GLUOCXEOGENIC ENZYMES* Neogenic Processes. The

96 BIOCHEMISTRY: WEBER ET AL. PROC. N. A. S.

tion yielded products which competitively inhibited the aminoacylation of native sRNA. The inhibition was specific for a given amino acid acceptor. Periodate oxidation of preparations enriched in tyrosyl-sRNA and poor in valyl-sRNA yielded an inhibitor effective in the tyrosine system and ineffective in the valine system. Similar specificity was observed with a periodate-treated fraction which had been enriched in valine acceptor activity. * USPHS postdoctoral International Fellow; Fellow del Instituto Nacional de la Investigacion Cientifica de Mexico. 1 Chapeville, F., F. Lipmann, G. von Ehrenstein, B. Weisblum, W. J. Ray, Jr., and S. Benzer, these PROCEEDINGS, 48, 1086 (1962). 2 Bergmann, F. H., P. Berg, and M. Dieckmann, J. Biol. Chem., 236, 1735 (1961). 3 Apgar, J., R. W. Holley, and S. H. Merrill, J. Biol. Chem., 237, 796 (1962). 4 Berg, P., F. H. Bergmann, E. J. Ofengand, and M. Dieckmann, J. Biol. Chem., 236, 1726 (1961). 6 Zubay, G., and M. Takanami, Biochem. Biophys. Res. Commun., 15, 207 (1964). 6Nihei, T., and G. L. Cantoni, J. Biol. Chem., 238, 3991 (1963). 7 Holley, R. W., J. Apgar, B. P. Doctor, J. Farrow, M. A. Marini, and S. H. Merrill, J. Biol. Chem., 236, 200 (1961). 8 Preiss, J., P. Berg, E. J. Ofengand, F. H. Bergmann, and M. Dieckmann, these PROCEEDINGS, 45, 319 (1959). 9 Whitfeld, P. R., and R. Markham, Nature, 171, 1151 (1953). 10 Lineweaver, H., and D. Burk, J. Am. Chem. Soc., 56, 658 (1934). 11 A comparison of the products of a 4% digestion with snake venom by the two procedures showed that the capacity to accept valine was reduced to about 1% by the technique of Zubay and Takanami (ref. 5), whereas digestion at pH 8.9 resulted in retention of 33% of valine acceptor activity. 12 The quantity of periodate-oxidized sRNA required to achieve a 50% inhibition in the tyrosine test system using fraction Tyr (Fig. 5) was about l/2o of that necessary for a 50% inhibition in the valine test system using fraction Val, (Fig. 4). This disparity can largely be accounted for by the facts that: (a) compared to crude sRNA, tyrosine acceptor sRNA was 6-fold enriched in fraction Tyr, while valine acceptor sRNA was enriched only 1.4-fold in fraction Val, (Table 2); and (b) the level of untreated sRNA used in the valine experiment contained three times more valine acceptor sRNA than the amount of tyrosine acceptor sRNA used in the tyrosine experiment and therefore would require a larger quantity of periodate-oxidized sRNA to achieve an equivalent inhibition. 13 Hecht, L. I., M. L. Stephenson, and P. C. Zamenenik, these PROCEEDINGS, 45, 505 (1959).

INSULIN: SUPPRESSOR OF BIOSYNTHESIS OF HEPATIC GLUOCXEOGENIC ENZYMES* BY GEORGE WEBER, R. L. SINGHAL, AND S. K. SRIVASTAVA

DEPARTMENT OF PHARMACOLOGY, INDIANA UNIVERSITY SCHOOL OF MEDICINE, INDIANAPOLIS, INDIANA Communicated by Charles Huggins, October 30, 1964 The homeostatic control of blood sugar depends in part on regulation of gluconeogenic processes. The "final common path of gluconeogenesis,"l the series of steps converting pyruvate into glucose, involves a number of reversible and 4 one- way reactions. From the point of view of metabolic control our attention is Downloaded by guest on September 28, 2021 VOL. 53, 1965 BIOCHEMISTRY: WEBER ET AL. 97

