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l -Tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo
ARCHIVES
OF
BIOCHEMISTRY
L-Tryptophan
McArdle
AND
Inhibition
166, 188-194
BIOPHYSICS
(1973)
of Tyrosine
Aminotransferase
in Rat Liver
in Viva’
ALOIS
CIHAKz,
CARLOS
LAMAR,
Memorial
Laboratory,
The Medical
School,
Received
JR.~, University
December
AND HENRY of Wisconsin,
Degradation
C. PITOT Madison,
Wisconsin
54706
1, 1972
The administration of L-tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of L-tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in viva. When L-tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-HydroxynL-tryptophan and indole administration in amounts equimolar to L-tryptophan also result in similar increases in activity whereas a-methyl-DL-tryptophan produces little or no increase. Utilizing pulse-labeling ipz viva with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.
1.13.1.12), by tryptophan and cortisone, which had been noted earlier by Knox and his associates (2) as well as Feigelson and Greengard (3), could be explained on the basis that cortisone induced an increased rate of synthesis of the enzyme whereas tryptophan stabilized the enzyme in vivo and prevented its degradation. Schimke’s report was perhaps the first concrete evidence that the level of an enzyme could be controlled in vivo by altering the dynamic turnover of the enzyme and preventing its destruction within the cell. Since those reports, other studies have indicated that the regulation of the rate of degradation of an enzyme is extremely important in the final determination of enzyme levels in vivo.
In 1965 Schimke and his associates (1) reported that the differences in the regulation of the enzyme, tryptophan oxygenase (L-tryptophan , oxygen oxidoreductase, EC 1 The work reported in this paper was supported in part by grants from the National Cancer Institute (CA-07175) and the American Cancer Society (P-314 and E-588). 2 The work reported in this paper was undertaken during the tenure of an Eleanor Roosevelt Cancer Fellowship of the International Union Against Cancer awarded to Dr. Alois Cihak. Permanent address : Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague-6, Czechoslovakia. 3 Present address: Department of Medicine, The Medical School, Texas Technological University, Lubbock, Texas. 188
Copyright 0 1973by AcademicPress,Inc. All rights of reproductionin my form reserved.
TYROSINE
AMINOTRANYFERASE
Studies on the heart and muscle forms of lactate dehydrogenase (4) have demonstrated that the rates of turnover of the two subunits in heart and liver are quite dissimilar and may, in fact, account for the differences in the levels of the lact’ate dehydrogcnase isozymes seen in these two tissues. In tissue culture, an inhibition of the degradation of glutaminc synthetase has been invoked as the mechanism whereby this enzyme increases in the absence of glutamine (5), and the high level of arginase stimulated by addition of manganese in vitro also appears to be the result of enzyme stabilization (6). Alt’hough the earlier studies by Schimke and his associates (1) were only concerned with the stabilization of tryptophan oxygenase, it is now apparent Ohat t’ryptophan has several other functions in enzyme synthesis (7-10). The data reported in this paprr indicate that at least one other enzyme, tyrosine aminotransferase (L-tyrosine, 2-oxoglutarate aminotransferase, EC 2.6.1.5), is also stabilized by the administration of L-tryptophan GL vivo. MATERIALS
AND
METHODS
In all the experiments reported in this paper, male rats (180-220 g) obtained from the Holtzmann Laboratories in Madison, WI were kept for 3-5 days either on a laboratory chow diet containing a minimum of 2O7? crude protein or on a 12.5% casein diet. The animals were fasted for lo-12 hr prior to initiating the experiments between 7:00 and 8:00 .41x. Adrenalectomized male rats (160-180 g) were obtained from the Endocrine Laboratories in Madison and were kept under the same conditions as described above with the addition of 1% sodium chloride in their drinking water. For determination of enzyme levels and incorporat,ion of labeled amino acids irk Y~ZJO,groups of four to six rats were used for each point. Individual experiments were repeated two to four times. At various t,ime intervals after injection and/or intubation, the animals were sacrificed by decapitation, the livers rapidly removed, weighed, and homogenized in 3 vol (w/v) of 0.25 M sucrose in 0.05 ~1 Tris-HCl buffer (pH 7.2) containing 10 rnbr MgC12, 25 mM KCl, 0.1 mM pyridoxal phosphate, and 1 rnzf dithiothreitol. Homogenates were centrifuged for 1 hr at 100, OOOg, and the SUpernatants were used for the determination of enzyme activities and later immunoprecipitation. Tryptophan oxygenase and tyrosine aminotrans-
STABILIZATION
189
ferase were assayed utilizing a combination automated unit previously described (11). For immunoprecipitation studies, L-[4,5-3H]leucine (sp act 50 pCi/l5 nmolelanimal) or L-[l14C]valine (sp act. 21 &i/O.9 rmolejrat) was injected intraperitoneally in a 0.25.ml volume 45 min prior to sacrifice of the animals. Aliquots of the 100,OOOg supernatant (0.2-0.3 ml) were added to immune and nonimmune rabbit serum (0.2-0.4 ml). Saline was added to a total volume of 0.5 ml and the antigen-antibody complex allowed to precipitate over a 4%hr period at 4°C. The immune precipitates were centrifuged, washed 3 times with 2 ml of 0.9(% NaCl, and dissolved in 0.2 ml of 99yA formic acid. Radioartivity was measured in a Packard Tri-Carb liquid scintillation counter utilizing Scintisol (Isolab) as a scintillation counting medium. In all instances, the absence of tyrosine aminotransferase in supernatants after immunoprecipitation was checked by means of an automated assay (11). Antibodies to tyrosine aminotransferase were prepared in rabbits and titrat,ed as reported in a previous publication (12). The immunological identity of enzyme induced by cortisone or tryptophan was also reported in the same paper. All immunoprecipitations were carried out in the region of antibody excess, nonspecific adsorption of counts was determined by the method of ChoChung and Pitot (13), and appropriate corrections were made. Cortisone acetate was obt,ained from the Upjohn Company. L-Tryptophan was obtained from General Biochemicals. L-[4,5-3H]Leucine (sp act 22.6 mCi/mmole) was obtained from Schwarz BioResearch. Dithiothreitol and L-kynurenine were obtained from the Calbiochem Corporation. 5-Hydroxy-oL-tryptophan and quinolinic acid were products of Sigma, while anthranilate and indole were from Eastman Chemicals. a-MethyluL-tryptophan was kindly provided by Dr. Henry Lardy. RESULTS
Previous studies by other workers (9, 10, 14) have demonstrated that t’he administration of L-tryptophan causes a marked increase in the activities of several enzymes, including tyrosine aminotransferase. The relationship of the amount of L-tryptophan administered to the hepatic activity of both tryptophan oxygenase and tyrosine aminotransferase measured 5 hr after tryptophan administration has been reported (15). From this data it may be noted that the induction of tyrosine aminotransferase is, in fact, much
190
CIHAK,
LAMAR,
-
z 2 $ 6> 300
90 y” 2 3.
