THE MECHANISM OF THE EFFECT OF CALCIUM SALTS ON THE SUCCINOXIDASE SYSTEM*

BY KARL F. SWINGLE, A. E. AXELROD, AND C. A. ELVEHJEM

(From the Department of Biochemistry, College of Agriculture, Iiniversity of Wisconsin, &fadison)

(Received for publication, August 8, 1942)

In a preliminary report (1) we described a strong stimulation of the oxidation of succinate by fresh tissues when traces of calcium salts were added. After a number of preliminary studies to determine the conditions under which the calcium effect may be observed, the following working hypotheses were developed to explain the stimulatory effect of calcium on the succinoxidase system. (1) C 1 a cium, by its well known influence on membrane permeability, in some manner causes the rate of succinate oxidation to increase. (2) Th e calcium ion might remove some inhibitory substance, either one formed by the reaction or one otherwise present in the tissue. This effect might be due either to combination with the in- hibitor or to the catalytic removal of the inhibitor. (3) The calcium ion might be a specific activator for succinic dehydrogenase, or some other component of the succinoxidase system. This activation could be accom- plished in either of two ways: Calcium could become an integral part of the enzyme, necessary for its activity; or, calcium could be catalytically involved in the activation of the enzyme. (4) The calcium ion might prevent the formation of some inhibitory substance.

The experiments reported in this paper show that calcium functions by preventing the formation of oxalacetate, a strong inhibitor for the succin- oxidase system.

Methods

Respiratory studies were conducted, for the most part, in Barcroft differential manometers (2). Details of the additions to the various flasks will be given with each experiment reported. Air was used in the gas phase and potassium hydroxide placed in the center cups in each experi- ment. All succinoxidase experiments were equilibrated 10 minutes before the cocks were closed. The bath temperature was 37”, and the flasks were shaken at about 100 cycles per minute. Results are expressed as Qo, (microliters of oxygen consumed per mg. of dry weight of tissue per hour).

* Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. These studies were aided by grants from the Rockefeller Foun- dation and the Wisconsin Alumni Research Foundation.

581

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582 CALCIUM AND SUCCINOXIDASE SYSTEM

The Qo, values given are the maximum values observed in any of the 10 or 15 minute periods between readings.

Tissues were obtained by decapitating white rats and removing the re- quired tissue immediately. The sample was blotted on a piece of moist filter paper to remove blood and excess interstitial fluid. If it was to be minced, the sample was run through a Seevers and Shideman (3) mincer and portions of the mince weighed on slips of cellophane and placed in the flasks. A tissue to be homogenized was weighed in a homogenizer tube, the appropriate amount of water or buffer added, and the tissue homo- genized by the Potter and Elvehjem (4) technique. Firm tissues, such as heart or skeletal muscle, were minced before being weighed into the homo- genizer tube. Aliquots of the homogenate were added by pipette to the manometer flasks or other reaction vessels.

Calcium analyses were performed by the dry ashing method, by pre- cipitating the calcium as tricalcium phosphate, and determining the phosphate in the precipitate by the Fiske and Subbarow method (5).

Cozymase assays were conducted by the method of Axelrod and Elve- hjem (6), modified to suit the particular apozymase preparation used, since for maximum carbon dioxide production, this apozymase required, in addition to the usual reagents, also muscle adenylic acid and cocarboxylase. An assay flask would thus contain Mg and Mn (1 mg. each, as chlorides, per ml.) 0.10 ml., glucose (40 per cent in 0.1 M phosphate buffer, pH 6.2) 0’20 ml., buffer (1 M phosphate, pH 6.2) 0.06 ml., hexose diphosphate (10 mg. of organic P per ml.) 0.4 ml., muscle adenylic acid (1 mg. per ml.) 0.2 ml., cocarboxylase (1 mg. per ml.) 0.02 ml., water 0.02 ml., sample plus water 1.0 ml., apozymase 50 mg.

