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The role of mitochondria in in vitro tryptophan peroxidase increments
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
32-45
84,
(19%)
The Role of Mitochondria in in Vitro Tryptophan Peroxidase Increment& Malcolm From
the Biochemical
and Irene J. Rydzie12
W. Gordon
Research
Laboratory,
Received
Institute
February
of Living,
Hartford,
Connecticut
19, 1959
Clouet and Gordon (1) reported that the rate of formation of kynurenine increases in cell-free, rat liver homogenates when supplemented with an amino acid mixture and ATP.3 They also demonstrated that the response of the system is not changed by the addition of an appropriate peroxidegenerating system and of kynurenine formamidase, both of which are required for optimal activity in the assay of purified TPO preparations (2). For these reasons they suggested that the in vitro synthesis of one or more of the components of the TPO complex might account for the observed activity increment. An aspect of this work which did not easily fit a hypothesis of TPO synthesis was the finding that mitochondria are required for the in vitro increment effect. The experiments reported in this paper were undertaken to investigate the mitochondrial requirement and to try to determine by direct methods whether or not in vitro protein synthesis did in fact account for the observed increment in TPO activity. It appears probable that under the conditions employed by Clouet and Gordon and in the experiments described here, the increment in TPO activity is a function of the permea1 This investigation was supported in part by the Medical Research and Development Board, Office of the Surgeon General, Department of the Army under Contract No. DA-49.007.MD-204, and in part by the U. S. Atomic Energy Commission under Contract No. AT(30-1).2026. 2 A preliminary account of some of this work was presented at the April, 1958 meeting of the American Chemical Society. 3 The following abbreviations are used: ATP, adenosine triphosphate; TPO, tryptophan peroxidase; EDTA, ethylenediamine tetraacetate; Tris, tris(hydroxymethyl)aminomethane; G-6-P, glucose g-phosphate; BRIJ, Brij 35 (Atlas Powder Co., Wilmington, Del.), a polyoxyethylene lauryl alcohol; ATPase, adenosinetriphosphatase; RNA, ribonucleic acid; RNase, ribonuclease, a 5X recrystallized bovine pancreas preparation, Sigma Laboratories; Cortef, a 500 mg.% solution of 17-hydroxycorticosterone in 50yo ethanol, prepared by the Upjohn Co. This solution was diluted 1:5O with distilled water before use; and TCA, trichloroacetic acid. 32
MITOCHONDRIA
AND TPO ACTIVITY
33
bility characteristics of subcellular particulates, and is not, to any significant degree, a manifestation of de novo synthesis of enzyme protein. METHODS
Preparation of Tissue Fractions Young Wistar strain rats, both male and female, weighing from 70 to 120 g. were used, although best results are obtained with animals weighing less than 100 g. The animals were killed by decapitation, the livers were quickly removed and, in most experiments, they were immediately immersed into an ice-cold solution of 0.44 M sucrose, 0.001 M with respect to EDTA, and containing sufficient Tris buffer to bring the pH to 7.4. The livers were removed from the sucrose solution in the cold room, blotted on filter paper, minced with a razor blade, and weighed. Twenty-five per cent homogenates were prepared in the sucrose solution described above, using a Teflon pestle, glass homogenizer. Care was taken to keep the homogenizing vessel ice-cold and not to permit the pestle speed to exceed 1300 r.p.m. The homogenate was centrifuged at 600 X g for 15 min. in a refrigerated centrifuge, and the nonsedimented fraction was decanted and used in all experiments. Microscopic examination of methylene blue-stained aliquots of this supernatant revealed only an occasional unbroken cell. This preparation will be referred to as the MtMcS fraction. In experiments designed to evaluate the effects of tonicity on the increment effects, a 0.25 M sucrose homogenate was prepared as above.
Incubation Medium and Methods All evaluations of TPO activity were made in an incubation medium consisting of G-6-P, 100 pmoles; MgCL , 120 pmoles; ATP, 126 rmoles; cytochrome c, 0.5 mg.; succinic acid, 260 rmoles; 0.25 M Tris buffer, pH 7.5, 2.5 ml.; Krebs-Ringer phosphate buffer, 0.0015 M with respect to EDTA, pH 7.2, 6.0 ml.; 25 mg. L-tryptophan; and sufficient water to bring the volume to 16.5 ml. In those experiments in which additional amino acids were used, 5 ml. of a solution containing 0.5 mg./ml. of the appropriate amino acids was added in place of water, bringing the final volume to 16.5 ml., as above. The incubation medium and 8.5 ml. of the 25% homogenate were either equilibrated separately at 37” for 510 min. before mixing or were mixed at 0” and agitated for 5-10 min. at 37” before the zero time sample was removed for analysis. Aliquots of 2-4 ml. of the above mixtures were pipetted into separate 20-ml. beakers and were shaken in an atmosphere of 100% 02 in a Dubnoff metabolic shaker for various lengths of time before stopping the reaction with an equal volume of 0.6 M perchloric acid. In some experiments other materials were added to this incubation medium. They are indicated under the appropriate experiments.
In Vivo TPO Induction A dose of 1 mg./g. rat body weight, dispersed in 2.5 ml. water, was injected intraperitoneally into animals fasted for at least 16 hr. In such experiments, a control group was always prepared into which 2.5 ml. water was injected. After 5 hr., the animals were killed and the livers prepared for experimentation as described above. (5) With Epinephrine. Animals were injected intraperitoneally with 30 pg. of an aqueous solution of epinephrine. These animals were killed 54 hr. after injection and the livers prepared for experiment as described above. (a)
With
L-Tryptopkan.
34
GORDON
AND
Kynurenine
RYDZIEL
Determination
TPO activity was estimated from the rate of kynurenine production, following the essentials of the procedure of Knox (a), except that 0.3 M perchloric acid was used to precipitate protein since clearer filtrates were obtained in the presence of detergents. After standing in the cold for at least 3 hr., the suspension was centrifuged, an aliquot of the supernatant was brought to neutrality with a standardized solution of NaOH, and the absorption of the resulting solution was determined at 365 ml.c. Enzyme activity is expressed in micromoles kynurenine formed/mg. N/hr. The molar absorption of kynurenine is taken as 4.54 X 103.
Radioactive Amino Acid Experiments Two kinds of radioactive amino acid experiments are described. In one group, the CWlabeled amino acids were prepared by acid hydrolysis of Chlorella vulgaris grown in the presence of NaHCi403 . After purification by ion-exchange chromatography, the specific activity of this combined group of amino acids (which contained no detectable quantities of tryptophan) was 34,600 counts/min./mg. in the thin-window, Geiger-Miiller counter employed. In the other group of experiments, nn-tryptophan3-CY4, specific activity 7.86 mc./mmole, was used. The preparation of the incubation medium and of the liver homogenate was identical to that described above, except that 120,000 counts of the appropriate amino acid solution was added to the incubation mixture prior to mixing with the homogenate. After a suitable period of incubation, the proteins were precipitated as described above, but otherwise prepared for counting as described by Sachs (3). Infinite thickness in this counting method was found to be at 12 mg. Results are expressed in specific activity, counts/min./mg. protein.
Chemical Methods Nitrogen
was determined
by a micro-Kjeldahl
method.