- 60 a a\ 3 c
i
a
4 i% 150 E a B f x
/ 0
I I 3
I 6
I 9
-
1 12
TIME (hrs) FIG. 1. Time course of tyrosine aminotransferase (pOHphenylpyruvate production, -o-) and tryptophan oxygenase (kynurenine production, -O-) activities after n-tryptophan administration. Holtzman male rats (2OG220 g) starved 14 hr were intubated with L-tryptophan [25 mg (-•-) and 100 mg (-O-) per 100 g, respectively] and killed at different time intervals thereafter.
more sensitive to the administration of L-tryptophan than is tryptophan oxygenase. In Fig. 1 is seen the time course of changes in tyrosine aminotransferase activity after a single dose of L-tryptophan. Although the amount of L-tryptophan given to induce the enzyme in this figure was only one-quarter that given to induce tryptophan oxygenase, the shapes of the curves are quite similar. As previous workers have demonstrated (14, 16, 17), tyrosine aminotransferase may be induced in the intact animal by a number of different materials, all of which have as their common denominator a stimulation of adrenocortical response. In Fig. 2a and b is noted the effect of the time after the administration of L-tryptophan on the level of tyrosine aminotransferase activity as well as the relation of the dose of tryptophan to enzyme activity in adrenalectomized animals. The time course of induction is almost identical to that seen in intact animals, and the sensitivity to the amount of L-tryptophan given also appears to be quite similar, although, at doses of tryptophan above 3040 mg/lOO gm body wt, animals die due to
AND
PITOT
the toxicity of the amino acid. Thus, it is apparent that the increases seen under these conditions are not mediated by the adrenal glands. In Table I is seen the effect of the administration of the analogue, m-(a-methyl) tryptophan, as well as several tryptophan metabolites on the activity of tyrosine aminotransferase in vivo. In all instances, the compounds were administered by intubation in amounts equimolar to 20 mg of L-tryptophan, which was administered to a control group of animals. If the compound was a mixt’ure of the DL forms, t,he racemate was made equimolar in the L form. It can be noted that the analogue, cr-methyl-mtrvptophan, produces essentially no increase in the level of tyrosine aminotransferase over the control activity. On the other hand, 5-hydroxy-m-tryptophan and indole produce an increase in tyrosine aminotransferase activity which is similar to that seen with L-tryptophan alone. In two animals given L-kynurenine, the increase also was essentially identical to that in animals given L-tryptophan. Quinolinate and anthranilate produced either no increase in the case of the former, or an intermediate increase in the case of the latter. While these studies do not establish a structure-response relationship, they do serve to indicate that the tryptophan nucleus itself probably does not stimulate the increase in tyrosine aminotransferase, since the analogue had no effect. Furthermore, metabolism beyond anthranilate also appears to destroy the stimulatory activity. On the other hand, indole, which forms the nucleus of the amino acid, appears to be equally as good as tryptophan in stimulating the response. When L-tryptophan was given to animals who had already received an injection of cortisone, there is a further increase in the level of both enzymes. The data may be seen in Figs. 3 and 4. Herein, cort’isone was administered at zero time, and at the peak of enzyme induction (5 hr later) a dose of L-tryptophan was administered. In contrast to the animals not receiving L-tryptophan, wherein the level of the enzyme decreased markedly for the next 7 hr, in those animals receiving L-tryptophan a continual increase
TYROSINE
AMINOTRANSFERASE I
1
STABILIZATION
I
191
-11
b
0 300
150
0
4 I3 TIME (hrs)
I2
40 60 20 Dosage af L-Tryplophan (mg per IOOgm body weight)
FIG. 2. Increase of liver t,yrosine aminotransferase in adrenalectomized rats after the intubation of 1,.tryptophan. Time (a) and dose (b) relations. Holtzman male adrenalectomized rats (160 g) kept 3 days on 12.5y0 protein diet were starved for 12 hr. L-Tryptophan was intubated in a maximal volume of 5 ml of saline. (a) L-tryptophan was administered in a dose of 0.2 g/kg; (b) animals were killed6 hr after intubation of the indicated dose of L-tryptophan, and tyrosine aminotransferase activities are expressed as the average of 46 values f SEM.
