Purification of Rat Liver Acetyl Coenzyme A Carboxylase and Immunochemical Studies on its Synthesis and Degradation

Transcription

1 Eur. J. Biochem. 16 (i970) Purification of Rat Liver Acetyl Coenzyme A Carboxylase and Immunochemical Studies on its Synthesis and Degradation Shigetada NAKANISHI and Shosaku NUMA Department of Medical Chemistry, Kyoto University Faculty of Medicine (Received April 30, 1970) Acetyl coenzyme A carboxylase from rat liver was isolated in homogeneous form as evidenced by sedimentation velocity experiments and Ouchterlony double diffusion analyses. The specific activity and biotin content of the purified enzyme preparation were similar to those of pure chicken liver enzyme isolated previously. Antibody against rat liver acetyl-coa carboxylase was prepared by injecting rabbits with the purified enzyme. With the use of this antibody, the mechanisms were studied by which the level of liver acetyl-coa carboxylase activity was varied in fasted rats, in those subsequently refed a fat-free diet and in diabetic rats. Immunochemical analysis demonstrated that the wide variations in the level of acetyl-coa carboxylase activity in liver extracts derived from rats under the different dietary and hormonal conditions were accompanied by proportionate changes in the quantity of immunochemically reactive protein. This indicates that the changes in the activity level are determined by changing quantities of the carboxylase protein. Isotopic leucine incorporation studies showed that the relative rate of synthesis of liver acetyl-coa carboxylase was decreased 1.9-fold and 1.7-fold by fasting and diabetes, respectively, whereas increased 4.0-fold by fat-free refeeding. The rate of degradation of the carboxylase, expressed as half-life, was found to be essentially the same in normal, refed and diabetic rats, i.e. 59, 55 and 59 h respectively, and accelerated in fasted rats, i.e. 31 h. Thus, the increase or decrease in the carboxylase quantity in refed or diabetic animals, which are presumably in a steady state, can be ascribed to a rise or fall in the rate of enzyme synthesis, whereas the decrease in the carboxylase quantity in fasted animals, which are not in a steady state, is due both to diminished enzyme synthesis and to accelerated enzyme degradation. On the basis of these results, it is discussed that the control of enzyme quantity by changes in the rate of enzyme degradation may play an important role when the animal deviates from a steady state for adjustment to a new environment. Acetyl coenzyme A carboxylase plays a critical role in the regulation of long-chain fatty acid synthesis (see [l]). The level of this enzyme activity in tissue extracts is influenced by different nutritional and hormonal states of the animal associated with increased or decreased lipogenesis. Numa et ae. [2], Wieland et al. [3] and Allmann et al. [4] demonstrated that the activity level in liver extract is lowered in fasted and alloxan-diabetic rats and elevated upon refeeding fasted animals a fat-free diet. These studies were based exclusively on measurements of catalytic activity, which did not definitely differentiate between changes in catalytic efficiency per enzyme molecule and changes in the number of enzyme - Enzymes. Acetyl-CoA carboxylase or acetyl-coa: CO, ligase (ADP)(EC ); pyruvate kinase or ATP:pyruvate phosphotransferase (EC ) ; lactate dehydrogenase or L-1actate:NAD oxidoreductase (EC ). 11 Eur. J. Biochem., Val. 16 molecules, i. e. enzyme quantity, It was suggested that both these mechanisms operate in the regulation of the acetyl-coa carboxylation step in vivo; changes in catalytic efficiency per enzyme molecule appear to be more responsible for the short-term control, while changes in enzyme quantity for the long-term one [1,5,6]. Work of our group and others revealed that the activity of acetyl-coa carboxylase is affected by various metabolites including citrate as an activator [7-101 and long-chain acyl-coa derivatives as inhibitors [ 11,121. This inhibition can be regarded as a negative feedback mechanism due to endproduct inhibition, since the inhibitors accumulate in the livers of fasted and diabetic animals [13,14]. On the other hand, Hicks et al. [15] showed that actinomycind or puromycin prevents the rise in the level of liver acetyl-coa carboxylase activity upon fat-free refeeding, suggesting a participation of

2 162 Immnnochemical Studies on Acettyl Coenzyme A Carboxylase Eur. J. Biochem. protein synthesis in this regulatory phenomenon. There were no further studies, however, on the mechanisms involved in the control of the quantity of acetyl-coa carboxylase. The present investigation concerns the questions, whether the varying levels of acetyl-coa carboxylase activity in liver extracts observed under the different dietary conditions and in diabetes are actually determined by changing quantities of enzyme protein, and if so, whether these changes are due to variations in the rate of enzyme synthesis or in that of enzyme degradation. In order to investigate these problems with immunochemical techniques, we purified rat liver acetyl-coa carboxylase to homogeneity and prepared antibody against this enzyme. With the use of the antibody, carboxylase protein in various rat liver extracts was titrated. Furthermore, the rates of enzyme synthesis and degradation were assessed by measuring the extent of isotopic leucine incorporation in vivo into liver acetyl-coa carboxylase isolated by precipitation with the antibody and the rate of loss of radioactivity from prelabeled enzyme. The results of this investigation were presented in part as preliminary accounts [ During the preparation of this manuscript, work of Majerus and Kilburn [19] appeared in which similar immunochemical studies with rats under different dietary conditions were carried out by employing antibody made against homogeneous chicken liver acetyl-coa carboxylase. MATERIALS AND METHODS Chemicals and Determinations CoA, NADPH, potassium phosphoenolpyruvate, pyruvate kinase and lactate dehydrogenase were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). ATP, NADH, GSH and crystalline bovine serum albumin were products of Sigma (St. Louis, U.S.A.). Calcium phosphate gel was prepared as described by Heppel [ZO]. DEAE-cellulose was obtained from Serva (Heidelberg, Germany) and Sepharose 2B and Sephadex G-25 from Pharmacia (Uppsala, Sweden). NaH14C0, and ~-[4,5-~H,]leucine were purchased from the Radiochemical Centre (Amersham, England). All other chemicals were of analytical grade. Preparations and determinations of acetyl-coa