FACTORS AFFECTING THE ACTIVITY OF ACONITASE* The ...

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FACTORS AFFECTING. THE ACTIVITY. OF ACONITASE* ... The enzyme which mediates the equilibrium among citrate, cis-aconi- tate, and d-isocitrate was ...
FACTORS

AFFECTING BY

SHERMAN

THE

ACTIVITY

R. DICKMAN

AND

OF ACONITASE*

A. A. CLOUTIER

(From the Department of BiologicalChemistry, University of Utah College of Medicine, Salt Lake City, Utah) (Received for publication,

June 26, 1950)

Determination

of Citric

Acid

The method described below is a modification of the methods of Natelson et al. (9, 10) and of Speck et al. (11). 0.5 ml. of the protein-free solution, containing 0.1 to 0.4 PM of citric acid, is placed in a test-tube-shaped Pyrex volumetric flask, fitted with a *This work was supported in part by a grant from the United States Public Health Service and the University of Utah Medical Resea.rch Fund. 379

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The enzyme which mediates the equilibrium among citrate, cis-aconitate, and d-isocitrate was initially described by Martius (1). It was designated aconitase by Breusch (2). Whether the equilibrium is catalyzed by one protein or two has been the subject of some discussion. Jacobsohn et al. (3) postulated two enzymes, whereas Martius and Leonhardt (4) considered that a single protein was involved. Buchanan and Anfinsen (5) achieved a 23-fold purification of aconitase and obtained no evidence for a separation of enzymes. Racker (6), on the other hand, reported an isocitrase-citrase ratio of 2.1 in crude heart extracts, while a purified preparation had a ratio of 7.5. It is obvious that an unequivocal answer to this question is dependent on a higher degree of purification of the protein or proteins than has yet been accomplished. Previous efforts in this direction have been greatly handicapped by the instability of the system. Krebs and Eggleston (7) found t,hat glycerol stabilized the aconitase activity of crude extracts, but Buchanan and Anfinsen (5) reported that glycerol was ineffectual in maintaining the stability of purified extracts. These authors also observed that cysteine stabilizes crude solutions of aconitase but strongly inhibits purified preparations. Our initial objective in approaching these problems has been to stabilize aconitase. It has been reported by Dickman and Cloutier (8) that ferrous ion in combination with cysteine appreciably stabilizes crude aconitase solutions and that the addition of these agents restores dialyzed solutions to their original activity. Further work, to be described below, has demonstrated that these two reagents are also efficacious in stabilizing and activating more highly purified aconitase preparations.

380

ACTIVITY

OF

ACONITASE

reground Silicone-greased standard taper stopper. To each flask 0.1 ml. of each of the following solutions is then added in order: 0.4 M KBr, 18 N H&O+ and 0.3 M KM&. The group of unstoppered flasks is placed in a Kahn rack and gently shaken for 10 minutes in a shaking machine. Excess KMn04 is destroyed by the addition of 0.1 ml. of 1.5 M NaN02 and the mixture is gently agitated.2 0.1 ml. of 2.0 M urea is added and the upright flasks shaken vigorously until the reaction subsides, generally about 4 to 5 minutes. 1.0 ml. of purified heptane (9) is added to each, and the flasks are stoppered and shaken in a horizontal position on the machine for 10 minutes. They are then centrifuged at 2500 r.p.m. to break the emulsion and the aqueous phase is removed with a syringe. 0.5

1. Quantitative

determination

of citric

acid.

Procedure

described

in the

ml. of distilled water is added, the tubes shaken for 30 seconds, centrifuged, and the aqueous layer again removed. A second water washing is carried out as described above. 1.5 ml. of 4 per cent thiourea in saturated borax solution are now added to the heptane solution and the mixture vigorously shaken in a horizontal position for 10 minutes.3 The contents are poured into optically calibrated 10 X 75 mm. soft glass tubes and centrifuged. The absorbancy at 4350 A is determined with a Coleman junior spectrophotometer.4 A standard curve, indicating that Beer’s law is followed up to 0.4 PM of citric acid is shown in Fig. 1. 1 Since other compounds recognized as forming pentabromoacetone are known to be absent under these conditions, the preliminary oxidation was routinely omitted. 2 The solution becomes cloudy at this step if more than 0.2 PM of citric acid is present. The formation of “clouds” serves as a rapid, semiquantitative test for citric acid under these conditions. 3 It has been found convenient to prepare a large volume of borax solution saturated at room temperature. Solid thiourea is added to this solution just prior to use. 4 We wish to thank Dr. Samuel Natelson for the information that the absorbancy at 4350 A is considerably more permanent than at 4500 A.

