Demonstration of the Occurrence of Inactive Fatty Acid Synthetase in Rat Liver by Immunotitration and Its in Vitro Partial Activation*

Direct immunotitrations of rat liver fatty acid synthetase in crude tissue homogenates with monospecific rabbit anti-rat liver fatty acid synthetase antibody en-abled us to make a comparison of fatty acid synthetase protein and activity (percentage of maximal activity) as a function of the nutritional state in normal, diabetic, and insulin- and glucagon-insulin treated animals. Previous results, in which large changes in fatty acid synthetase activity were related to protein synthesis and degradation rather than to enzyme activation, were confirmed. It was also shown that fatty acid synthetase activation does not occur immediately on synthesis but follows the synthesis of fatty acid synthetase protein. In order to characterize the enzymatically inactive protein found on immunotitration and to develop an in vitro system for fatty acid synthetase activation, con- ditions were sought to obtain large amounts of fatty acid synthetase protein free from, or low in, activity. It was found that treatment of hypophysectomized rats with triiodothyronine meets these requirements, yield- ing milligram quantities of inactive fatty acid synthetase protein with less than 2% of maximal activity. A part of the inactive fatty acid synthetase was found to be the apoenzyme as indicated by &ketoreductase and thioesterase activities, by its ability to incorporate label from [G3H]CoA, and by its partial in vitro activation, which Ied to an increase in overall synthetase activity in crude and partially purified cell-free systems. The components required for activation include magnesium

Direct immunotitrations of rat liver fatty acid synthetase in crude tissue homogenates with monospecific rabbit anti-rat liver fatty acid synthetase antibody enabled us to make a comparison of fatty acid synthetase protein and activity (percentage of maximal activity) as a function of the nutritional state in normal, diabetic, and insulin-and glucagon-insulin treated animals. Previous results, in which large changes in fatty acid synthetase activity were related to protein synthesis and degradation rather than to enzyme activation, were confirmed. It was also shown that fatty acid synthetase activation does not occur immediately on synthesis but follows the synthesis of fatty acid synthetase protein.
In order to characterize the enzymatically inactive protein found on immunotitration and to develop an in vitro system for fatty acid synthetase activation, conditions were sought to obtain large amounts of fatty acid synthetase protein free from, or low in, activity. It was found that treatment of hypophysectomized rats with triiodothyronine meets these requirements, yielding milligram quantities of inactive fatty acid synthetase protein with less than 2% of maximal activity. A part of the inactive fatty acid synthetase was found to be the apoenzyme as indicated by &ketoreductase and thioesterase activities, by its ability to incorporate label from [G3H]CoA, and by its partial in vitro activation, which Ied to an increase in overall synthetase activity in crude and partially purified cell-free systems. The components required for activation include magnesium ion and a transferase fraction prepared from livers of 48-h fasted, 12-h refed rats.
The activity of mammalian liver fatty acid synthetase is known to be a function of the nutritional state of the animal (l,2). For example, fasting of the animal results in a reduction of the activity of this enzyme (3, 4). Under refeeding, the activity of the enzyme returns to normal, but feeding a high carbohydrate, fat-free diet causes the enzyme activity to rise to above normal levels. In recent years, hormonal factors, particularly insulin, have been shown to stimulate fatty acid synthetase activity to supranormal levels during fat-free diet feeding of diabetic rats previously fasted for 48 h (3, 5, 6).
Lakshmanan et al. (6) have demonstrated that the relative * This investigation was supported in part by Grants AM 01383 and AM 21148 from the National Institute of Arthritis, Metabolism, and Digestive Diseases of the National Institutes of Health, United States Public Health Service, and by a grant from the Medical Research Service of the Veterans Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with If3 U.S.C. Section 1734 solely to indicate this fact. rate of synthesis of fatty acid synthetase for diabetic rats treated with insulin was about IO-fold higher than that for untreated diabetic animals. On the other hand, Volpe and Vagelos (7) found that feeding a fructose diet to diabetic rats considerably increased the hepatic fatty acid synthetase level, and this stimulation was not due to enhanced plasma insulin levels. The rates of synthesis of enzyme and the levels achieved were quite different, though, from those found in the experiments with insulin. The effect of thyroid hormones on fatty acid synthetase and other lipogenic enzyme systems has also been investigated (8,9).