and malonyl-coa were described previously [21]. Protein was determined by the method of Lowry et al. [22] with crystalline bovine serum albumin as standard or for the purified rat liver acetyl-coa carboxylase from the absorbance at 280 nm. The relation between both values for the purified enzyme was A&'& x 0.70 = mg protein/ml. The protein content in antigen-antibody precipitates were determined by the method of Lowry et al. after the precipitates were dissolved in 0.5 N NaOH. (+)-Biotin was determined by bioassay with Lactobacillus arabinosus as described by Wright et al. [23], and blood sugars by the method of Somogyi [24]. Treatment of Animals Male Wistar strain rats were employed in all experiments except that both male and female rats were used for purification of liver acetyl-coa carboxylase. Animals were fed either a balanced diet or a fatfree diet, both of which were obtained from Clea (Tokyo, Japan).To produce diabetes, animals weighing g, which had been fasted overnight, were given a single intraperitoneal injection of 230 mg per kilogram of body weight of alloxan monohydrate (Eastman Kodak, Rochester, U.S.A.) dissolved in 0.15 M NaCl and were subsequently fed a balanced diet ad libitum. Only those rats were employed which showed continuous and marked glycosuria (more than 2 Olio as estimated with Tes-tape, Lilly, Indianapolis, U.S.A.) for days following the injection of alloxan.these animals exhibited also typical diabetic symptoms, such as retarded growth, polyuria, polydipsia and polyphagia. When they were killed, the concentration of blood sugars (not fasting) ranged from 440 to 610 mg/loo ml. Assay of Enzymes Acetyl-CoA carboxylase activity was determined by the H14C03- fixation assay or by one of the two coupled spectrophotometric assays. The H14C0,- fixation assay follows the rate of acetyl-coa-dependent H14C03- incorporation into acid-stable material, i. e. malonyl-coa. Since rat liver acetyl- CoA carboxylase required preincubation with citrate in order to attain full activity [25], it was first preincubated at 37" for 30min in a mixture (final volume, 1.O ml) containing 50 mm Tris-C1 ph 7.5, 10 mm potassium citrate, 10 mm MgCl,, 3.75 mm GSH and 0.75 mg/ml bovine serum albumin. The reaction was then initiated by adding an aliquot of the preincubated enzyme to an assay mixture (final volume, 0.8 ml) containing 50 mm Tris-C1 ph 7.5, I0 mm potassium citrate, 10 mm MgCl,, 3.75 mm ATP, mm acetyl-coa, 3.75 mm GSH, 0.75 mg/ ml bovine serum albumin and 12.5 mm KHI4CO, (specific activity, 3.0 x lo5 counts x min-1 x pmole-l). Following incubation at 37" for 10 min, the reaction was terminated with 0.2 ml of 5 N HC1. After centrifugation, an aliquot of the supernatant fluid was taken to dryness in a scintillation counting vial at 80"for 45-60min. 0.5ml ofwater and 10ml of Bray's scintillator solution [26] were added, and 14C activity was determined with the use of a liquid scintillation spectrometer. Both coupled spectrophotornetric assays were performed at 25" and used for purification of carboxy-

3 Vol. 16, No. 1, 1970 S. NAKANISHC and S. NUMA 163 lase and for sucrose density gradient centrifugation studies. The first assay follows the rate of malonyl- CoA formation by coupling the carboxylase reaction with the fatty acid synthetase reaction and measuring the oxidation of NADPH. The assay procedure was the same as described previously [27], except that yeast fatty acid synthetase was substituted by rat liver synthetase, which was purified by a modification of the method of Hsu et al. [28]. Alternatively, the rate of acetyl-coa-dependent ADP formation is followed by coupling with the pyruvate kinase-lactate dehydrogenase system and measuring the oxidation of NADH. For this assay, NADPH and fatty acid synthetase used for the first assay were replaced by 0.50 mm potassium phosphoenolpyruvate, mm NADH, 12.5 pg/ml pyruvate kinase and 12.5 pg/ml lactate dehydrogenase. One unit of acetyl-coa carboxylase is defined as that amount which catalyzes the carboxylation of 1 pmole of acetyl-coa per min. Both coupled spectrophotometric assays gave the same values for the activity expressed as unit. The ratio of the activity found at 25" by the coupled spectrophotometric assays to that found at 37" by the H14C0,- fixation assay was 0.5. Fatty acid synthetase was assayed spectrophotometrically as described by Lynen [291. Purification of Rat Liver Acetyl-CoA Carboxylase The results of a typical purification of rat liver acetyl-coa carboxylase are summarized in Table 1. All operations through the second ammonium sulfate fractionation step were carried out at 0-4", whereas subsequent operations at 24" except ammonium sulfate precipitations. All phosphate buffers employed were potassium phosphate buffer ph 7.5 containing 5 mm 2-mercaptoethanol and 1 mm EDTA, unless otherwise specified. Preparation of Crude Extract. 200 rats weighing approximately 250g were fasted for 48h and subsequently refed a fat-free diet for 48 h prior to removal of the livers. The animals were killed by decapitation, and the livers (1660 g) were quickly removed and homogenized in 1.5 volumes of 0.1 M phosphate buffer for 60 sec with a Waring blendor at top speed. The homogenate was centrifuged at x g for 90 min. The supernatant fluid was collected and filtered through cheesecloth (2440 ml). First Ammonium Sulfate Fractionation (0-30 Saturation). The crude extract was diluted to 3320ml (2-fold liver wet weight) with 0.1 M phosphate buffer and brought to 3001, saturation by addition of 176 g of solid ammonium sulfate per liter of extract; the ph was maintained at by addition of 5 N KOH. After further stirring for 30 min, the resulting precipitate was collected by centrifugation at xg for 30 rnin and dissolved in 20 mm phosphate buffer. The turbid solution (815 ml) was frozen at -15". Calcium Phosphate Gel Fractionation and Second Ammonium Sulfate Fractionation (0-25 Ole). The first ammonium sulfate fraction was thawed and diluted with distilled water containing 5 mm 2-mercaptoethanol and 1 mm EDTA to give a protein concentration of 5.7 mg/ml. The diluted enzyme solution (7620 ml) was stirred into 4500 ml of calcium phosphate gel suspension (17.0 mg/ml) (protein: gel = 1 : 1.8). After a 20 min stirring, the gel was collected by centrifugation at 2600 xg for 10 min and washed 3 times, each time with 3150 ml (0.7 volume of calcium phosphate gel suspension added) of 33 mm phosphate buffer. The enzyme was then eluted twice, each time with 3150 ml of 0.2 M phosphate buffer. The enzyme in the combined eluates (6300 ml) was precipitated