Thyroxine Benjamin From the Biological
and Succinate Oxidation J. Kripkel
and Arley T. Beve?
Received June 20, 1955 INTRODUCTION
Gemmill, in 1952, stated that the addition of thyroxine to a cell-free succinoxidase preparation from a rat heart homogenate permitted an increase in oxygen uptake (1,2). His efforts have since been duplicated (3, 4). Attempts to isolate the point or points of this action were not conclusive. The possibility that the role of thyroxine in such an ensymic phenomenon may involve an oxidation-reduction mechanism has yielded interesting data in recent years. In 1941, it was shown that only those isomers of thyroxine which could form a quinoid through the hydroxyl group were capable of physiological activity (2,5). Evert has shown that thyroxine may act as a reducing agent to certain metallic ions present in a higher valence state (6). The experiments described in the present paper are believed to support a reductive role for thyroxine in its effect upon the succinoxidase system. MATERIALS AND METHODS The following materials were used in these experiments, as stock solutions, in the concentrations given. 1. 0.185 M sodium succinate. 2. 0.1 M NazHPOa-KHzPOa buffer, pH 7.2. 3. 1% KCN, adjusted to pH 7.2 with 1 N acetic acid. 4. 1% cytochrome c in 0.570 N&l. 5. p-Chloromercuribenzoic acid (PCMB), molarities as indicated. 6. Lapachol [2-hydroxy-3(3-methyl-2-butenyl)-1,4-naphthoquinonel, molarities as indicated. 7. 0.4% neotetrazolium chloride (NTZ). 1 Present address: Tufts University Medical School, Boston, Mass. 2 Present address: Dept. of Biochemistry, School of Medicine, Univ. of Oklahoma, Oklahoma City, Oklahoma. 326
8. 3.0 X 1OP M L-thyroxine (Squibb), in 0.005 31 NaOH, prepared shortly before each experiment. 9. All solutions above were prepared with glass-distilled water. 10. 50% trichloroacetic acid (TCA). The animals used u-ere loo-day or older albino rats from the Harvard colony. ?;o distinction was made as t.o the sex of animals selected. The animals were killed by a sharp blow to the head, and the hearts were removed as rapidly as possible. The enzyme preparat’ion w-as made by homogenizat,ion of 0.5 g. of rat heart in 10 ml. of cold phosphate buffer. The homogenate was centrifuged for 4 min. in :L clinical centrifuge to remove whole cells and debris. This is essentially the s:mw preparation used by Gemmill as succinoxidase source. The supernatant was used as the succinateecytochrome c reductase source. Thcl supernatant was routinely diluted by adding 1 vol. of phosphate buffer and 1 part of 0.005 M NaOH. When the effect of thyroxine was to be studied, it was added in 0.005 111 NaOH. The activity of the succinate-cytochrome c reductase @CR) preparation, modified as above, was measured by recording the rate of redurt,ion of cytochrome c at 550 rnp in the Beckman DU spectrophotometer with succinatc as substrate and with cytochrome oxidase inhibited by cyanide ion. The observed optical density was recorded at 15.sec. intervals after the addition of the enzymcl for a 2-min. period, and the average optical density change per minute was C:I~VIII:rted. The amount of cytochrome c’ reduced was calculated by employing the molecular extinction coefficient for the difference in absorption between oxidized and reduced cytochrome c (~a60 = 1.71 X l(F). EXPERIMENTAL
Experiment 1: The h’flect of Incubation upon ObservedRate in the Presenceand Absence of Thyroxine An SCR preparation, prepared as described above, in duplicate alicluots, one containing thyroxine, the other omitting thyroxine, was removed from the ice bath immediately after preparation and incubated at 25°C. for 5 min. for thermal equilibrium in a constant-temperakre water bath. Thyroxine concentration in the incubat’ion mixture was 1.0 X lOwA AI. At the end of 5 min. incubation, t’he initial zero-t,ime activity for the two preparations was determined. The activities of the t’wo were then determined at 15 and 30 min. from t’he initial measurement. The results are shown in Table 1. Experiment 2: The E#ect of Oxygen The original activities were determined of SCR preparations, prepared and diluted as described to furnish a thyroxine-containing preparation and a t,hyroxine-less control. These t’wo preparations were divided int’o two aliquots. A%llfour tubes were incubated in an ite bath. During the
TABLE I Protection by Thyroxine Against Incubation Loss of SCR Activitya Reaction mixture: Both tubes contained the following: succinate, 0.2 ml.; 1.6 ml. buffer, 0.1 ml. cyanide, 0.6 ml. water, 0.4 ml. cytochrome c, 0.1 ml. enzyme. Final concentration of thyroxine in the cuvette mixture was 3.3 X 10-“&f; control and thyroxine cuvettes were measured against a blank containing 0.4 ml. cytochrome c, 2.5 ml. buffer, and 0.1 ml. enzyme. 15 Time
0, % S.D.