focused on the one-way reactions catalyzed by the key enzymes of gluconeogenesis, glucose 6-phosphatase, fructose 1,6-diphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate carboxylase. The limiting role of these enzymes is emphasized by the following properties relevant to regulation. The enzymes are involved in circumventing thermodynamic barriers,2 they are all one-way reactions, their activities are the lowest in the gluconeogenic sequence,' and they are organ- specific since they occur only in liver and kidney where gluconeogenesis can take place. The genetic code of the key gluconeogenic enzymes probably is in the same region or on the same genetic chain, since these are the only enzymes of carbo- hydrate metabolism which are progressively deleted with increasing growth rate in hepatomas and are completely absent in rapidly growing liver tumors.3 4 In searching for regulatory influences that could selectively and differentially control the enzymatic processes of gluconeogenesis, we investigated the action of glucocorticoid hormone and insulin. Evidence will be presented indicating that the genic expression of the key gluconeogenic enzymes is regulated by the action of glucocorticoid as an inducer and insulin as a suppressor of the biosynthesis of these hepatic enzymes. Methods.-Male Wistar rats of 90-110 gm were kept in separate cages, with Purina laboratory chow and water ad libitum unless otherwise specified. The tech- niques for preparation of tissue homogenate, supernatant fluid, counting of cell nuclei, assaying glucose 6-phosphatase and fructose 1,6-diphosphatase were described previously.5' 6 The determination of RNA amount and specific activity was cited elsewhere.7 Orotic acid-6-C14 (New England Nuclear Corp.) had a specific activity of 6.5 mc/mM, and 3 jAC/100 gm rat was injected intraperitoneally 2 hr before animals were killed. Triamcinolone (Lederle) was purchased as a commercial preparation. Actinomycin D was a gift from Merck, Sharp & Dohme. Enzyme activities were expressed per cell as micromoles of substrate X 107 metabolized per hr at 370C. The RNA specific activity was calculated as cpm per mg RNA. The data were given for convenient comparison as percentages of values found in normal untreated rats. Alloxan diabetes was induced, in animals starved for 30 hr, by intraperitoneal injection of 12 mg per 100 gm rat alloxan monohydrate (Eastman). Blood sugar was determined according to Nelson's adaptation of the Somogyi method for glucose.8 Results and Discussion. -Evidence for the action of glucocorticoid hormone as inducer of biosynthesis of key gluconeogenic enzymes: For the definition of an inducer, the one recommended in the "Report of the Commission on Enzymes" is used.9 "A relative increase in the rate of synthesis of a specific apoenzyme resulting from ex- posure to a chemical substance will be called enzyme induction. The substance inducing such a synthesis is an enzyme inducer." The following evidence has ac- cumulated to show that this concept applies to the action of glucocorticoid hor- nmones on hepatic gluconeogenic enzymes. Glucocorticoid hormones increased the activities of rat liver glucose 6-phosphatase,5' 10. "fructose 1,6-diphosphatase," 12phosphoenolpyruvate carboxykinase,3 14 and pyruvate carboxylase. 1" A more marked response for the specific phosphatases was obtained with triamcinolonel which was also more effective in promoting gluconeogenesis."6 Graded effects occurred with increasing hormone dosage." 13, 17 The enzyme increases were detectable in a few hours. 1, 17. 18 The increases are Downloaded by guest on September 28, 2021 98 BIOCHEMISTRY: WEBER ET AL. PROC. N. A. S.