by addition of 144 g of solid ammonium sulfate per liter of eluate; the ph was kept at 7.3 to 7.4 by addition of 5 N KOH. After further stirring for 30 min, the resulting precipitate was collected by centrifugation at xg for 30 rnin and dissolved in 10 mm phosphate buffer (final volume, 105 ml). Dialysis at 24" in the Presence of Citrate.The second ammonium sulfate fraction was dialyzed at 24" for approximately 14 h against 20 volumes of 10 mm phosphate buffer containing 10 mm potassium citrate with a change of buffer. The dialyzed solution was centrifuged at xg for 10 min. The precipitate was resuspended in 10 ml of the same buffer, and the suspension centrifuged at xg for 10 min. Both supernatant solutions were combined (1 12 ml). DEAE-Cellulose Chromatography. The dialyzed enzyme solution was applied to a DEAE-cellulose column (3.6 x 20 cm; bed volume, 200 ml) equilibrated previously with 20 mm phosphate buffer containing 10 mm potassium citrate. The column was washed with 1.2 column volumes of 20 mm phosphate buffer containing 10 mm potassium citrate. Then, elution was accomplished with a linear concentration gradient established between I.6 column volumes of 20 mm phosphate buffer containing 10 mm potassium citrate and 1.6 column volumes of 0.75 M phosphate buffer containing I0 mm potassium citrate. Carboxylase appeared in the eluate when the phosphate concentration reached approximately 0.15 M. The fractions containing enzyme of specific activities higher than 1.3 units/mg protein were pooled (104 ml). Sepharose 2B Chromatography. Enzyme in the pooled eluate from DEAE-cellulose chromatography was precipitated at 0" by adding 0.67 volume of saturated (0') ammonium sulfate solution ph 7.4. After a 30 min centrifugation at x g, the pellet was suspended in approximately 5 ml of 0.1 M phosphate buffer containing 10 mm potassium citrate and dialyzed at 24" for approximately 6 h against 250 vol- 11*

4 164 Immunochemical Studies on Acetyl Coenzyme A Carboxylasc Eur. J. Biochem. v 0 100xo Volume of effluent (mi) Fig. 1. Sepharose 2B chromatography of rat liver acetyl-coa carboxylase. For experimental details, see Materials and Methods. Elution of protein was followed by absorbance at 280nm (0). Carboxylase activity was determined at 25" by the spectrophotonietric method by coupling with the fatty acid synthetase reaction; 0, activity per milliliter of effluent; B, specific activity umes of the same buffer, until the precipitate was completely dissolved. The dialyzed solution (7.7 ml) was applied to a Sepharose 2B column (2.7 x 87 cm; bed volume, 500 ml) equilibrated previously with 0.1 M phosphate buffer containing 10 mm potassium citrate. Elution was carried out with the same buffer. As shown in Fig. 1, three protein peaks appeared; the second peak corresponded to carboxylase, whereas the other two fractions were contaminants. The fractions containing enzyme of specific activities higher than 7.0 units/mg protein were pooled (55 ml). To concentrate the purified enzyme solution, an equal volume of saturated (0") ammonium sulfate solution ph 7.4 was added at 0" to the pooled eluate, and the precipitated enzyme was collected by centrifugation at x g for 30 min, suspended in a minimal volume of 0.1 M phosphate buffer containing 10 mm potassium citrate and then dialyzed at 24" for approximately 4 h against I00 volumes of the same buffer, until the precipitate was completely dissolved. This enzyme preparation was stored at -15" for periods up to one month without loss of activity. Preparation of Antibody Antiacetyl-CoA carboxylase serum was prepared according essentially to the procedure of Marshall and Cohen [30]. The purified a d concentrated rat liver acetyl-coa carboxylase preparation from the Sepharose 2B chromatography step was diluted with 50 mm phosphate buffer to give a concentration of 0.5mg/ml and mixed with an equal volume of complete Breund's adjuvant (Difco, Detroit, U.S.A.). This mixture was injected into the footpads of rabbits, each rabbit receiving 0.5 mg of carboxylase. Four weeks later, 1 mg of enzyme dissolved in 0.1 M phosphate buffer was injected intravenously, and blood was removed by cardiac puncture in 8 days following the injection. Two to three more intravenous injections of increasing amounts of enzyme (up to 3 mg) were given at 3 weeks' intervals, and cardiac puncture was carried out in 4-8 days following each injection, except that the last bleeding was accomplished by exsanguination. Maximal titers were attained in about 8 weeks following the first injection and maintained thereafter. Sera of high titers were combined, and the y-globulin fraction was isolated as described by Campbell et al. [31], except that the final precipitate was dissolved in 20 mm Tris-C1 buffer ph 7.5 containing 0.15 M NaCI, 5 mm 2-mercaptoethanol and 1 mm EDTA. Control y-globulin was prepared from rabbits injected with buffer instead of enzyme. Enzyme Preparations for Immunochemical Procedures All operations were carried out at 0-4". Immunochemical Titrations. Liver extracts employed for immunochemical titrations were prepared as follows. Two to five rats were killed, and the livers removed quickly and pooled. Tissues were minced and homogenized in 3 volumes of 0.25M sucrose with 3 strokes in a Potter-Elvehjem homogenizer. The homogenate was centrifuged at x g for IOmin, and the supernatant fluid was further centrifuged at x g for 45 min. An aliquot of the soluble supernatant solution was filtered through a 10-fold volume of Sephadex (4-25 equilibrated with 20 mm Tris-C1 buffer ph 7.5 containing 0.15 M NaCl, 5 mm 2-mercaptoethanol and I mm EDTA. The gel-filtration was necessary for the HI4CO,- fixation assay, since an appreciable amount of H14C03- fixation independent of acetyl- CoA occurred when the non-treated x g supernatant solution was employed. Ouchterlony Double Diffusion Analyses. Since the carboxylase content in the xg supernatant solution was too low to give definite precipitin bands in Ouchterlony double diffusion analyses, the enzyme was concentrated by ammonium sulfate fractionation (0-30 /, saturation) as follows. To the xg supernatant solution was added 0.43 volume of saturated (0') ammonium sulfate solution ; the ph was maintained at by addition of 2 N KOH. After further stirring for 20 min, the resulting precipitate was collected by centrifugation at I0000 x g for 20 min and dissolved in a minimal volume of 20 mm Tris-C1 buffer ph 7.5 containing 0.15 M NaC1, 5 mm 2-mercaptoethanol and 1 mm EDTA. Ouchterlony double diffusion analyses were performed as described by Ouchterlony [32].

