ARCHIVES
OF BIOCHEMISTRY
Investigation
AND
BIOPHYSICS
of the Role of Lysine in the Subunit Regions
GREGORY Department
of Chemistry
168, 525-530 (1975)
of Rabbit
Muscle
C. SAUNDERS
and Institute
AND
Aldolase’
BRUCE
of Molecular Biology, California Fullerton, California 92634
Received October
Contact
H. WEBER* State
Uniuersity,
Fullerton,
11, 1974
Rabbit muscle aldolase (E.C. 4. 1. 2. 13) was guanidinated by reaction with Omethylisourea. Up to 60% of the lysine residues can be guanidinated without any dissociation of the tetramer but with a complete loss of enzymatic activity. Native and guanidinated aldolase can be dissociated into monomers in 2.4 M MgCl, with only slight change in conformation of the subunit. Nitrotroponylation of guanidinated aldolase in dilute buffer gives no reaction whereas in 2.4 M MgCl, nitrotroponlylation modifies another 8-12% of the lysine residues. Removal of MgCl, by dialysis affords 100% recovery of activity and tetrameric structure for native aldolase and 100% recovery of tetrameric structure for guanidinated aldolase. In contrast nitrotroponylated and guanidinated aldolase remains monomeric before precipitating as the MgCl, concentration is lowered. It is concluded that lysine may be involved in the protein-protein interaction of the subunit contact domains of muscle aldolase.
equilibrium can be obtained in 1.2 M MgCl, (6); increasing the magnesium chloride concentration to 2.4 M produces monomers of aldolase in a near native conformation (10). These facts suggest that it should be possible to chemically label residues that are in the subunit contact domains of aldolase since surface residues can be blocked on the stable tetramer and then by dissociation in magnesium chloride “cryptic” residues can be exposed that were involved in the subunit contacts. In this paper experiments are described in which the role of one amino acid, lysine, in subunit interactions of aldolase is studied by guanidinating surface lysines followed by nitrotroponylating “cryptic” lysines in 2.4 M MgCl,.
Aldolase (E.C. 4. 1. 2. 13) from rabbit muscle has btien demonstrated to consist of four subunits (1) that are chemically identical except for the deamination of a single asparagine in two of the subunits (2, 3). Crystallographically the subunits appear identical and are arrayed tetrahedrally (4, 5). Hsu and Neet (6) have demonstrated that the interactions between aldolase subunits are isologous. Thus there are three contact domains between each subunit and the other subunits (7) but these appear to be of unequal strength as the assembly from monomer appears to occur as a dimerization of dimers (6, 8). The formation of tetramer is irreversible under the usual conditions of reassociation (8, 9) and there is no evidence of a monomer = tetramer equilibrium under normal conditions. This ’ Supported by a grant from the National Institute of Health (AM-15055. B. H. Weber, principal investigator). Taken in part from the Thesis of G. C. Saunders submitted in partial fulfillment of a master’s degree, California State University, Fullerton. 2 To whom inquiries and reprint requests should be addressed.
MATERIALS
All
rights
0 1975 by Academic Press. of reproduction in any form
Inc. reserved.
METHODS
Materials Rabbit muscle aldolase was obtained as a crystalline suspension in 2.0 M ammonium sulfate, 0.001 M EDTA, pH approximately 6, and fructose-l, 6.diphosphate, NADH, glycerol-3-phosphate (E.C.l.l.l,8)/tri-
525 Copyright
AND
526
SAUNDERS
osephosphate isomerase (E.C.5.3.1.1) crystalline suspension were purchased from Boehringer-Mannheim Corporation. 0-Methylisourea was obtained from Aldrich Chemical Co.; 2-methoxy-5-nitrotropone was obtained from Calbiochem; and magnesium chloride was obtained from Fischer Chemical. All other chemicals were of analytical grade. All water used was double distilled and dionized.