Previous investigations were undertaken to understand the mechanism of enzyme regulation in vivo. Attempts were made to determine whether nutritional and hormonal variation in fatty acid synthetase activity are due to protein synthesis or modification, or both. Burton et al. (3) found a considerable increase in the rate of incorporation of ['4C]leucine into fatty acid synthetase and an increase in specific activity of fatty acid synthetase between 3 and 12 h after the start of refeeding.
Later, Craig et a2. (lo), using immunochemical studies, showed that it is the rate of synthesis and not that of degradation that is the major parameter in controlling the liver fatty acid synthetase content in rats subjected to nutritional stress. The role of insulin in the regulation of the rate of synthesis of fatty acid synthetase was unequivocally established (6) when a 20fold increase was found in the actual amount of purified fatty acid synthetase from diabetics treated with insulin as compared to untreated rats. On the basis of these results, it was concluded that the diet-and insulin-induced changes in the level of fatty acid synthetase are due primarily to an adaptive increase in the rate of synthesis of this enzyme rather than an activation of previously existing enzyme.
In earlier investigations, the ratio of active to inactive enzyme species could not be determined directly in liver homogenates but had to be inferred after several purification steps from the specific activity of the enzyme. This involved the tenuous assumption that active and inactive forms of the same enzyme co-purified in the same ratio as was present in the crude extract. In the present investigation, we have estimated the amounts of active and inactive species of fatty acid synthetase by titrating a specified enzyme activity with monospecific anti-rat liver fatty acid synthetase antibody. In this procedure, equivalent units of enzyme activity will give the same endpoints on immunotitration if all enzyme molecules have the same activity, whereas in the presence of inactive species, the endpoint will be different. The immunotitration method is fast, reproducible, and sensitive. These studies have confirmed that the rate of synthesis is the major parameter in regulating the concentration of fatty acid synthetase in rats subjected to nutritional stress and insulin administration. However, administration of triiodothyronine to ad libitumfed hypophysectomized rats resulted in the accumulation of an amount of inactive species of the synthetase which was much greater than observed for other dietary or hormonal regimens. The large yield of inactive species of fatty acid synthetase allowed us to partially establish the identity of this species and to develop a system which partially reactivates the inactive species in vitro.

RESULTS
Effect of T3 and Hydrocortisone on the Ratio of Enzymatically Active to Inactive Fatty Acid Synthetase in Hypophysectomized (Ad libitum-fed) Rats-The administration of TB to hypophysectomized animals did not result in any significant change in fatty acid synthetase activity over a period of 48 h. Immunotitrations confirmed these results by showing very low levels of active enzyme which remained almost constant up to 48 h after T3 injection. However, there was a dramatic increase in the levels of inactive fatty acid synthetase species at 16, 24, and 32 h after T3 injection (100 pg/100 g of body weight) (Table VI). These data are in agreement with earlier reports (5, 22), inasmuch as there is an increase in protein synthesis in response to TI administration. It may be pointed out here that Tata (23) has shown that the increase in protein synthesis on T3 administration is dependent on the dose and that there is a lag period of 12 to 48 h. In the present studies, a T3 dosage level of 100 pg/100 g of body weight greatly stimulated the synthesis of the fatty acid synthetase complex, whereas 10 pg/lOO g of body weight produced a much smaller stimulation. However, if one had followed the increase in enzyme activity as an indicator of protein synthesis, one would have concluded the absence of enzyme synthesis and missed the inactive enzyme species present. Interestingly, hydrocortisone administration had no effect on the induction process. There was no difference in the levels of active and inactive enzyme over a time period of 0 to 48 h from the time of injection.