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FIG.

text.

S.

R.

DICKMAN

AND

Determination

A.

A.

381

CLOUTIER

of Aconitase Activity

4.5 ml. of water in each of a series of thick walled 100 X 14 mm. Pyrex test-tubes are brought to 30”. 0.1 ml. of enzyme solution is added to each. The reaction is begun by the addition of 0.5 ml. of a solution containing 20 PM of cis-aconitate at pH 7.4 and 30”. After 10.0 minutes in the bath, the reaction is stopped and proteins precipitated by the addition of 0.5 ml. of 50 per cent (weight to volume) trichloroacetic acid. After mixing and cooling the solutions in the refrigerator, the tubes are centrifuged and a suitable aliquot (usually 0.5 ml.) taken for citric acid analysis. Fig. 2 demonstrates that under these conditions there is found a quantitative

of aconitase. determined

Each tube contained as described in the text.

20 PM of

relationship between the amount of enzyme added and the quant,ity of citric acid formed. Dejhition of Aconitase Activity Unit-l unit of aconitase activity has been taken as that amount of enzyme which forms 1.0 pin of citric acid per tube in 10 minutes at 30” and pH 7.4. Determination

of Protein Concentration

The protein concentration of aconitase solutions has been determined by measurement of the absorbancy at 2800 A after dilution with 0.08 N KOH. A solution containing 1 mg. of protein per ml. as determined by micro-Kjeldahl analysis was found to have an Azsoo of 1.5. Materials cis-Aconitic anhydride was prepared acid by the procedure of Malachowski

from commerical and Maslowski

trans-aconitic (12). It was

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FIG. 2. Quantitative determination Citric acid cis-aconitate, pH 7.4, 30”.

382

ACTIVITY

OF

ACONITASE

recrystallized four times from benzene and melted at 77” (uncorrected). o-Phenanthroline, a, Lu’-bipyridyl, ascorbic acid, and glutathione were commercial preparations. Preparation

of Aconitase from Frozen Pork Heart

Activation

of Aconitase

Solutions of aconitase were “activated” by the addition of ferrous ammonium sulfate and a reducing agent prior to the determination of activity. Reducing agents were neutralized before addition; Fe++ solutions were not. The cold mixture was immediately adjusted to the desired pH and stored at 5’ until its aconitase activity was determined. In this paper the concentration of each activating or inhibiting agent in the incubation 6 Obtained

from Johns-Manville.

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Active aconitase solutions have been obtained from fresh frozen pork heart or from frozen cornminuted pork heart. With the former material the heart was freed from fat, cut into small pieces before freezing, and then ground twice in the frozen condition with a manually operated meat grinder. A typical preparation is described. 200 gm. of frozen heart are stirred mechanically at 0 to-2” with 600 ml. of 19 per cent EtOH for 30 minutes. The suspension is filtered by suction through cloth on a precooled Biichner funnel. The residue is again extracted with 600 ml. of alcohol at 0 to -2” and filtered. The extractions serve to remove considerable pigmented material, but these alcoholic solutions are devoid of aconitase activity. The cold residue is next placed in a precooled Waring blendor and blended 30 seconds with 600 ml. of 0.05 M phosphate, pH 7.0, and 150 ml. of CHCI,. The emulsion is centrifuged at 2500 r.p.m. in a refrigerated centrifuge. The pale pink supernatant is brought to 0.4 saturation with solid (NH&S04, the ice-cold solution being maintained at pH 7.4. The suspension is filtered by suction through cloth covered with a layer of Hyflo Super-Ce15 or centrifuged in the cold. The clear filtrate is brought to 0.7 saturation with solid (NH&SO+ the solution being maintained at pH 7.4. The precipitate can be centrifuged in the cold or filtered by suction through Whatman No. 1 paper. The (NH&S04 treatment results in a preparation 6 times as pure as the blend supernatant, with an over-all recovery of 77 per cent. The precipitate can be stored at -20” for a week with little loss of activity. Most of the studies reported here have been accomplished with this “70 per cent precipitate.” Table I lists the results of a typical preparation, as well as the specific activity of a direct blend of heart.