Methods Enzyme preparation. Crystalline aldolase was desalted at 4°C by either conventional or pressure dialysis against the desired buffer. Following dialysis the protein solution if necessary was clarified by centrifugation. Dialyses following chemical modification were performed in a like manner. Protein determination. Aldolase concentration was determined spectrophotometrically using an extinction coefficient e :L (280 nm) = 9.1 (11). Protein concentrations were adjusted to the desired level by dilution with the appropriate buffer or by concentration by ultrafiltration. Enzyme assay. Aldolase activity was determined by the method of Blostein and Rutter (12) using a Beckman Acta III recording spectrophotometer. Assays were performed at 30°C in 0.1 M triethanol amine buffer pH 7.9 with a final volume of 1 ml. The initial concentrations of fructose-l, 6-diphosphate and NADH were 0.51 M and 0.19 mM, respectively. The mixture of coupling enzymes was present at a concentration 7 pg/ml. The specific activity of the aldolase used in these studies was 9913 units/mg. Magnesium chloride dissociation-reassociation. The dissociation of native or modified aldolase was performed at room temperature in 2.4 M MgCl, (millipore filtered 4.0 M MgCl, stock solutions were used) in 0.2 M Tris-Cl buffer pH 8.2 at a protein concentration of 0.4 mg/ml. Reassociation was achieved by dialysis at room temperature against 0.2 M Tris-Cl pH 8.2. Guanidination of native aldolase. An equal volume of freshly prepared 0.6 M 0-methylisourea, pH 9.8, was combined with aldolase (1.0 mg/ml) in 0.1 M phosphate buffer pH 6.9 at 30°C. Aliquots were removed at timed intervals and dialyzed against 0.1 M phosphate pH 6.9. Quantitation was .achieved by amino acid analysis by monitoring the disappearance of lysine and the concomitant appearance of homoarginine. Protein concentration was determined from the yields of histidine and arginine. Nitrotroponlylntion of guanidinated aldolase. To solutions of guanidinated aldolase (1.0-10.0 mg/ml), pH 8.2, 0.1 M Tris-Cl, was added 6.5% by volume of 30.8 mM solution of 2-methoxy-5-nitrotropone in dimethylformamide. The reaction was allowed to proceed for two hours at 20°C and was then subjected to gel-filtration. The absorbance at 280 nm and 420 nm was checked for the protein fraction to see if there had been any modification due to nitrotroponylation.
AND WEBER Nitrotroponylation of dissociated guanidinated aldolase. To a solution of guanidinated aldolase (0.4 mg/ml) in 2.4 M MgCl, pH 8.2, 0.2 M Tris-Cl, was added 6.5% by volume of 30.8 M solution of 2methoxy-5-nitrotropone in dimethylformamide. The reaction was carried out at 20°C with aliquots removed at time intervals for dialysis against 0.2 M Tris-Cl pH 8.2. Quantitation was achieved spectrophotometrically using an extinction coefficient of c,+, (420 nm) = 20,700 (13). Protein concentration was determined using t :L (277 nm) = 8.32 for dissociated aldolase (14). Amino acid analysis. Analyses were performed by the method of Spackman et al. (15) and Moore and Stein (16) using an accelerated system (17) employing Beckman model 120C amino acid analyzer. Sedimentation velocity studies. Sedimentation coefficients for native, dissociated, reassociated and the modified aldolases were determined using a Spinco Model E analytical ultracentrifuge using schlieren or ultraviolet optics depending on the protein concentration at a rotor speed of 60,000 rpm. The observed sedimentation coefficients were corrected for solvent density and temperature. Spectrofluorometry. Fluorescence measurements were performed at 20°C with an Aminco-Bowman spectrophotofluorimeter with a ratio recorder with an excitation wavelength of 282 nm. The emission spectra were examined in the region of tryptophan emission for evidence of possible conformational changes. Optical rotatory Dispersion. Optical rotatory dispersion measurements for native and modified aldolase were performed on a Cary 61 spectropolarimeter, at a protein concentration of 0.4 mg/ml, over the region of 300-220 nm. Myoglobin was used as a standard for calibration of the instrument. RESULTS
Guanidination of Aldolase In order to determine the optimal conditions for the guanidination of aldolase preliminary sedimentation and enzyme activity experiments were performed to ascertain the pH and temperature stability of native aldolase over the pH range of 8.5-11.0 and the temperature range of 4-50°C. These studies indicate that at pH 10.0 at 30°C the aldolase tetramer is stable and fully active for periods of at least twelve hours. Our results agree with those recently reported by Gibbons and Perham (18). Under these conditions up to 60 lysine residues react with 0-methylisourea within 12 h (Fig. 1) and there is complete loss of activity (Fig. 2) indicating that at least one and possibly both the lysines known to be
