Synthesis and Degradation of Malic Enzyme in Chick Liver*

SUMMARY The role of synthesis and of degradation in regulation of malic enzyme concentration in the liver of growing chicks has been studied. Malic enzyme was assayed by enzymic activity and by immunological analysis with immunoglobulin containing specific anti-malic enzyme. Quantitative immu-noprecipitin curves and equivalence point determinations indicated that there was a constant relationship between immunologically precipitable protein and units of enzyme activity in livers of neonatal and growing chicks during a time period in which total enzyme activity increased 60-fold. Hence, changes in malic enzyme activity were due to changes in enzyme content of the liver rather than to activation or inhibition of preformed enzyme. The relative rate of malic enzyme synthesis was determined by measuring incorporation of counts into malic enzyme protein after pulse labeling total protein with L-[4,5-3H?]leucine. followed

The role of synthesis and of degradation in regulation of malic enzyme concentration in the liver of growing chicks has been studied.
Malic enzyme was assayed by enzymic activity and by immunological analysis with immunoglobulin containing specific anti-malic enzyme. Quantitative immunoprecipitin curves and equivalence point determinations indicated that there was a constant relationship between immunologically precipitable protein and units of enzyme activity in livers of neonatal and growing chicks during a time period in which total enzyme activity increased 60-fold. Hence, changes in malic enzyme activity were due to changes in enzyme content of the liver rather than to activation or inhibition of preformed enzyme. The relative rate of malic enzyme synthesis was determined by measuring incorporation of counts into malic enzyme protein after pulse labeling total protein with L- [4,5-3H?]leucine.
Malic enzyme protein was purified by quantitative precipitation of the enzyme by antibody followed by disc gel electrophoresis in the presence of sodium dodecyl sulfate.
Radioactivity in the band corresponding to malic enzyme was estimated after electrophoresis of the immunoprecipitate.
Relative to synthesis of total protein, the rate of synthesis of malic enzyme increased more than 50-fold when neonatal chicks were fed. Starvation of week-old chicks for 2 days caused a 60 to 70% decrease in synthesis of malic enzyme.
Feeding, which increased the synthesis and concentration of malic enzyme also caused an increase in the rate of degradation.
In 8-and 11-day-old chicks degradation of malic enzyme was found to be first order with a fg of 55 hours.
In fasting chicks a f+ of 28 hours was observed, but there was also an increase in the rate of degradation of total liver protein suggesting that part of the increased rate of degradation of malic enzyme was due to a general increase in protein breakdown.
The temporal relationship between synthesis of malic enzyme and synthesis of fatty acids was studied in unfed neonatal chicks given a single glucose meal. Fatty acid synthesis from [lJ4C]acetate was increased 7-fold at 13 hours and 20-fold at 3 hours after the glucose meal.
Neither the activity nor the synthesis of malic enzyme were increased at 3 hours but both were significantly increased at 6 hours * This investigation was supported by Grant MA-3332 from the Medical Research Council of Canada.
These results suggest that the increased synthesis of malic enzyme may have been initiated by an increased flux through the pathway for fatty acid synthesis.
This correlation between fatty acid synthesis and the activity of malic enzyme, whose function appears to be to provide NADPH for de no~o fatty acid synthesis (1, 2), suggests a possible causal relationship.
This paper reports the rates of synthesis and degradation of malic enzyme in the liver of neonatal and growing chicks. In the embryonic and unfed neonatal chick, malic enzyme and fatty acid synthesis are very low; both are increased manyfold when neonatal chicks are fed but show no change if the chicks are not fed (4,5). In older chicks, fasting reduces and refeeding increases both activities (6,8).
With the use of immunological techniques, we have investigated the following questions.
(a) Are changes in the activity of malic enzyme the consequence of changes in the concentration of malic enzyme protein? (6) What are the roles of synthesis and degradation in the regulation of malic enzyme concentration?
(c) What is the temporal relationship between an increased rate of fatty acid synthesis and an increased rate of malic enzyme synthesis? EXPERIMENTAL PROCEDURE Animal Care-Unincubated embryonated eggs from white Leghorn chickens were obtained from a commercial supplier and incubated in an electric forced draft incubator at 35.5 f 0.5 and 60% relative humidity.