S.

R.

DICKMAN

AND

A.

A.

383

CLOUTIER

solution will be stated rather than its concentration in the activity determination. The presence of additional Fe++ and ascorbate in the activity determination did not affect the amount of citrate formed by a given aconitase preparation. For example, one-half of a water solution of the 70 per cent precipitate was brought to Fe++, 5 X 10V3M and ascorbate, 10m2M. No additions were made to the other half. The aconitase activity of each of these solutions was determined by the usual assay. Citric acid formation was also measured in other tubes which contained Fe++, lop4 M, and ascorbate, 1O-2 M, in addition to the cis-aconitate. The non-activated aconitase solution showed an activity of 1.1 units per ml. and the Feft-ascorbate-

Aconitase with 0.01 of activity. -

M

fractions cysteine,

of Aconitase

obtained 5 X IOV

as described M Fe++, pH

from

Frozen

Pork

Heart

in the text. All solutions incubated 7.4, 5” for 1 hour before determination

-

-

Protein specific Total Volume t’Lconitase activity aconitase concentration activity

Fraction

.units per ml.

ml. 1st 19% EtOH filtrate.. .. . 2nd 19% “ “ . Blend supernatant.. 0.4 ammonium sulfate supernatant.. 0.7 “ “ ppt. dissolved in HzO. . . ... . . .. Direct blend supernatant..

600 600 550 660

0 0 33.6 23.5

364

39.2 28.0

-

ulziis 0 0 18,480 15,510 14,270

-

mg;pr

units per nag.

5.2 2.5 7.3 4.9

0 0 4.3 4.8

1.5 12.0

26.2 2.3

treated aconitase solution, 24.6 units per ml. in both types of substrate tubes. These results are apparently due to a relatively slow formation of an Fe++protein complex. Since even the presence of phosphate or bicarbonate buffer in the activity run did not decreasethe aconitase activity of an activated preparation, it would appear that once the Fe++-protein complex had formed it did not dissociate rapidly. Results Factors Affecting Activation of Aconitase As shown in Fig. 3, addition of cysteine trebled the activity of a solution of the 70 per cent saturated (NH&S04 precipitate. The fact that addition of Fe++ did not activate this preparation indicates that the protein must also be present in a reduced condition in order to exhibit maximum activity. The addition of cysteine thus accomplishes a double function:

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TABLE I Preparation

384

ACTIVITY

OF

ACONITASE

maintenance of iron as Fe++ and maintenance of protein reducing groups. The addition of Fe++ and cysteine together approximately doubled the activity obtained with cysteine alone. Evidently this enzyme preparation was about half saturated with Fe++ for maximum activity. When the 70 per cent precipitate was dissolved in 0.1 M bicarbonate, cysteine activation was again obtained, but further activation owing to addition of Fe++ was prevented. Apparently the bicarbonate did not remove Fe++ bound to the protein but did prevent the added Fe++ from combining with the enzyme. When the precipitated protein was dissolved in phosphate buffer, neither cysteine alone nor the cysteine-Fe++ combination exerted an activating ADDITION

mO.01

M. CYSTEINE

~5Xt0-4hl.

Fe++

5X10-4M.Fe++ + 0.01 M. CYSTEINE

WATER

0.1

M. HC03

0.1

M.