5 Vol. 16, No. 1, 1970 S. NAKANISHI and S. NIJMA 165 Quantitative Precipitin Reactions. For quantitative precipitin reactions and isotopic leucine incorporation studies described below, neither the x g supernatant solution nor the fraction precipitated between 0-30 Oi0 ammonium sulfate saturation was suited, since these enzyme preparations, when incubated at 37" with control y-globulin and stored at 4" overnight, gave non-specific precipitation. For these studies, therefore, the enzyme was purified further as follows. The fraction precipitated between 0-30 Oi0 ammonium sulfate saturation was prepared as described above, except that the precipitate was dissolved in 50 mm Tris-C1 buffer ph 7.5 containing 5 mm 2-mercaptoethanol and 1 mm EDTA, the final volume being approximately the same as the volume of the xg supernatant solution employed. The enzyme solution was applied to a column of DEAEcellulose equilibrated previously with the same buffer; the column load was 4 ml of the enzyme solution per ml of bed volume. After the column was washed with one column volume of the same buffer, carboxylase was eluted with 1.2 column volumes of the same buffer containing 0.2 M NaC1. This entire procedure yielded /, of the initial carboxylase activity present in the x g supernatant solution and 20- to 30-fold purification. Quantitative precipitin reactions were performed as described by Kabat and Mayer [331. [3H]Leucine Incorporation Studies Each rat received a single intraperitoneal injection of 0.9 mc of ~-[4,5-~H,]leucine (specific activity, 57.6 Cimmole) dissolved in S.0 ml of 0.15 M NaC1. At the times specified in the legends to figures and tables, two or three rats were killed, and the livers were removed, pooled and homogenized in 0.25 M sucrose as described above. A 1 ml aliquot of the xg supernatant solution was filtered through a Sephadex G-25 column (1.0 x 13 cm) equilibrated with 50 mm Tris-CI buffer ph 7.5 containing 5 mm 2-mercaptoethanol and 1 mm EDTA. The acetyl-coa carboxylase activity in the gel-filtered extract was determined at 37" by the H14C03- fixation assay. Another aliquot of the SO5000 xg supernatant solution was used for determination of the radioactivity incorporated into total soluble protein. For this purpose, the protein was precipitated with 10 Ol0 trichloroacetic acid, washed 3 times with the same acid, then dissolved in BOO/, formic acid and assayed for radioactivity with a liquid scintillation spectrometer. The remainder of the xg supernatant solution was subjected to the purification by ammonium sulfate fractionation and DEAE-cellulose chromatography as described above, and this partially purified preparation was employed to determine the radioactivity incorporated into protein precipitated by antiacetyl- COB carboxylase. This determination, together with corrections for non-specific radioactive precipitates, was carried out according to Cho-Chung and Pitot [34] as follows. The DEAE-cellulose eluate was assayed for acetyl-coa carboxylase activity at 37" by the HI4CO3- fixation method and then divided into 7 aliquots, each of which contained 0.25 unit of labeled carboxylase, except that each aliquot derived from fasted animals contained 0.07 unit of labeled carboxylase and 0.18 unit of unlabeled carrier enzyme (specific activity at 37", 1.4 units/mg protein) added. To three of the 7 aliquots was added sufficient antiacetyl-coa carboxylase y-globulin to precipitate twice the amount of carboxylase present in the eluate, and to one aliquot the same amount of controi y-globulin to determine non-specific precipitation. To the remaining 3 aliquots were added 0.25 unit of unlabeled carrier enzyme and sufficient antiacetyl-coa carboxylase y-globulin to precipitate twice the amount of carboxylase present; these aliquots were used to determine the extent of nonspecific adsorption of radioactivity to the antigenantibody precipitate. The samples were incubated at 37" for 15 min and stored at 4" overnight. Precipitates were collected by centrifugation at 1500 xg for 15min and washed 3 times with chilled 0.15 M NaCI. One sample containing 0.25 unit of carboxylase and antibody, and one containing 0.50 unit of carboxylase and antibody were used to determine the protein content in the precipitate ; the latter sample yielded twice as much protein as the former. The precipitates from the remaining 5 aliquots were dissolved in 60 /, formic acid and assayed for radioactivity with a liquid scintillation spectrometer. The extent of non-specific precipitation found by this method was less than 20 OIO of the total radioactivity precipitated by antibody. RESULTS Purity of Rat Liver Acetyl-CoA Carboxylase As shown in Table 1, the enzyme preparation from the Sepharose 2B chromatography step was purified 1700-fold over crude liver extract, the yield being 17O/,. The specific activity of the purified rat liver acetyl-coa carboxylase, which ranged from 6.2 to 7.5 units/mg protein at 25", was somewhat higher than that of pure chickenliver enzyme, i. e. 3-5unitsi mg protein at 25" [27,35,36] or 8-11 unitsimg protein at 37" [37,38]. The purified rat liver enzyme contained 0.50 pg of (+)-biotin per milligram of protein determined by the method of Lowry et al. [22] with crystalline bovine serum albumin as standard. This biotin content was comparable to that of pure chicken liver enzyme, i. e pg/mg protein dry weight [35,36] (corresponding to 0.74 pg/mg protein determined by the method of Lowry et al.) or 0.63 pg/mg protein determined by the method of Lowry et al. [37J.

6 166 Immunochemical Studies on AcetyI Coenzyme A Carboxylase Eur. J. Biochem. Fig. 2. Sedimentation patterns of rat liver acetyl-coa carboxylase. Enzyme from the Sepharose 2B chromatography step (concentrated) was dialyzed at 4" overnight against 50 mm Tris-C1 buffer ph 8.0 containing 5 mm 2-mercaptoethanol and 5 mm EDTA. The dialyzed enzyme solution was preincubated at 24" for 30 min in the presence (A) or absence (B) of 10 mm potassium citrate and centrifuged at 25.0" (A) or 25.3" (B) and 31410rev./min in a Spinco model E ultracentrifuge; protein concentration, 1.0 mg/ml; direction of centrifugation, left to right; photographs taken at 4, 8 and 12 min (A) or at 8, 12 and 16 min (B) after reaching speed Table 1. Purification of acetyl-coa carboxylase from rat liver 200 livers (1660 g) were used. Enzyme activity was determined at 25" by the spectrophotometric method by coupling with the fatty acid synthetase reaction, except for crude extract and 1st (NH4)&30, fraction whose activities were determined at 37" by the H14C0,- fixation method and corrected to the conditions for the spectrophotometric assay at 25". Enzyme from the Sepharose 2B chromatography stf,p was assayed also by the spectrophotometric method by coupling with the pyruvate kinase-lactate dehydrogenase system 15 r 43 s Fraction Volume Protein Total activity Specific activity at 25" at 25" ml mg units unitsimg Crude extract st (NH&SO, Ce,(PO,), gel and 2nd (NH,),SO, Dialysis at 24" DEAE-cellulose Sepharose 2B Fig. 3. Sucrose density gradient centrifugation of rat Ever acetyl-coa carboxylase. The method of Martin and Ames [39] was employed with a gradient from 5 to 20 /, (w/v) sucrose. Enzyme from the Sepharose 2B chromatography step (concentrated) was dialyzed and preincubated in the presence (0-0) or absence (0---9) of citrate as described in the legend to Fig ml of each enzyme solution (80 m- units) was applied on sucrose gradients containing the same additions as the respective preincubation medium. The gradient tubes were centrifuged at 25" and rev./min for 84min in a SW-39 rotor in a Spinco model L ultracentrifuge. 21 fractions were collected, and aliquots assayed for carboxylase activity at 25" in the presence of citrate by the spectrophotometric method by coupling with the fatty acid synthetase reaction. Yeast fatty acid synthetase (sz~,~ = 43 S) was used as external marker. Direction of centrifugation, right to left