thought to represent de novo enzyme synthesis because the rise can be blocked or prevented by inhibitors of protein synthesis such as actinomycin," 7, 18-20 puromycin," 13 or ethionine." 12, 13, 21 The blockers of protein synthesis were capable of interrupting the glucocorticoid-induced synthesis and then enzyme activities decreased to normal in 24 hr." 18, 22 In acute starvation these enzymes were preferentially maintained near normal range.'3' 23-25 However, when hypophysectomized rats were fasted, the enzyme activities rapidly declined.6' 25 The preferential maintenance of specific phosphatases in starvation also can be interrupted by actinomycin or ethionine and then activities rapidly decrease.22 Adrenalectomy returned the highly increased enzyme activities to normal in diabetic rats." 11 13 In rats carrying ACTH-secreting pituitary tumors the glucose 6-phosphatase and fructose 1,6-diphosphatase activities increased and upon adrenalectomy they returned to normal range.26 Thus, endogenous hormones exert biosynthetic effects similar to those observed with exogenous synthetic glucocorticoid derivatives. The role of RNA metabolism in glucocorticoid-induced metabolic responses was shown by the stimulation in the rates of incorporation of radioactive precursors into RNA.27-29 Since it was observed that actinomycin, a selective inhibitor of DNA- directed RNA synthesis,30 inhibited the cortisone-induced elevation of certain enzymes, it was suggested that the stimulation of precursor incorporation into RNA is an integral part of the hormone action.3' Similar concepts were formed by in- vestigators who brought evidence that the early effects of corticosteroid hormones involve stimulation of RNA polymerase activity and messenger RNA synthesis in rat liver nucleus.32-36 The role of RNA metabolism in glucocorticoid action is further emphasized by the graded dose response of an increase in hepatic RNA amount and by the rise in specific activity with orotate or uracil as precursors in response to increasing doses of triamcinolone.20 22 A correlation of the glucocorticoid-induced rise in liver enzyme biosynthesis, RNA metabolism, and amino acid levels was shown and the increases found with triamcinolone were more marked than those obtained with cortisone.7' 20 This accords with the higher gluconeogenic potency of the fluori- nated steroid.'6 The involvement of DNA-directed RNA synthesis in gluconeogenic events is indicated by the complete blocking of the triamcinolone or cortisone- induced rise in the biosynthesis of hepatic gluconeogenic enzymes'7' 18-20 and in the increase of RNA metabolism by actinomycin.7 20 The enumerated evidence favors the concept that the glucocorticoid hormones act as inducers of biosynthesis of the key gluconeogenic enzymes. Thus, glucocorticoids act positively on the transcription of structural genes into specific cata- lytic proteins, namely, key enzymes in the gluconeogenic sequence. Evidence for the action of insulin as suppressor of biosynthesis of key gluconeogenic enzymes: In contrast to the function of an inducer, a suppressor acts negatively o11 the transcription of structural genes into enzyme proteins. The antagonistic action of insulin to the function of glucocorticoid hormone has been observed at both clinical and physiological levels. The suggestion that the action of insulin on gluconeogenesis is explained, in part, at the molecular level by its role as a suppressor of the biosynthesis of hepatic gluconeogenic enzymes is supported by the following experimental evidence. Downloaded by guest on September 28, 2021 VOL. 53, 1965 BIOCHEMISTRY: WEBER ET AL. 99

260 FGLUCOSE - 6- PHOSPHATASE 227' 45 FRUCTOSE - 1.6- DIPHOSPHATASE 231 220 214'

.i180 166' 8'20

0 140 a4 .4S- 1 o126' 140 124' 138' 13 . 106- ~~~~~~~~~~~~~~~~~~~~~200'127' 133' 107 " 131 10-40 113'16 5'86 FHOMOGENATE NITROGENATNITOG1

20 INJECTION IN ION

0 30hrs of 0 24 48 72 96 030hrs of 24 48 72 96

Fasting I| FFasting IHOURS AFTER ALLOXAN INJECTION HOURS AFTER ALLOXAN INJECTION1 FIG. 1.-Sequence of events in acute diabetes-induced synthesis of hepatic gluconeogenic enzymes. (Enzyme activities and nitrogen levels were calculated per average cell.) Rats were starved for 30 hr and then injected with alloxan. Groups of animals were killed at subsequent intervals and enzyme activities were determined. Asterisks denote alterations statistically dif- ferent from values of normal fed rats. The findings which first implicated the influence of insulin on liver glucose 6- phosphatase were the reports of Ashmore et al. showing that this enzyme was increased in alloxan diabetic rats and the activity returned to normal by insulin administration."' 23 Insulin further decreased the enzyme activity in hypophysectomized rats,23 and adrenalectomy was capable of returning the high enzyme activity in diabetes to normal range." 23 The importance of these findings is emphasized by the data obtained in this laboratory on the effects of insulin on liver gluconeogenic enzyme biosynthesis. The new evidence summarized below suggests that the attacking point of insulin may be identified at the level of the enzyme-form- ing systems. (1) Acute diabetes-induced sequence of events in enzyme biosynthesis: When acute diabetes is induced by alloxan injection and the insulin level is suddenly decreased or disappears from circulation, hepatic glucose 6-phosphatase and fructose 1,6-diphosphatase activities steadily rise and in 72-96 hr increase more than two- fold. The increase is much higher than that in nitrogen content, indicating a preferential synthesis of these enzyme proteins (Fig. 1). The activities of phosphoenolpyruvate carboxykinase'3 14, 18, 37 and pyruvate carboxylase38 39 were also increased in diabetes. (2) Blocking of diabetes-induced enzyme synthesis by inhibitors of protein synthesis: That in acute diabetes the increases in liver glucose 6-phosphatase and fructose 1,6-diphosphatase activities were due to synthesis is suggested by the fact that the rises were blocked by injection of inhibitors of protein synthesis, actinomycin, puromycin, or ethionine." 40 (3) Release from insulin suppression of the enzyme biosynthetic processes: If insulin can act as a suppressor, hormone replacement therapy in diabetes should keep enzyme activities in normal range. However, when the suppressor is suddenly re- moved, enzyme activities should abruptly rise. That this is indeed the case is shown in the experimental series given in Figure 2. Insulin administration was able to keep the gluconeogenic enzyme activity in normal range in acutely diabetic rats. Downloaded by guest on September 28, 2021 100 BIOCHEMISTRY: WEBER ET AL. PROC. N. A. S.