Reagents other than those used in the cozymase assay were prepared as nearly calcium-free as possible, and analyses of them showed no detectable calcium.

EXPERIMENTAL

To determine the correctness of hypothesis (l), a comparison of the effect of calcium in minced and homogenized tissues was made. In the mince, only a portion of the cells is ruptured, while in a carefully prepared homo- genate, almost all of them are destroyed. The results are given in Table I.

It is thus apparent that though the homogenized tissues give much higher rates of respiration than do the minced tissues, the calcium effect is present in both types and therefore is not dependent upon a permeability effect.

To test hypothesis (Z), it was decided to see if the presence of calcium relieved the inhibition due to any of the known inhibitors of succinoxidase which might be present in the system. The three inhibitors tested were

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SWINGLE, AXELROD, AND ELVEHJEM 583

TABLE I

Comparison of Calcium Effect on Minced and Homogenized Tissues

Buffer, ~/60 Na and K phosphate, pH 7.4; substrate, 90 micromoles of sodium succinate per flask; tissue, 50 mg. of rat liver per flask; total volume, 2.0 ml.

Flask No. Preparation of tissue Calci;~dn~loride Qos

micromole

1 Minced 0.025 19.4 2 “ 0.125 24.4 3 ‘I 0.250 24.5 4 Homogenized 0.025 43.9 5 ‘I 0.125 52.1 6 “ 0.250 53.7

TABLE II

Effect of Calcium on Inhibition of Succinoxidase System by Fumarate, Malate, and Oxalacetate

Buffer, ~/45 Na and K phosphate, pH 7.4; cytochrome c, 0.02 micromole per flask; substrate, 90 micromoles of sodium succinate per flask (tipped from Keilin cups after the 10 minute equilibration period in order to measure the true initial &02); tissue, 20 mg. of homogenized rat heart.

Experiment Flask No.

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

I- Inhibitor %lciumchlorid<

nzicromoles

None “

5 fumarate 5 “

5 malate 5 L‘

None “

1 .O oxalacetate 1.0 “ 0.1 “ 0.1 “ None

“ “

0.2 oxalacetate 0.2 “ 0.2 “

-

0.0 40.5 0.5 120.5 0.0 17 0.5 83 0.0 17.5 0.5 86 0.0 40.5 0.5 127 0.0 5 0.5 2.5 0.0 29 0.5 99 0.0 68.5 0.5 80 2.5 78.5 0.0 28.5 0.5 27.5 2.5 31

@02

malate, fumarate, and oxalacetate, as illustrated in Experiments A and B, Table II. Oxalacetate was prepared from the commercial sodium ethyl oxalacetate (7).

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584 CALCIUM AND SUCCINOXIDASE SYSTEM

These experiments are not conclusive. Although it is true that the calcium level used was insufficient to overcome the effect of the added inhibitor, it is possible that higher levels of calcium might do so, if the calcium actually combines with the inhibitor. Experiment C (Table II) tests this point.

The fact is here demonstrated that the use of even 10 times as much calcium as oxalacetate does not relieve the oxalacetate inhibition at all. Oxalacetate was chosen for this experiment, rather than.fumarate or malat,e, because it is a very potent inhibitor of the succinoxidase system; so that very small amounts, of the same order as the amounts of calcium used, gave inhibitions of the proper magnitude to compare with the inhibition produced by omission of calcium from the system. We may conclude that calcium does not act by removing any of these compounds as inhibitors.

TABLE III

Variations in Time Relationships of Substrate and Calcium Additions

Buffer, M/45 Na and K phosphate buffer, pH 7.4; cytochrome c, 0.02 micromole per flask; tissue, 20 mg. of homogenized rat heart per flask; substrate, 90 micromoles of sodium succinate per flask; calcium chloride (when used), 0.05 micromole per flask.

