were fixed by holding the moist grid over 2% osmic acid solution for 20 seconds. All preparations were examined in a Siemens Elmiskop lA, using a mean magnification of 43,360, an accelerating voltage of 80 kv, a 200-p condenser aperture and a, 50-p objective aperture. Molecular dimensions were estimated by measuring the projected width of a large number of particles, in a direction normal to an arbitrarily defined direction. This method of measurement, which has been applied to shadowed and negatively contrasted macromolecules (Feinstein & Rowe, 1965; Gibbons & Rowe, 1965), minimizes subjectivity in interpretation. In the Tris-EDTA buffer, citrate synthase molecules appear as nearly symmetrical structures (Plate I(a)). Their mean diameter for this preparation was 98.7&0*6 A (standard error of mean) for 100 particles. A spherical, close-packed sphere of protein (@= O-73) of this diameter would have a molecular weight of 398,000. The real molecular weight may be less than this, since the molecule does not always appear to be close-packed and, since one molecular dimension (height) is unknown, could be oblate in shape. Results from shadowing do indeed suggest this (see below). In the presence of NADH (0.5 mM), the molecules have an enlarged mean diameter (Plate I(c) ). The difference is not readily noticed by the naked eye, but is very apparent upon measurement (Plate I(c), histogram). The mean diameter is now 122*7&1*2 A (loo), a highly significant increase (P < 0~0000002 for this and for all differences here reported, by Student’s t test). There is now also a qualita,tive distinction, in that the enzyme can now only be successfully visualized after fixation, whereas enzyme without NADH can readily be visualized with or without fixation. The use of a fixative does not of itself affect the measured diameters. For example, the following diameters were measured on six different specimens of plain enzyme, negatively contrasted with many1 acetate after fixation: 98*3-&1-O A (50); 96.1+0*9 A (50); 96.9f1.3 b (50); 97.9&O-9 A (50); 98.2kO.8 ii (50); 97.3*1-O A (50). As described above, neutral salt relieves the inhibition by NADH. In the electron microscope, the effect of addition of 0.2 ~-Kc1 is to decrease the mean diameter to a value of 109*8*0*5 A (100). Fixation is now no longer a necessary pre-condition for visualization. This reduced value is identical with that found in the presence of O-2 ~-Kc1 alone, 109*3&0*5 A (loo), see Plate I(b) and (d), but is larger than that of enzyme in Tris-EDTA solution. For another preparation of enzyme, the following mean diameters were estimated: enzyme in Tris-EDTA, 97*1&0*4 A (227); enzyme in O-5 m&r-NADH, 12O*ljl*O A (123). The specificity of NADH action previously observed by kinetic studies appears to be borne out by electron microscopy: neither NAD nor NADPH affects the molecular diameter of the enzyme. In very recent work, we have found that the use of ammonium molybdate (pH 6.6) as the negative contrast medium enables the enzyme to be seen without fixation, even in the presence of NADH. For eight specimens from a new preparation of enzyme, mean projected widths measured as 98.4fl.O A (50); 99.5f0.8 A (50); lOO~S&l~O A (50); 99.7fl.l A (50) (plain enzyme); 116*8&-1*4A (50); 117*0*1*2 d (50); 1195f1.5 A (50); 116*2*1*4 A (50) (enzyme+1 m&r-NADH). These values agree adequately with the earlier results. Close examination of the enzyme molecules in ammonium molybdate reveals a minority of molecules (- 10%) showing a definite S-fold symmetry. The over-all
NADH
(iv)
(iii)
5
PLATE III. Pictures obtained by, super-imposition of micrographs of enzyme molecules, ilk ,gwups of eight.. The groups correspond to the rows in Plate II. .A film negative corresponding exart,ly with Plate II was prepared, and t,he individual molecules were cut out and super-imposed to give compound negatives. The apparent over-all diameters seen in the final print depend someirhat on thr printing exposure, but the rhnnges seen in the presence of SADH and referred t,o irl tkw text are not dependent upw~ photographic conditiorls. “: l,OOO,OOO.
(ii)
(i)
I
0)
(ii)
(iii)
PLATE IV. Electron micrographs of shadow-cast molecules. (i) Plain enzyme, x 130,000. (ii) Enzyme + 1 mM-NADH + 0.5 mnr-AMP, x 430,000. (iii) Enz,yme + 1 mn~-NADH. x 430,000.