7 Vol. 16, No. 1, NAKANISHI and S. NUMA 167 Fig.4. Ouchterlony double diffusion patterns of rat liver acetyl-coa carboxylase. Agar gels (1.0 o/o) contained 20 mm Tris-C1 ph 7.5, 0.15 &I NaCl and for C and E, lomm potassium citrate. The carboxylase preparations used were made as described in Materials and Methods, unless otherwise specified. The plates were developed at 24" for 12 h and then at 4" for 2 days before the photographs were taken. The figures in the following parentheses represent specific activities at 37" expressed as m-units/mg protein. A, well 1, refed (82); well 2, normal (41); well 3, fasted (7.8); well 4, enzyme from the Sepharose 2B chromatography step (citrate removed) (12400) ; well 5, enzyme from the second ammonium sulfate fractionation step (230); well 6, NaC1. B and C, wells 1 and 4, refed (82); wells 2 and 5, normal (41); wells 3 and 6, fasted (7.8). D and E, wells 1, 3 and 5, normal (44); wells 2, 4 and 6, diabetic (31). The center wells contained antiacetyl-coa carboxylase y-globulin Figs.2A and 2B reveal that the purified rat liver enzyme sedimented as a single, sharp, symmetrical boundary in the analytical ultracentrifuge with no evidence of impurity. The ~ 2 0 value, ~ was found to be 57 S in the presence of citrate and 46 S in its absence. In sucrose density gradient centrifugation studies shown in Fig.3, however, the enzyme sedimented more slowly in the absence of citrate than

8 168 Immunochemical Studies on Acetyl Coenzyme A Carboxylase Bur. J. Biochem. in its presence; the SZO,~ values were estimated to be about 25 S and 51 S, respectively. Acetyl-CoA carboxylase from animal sources is known to exist both as an inactive protomeric form and as an active polymeric form, the former being converted to the latter in the presence of citrate [12,25,35,37,40 to 431. By analogy with chicken liver acetyl-coa carboxylase, the higher sedimentation coefficient found in the analytical ultracentrifuge in the absence of citrate appears to be due to aggregation of carboxylase molecules at higher protein concentrations [35,41]. As documented below (Fig.4), immunological evidence also indicated the homogeneity of the purified rat liver acetyl-coa carboxylase. Ouchterlony Double Diffusion Analyses Fig.4A shows an Ouchterlony double diffusion pattern of antibody made against the purified rat liver acetyl-coa carboxylase in the center well and carboxylase preparations of widely differing specific activities. A single connecting band of precipitation was observed, indicating that the enzyme preparation employed as antigen was homogeneous. Figs.4B to 4E represent Ouchterlony double diffusion analyses in the absence and presence of aitrate with carboxylase preparations derived from livers of normal, fasted, refed and diabetic rats. The appearance of a single precipitin band in the absence and presence of citrate indicated that both protomeric and polymeric forms of carboxylase were precipitated by antibody. The band of precipitation under addition of citrate was located more closely to the antigen wells, corresponding to slower diffusion of aggregated carboxylase molecules in the presence of citrate. Furthermore, the completeness of connections of the precipitin bands showed that carboxylase molecules obtained from livers of fasted, refed and diabetic rats were immunologically similar to those from normal animals. Immunochemical Titration of Acetyl-CoA Carboxylase in Liver Extracts In order to estimate the quantity of liver acetyl- CoA carboxylase immunochemically, it is required that a stoichiometric relationship exist between antigen and antibody. Fig.5 shows that the properties of the system employed for immunochemical titrations of carboxylase satisfied this requirement. It is evident from the breaking points illustrated that the amount of enzyme removed in the carboxylase-antibody precipitate was proportional to the amount of antibody added. No carboxylase was removed by control y-globulin. Fig. 6 represents immunochemical titrations of liver extracts obtained from normal, fasted, refed and diabetic rats. Despite the fact that the level of carboxylase activity derived from 1 g of liver fell Liver extract (mi) Fig.5. Relationship between amount of antibody added and amount of acetyl-coa carboxylase removed. Increasing amounts of gel-filtered x g liver supernatant solution from normal rats (see Materials and Methods, carboxylase activity at 37", 173 m-units/ml) were added to 40pg (0-o), 80 pg ( x -x ) or 120 pg (0-0) of antiacetyl-coa carboxylase y-globulin or to 120 pg of control y-globulin (o----o). The precipitin reaction mixtures contained in addition final concentrations of 20 mm Tris-C1 ph 7.5, 0.15 M NaCI, 5 mm 2-mercaptoethanol and 1 mm EDTA. The samples were incubated at 37" for 15min and stored at 4" overnight. The antigen-antibody precipitate was removed by centrifugation at 1500xg for 15 min, and the resulting supernatant fluid was assayed for carboxylase activity at 37' by the H14C0,- fixation method. Since partial loss of carboxylase activity occurred during the incubation and storage overnight (generally /, lost), the activities given on the ordinate are the values corrected for this inactivation by multiplying by a factor which was found when the same procedure was carried out with control y-globulin instead of antibody Activity added (m- units) Fig. 6. Irnmunochernical aizalysis of levels of acetyl-coa carboxylase activity in liver extracts from normal, fasted, refed and diabetic rats. Increasing amounts of gel-filtered x g liver supernatant solutions (see Materials and Methods) containing carboxylase activities indicated were added to 50 pg of antiacetyl-coil carboxylase y-globulin. Following completion of precipitation, the supernatant fluids were assayed for carboxylase activity at 37". Other details were the same as described for Fig.5. 0, rats fed a balanced diet (specific activity at 37", 9.9 m-units/mg protein); A, rats fasted for 48 h (2.5 m-units/mg protein); x, rats fasted for 48 h and subsequently refed a fat-free diet for 48 h (25.4 m- units/mg protein); 0, diabetic rats (3.5 m-units/mg protein)