53 f8.5 97 f3
a Per cent (%) refers to percentage of the original zero time activity remaining. S.D. refers to standard deviation of the mean of 18 experiments. 100% Control = 3.34 X 10-z &f cyt. c reduced/min. 100~o Thyroxine = 3.21 X lo-% PM cyt. c reduced/min.
5-min. incubation one of the tubes containing thyroxine and one of the tubes without thyroxine were subjected to pure oxygen gas introduced by slow controlled bubbling of the gas through a capillary pipet. The activities of all four preparations were determined at the end of the incubation. The data are presented in Table II. Experiment 5: Nonprotection of Calcium Phosphate Gel The standard diluted enzyme preparation was modified by the addition of aged calcium phosphate gel to make a solution 0.66 % with respect to the gel. The preparation was incubated at 25”C., and the activity levels were measured at zero time after a 5-min. thermal equilibrium, at 15 min. and at 30 min. The test conditions of Expt. 1 were employed. The standard control range of Expt. 1 is shown for comparison. The results are given in Table III. TABLE II Protection by Thyroxine Against Oxygen Exposure Reaction mixture same as in Table I Treatment
Control Plus thyroxine @ Activity to percentage
3.042 (100%) 2.330 (100%)
1.746 (57%) 2.520 (87%)
expressed in units X 10ee p&f cytochrome of original activity remaining.
2.84 (93%) 2.782 (97%)
4: Thyroxine and Succinic Dehydrogenass Actior~
Triplicate tubes containing succinate, the standard diluted enzyme preparations, buffer, cyanide, and NTZ, in the proportions listed in Table IV, were incubated for 20 min. at 25°C. One milliliter of 50 % TCA was added to each tube, and the contents were centrifuged in the cold at 2500 r.p.m. for 15 min. The precipitated protein and adsorbed colored diformazan were in the residue. The supernatant was discarded. Five milliliters of cold acetone was added to each tube, and the pigment was extracted by stirring and recentrifuging. The amount of reduced NT2 was determined by measurement in the Beckman DU spectrophotometer at 490 ml.r. The extinction coefficient for reduced NTZ in acetone was determined from twice-recrystallized diformazan made up as a standard TABLE
100% activity of the calcium phosphate IOF p-W cytochrome c reduced/min. Control Time
of Thyroxine in units
‘I Molarit,ies mixtures.
3.3 X 10-C fir of PCMB
X 10ee PM cytochrome Without
0.25 ml. sodium succinate, 0.10 enzyme with enz,vme containing
Incubation mixtures contained 0.25 ml. NTZ, ml. sodium cyanide, 0.90 ml. buffer, and 0.50 ml. thyroxine or 0.005 211 NaOH as control.
was 3.5 X I.
Against Succinic on Incubation
gel containing preparation activity same as in Table
1.7 X 10-Y
c reduced/min. PCMB”
(66%) (68% )
6.6 X 10”
0.306 0.180 in final
(11%) (7%) reaction
None 3.3 x 10-S 6.6 x 10-h 1.0 x 10-d
3.510 2.646 2.142 1.692
100 75 61 48
3.420 3.096 2.646 2.214
100 90 78 64
a Lapachol concentrations refer to final cuvette test mixture. * Concentration of thyroxine in cuvette test mixture was 3.3 X 1OF M. Activity is expressed in units X 10W2PM cytochrome c reduced/min.