RESULTS
The Effect of Added Amino Acids on the Increment in TPO Activity Reports on in vitro increments in the activity of amylase point out that this effect is dependent on the uptake of only two amino acids, glycine and tyrosine (4). A planned systematic investigation of the amino acid requirements of the TPO increment was therefore begun with a medium unsupplemented with amino acids other than the substrate of the reaction, Ltryptophan. Figure 1 compares the TPO increment obtained in media which differed only in their amino acid composition. Contrary to expectation, it was found that a complete amino acid supplement inhibited TPO activity, while the effect of the essential amino acid mixture was small. While the possibility was considered that addition of amino acids competitively promoted the synthesis of protein other than TPO, or that a particular amino acid was exerting a specific inhibitory effect, it seemed advisable at this point to attempt a direct evaluation of the over-all rate of protein synthesis in this medium. Further investigation of the effects
MITOCHONDRIA
.4ND
TPO
35
ACTIVITY
Bcontrol OEsssntid
Amino
Acids
INCUBATION
TIME
1. Effect of added amino acids on TPO activity increments. The incubations were carried out as described in the text. The plotted values are the activities of the time intervals indicated from which zero-time values have been subtracted, so that direct comparison of activity per time interval is possible. Essential amino acids, equal weights of the L-forms of the following amino acids were added in the amounts indicated in the text: histidine, lysine, phenylalanine, leucine, isoleucine, threonine, methionine, and valine. For the “complete” amino acids, in addition to the above, the L-forms of the following amino acids were added in the same amounts: glycine, alanine, serine, aspartic acid, citrulline, tyrosine, glutamic acid, proline, hydroxyproline, arginine, ornithine, and cysteine. FIG.
of individual amino acids was therefore deferred. As the work developed, such a study did not seem crucial to the problem of the in vitro increments and was, therefore, not undertaken. The E$ect of RNase on the in Vitro Increments
of TPO Activity
While RNA or some of its precursors undoubtedly play some role in the synthesis of enzymes, the crucial intermedia.tes in this process have nob yet been determined (5). Nevertheless, on the basis of current information, it seems highly probable that gross interference with RNA metabolism would somehow affect the rate of synthesis of new enzyme protein. Accordingly, the MtMcS fraction was incubated at 37” with and without added RNase. This incubation mixture, which contained no added tryptophan, was aliquoted at several time intervals and the activity of TPO determined in the fortified medium previously described. The results are shown in Table I. No significant effect of RNase was observed. The E$ect of Ethionine and p-Fluoro-Lsphenylalanine in Vitro Increments of TPO Activity
on the
The first direct indication that de novo protein synthesis probably did not account for the observed increments in TPO activity was obtained by studying the effects of these two protein synthesis inhibitors on the in
36
GORDON
AND
TABLE Effect
-
of Ribonuclease
on TPO
TPO activity Incubation time of YtMcS prior to TPO assay, in minutes
I Activity
(in supplemented
Increments medium
after
in Viva
incubation
of YtMcS)
20
Activitya
With 0.1 mg./ml. RNase 0 5 10 15 Untreated MtMcS 0 5 10 15
RYDZIEL
Initial rateb
Activitya
Rate incrementC
Activity5
Rate incrementC
0.108 0.153 0.065 0.053
1.0 1.4 0.6 0.5
0.523 0.440 0.386 0.237
3.8 2.5 3.0 1.7
1.14 0.773 0.570 0.346
5.7 3.1 1.7 1.0
0.085 0.071 0.061 0.044
1.0 0.6 0.7 0.5
0.493 0.450 0.323 0.207
4.8 3.2 3.1 1.9
1.02 0.727 0.526 0.303
6.2 3.2 2.4 1.1
* Activity = pmoles kynurenine/mg. N/hr. b Initial rate = O-20 min. activity = 1.0. c Rate increment = (pmoles kynurenine formed in 20-min. interval/pmoles renine formed in initial 20-min. interval).
kynu-
increment. Both ethionine and p-fluoro-nn-phenylalanine have been shown to inhibit protein synthesis (6, 7), and, in particular, ethionine has been shown to inhibit the in viva induction of TPO activity when administered to the whole animal and to inhibit the induction of TPO activity in perfused livers (8) and the induction of TPO activity in liver slices (9). Figure 2 shows that the small changes in TPO increments due to ethionine fall close to the experimental error of the analytical method. Most important, the effect is not appreciably diminished with increasing concentrations of the inhibitor. It remains possible that ethionine requires activation before it can partake in the reactions in which it manifests its inhibitory properties. As can be seen in Fig. 3, p-fluoro-nn-phenylalanine differs considerably from ethionine in its effects on the activity increments. After 30 min., significant inhibition of the increment occurs, but no further reduction in TPO activity is noted after this time. Furthermore this 30-min. value approximates the value that is characteristic of the control preparation after 45 min. of incubation. The gross appearance of the p-fluoro-mphenylalanine mixture may provide the explanation for this effect. Gross agglomeration of the incubation mixture began to become evident after 30 min. of incubation. This phenomena will be discussedlater in terms of the
vitro
MITOCHONDRIA
J
AND
TPO
37
ACTIVITY
No Et hionine
- .INCUBATION
TIME
2. Effect of ethionine on TPO activity. Kynurenine scribed in the text. The plotted values represent the activity N/hr.) per indicated time interval. FIG.
I 1-45
INCUBATION
was determined as debmoles kynurenine/mg.
45-6 ‘0
TIME
FIG. 3. Effect of p-fluoro-nn-phenylalanine on TPO activity. Kynurenine termined as described in the text. The plotted values represent the activity kynurenine/mg. N/hr.) per indicated time interval.
was debmoles
mechanism which is thought to be operating in the production of in vitro TPO increments.
general
The E$ect of the Nonionic Detergent, BRIJ, on the in Vitro Increments in TPO Activity At this point an analogy between the observed TPO increments and the increments in activity of several other enzymes in aged mitochondrial preparations was considered. Among others, it is known that Mg++-activated ATPase (10) and catalase (11) increase their activity as the integrity of the mitochondria is compromised. The data from the experiments thus
38
GORDON
HNo
AND
RYDZIEL
Brij
I .35 mg./ml.
O-IO
IO-20 20-30 INCUBATION
30-40 TIME
FIG. 4. Effect of BRIJ on TPO activity. Kynurenine in the text. The plotted values represent the activity per indicated time interval.
was determined as described (pmoles kynurenine/mg. N/hr.)
far reported would be consistent with such an interpretation. Accordingly, the effect of BRIJ on the reaction was studied, since similar detergents have been shown to modify the physical properties of mitochondria (12). The response of the system to BRIJ is shown in Fig. 4. The increase in TPO activity in the presence of BRIJ is greater than found with the controls. Microscopic examination of the reaction mixtures in the presence and absence of BRIJ, reveal that the number of particles which stain with Janus Green B are markedly reduced in the BRIJ-treated mixtures. The qualitative microscopic impressions are consistent with the assumption that the increment in TPO activity follows the disappearance of particles which stain as mitochondria. Unfortunately, the centrifuge equipment available did not permit studies of the changes in the microsomal fraction. Experiments are planned to determine whether or not these subcellular particles also change in number or character in the course of the experiments. Uptake
of Radioactive
Amino
Acids
The uptake of radioactive amino acids for the Chlorella vulgaris hydrolyzate is shown in Table II. No significant increment of radioactive amino acid uptake is found in any of the mixtures which promote the TPO activity increment. As an example is plotted the increments in TPO activity obtained in this experiment with the control preparation and with the identical preparation supplemented with BRIJ. The possibility was considered that the appearance of TPO activity might involve a dual role for tryptophan: one, as the substrate of the reaction, the other, as the stimulus for the reaction, perhaps involving the
MITOCHONDRIA
Incrementation Incubation of MthfcS radioactive medium
min. 0
20 40
time in
TPO
39
ACTIVITY
TABLE II Amino Acids in TCA-Insoluble Fractions during of TPO Activity under Two Conditions
of Radioactive
Incorporation
AND
Specific Control
radioactivity BRIJ
counts/min./mg.