ROLE OF LYSINE
IN ALDOLASE
SUBUNIT
CONTACTS
527
rotatory dispersion spectrum of both native and modified aldolase are very similar with WI 233= -5606 for native aldolase and [m’]233 = -5278 for guanidinated aldolase. This difference is slightly larger than experimental error. This suggests that a slight conformational change may have occured as a result of guanidination of 60 lysine residues per tetramer. Magnesium Chloride Dissociation-Reassociation Studies
The sedimentation velocity and spectrofluorescence results for native and guanidinated aldolase are presented in Table I. Both the native and guanidinated aldolases appear to exist as monomers in 2.4 MgCl, as judged by the sedimentation coefficient of s20,w= 2.5-2.6, which is close to that reported by Hsu and Neet (6, 10) for aldolase monomers in MgCl, and to those values seen for aldolase subunits in acid or alkaline solutions (20, 21). The spectrofluorescence data show that in 2.4 M MgCl, there is a slight red shift of 3 nm suggesting at most a slight conformational change, consistent with the observations of Hsu and Neet (6, lo), and not a major unfolding of the monomer as happens in other methods of dissociation. Removal of magnesium chloride by dialysis provides reassociation to the tetrameric structure and a blue shift FIG. 2. Activity time course for the guanidination of back to the native fluorescence emission aldolase. See text for details. maximum. Assays of the reconstituted native aldolase showed 100% recovery of acin the active site (19) have also reacted since there appears to be little or no tivity. conformational change upon guanidination Nitrotroponylation of Guanidinated (see below). Attempts to react even more Aldolase lysines lead to denaturation and precipitation of the protein. This suggests that 15 When guanidinated tetrameric aldolase out of the 26 lysines per subunit are “ex- was exposed to 2-methoxy-5-nitrotropone, posed” enough to react with O- and the excess reagent removed by gel methylisourea, and that up to 11 lysines filtration, there was no protein precipitaper subunit may be “buried” inside the tion observed and no incorporation of nimonomer or be in the binding domains trotroponyl moieties as judged by spectrobetween the subunits. The guanidinated photometric measurements at 420 nm. aldolase has a sedimentation coefficient of In contrast, dissociation of the fully guas20.w= 7.61 which is virtually identical to nidinated aldolase in 2.4 M MgCl, followed that obtained for native aldolase, sZO.w = by nitrotroponylation of the newly exposed 7.60. Examination of the fluorescence em- “cryptic” lysine residues does lead to modmission spectrum shows no alteration with ification of additional lysine residues x max (emission) = 327 nm for both native (Table II). Quantitation of the extent of and guanidinated aldolase. The optical nitrotroponylation and characterization of
FIG. 1. Stoichiometry time course for the guanidination of aldolase; (-A-l represents moles of homoarginine per mole of aldolase and (-W-) represents moles of residual lysine per mole of aldolase. See text for details.
528
SAUNDERS AND WEBER TABLE I
SEDIMENTATION-VELOCITY AND SPECTROFLLJORESCENCE ANALYSIS OF DISSOCIATEDAND REASSOCIATEDALDOLASE FOR THE NATIVE AND GUANIDINATED ENZYME
Sample
Medium”
sz0.w
x maxof emission (nm)
Native aldolase Dissociated native aldolase Reassociated native aldolase Guanidinated aldolase Dissociated guanidinated aldolase Reassociated guanidinated aldolase
0.2 M Tris-Cl 2.4 M MgCl, 0.2 M Tris-Cl 0.2 M Tris-Cl 2.4 M MgCl,
7.60 2.50 7.56 7.61 2.63
327 330 327 327 330
0.2 M Tris-Cl
7.67
326
DFor all samples pH = 8.2. TABLE II EXAMPLE OF QUANTITATION OF NITROTROPONYLATION OF GUANIDINATED ALDOLASE FOR Two INDEPENDENT MODIFICATIONS UNDER SIMILAR REACTION CONDITIONS’
Control Aldolase concentration (mg/ml) Absorbance at 420nm (corrected) Moles of nitrotroponylhetramer Moles of nitrotroponyl/monomer
0.32 0.000 0 0
Modification 1 0.94 0.897 7.36 1.84
Modification 2 0.52 0.686 10.18 2.55
DSee text for details.
the doubly modified aldolase was performed in 1.2 M MgCl, as attempts to completely remove magnesium chloride result in complete precipitation of the protein. It appears that 2-3 mol of lysine can be modified by 2-methoxy-5-nitrotropone per mole of dissociated guanidinated monomer. Sedimentation velocity analysis of the nitrotroponylated guanidinated aldolase in 1.2 M MgCl, gives a sedimentation coefficient of s2,,+. = 2.31 indicating that the modification of the “cryptic” lysines prevents reassociation to the dimeric form which Hsu and Neet (6) have demonstrated predominates under these conditions.