in enzyme activity, although at a slightly lower rate, was demonstrated. The administration of actinomycin subsequent to corticosteroid administration produces a similar effect (18, 19) in the case of both tyrosine aminotransferase and tryptophan oxygenase. The administration of 8-azaguanine (20) and of 5-fluoroorotic acid or 5-azacytidine (15) after corticosteroid administration also mimics the effects of tryptophan or actinomycin. In order to determine the relative roles of enzyme synthesis and degradation in the effect of L-tryptophan on tyrosine aminotransferase, pulse-labcling of the enzyme in vivo coupled with quantitative immunoprecipitation techniques was utilized (1, 13). Animals were given either cortisone or L-tryptophan, and 4 hr and 15 min later a dose of tritiated lcucine was injected as described in the Methods. These animals were sacrificed 45 min later, high-speed supernatants of the liver prepared, enzyme activities determined, and the tyrosinc aminotransferase quantitatively precipitated by its specific antibody. The antigen-antibody precipitate was then counted as a measure of the pulse-labeled synthesis of the enzyme in vivo. Earlier studies (15) had dcmon-
&rated that although there was a marked increase in the activity of the enzyme after the administration of cortisone or L-tryptophan, both in intact and adrenalectomized animals there was a significant increase in label in the enzyme antigen only after the administration of cortisone. Cortisone administration resulted in twice the activity of tyrosine aminotransfcrase of that seen after L-tryptophan administration. This might be expected if the hormone actually induces an increased rate of synthesis while the amino acid inhibits enzyme degradation (1). The labeling of total liver soluble protein was at a level of 200,000-300,000 cpm/mg protein and did not change significantly under the conditions utilized, indicating a selective induction of the synthesis of tyrosine aminotransferase. It was apparent, however, that the administration of L-tryptophan, either to intact or adrenalectomized animals, did not cause an increased labeling of total protein under the conditions of the experiment. In Fig. 5 is seen the result of an experiment wherein tyrosine aminotransferase was induced by the administration of cortisone; 4 hr later [lJ4C]valine was injected, and animals were sacrificed at the times after the
192
CIHAK, TABLE
LAMAR,
AND PITOT
I
EFFECT OF ADMINISTRATION OF ANALOGUBX AND METABOLITES OF TRYPTOPHAN ON TYROSINI.: AMINOTRANSFERASE ACTIVITY Z'YLVivoa
Compound
No. of rats
Tyrosine aminotransferase activity
Saline L-Tryptophan (20 mg) 5-Hydroxy-nL-tryptophan (44 md L-Kynurenine SO4 (42 mg) Quinolinate (17 mg) DL-(a-methyl)Tryptophan (44 mg) Anthranilate (14 mg) Indole (12 mg)
9 4 3
89.0 f 230.1 f 239.9 f
13.3 16.5 31.3
2 6 5
218.2 f 114.8 f 130.9 f
17.1 13.1 13.4
6 5
183.3 f 258.6 f
19.4 18.1
(1All compounds were administered by intubation of the amounts shown in parentheses dissolved in 5 ml Hz0 at zero time to adrenalectomized male rats (140-160 g) maintained for 3 days after adrenalectomy on a 12.5% protein diet. Animals were sacrificed 5 hr after intubation, and the enzyme activity assayed as described in the Methods. Values are averages f SEM.
r,
5
300 I P
2 I a
I
I
I
TYROSINE \ AMINOTRANSFERASE
$,
/ a
I 0
I 8
I 4 TIME
I 12
(HRS.)
FIG. 3. Increase of cortisone-induced tyrosine aminotransferase activity by the delayed administration of L-tryptophan (L-TRY). Holtzman male rats starved 14 hr were injected ip with cortisone (5 mg per animal, -O-), and 5 hr later a group of 4-6 of these animals were intubated with L-tryptophan (100 mg/lOO g, -0-). All points represent the average values of 4-6 animals f SEM.
0
4
8
12
TIME (HRS.1 FIG. 4. Increase of cortisone-induced tryptophan oxygenase activity, as measured by kynurenine production, by the delayed administration of L-tryptophan (L-TRY).
initial
administration
of cortisone
shown in
Fig. 5. The X’s and solid line denote the changes in radioactivity and enzyme activity of tyrosine aminotransferase after the administration of cortisone. The solid circles
and dashed line denote the decay in radioactivity and enzyme activity of tyrosine aminotransferase in animals given cortisone at zero time and L-tryptophan (170 mg/kg/ dose) by intubation at 5 hr and 7 hr after the administration of cortisone. As can be seen in Fig. 5, while there was a decay in both radioactivity and enzyme activity between 6% and 945 hr after the administration of cortisone only, those animals receiving tryptophan in addition at 5 and 7 hr showed a marked inhibition of the rate of enzyme decay. These data, coupled with those previously reported (15), argue that the increases in tyrosine aminotransferase brought about by the administration of L-tryptophan are predominantly the result of an inhibition of enzyme degradation rather than a stimulation of enzyme syntheSlS.
DISCUSSION
The phenomenon of enzyme and protein turnover has been known for many years
TYROSINE
Time
in Hours after
AMINOTItANSFE:RASE
Cortisone
5. Loss of activity and radioactivity of tyrosine aminotransferase (TAKG) in livers of animals induced with cortisone and given L-tryptophan 5 and 7 hr after administration of the steroid. All animals received cortisone acetate (5 mg/rat ip) at zero time. [lJ4C]valine (21 pCi/O.S pmolelrat) was injected ip 4 hr after cortisone administration. L-tryptophan (170 mg/kg/dose, dashed line) or saline (0.5 ml, solid line) were administered by intubation in two doses at 5 and 7 hr after cortisone. Animals were sacrificed at the time intervals shown after cortisone administration. The radioactivity of the enzyme-antigen complex was determined as described in the Methods. All values are averages from five animals. The standard error of the mean did not exceed 50 pmoles/g liver/hr or 150 counts per 100,000 counts. FIG.