77.5 152

Flask No. Time relationships Q02

1 Ca and succinate added at beginning of equilibration period 2 “ added at beginning of equilibration and succinate added 20

min. after closing cocks 3 Succinate added at beginning of equilibration and Ca added 20

min. after closing cocks 4 Succinate and Ca both added 20 min. after closing cocks 5 “ added at start of equilibration; no Ca added 6 “ “ 20 min. after closing cocks; no Ca added

14.5

91 15 68

Assuming for the moment that hypothesis (2) is ruled out, it should be possible to distinguish between hypotheses (3) and (4) by an experiment in which the time of adding the calcium to the system is varied.

If calcium acts as a specific activator for some component of the suc- cinoxidase system, a reaction which is proceeding slowly because calcium has been omitted should be accelerated on the addition of calcium. On the other hand, if calcium acts only by preventing the formation of an inhibi- tory substance, the reaction taking place in the absence of calcium will already have formed the inhibitory substance and the subsequent addition of calcium will have no stimulatory action. The next experiment was designed to distinguish between these t,wo modes of action of the calcium ion and the results arc given in Table III.

These data are entirely in support of hypothesis (4), in that calcium

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SWINGLE, AXELROD, AND ELVEHJEM 585

has no effect when added after the reaction has proceeded for 30 minutes (compare Flasks 3 and 5). Calcium therefore cannot be an activator for the succinoxidase enzymes. In addition, the oxygen uptake is much greater if the calcium chloride is added before the succinate than it is if the two are added simultaneously (compare Flasks 2 and 5). This would indicate that the inhibitory material is formed from the succinate by a sys- tem which is inactivated by calcium.

Inactive enzyme X

I Ca

(enzyme X) Succinate -----+ inhibitor

When the succinate is added at the same time as the calcium, the inhibi- tory material is formed from it before the calcium has had a chance to inactivate the system forming it, while, if the calcium is added to the system before any of the succinate, the inhibitor-forming system is inacti- vated before any inhibitor is formed. Flask 6 shows that this inactivation of the inhibitor-forming system takes place wit.hout the addition of calcium but at a much slower rate. Elliott and Greig (8) found similarly an in- crease in the succinoxidase activity of tissues stored in the refrigerator.

From these data it appeared probable that the inhibitor sought would be one which is a product of succinate metabolism and the formation of which is conducted by a system which is spontaneously inactivated in the tissue preparations, but which is more rapidly inactivated in the presence of calcium ions.

Of the known inhibitors for the succinoxidase system, the only ones which are breakdown products of succinate are fumarate, malate, and oxalacetate. Fumarate may be eliminated from consideration because it must of necessity be formed by the oxidation of succinate. Malate, being no more inhibitory than the fumarate from which it is derived, could not be the inhibitor sought. Oxalacetate, however, is a very much more potent inhibitor than fumarate or malate; so if a small amount of oxal- acetate were formed, we might expect a considerable inhibition of the succinoxidase system. Oxalacetate is formed from malate by a malic dehydrogenase linked with coenzyme I (cozymase). It is well known that cozymase rapidly disappears from minced tissues (9-12); so if the cozymase is destroyed, no oxalacetate should be formed, and the succinoxidase system should not be inhibited. On this basis it is postulated that the calcium e$ect is one of accelerating the destruction of cozymase in the tissues so that oxalacetate may not be formed to inhibit the succinoxidase system.

This hypothesis was tested against all the data accumulated to date and

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586 CALCIUM AND SUCCINOXIDASE SYSTEM

found to fit perfectly. The results given in Table IV, which show that calcium inhibited the oxidation of lactate, pyruvate, glucose, and the en- dogenous substrates, give particular support to this view. This result would be expected, since cozymase is involved in all of these respirations.

TABLE IV

E$ect of Calcium Chloride on Oxidation of Various Substrates

Buffer, ~/30 Na and K phosphate, pH 7.4; tissue, 50 mg. of minced liver per flask (minced, rather than homogenized tissues were employed because the homogeniza- tion reduced almost to zero the power of these tissues to oxidize this group of sub- strates); substrates, lactate and pyruvate used as the sodium salts.