electrostatic repulsion and a conformational change. Meighen and Schachman (22) found that attempts to succinylate more than 40% of the lysines of aldolase produced dissociation, presumably through a conformational change. Gibbons and Perham (23) observed that dissociation begins to occur for aldolase when about 50% of the lysines of aldolase are citraconylated. In contrast guanidination allows the modification of 60% of the lysines of aldolase without any dissociation and with little, if any, conformational change in the subunits of the tetramer. Further, the guanidinated aldolase behaves identically to native aldolase in the dissociation-reassociation experiments. DISCUSSION Initially the dissociation-reassociation Guanidination permits modification of experiments were performed in 4 M urea, lysine residues while preserving the posi- which allowed 100% recovery of aldolase tion charge in contrast to succinylation and activity upon urea removal. However, opticitraconylation which modify lysines while cal rotatory dispersion and spectrofluorescence analysis of the dissociated monomers converting the positive charge to a negative charge. The conversion of a number of showed that they had undergone a significhange in 4 M urea positive charges to negative on the surface cant conformational of a protein would be expected to lead to and behaved as if they were “randon coils”
ROLE
OF LYSINE
IN ALDOLASE
(Saunders and Weber, unpublished results). The studies of Hsu and Neet (6, 10) suggested an alternative method of dissociation employing MgCl, that appeared to produce aldolase monomers of near native conformation. In the present study we have confirmed their results and observe that 2.4 M MgCl, does form monomers of aldolase that by criterion of spectrofluorescence appear to be very similar to the native conformation of the subunit in the tetramer. Since the guanidinated aldolase behaves identically to the native aldolase in these experiments, it is possible to expose and modify, in 2.4 M MgCl,, lysines that may be involved in subunit-subunit interactions with the majority if not all of the surface lysines blocked from further chemical modification. Nitrotroponylation of the dissociated monomers of guanidinated aldolase does modify two to three moles of lysine per monomer that presumably were “buried” between the subunits in the tetramer. As there are 11 lysines per subunit that are not guanidinated when the tetramer is modified, nor are reactive with 2-methoxy-5nitrotropone for intact guanidinated tetramers, the observation of nitrotroponylation of only up to 3 mol of lysine may mean that the other lysines are buried inside the monomer, that they remain unreactive in 2.4 M MgCl, or that it could be that under the conditions employed only 30% reaction was achieved with all 11 “cryptic” lysines. Nor can it be determined at present how many of these 3-11 lysines are “essential” for the subunit-subunit interactions. Additionally, some of the cryptic lysines may be surface residues that are unreactive due to their microenvironment and that the slight conformational change observed in 2.4 M MgCl, may have allowed these residues to react with 2-methoxy-5-nitrotropone. The fact that the dissociated nitrotroponylated and guanidinated aldolase does not appear to form dimers in 1.2 M MgCl, whereas dissociated guanidinated aldolase does suggests that modification of cryptic lysines has blocked the reassociation either through a direct steric interference or by a changing in conformation concomitant with the nitrotroponylation. In either event, one or more of the cryptic
SUBUNIT
529
CONTACTS
lysines per monomer seems to be essential for assembly of the oligomeric structure. The precipitation observed for a dissociated nitrotroponylated and guanidinated aldolase in low ionic strength may be due to random aggregation caused by the hydrophobic character of the added nitrotroponyl groups or to a conformational change caused by the nitrotroponylation. Proteolytic modification of yeast hexokinase has resulted in the identification of an eleven amino-acid peptide, containing two lysines and one arginine, that appears to be necessary for subunit association (24). Lysine and arginine have been shown to be involved in subunit contacts in hemoglobin by x-ray crystallography (25). It seems possible that lysine may be functioning in a similar manner in aldolase, although further experimentation is needed to establish with certainty the precise role of the lysine. The procedure presented in this paper provides one alternative to x-ray crystallography for investigating the nature of the subunit contacts in aldolase. REFERENCES 1. KAWAHARA,
K., AND TANFORD,
C. (1966)
Biochem-
istry 5, 1578. 2. LAI, C. Y., CHEN, C., AND HORECKER, B. L. (1970) Biochem. Biophys. Res. Commun. 40, 461. 3. LAI, C. Y., NAKAI, N., AND CHANG, P. (1974) Science
183, 1204.
4. EAGLES,
P., JOHNSON, L. N., JOYNSON, M. A., MCMURRAY, C. H., AND GUTFREUND, H. (1969) J.
Mol. Biol. 45, 533. 5. HEIDNER, E. G., WEBER, B. H., AND EISENBERG, D. (1971)
Science
171, 677.
6. Hsu, L. S., AND NEET, K. E. (1973) Biochemistr.y 12, 586.
7. CORNISH-BOWDEN, 8. 9. LO. 11.
12.
A., AND KOSHLAND, D. E., JR. (1971) J. Bio2. Chem. 246, 3092. TEIPEL, J. W. (1972) Biochemistry 11, 4100. CHAN, W. W. -C., AND MAWER, H. M. (1972) Arch. Biochem. Biophys. 149, 136. Hsu, L. S., AND NEET, K. E. Biochemistry, in press. TAYLOR, J. F. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, p. 310, Academic Press, New York. BLOSTEIN, R., AND RLJTTER, W. J. (1963) J. Biol.
Chem. 238, 3280. 13. TAMAOKI, H., MURASE, Y., MINATO, S., NAKAMSHI, K. (1967) J. Biochem. 62, 7. 14. DONOVAN, J. W. (1964) 2Gochemistr.v 3, 67.
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SAUNDERS
15. SPACKMANN, D. H. STEIN, W. H., AND MOORE, S. (1958) Anal. Chem. 30, 1190. 16. MOORE, S. AND STEIN, W. H. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 6, p. 819, Academic Press, New York. 17. SPACKMAN, D. H. (1967) in Methods in Enzymoiogy (Hirs, C. H. W., ed.), Vol. 11, p. 3, Academic Press, New York. 18. GIBBONS, I., AND PERHAM, R. N. (1974) Biochem. J. 139, 331. 19. HORECKER, B. L., TSOLAS, O., AND LAI, C. Y. (1972) in The Enzymes (Boyer, P. D., ed.), Third Edition, Vol. 7, p. 213, Academic Press, N. Y.