PO,

FIG. 3. Effect of buffers on activation of aconitase. The 70 per cent precipitate was dissolved in the minimum volume of HsO, then diluted with the indicated solution. Aconitase activity determined after 15 hours incubation at 5’, pH 7.4.

effect. Phosphate thus competed with the protein for the iron in the system, as well as prevented the added Fe ++ from combining with the enzyme. With another preparation appreciable activation was obtained in the presence of 0.1 M phosphate when the Fe++ concentration was increased to 5 X 10h3M. These results explain in large part the instability of aconitase prepared with phosphate buffer. Stability of Aconitase The addition of ferrous ammonium sulfate and cysteine to aconitase solutions maintained at pH 7.4 appreciably activated and stabilized the enzyme, as demonstrated in Fig. 4. Supplemental additions of cysteine at the end of the 1st and 3rd days appear to have been helpful in maintaining activity. The addition of cysteine alone did not result in nearly

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INO

S.

R.

as large an activation Fe++ combination.

DICKMAN

AND

or maintain

A.

A.

as high an activity o---O5 o----o

385

CLOUTIER

as did the cysteine-

XIO-4M.Fe,++0.01M. O.OlM. CYSTEINE NO ADDITION

CYSTEINE

I

30 O

2

4

5

6

DAYS” FIG. 4. Effect of cysteine and Fe++ on aconitase stability. The 70 per cent precipitate was dissolved in water and aliquots brought to a concentration of 0.01 M cysteine plus 5 X lo-* M Fe+ and adjusted to pH 7.3. 30 minutes were required for these treatments, as indicated on the chart. The solutions were stored at 5” and aliquots removed for determination of activity at the indicated times. Cysteine additions at 24 and 72 hours were made to bring the concentration to 0.01 M, exclusive of previous additions. min.

TABLE

II

of Reducing Agents on Aconitase Activity The 70 per cent precipitate was dissolved in HzO, brought to 5 X 10-d M Fe++, and incubated 6 hours at 5”, pH 7.3, with the reducing agents prior to determination of the activity. Ej’ect

Reducing agent

Concentration

None ....................................... Cysteine .................................... Glutathione ................................. Ascorbic acid ...............................

0.01 0.01 0.01

Aconitase activity

4.6 26.9 15.7 25.8

Reducing Agents The data of Table II indicate that cysteine can be replaced by ascorbic acid. Glutathione, however, was not as effective in activating aconitase.

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CYSTEINE

386

ACTIVITY

OF

Inhibitors

ACONITASE

of Aconitase

DISCUSSION

The effects of additions of Fe* on aconitase activity which have been presented above deal primarily with Fe++-protein complex formation, Fe++ might also be expected to form stable complexes with the tricarboxylic acids. While studies of metal-hydroxy acid complexes are quite numerous, few have dealt with Fe++ complexes. Polarographic, spectrophotometric, and titrimetric data obtained in this laboratory definitely indicate that Fe++-tricarboxylic acid complexes are formed under conditions of optimum aconitase activity. This work will subsequently be reported in detail. The data at hand, although qualitative, are indicative of two different complexes: Few-substrate and Fe++-protein. It is probable that a mixIn this ture of citrate, Fe++, and enzyme would form a third complex.

‘CH&OO’ (1)

formulation, Fe++ is considered as an integral component of the citrate-

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As shown in Table III, the addition of an agent which forms metal complexes, o-phenanthroline, effectively inhibited aconitase. In confirmation of the results of Warburg (13) on yeast aldolase, it was found that o-phenThe anthroline was a more effective inhibitor than was a,a’-bipyridyl. inhibition of the former agent was reversed by the addition of excess ferrous ion. Since o-phenanthroline forms complexes with other cations than Fe++, Cd++ and Zn+ have been tested as aconitase-activating agents in addition to those tested previously (8). Fe++ is the only cation tested to date by which consistent activation of aconitase has been secured. The results of a study of aconitase inhibition by a,a’-bipyridyl are presented in Fig. 5. The 70 per cent precipitate was dissolved in a solution containing 0.01 M ascorbate and an aliquot was brought to 10e3 M bipyridyl. The inhibition obtained with this agent, which forms iron complexes, increased with time; complete inhibition was found at the end of 5 days. An aliquot of the bipyridyl-treated enzyme was brought to 5 X 1O-4 M Fe++ on the 2nd day and the aconitase activity of the solution determined on the 5th day. Aconitase activity was brought to the level of non-inhibited enzyme by this treatment.