3.0 10.5 17.0
TPO activity (0.35 mg./ml.)
protein
4.0 7.5 12.5
in Vitro
increment
Control
BRIJ
,moles
kynureninejmg.
0.105 0.170
(0.35 mg./ml.) N/hr.
0.125 0.280
fixing of tryptophan into some protein TPO precursor. Experiments with tryptophan-Cl4 gave no indication that this compound was fixed at a greater rate in the more active preparations compared to the less active ones. Furthermore, even at the earliest stage of the reaction, no evidence was obtained that any product other than the ones already described (2) was formed in the reaction. This finding appears to eliminate from consideration the possibility that TPO, like Mg++-activated ATPase (lo), has some other function analogous in principle to the postulated transphosphorylase role of Mg++-activated ATPase in intact mitochondria. The Efect of Ascorbic Acid on the TPO Increment Even though it had been shown previously (1) that the addition of a peroxide-generating system and catalase did not influence the increment in TPO activity, it seemedadvisable to test the effect of reducing substances which might be present in the homogenate. Furthermore, it has been reported that ascorbic acid will delay the manifestation of amine oxidase activity (13) and that its presence is important for the in vitro inductions of amylase (7). Figure 5 showsthat ascorbic acid in a concentration of 12.5 mg./25 ml. incubation mixture is without effect in either stimulating or inhibiting the TPO increment. The insensitivity of the system to ascorbic acid is manifested under two different experimental conditions. In the first, the ascorbic acid is mixed with the incubation medium and the MtMcS and the mixture is shaken in air for 10 min. at 37” before a zero-time sample is obtained. In the second experiment, with the identical homogenate and incubation medium, the MtMcS and the incubation medium containing added ascorbic acid are prewarmed separately at 37” for 10 min. with shaking. They are then mixed and a zero-time sample is removed at once. If the lag in the manifestation of TPO activity is a reflection of unchanged ascorbic acid, as in the systems referred to above, a greater lag in full TPO activity would be expected in the second case where the ascorbic acid cannot react with the homogenate during the initial 10 min. In fact,
40
GORDON
AND
RYDZIEL
1 Mired
.25
Cold
fl M ixed After Pro - Warming
-?.2C z : \ f .IC Y I a C
IO-IO 0
INCUBATION
TIME
Fro. 5. Effect of ascorbic acid on TPO activity. Kynurenine was determined as described in the text. The plotted values represent the activity (pmoles kynurenine/ mg. N/hr.) per indicated time interval.
however, both react identically, suggesting that changes in ascorbic acid are not critical for the increase in TPO activity. The E$ect of Tonicity on TPO Increments The homogenizing medium employed in these experiments was chosen because of its ability to maintain mitochondrial integrity over prolonged periods (14). If the assumption that in vitro increments in TPO are a function of changes in mitochondrial integrity is correct, it should be possible to demonstrate greater increments in TPO activity if the mitochondrial integrity is deliberately interfered with. Since the experiments with BRIJ seemed to support this view, confirmatory experiments, involving the manipulation of the tonicity of the medium, were undertaken. Supernatants were prepared much like those described previously. Two modifications in the original homogenizing medium were made: 0.25 M sucrose was substituted for 0.44 M, so that, in subsequent manipulation, frankly hypotonic preparations could be made without excessive dilution; and, EDTA was omitted from the homogenizing medium, to eliminate the mitochondrial stabilizing effect of this compound. After centrifugation under the conditions described previously, the supernatant was divided into two fractions. One was incubated with an equal volume of water at 37” for 10 min.; the other was incubated with an equal volume of the Krebs-Ringer-EDTA solution described previously, at the same temperature and for the sametime. Both contained a small quantity of L-tryptophan (0.6 mg. in 12 ml.) to stabilize the TPO during this brief incubation. After incubation, the two differently treated preparations were mixed with
MITOCHONDRIA
. .3
FIG.
41
ACTIVITY
0 Isotonic i
0 2( INCUBATION scribed N/hr.)
TPO
~Hypotonic
5 \
AND
6. Effect of tonicity on TPO in the text. The plotted values per indicated time interval.
activity. represent
I
1 30-40 TIME Kynurenine the activity
was determined as de&moles kynurenine/mg.
buffer, salts, and the other addenda described previously so that they were once more identical to each other and to the other preparations described in this report except for the somewhat lowered sucrose concentration. Figure 6 describes the changes in TPO activity from these two preparations measured from the time that they were once again made identical to one another. Contrary to the results of the experiments with BRIJ, the increment in activity in the preparation treated with water is markedly less than from the preparations treated with Krebs-Ringer-EDTA during the IO-min. preincubation. This difference between observed and predicted * behavior will be referred to in the Discussion. The E$ect of in Vivo Injections of Epinephrine Increments in TPO Activity
on in Vitro
A sometimes distressing feature of the experiments reported here is the variability of the TPO response. With animals selected as described earlier, some response was always demonstrable. However, the response varies over wide limits. In some experiments up to sixfold increments in activity were obtained (see Table I) ; and in others, less than twofold (see Table III). An explanation of these findings may be found in a comparison of the in vitro inductions obtained from animals that had been disturbed by placing them in a shaker for 30 min. prior to sacrifice, with animals that were injected with epinephrine, and with a control group in which particular pains were taken not to introduce disturbing environmental changes prior to sacrifice. It is apparent from Table III that early sacrifice after injection with epinephrine, or after shaking, does not lead to significant changes in zero-time TPO activity, even though permitting a longer period of reaction
42
GORDOS
AND
RYDZIEL
TABLE Effect
of in Viva
Injection TPO
Activity
10 min. Treatment
of animal to sacrifice
III
of Epinephrine (after
on in Vitro indicated
period
20 min.
Increments of incubation
30 min.
in
TPO
Activity
in supplemented
medium)
40 min.
50 min.
prior “A c .A 2
% 3
a. 2
b 2% .- E au E 2 _____
Controld 3Opg. epinephrineintraperitoneally 36 hr. prior to sacrifice 30 min. of shaking prior to sacrifice Controld + 100 pg. epinephrine in vitro Controld + 100 pg. Cortef in vitro 8 Activity = pmoles b Rate = activity of c Rate increment = renine formed in initial d These are separate
Q.