(21). As indicated in the introduction, however, specific environmental controls of enzyme turnover have only recently become apparent. The studies described herein expand a previous investigation (1) showing that the essential amino acid, L-tryptophan, is capable of stabilizing tryptophan oxygenase, demonstrate that at least one other enzyme, tyrosine aminotransferasc, is also stabilized, and its degradation in viva prevented by the administration of the amino The data described acid, L-tryptophan. herein actually demonstrate that the turnover of this enzyme is considerably more
STABILIZATION
193
sensitive to L-tryptophan administration than is that of tryptophan oxygenase. Several investigators, including Munro (7), Sidransky (8), and others, have shown that L-tryptophan appears to have a relatively unique role in the regulation of protein synthesis in liver. Munro and his associates (7) have demonstrated that, when tryptophan is absent from a mixture of amino acids, the administration of such tryptophan-devoid solutions in vivo causes a marked decrease in the polysomes of normal liver. Sidransky et al. (8) has shown a similar effect, although it is appa,rent that the dccreascd polysome pattern is dependent on stimulation of new protein synthesis. In recent studies by Sidransky’s group (unpublished observations), the loss of polysome patterns in liver resulting from puromycin administration in vivo can be reversed by the administration of tryptophan alone in vivo. Lardy and his associates (10) have also demonstrated that, the administrat,ion of tryptophan causes an increase in the hepatic enzyme, phosphoenolpyruvate carboxykinase (phosphoenolpyruvate oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32). Tryptophan has a marked effect, on gluconeogenesis in liver that appears to be related to its degradat’ion to quinolinic acid; this compound acts as a chelator of essential metal ions required in the activity of certain enzymes, notably the carboxykinase. Recently it was observed that administration of L-trypt’ophan results in a marked increase of DNA-dependent RNA polymerase isolated from rat liver nuclei (22) and that the synthesis of liver messenger RNA is enhanced by this t’reatmcnt (23). Other studies by Oravec and Korner extend these investigat’ions showing that tryptophan administration also st’imulates the synthesis of ribosomal RtNA (24). Studies by Allen et al. have demonstrated that tryptophan deficiency causes a marked decrease in the level of charged tryptophanyl tRNA. This decrcasr is much greater than that seen u-ith other specific aminoacyl t,RNA’s in the livers of animals fed diets deficient
in other amino acids (25).
Therefore, it is apparent that tryptophan plays a wntral role in protein synthesis, as
194
CIHAK,
LAMAR,
well as in the regulation of enzyme levels in normal rat liver. The fact that tryptophan stabilizes tryptophan oxygenase and tyrosine aminotransferase is another facet of this rather complex picture. As described in this paper (Table I), metabolites of tryptophan also appear to produce the stabilizing effect on tyrosine aminotransferase. Hardeland (26) described experiments in which quinolmate, when given in doses substantially higher than those administered here, produced a significant increase in tyrosine aminotransferase and tryptophan oxygenase. Deguchi and Barchas (27) also demonstrated that the administration of 5-hydroxytryptophan caused a significant increase in tyrosine aminotransferase in rat liver ad did earlier studies by Rosen and Milholland (28). The fact that the analoguc, ar-methyltryptophan, produced no such increase does suggest that some sort of metabolism of the amino acid must occur for the stabilizing effect to be seen. On the other hand, indole, a rather distant metabolite, does produce the stabilizing effect, at least for tyrosine aminotransferase. In a previous paper (12), the administration of 5-fluoroorotate to animals subsequently given tryptophan produced a further stimulation over that by the amino acid alone. Since those studies demonstrated that the analogue also caused a stabilization of the enzyme by inhibiting its degradation in vivo, it would appear that the mechanism of the pyrimidine analogues and tryptophan in stabilizing tyrosine aminotransforase are basically different. Since tryptophan plays a central role in protein synthesis, it is tcmpting to speculate that the stabilization of an enzyme by this amino acid is related to its role in translation. On the other hand, the base analogues and actinomycin D, having in common the inhibition of RNA synthesis, may exert their effects by a mechanism resulting from or associated with this inhibition. REFERENCES 1. SCHIMKE, R. T., SWEENEY, E. W., AND BERLIN, C. M. (1965) J. Biol. Chem. 240,322. 2. KNOX, W. E., AND AUERBACH, V. H. (1955) J. Biol. Chem. 214, 307.
AND
PITOT
3. GREENGARD, O., AND FEIGELSON, P. F. (1961) Nature (London) 190, 446. 4. PITOT, H. C., P*XUINO, C., LAMAR, C., AND KENNAN, A. L. (1965) Proc. Nat. Acad. Sci. USA 64, 845. 5. PAUL, J., AND FOTTRELL, P. F. (1963) Biochim. Biophys. Acla 67, 334. 6. SCHIMICE, R. T. (1964) J. Biol. Chem. 239, 3808. WUNNER, W. H., B~:LL, J., AND MUNRO, H. N. (1966) Biochem. J. 101,417. 8. SIDRANSKY, H., SARMA, 1). S. R., BONGIORNA, AND VICRNEY, (1968) J. Biol. Chem. 243, 1123. 9. PERAINO, C., BLAKE, R. L., AND PITOT, H. C. (1965) J. Biol. Chem. 240, 3039. 10. FOSTER, D. O., RAY, P. D. AND LARDY, H. A. (1966) Biochemistry 6, 563. 11. PITOT, H. C., WRATTEN, N., AND POIRIER, M. (1968) Anal. Biochem. 22, 359. 12. CIHAK, A., LAMAR, C., AND PITOT, H. C. (1973) Arch. Biochem. Biophys. 166, 176. 13. CHO-CHUNG, Y. S., AND PITOT, H. C. (1968) Eur. J. Biochem. 3, 401. 14. ROSEN, F., AND NICHOL, C. A. (1968) Advan. Enzyme Regul. 2, 115. 15. CIHAK, A., WILKINSON, I>., AND PITOT, H. C. (1971) Advan. Enzyme Regul. 9, 267. 16. KENNEY, F. T. (1962) J. Biol. Chem. 237, 3495. 17. LIN, E. C. C., AND KNOX, W. E. (1957) Biochim. Biophys. Acta 26, 85. 18. GARREN, L. D., HOWELL, R. R., TOMKINS, G. M., AND CROCCO, R. M. (1964) Proc. Nat. Acad. Sci. USA 62, 1121. 19. REEL, J. R., AND KENNEY, F. T. (1968) Biochemistry 61, 200. 20. LEVITAN, I. B., AND WISBB, T. E. (1969) J. Biol. Chem. 244, 341. 21. SCHOENHEIM&R, It. (1964) The Dynamic State of Body Constituents, Harvard Univ. Press, Cambridge. 22. VESELY, J., AND CIH~K, A. (1970) Biochim. Biophys. Acta 204, 614. 23. MURTY, C. N., AND SEDRAUSKY, N. (1972) Biochim. Biophys. Acta 262, 328. 24. ~)I~Av~E, M., AND KORNER, A. (1971) Biochim. Biophys. Acta 247, 404. 25. ALT.EN, R. E., RAINES, P. L., AND REGEN, U. M. (1969) Biochim. Biophys. Acta 190, 323. 26. HARDELAND, R. (1970) Life Sci. 9, 901. 27. SF,GUCHI, T., AND BARCHAS, J. (1971) J. Biol. Chem. 246, 7217. 28. ROSEN, F., AND MILHOLLAND, V. (1963) J. Biol. Chem. 238, 2730.