One-day-old chicks' were removed from the incubator and placed in battery brooders which had thermostatically controlled heaters and wire mesh floors. Commercial chicken mash (Master Starter Krumbs, Maple Leaf Mills Ltd., Toronto, Canada) and water were available ad Zibitum. For the in viva isotope incorporation experiments, the chicks were placed in an incubator (31 f 0.5") which was vented into a fume hood. In the forced feeding experiments 20% glucose (2 ml) was administered directly into the cardiac stomach of unanesthetized chicks. ; and L- [4,leucine (New England Nuclear) were obtained from the designated sources.
Purijication of Malic Enzyme-Malic enzyme was isolated from the livers of 4-to g-week-old white Leghorn chicks by the method used by Hsu and Lardy (9) to purify pigeon liver malic enzyme.
The following modifications were made: (a) DTT2 (1 mM) was used instead of P-mercaptoethanol throughout the procedure; (b) the first and second ethanol fractionations were from 21% to 367, and 23% to 46% ethanol, respectively; (c) the zinc acetate step was omitted because it completely inactivated the enzyme; and (d) a Sephadex G-200 chromatography step was added between the ammonium sulfate fractionation and DEAE-cellulose chromatography. A typical recovery sheet is shown in Table I. All subsequent references to protein fractions refer to Table I.
For the Sephadex G-200 chromatography step, Fraction V was dialyzed overnight against 0.03 M Tris-HCl-1 mM DTT, pH 7.4. Dialyzed Fraction V (2 or 3 ml) was applied to a column of Sephadex G-200 (1.5 x 90 cm) which had been equilibrated with 0.03 M Tris-HCI-1 mM DTT, pH 7.4. The enzyme was eluted in the void volume with t,he same buffer. Fractions containing malic enzyme of high specific activity were pooled and concentrated by ultrafiltration (Amicon Corporation, Cambridge, Massachusetts). Concentrated Fraction VI was dialyzed against 0.03 M Tris-HCI-1 mM DTT, pH 7.7, prior to DEAEcellulose chromatography.
After DEAE-cellulose chromatography, the fractions containing activity were pooled, concentrated by ultrafiltration (Amicon), and dialyzed against 0.05 M Tris-HCl-1 mM DTT, pH 7.4. The enzyme could be stored in this form (Fraction VII) at -15" for a few months without any detectable loss of activity.
Malic enzyme was assayed at 40" by the method of Wise and Ball (1). All assays were linear with respect to time and protein concentration.
A unit of enzyme activity is defined as 1 pmole of NADP reduced per min. Protein was determined by the method of Lowry et al. (10) with crystalline bovine serum albumin as a standard.

Polyacrylamide
Disc Gel Electrophoresis-Polyacrylamide gels were prepared by the method of Davis (11) in buffer containing 0.05 M Tris-HCl-0.4 M glycine, pH 8.5. The running gels contained 5% acrylamide and 1.5 M urea. Electrophoresis was carried out in glass tubes (0.5 x 11 cm) at 2 ma per tube for 45 min at 4". Protein in the gels was stained with Amido black. Malic enzyme activity in the gels after electrophoresis was determined by the method of Henderson (12). For the latter experiments the gels underwent electrophoresis for 45 min prior to adding the protein sample in order to remove contaminants which might inhibit enzyme activity. Molecular weight determination by SDS polyacrylamide gel electrophoresis was carried out as described by Weber and Osborn (13). The samples were incubated at 37" for 2 hours in 2 The abbreviations used are: DTT, dithiothreitol; SDS, sodium dodecyl sulfate. where R is the gas constant, t is the absolute temperature, w is the rotor speed in radians per set, V is the partial specific volume, p is the density of the buffer solution, and c is the concentration (milligrams per ml) at radial distance r from the axis of rotation.
Immunological Procedures-Fraction VII was thawed and centrifuged to remove traces of denatured protein.
The preparation was diluted to a protein concentration of 1 to 2 mg per ml with 0.05 M Tris-HCI-1 mM DTT, pH 7.4. The diluted enzyme was mixed with an equal,volume of Freund's complete adjuvant and was ejected from a ~-CC glass syringe forcibly and repeatedly until a thick smooth emulsion was obtained.