L : 2% 3’”
2
2
E
0. 2
E Se .d 0 %B Ps
0. 2
e 20, .- c $2 Fd
0.091 0.083
1.0 1.0
0.176 0.207
0.93 1.50
0.321 0.380
1.59 2.09
0.500 0.586
1.97 2.48
0.631 0.886
1.44 3.62
0.103
1.0
0.215
0.99
0.396
1.76
0.586
1.84
0.804
2.12
0.087
1.0
0.182
1.09
0.323
1.62
0.499
2.02
0.616
1.35
0.089
1.0
0.175
0.97
0.340
1.86
0.512
1.93,
0.632
1.35
kynurenine/mg. N/hr. initial 10.min. assay = 1.0. bmoles’ kynurenine formed in IO-min. IO-min. interval). aliquots of the same homogenate.
interval/pmoles
kynu-
in vivo would increase the zero-time activity also (15). However, in the epinephripjzed animals and in the animals that were shaken, the increase in TPO activity is much more marked after prolonged incubation than is characteristic of the control group. On the basis of these experiments, it seems likely that the varia.bility in response is a function of uncontrolled factors in the environment which by some undetermined mechanism may act to prime the test system for rather high increments in TPO activity, compared to the increments obtainable in animals that have not been agitated. Data on in vitro additions of epinephrine and Cortef are included to demonstrate that these hormones, both of which induce TPO activity in vivo (16), are without effect in vitro. DISCUSSION There seems little doubt, based on the results reported here, that the in vitro increments in TPO activity, which can be demonstrated under our conditions, do not reflect protein synthesis. It is, of course, possible that protein synthesis, in the very limited sense reported for the in vitro increments in amylase activity, i.e., the uptake of only two amino acids (4), also occurs in this system. However, the lack of correspondence between
MITOCHONDRIA
AND
TPO
ACTIVITY
43
activity increment and radioactive amino acid uptake makes even this possibility somewhat remote. Another, perhaps more compelling argument against protein synthesis, stems from a consideration of the increment and later fall in TPO activity in vitro. TPO activity under our conditions rises to levels which are three and four times greater than the activities reported by others for this enzyme (2, 16). However, after 30-40 min. of incubation, the activity falls and after an hour tends to reach values consistent with those reported previously for TPO activity. In addition to denaturation, an added explanation of this rise and fall in activity is suggested by the mitochondrial requirement for act,ivity increments. If part of the enzyme complex is initially organized in the mitochondria, and if this organization maintains its integrity while the permeability of the mitochondria increases, a more and more rapid reaction of the tryptophan might be expected. The maximal activity, however, falls as the elements of the TPO complex become solubilized. Under these conditions the kinetics of the reaction are determined by the probabilities of chance encounters between soluble enzymes and soluble substrates. Such a mechanism has been invoked by Green (17) to account for the rapid oxidation of Krebs-cycle intermediates in mitochondria. This mechanism is also supported by the difference in responseto BRIJ and hypotonic solutions. If the increment in activity comesabout by mitochondrial disruption and the consequent release of enzymic activity, the response to hypotonicity and to BRIJ would be expected to be similar. However, hypotonic solutions probably promote a more rapid disruption of mitochondria than they do changes in membrane permeability in nondisrupted mitochondria, while BRIJ very probably influences permeability to a greater extent than disruption since its effects were determined in isotonic solutions. Recknagel and Malamed have recently discussed these two aspects of mitochondrial swelling (18). Further support for this suggestion is found in the p-fluoro-m-phenylalanine experiment. Under our condition the p-fluoro-m-phenylalanine promotes, even under isotonic conditions, protein denaturation which is visible as a settling-out of gross agglomerated particles in the incubation medium. If this denaturation is assumed to occur by a process which simultaneously releases the components of the TPO complex into the medium, the rate of reaction would tend to fall to values characteristic of the concentration of the free enzyme components rather than the higher rates characteristic of their postulated organized forms. The TPO activity obtainable from older animals is as great per milligram N as that from younger animals. However, no consistent increment in activity can be demonstrated under our conditions with animals above 120
44
GORDON
ASD
RYDZIEL
g., while the response can always be elicited from animals below 100 g. The role of the mitochondria in the production of the in vitro increments may explain the relation of age to increment. Cooper and Tapley (19) have demonstrated that the degree of mitochondrial swelling under a given experimental condition is a function of age, nutrition, and the organ from which the mitochondria are obtained. It seems possible that the balance between enzyme complex disruption and changes in mitochondrial permeability also differs as a function of animal age. This aspect of the problem is under investigation in another connection and will be reported separately. The epinephrine data and the wide variety of hormones which stimulate TPO activity (15, 16) make it necessary to consider that the increments reported here are all a function of the hormonal changes produced by manipulating the animals prior to sacrifice. However, here too, changes in mitochondrial permeability must be considered, since it is known that various hormones influence this property of subcellular particulates also (14). This problem is an aspect of the one mentioned above and is also now under investigation. It is nevertheless interesting to speculate, at this point, whether tryptophan and hormones which do stimulate in vivo TPO increments, do so in the first instance by affecting mitochondrial permeability. It should be emphasized that the experiments reported here do not contradict evidence for in vivo protein synthesis of TPO under appropriate stimulation. The fact that the same kind of in vitro increments may be obtained both with adapted and nonadapted preparations (1) suggests that more enzyme is available for reaction in the adapted preparations, and presumably this added activity reflects additional enzyme. SUMMARY
In vitro increments of tryptophan peroxidase activity in cell-free preparations from rat liver are shown to be correlated with the permeability of mitochondria. Disruption of these particulates reduces the in vitro increment, but isotonic solutions containing substances which change the mitochondrial permeability promote such increments. REFERENCES 1. CLOUET, D. H., AND GORDON, M. W., Arch. Biochem. Biophys. 84, 22 (1959). 2. KNOX, W. E., in “Methods in Enzymology” (Colowick, S. P., and Kaplan, N. O., eds.) Vol. 2, p. 242. Academic Press, New York, 1955. 3. SACHS, H., J. Biol. Chem. 228, 23 (1957). 4. GARZO, T., PERL, K., T.-SZABO, M., ULMAN, A., AND STRAUB, F. B., Actu Physiol. Acud. Sci. Hung. 9,23 (1957). 5. CHANTRENNE, H., Ann. Rev. Biochem. 27, 35 (1958). 6. LEE, N. D., AND WILLIAMS, R. H., Biochim. et Biophys. Acta 9, 698 (1952).