OF
BIOCHEMISTRY
L-Tryptophan
McArdle
AND
Inhibition
166, 188-194
BIOPHYSICS
(1973)
of Tyrosine
Aminotransferase
in Rat Liver
in Viva’
ALOIS
CIHAKz,
CARLOS
LAMAR,
Memorial
Laboratory,
The Medical
School,
Received
JR.~, University
December
AND HENRY of Wisconsin,
Degradation
C. PITOT Madison,
Wisconsin
54706
1, 1972
The administration of L-tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of L-tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in viva. When L-tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-HydroxynL-tryptophan and indole administration in amounts equimolar to L-tryptophan also result in similar increases in activity whereas a-methyl-DL-tryptophan produces little or no increase. Utilizing pulse-labeling ipz viva with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.
1.13.1.12), by tryptophan and cortisone, which had been noted earlier by Knox and his associates (2) as well as Feigelson and Greengard (3), could be explained on the basis that cortisone induced an increased rate of synthesis of the enzyme whereas tryptophan stabilized the enzyme in vivo and prevented its degradation. Schimke’s report was perhaps the first concrete evidence that the level of an enzyme could be controlled in vivo by altering the dynamic turnover of the enzyme and preventing its destruction within the cell. Since those reports, other studies have indicated that the regulation of the rate of degradation of an enzyme is extremely important in the final determination of enzyme levels in vivo.
In 1965 Schimke and his associates (1) reported that the differences in the regulation of the enzyme, tryptophan oxygenase (L-tryptophan , oxygen oxidoreductase, EC 1 The work reported in this paper was supported in part by grants from the National Cancer Institute (CA-07175) and the American Cancer Society (P-314 and E-588). 2 The work reported in this paper was undertaken during the tenure of an Eleanor Roosevelt Cancer Fellowship of the International Union Against Cancer awarded to Dr. Alois Cihak. Permanent address : Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague-6, Czechoslovakia. 3 Present address: Department of Medicine, The Medical School, Texas Technological University, Lubbock, Texas. 188
Copyright 0 1973by AcademicPress,Inc. All rights of reproductionin my form reserved.
TYROSINE
AMINOTRANYFERASE
Studies on the heart and muscle forms of lactate dehydrogenase (4) have demonstrated that the rates of turnover of the two subunits in heart and liver are quite dissimilar and may, in fact, account for the differences in the levels of the lact’ate dehydrogcnase isozymes seen in these two tissues. In tissue culture, an inhibition of the degradation of glutaminc synthetase has been invoked as the mechanism whereby this enzyme increases in the absence of glutamine (5), and the high level of arginase stimulated by addition of manganese in vitro also appears to be the result of enzyme stabilization (6). Alt’hough the earlier studies by Schimke and his associates (1) were only concerned with the stabilization of tryptophan oxygenase, it is now apparent Ohat t’ryptophan has several other functions in enzyme synthesis (7-10). The data reported in this paprr indicate that at least one other enzyme, tyrosine aminotransferase (L-tyrosine, 2-oxoglutarate aminotransferase, EC 2.6.1.5), is also stabilized by the administration of L-tryptophan GL vivo. MATERIALS
AND
METHODS
In all the experiments reported in this paper, male rats (180-220 g) obtained from the Holtzmann Laboratories in Madison, WI were kept for 3-5 days either on a laboratory chow diet containing a minimum of 2O7? crude protein or on a 12.5% casein diet. The animals were fasted for lo-12 hr prior to initiating the experiments between 7:00 and 8:00 .41x. Adrenalectomized male rats (160-180 g) were obtained from the Endocrine Laboratories in Madison and were kept under the same conditions as described above with the addition of 1% sodium chloride in their drinking water. For determination of enzyme levels and incorporat,ion of labeled amino acids irk Y~ZJO,groups of four to six rats were used for each point. Individual experiments were repeated two to four times. At various t,ime intervals after injection and/or intubation, the animals were sacrificed by decapitation, the livers rapidly removed, weighed, and homogenized in 3 vol (w/v) of 0.25 M sucrose in 0.05 ~1 Tris-HCl buffer (pH 7.2) containing 10 rnbr MgC12, 25 mM KCl, 0.1 mM pyridoxal phosphate, and 1 rnzf dithiothreitol. Homogenates were centrifuged for 1 hr at 100, OOOg, and the SUpernatants were used for the determination of enzyme activities and later immunoprecipitation. Tryptophan oxygenase and tyrosine aminotrans-
STABILIZATION
189
ferase were assayed utilizing a combination automated unit previously described (11). For immunoprecipitation studies, L-[4,5-3H]leucine (sp act 50 pCi/l5 nmolelanimal) or L-[l14C]valine (sp act. 21 &i/O.9 rmolejrat) was injected intraperitoneally in a 0.25.ml volume 45 min prior to sacrifice of the animals. Aliquots of the 100,OOOg supernatant (0.2-0.3 ml) were added to immune and nonimmune rabbit serum (0.2-0.4 ml). Saline was added to a total volume of 0.5 ml and the antigen-antibody complex allowed to precipitate over a 4%hr period at 4°C. The immune precipitates were centrifuged, washed 3 times with 2 ml of 0.9(% NaCl, and dissolved in 0.2 ml of 99yA formic acid. Radioartivity was measured in a Packard Tri-Carb liquid scintillation counter utilizing Scintisol (Isolab) as a scintillation counting medium. In all instances, the absence of tyrosine aminotransferase in supernatants after immunoprecipitation was checked by means of an automated assay (11). Antibodies to tyrosine aminotransferase were prepared in rabbits and titrat,ed as reported in a previous publication (12). The immunological identity of enzyme induced by cortisone or tryptophan was also reported in the same paper. All immunoprecipitations were carried out in the region of antibody excess, nonspecific adsorption of counts was determined by the method of ChoChung and Pitot (13), and appropriate corrections were made. Cortisone acetate was obt,ained from the Upjohn Company. L-Tryptophan was obtained from General Biochemicals. L-[4,5-3H]Leucine (sp act 22.6 mCi/mmole) was obtained from Schwarz BioResearch. Dithiothreitol and L-kynurenine were obtained from the Calbiochem Corporation. 5-Hydroxy-oL-tryptophan and quinolinic acid were products of Sigma, while anthranilate and indole were from Eastman Chemicals. a-MethyluL-tryptophan was kindly provided by Dr. Henry Lardy. RESULTS
Previous studies by other workers (9, 10, 14) have demonstrated that t’he administration of L-tryptophan causes a marked increase in the activities of several enzymes, including tyrosine aminotransferase. The relationship of the amount of L-tryptophan administered to the hepatic activity of both tryptophan oxygenase and tyrosine aminotransferase measured 5 hr after tryptophan administration has been reported (15). From this data it may be noted that the induction of tyrosine aminotransferase is, in fact, much
190
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TIME (hrs) FIG. 1. Time course of tyrosine aminotransferase (pOHphenylpyruvate production, -o-) and tryptophan oxygenase (kynurenine production, -O-) activities after n-tryptophan administration. Holtzman male rats (2OG220 g) starved 14 hr were intubated with L-tryptophan [25 mg (-•-) and 100 mg (-O-) per 100 g, respectively] and killed at different time intervals thereafter.