FRUCTOSE-1, 6-DIPHOSPHATASE --% F. 250300--

U~~~~~~~~~~~~~~~~~~~~~~~~~

O I. DIABETIC l 0 1

DAYS -_ 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 30 Hrs. Fasted 1tt t t t t t t t t t t t t t t t t t t t t t t I INSULIN INJEIONS INSULIN I INIJECTIONS I ALLOXAN . l (4 Units) WITHDRAWN (4 Units) (8 Units) FIG. 2.-Effect of insulin administration on hepatic fructose 1,6-diphosphatase activity in alloxan diabetic rats. Hepatic fructose 1,6-diphosphatase activity rose in 5-6 days to a plateau of high activity which was maintained throughout the experiment. Insulin administration lowered the increased enzyme level to normal range; however, when insulin was stopped, fructose 1,6-diphosphatase activity quickly rose in 1 day to 2.5-fold of normal activity. Reinstatement of insulin therapy was of little effect, but double doses of insulin brought the enzyme activity down to normal. However, as soon as insulin injections were stopped, the specific phosphatase rapidly rose to high activity levels. The dose regimen of insulin which was adequate in acute diabetes was not effective in chronically diabetic rats; increasing the dose brought down the enzyme activity to normal. Glucose 6-phosphatase showed similar behavior.22 That the end product of gluconeogenesis, glucose, is not capable of suppressing the enzymes involved in its production is shown by the fact that in diabetic rats where blood sugar is very high, enzyme synthesis is not inhibited. In contrast, enzymes of gluconeogenesis are markedly increased. Since the liver is freely per- meable to glucose,41 it may be concluded that there is no evidence to suggest that glucose as an end product is capable of suppressing the enzymes involved in its production in this system.22 (4) Insulin-induced enzyme decay rates in acute and chronic diabetes: Insulin administration in diabetic rats was capable of returning the markedly increased gluconeogenic enzyme activities to normal range."' 13, 18, 23 The behavior of decay rates in acutely (4-day) and chronically (14-day) diabetic rats was studied in further detail during the influence of insulin suppression. It was observed that in acutely diabetic rats the half-life of both enzymes was 1 day. However, in chronically diabetic animals it was 2-3 days for glucose 6-phosphatase and 4-5 days for fructose 1 ,6-diphosphatase. The slower response of chronically diabetic rats to insulin blocking of enzyme synthesis may be interpreted as a stabilization of the template at the ribosomal sur- face. This concept is supported by the observation that under these conditions the blockers of protein synthesis, in concentrations previously adequate in acutely diabetic rats, were ineffective or only partially effective.' Downloaded by guest on September 28, 2021 VOL. 53, 1965 BIOCHEMISTRY: WEBER ET AL. 101

350 FG-6-PFASE] 01F-D-PASE 300-

2250

200-

150 ETHLI9ONINE j~

100 4 [4 1 3

.3 200 H' HOMGENTESUPERNATANT1~~~~~~NITROGEN NITROGEN

0150

41 100 - - -

1 2 3 4 1 2 3 4 D A Y S O F T R E A T M E N T

FIG. 3.-Prevention of triamcinolone (TAC)-induced synthesis of hepatic gluconeogenic enzymes Liver glucose 6-phosphatase and fructose 1,6-diphosphatase activities rose to more than threefold of normal values upon daily injection of 1 mg triamcinolone. In steroid-treated animals which were injected concurrently with ethionine or actinomycin, enzyme activities failed to increase. When animals were pretreated for 2 days with 4 units of insulin per day, and then 4 units per day were given concurrently with steroid administration, the new synthesis of gluconeogenic enzymes was completely blocked. Similar effects were found for total nitrogen content.