9 Vol.16, No.1, 1970 S. NAKANISHI and S. NUMA fold and 2.7-fold in starvation and diabetes, respectively, and rose 2.0-fold following refeeding, the equivalence point, i. e. the point at which enzyme activity first appeared in the supernatant fluid, was the same for all four types of liver extracts when based on the amount of enzyme activity added. This showed that the observed changes in the level of carboxylase activity were accompanied by proportionate changes in the quantity of immunochemically reactive protein. In these studies, the amount of antibody was held constant. Another titration procedure, in which the amount of liver extract was kept constant, was carried out in an experiment represented in Fig. 7. The amount of antibody required to precipitate completely the carboxylase activity in a given amount of liver extracts, as estimated by extrapolations of the linear portions of the titration curves to zero carboxylase activity, was decreased 2.2-fold by fasting and increased 2.7-fold by refeeding. Since the levels of carboxylase activity in normal, fasted and refed rats were 95, 43 and 241 m-units/ml liver extract, respectively, the proportionality between catalytic activity and immunochemically estimated enzyme protein was confirmed. These results indicate that the catalytic efficiency per acetyl-coa carboxylase molecule is not changed despite the wide variations in the level of carboxylase activity in liver extract seen under the different alimentary and hormonal conditions. Thus, the changes in the activity level are actually determined by changing quantities of enzyme protein. Anticarboxylase y -globulin (pg) Fig. 7. Irnmunochemical titration of liver acetyl-coa carboxylase from normal, fasted and refed rats. Increasing amounts of antiacetyl-coa carboxylase y-globulin were added to 1 ml of gel-filtered ~ g liver supernatant solutions (see Materials and Methods). Following completion of precipitation, the supernatant fluids were assayed for carboxylase activity at 37". Other details were the same as described for Fig.5. -0, rats fed a balanced diet (specific activity at 37O, 6.9 m-units/mg protein); A-A, rats fasted for 48 h (2.9 m-units/mg protein); x x, rats fasted for 48 h and subsequently refed a fat-free diet for 48 h (20.4 m-units/mg protein) Quantitative Precipitin Reactions Fig.8 shows that the protein content of the immunoprecipitates plotted against added activity gave a typical curve corresponding to the equivalence point. The enzyme preparation employed in this experiment was purified partially by ammonium sulfate fractionation and DEAE-cellulose chromatography as described in Materials and Methods. This partially purified preparation, in contrast to liver extract, produced essentially no precipitation with control y -globulin and was therefore suited for quantitative precipitin reactions. It is also Seen from Fig.8 that the catalytic efficiency per immunochemically reactive protein molecule remained unchanged during this purification procedure ; 270 pg of antibody precipitated 160 m-units of the partially purified carboxylase, while, as shown in Figs.5 and 6, this amount of antibody corresponded to m-units of carboxylase in liver extract. This fact, together with the absence of non-specific precipitation, justifies the use of this partially purified preparation for isolation of enzyme protein for the isotopic leucine incorporation studies described below. 0 m Activity added (rn-units) Big. 8. Quantitative precipitin reactions of rat liver acetyl-coa carboxylase. Increasing amounts of DEAE-cellulose eluate (see Materials and Methods) containing carboxylase activities indicated were added to 270 pg of antiacetyl-coa carboxylase y-globulin. The precipitin reaction mixtures contained in addition final concentrations of 50 mm Tris-C1 ph 7.5, 0.20 M NaC1, 5 mm 2-mercaptoethanol and 1 mm EDTA. Following completion of precipitation, the supernatant fluids were assayed for carboxylase activity at 37", and the precipitates washed 3 times with chilled 0.15M NaCl for determination of protein content,. Other details were the same as described for Fig.5. o----o, protein precipitated; e-e, carboxylase activity in supernatant fluid Isotopic Leucine Incorporation into Liver Acetyl-CoA Carboxylase In order to see whether the variations in the quantity of liver acetyl-coa carboxylase following

10 170 Immunochemical Studies on Acetyl Coenzyme A Carboxylase Eur. J. Biochem. Table 2. Relative rates of liver acetyl-coa carboqlase synthesis in normal, refed, fasted and diabetic rats Rats treated as indicated were given a single intraperitoneal injection of 0.9 mc of [3H]leucine. Three h later, two rats from each group were killed, except that three fasted animals were used. For experimental details, see Materials and Methods Treatment Acetyl-CoA carboxylase in liver extract [PH]Leucine incorporation Xean (4 (b) weight Specific activity Total activity * Acetyl-CoA Total soluble alb at 37" at 37" carboxylase totals protein I3 mu/mg U countslmin counts x min-' x mg-' None Fasted for 48 h and subsequently refed a fat-free diet for 72 h Fasted for 48 h Alloxan-diabetes * Per two rats Time after [3H]leucine injection (h) Fig. 9. Turnover of liver acetyl-coa carboxylase in normal, refed and diabetic rats. Rats were treated as specified in Table 2. Normal (0) and refed rats ( x ) weighing 180 g and diabetic rats (0) weighing g were given a single intraperitoneal injection of 0.9 mc of [3H]leucine at zero time. Two animals from each group were killed at the indicated times, and the radioactivity incorporated into liver acetyl-coa carboxylase was determined as described in Materiais and Methods. The results are expressed as radioactivity per unit of acetyl-coa carboxylase dietary manipulation or in diabetes are due to changes in the rate of enzyme synthesis or in that of enzyme degradation, studies on the incorporation of isotopic leucine into carboxylase were undertaken. As a measure of the rate of enzyme synthesis, the extent of [3H]leucine incorporation into protein precipitated by antiacetyl-coa carboxylase following pulselabeling was determined. The results of such experiments with normal, fasted, refed and diabetic rats are shown in Table 2. The specific radioactivity of total soluble liver protein differed considerably in the four types of rats due probably in part to different free amino acid pools present in various animals. Since the extent of labeling of total soluble protein is reflected in carboxylase labeling, the ratio of the radioactivity incorporated into enzyme to that incorporated into total soluble protein (a/b) was cal- I culated as a measure of the rate of enzyme synthesis. The data indicate