more sensitive to the administration of L-tryptophan than is tryptophan oxygenase. In Fig. 1 is seen the time course of changes in tyrosine aminotransferase activity after a single dose of L-tryptophan. Although the amount of L-tryptophan given to induce the enzyme in this figure was only one-quarter that given to induce tryptophan oxygenase, the shapes of the curves are quite similar. As previous workers have demonstrated (14, 16, 17), tyrosine aminotransferase may be induced in the intact animal by a number of different materials, all of which have as their common denominator a stimulation of adrenocortical response. In Fig. 2a and b is noted the effect of the time after the administration of L-tryptophan on the level of tyrosine aminotransferase activity as well as the relation of the dose of tryptophan to enzyme activity in adrenalectomized animals. The time course of induction is almost identical to that seen in intact animals, and the sensitivity to the amount of L-tryptophan given also appears to be quite similar, although, at doses of tryptophan above 3040 mg/lOO gm body wt, animals die due to
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the toxicity of the amino acid. Thus, it is apparent that the increases seen under these conditions are not mediated by the adrenal glands. In Table I is seen the effect of the administration of the analogue, m-(a-methyl) tryptophan, as well as several tryptophan metabolites on the activity of tyrosine aminotransferase in vivo. In all instances, the compounds were administered by intubation in amounts equimolar to 20 mg of L-tryptophan, which was administered to a control group of animals. If the compound was a mixt’ure of the DL forms, t,he racemate was made equimolar in the L form. It can be noted that the analogue, cr-methyl-mtrvptophan, produces essentially no increase in the level of tyrosine aminotransferase over the control activity. On the other hand, 5-hydroxy-m-tryptophan and indole produce an increase in tyrosine aminotransferase activity which is similar to that seen with L-tryptophan alone. In two animals given L-kynurenine, the increase also was essentially identical to that in animals given L-tryptophan. Quinolinate and anthranilate produced either no increase in the case of the former, or an intermediate increase in the case of the latter. While these studies do not establish a structure-response relationship, they do serve to indicate that the tryptophan nucleus itself probably does not stimulate the increase in tyrosine aminotransferase, since the analogue had no effect. Furthermore, metabolism beyond anthranilate also appears to destroy the stimulatory activity. On the other hand, indole, which forms the nucleus of the amino acid, appears to be equally as good as tryptophan in stimulating the response. When L-tryptophan was given to animals who had already received an injection of cortisone, there is a further increase in the level of both enzymes. The data may be seen in Figs. 3 and 4. Herein, cort’isone was administered at zero time, and at the peak of enzyme induction (5 hr later) a dose of L-tryptophan was administered. In contrast to the animals not receiving L-tryptophan, wherein the level of the enzyme decreased markedly for the next 7 hr, in those animals receiving L-tryptophan a continual increase
TYROSINE
AMINOTRANSFERASE I
1
STABILIZATION
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191
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b
0 300
150
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4 I3 TIME (hrs)
I2
40 60 20 Dosage af L-Tryplophan (mg per IOOgm body weight)
FIG. 2. Increase of liver t,yrosine aminotransferase in adrenalectomized rats after the intubation of 1,.tryptophan. Time (a) and dose (b) relations. Holtzman male adrenalectomized rats (160 g) kept 3 days on 12.5y0 protein diet were starved for 12 hr. L-Tryptophan was intubated in a maximal volume of 5 ml of saline. (a) L-tryptophan was administered in a dose of 0.2 g/kg; (b) animals were killed6 hr after intubation of the indicated dose of L-tryptophan, and tyrosine aminotransferase activities are expressed as the average of 46 values f SEM.