AND WEBER 20. DEAL, W. C., AND VAN HOLDE, K. E. (1962) Fed. Proc. 21, 254. 21. HAAS, L. F., AND LEWIS, M. S. (1963) Biochemistry 2, 1368. 22. MEIGHEN, E. A., AND SCHACHMAN, H. K. (1970) Biochemistry 9, 1163. 23. GIBBONS, I., AND PERHAM, R. N. (1970) &o&em. J. 116, 843. 24. SCHMIDT, J. J., AND COLOWICK, S. P. (1973) Arch. Biochem. Biophys. 158, 471. 25. MCLACHLAN, A. D., PERUTZ, M. F., AND PULSINELLI, P. D. (1972) in Protein-Protein Interactions (Joenicke, R., and Helmreich, E., eds.,) p. 91, Springer-Verlag, New York.
OF BIOCHEMISTRY
Investigation
AND
BIOPHYSICS
of the Role of Lysine in the Subunit Regions
GREGORY Department
of Chemistry
168, 525-530 (1975)
of Rabbit
Muscle
C. SAUNDERS
and Institute
AND
Aldolase’
BRUCE
of Molecular Biology, California Fullerton, California 92634
Received October
Contact
H. WEBER* State
Uniuersity,
Fullerton,
11, 1974
Rabbit muscle aldolase (E.C. 4. 1. 2. 13) was guanidinated by reaction with Omethylisourea. Up to 60% of the lysine residues can be guanidinated without any dissociation of the tetramer but with a complete loss of enzymatic activity. Native and guanidinated aldolase can be dissociated into monomers in 2.4 M MgCl, with only slight change in conformation of the subunit. Nitrotroponylation of guanidinated aldolase in dilute buffer gives no reaction whereas in 2.4 M MgCl, nitrotroponlylation modifies another 8-12% of the lysine residues. Removal of MgCl, by dialysis affords 100% recovery of activity and tetrameric structure for native aldolase and 100% recovery of tetrameric structure for guanidinated aldolase. In contrast nitrotroponylated and guanidinated aldolase remains monomeric before precipitating as the MgCl, concentration is lowered. It is concluded that lysine may be involved in the protein-protein interaction of the subunit contact domains of muscle aldolase.
equilibrium can be obtained in 1.2 M MgCl, (6); increasing the magnesium chloride concentration to 2.4 M produces monomers of aldolase in a near native conformation (10). These facts suggest that it should be possible to chemically label residues that are in the subunit contact domains of aldolase since surface residues can be blocked on the stable tetramer and then by dissociation in magnesium chloride “cryptic” residues can be exposed that were involved in the subunit contacts. In this paper experiments are described in which the role of one amino acid, lysine, in subunit interactions of aldolase is studied by guanidinating surface lysines followed by nitrotroponylating “cryptic” lysines in 2.4 M MgCl,.
Aldolase (E.C. 4. 1. 2. 13) from rabbit muscle has btien demonstrated to consist of four subunits (1) that are chemically identical except for the deamination of a single asparagine in two of the subunits (2, 3). Crystallographically the subunits appear identical and are arrayed tetrahedrally (4, 5). Hsu and Neet (6) have demonstrated that the interactions between aldolase subunits are isologous. Thus there are three contact domains between each subunit and the other subunits (7) but these appear to be of unequal strength as the assembly from monomer appears to occur as a dimerization of dimers (6, 8). The formation of tetramer is irreversible under the usual conditions of reassociation (8, 9) and there is no evidence of a monomer = tetramer equilibrium under normal conditions. This ’ Supported by a grant from the National Institute of Health (AM-15055. B. H. Weber, principal investigator). Taken in part from the Thesis of G. C. Saunders submitted in partial fulfillment of a master’s degree, California State University, Fullerton. 2 To whom inquiries and reprint requests should be addressed.
MATERIALS
All
rights
0 1975 by Academic Press. of reproduction in any form
Inc. reserved.
METHODS
Materials Rabbit muscle aldolase was obtained as a crystalline suspension in 2.0 M ammonium sulfate, 0.001 M EDTA, pH approximately 6, and fructose-l, 6.diphosphate, NADH, glycerol-3-phosphate (E.C.l.l.l,8)/tri-
525 Copyright
AND
526
SAUNDERS
osephosphate isomerase (E.C.5.3.1.1) crystalline suspension were purchased from Boehringer-Mannheim Corporation. 0-Methylisourea was obtained from Aldrich Chemical Co.; 2-methoxy-5-nitrotropone was obtained from Calbiochem; and magnesium chloride was obtained from Fischer Chemical. All other chemicals were of analytical grade. All water used was double distilled and dionized.