that this relative rate of enzyme synthesis was decreased 1.9-fold and 1.7-fold by fasting and diabetes, respectively, and increased 4.0-fold by refeeding. As a measure of the rate of enzyme degradation, the rate of loss of radioactivity from prelabeled carboxylase was determined after injection of [3H]- leucine. Fig.9 represents the results of such experiments with normal, refed and diabetic rats, which are presumably in a steady state, exhibiting different but constant levels of carboxylase activity. The decay of specific radioactivity of carboxylase followed a first order reaction. The rate of loss of isotope, expressed as half-life, was essentially the same under all three conditions, i. e. 59, 55 and 59 h in normal, refed and diabetic animals, respectively. Under steady state conditions, the content of an enzyme is related to the rates of its synthesis and degradation as follows: E = k,/kd, where E is the content of enzyme per mass, k, is a zero order rate constant of synthesis per mass, and kd is a first order rate constant of degradation expressed as time-1 (see [44]). The content of carboxylase can be expressed as the level of its activity as concluded above. Since the rate constant of enzyme degradation was essentially the same in the three types of animals, the 3.8-fold increase in enzyme content observed in refed rats and the 1.9-fold decrease in it seen in diabetic rats (see Table 2) should be reflected by changes in the rate constant of enzyme synthesis. In fact, the relative rate of enzyme synthesis (a/b) was found to rise 4.0-fold in refed animals and to fall 1.7-fold in diabetic animals. Thus, the increased or decreased carboxylase content in refed or diabetic rats can be ascribed to an increased or decreased rate of enzyme synthesis. Fig. 10 shows the results of studies on enzyme degradation in fasted animals. Under the experimental conditions employed, fasted rats were not in a steady

11 Vol. 16, No. 1, 1970 S. NAKANISHI and S. NUMA t Time after [ 3H] lwiw injection (h) Fig. 10. Turnover of liver acetyl-coa carboxytylase in fasted rats. Rats fasted for 48 h and weighing 180 g were given a single intraperitoneal injection of 0.9 mc of [3H]leucine. Three animals were killed at the indicated times, and the radioactivity incorporated into liver acetyl-coa carboxylase was determined as described in Materials and Methods. The results are expressed as total radioactivity incorporated per three animals state, since both the weight of liver and the content of carboxylase per rat were diminished gradually during the experimental period. In contrast to steady state conditions, the rate of degradation of protein under conditions of changing contents is given by the change in total radioactivity in the protein, not by the change in specific radioactivity [45]. The decay of total radioactivity precipitated by antiacetyl-coa carboxylase followed a first order reaction, the halflife being 31 h. Thus, the 3.6-fold decrease in enzyme content observed following 2 days starvation was accompanied by a 1.9-fold rise in the rate of enzyme degradation and a 1.9-fold fall in the relative rate of enzyme synthesis (~16). Although calculations according to the above equation can not be made in this instance, it is concluded that the decrease in enzyme content following fasting is due both to diminished enzyme synthesis and to accelerated enzyme degradation. In the experiments to determine the rate constant of enzyme degradation, reutilization of [3H]leucine may lead to an overestimation of the half-life [45]. However, this appears not to be of major significance in our experiments for the following reasons : the halflife for total soluble liver protein estimated simultaneously by means of this isotope was 3.8, 3.7, 3.4 and 2.9 days in normal, refed, diabetic and fasted rats, respectively, while the half-life found by the use of guanido-labeled arginine, which is not reutilize& is 5.1 days in normal rats as reported by Arias et al. [MI * DISCUSSION Acetyl-CoA carboxylase from chicken liver was isolated in homogeneous form in Lane s laboratory b [37,38] as well as in our group and also crystallized [27,35,36]. In the present investigation, rat liver acetyl-coa carboxylase has been purified to homogeneity, as evidenced by sedimentation velocity experiments in the analytical ultracentrifuge and Ouchterlony double diffusion analyses. Its specific activity and biotin content are similar to those of pure chicken liver enzyme. The isolation of homogeneous rat liver acetyl-coa carboxylase has permitted immunochemical studies on the changes in the quantity of enzyme protein as well as on its synthesis and degradation under various metabolic conditions. The results of immunochemical analysis indicate that the changes in the level of acetyl-coa carboxylase activity in liver extract seen under the different dietary and hormonal conditions studied are determined by changing quantities of enzyme protein. However, this finding in vitro excludes by no means that the control by changes in catalytic efficiency per enzyme molecule due to various metabolites (see [I]) is also involved in the regulation of the acetyl-coa carboxylation step in vivo. The contents of some effectors in the liver are known to vary under different metabolic conditions associated with increased or decreased fatty acid synthesis ; the content of long-chain acyl-coa derivatives is elevated 2- to 4-fold in fasted and diabetic rats and lowered about 2-fold in refed animals [13,14], while the citrate content is diminished to about one half in starvation and diabetes [46,47]. Moreover, Korchak and Masoro [5] and Wieland and Eger-Neufeldt [6] demonstrated that in an earlier stage of fasting, i.e. after 24h fasting or in acute decompensated diabetes, the fatty acid-synthesizing capacity of liver slices is more depressed than can be accounted for by the level of acetyl-coa carboxylase activity in liver extract. It is evident from the half-lives of liver acetyl-coa carboxylase found in the present studies that the enzyme content can not fall very rapidly. Therefore, the control mechanisms by inhibition or activation of carboxylase activity may play a more important role, when the rate of fatty acid synthesis must be adjusted promptly, whereas the control mechanisms by changes in carboxylase quantity may make a greater contribution to the long-term regulation of fatty acid synthesis. It is of interest to note that the synthesis and degradation of liver acetyl-coa carboxylase are controlled independently. In refed and diabetic rats, the rate of enzyme synthesis is increased or decreased, whereas the rate of enzyme degradation remains unchanged. On the other hand, in fasted rats not only the rate of enzyme synthesis is decreased, but also the rate of enzyme degradation is increased. Of particular interest is a comparison of the situations in diabetic and fasted animals. Although the enzyme content is diminished in both types of animals, an