in enzyme activity, although at a slightly lower rate, was demonstrated. The administration of actinomycin subsequent to corticosteroid administration produces a similar effect (18, 19) in the case of both tyrosine aminotransferase and tryptophan oxygenase. The administration of 8-azaguanine (20) and of 5-fluoroorotic acid or 5-azacytidine (15) after corticosteroid administration also mimics the effects of tryptophan or actinomycin. In order to determine the relative roles of enzyme synthesis and degradation in the effect of L-tryptophan on tyrosine aminotransferase, pulse-labcling of the enzyme in vivo coupled with quantitative immunoprecipitation techniques was utilized (1, 13). Animals were given either cortisone or L-tryptophan, and 4 hr and 15 min later a dose of tritiated lcucine was injected as described in the Methods. These animals were sacrificed 45 min later, high-speed supernatants of the liver prepared, enzyme activities determined, and the tyrosinc aminotransferase quantitatively precipitated by its specific antibody. The antigen-antibody precipitate was then counted as a measure of the pulse-labeled synthesis of the enzyme in vivo. Earlier studies (15) had dcmon-
&rated that although there was a marked increase in the activity of the enzyme after the administration of cortisone or L-tryptophan, both in intact and adrenalectomized animals there was a significant increase in label in the enzyme antigen only after the administration of cortisone. Cortisone administration resulted in twice the activity of tyrosine aminotransfcrase of that seen after L-tryptophan administration. This might be expected if the hormone actually induces an increased rate of synthesis while the amino acid inhibits enzyme degradation (1). The labeling of total liver soluble protein was at a level of 200,000-300,000 cpm/mg protein and did not change significantly under the conditions utilized, indicating a selective induction of the synthesis of tyrosine aminotransferase. It was apparent, however, that the administration of L-tryptophan, either to intact or adrenalectomized animals, did not cause an increased labeling of total protein under the conditions of the experiment. In Fig. 5 is seen the result of an experiment wherein tyrosine aminotransferase was induced by the administration of cortisone; 4 hr later [lJ4C]valine was injected, and animals were sacrificed at the times after the
192
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LAMAR,
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EFFECT OF ADMINISTRATION OF ANALOGUBX AND METABOLITES OF TRYPTOPHAN ON TYROSINI.: AMINOTRANSFERASE ACTIVITY Z'YLVivoa
Compound
No. of rats
Tyrosine aminotransferase activity
Saline L-Tryptophan (20 mg) 5-Hydroxy-nL-tryptophan (44 md L-Kynurenine SO4 (42 mg) Quinolinate (17 mg) DL-(a-methyl)Tryptophan (44 mg) Anthranilate (14 mg) Indole (12 mg)
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19.4 18.1
(1All compounds were administered by intubation of the amounts shown in parentheses dissolved in 5 ml Hz0 at zero time to adrenalectomized male rats (140-160 g) maintained for 3 days after adrenalectomy on a 12.5% protein diet. Animals were sacrificed 5 hr after intubation, and the enzyme activity assayed as described in the Methods. Values are averages f SEM.
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FIG. 3. Increase of cortisone-induced tyrosine aminotransferase activity by the delayed administration of L-tryptophan (L-TRY). Holtzman male rats starved 14 hr were injected ip with cortisone (5 mg per animal, -O-), and 5 hr later a group of 4-6 of these animals were intubated with L-tryptophan (100 mg/lOO g, -0-). All points represent the average values of 4-6 animals f SEM.
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TIME (HRS.1 FIG. 4. Increase of cortisone-induced tryptophan oxygenase activity, as measured by kynurenine production, by the delayed administration of L-tryptophan (L-TRY).
initial
administration
of cortisone
shown in
Fig. 5. The X’s and solid line denote the changes in radioactivity and enzyme activity of tyrosine aminotransferase after the administration of cortisone. The solid circles
and dashed line denote the decay in radioactivity and enzyme activity of tyrosine aminotransferase in animals given cortisone at zero time and L-tryptophan (170 mg/kg/ dose) by intubation at 5 hr and 7 hr after the administration of cortisone. As can be seen in Fig. 5, while there was a decay in both radioactivity and enzyme activity between 6% and 945 hr after the administration of cortisone only, those animals receiving tryptophan in addition at 5 and 7 hr showed a marked inhibition of the rate of enzyme decay. These data, coupled with those previously reported (15), argue that the increases in tyrosine aminotransferase brought about by the administration of L-tryptophan are predominantly the result of an inhibition of enzyme degradation rather than a stimulation of enzyme syntheSlS.
DISCUSSION
The phenomenon of enzyme and protein turnover has been known for many years
TYROSINE
Time
in Hours after
AMINOTItANSFE:RASE
Cortisone
5. Loss of activity and radioactivity of tyrosine aminotransferase (TAKG) in livers of animals induced with cortisone and given L-tryptophan 5 and 7 hr after administration of the steroid. All animals received cortisone acetate (5 mg/rat ip) at zero time. [lJ4C]valine (21 pCi/O.S pmolelrat) was injected ip 4 hr after cortisone administration. L-tryptophan (170 mg/kg/dose, dashed line) or saline (0.5 ml, solid line) were administered by intubation in two doses at 5 and 7 hr after cortisone. Animals were sacrificed at the time intervals shown after cortisone administration. The radioactivity of the enzyme-antigen complex was determined as described in the Methods. All values are averages from five animals. The standard error of the mean did not exceed 50 pmoles/g liver/hr or 150 counts per 100,000 counts. FIG.
(21). As indicated in the introduction, however, specific environmental controls of enzyme turnover have only recently become apparent. The studies described herein expand a previous investigation (1) showing that the essential amino acid, L-tryptophan, is capable of stabilizing tryptophan oxygenase, demonstrate that at least one other enzyme, tyrosine aminotransferasc, is also stabilized, and its degradation in viva prevented by the administration of the amino The data described acid, L-tryptophan. herein actually demonstrate that the turnover of this enzyme is considerably more
STABILIZATION
193
sensitive to L-tryptophan administration than is that of tryptophan oxygenase. Several investigators, including Munro (7), Sidransky (8), and others, have shown that L-tryptophan appears to have a relatively unique role in the regulation of protein synthesis in liver. Munro and his associates (7) have demonstrated that, when tryptophan is absent from a mixture of amino acids, the administration of such tryptophan-devoid solutions in vivo causes a marked decrease in the polysomes of normal liver. Sidransky et al. (8) has shown a similar effect, although it is appa,rent that the dccreascd polysome pattern is dependent on stimulation of new protein synthesis. In recent studies by Sidransky’s group (unpublished observations), the loss of polysome patterns in liver resulting from puromycin administration in vivo can be reversed by the administration of tryptophan alone in vivo. Lardy and his associates (10) have also demonstrated that, the administrat,ion of tryptophan causes an increase in the hepatic enzyme, phosphoenolpyruvate carboxykinase (phosphoenolpyruvate oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32). Tryptophan has a marked effect, on gluconeogenesis in liver that appears to be related to its degradat’ion to quinolinic acid; this compound acts as a chelator of essential metal ions required in the activity of certain enzymes, notably the carboxykinase. Recently it was observed that administration of L-trypt’ophan results in a marked increase of DNA-dependent RNA polymerase isolated from rat liver nuclei (22) and that the synthesis of liver messenger RNA is enhanced by this t’reatmcnt (23). Other studies by Oravec and Korner extend these investigat’ions showing that tryptophan administration also st’imulates the synthesis of ribosomal RtNA (24). Studies by Allen et al. have demonstrated that tryptophan deficiency causes a marked decrease in the level of charged tryptophanyl tRNA. This decrcasr is much greater than that seen u-ith other specific aminoacyl t,RNA’s in the livers of animals fed diets deficient
in other amino acids (25).