Incorporation of ['4C]Pantetheine into Active and Inactive Fatty Acid Synthetase-Aliquots of the dialyzed DEAE-cellulose-purified fatty acid synthetase proteins were mixed with equal volumes of 4 N KOH in small test tubes. The contents of the tubes were heated to boiling over a small flame, cooled to room temperature, and neutralized with a slight excess of 2 N acetic acid. The mixtures were assayed in dioxane for radioactivity. Assays were corrected for error by adding [14C]toluene as an internal standard and again assaying for radioactivity. The results shown in Table VI1 indicate that the incorporation of ['4C]pantetheine into the fatty acid synthetase of triiodothyronine-treated hypophysectomized rats is less by an order of 10-fold that in normal rats.
Retention of the f4C]Pantothenate Pool-The cytosols were counted for radioactivity in order to determine whether nonincorporation of [I4C]pantetheine into inactive fatty acid synthetase could be accounted for by a failure of the hypophysectomized rats to retain ['4C]pantothenate. The 100, OOO X g supernatant solution of liver homogenate from normal rats 24 h after refeeding and 21 h after the first injection of [I4C]pantothenate contained 68,000 dpm/ml or a total of      675,000 dpm per rat liver (average of 4 rats). The 100, OOO X g supernatant solution of liver homogenate from hypophysectomized T3-injected rats contained 93,000 dpm/ml or 450,000 dpm per rat liver (average of 2 rats). Thus, the retention of the ['*C]pantothenate pool in the two sets of animals is quite similar, which means that the nonincorporation of ["Clpantetheine into fatty acid synthetase could not be attributed to a premature excretion of substrate. The results indicate, therefore, that the inactivity of the fatty acid synthetase in TStreated hypophysectomized rats is due to a lack of 4"phosphopantetheine, and that the inactive enzyme complex may, at least in part, consist of the apo form of the enzyme.
Evidence of Enzymic Transfer of 4'-Phosphopantetheine from CoA into Inactive Fatty Acid Synthetase-The incorporation of radioactivity from [G-3H]CoA into inactive rat liver fatty acid synthetase is shown in  Table I1 yields the result of -0.11 nmol of pante-"transferase" activity precipitates between 20 and 35% of theine incorporated per 0.125 nmol of fatty acid synthetase saturation (data not shown). phosphate, pH 7.0, containing 1 mM EDTA. The fractions containing protein were combined (total, 0.8 pl), and sufficient 2 M potassium phosphate, pH 7.0 (0.133 pl), was added to bring the phosphate concentration to 0.5 EX. The samples were brought to 10 mM with respect to dithiothreitol and incubated at 30 "C for 45 min. A 0.450ml aliquot of each incubation mixture was assayed for fatty acid synthetase activity as described for A. 0, increase in units of fatty acid synthetase activity versus quantity of inactive fatty acid synthetase. For each determination, a control without transferase (heattreated 20 to 35% ammonium sulfate-precipitated fraction of 48-h fasted, 12-h refed rats) was run. Assays with the complete system included 30 pl (20 pg of protein) of the transferase fraction, 10 mM magnesium chloride, 1.5 X M CoA, and designated amounts of the 20 to 30% ammonium sulfate-precipitated fraction of inactive fatty acid synthetase (0.6 mg of protein/ml), and the reaction buffer, in a volume of 0.1 mi. Reactions were initiated by adding CoA, carried out at 30 "C for 60 min, and then terminated by pipetting the mixtures into 0.350 ml of the reassociation buffer. The remaining steps of the assays were carried out as described for A.