This emulsion was injected into rabbits at multiple subcutaneous sites. Injections (0.5 to 1.0 mg of malic enzyme per rabbit) were made three times at 7-to lo-day intervals.
The rabbits were bled weekly after the last injection.
Peak antibody levels were achieved 2 to 4 weeks after the last injection.
Sera with high antibody titer were pooled and frozen at -15". Immunoglobulins were purified from undialyzed serum by a batch DEAE-Sephadex procedure (15). Ouchterlony double diffusion patterns and quantitative precipitin tests were performed as outlined by Ouchterlony (16) and by Kabat and Mayer (17).

Synthesis and Degradation of Malic Enzyme-Chicks
were injected intraperitoneally with 0.5 mCi of L- [4, twice with 5% trichloroacetic acid containing 0.5% unlabeled leucine and once with chloroform-methanol (2:l) and then dissolved in 0.5 ml of formic acid (90%).
FIG. 2. Polyacrvlamide gel electrophoresis of chicken liver malic enzyme. The running gel contained 5% acrylamide, 1.5 M urea. 0.05 M Tris-HCl. and 0.4 M alvcine. nH 8.5. Twentv micrograms of protein were applied to&&h gel: a, stained forenzyme activity with tetrazolium blue. The gels were incubated at 37" for about 15 min in 0.14 M Tris-HCl, pH 8.0, containing nitrotetrazolium blue, 80 pg per ml; phenazine methosulfate, 120 pg per ml; L-malic acid, 60 mu; NADP, 15 pg per ml; and MnCle, 0.8 111~. b, stained for protein with Amido black. The same patterns were observed when urea was omitted from the gels. diphenyloxazole, 0.5%, 1 ,Cbis(5-phenyloxazol-2-yl)benzene, 0.025%) was added and radioactivity measured in a Nuclear-Chicago Unilux II liquid scintillation spectrometer. The remainder of the homogenate was purified to Fraction II (Table I). In samples from all but the unfed, neonatal chicks, Fraction II was diluted with 0.05 M Tris-HCl-0.15 M NaCl-1 mM DTT, pH 7.4, to give a malic enzyme concentration of 0.35 to 0.40 unit per 0.2 ml. In samples from unfed, neonatal chicks, nonradioactive Fraction VII malic enzyme was added to Fraction II to bring the malic enzyme concentration up to 0.35 to 0.40 unit per 0.1 ml, thus maintaining a constant ratio of antibody to antigen in all preparations.
Malic enzyme (0.35 to 0.40 unit in 0.1 or 0.2 ml as noted above) was precipitated with 10 mg of immunoglobulin containing specific malic enzyme antibody (an amount sufficient to precipitate 1 unit of malic enzyme).
The reaction mixture contained 0.05 M Tris-HCl-0.15 M NaCl-1 mM DTT, pH 7.4, in a total volume of 2 ml and was incubated at 37" for 30 min and stored overnight at 4". The precipitate was collected by centrifugation at 2000 x g for 10 min and washed twice with cold 0.9% NaCl.
The washed precipitate was dissolved in 0.2 to 0.5 ml of 1 M acetic acid. After 30 min at room temperature, the sample was lyophilized.
The entire sample (approximately 100 pg of protein) was subjected to SDS polyacrylamide gel electrophoresis at 5 ma per tube for 4 hours. After electrophoresis the gels were removed from the tubes and cut into 1.0 to 1.5-mm fractions.
After 30 min the birds were killed and their livers removed and frozen on solid COz. Fatty acids were extracted and their radioactivity measured as previously described (4). At 0 and 3 hours after the glucose meal, incorporation into fatty acids increased for at least 30 min. At 6 hours after the glucose meal, incorporation leveled off at 15 min, suggesting an increased output of newly synthesized fatty acids into the blood.

RESULTS
Homogeneity and Molecular Weight- Fig.   1 shows the sedimentation pattern of purified malic enzyme in the analytical ultracentrifuge.
A single symmetrical peak with an &bs value of 9.5 was observed.
This indicates the enzyme was homogeneous with respect to size. Fig. 2 shows the results of polyacrylamide gel electrophoresis of the purified enzyme.
Protein was stained on one gel (Fig. 2a), enzyme activity on the other (Fig. 2b). Three protein bands, each staining for malic enzyme activity, are visible.