Therefore, it is apparent that tryptophan plays a wntral role in protein synthesis, as
194
CIHAK,
LAMAR,
well as in the regulation of enzyme levels in normal rat liver. The fact that tryptophan stabilizes tryptophan oxygenase and tyrosine aminotransferase is another facet of this rather complex picture. As described in this paper (Table I), metabolites of tryptophan also appear to produce the stabilizing effect on tyrosine aminotransferase. Hardeland (26) described experiments in which quinolmate, when given in doses substantially higher than those administered here, produced a significant increase in tyrosine aminotransferase and tryptophan oxygenase. Deguchi and Barchas (27) also demonstrated that the administration of 5-hydroxytryptophan caused a significant increase in tyrosine aminotransferase in rat liver ad did earlier studies by Rosen and Milholland (28). The fact that the analoguc, ar-methyltryptophan, produced no such increase does suggest that some sort of metabolism of the amino acid must occur for the stabilizing effect to be seen. On the other hand, indole, a rather distant metabolite, does produce the stabilizing effect, at least for tyrosine aminotransferase. In a previous paper (12), the administration of 5-fluoroorotate to animals subsequently given tryptophan produced a further stimulation over that by the amino acid alone. Since those studies demonstrated that the analogue also caused a stabilization of the enzyme by inhibiting its degradation in vivo, it would appear that the mechanism of the pyrimidine analogues and tryptophan in stabilizing tyrosine aminotransforase are basically different. Since tryptophan plays a central role in protein synthesis, it is tcmpting to speculate that the stabilization of an enzyme by this amino acid is related to its role in translation. On the other hand, the base analogues and actinomycin D, having in common the inhibition of RNA synthesis, may exert their effects by a mechanism resulting from or associated with this inhibition. REFERENCES 1. SCHIMKE, R. T., SWEENEY, E. W., AND BERLIN, C. M. (1965) J. Biol. Chem. 240,322. 2. KNOX, W. E., AND AUERBACH, V. H. (1955) J. Biol. Chem. 214, 307.
AND
PITOT
3. GREENGARD, O., AND FEIGELSON, P. F. (1961) Nature (London) 190, 446. 4. PITOT, H. C., P*XUINO, C., LAMAR, C., AND KENNAN, A. L. (1965) Proc. Nat. Acad. Sci. USA 64, 845. 5. PAUL, J., AND FOTTRELL, P. F. (1963) Biochim. Biophys. Acla 67, 334. 6. SCHIMICE, R. T. (1964) J. Biol. Chem. 239, 3808. WUNNER, W. H., B~:LL, J., AND MUNRO, H. N. (1966) Biochem. J. 101,417. 8. SIDRANSKY, H., SARMA, 1). S. R., BONGIORNA, AND VICRNEY, (1968) J. Biol. Chem. 243, 1123. 9. PERAINO, C., BLAKE, R. L., AND PITOT, H. C. (1965) J. Biol. Chem. 240, 3039. 10. FOSTER, D. O., RAY, P. D. AND LARDY, H. A. (1966) Biochemistry 6, 563. 11. PITOT, H. C., WRATTEN, N., AND POIRIER, M. (1968) Anal. Biochem. 22, 359. 12. CIHAK, A., LAMAR, C., AND PITOT, H. C. (1973) Arch. Biochem. Biophys. 166, 176. 13. CHO-CHUNG, Y. S., AND PITOT, H. C. (1968) Eur. J. Biochem. 3, 401. 14. ROSEN, F., AND NICHOL, C. A. (1968) Advan. Enzyme Regul. 2, 115. 15. CIHAK, A., WILKINSON, I>., AND PITOT, H. C. (1971) Advan. Enzyme Regul. 9, 267. 16. KENNEY, F. T. (1962) J. Biol. Chem. 237, 3495. 17. LIN, E. C. C., AND KNOX, W. E. (1957) Biochim. Biophys. Acta 26, 85. 18. GARREN, L. D., HOWELL, R. R., TOMKINS, G. M., AND CROCCO, R. M. (1964) Proc. Nat. Acad. Sci. USA 62, 1121. 19. REEL, J. R., AND KENNEY, F. T. (1968) Biochemistry 61, 200. 20. LEVITAN, I. B., AND WISBB, T. E. (1969) J. Biol. Chem. 244, 341. 21. SCHOENHEIM&R, It. (1964) The Dynamic State of Body Constituents, Harvard Univ. Press, Cambridge. 22. VESELY, J., AND CIH~K, A. (1970) Biochim. Biophys. Acta 204, 614. 23. MURTY, C. N., AND SEDRAUSKY, N. (1972) Biochim. Biophys. Acta 262, 328. 24. ~)I~Av~E, M., AND KORNER, A. (1971) Biochim. Biophys. Acta 247, 404. 25. ALT.EN, R. E., RAINES, P. L., AND REGEN, U. M. (1969) Biochim. Biophys. Acta 190, 323. 26. HARDELAND, R. (1970) Life Sci. 9, 901. 27. SF,GUCHI, T., AND BARCHAS, J. (1971) J. Biol. Chem. 246, 7217. 28. ROSEN, F., AND MILHOLLAND, V. (1963) J. Biol. Chem. 238, 2730.