Stability of Apo-Fatty Acid Synthetase-Assays of mixtures of the apoenzyme before and after activation of fatty acid synthesis by the transferase reaction indicated that the specific activity of the partial reaction, P-ketoacyl reductase, did not change. Therefore, the total P-ketoacyl reductase activity represents the sum of the ketoreductase activities of apo-and holoenzymes and could be used to determine the quantity of apoenzyme (which could be converted to active enzyme) in a sample in conjunction with assays for fatty acid synthetase activity. However, it was found that apoenzyme loses its ,&-ketoreductase activity after one or two freezings and thawings unless it is stored in 15% glycerol. The fist ammonium sulfate step also causes a decrease in ,&-ketoreductase activity of the apoenzyme, even though the recovery of fatty acid synthetase-related protein is quantitative.
Dependence of Reactivation of Fatty Acid Synthetase on Quantity of Transferase Fraction-A linear dependence in the increase in fatty acid synthetase activity on the volume of the transferase fraction added is shown in Fig. 5A.
Dependence of Reactivation of Fatty Acid Synthetase on Time of Reaction-A linear increase in fatty acid synthetase activity with time of transferase action is shown in Fig. 5B.
Dependence of Reactivation of Fatty Acid Synthetase on Concentration of CoA-In this experiment, CoA was removed from each reaction mixture by molecular filtration before assay of fatty acid synthetase activity. The experiment was designed so that the time of reaction at the highest level of CoA concentration permitted the reaction of no more than one-third of the apoenzyme. Under these conditions, the rate of activation of the apoenzyme showed a linear dependence on the concentration of CoA substrate at low levels. At higher concentrations, the reaction rate was nonlinear with CoA ( Fig.  5C). An apparent K , (pseudo-first order reaction) of approximately 1.5 X M for CoA was obtained. Dependence of Increase of Fatty Acid Synthetase Activity on Quantity of Apoenzyme-The reaction for each level of apoenzyme concentration was allowed to go to completion. Comparison of NADPH Disappearance with Long Chain Fatty Acid Formation due to Increase in Fatty Acid Synthetase Activity-The increase in NADPH oxidized in a specified time interval (determined spectrophotometrically) and the increase in fatty acid synthesis in the same time interval (determined radiochemically) are plotted against time of transferase reaction in Fig. 6A. Fig. 6B is a plot of the increase in fatty acids synthesized against the increase in NADPH oxidized. NADPH disappearance and fatty acids formed are related by the stoichiometry of the following equation: These plots indicate a linear relationship between NADPH disappearance and fatty acid formation with time. The plots also indicate the stoichiometric equivalence between substrate consumed (NADPH) and product formed (long chain fatty acids considered as palmitate) due to newly activated fatty acid synthetase.

DISCUSSION
The preparation of monospecific antirat liver fatty acid synthetase antibody by afiinity chromatography on a stationary phase containing homogeneous antigen makes it possible to estimate the antigen directly in crude homogenates. It had been established earlier (14) and in this work that immunoti-tration is a suitable method for determining the amount of active and inactive enzyme protein in 100, OOO X g supernatant solutions of animals subjected to different hormonal and dietary regimens. In this report, we have shown that under conditions of partial inhibition of fatty acid synthetase activity by the monospecific immunoglobulin, the quantity of immunoprecipitate formed by the antibody is a finction of quantity of the antibody and not antigen. The quantity of antigen removed from an excess by a given amount of antibody is constant. These results further c o n f i the validity of Equations 1 and 2. Determination of the endpoint in the titration (Fig. 2) depends on a determination of the intercept on the y axis. The use of linear regression analysis for determining the intercepts gave standard deviations of less than k0.3 nmol/ min. This corresponds to a value of &0.5 mg of antigen protein/ml for 48-h refed liver supernatant solution and k0.025 mg of antigen protein/ml for 0-h refed animals.