Methods Enzyme preparation. Crystalline aldolase was desalted at 4°C by either conventional or pressure dialysis against the desired buffer. Following dialysis the protein solution if necessary was clarified by centrifugation. Dialyses following chemical modification were performed in a like manner. Protein determination. Aldolase concentration was determined spectrophotometrically using an extinction coefficient e :L (280 nm) = 9.1 (11). Protein concentrations were adjusted to the desired level by dilution with the appropriate buffer or by concentration by ultrafiltration. Enzyme assay. Aldolase activity was determined by the method of Blostein and Rutter (12) using a Beckman Acta III recording spectrophotometer. Assays were performed at 30°C in 0.1 M triethanol amine buffer pH 7.9 with a final volume of 1 ml. The initial concentrations of fructose-l, 6-diphosphate and NADH were 0.51 M and 0.19 mM, respectively. The mixture of coupling enzymes was present at a concentration 7 pg/ml. The specific activity of the aldolase used in these studies was 9913 units/mg. Magnesium chloride dissociation-reassociation. The dissociation of native or modified aldolase was performed at room temperature in 2.4 M MgCl, (millipore filtered 4.0 M MgCl, stock solutions were used) in 0.2 M Tris-Cl buffer pH 8.2 at a protein concentration of 0.4 mg/ml. Reassociation was achieved by dialysis at room temperature against 0.2 M Tris-Cl pH 8.2. Guanidination of native aldolase. An equal volume of freshly prepared 0.6 M 0-methylisourea, pH 9.8, was combined with aldolase (1.0 mg/ml) in 0.1 M phosphate buffer pH 6.9 at 30°C. Aliquots were removed at timed intervals and dialyzed against 0.1 M phosphate pH 6.9. Quantitation was .achieved by amino acid analysis by monitoring the disappearance of lysine and the concomitant appearance of homoarginine. Protein concentration was determined from the yields of histidine and arginine. Nitrotroponlylntion of guanidinated aldolase. To solutions of guanidinated aldolase (1.0-10.0 mg/ml), pH 8.2, 0.1 M Tris-Cl, was added 6.5% by volume of 30.8 mM solution of 2-methoxy-5-nitrotropone in dimethylformamide. The reaction was allowed to proceed for two hours at 20°C and was then subjected to gel-filtration. The absorbance at 280 nm and 420 nm was checked for the protein fraction to see if there had been any modification due to nitrotroponylation.
AND WEBER Nitrotroponylation of dissociated guanidinated aldolase. To a solution of guanidinated aldolase (0.4 mg/ml) in 2.4 M MgCl, pH 8.2, 0.2 M Tris-Cl, was added 6.5% by volume of 30.8 M solution of 2methoxy-5-nitrotropone in dimethylformamide. The reaction was carried out at 20°C with aliquots removed at time intervals for dialysis against 0.2 M Tris-Cl pH 8.2. Quantitation was achieved spectrophotometrically using an extinction coefficient of c,+, (420 nm) = 20,700 (13). Protein concentration was determined using t :L (277 nm) = 8.32 for dissociated aldolase (14). Amino acid analysis. Analyses were performed by the method of Spackman et al. (15) and Moore and Stein (16) using an accelerated system (17) employing Beckman model 120C amino acid analyzer. Sedimentation velocity studies. Sedimentation coefficients for native, dissociated, reassociated and the modified aldolases were determined using a Spinco Model E analytical ultracentrifuge using schlieren or ultraviolet optics depending on the protein concentration at a rotor speed of 60,000 rpm. The observed sedimentation coefficients were corrected for solvent density and temperature. Spectrofluorometry. Fluorescence measurements were performed at 20°C with an Aminco-Bowman spectrophotofluorimeter with a ratio recorder with an excitation wavelength of 282 nm. The emission spectra were examined in the region of tryptophan emission for evidence of possible conformational changes. Optical rotatory Dispersion. Optical rotatory dispersion measurements for native and modified aldolase were performed on a Cary 61 spectropolarimeter, at a protein concentration of 0.4 mg/ml, over the region of 300-220 nm. Myoglobin was used as a standard for calibration of the instrument. RESULTS
Guanidination of Aldolase In order to determine the optimal conditions for the guanidination of aldolase preliminary sedimentation and enzyme activity experiments were performed to ascertain the pH and temperature stability of native aldolase over the pH range of 8.5-11.0 and the temperature range of 4-50°C. These studies indicate that at pH 10.0 at 30°C the aldolase tetramer is stable and fully active for periods of at least twelve hours. Our results agree with those recently reported by Gibbons and Perham (18). Under these conditions up to 60 lysine residues react with 0-methylisourea within 12 h (Fig. 1) and there is complete loss of activity (Fig. 2) indicating that at least one and possibly both the lysines known to be