in acetone. The observed molecular extinction coefficient was ~4~0= 1.94 X 104. The results of three experiments are combined (Table IV). Final concentration of thyroxine in the incubation mixture was 2.5 X lO+ M. Experiment 5: E$ect of Thyroxine upon PCMB Inhibition
The experimental method of Expt. 1 was repeated, with the concentrations of PCMB added to the enzyme preparation in 0.005 M NaOH in the dilution step of preparation. The inhibition produced by two concentrations of PCMB and the inability of thyroxine to prevent the inhibition is shown in Table V. Activity of SCR.was recorded immediately upon addition of PCMB. Experiment 6: E$ect of Thyroxine upon Lapachol Inhibition
Lapachol was added to the enzyme preparation at the dilution step in 0.005 M NaOH to make the final concentrations indicated in Table VI. The immediate SCR activity resulting in the presence and absence of thyroxine was determined. DISCUSSION
Experiment 1 indicates that incubation of the enzyme with thyroxine under the mild oxidizing conditions present in the experiment permits the maintenance of the original enzyme activity level. In the absence of thyroxine the succinate-cytochrome c reductase level falls off rapidly. If measured manometrically with the attendant shaking in a Warburg apparatus, a difference between the succinoxidase activities of the two
preparations would be observed. It would seem likely that t,hc report,ed differences in oxygen uptake in vitro between thyroxine and cont’rol (l--4) resulted not from a,n activation over the original enzyme capnbility, but by means of a preservation of the original activit)y of tht: preparation. The preservation is pH-sensitive. It can be observed over a pH range of 6.9-7.3. At a pH of 8.2 the SCR activity in the presence and absence of thyroxine was the same and remained steady over a period of an hou:r at 25°C. with no loss of activity. Keilin and Hartree have shown that the succinic dehydrogenase-cytochrome system of a cell-free extract behaves similarly to that of the intact cell (7). The acbivity and efficiency are affected by the same inhibitors and to the same degree. Slater demonstrated that the succinoxidase system is a colloidal aggregation of particles which, for the complete oxidation of succinate, requires a chain of hydrogen or electron carriers that appear to be firmly attached to these particles and thus exist in an intimate spatial relationship (8). Inhibition of such a system can take one of two forms: a chemical one, whereby certain essential groups are specifically affected, or a physical one where the organization of the particles and their mutual accessibility are disturbed. It is important to determine then, the cause of the loss of activity of the control during the incubation period as in Expt. 1, since t’hyroxine addition appears to protect the system. Experiment 2 shows that thyroxine protects against the specific inhibiting effect of bubbled oxygen, inferring that an oxygen-labile system is protected. Slater has stated that irreversibility of inhibition in the presence of calcium phosphate gel is presumptive indication of a true chemical inhibition (8). The final concentration of the gel (0.66%) employed in Expt. 3 is at least 25 times that of the usual thyroxine level expressed on a percentage basis. The inability of the gel to reverse or prevent the inhibition of mild oxidizing conditions during the air exposure would indicate that thyroxine prevents a chemical inhibition rather t,han a physical one, although this criterion alone cannot be taken as final. An effort was made in Expt. 4 to determine the site of preservation at the level of the succinic dehydrogenase complex. For the reduction of redox dyes such as methylene blue and neotetrazolium, it is likely that reduction depends upon succinic dehydrogenase, i.e., the specific substrate dehydrogenating enzyme, and upon cytochrome b (7). Whether or not NTZ reduction depends only upon succinic dehydrogenase or upon
both has not been conclusively determined since the redox potential of NT2 is somewhat less than that of methylene blue. However, if the protective effect of thyroxine could not be demonstrated with NT2 reduction, the site of protection at the dehydrogenase complex level could be eliminated, and the steps above it leading to cytochrome c reduction be more positively implicated. The results of the experiment suggest that thyroxine acts upon the initial substrate dehydrogenase to stabilize it during the incubation since the amount of protection and percentage difference between control and thyroxine-protected systems are fully as great as that observed in the succinate-cytochrome c reductase protection. Hopkins has repeatedly shown that the dehydrogenation enzyme of the complex has an oxygen-labile sulfhydryl group, and that biological activity of the enzyme is dependent upon preservation of a reduced state for the group (10). Slater has divided the complex into an oxidative (dehydrogenating) enzyme and cytochrome b portion with methylene blue measurements, while succinoxidase measurements include the total complex including cytochrome c and cytochrome oxidase (11). Using both procedures, he has shown that the succinoxidase complex is affected by inhibition by an oxidizing agent more than the dehydrogenase-cytochrome b portion (12). Since cytochrome c and cytochrome oxidase are not oxygen-labile, Slater concludes that Slater’s factor also contains an oxygen-labile group, but not necessarily an -SH group. He also has found that this group is released from inhibition with more difficulty than the -SH group of the dehydrogenating enzyme. Gemmill concluded that cytochrome oxidase was not involved in the activation by thyroxine of the succinoxidase system (2). Experiment 5 demonstrates that the inhibition by PCMB, presumably upon sulfhydryl groups of the succinate-cytochrome c complex, cannot be prevented by the presence of thyroxine, added simultaneously. Experiment 6, however, would indicate that the degree of inhibition by lapachol, an oxidizing agent, can be reduced in the presence of thyroxine. A possible explanation of the above findings can be revealed through a consideration of the mechanisms of the inhibitions involved. PCMB, an organic mercurial, combines with essential sulfhydryl groups of succinic dehydrogenase producing a stable bond, and, by blocking the formation of an enzyme-substrate complex, renders the enzyme inactive. Lapachol, as an oxidizing agent (13, 14), would not combine with the sulfhydryl
AND SUCCIRTrlTE OXIDATIOS
group I)ut would oxidize it to t.he disulfide, --S--S-, linkage and thrw lkng al)outsinhibition. In its oxidizing capacity ib could also bring about inhibition through dwtnlctlion of the 1mknow-n osygrn-lahilc~ group (II’ Slatcr’s factor. Thyroxine may then act to protect the labile forms from oxidat’ion, serving as a reducing agent. It may be thought of as being preferentially oxidized in place of the essential --SH groups of the dehydrogenase or of similarly labile groups of Slater’s factor, arid thus prevent inhibition. ,\ reducing agent without an -SH group, such as thyroxine, would not reverse PCMB inhibition. Thyroxine would, however, louver the degree of inhibition produced by lapachol oxidation since lapachol does not combine with sulfhydryl groups. The experimental results indicate such an action, although the presence of an unknown oxygen-labile group in Slater’s factor prevents direct confirmation. To what extent the protection by thyroxine of succinabe-cytochrome c reductase preparations in vitro can be transferred to a physiological role is not possible t’o evaluate. The experiments of Wiswell e2al. (3) in confirming and extending the original findings by Gemmill (1,2) indicate a positive correlation between thyroxine stimulation of succinoxidase irr &fro and stimulation of the enzyme system in the same tissues aftw thyroxine administration. These findings would be unitized if the effects observed in vitro as a preservation phenomenon in our experiments operate through the same mechanism physiologically to regulate the amount, of active enzyme present’ at the time of tissue csamination in inject’ion st’udies. ACKNOWLEDGMENTS The authors take this opportunity to thank Professor Frederick L. Hisaw for the kind use of his laboratory facilities and for his generosity and advice. This project was supported in part by an institutional grant to Prof. Hisaw hy Harvard I!niversit(y from funds made available by the American Cancer Society. SUMMARY
1. Thyroxine preserves the activity of succinate-cytochrome c reductase act’ivity in preparations from rat heart homogenat’e, but does not increase the original activity. 2. The preservation appears to be a chemical rather than physical effect. 3. At least one site of action is in the succinic dehydrogenase complex where sulfhydryl groups are protected from oxidat’ion.
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GEMMILL, C. L., hz. J. Physiol. 170, 502 (1952). GEMMILL, C. I,., J. Clin. Endocrinol. and Metabolism 12, 1300 (1952). REID, C., AND KOSSA, J., Arch. Biochem. and Biophys. 63, 321 (1954). WISWELL, J. G., ZIERLER, K. I,., FASAR’O, M. B., AND ASPER, S. P., JR., Bull. Johns Hopkins Hosp. 94, 94 (1954). NIEMANN, C., AND REDMANN, C. E., J. Am. Chem. Sot. 63, 2685 (1941). EVERT, H. E., Federation Proc. 12, 657 (1953). KEILIN, D., AND HARTREE, E. T., Proc. Roy. Sot. (London) Bl29, 277 (1940). SLATER, E. G., Biochem. J. 46,9 (1949). SHELTON, E., AND SCHNEIDER, W. C., Anat. Record 112, 61 (1952). HOPKINS, F. G., AND MORGAN, E. J., Biochem. J. 33, 611 (1938). SLATER, E. G., Biochem. J. 46, 1 (1949). SLATER, E. G., Biochem. J. 46, 130 (1949). WENDEL, W. B., Federation Proc. 6, 406 (1946). BALL, E. G., ANFINSEN, C. B., AND COOPER, C., J. Biol. Chem. 168,257 (1947).