There is a rapid increase in the ratio of active enzyme to total enzyme in the initial 6-to 12-h period after the commencement of refeeding fasted rats a high carbohydrate, fatfree diet. Similarly, the refeeding of diabetic rats treated with insulin resulted in the production of rapidly increasing amounts of enzymatically active hepatic fatty acid synthetase. This shows that feeding of a fat-free diet and administration of insulin increases the rate of synthesis of the fatty acid synthetase. This c o n f i i s the earlier work (6,10) and for the f i t time provides a means to quantitate directly the actual amount of active enzyme present in a particular hormonal or nutritioaal state. However, the amount of inactive species in the animals in these nutritional and hormonal states remains almost at the same level during the initial 8-h period. It seems probable that the accumulation of low levels of inactive species in the initial period is due to the low rate of conversion of inactive species into active species, which implies the absence or low level of an enzyme involved in such a conversion in the initial period. The disappearance of the inactive species after an 8-h period would, then, suggest that the transferase activity involved in the conversion of the enzymatically inactive to active species is being generated at a later time than the fatty acid synthetase. It is tempting to suggest that the inactive species is apo-fatty acid synthetase. Earlier investigators (10,24,25) have shown the presence of apoenzyme in rat liver. Yu and Burton (24) found that in fasted, refed animals, incorporation of [14C]pantetheine into fatty acid synthetase does not occur until approximately 4 h from the start of refeeding, whereas the incorporation of 3H-labeled amino acids into immunoprecipitable protein commences with the time of refeeding. Consequently, it was suggested that the enzyme converting the apoenzyme to holoenzyme, viz. 4"phosphopantehtheine transferase may be present in the 100,000 X g supernatant solution of liver homogenates from 12-h refed rats.
The role of glucagon in inhibiting the induction of fatty acid synthetase in insulin-treated animals was demonstrated earlier (6). Our data obtained by immunotitration demonstrate the absence of active enzyme in glucagon-treated animals, whereas the amount of inactive species stays constant (Table  V). It is clear from our results that immunotitration gives a reliable estimate of the amount of active and inactive enzyme in the 100,000 X g supernatant solution obtained from animals in different hormonal and nutritional states. This method is fast, reproducible, and sensitive.
The only hormonal state in which large amounts of inactive fatty acid synthetase species were found was in hypophysectomized rats 24 h after a TS injection. Even though growth hormone was absent in these animals (as shown by an average of zero weight gain in one week), TS injection at the level of 10 Immunotitration of Rat Liver Fatty Acid Synthetase and 100 pg/100 g of body weight stimulated an approximately 2-and 10-to 20-fold increase, respectively, in fatty acid synthetase protein. It is of significance to note that even though there is not a large increase in fatty acid synthetase protein in liver during refeeding fasted normal rats a regular diet, the percentage of maximal activity increases sharply.
Only when normal rats are refed a fat-free diet after fasting are high levels reached for both fatty acid synthetase protein and percentage of maximal activity for that protein. This results in a 10-to 20-fold increase in capacity to produce fatty acids.
The rationale for feeding the hypophysectomized rats on a regular, instead of a fat-free, diet is based on the fact that the rats are unable to synthesize endogenous fatty acids. This will ultimately result in fatal consequences if fatty acids are not externally replenished. Hypophysectomized animals eat very little, and many lose 5 to 10% in weight during the week after the operation. As a result, they are virtually in a state of fasting. Hypophysectomy causes a condition of thyroid and adrenal deficiency, which in turn affects lipid metabolism. It is known that thyroid hormones enhance lipid synthesis (21-23), though the mechanism of stimulation is not known. That factors in addition to thyroid hormones appear necessary is shown by the fact that the amount of active fatty acid synthetase is reduced in hypophysectomized animals to about 1% of that in 48-h fasted, 48-h refed normal animals. Similar decreases in the specific and total activity of fatty acid synthetase after hypophysectomy were reported earlier (9). On the other hand, in the T3-treated hypophysectomized rats, the amount of inactive enzyme increases, whereas the amount of active fatty acid synthetase decreases during the period of protein synthesis (Table VI). Kumar et al. (9) reported that T3 at 100 pg/lOO g of body weight elevates the specific activity of fatty acid synthetase in the hypophysectomized animals 8to %fold over control animals 3 days after the injection. However, the data reported by these authors indicate that, even after this increase, the level of active enzyme reached only 1 mg/g of liver tissue or 0.4 mg/ml of homogenate.