Flask No. Substrate

-I- micromoles

None “

180 dl-lactate 180 “ 90 pyruvate 90 “ 45 glucose 45 ‘(

Calcium chloride added

micronzole

0.5

0.5

0.5

0.5

00,

4.2 2.15 9.9 6.1 5.4 3.2 3.7 1.1

TABLE V

Calcium Chloride and Mixed Lactate and Succinate Systems

Buffer, ~/30 Na and K phosphate buffer, pH 7.4; tissue, 50 mg. of minced liver per flask; the substrates were added as the sodium salts.

Flask No. Substrate

6

micromoles

90 dl-lactate 90 “ 90 succinate 90 “ 90 “ + 90 dl-

lactate “ “

Calcium chloride

?tiicronzole

0.5

0.5

QO,

13.8 8.3

15.8 50.4

( 0.5 1 ::::

Another experiment in agreement with this postulate, if, in some manner, the lactate or its oxidation product, pyruvate, removes the oxalacetate formed, or ties up the cozymase so that it is not available to participate in the oxidation of malate to oxalacetate, is given in Table V.

In the absence of calcium chloride, lactate stimulates the succinoxidase system more than can be accounted for by the respiration of the lactate

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SWINGLE, AXELROD, AND ELVEHJEM 587

itself. On the other hand, in the presence of calcium chloride, lactate has no influence on the succinoxidase system. It appears as if lactate had an effect almost like that of calcium itself. Analysis of the lactate solution showed it to be free of calcium.

Mann and Quastel (12) have studied the system in tissues which destroys cozymase. Our postulate supports their explanation of the inhibition of succinoxidase by cozymase (13) as being a matter of oxalacetate formation. These authors found that nicotinamide would prevent the destruction of cozymase by the enzyme which they have called “cozymase nucleotidase.” It was therefore decided to study the effect of nicotinamide on the suc- cinoxidase system. See Table VI.

TABLE VI

Effect of Nicotinamide on Succinoxidase System

Buffer, ~/45 Na and K phosphate, pH 7.4; cytochrome c, 0.02 micromole per flask; substrate, 90 micromoles of sodium succinate per flask (added after equilibration); tissue, 22 mg. of homogenized heart.

Flask No. Preparation

Tissue homogenized in CaClz solution “ “ “ water; Ca added before equilibration “ “ “ nicotinamide solution “ “ “ water; nicotinamide added before

equilibration Tissue homogenized in CaCl*; nicotinamide added before

equilibration Tissue homogenized in nicotinamide; CaClz added before

equilibration

QO2

92.5 85 41 68.5

81.5

45

When Flasks 3 and 4 are compared, it is apparent that the earlier addition of nicotinamide to Flask 3 protected the cozymase from destruction, lowering the succinoxidase activity.

When Flasks 3 and 6 are compared, the figures show that even though the calcium chloride was added to Flask 6 before the succinate, it had no stimulatory effect on the succinoxidase, because the nicotinamide was pro- tecting the cozymase.

A comparison of Flasks 5 and 6, each of which contained both calcium chloride and nicotinamide, but added in reverse order, shows that in Flask 5 the calcium chloride had largely completed the destruction of the cozymase (cf. Flask 2) by the time the nicotinamide was added, while in Flask 6 the cozymase was protected from the action of calcium chloride by the previous addition of nicotinamide.

The final test of this theory thus lies in actually determining the rate of destruction of cozymase in the presence and absence of calcium.