S. R.

DICKMAN

AND

A.

A.

387

CLOUTIER

TABLE III Inhibition and Reactivation of Aconitase The aconitase solution contained lo+ M ascorbate, 5 X 1O-4 M Fe++, and was incubated with p-chloromercuribenzoate for 30 minutes prior to determination of the activity. o-Phenanthroline inhibition was determined on an aconitase solution to which ascorbate but no Fe ++ had been added. All solutions maintained at pH 7.3, 5’. Aconitase activity determined as described in the text.

Aconitase j Concentration activity -(units per ml.

Agent

Inhibition #CT

dl

I

10-4

100 100

10-4 x 10-s

17

26

10-e 10-s

!-

2

cent

23 16 0 0

5

3

4

5

30

6

DAYS

Curve FIG. 5. Inhibition of aconitase by&,&-bipyridyl and reactivation by Fe*. Curve 2,70 per cent 1,70 per cent precipitate dissolved in 0.01 M ascorbate solution. Curve 3, aliquot of precipitate dissolved in 0.01 M ascorbate, and 10-s M bipyridyl. solution containing bipyridyl brought to 5 X 10e4 M Fe* on the 2nd day. All solutions maintained at pH 7.3, 5”. Aconitase activity determined as described in text.

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None ............................... ........... p-Chloromercuribenzoate ‘I ........... o-Phenanthroline ................... I‘ ................... + Fe* ...........................

388

ACTIVITY

OF

ACONITASE

enzyme complex and accordingly structure I differs from the structures of Ogston (14) and of Martius and Lynen (15). It that the “three-point combination” postulated by Ogston mechanism of forming an asymmetrical citrate is mediated ferro complex.

hypothetical is suggested (14) as the through the

SUMMARY

The authors wish to acknowledge generous gifts of comminuted heart from Armour and Company and a sample of trans-aconitic acid from Godchaux Sugars, Inc. BIBLIOGRAPHY

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Martius, C., Z. physiol. Chem., 247, 194 (1937). Breusch, F. L., Z. physiol. Chem., 260, 262 (1937). Jacobsohn, K. P., and Tapadinhas, J., Enzymologia, 6, 388 (1939). Martius, C., and Leonhardt, H., Z. physiol. Chem., 278, 208 (1939). Buchanan, J. M., and Anfinsen, C. B., J. Biol. Chem., 180, 47 (1949). Racker, E., Biochim. et biophys. acta, 4, 211 (1950). Krebs, H. A., and Eggleston, L. V., Biochem. J., 38,426 (1944). Dickman, S. R., and Cloutier, A. A., Arch. Biochem., 26, 229 (1950). Natelson, S., Lugovoy, J. K., and P&us, J. B., J. Biol. Chem., 170, 597 (1947). Natelson, S., Pincus, J. B., and Lugovoy, J: K., J.“BioZ. Chem., 176, 745 (1948). Speck, J. F., Moulder, J. W., and Evans, E. A., Jr., J, Biol. Chem., 164,123 (1946) Malachowski, R., and Maslowski, M., Ber. them Ges., 61, 2521 (1928). Warburg, O., Heavy metal prosthetic groups and enzyme action, Oxford, 190 (1949). 14. Ogston, A. G., Nature, 162, 4129 (1948) 15. Martius, C., and Lynen, F , Advances zn.Enzymol., 10, 198 (1950).

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1. A micromodification of the pentabromacetone method for citric acid has been described. 2. An aconitase enzyme-concentration curve has been demonstrated. 3. Evidence of the activating and stabilizing effect of Fe* and reducing agents on aconitase has been presented. 4. Aconitase can be inhibited by dilute solutions of o-phenanthroline and (Y, a’-bipyridyl and reactivated by subseqdent additions of Fe++. It thus appears that Fe++ acts as a cofactor in the cis-aconitic to citric acid reaction.

FACTORS AFFECTING THE ACTIVITY OF ACONITASE Sherman R. Dickman and A. A. Cloutier J. Biol. Chem. 1951, 188:379-388.

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