The inactive enzyme found in our work appears to be apofatty acid synthetase, as characterized by the following experiments: (a) approximately quantitative transfer of the label from [G-3H]CoA to inactive fatty acid synthetase in the presence of M e and a fraction from 48-h fasted, 12-h refed rats, and ( b ) increase in overall fatty acid synthetase activity in the presence of CoA, Mg2+, and the transferase fraction. Sucrose density and sodium dodecyl sulfate-gel electrophoresis indicate that the inactive protein has a molecular weight close to that of the active protein and is homogeneous, as shown by a single peak corresponding to that of the active complex and a single band, respectively, corresponding to that of the halfmolecular weight subunit.
While it appears that all of the inactive protein immunologically cross-reacting with fatty acid synthetase is apo-fatty acid synthetase within the uncertainty of the distribution and exchange with H20 of the label in [3H]CoA, the recovery of overall activity of the enzyme upon incorporation of pantetheine is incomplete. The generation of overall activity is, however, dependent upon the parameters which characterize a transformation from apo-to holoenzyme. It remains to be shown whether another step in modification of the enzyme protein is needed to bring about complete reactivation or whether, in fact, some of the material, while of high molecular weight, is in the initial stage of degradation. Indeed, the reductase and thioesterase partial reactions of the apoenzyme are much less stable than those in the holoenzyme, indicating that the presence of 4'-phosphopantetheine is needed for stability of some portions of the complex as well as for the overall reaction mechanism. Our results are similar to those of Werkmeister et al. (26), who recently reported that only a 20% increase in the specific activity of the in vitro-prepared holoenzyme was obtained, as compared to in uzuo-synthesized enzyme, when partially purified yeast cell extract was utilized as a source of apoenzyme 4'-phosphopantetheine transferase activity.
Further work is being carried out on the characterization of the apoenzyme and on the quantitative determination of the prosthetic group in both the apo-and holoenzyme, as well as in the product of the transferase reaction. feeding, one group of diabeelc rats was ~nlected SUbCutaneOUSly wkth insulin at a dose of 3 unlts/day/lOO g Of body weight. Iletzn lnsvlin was administered along with the longer-acting protamme-zlnc ~nsulin 1" order to minimize any lag in response to ~nsulln. For Short feedmg times, Ilet~n msulin at a dose Of 1 unit/lOO g Of body welght was admmistered 2, 4 and 8 h before Sacrifrce. Another group of diabetlc rats was Injected with inaulm and glucagon fore the insvlln administration. A third group Of diabetic rats was not 1200 ug/lOO g body weight). Glucagon was admlnrstered subcutaneously 2 h betreated w t h h o m n e s .

DEHONSTWTION OF THE OCCURRENCE OF INACTIVE FATTY ACID SKNTHETASP IW RRT
by Altech laboratories, Inc., were accl-tiled to laboratory conditions for one week. Only those rats that showed an mcrease of less than 6% in body velght over a perlad of 7 days were Used I" theae experiments. The hypophysectomized animals Were fed a n o m 1 dlet . d IibiLu..
Two groups of animals recelved Subcutaneous mlectlons of triiodothyronlne (Tz) at dosages of 10 v g and 100 u g / l O O g of body welght. These animals were sacrificed at specified p e r m d a following T3 administratLon. A third group Of hypophysectomized rats was mlected with hydrocortisone sodium succinate at a dosage of 2.5 mg/100 g body weight. Two ~n)ectione were admmistered 12 h apart, and the rats were eacnficed 12 h after the last injectlo".