MITOCHONDRIA
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
AND
TPO
ACTIVITY
45
STRAUB, F. B., AND ULMAN, A., Biochim. et Biophys. Acta 23, 665 (1957). PRICE, J. B., JR., AND DIETRICH, L. S., J. Biol. Chem. 337, 633 (1957). GIVEN, M., AND KNOX, W. E., Federation Proc. 16, 165 (1957). POTTER, V. R., AND RECKNAGEL, R. O., in “Phosphorus Metabolism” (McElroy, W. D., and Glass, B., eds.), p. 377. Johns Hopkins Press, Baltimore, 1951. ADAMS, D. H., AND BURGESS, E. A., Brit. J. Cancer 11, 310 (1957). WITTER, R. F., AND MINK, W., J. Biophys. Biochem. Cytol. $73 (1958). ACKERFELDT, S., Science 126, 117 (1957). TAPLEY, D. F., J. Biol. Chem. 222, 326 (1956). KNOX, W. E., Brit. J. Ezptl. Pathol. 32, 462 (1951). SCHLOR, J. M., AND FRIEDEN, E., J. Biol. Chem. 233, 612 (1958). GREEN, D. E., in “Chemical Pathways of Metabolism” (Greenberg, D. M., ed.), p. 27. Academic Press, New York, 1954. RECKNAGEL, R. O., AND MALAMED, S., J. Biol. Chem. 232, 705 (1958). COOPER, C., AND TAPLEY, D. F., Biochim. et Biophys. A& 26,426 (1957).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
32-45
84,
(19%)
The Role of Mitochondria in in Vitro Tryptophan Peroxidase Increment& Malcolm From
the Biochemical
and Irene J. Rydzie12
W. Gordon
Research
Laboratory,
Received
Institute
February
of Living,
Hartford,
Connecticut
19, 1959
Clouet and Gordon (1) reported that the rate of formation of kynurenine increases in cell-free, rat liver homogenates when supplemented with an amino acid mixture and ATP.3 They also demonstrated that the response of the system is not changed by the addition of an appropriate peroxidegenerating system and of kynurenine formamidase, both of which are required for optimal activity in the assay of purified TPO preparations (2). For these reasons they suggested that the in vitro synthesis of one or more of the components of the TPO complex might account for the observed activity increment. An aspect of this work which did not easily fit a hypothesis of TPO synthesis was the finding that mitochondria are required for the in vitro increment effect. The experiments reported in this paper were undertaken to investigate the mitochondrial requirement and to try to determine by direct methods whether or not in vitro protein synthesis did in fact account for the observed increment in TPO activity. It appears probable that under the conditions employed by Clouet and Gordon and in the experiments described here, the increment in TPO activity is a function of the permea1 This investigation was supported in part by the Medical Research and Development Board, Office of the Surgeon General, Department of the Army under Contract No. DA-49.007.MD-204, and in part by the U. S. Atomic Energy Commission under Contract No. AT(30-1).2026. 2 A preliminary account of some of this work was presented at the April, 1958 meeting of the American Chemical Society. 3 The following abbreviations are used: ATP, adenosine triphosphate; TPO, tryptophan peroxidase; EDTA, ethylenediamine tetraacetate; Tris, tris(hydroxymethyl)aminomethane; G-6-P, glucose g-phosphate; BRIJ, Brij 35 (Atlas Powder Co., Wilmington, Del.), a polyoxyethylene lauryl alcohol; ATPase, adenosinetriphosphatase; RNA, ribonucleic acid; RNase, ribonuclease, a 5X recrystallized bovine pancreas preparation, Sigma Laboratories; Cortef, a 500 mg.% solution of 17-hydroxycorticosterone in 50yo ethanol, prepared by the Upjohn Co. This solution was diluted 1:5O with distilled water before use; and TCA, trichloroacetic acid. 32
MITOCHONDRIA
AND TPO ACTIVITY
33
bility characteristics of subcellular particulates, and is not, to any significant degree, a manifestation of de novo synthesis of enzyme protein. METHODS
Preparation of Tissue Fractions Young Wistar strain rats, both male and female, weighing from 70 to 120 g. were used, although best results are obtained with animals weighing less than 100 g. The animals were killed by decapitation, the livers were quickly removed and, in most experiments, they were immediately immersed into an ice-cold solution of 0.44 M sucrose, 0.001 M with respect to EDTA, and containing sufficient Tris buffer to bring the pH to 7.4. The livers were removed from the sucrose solution in the cold room, blotted on filter paper, minced with a razor blade, and weighed. Twenty-five per cent homogenates were prepared in the sucrose solution described above, using a Teflon pestle, glass homogenizer. Care was taken to keep the homogenizing vessel ice-cold and not to permit the pestle speed to exceed 1300 r.p.m. The homogenate was centrifuged at 600 X g for 15 min. in a refrigerated centrifuge, and the nonsedimented fraction was decanted and used in all experiments. Microscopic examination of methylene blue-stained aliquots of this supernatant revealed only an occasional unbroken cell. This preparation will be referred to as the MtMcS fraction. In experiments designed to evaluate the effects of tonicity on the increment effects, a 0.25 M sucrose homogenate was prepared as above.
Incubation Medium and Methods All evaluations of TPO activity were made in an incubation medium consisting of G-6-P, 100 pmoles; MgCL , 120 pmoles; ATP, 126 rmoles; cytochrome c, 0.5 mg.; succinic acid, 260 rmoles; 0.25 M Tris buffer, pH 7.5, 2.5 ml.; Krebs-Ringer phosphate buffer, 0.0015 M with respect to EDTA, pH 7.2, 6.0 ml.; 25 mg. L-tryptophan; and sufficient water to bring the volume to 16.5 ml. In those experiments in which additional amino acids were used, 5 ml. of a solution containing 0.5 mg./ml. of the appropriate amino acids was added in place of water, bringing the final volume to 16.5 ml., as above. The incubation medium and 8.5 ml. of the 25% homogenate were either equilibrated separately at 37” for 510 min. before mixing or were mixed at 0” and agitated for 5-10 min. at 37” before the zero time sample was removed for analysis. Aliquots of 2-4 ml. of the above mixtures were pipetted into separate 20-ml. beakers and were shaken in an atmosphere of 100% 02 in a Dubnoff metabolic shaker for various lengths of time before stopping the reaction with an equal volume of 0.6 M perchloric acid. In some experiments other materials were added to this incubation medium. They are indicated under the appropriate experiments.
In Vivo TPO Induction A dose of 1 mg./g. rat body weight, dispersed in 2.5 ml. water, was injected intraperitoneally into animals fasted for at least 16 hr. In such experiments, a control group was always prepared into which 2.5 ml. water was injected. After 5 hr., the animals were killed and the livers prepared for experimentation as described above. (5) With Epinephrine. Animals were injected intraperitoneally with 30 pg. of an aqueous solution of epinephrine. These animals were killed 54 hr. after injection and the livers prepared for experiment as described above. (a)
With
L-Tryptopkan.
34
GORDON
AND
Kynurenine
RYDZIEL
Determination
TPO activity was estimated from the rate of kynurenine production, following the essentials of the procedure of Knox (a), except that 0.3 M perchloric acid was used to precipitate protein since clearer filtrates were obtained in the presence of detergents. After standing in the cold for at least 3 hr., the suspension was centrifuged, an aliquot of the supernatant was brought to neutrality with a standardized solution of NaOH, and the absorption of the resulting solution was determined at 365 ml.c. Enzyme activity is expressed in micromoles kynurenine formed/mg. N/hr. The molar absorption of kynurenine is taken as 4.54 X 103.
Radioactive Amino Acid Experiments Two kinds of radioactive amino acid experiments are described. In one group, the CWlabeled amino acids were prepared by acid hydrolysis of Chlorella vulgaris grown in the presence of NaHCi403 . After purification by ion-exchange chromatography, the specific activity of this combined group of amino acids (which contained no detectable quantities of tryptophan) was 34,600 counts/min./mg. in the thin-window, Geiger-Miiller counter employed. In the other group of experiments, nn-tryptophan3-CY4, specific activity 7.86 mc./mmole, was used. The preparation of the incubation medium and of the liver homogenate was identical to that described above, except that 120,000 counts of the appropriate amino acid solution was added to the incubation mixture prior to mixing with the homogenate. After a suitable period of incubation, the proteins were precipitated as described above, but otherwise prepared for counting as described by Sachs (3). Infinite thickness in this counting method was found to be at 12 mg. Results are expressed in specific activity, counts/min./mg. protein.
Chemical Methods Nitrogen
was determined
by a micro-Kjeldahl
method.