Thus, chick liver may contain three or more isozymes of malic enzyme.
Isozymes of malic enzyme have been described in mouse tissues (12). The multiple bands also could represent subunits or polymers of malic enzyme.
The molecular weight of undissociated chicken liver malic enzyme was determined by the sedimentation equilibrium method (14). By assuming a partial specific volume of 0.74 (9) Malic enzyme (t ), crystalline bovine serum albumin (a), glutamic dehydrogenase (X), and aldolase (A) were incubated in 0.01 M sodium Dhosphate (pH 7.0) containing SDS (1%) and p-mercaptoethanol*(l%) for 2 hours at 37' and subjected to polyacrylamide gel electrophoresis in the presence of 0.1% SDS as described by Weber and Osborn (13). About 50 pg of each protein were used. (e) lo-day-old chicks which had been starved for 2 days (specific activity, 0.4 units per mg of protein), a---0.
The upper curve is protein; the lower curve is activity.
Precipitation reaction mixtures contained 2 mg of anti-malic enzyme immunoglobulin and various amounts of Fraction 11 extracts in 2 ml of 0.05 M Tris-HCl-0.15 M NaCl-1 mM DTT, pH 7.4. This mixture was incubated at 37" for 30 min and 4' overnight.
The precipitates were collected by centrifugation and washed twice with ice-cold 0.9% NaCl. When immunoglobulin from nonimmunized rabbits was used, there was no precipitate. tion VII) or malic enzyme from Fraction II of unfed, neonatal chicks and 3-and %day-old chicks (Fig. 4). The results of quantitative precipitin reactions between anti-malic enzyme and Fraction II preparations from chicks of different ages are shown in Fig. 5. By measuring enzyme activity in the supernatants after removal of the antibody-antigen complexes, equivalence points were established. The same equivalence point, namely 0.1 unit per mg of immunoglobulin, was observed for the Fraction II preparations from (a) unfed, neonatal chicks, (6) chicks fed for 2, 7, or 10 days, and (c) lo-day-old chicks which had been fasted for 2 days. The same equivalence point also was observed for purified malic enzyme (Fraction VII). The amount of protein precipitated by 2 mg of antibody also is shown in Fig. 5. Similar curves were observed for Fraction II preparations from all the kinds of chicks. In sum, these results indicate that the antibody precipitated a protein immunologically identical with purified malic enzyme no matter what kind of chick liver was the source of enzyme. In addition, the precipitin reactions show that the changes in enzyme activity which followed the feeding of neonatal chicks or the fasting of older chicks (Table II)  For all other chicks one liver was used per experiment.
Synthesis is expressed as disintegrations per min incorporated into malic enzyme per 10,000 dpm incorporated into total liver protein.
In all experiments the immunoprecipitate was subjected to SDS-polyacrylamide electrophoresis as outlined under "Experimental Procedure," and only the malic enzyme ies of enzyme synthesis and degradation have failed to identify rigorously the radioactive product in the antibody-antigen complex (19)(20)(21)(22).
Nonspecific adsorption and trapping have generally been determined by a second precipitation technique (21,22) in which nonradioactive enzyme is added to the sample after the original antibody-antigen precipitate has been removed. The nonradioactive enzyme is then precipitated by an amount of antibody equivalent to that used in the first precipitation.
A second correction for nonspecific precipitation is determined by counting the precipitate formed by adding nonimmune serum to a duplicate of each sample. The sum of counts in the second precipitate and counts in the nonimmune precipitate is subt,racted from the value for the first precipitate.
The difference is taken to represent incorporation into the specific antigen being studied.
In order to increase our confidence that the radioactivity precipitated by immunoglobulin was pure malic enzyme, we dissociated the antibody-antigen precipitate in 1 M acetic acid and separated the component proteins by polyacrylamide gel electrophoresis in the presence of SDS. Four major radioactive peaks were present (Fig. 6~). Pure malic enzyme which underwent. electrophoresis on a separate gel migrated to the same position as the peak at Fraction 20 (Fig. 6~). When excess unlabeled enzyme was added prior to immunoprecipitation, only the peak at Fraction 20 was displaced, thus identifying it as malic enzyme (Fig. 6b). The possibility remained that the other peaks represented proteins which could be cleaved to form active malic enzyme. In the above experiments S-day-old birds were killed 3 hours after administration of the [3H]leucine. The relative distribution of radioactivity among the major peaks remained the same in another serie? of experiments in which the birds were killed 6 hours after administration of the leucine. This suggested that the nonmalic enzyme peaks were not precursors of malic enzyme.