Rats welghlng 120 to I40 g . obtalned from Holtzman and hypophysectomized ASSaV for Enzyme Activity -The fatty acid synthetase was assayed speccarrled Out on rat llver h-genates (100.000 9 supernatant solutions) and on DERE-cellUlohe-pU=ified rat llver fatty acid synthetase in a manner similar to that for pigeon liver enzyme 1141. Prior to 1munOtitIation. the homagenates were assayed for NADPH oxidation in the absence of Other substrates. If a homogenate gave a blank of greater than one-half the 0veez.11 rate, it was Passed through a Sephadex G-25 column. preequilrbrated wlth 0.5 M potassium Phoqhate, and eluted with the s a buffer. This reduced the blank NADPB oxml. Titratmnn were performed by incubating the afflnxty-purlfled fatty acld idation by 60 to 75%. The volme Of the assay mixture Was elther 0.50 or1.00 Or In aliquot of high-speed liver supernatant SOlUtlonl 1" 0.5 M pOtaDSium synthetase antibody and Yaryln9 amDunfs Of antlqen (pure fatty acid synthetase phosphate buffer (pH 7.01. 1 mM EDTA, and 1 mM dlthlothreltol for 45 m l n at 30-c. In a typical titratron, 4 to 32 ug of rat l~v e r enzyme were added to 2 . 6 8 pg of the monospecific antibody ln Separate IncubatLons I" 0.5 M potar-si= phoSphate buffer (pH 7.0).
After 45 min Of 1ncuhaCLon at 30-C the residual activities for fatty acid synthesis Yere determmed. The data were plotted as the m Y n t of enzyme or volume of homogenate (contalnlng known Ynlts of enzyme actlvltyl added Y B I S I Y~ the Units Of fatty acld actlvlty found. When the overall Synthetase actlvlty was too low to be accurately determined, or was completely absent in rat liver homogenates, known qllantltles of standard DERE-cellulose-purifred rat liver fatty a c i d synthetase of *noun specific activity were added to the sample. The amount of inactive fatty body1 was calculated from the decrease I" inhibztmn of active fatty acld acid synthetase-related proteln (cross reacting wlth the mnospeclfx antl- tometrically. The IdRe assay m~x t u r e s were kept overnight at O ' C and then s a l i n e solution, centrifuged agaln, and dissolved in 25 v1 of 80% formic acid by heating at 1 0 0 D C for 2 min. The Samples so obtalned were then Sublected to protein determinatmns as described below.
InCDrWratlon Of ['*ClPantetheme Into Fatty Acld Synthetase by Normal and Hypophysectomized Rats after Ta Admlnistratlon -Hypophysectomized rats were injected with triiodothyronine (1 yg/g body weight), fed a noma1 dlet (see discussionl. and inlected with 10 yCi of [ 'Clpantothen~c acid in 0.2 rnl Of 0.9% Saline rolutlon at 3 h and 10 h after the T g inJectlon. After 24 h ogenares and DFAE-cellulase-purlfled fatty acld Synthetase were prepared by the rats Were Sacrlflced and the 100,000 g Supernatant SOlUtlOnS Of liver homstandard procedures. A group Of Control albino male zatS I140 to 160 9 1 were fasted for 48 h and then refed a fat-free dxet (see D16CuSs1onl. The rats were injected with 10 YCI of ['*Cbantothenle acld ~n 0.2 ml of 0.9% sallne SOlUtlOn at 3 h and 12 h after Commencement Of refeedlng. At 2 4 h after the comncement of refeeding, the rats were sacrificed and DFAE-cellulose-purlfied fatty aczd Synthetase was pzepared from the liver homogenate as described previously 1111. . .  P r e a n t or addad t o t h e s w l e being t i t r a t e d . and x is the eoncentra-

Honospeclflc Antirat Llver
The p a n t i t i e .    Table Iv (see also  SeCtlOn on Nethods).