RESULTS
The Effect of Added Amino Acids on the Increment in TPO Activity Reports on in vitro increments in the activity of amylase point out that this effect is dependent on the uptake of only two amino acids, glycine and tyrosine (4). A planned systematic investigation of the amino acid requirements of the TPO increment was therefore begun with a medium unsupplemented with amino acids other than the substrate of the reaction, Ltryptophan. Figure 1 compares the TPO increment obtained in media which differed only in their amino acid composition. Contrary to expectation, it was found that a complete amino acid supplement inhibited TPO activity, while the effect of the essential amino acid mixture was small. While the possibility was considered that addition of amino acids competitively promoted the synthesis of protein other than TPO, or that a particular amino acid was exerting a specific inhibitory effect, it seemed advisable at this point to attempt a direct evaluation of the over-all rate of protein synthesis in this medium. Further investigation of the effects
MITOCHONDRIA
.4ND
TPO
35
ACTIVITY
Bcontrol OEsssntid
Amino
Acids
INCUBATION
TIME
1. Effect of added amino acids on TPO activity increments. The incubations were carried out as described in the text. The plotted values are the activities of the time intervals indicated from which zero-time values have been subtracted, so that direct comparison of activity per time interval is possible. Essential amino acids, equal weights of the L-forms of the following amino acids were added in the amounts indicated in the text: histidine, lysine, phenylalanine, leucine, isoleucine, threonine, methionine, and valine. For the “complete” amino acids, in addition to the above, the L-forms of the following amino acids were added in the same amounts: glycine, alanine, serine, aspartic acid, citrulline, tyrosine, glutamic acid, proline, hydroxyproline, arginine, ornithine, and cysteine. FIG.
of individual amino acids was therefore deferred. As the work developed, such a study did not seem crucial to the problem of the in vitro increments and was, therefore, not undertaken. The E$ect of RNase on the in Vitro Increments
of TPO Activity
While RNA or some of its precursors undoubtedly play some role in the synthesis of enzymes, the crucial intermedia.tes in this process have nob yet been determined (5). Nevertheless, on the basis of current information, it seems highly probable that gross interference with RNA metabolism would somehow affect the rate of synthesis of new enzyme protein. Accordingly, the MtMcS fraction was incubated at 37” with and without added RNase. This incubation mixture, which contained no added tryptophan, was aliquoted at several time intervals and the activity of TPO determined in the fortified medium previously described. The results are shown in Table I. No significant effect of RNase was observed. The E$ect of Ethionine and p-Fluoro-Lsphenylalanine in Vitro Increments of TPO Activity
on the
The first direct indication that de novo protein synthesis probably did not account for the observed increments in TPO activity was obtained by studying the effects of these two protein synthesis inhibitors on the in
36
GORDON
AND
TABLE Effect
-
of Ribonuclease
on TPO
TPO activity Incubation time of YtMcS prior to TPO assay, in minutes
I Activity
(in supplemented
Increments medium
after
in Viva
incubation
of YtMcS)
20
Activitya
With 0.1 mg./ml. RNase 0 5 10 15 Untreated MtMcS 0 5 10 15
RYDZIEL
Initial rateb
Activitya
Rate incrementC
Activity5
Rate incrementC
0.108 0.153 0.065 0.053
1.0 1.4 0.6 0.5
0.523 0.440 0.386 0.237
3.8 2.5 3.0 1.7
1.14 0.773 0.570 0.346
5.7 3.1 1.7 1.0
0.085 0.071 0.061 0.044
1.0 0.6 0.7 0.5
0.493 0.450 0.323 0.207
4.8 3.2 3.1 1.9
1.02 0.727 0.526 0.303
6.2 3.2 2.4 1.1
* Activity = pmoles kynurenine/mg. N/hr. b Initial rate = O-20 min. activity = 1.0. c Rate increment = (pmoles kynurenine formed in 20-min. interval/pmoles renine formed in initial 20-min. interval).
kynu-
increment. Both ethionine and p-fluoro-nn-phenylalanine have been shown to inhibit protein synthesis (6, 7), and, in particular, ethionine has been shown to inhibit the in viva induction of TPO activity when administered to the whole animal and to inhibit the induction of TPO activity in perfused livers (8) and the induction of TPO activity in liver slices (9). Figure 2 shows that the small changes in TPO increments due to ethionine fall close to the experimental error of the analytical method. Most important, the effect is not appreciably diminished with increasing concentrations of the inhibitor. It remains possible that ethionine requires activation before it can partake in the reactions in which it manifests its inhibitory properties. As can be seen in Fig. 3, p-fluoro-nn-phenylalanine differs considerably from ethionine in its effects on the activity increments. After 30 min., significant inhibition of the increment occurs, but no further reduction in TPO activity is noted after this time. Furthermore this 30-min. value approximates the value that is characteristic of the control preparation after 45 min. of incubation. The gross appearance of the p-fluoro-mphenylalanine mixture may provide the explanation for this effect. Gross agglomeration of the incubation mixture began to become evident after 30 min. of incubation. This phenomena will be discussedlater in terms of the
vitro
MITOCHONDRIA
J
AND
TPO
37
ACTIVITY
No Et hionine
- .INCUBATION
TIME
2. Effect of ethionine on TPO activity. Kynurenine scribed in the text. The plotted values represent the activity N/hr.) per indicated time interval. FIG.
I 1-45
INCUBATION
was determined as debmoles kynurenine/mg.
45-6 ‘0
TIME
FIG. 3. Effect of p-fluoro-nn-phenylalanine on TPO activity. Kynurenine termined as described in the text. The plotted values represent the activity kynurenine/mg. N/hr.) per indicated time interval.
was debmoles
mechanism which is thought to be operating in the production of in vitro TPO increments.
general
The E$ect of the Nonionic Detergent, BRIJ, on the in Vitro Increments in TPO Activity At this point an analogy between the observed TPO increments and the increments in activity of several other enzymes in aged mitochondrial preparations was considered. Among others, it is known that Mg++-activated ATPase (10) and catalase (11) increase their activity as the integrity of the mitochondria is compromised. The data from the experiments thus
38
GORDON
HNo
AND
RYDZIEL
Brij
I .35 mg./ml.
O-IO
IO-20 20-30 INCUBATION
30-40 TIME
FIG. 4. Effect of BRIJ on TPO activity. Kynurenine in the text. The plotted values represent the activity per indicated time interval.
was determined as described (pmoles kynurenine/mg. N/hr.)
far reported would be consistent with such an interpretation. Accordingly, the effect of BRIJ on the reaction was studied, since similar detergents have been shown to modify the physical properties of mitochondria (12). The response of the system to BRIJ is shown in Fig. 4. The increase in TPO activity in the presence of BRIJ is greater than found with the controls. Microscopic examination of the reaction mixtures in the presence and absence of BRIJ, reveal that the number of particles which stain with Janus Green B are markedly reduced in the BRIJ-treated mixtures. The qualitative microscopic impressions are consistent with the assumption that the increment in TPO activity follows the disappearance of particles which stain as mitochondria. Unfortunately, the centrifuge equipment available did not permit studies of the changes in the microsomal fraction. Experiments are planned to determine whether or not these subcellular particles also change in number or character in the course of the experiments. Uptake
of Radioactive
Amino
Acids
The uptake of radioactive amino acids for the Chlorella vulgaris hydrolyzate is shown in Table II. No significant increment of radioactive amino acid uptake is found in any of the mixtures which promote the TPO activity increment. As an example is plotted the increments in TPO activity obtained in this experiment with the control preparation and with the identical preparation supplemented with BRIJ. The possibility was considered that the appearance of TPO activity might involve a dual role for tryptophan: one, as the substrate of the reaction, the other, as the stimulus for the reaction, perhaps involving the
MITOCHONDRIA
Incrementation Incubation of MthfcS radioactive medium
min. 0
20 40
time in
TPO
39
ACTIVITY
TABLE II Amino Acids in TCA-Insoluble Fractions during of TPO Activity under Two Conditions
of Radioactive
Incorporation
AND
Specific Control
radioactivity BRIJ
counts/min./mg.