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588 CALCIUM AND SUCCINOXIDASE SYSTEM

A piece of fresh rat liver was dropped into 10 cc. of redistilled ice water in a homogenizer tube and homogenized quickly. 5.0 ml. samples were transferred to each of the following: (a) 5.0 ml. of water at 30”, and (b) 5.0 ml. of calcium chloride solution (100 y of calcium per ml.) at 30”. Sam- ples of each of these were withdrawn at noted intervals, brought to a boil, and placed in a boiling water bath for 2 minutes to destroy the cozymase nucleotidase, and then cooled in an ice bath. These extracts were assayed for cozymase by the yeast fermentation method described above. The

TABLE VII

&feet of Calcium on Destruction of Cozymase

Sample taken at

nzin.

4 10 20

Cozymase remaining

No calcium added Plus added calcium

Y 7

7.7 7.4 5.5 4.2 4.8 1.5

TABLE VIII

Glutamate and Succinoxidase System

Buffer, ~/30 potassium phosphate, pH 7.4; cytochrome c, 0.02 micromole per flask; substrate, 90 micromoles of potassium succinate per flask; tissue, 50 mg. of homogenized heart.

Flask No. Oxalacetate ______

micromole

0.2 0.2

Calcium chloride

aicromole

0.4 0.4 0.4

0.4

0.4

Glutamate QOZ

micromoles

49 73 35

6 65 47 69

6 63 6 89

results are given in Table VII. Each assay represents 36 mg. of fresh liver.

This confirms our theory that the calcium ion stimulates the destruction of cozymase by animal tissues.

The other piece of direct evidence desired to support this theory would be to measure the amounts of oxalacetate formed in the Barcroft flasks in the presence and absence of calcium. Unfortunately, the amounts are too small for any known method of analysis. Approximately 0.2 micromole

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SWINGLE, AXELROD, AND ELVEHJEM 589

of oxalacetate gives the inhibition of the succinoxidase system obtained by the omission of calcium from that system. If this were assayed by measur- ing the carbon dioxide formed by decarboxylation (14, 15), the amount formed would be only 4.5 microliters, which is not measurable in the Barcroft apparatus with any degree of precision.

However, an indirect bit of evidence for the presence of oxalacetate as the inhibitor involved is given in Table VIII. These experiments were suggested by Dr. V. R. Potter. Oxalacetate, in the presence of glutamate, will transaminate with the latter to give cr-ketoglutarate and aspartate (16) ; so the addition of glutamate should remove at least a portion of the oxalacetate and accelerate the succinoxidase system.

This shows that glutamate will, to a large extent, relieve the inhibition due to added oxalacetate.

Table VIII shows that glutamate is stimulatory to the succinoxidase sys- tem, approaching t,he activity of calcium chloride itself.

DISCUSSION

This entire study has been concerned with the effect of the calcium ion, the other ions which were present in the solution being ignored. To study the influence of a single ion directly is quite impossible, since at least one anion must be added with the cation. Also, buffers are essential to main- tain the reactions at more or less constant rates, and the substrate must be added as a salt.

A few studies on the effects of other ions on the cozymase nucleotidase activity and on the succinoxidase activity of tissues were made, but though general correlation existed, the evidence is not sufficiently well developed to bc presented here. An interesting paper in this regard is that of Das (9) who reports activation of his cozymase-splitting enzyme by magnesium ion, &den (17) reports a stimulation by magnesium of the succinoxidase activity of minced pigeon breast muscle.

With regard to the stimulatory effect of lactate on the succinoxidase system, a paper by von Euler and Heiwinkel (11) may be of interest. They report that reduced cozymase is much more rapidly destroyed by tissues than is oxidized cozymase. Thus, in the presence of lactate, it is con- ceivable that the cozymase is reduced through the lactic dehydrogenase and is therefore more quickly destroyed; so that it cannot enter into the conversion of malate to oxalacetate and the succinate system proceeds unimpeded.

Inhibition of respiratory systems by nucleotidases when the respiratory systems require cozymase has been described for the nucleotidase found in snake venoms by Chain (18) and by Mann and Quastel (12). The former author did not find any stimulation for the succinoxidase system by

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590 CALCIUM AND SUCCINOXIDASE SYSTEM

his snake venom, but he used very high levels which apparently inhibited some component of the succinoxidase by some other mechanism. The inhibition of the cozymase-requiring systems was complete, while that of the succinoxidase system was only partial.