The size and distribution of the nonmalic enzyme peaks varied somewhat from experiment to experiment suggesting that they were nonspecific in nature.
Measurements of radioactivity in immunoprecipitable malic enzyme by the SDS electrophoresis method and the nonspecific adsorption method described above are compared in Table III. Recovery of radioactivity from the gel was 98% or more (compare Columns a and d, counting efficiency varied by less than 5%). In 3-day-old birds, actual incorporation into malic enzyme (Column e) was 71% of that calculated from the nonspecific adsorption method., In our experiments, we could not detect any precipitate when immunoglobulin prepared from unimmunized rabbits was added to the samples. Hence, that correction was not applied.
The difference between the non specific adsorption and electrophoresis methods was even greater when Fraction II prepared from unfed, neonatal chicks was used. In this case incorporation into malic enzyme was only 25% of the value predicted by the nonspecific adsorption method.
Attempts to reduce nonspecific adsorption by further purification of the malic enzyme in liver extracts from neonatal chicks were unsuccessful.
In view of these results, in all experiments described below, we isolated radioactive malic enzyme from the antibody-antigen precipitate by SDS electrophoresis and counted only the peak corresponding to malic enzyme. Identity of the enzyme peak was frequently checked by competition with purified nonradioactive enzyme (as in Fig. 6). L- [4, 5-3H2]Leucine Incorporation Xtudies-Incorporation of leucine into total protein reached a maximum at about 1 hour after the isotope injection (Fig. 7). Radioactivity remaining in the free amino acid pool (counts soluble in trichloroacetic acid) decreased to a minimum at 1 hour (Fig. 7). The quantity of radioactivity actually in leucine was determined by subjecting a portion of the trichloroacetic acid ext,ract to high voltage paper electrophoresis.
Radioactivity in the spot corresponding to free leucine was barely detectable (Fig. 7). The time courses shown in Fig. 7 were obtained with chicks which had been fed for 2 days. Similar patterns were obtained with unfed, neonatal chicks. Therefore, we selected a l-hour incorporation period to estimate synthesis of malic enzyme and I-, 24., and 4%hour periods to estimate degradation.
When neonatal chicks were fed there was a rapid increase in the rate of synthesis of malic enzyme relative to the synthesis of total protein (Table II).
The 54.fold increase in the rat,e of synthesis was comparable to the 63-fold increase in total activity of the enzyme, suggesting that increased synthesis played a major role in increasing t,he concent,ration of malic enzyme. This finding was confirmed when the degradation of malic enzyme was investigated (Fig. 8). Degradation of malic enzyme was barely detectable in unfed, neonatal chicks. After the chicks had been fed for 7 or 10 days the rate of degradation increased, a phenomenon which w-ould t,end to oppose the observed increase in activity.
The t+ for degradation of malic enzyme in neonatal chicks was estimat,ed to be 350 hours. In 8-and II-day-old chicks, the f+ was about 55 hours. Incorporation of [3H]leucine into t.otal liver protein (per bird) decreased by about 40% during the feeding experiment (Fig. 9). This decrease was probably due to the increased body size and hence increased leucine pool size of the fed birds. By expressing the results for synthesis of malic enzyme relative to synthesis of total protein (2s22), we eliminated this factor in the interpretation of the measurements of malic enzyme synthesis.
Degradation of total liver protein was difficult to estimate because it did not appear to be a first order reaction (Fig. 9). Since secretion of protein is an important, function of the liver, we The glucose solution (20yo) was administered intragastrically (2 ml) into unfed, neonatal chicks. At the times specified, the birds were injected intraperitoneally with [1-i%]acetate or [3H]leucine. In the fatty acid synthesis experiments the chicks were killed 30 min after the injection of acetate. Livers were removed and frozen in solid COZ. Total fatty acids were extracted and their radioactivity measured as previously described (4). In the malic enzyme synthesis experiments, the birds were killed 60 min after the leucine injection.