U.3~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~340- 300 [I-

H 260 E TRLZMCINOLOEt INSULIN IZ 20I2 I||

W 180 U -< U 0H140 %Ir'6

O 100 0

I 1111111~~tlitIItI 1. 1 3 ~~~~~~~~5 TRAM0NLONE INSULIN fTRIAMAINOLONE INSULIN INJECTIONS INJECTIONS J INJECTIONS INJECTIONS J DAYS OF TREATMEN T

FIG. 4.-Inhibition of triamcinolone-induced synthesis of hepatic gluconeogenic enzymes. When animals were given daily injections of 1 mg triamcinolone, the gluconeogenic enzyme activities rose to more than threefold in 3 days. In groups of animals which received insulin on the 3rd and 4th days concurrently with steroid administration the enzyme activities returned to normal range in 2 days. The half-life of glucose 6-phosphatase and fructose 1,6-diphosphatase was approximately 1 day, as estimated from the insulin-induced decay curve. Downloaded by guest on September 28, 2021 102 BIOCHEMISTRY: WEBER ET AL. PROC. N. A. S.

(5) Insulin prevention of glucocorticoid-induced enzyme biosynthesis: Insulin was capable of preventing the triameinolone-induced synthesis of hepatic gluconeogenic enzymes. In consequence, enzyme activities failed to rise in glucocorticoid- treated rats. This is similar to the blocking effects achieved by actinomycin or ethionine when given concurrently with injections of adrenocortical hormones (Fig. 3). (6) Insulin blocking of glucocorticoid-induced enzyme biosynthesis: Insulin was also capable of blocking enzyme synthesis in animals pretreated with triamcinolone for 3 days; the already elevated glucose 6-phosphatase and fructose 1,6-diphosphatase activities promptly began to decrease on insulin injection and activities returned to normal range in 2 days (Fig. 4). (7) Insulin-mediated reversal of RNA metabolic changes in acute diabetes: In acutely alloxan diabetic rats the RNA specific activity increased to 140 per cent and injection of insulin returned the specific activity to normal in 1 day. Further insulin treatment brought down RNA specific activity to 60 per cent of normal values where it was stabilized until the 8th day. The RNA amount increased to 140 per cent and then slowly decreased to 125 per cent by the 8th day (Fig. 5). The available data are in accord with the PRECURSOR: OROTIC ACID-6-C14 present suggestion that insulin is capable 13- Rof acting as a suppressor of the de novo Ac14tivity, ,, synthesis of hepatic glueoneogen ic en-

120- zymes. The word "suppressor" is recom- 120 mended for this action of the hormone to o 110r Do' ii |distinguish it sharply from the z100 "repressor" W 90 ax # \] concept used in microorganisms. 980 F \ In unicellular organisms the end product 70- _i\ of a reaction sequence may be able to 60 1 repress synthesis of the enzymes which Q50; _ produced it. Furthermore, specific re-

fz, AN | pressor substances in microorganisms are 4 5 6 7 8 tNORMALORL DAYS the products of the same cell coming from t l t t t t t t the regulator gene. In multicellular or- Alloxan Diabetes INSULIN INJECTIONS ganisms, such as the rat, the end product of FIG. 5.-Effect of insulin treatment on gluconeogenesis, glucose, does not inhibit metabolism of hepatic RNA in acute diabetes. Alloxan diabetic animals were in- the synthesis of enzymes involved in its jected with insulin (4 units per day) and production. 22 However, glucose causes groups of rats were killed subsequently. Orotic acid 6-C14 (3 lsc/100 gm) was injected the release of the hormone insulin from 2 hr before death. The means and standard another organ, the pancreas. Insulin then errors, representing 3 or more animals in each group, are indicated on the curve. The RNA acts to suppress the biosynthesis of key amount decreased to 83%, whereas the liver enzymes involved in glucose produc- specific activity increased to 143%. Insulin brought RNA values to normal in 1 day. A tion. Furthermore, insulin by transferring continuation of insulin administration resulted glucose into peripheral cells, by promoting in a rise in RNA amount and a marked decrease in specific activity. glycogenesis through increasing glycogen synthetase,42 and by inducing synthesis of glucokinase43 promotes the removal of glucose from the blood. In consequence, insulin decreases the level of its own evoker and turns off the stimulus for its own release. Downloaded by guest on September 28, 2021 VOL. 53, 1965 BIOCHEMISTRY: WEBER ET AL. 103