SUMMARY

1. The succinoxidase activity of several minced and homogenized tissues is increased by the addition of traces of calcium chloride to the medium, but the endogenous respiration and respiration in the presence of lactate, py- ruvate, and glucose are inhibited by the same concentration of calcium.

2. Lactate, in the absence of added calcium, stimulates the respiration of tissue in the succinoxidase system more than can be accounted for by the respiration of the lactate itself. Lactate has an influence on the suc- cinoxidase system similar to that of calcium.

3. Calcium chloride does not relieve the inhibition of the succinoxidase system caused by adding oxalacetate.

4. Calcium chloride added a few minutes after the addition of succinate to a tissue suspension fails to show any stimulation. Maximum stimula- tion is secured by adding the calcium a few minutes before the succinate.

5. Tissues standing in the absence of added calcium are gradually acti- vated until they attain the same succinoxidase activity as tissues to which c.alcium has been added, and these spontaneously activated tissues no longer respond to added calcium.

6. Nicotinamide, added to the tissue a few minutes before the calcium is added, inhibits the succinoxidase system and prevents any stimulation by calcium. If the calcium is added first, the usual stimulation is observed and the nicotinamide has no inhibitory effect.

7. Cozymase is destroyed by macerated tissue more rapidly in the presence of added calcium chloride than in its absence.

8. Glutamate will relieve the inhibition of the succinoxidase system due to oxalacetate and will also stimulate the succinoxidase system to approxi- mately the same extent as does calcium.

9. All of this evidence is in support of the hypothesis that calcium stimu- lates the succinoxidase system by activating the cozymase nucleotidase present in the tissue so that cozymase is destroyed and cannot function in the dehydrogenation of malate to oxalacetate, which is strongly inhibitory to the succinoxidase system.

BIBLIOGRAPHY

1. Axelrod, A. E., Swingle, K. F., and Elvehjem, C. A., J. Biol. Chem., 140, 931 (1941).

2. Dixon, M., Manometric methods, Cambridge (1934).

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SWIKGLE, AXELROD, AND ELVEHJEM 591

3. Seevers, M. H., and Shideman, F. E., Science, 94, 351 (1941). 4. Potter, V. R., and Elvehjem, C. A., J. Biol. Chem., 114,495 (1936). 5. Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 66,375 (1925). 6. Axelrod, A. E., and Elvehjem, C. A., J. Biol. Chem., 131,77 (1939). 7. Simon, L. S., Compt. rend. Acad., 13’7, 855 (1903). 8. Elliott, K. A. C., and Greig, M. E., Biochem. J., 32, 1407 (1938). 9. Das, N. B., Ark. Kemi, IMineral. o. Geol., 13 A, No. 7 (1938).

10. von Euler, H., and Gunther, C., 2. physiol. Chem., 243, 1 (1936). 11. von Euler, H., and Heiwinkel, H., Naturwissenschuften, 26, 269 (1937). 12. Mann, J. G., and Quastel, J. H., Biochem. J., 36, 502 (1941). 13. Potter, V. R., Ark. Kemi, Mineral. o. Geol., 13 B, No. 7 (1939). 14. Edson, N. L., Biochem. J., 29, 2082 (1935). 15. Ostern, P. Z., 2. physiol. Chem., 218, 160 (1933). 16. Cohen, P. P., J. BioZ. Chem., 136, 565 (1940). 17. Elsden, S., Biochem. J., 33, 1890 (1939). 18. Chain, E., B&hem. J., 33, 407 (1939).

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ElvehjemKarl F. Swingle, A. E. Axelrod and C. A.

SUCCINOXIDASE SYSTEMCALCIUM SALTS ON THE

THE MECHANISM OF THE EFFECT OF

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