The livers were removed and homogenized and incorporation into total protein and malic enzyme determined as described under "Experimental Procedure." The results are expressed as mean f S.E. of the numbers of experiments in parentheses. 0 0.06 f 0.02 (7) 0.5 0.10 f 0.02 (7)  1 0.42 f 0.16 (8)  3 1.18 f 0.19 (7) (4) may represent secretion of blood proteins.
In any event, turnover of total liver protein was very similar in unfed, neonatal chicks or chicks fed for 7 to 10 days, suggesting that the increased rate of degradation of malic enzyme which occurred with feeding was not an artifact due to isotope reutilizat,ion in unfed, neonatal chicks nor was it simply a reflection of changes occurring in all liver proteins.
Synthesis also played a major role in the regulation of malic enzyme concentration during starvation. Chicks (10 days old) which had been fasted for 2 days had an enzyme activity which was about one-half that of normally fed controls (Table II). Relative synthesis of malic enzyme was inhibited by about 60% by fasting (Table II).
The rate of degradation of enzyme was increased by starvation (Fig. S), but the increased rate of degradation was accompanied by an increase in the degradation of all liver protein (Fig. 9). Part of the increased degradation of malic enzyme, therefore, was due to a general increase in the breakdown of liver protein.
Temporal Relationship Between Fatty Acid Synthesis and Synthesis of Malic Enzyme-A single glucose meal stimulates hepatic fatty acid synthesis in the unfed, neonatal chick (23). In liver slices incorporation of glucose and acetate into total fatty acids increased at 1 and 3 hours, respectively, after the glucose meal (23). In z&o, incorporation of [1-i4C]acetate into total fatty acids increased by 1 to 12 hours after the glucose meal (Table IV).
At 3 hours fatty acid synthesis in vivo increased by 20.fold while neither the activity nor the rate of synthesis of malic enzyme increased (Table IV).
Six hours after the meal, fatty acid synthesis began to fall off (possibly accompanied by increased release of fatty acids to the blood), but the activity and rate of synthesis of malic enzyme increased 100% and ISO%, respectively (Table IV). DISCUSSION Total activity of malic enzyme in the livers of neonatal and growing chicks was regulat,ed almost exclusively by regulating the concentration of the enzyme. Concentration, in turn, was regulated by varying the rate of synthesis.
Activation or inhibition of existing malic enzyme has been virtually eliminated as as a possible explanation for the observed changes in total enzyme activity.
However, this conclusion is subject to the reservation that neonatal chick liver does not contain a form of malic enzyme which is enzymatically inactive and immunologically unreactive.
To our knowledge, no such form of malic enzyme has been described or postulated.
Degradation of malic enzyme appeared to play a minor role in controlling the concentration of malic enzyme. The rate of degradation (tg) for liver malic enzyme was 55 hours in normally fed birds. This rate of degradation is much slower than degradation rates of enzymes such as tryptophan pyrrolase (t+ = 2) hours) or tyrosine aminotransferase (t+ = 2 hours) (20, 24) but about the same as that reported for acet.yl-CoA carboxylase in fed rats (t, = 48 to 59 hours) (22,25). The degradation of acetyl-CoA carboxylase is increased by starvation (t+ = 18 to 30 hours) (22,25) but neither Majerus and Kilburn nor Nakanishi and Numa (22,25) indicated whether or not this was specific for malic enzyme or general for all liver proteins.
Malic enzyme in starved chicks was degraded with a t+ of 28 hours but much of the increased breakdown was due to the increased rate of breakdown of all liver proteins. The extremely long halflife of malic enzyme found in the liver of unfed, neonatal chicks (tq = 350 hours) is grossly similar to the finding that arginase is not degraded in livers from fasted rats (19). However, the biological significance of such a low turnover rate for malic enzyme is difficult to assess. It may represent a means of conserving energy during embryonic life, as the potential for adaptive regulation of this enzyme would appear to be of little functional value to the embryo.
Under steady state conditions it is possible to calculate a rate constant for enzyme synthesis (26). In our experiments, steady state conditions were approximated in the unfed, neonatal chicks and in the g-day-old chicks. By means of the enzyme activities actually measured (0.28 and 25 units ner livctr for neo-