ROLE OF LYSINE
IN ALDOLASE
SUBUNIT
CONTACTS
527
rotatory dispersion spectrum of both native and modified aldolase are very similar with WI 233= -5606 for native aldolase and [m’]233 = -5278 for guanidinated aldolase. This difference is slightly larger than experimental error. This suggests that a slight conformational change may have occured as a result of guanidination of 60 lysine residues per tetramer. Magnesium Chloride Dissociation-Reassociation Studies
The sedimentation velocity and spectrofluorescence results for native and guanidinated aldolase are presented in Table I. Both the native and guanidinated aldolases appear to exist as monomers in 2.4 MgCl, as judged by the sedimentation coefficient of s20,w= 2.5-2.6, which is close to that reported by Hsu and Neet (6, 10) for aldolase monomers in MgCl, and to those values seen for aldolase subunits in acid or alkaline solutions (20, 21). The spectrofluorescence data show that in 2.4 M MgCl, there is a slight red shift of 3 nm suggesting at most a slight conformational change, consistent with the observations of Hsu and Neet (6, lo), and not a major unfolding of the monomer as happens in other methods of dissociation. Removal of magnesium chloride by dialysis provides reassociation to the tetrameric structure and a blue shift FIG. 2. Activity time course for the guanidination of back to the native fluorescence emission aldolase. See text for details. maximum. Assays of the reconstituted native aldolase showed 100% recovery of acin the active site (19) have also reacted since there appears to be little or no tivity. conformational change upon guanidination Nitrotroponylation of Guanidinated (see below). Attempts to react even more Aldolase lysines lead to denaturation and precipitation of the protein. This suggests that 15 When guanidinated tetrameric aldolase out of the 26 lysines per subunit are “ex- was exposed to 2-methoxy-5-nitrotropone, posed” enough to react with O- and the excess reagent removed by gel methylisourea, and that up to 11 lysines filtration, there was no protein precipitaper subunit may be “buried” inside the tion observed and no incorporation of nimonomer or be in the binding domains trotroponyl moieties as judged by spectrobetween the subunits. The guanidinated photometric measurements at 420 nm. aldolase has a sedimentation coefficient of In contrast, dissociation of the fully guas20.w= 7.61 which is virtually identical to nidinated aldolase in 2.4 M MgCl, followed that obtained for native aldolase, sZO.w = by nitrotroponylation of the newly exposed 7.60. Examination of the fluorescence em- “cryptic” lysine residues does lead to modmission spectrum shows no alteration with ification of additional lysine residues x max (emission) = 327 nm for both native (Table II). Quantitation of the extent of and guanidinated aldolase. The optical nitrotroponylation and characterization of
FIG. 1. Stoichiometry time course for the guanidination of aldolase; (-A-l represents moles of homoarginine per mole of aldolase and (-W-) represents moles of residual lysine per mole of aldolase. See text for details.
528
SAUNDERS AND WEBER TABLE I
SEDIMENTATION-VELOCITY AND SPECTROFLLJORESCENCE ANALYSIS OF DISSOCIATEDAND REASSOCIATEDALDOLASE FOR THE NATIVE AND GUANIDINATED ENZYME
Sample
Medium”
sz0.w
x maxof emission (nm)
Native aldolase Dissociated native aldolase Reassociated native aldolase Guanidinated aldolase Dissociated guanidinated aldolase Reassociated guanidinated aldolase
0.2 M Tris-Cl 2.4 M MgCl, 0.2 M Tris-Cl 0.2 M Tris-Cl 2.4 M MgCl,
7.60 2.50 7.56 7.61 2.63
327 330 327 327 330
0.2 M Tris-Cl
7.67
326
DFor all samples pH = 8.2. TABLE II EXAMPLE OF QUANTITATION OF NITROTROPONYLATION OF GUANIDINATED ALDOLASE FOR Two INDEPENDENT MODIFICATIONS UNDER SIMILAR REACTION CONDITIONS’
Control Aldolase concentration (mg/ml) Absorbance at 420nm (corrected) Moles of nitrotroponylhetramer Moles of nitrotroponyl/monomer
0.32 0.000 0 0
Modification 1 0.94 0.897 7.36 1.84
Modification 2 0.52 0.686 10.18 2.55
DSee text for details.
the doubly modified aldolase was performed in 1.2 M MgCl, as attempts to completely remove magnesium chloride result in complete precipitation of the protein. It appears that 2-3 mol of lysine can be modified by 2-methoxy-5-nitrotropone per mole of dissociated guanidinated monomer. Sedimentation velocity analysis of the nitrotroponylated guanidinated aldolase in 1.2 M MgCl, gives a sedimentation coefficient of s2,,+. = 2.31 indicating that the modification of the “cryptic” lysines prevents reassociation to the dimeric form which Hsu and Neet (6) have demonstrated predominates under these conditions.