12 172 Immunochemical Studies on Acetyl Coenzyme A Carboxylase Eur. J. Biochem. increased rate of enzyme degradation is encountered in fasted rats, but not in diabetic rats. Animals suffering from prolonged diabetes, like normal animals maintained on a balanced diet and those refed a fatfree diet for more than 72h, are presumably in a steady state, each type of animals exhibiting different steady state carboxylase contents, whereas fasted animals are not in a steady state. The above findings are consistent therefore with the assumption that the control of enzyme content by changes in the rate of enzyme degradation may play an important role only when the animal deviates from a steady state for adjustment to a new environment. This view is supported also by the following findings of Schimke [48] on liver arginase. Rats maintained on diets containing 8, 30 and 70 /, casein show different steady state arginase contents but essentially the same rates of arginase degradation. Upon changing rats from a diet containing 70 1, protein to one containing 8,Ilo protein, the rate of arginase degradation is increased during the first 3 days. However, it then gradually approaches the rate seen in the steady state, as the enzyme content attains a new steady state level lower than the initial one. When rats are fasted, the arginase degradation ceases, and the enzyme content rises concomitantly. The factors responsible for independent controls of the synthesis and degradation of liver acetyl-coa carboxylase are unknown. In this connection, Allmann et al. [4] showed that addition of linoleate to fat-free diet tends to suppress the elevation of fatty acid synthesis seen when fat-free diet alone is fed. Moreover, the relative linoleate content was found to fall progressively during refeeding of fat-free diet coincidently with a rise in fatty acid synthesis. The mechanisms of the linoleate effect, however, remain to be elucidated. In a very recent report, Majerus and Kilburn [19], employing antibody prepared against homogeneous chicken liver acetyl-coa carboxylase, which was found to cross-react with rat liver enzyme, made similar studies on the synthesis and degradation of carboxylase in rats under different dietary conditions. Although their conclusions were in general agreement with ours, the amount of isotopic leucine incorporation into enzyme was markedly lower in their studies than in ours. The reason for this discrepancy is as yet unknown, but it might be due to the use of different antibodies. We are indebted to Drs. F. Lynen and D. Oesterhelt for a generous gift of yeast fatty acid synthetase and to Dr. 0. Midorikawa for his helpful advice in producing alloxandiabetes. We thank also Drs. K. Ogata and Y. Izumi for kindly performing determinations of biotin and Dr. S. Nakamura for determinations of blood sugars. This investigation was supported in part by research grants from the Ministry of Education of Japan, the Japan Waksman Foundation and the Japanese Medical Association. S. Nakanishi is a recipient of a Sigma Chemical Postgraduate Fellowship. REFERENCES 1. Numa. S.. Bortz. W. M.. and Lvnen, " - F., - Adwan. Enzqme Regk 3 (1965) Numa, S., Matsuhashi, M., and Lynen, F., Biochem. Z. 334 (1961) Wieland. 0.. Eeer-Neufeldt, I., Numa, S., and Lynen, F,, Biochem (1963) Allmann, D. W., Hubbard, D. D., and Gibson, D. M., J. Lipid Res. 6 (1965) Korchak, H. M., and Masoro, E. J., Biochim. Biophys. Acta, 58 (1962) Wieland, O., and Eger-Neufeldt, I., Biochem (1963) Matsuhashi, M., Matsuhashi, S., Numa, S., and Lynen, F., Fed. Proc. 21 (1962) Martin, D. B., and Vagelos, P. R., Fed. Proc. 21 ( 1962) Waite, M., Fed. Proc. 21 (1962) Kallen, R., and Lowenstein, J. M., Arch. Biochem. Bio Bray, G. A., Anal. Biochem. 1 (1960) Numa, S., In Methods in Enzymology (edited by S. P. Colowick, N. 0. Kaplan, and J. M. Lowenstein), Academic Press, New York 1969, Vol. XIV, p Hsu, R. Y., Wasson, G., and Porter, J. W., J. Biol. Chem. 240 (1965) Lynen, F., In Methods in Enzymology (edited by S. P. Colowick, N. 0. Kaplan, and J. M. Lowenstein), Academic Press, New York 1969, Vol. XIV, p Marshall, M., and Cohen, P. P., J. Biol. Chem. 236 (1961) Campbell, D. H., Gamey, J. S., Cremer, N. E., and Sussdorf, D. H., MetRods in Immunology, W. A. Benjamin, New York 1963, p Ouchterlony, O., Arkiv Kemi Mineral. Geol. 26 B (1949) phys. 96 (1962) 188. Bortz, W. M., and Lynen, F., Biochem (1963) 505. Numa, S., Ringelmann, E., and Lynen, F., Biochem (1965) 243. Bortz, W. M., and Lynen, I?., Biochem (1963) 77. Tubbs, P. K., and Garland, P. B., Biochem. J. 93 (1964) 550. Hicks, S. E., Allmann, D. W., and Gibson, D. M., Biochim. Biophys. Acta, 106 (1965) 441. Numa, S., Nakanishi, S., and Iritani, N., Abstracts of Twelfth Japan Diabetic Society Veeting, Kyoto, July 1969, p Numa, S., Nakanishi, S., and Iritani, N., Proceedings of Eleventh Japanese Conferences on Biochemistry of Lipids Meeting, Sapporo, July 1969, p Nakanishi, S., Ohtsu, E., and Numa, S., Seikaguku, 41 (1969) 492. Majerus, P. W., and Kilburn, E., J. Biol. Chem. 244 (1969) Heppel, L. A., In Methods in Enzymology (edited by S. P. Colowick and N. 0. Kaplan), Academic Press, New York 1955, Vol. 11, p Kanegasaki, S., and Numa, S., Biochim. Biophys. dcta, 202 (1970) 436. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193 (1951) 265. Wright, L. D., Skeggs, H. R., and Cresson, E. L., J. Amer. Chem. Soc. 73 (1951) Somogyi, M., J. Biol. Chem. 195 (1952) 19. Numa, S., and Ringelmann, E., Biochem (1965) Kabat, E. A., and Maver, M. M., Experimental Immunochemistry, Charles "C..Thomas, gpringfield, Illinois, 1961, p Cho-Chung, Y. S., and Pitot, H. C., Eur. J. Biochem. 3 (1968) 401. Numa, S., Rineelmann. E., and Riedel. B.. Biochem., I Biophys. Res.-Commun. 24 (1966) 750.

13 Vol. 16, No. 1, 1970 S. NAKANISRI and S. NUMA Goto, T., Ringelmann, E., Riedel, B., and Numa, S., Life Sci. 6 (1967) Gregolin, C., Ryder, E., Kleinschmidt, A. K., Warner, R. C., and Lane, M. D., Proc. Nat. Acad. Sci. U. 8. A. 56 (1966) Gregolin, C., Ryder, E., and Lane, M. D., J. Biol. Chem. 243 (1968) Martin, R. G., and Ames, B. N., J. Biol. Chem. 236 (1961) Vagelos, P. R., Alberts, A. W., and Martin, D. B., J. Biol. Chem. 238 (1963) Gregolin, C., Ryder, E., Warner, R. C., Kleinschmidt, A. K., and Lane, M. D., Proc. Nat. Acad. Sci. U. S. A. 56 (1966) Numa, S., Goto, T., Ringelmann, E., and Riedel, B., Ew. J. Biochem. 3 (1967) Gregolin, C., Ryder, E., Warner, R. C., Kleinschmidt, A. K., Chang, H. C., and Lane, M. D., J. Biol. Chem. 243 (1968) Arias, I. M., Doyle, D., and Schimke, R. T., J. Biol. Chem. 244 (1969) Koch, B. L., J. Th.eor. Biol. 3 (1962) Lynen, F., Progr. Biochem. Pharmacol. 3 (1967) Start, C., and Newsholme, E. A., Biochem. J. 107 (1968) Schimke, R. T., J. Biol. Chem. 239 (1964) S. Nakanishi and S. Numa Department of Medical Chemistry Kyoto University Faculty of Medicine Yoshida, Sakyo-ku, Kyoto, Japan