The attacking point of hormones at the genetic level may involve two economy arrangements simplifying the regulatory processes. (a) It appears that the most economic regulatory control should act at the gen- ome since at the level of the chromosome a single molecule may activate or inhibit a gene. In contrast, the subsequent steps in enzyme synthesis involve many molecules in many locations and thus a number of regulatory signals would be required to affect the enzymatic expression of a functioning gene. In consequence, turning the gene on or off seems to be the most satisfactory method of regulating the amount of enzyme produced. The quantitative control of gene function may then be determined by the duration of gene activation which in turn is a function of the number of unstable activating or inactivating molecules." Thus, the role of the antagonistic hormones would be explained by an action on receptor sites at the fountainhead of enzyme production. (b) The economy of turning on and off a whole genetic chain of key enzymes, such as the 4 gluconeogenic enzyme unit, is also attractive. Evidence presented in support of the concept of the action of glucocorticoids and insulin as inducer and suppressor of the biosynthesis of the 4 key gluconeogenic enzymes is in agreement with such a concept.

* This investigation was supported by grants to one of us (G. W.) from US PHS, research grant CA-05034-05 from the National Cancer Institute, the American Cancer Society, and Damon Runyon Memorial Fund, Inc. 1Weber, G., R. L. Singhal, N. B. Stamm, E. A. Fisher, and M. A. Mentendiek, Advan. Enzyme Regulation, 2, 1 (1964). 2 Krebs, H. A., Bull. Johns Hopkins Hosp., 95, 19 (1954). 3 Weber, G., M. C. Henry, S. R. Wagle, and D. S. Wagle, Advan. Enzyme Regulation, 2, 335 (1964). 4 Weber, G., R. L. Singhal, and S. K. Srivastava, Advan. Enzyme Regulation, 3, in press. 5 Weber, G., G. Banerjee, and S. B. Bronstein, J. Biol. Chem., 236, 3106 (1961). 6 Weber, G., and A. Cantero, Am. J. Physiol., 197, 699 (1959). 7 Weber, G., S. K. Srivastava, and R. L. Singhal, Life Sci., 3, 829 (1964). 8 Nelson, N., J. Biol. Chem., 153, 375 (1944). 9 Report of the Commission on Enzymes of the International Union of Biochemistry (New York: Pergamon Press, 1961), p. 41. 10 Weber, G., C. Allard, G. De Lamirande, and A. Cantero, Biochim. Biophys. Acta, 16, 618 (1955). 11 Ashmore, J., A. B. Hastings, F. B. Nesbett, and A. E. Renold, J. Biol. Chem., 218, 77 (1956). 12 Kvam, D. C., and R. E. Parks, Jr., Am. J. Physiol., 198, 21 (1960). 13 Shrago, E., H. A. Lardy, R. C. Nordlie, and D. 0. Foster, J. Biol. Chem., 238, 3188 (1963). 14 Ashmore, J., S. R. Wagle, and T. Uete, Advan. Enzyme Regulation, 2, 101 (1964). 15 Henning, H. V., I. Seiffert, and W. Seubert, Biochim. Biophys. Acta, 77, 345 (1963). 16 West, K. M., Metab. Clin. Exptl., 7, 441 (1958). 17Weber, G., and R. L. Singhal, Biochemn. Pharmacol., 13, 1173 (1964). 18 Lardy, H. A., D. 0. Foster, E. Shrago, and P. D. Ray, Advan. Enzyme Regulation, 2, 39 (1964). 19 Weber, G., R. L. Singhal, and N. B. Stamm, Science, 142, 390 (1963). 20 Weber, G., S. K. Srivastava, and R. L. Singhal, J. Biol. Chem., in press. 21 Weber, G., G. Banerjee, and S. B. Bronstein, Am. J. Physiol., 202, 137 (1962). 22 Weber, G., R. L. Singhal, and S. K. Srivastava, Advan. Enzyme Regulation, 3, in press. 23Ashmore, J., A. B. Hastings, and F. B. Nesbett, these PROCEEDINGS, 40, 673 (1954). 24 Weber, G., and A. Cantero, Science, 120, 851 (1954). 25 Weber, G., Rev. Can. Biol., 18, 245 (1959). 26 Weber, G., R. L. Singhal, S. K. Srivastava, H. J. Hird, and J. Furth, Endocrinology, in press. Downloaded by guest on September 28, 2021 104 BIOCHEMISTRY: TATIBANA AND COHEN PROC. N. A. S.