electrostatic repulsion and a conformational change. Meighen and Schachman (22) found that attempts to succinylate more than 40% of the lysines of aldolase produced dissociation, presumably through a conformational change. Gibbons and Perham (23) observed that dissociation begins to occur for aldolase when about 50% of the lysines of aldolase are citraconylated. In contrast guanidination allows the modification of 60% of the lysines of aldolase without any dissociation and with little, if any, conformational change in the subunits of the tetramer. Further, the guanidinated aldolase behaves identically to native aldolase in the dissociation-reassociation experiments. DISCUSSION Initially the dissociation-reassociation Guanidination permits modification of experiments were performed in 4 M urea, lysine residues while preserving the posi- which allowed 100% recovery of aldolase tion charge in contrast to succinylation and activity upon urea removal. However, opticitraconylation which modify lysines while cal rotatory dispersion and spectrofluorescence analysis of the dissociated monomers converting the positive charge to a negative charge. The conversion of a number of showed that they had undergone a significhange in 4 M urea positive charges to negative on the surface cant conformational of a protein would be expected to lead to and behaved as if they were “randon coils”
ROLE
OF LYSINE
IN ALDOLASE
(Saunders and Weber, unpublished results). The studies of Hsu and Neet (6, 10) suggested an alternative method of dissociation employing MgCl, that appeared to produce aldolase monomers of near native conformation. In the present study we have confirmed their results and observe that 2.4 M MgCl, does form monomers of aldolase that by criterion of spectrofluorescence appear to be very similar to the native conformation of the subunit in the tetramer. Since the guanidinated aldolase behaves identically to the native aldolase in these experiments, it is possible to expose and modify, in 2.4 M MgCl,, lysines that may be involved in subunit-subunit interactions with the majority if not all of the surface lysines blocked from further chemical modification. Nitrotroponylation of the dissociated monomers of guanidinated aldolase does modify two to three moles of lysine per monomer that presumably were “buried” between the subunits in the tetramer. As there are 11 lysines per subunit that are not guanidinated when the tetramer is modified, nor are reactive with 2-methoxy-5nitrotropone for intact guanidinated tetramers, the observation of nitrotroponylation of only up to 3 mol of lysine may mean that the other lysines are buried inside the monomer, that they remain unreactive in 2.4 M MgCl, or that it could be that under the conditions employed only 30% reaction was achieved with all 11 “cryptic” lysines. Nor can it be determined at present how many of these 3-11 lysines are “essential” for the subunit-subunit interactions. Additionally, some of the cryptic lysines may be surface residues that are unreactive due to their microenvironment and that the slight conformational change observed in 2.4 M MgCl, may have allowed these residues to react with 2-methoxy-5-nitrotropone. The fact that the dissociated nitrotroponylated and guanidinated aldolase does not appear to form dimers in 1.2 M MgCl, whereas dissociated guanidinated aldolase does suggests that modification of cryptic lysines has blocked the reassociation either through a direct steric interference or by a changing in conformation concomitant with the nitrotroponylation. In either event, one or more of the cryptic
SUBUNIT
529
CONTACTS
lysines per monomer seems to be essential for assembly of the oligomeric structure. The precipitation observed for a dissociated nitrotroponylated and guanidinated aldolase in low ionic strength may be due to random aggregation caused by the hydrophobic character of the added nitrotroponyl groups or to a conformational change caused by the nitrotroponylation. Proteolytic modification of yeast hexokinase has resulted in the identification of an eleven amino-acid peptide, containing two lysines and one arginine, that appears to be necessary for subunit association (24). Lysine and arginine have been shown to be involved in subunit contacts in hemoglobin by x-ray crystallography (25). It seems possible that lysine may be functioning in a similar manner in aldolase, although further experimentation is needed to establish with certainty the precise role of the lysine. The procedure presented in this paper provides one alternative to x-ray crystallography for investigating the nature of the subunit contacts in aldolase. REFERENCES 1. KAWAHARA,
K., AND TANFORD,
C. (1966)
Biochem-
istry 5, 1578. 2. LAI, C. Y., CHEN, C., AND HORECKER, B. L. (1970) Biochem. Biophys. Res. Commun. 40, 461. 3. LAI, C. Y., NAKAI, N., AND CHANG, P. (1974) Science
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4. EAGLES,
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15. SPACKMANN, D. H. STEIN, W. H., AND MOORE, S. (1958) Anal. Chem. 30, 1190. 16. MOORE, S. AND STEIN, W. H. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 6, p. 819, Academic Press, New York. 17. SPACKMAN, D. H. (1967) in Methods in Enzymoiogy (Hirs, C. H. W., ed.), Vol. 11, p. 3, Academic Press, New York. 18. GIBBONS, I., AND PERHAM, R. N. (1974) Biochem. J. 139, 331. 19. HORECKER, B. L., TSOLAS, O., AND LAI, C. Y. (1972) in The Enzymes (Boyer, P. D., ed.), Third Edition, Vol. 7, p. 213, Academic Press, N. Y.
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