LETTERS
TO
THE
EDITOR
347
dimensions of these molecules can be shown to be within the normal range of the population, and analysis on gel electrophoresis does not suggest the presence of EO appreciable an impurity. It may be, therefore, that t’hese are indeed typical enzyme molecules, in which either the orientation or the degree of penetration is favourable for visualization of substructure. Plate II illustrates 32 such molecules, chosen at random by an independent party, from fields with and without NADH. The centre-to-centre separation of the three substructures was measured, and averaged (mean of 3) for each molecule. A clear difference is seen in the substructural separation: plain enzyme, 51.7k3.4 d; enzyme+NADH 61&3*5 A (standard deviation given). This difference is consistent with the observed changes in gross conformation. The scatter is only marginally too large to enable individual molecules to be classified as “large” or “small”. Superimposition of images (eight at a time) makes the change readily apparent to the eye (Plate III). In the presence of NADH, the substructures move apart, allowing penetration of electron-dense salt towards the “centre”. It should be said that the visualization of S-fold symmetry does not necessarily imply the presence of three physical subunits. For example, a tetrahedron of four spherical subunits could easily give such an appearance in a minority of cases. If each subunit is taken to have a diameter of 51 8, corresponding to a molecular weigh M = 58,000 (fi = O-73), then the possible number of subunits is limited. We do not yet have sufficient enzyme to determine M for the enzyme exactly, but the presence of four subunits, yielding M = 232,000, would be consistent with the value estimated from the shadowing procedure (see below). These results from the negative-contrast technique led us to consider using metalshadowing as an alternative way of visualizing this change in conformation. Although the presence of relatively large amounts of non-volatile salts in preparations to be metal-shadowed is undesirable in principle, and proved a moderate nuisance in practice, nonetheless very satisfactory pictures were obtained (Plate IV), both with a,nd without NADH. Preparations were also examined in the presence of NADH and AMP, since it has been shown that the inhibition by NADH of the citrate synthase of A. lwofi is specifically overcome by low concentrations of AMP (Weitzman & Jones, 1968; Weitzman, manuscript in preparation). The micrographs were measured and interpreted by a method to be described in detail elsewhere (Rowe & Rowe, manuscript in preparation). Figure 1 summarizes the shadow-width measurements obtained under the three conditions. The correction for metal cap has not been applied to these measurements. The general similarity to the negative-contrast results is obvious. The values inferred for the principal axes of the molecule in the plane of the substrate are as follows: plain enzyme, 107 x 107 A; enzyme+NADH, 96 x 137 A; cnzyme+NADH+AMP, 105 x 105 b. The molecular height, calculated from shadow lengths, showed a considerable degree of scatter in all cases, possibly due to the presence of salt on the substrate. Excluding those data for which salt contamination was grossly apparent, the heights did not significantly differ, and were: plain enzyme, 46 A; enzyme+NADH, 49 A; enzyme+NADH+AMP, 51 A. For an assumed 8 = 0.73, these dimensions correspond to molecular weights of 223,000, 277,000 and 242,000, respectively. These values are consistent with the elution behaviour of the enzyme on Sephadex gels (Weitzman & Dunmore, 1969). 23
348
A. J. ROWE
AND
P. D. J. WEITZMAN
Enzyme
(ii) Enzymet NADH
4
(iii) Enzymet NADH + AMP
50
125 FIG. 1. Histograms showing The widths are not, corrected -46 A (ii).
150
175
200
distribution of shadow widths for metal csp: the correction
with and without effecters present. amounts to -36 A ((i) and (iii)),
It is clear that, within experimental error, these results are in excellent accord with those obtained by the negative contrast technique. Further confirmation of the reality of the change of shape has been obtained from preliminary experiments in the analytical ultracentrifuge. In the presence of 1 ITLMNADH, a retardation of 7% was observed in the sedimentation rate, a,s compared with the control (1 mM-NADPH present). No such rekdation was observed in the additional presence of O-5 mM-AMP. More detailed studies are continuing on this and other aspects of the system. It is useful to consider, at this stage, the exact null hypotheses which our electron microscope data appear to contradict. The$mt null hypothesis is that all the molecules measured, under whatever conditions, constitute a random population. Very adequate data are available to support the rejection of this hypothesis. The second null hypothmis accepts that the molecules in a given preparation may differ in apparent width from those in other preparations, but attributes this to uncontrolled effects occurring during drying down. In other words, there is a random population offields of molecules, from which we may select fields of Yarge” or “small” molecules. The chances that a fortuitous correlation with the presence or absence of NADH would be obtained will be 1 in 2”, where 7~is equal to the number of specimen preparations examined (assuming “large” and “small” to be equally probable). The data quoted in this communication refer to 32 non-selected specimen preparations (23 negative-contrast, 9 shadowed), all of which could unambiguously be classified as “large” or “small”, all in a manner
LETTERS
TO
THE
349
EDITOR
consistent with the non-null hypothesis. Thus for n = 32, the null hypothesis may be rejected. The third null hypothesis is that although the altered conformation is correlated with the presence of NADH during drying down, it is not directly related to an effect of NADH in free solution; i.e. in the presence of NADH, the enzyme becomes deformable, but not actually deformed. Against this we can only urge the sedimentation evidence, which does strongly imply a significant shape change in solution. The degree of confidence which we have in this last case cannot readily, however, be quantified. We can state, however, with a considerable degree of confidence, that our results show that citrate synthase undergoes reversible changes in molecular conformation, in particular in the presence of the allosteric effeotors NADH and AMP. This clearly supports current views of the importance of conformational changes in regulatory proteins. We thank Mr J. Baker, Mm D. Duncan and Mm P. Dunmore for expert technical assistance, and M.S.E. Ltd. for kind assistance with facilities. This work was supported by Science Research Council grants B/SR/3376 and B/SR/652. Department of Biochemistry Adrian School of Biology University of Leicester L&ester, England Received
A. J. ROWE P. D. J. WEITZMAN
13 March 1967, and in revised form 8 May 1969
REFERENCES Feinstein, A. & Rowe, A. J. (1965). Nature, 205, 147. Gibbons, I. R. & Rowe, A. J. (1965). Science, 149, 424. Monod, J., Wyman, J. & Changeux, J. P. (1966). J. Mol. Biol. 12. 88. Weiteman, P. D. J. (1966a). Biochim. biophya. Acta, 128, 213. Weitzman, P. D. J. (1966b). B&hem. J. 101, 44~. Weitzman, P. D. J. BEDunmore, P. (1969). B&him. biophye. Acta, 171, 198. Weitzman, P. D. J. AZJones, D. (1968). Nature, 219, 270.