3.0 10.5 17.0
TPO activity (0.35 mg./ml.)
protein
4.0 7.5 12.5
in Vitro
increment
Control
BRIJ
,moles
kynureninejmg.
0.105 0.170
(0.35 mg./ml.) N/hr.
0.125 0.280
fixing of tryptophan into some protein TPO precursor. Experiments with tryptophan-Cl4 gave no indication that this compound was fixed at a greater rate in the more active preparations compared to the less active ones. Furthermore, even at the earliest stage of the reaction, no evidence was obtained that any product other than the ones already described (2) was formed in the reaction. This finding appears to eliminate from consideration the possibility that TPO, like Mg++-activated ATPase (lo), has some other function analogous in principle to the postulated transphosphorylase role of Mg++-activated ATPase in intact mitochondria. The Efect of Ascorbic Acid on the TPO Increment Even though it had been shown previously (1) that the addition of a peroxide-generating system and catalase did not influence the increment in TPO activity, it seemedadvisable to test the effect of reducing substances which might be present in the homogenate. Furthermore, it has been reported that ascorbic acid will delay the manifestation of amine oxidase activity (13) and that its presence is important for the in vitro inductions of amylase (7). Figure 5 showsthat ascorbic acid in a concentration of 12.5 mg./25 ml. incubation mixture is without effect in either stimulating or inhibiting the TPO increment. The insensitivity of the system to ascorbic acid is manifested under two different experimental conditions. In the first, the ascorbic acid is mixed with the incubation medium and the MtMcS and the mixture is shaken in air for 10 min. at 37” before a zero-time sample is obtained. In the second experiment, with the identical homogenate and incubation medium, the MtMcS and the incubation medium containing added ascorbic acid are prewarmed separately at 37” for 10 min. with shaking. They are then mixed and a zero-time sample is removed at once. If the lag in the manifestation of TPO activity is a reflection of unchanged ascorbic acid, as in the systems referred to above, a greater lag in full TPO activity would be expected in the second case where the ascorbic acid cannot react with the homogenate during the initial 10 min. In fact,
40
GORDON
AND
RYDZIEL
1 Mired
.25
Cold
fl M ixed After Pro - Warming
-?.2C z : \ f .IC Y I a C
IO-IO 0
INCUBATION
TIME
Fro. 5. Effect of ascorbic acid on TPO activity. Kynurenine was determined as described in the text. The plotted values represent the activity (pmoles kynurenine/ mg. N/hr.) per indicated time interval.
however, both react identically, suggesting that changes in ascorbic acid are not critical for the increase in TPO activity. The E$ect of Tonicity on TPO Increments The homogenizing medium employed in these experiments was chosen because of its ability to maintain mitochondrial integrity over prolonged periods (14). If the assumption that in vitro increments in TPO are a function of changes in mitochondrial integrity is correct, it should be possible to demonstrate greater increments in TPO activity if the mitochondrial integrity is deliberately interfered with. Since the experiments with BRIJ seemed to support this view, confirmatory experiments, involving the manipulation of the tonicity of the medium, were undertaken. Supernatants were prepared much like those described previously. Two modifications in the original homogenizing medium were made: 0.25 M sucrose was substituted for 0.44 M, so that, in subsequent manipulation, frankly hypotonic preparations could be made without excessive dilution; and, EDTA was omitted from the homogenizing medium, to eliminate the mitochondrial stabilizing effect of this compound. After centrifugation under the conditions described previously, the supernatant was divided into two fractions. One was incubated with an equal volume of water at 37” for 10 min.; the other was incubated with an equal volume of the Krebs-Ringer-EDTA solution described previously, at the same temperature and for the sametime. Both contained a small quantity of L-tryptophan (0.6 mg. in 12 ml.) to stabilize the TPO during this brief incubation. After incubation, the two differently treated preparations were mixed with
MITOCHONDRIA
. .3
FIG.
41
ACTIVITY
0 Isotonic i
0 2( INCUBATION scribed N/hr.)
TPO
~Hypotonic
5 \
AND
6. Effect of tonicity on TPO in the text. The plotted values per indicated time interval.
activity. represent
I
1 30-40 TIME Kynurenine the activity
was determined as de&moles kynurenine/mg.
buffer, salts, and the other addenda described previously so that they were once more identical to each other and to the other preparations described in this report except for the somewhat lowered sucrose concentration. Figure 6 describes the changes in TPO activity from these two preparations measured from the time that they were once again made identical to one another. Contrary to the results of the experiments with BRIJ, the increment in activity in the preparation treated with water is markedly less than from the preparations treated with Krebs-Ringer-EDTA during the IO-min. preincubation. This difference between observed and predicted * behavior will be referred to in the Discussion. The E$ect of in Vivo Injections of Epinephrine Increments in TPO Activity
on in Vitro
A sometimes distressing feature of the experiments reported here is the variability of the TPO response. With animals selected as described earlier, some response was always demonstrable. However, the response varies over wide limits. In some experiments up to sixfold increments in activity were obtained (see Table I) ; and in others, less than twofold (see Table III). An explanation of these findings may be found in a comparison of the in vitro inductions obtained from animals that had been disturbed by placing them in a shaker for 30 min. prior to sacrifice, with animals that were injected with epinephrine, and with a control group in which particular pains were taken not to introduce disturbing environmental changes prior to sacrifice. It is apparent from Table III that early sacrifice after injection with epinephrine, or after shaking, does not lead to significant changes in zero-time TPO activity, even though permitting a longer period of reaction
42
GORDOS
AND
RYDZIEL
TABLE Effect
of in Viva
Injection TPO
Activity
10 min. Treatment
of animal to sacrifice
III
of Epinephrine (after
on in Vitro indicated
period
20 min.
Increments of incubation
30 min.
in
TPO
Activity
in supplemented
medium)
40 min.
50 min.
prior “A c .A 2
% 3
a. 2
b 2% .- E au E 2 _____
Controld 3Opg. epinephrineintraperitoneally 36 hr. prior to sacrifice 30 min. of shaking prior to sacrifice Controld + 100 pg. epinephrine in vitro Controld + 100 pg. Cortef in vitro 8 Activity = pmoles b Rate = activity of c Rate increment = renine formed in initial d These are separate
Q.