27Feigelson, P., M. Feigelson, and C. Fancher, Biochim. Biophys. Acta, 32, 133 (1959). 28 Feigelson, P., M. Feigelson, and 0. Greengard, Recent Progr. Hormone Res., 18, 491 (1962). 29Feigelson, P., and M. Feigelson, J. Biol. Chem., 238, 1073 (1963). 30 Reich, E., R. M. Franklin, A. J. Shatkin, and E. L. Tatum, Science, 134, 556 (1961). 31 Greengard, O., M. A. Smith, and G. Acs, J. Biol. Chem., 238, 1548 (1963). 32 Jervell, K. F., Acta Endocrinol., 44, 3 (1963). 33 Lang, N., and C. E. Sekeris, Life Sci., 3, 391 (1964). 34 Sekeris, C. E., and N. Lang, Life Sci., 3, 169 (1964). 35 Kidson, C., and K. S. Kirby, Nature, 203, 599 (1964). 36 Garren, L. D., R. R. Howell, and G. M. Tomkins, J. Mol. Biol., 9, 100 (1964). 37Wagle, S. R., and J. Ashmore, Biochim. Biophys. Acta, 74, 564 (1963). 38 Wagle, S. R., Biochem. Biophys. Res. Commun., 14, 533 (1964). 39 Freedman, A. D., and L. Kohn, Science, 145, 58 (1964). 40 Weber, G., and R. L. Singhal, Metab., Clin. Exptl., 13, 8 (1964). 41 Cahill, G. F., Jr., J. Ashmore, A. S. Earle, and S. Zottu, Am. J. Physiol., 192, 491 (1958). 42 Steiner, D. F., and J. King, J. Biol. Chem., 239, 1292 (1963). 32Weinhouse, S., V. Cristofalo, C. Sharma, and H. P. Morris, Advan. Enzyme Regulation, 1, 363 (1963). 44Markert, C. L., in Cytodifferentiation and Macromolecular Synthesis, ed. M. Locke (New York: Academic Press, Inc., 1963), p. 65.

FORMATION AND CONVERSION OF MACROMOLECULAR PRECURSOR(S) IN THE BIOSYNTHESIS OF CARBAMYL PHOSPHATE SYNTHETASE* BY MASAMITI TATIBANAt AND PHILIP P. COHEN DEPARTMENT OF PHYSIOLOGICAL CHEMISTRY, UNIVERSITY OF WISCONSIN, MADISON Communicated by Karl Paul Link, October 27, 1964 Carbamyl phosphate synthetase activity is markedly increased in tadpole liver during the early stages of thyroxine-induced metamorphosis.' Experiments using whole animals2 and liver slices3 established that the increase was the result of de novo net synthesis of the enzyme. In the present paper, experiments are reported which bear on the mechanism of synthesis of carbamyl phosphate synthetase. Materials and Methods.-Preparation of tadpole liver slices, incubation, isolation of carbamyl phosphate synthetase, and radioactivity counting were carried out essentially as described pre- viously3 with a few modifications as described. In most of the experiments, carbamyl phosphate synthetase was extracted only from the fraction that was sedimentable at 6,000 X g for 10 min directly from the whole homogenate in 0.25 M sucrose, and includes cell debris, nuclei, an un- identified particle fraction rich in melanin-containing granules, and mitochondria.4 This fraction is referred to as "F-6." Because of the smaller amount of contaminating protein in fraction "F-6" as compared with the supernatant or extract from the microsomal fraction, enzyme purifi- cation did not require chromatography on DEAE-cellulose nor addition of carrier enzyme. The microsomal fraction was discarded unless otherwise stated, since the fraction, as will be described below, contained only insignificant amounts of the enzyme, and showed no preferential radioactivity incorporation. The F-6 fraction, generally kept at -18° until used, was thawed and extracted with 0.1% cetyltrimethylammonium bromide (CTB) as previously described.3 The extract was equilibrated with 0.005 M sodium succinate, pH 5.9, by means of a column of Sephadex G-25 (1.0 X 15 cm), diluted 1.5 times with cold water and applied on a column of phosphocellulose, 0.5 cm in diameter, with a bed volume equal to 2 times the original tissue weight. The column was washed with 1.0 column bed volume of 0.005 M sodium succinate solution followed by elution Downloaded by guest on September 28, 2021