L : 2% 3’”
2
2
E
0. 2
E Se .d 0 %B Ps
0. 2
e 20, .- c $2 Fd
0.091 0.083
1.0 1.0
0.176 0.207
0.93 1.50
0.321 0.380
1.59 2.09
0.500 0.586
1.97 2.48
0.631 0.886
1.44 3.62
0.103
1.0
0.215
0.99
0.396
1.76
0.586
1.84
0.804
2.12
0.087
1.0
0.182
1.09
0.323
1.62
0.499
2.02
0.616
1.35
0.089
1.0
0.175
0.97
0.340
1.86
0.512
1.93,
0.632
1.35
kynurenine/mg. N/hr. initial 10.min. assay = 1.0. bmoles’ kynurenine formed in IO-min. IO-min. interval). aliquots of the same homogenate.
interval/pmoles
kynu-
in vivo would increase the zero-time activity also (15). However, in the epinephripjzed animals and in the animals that were shaken, the increase in TPO activity is much more marked after prolonged incubation than is characteristic of the control group. On the basis of these experiments, it seems likely that the varia.bility in response is a function of uncontrolled factors in the environment which by some undetermined mechanism may act to prime the test system for rather high increments in TPO activity, compared to the increments obtainable in animals that have not been agitated. Data on in vitro additions of epinephrine and Cortef are included to demonstrate that these hormones, both of which induce TPO activity in vivo (16), are without effect in vitro. DISCUSSION There seems little doubt, based on the results reported here, that the in vitro increments in TPO activity, which can be demonstrated under our conditions, do not reflect protein synthesis. It is, of course, possible that protein synthesis, in the very limited sense reported for the in vitro increments in amylase activity, i.e., the uptake of only two amino acids (4), also occurs in this system. However, the lack of correspondence between
MITOCHONDRIA
AND
TPO
ACTIVITY
43
activity increment and radioactive amino acid uptake makes even this possibility somewhat remote. Another, perhaps more compelling argument against protein synthesis, stems from a consideration of the increment and later fall in TPO activity in vitro. TPO activity under our conditions rises to levels which are three and four times greater than the activities reported by others for this enzyme (2, 16). However, after 30-40 min. of incubation, the activity falls and after an hour tends to reach values consistent with those reported previously for TPO activity. In addition to denaturation, an added explanation of this rise and fall in activity is suggested by the mitochondrial requirement for act,ivity increments. If part of the enzyme complex is initially organized in the mitochondria, and if this organization maintains its integrity while the permeability of the mitochondria increases, a more and more rapid reaction of the tryptophan might be expected. The maximal activity, however, falls as the elements of the TPO complex become solubilized. Under these conditions the kinetics of the reaction are determined by the probabilities of chance encounters between soluble enzymes and soluble substrates. Such a mechanism has been invoked by Green (17) to account for the rapid oxidation of Krebs-cycle intermediates in mitochondria. This mechanism is also supported by the difference in responseto BRIJ and hypotonic solutions. If the increment in activity comesabout by mitochondrial disruption and the consequent release of enzymic activity, the response to hypotonicity and to BRIJ would be expected to be similar. However, hypotonic solutions probably promote a more rapid disruption of mitochondria than they do changes in membrane permeability in nondisrupted mitochondria, while BRIJ very probably influences permeability to a greater extent than disruption since its effects were determined in isotonic solutions. Recknagel and Malamed have recently discussed these two aspects of mitochondrial swelling (18). Further support for this suggestion is found in the p-fluoro-m-phenylalanine experiment. Under our condition the p-fluoro-m-phenylalanine promotes, even under isotonic conditions, protein denaturation which is visible as a settling-out of gross agglomerated particles in the incubation medium. If this denaturation is assumed to occur by a process which simultaneously releases the components of the TPO complex into the medium, the rate of reaction would tend to fall to values characteristic of the concentration of the free enzyme components rather than the higher rates characteristic of their postulated organized forms. The TPO activity obtainable from older animals is as great per milligram N as that from younger animals. However, no consistent increment in activity can be demonstrated under our conditions with animals above 120
44
GORDON
ASD
RYDZIEL
g., while the response can always be elicited from animals below 100 g. The role of the mitochondria in the production of the in vitro increments may explain the relation of age to increment. Cooper and Tapley (19) have demonstrated that the degree of mitochondrial swelling under a given experimental condition is a function of age, nutrition, and the organ from which the mitochondria are obtained. It seems possible that the balance between enzyme complex disruption and changes in mitochondrial permeability also differs as a function of animal age. This aspect of the problem is under investigation in another connection and will be reported separately. The epinephrine data and the wide variety of hormones which stimulate TPO activity (15, 16) make it necessary to consider that the increments reported here are all a function of the hormonal changes produced by manipulating the animals prior to sacrifice. However, here too, changes in mitochondrial permeability must be considered, since it is known that various hormones influence this property of subcellular particulates also (14). This problem is an aspect of the one mentioned above and is also now under investigation. It is nevertheless interesting to speculate, at this point, whether tryptophan and hormones which do stimulate in vivo TPO increments, do so in the first instance by affecting mitochondrial permeability. It should be emphasized that the experiments reported here do not contradict evidence for in vivo protein synthesis of TPO under appropriate stimulation. The fact that the same kind of in vitro increments may be obtained both with adapted and nonadapted preparations (1) suggests that more enzyme is available for reaction in the adapted preparations, and presumably this added activity reflects additional enzyme. SUMMARY
In vitro increments of tryptophan peroxidase activity in cell-free preparations from rat liver are shown to be correlated with the permeability of mitochondria. Disruption of these particulates reduces the in vitro increment, but isotonic solutions containing substances which change the mitochondrial permeability promote such increments. REFERENCES 1. CLOUET, D. H., AND GORDON, M. W., Arch. Biochem. Biophys. 84, 22 (1959). 2. KNOX, W. E., in “Methods in Enzymology” (Colowick, S. P., and Kaplan, N. O., eds.) Vol. 2, p. 242. Academic Press, New York, 1955. 3. SACHS, H., J. Biol. Chem. 228, 23 (1957). 4. GARZO, T., PERL, K., T.-SZABO, M., ULMAN, A., AND STRAUB, F. B., Actu Physiol. Acud. Sci. Hung. 9,23 (1957). 5. CHANTRENNE, H., Ann. Rev. Biochem. 27, 35 (1958). 6. LEE, N. D., AND WILLIAMS, R. H., Biochim. et Biophys. Acta 9, 698 (1952).
MITOCHONDRIA
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
AND
TPO
ACTIVITY
45
STRAUB, F. B., AND ULMAN, A., Biochim. et Biophys. Acta 23, 665 (1957). PRICE, J. B., JR., AND DIETRICH, L. S., J. Biol. Chem. 337, 633 (1957). GIVEN, M., AND KNOX, W. E., Federation Proc. 16, 165 (1957). POTTER, V. R., AND RECKNAGEL, R. O., in “Phosphorus Metabolism” (McElroy, W. D., and Glass, B., eds.), p. 377. Johns Hopkins Press, Baltimore, 1951. ADAMS, D. H., AND BURGESS, E. A., Brit. J. Cancer 11, 310 (1957). WITTER, R. F., AND MINK, W., J. Biophys. Biochem. Cytol. $73 (1958). ACKERFELDT, S., Science 126, 117 (1957). TAPLEY, D. F., J. Biol. Chem. 222, 326 (1956). KNOX, W. E., Brit. J. Ezptl. Pathol. 32, 462 (1951). SCHLOR, J. M., AND FRIEDEN, E., J. Biol. Chem. 233, 612 (1958). GREEN, D. E., in “Chemical Pathways of Metabolism” (Greenberg, D. M., ed.), p. 27. Academic Press, New York, 1954. RECKNAGEL, R. O., AND MALAMED, S., J. Biol. Chem. 232, 705 (1958). COOPER, C., AND TAPLEY, D. F., Biochim. et Biophys. A& 26,426 (1957).