THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 244, No. 20, Issue of October 25, pp. 6626-6630, 1969

Printed in U.S.A.

Purification of Phosphoenolpyruvate Carboxykinase from the

Cytosol Fraction of Rat Liver and the Immunochemical Demonstration of Differences between This

Enzyme and the Mitochondrial Phosphoenolpyruvate Carboxykinase*

(Received for publication, May 16, 1969)

F. J. BALLARD~ AND R. W. HANSON

From the Fels Research Institute and the Department of Biochemistry, Temple Univer&y fkhool of Medicine, Philadelphia, Pennsylvania 19140

SUMMARY

Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) has been isolated and purified from the cytosol fraction of rat liver to give a specific activity of 14 to 16 moles of bicarbon- ate tied per min per mg of protein. The enzyme is homo- geneous on disc electrophoresis and Sephadex G-100 chro- matography and has a molecular weight of 74,500 with an isoelectric point of 5.04. Antibodies prepared in rabbits against the pure enzyme show identical titration curves against the cytosol P-enolpyruvate carboxykinase of liver and adipose tissue. A single continuous precipitation line is obtained on Ouchterlony double dausion precipitation analy- sis when pure enzyme and the cytosol P-enolpyruvate car- boxykinase of liver and adipose tissue are compared. On the other hand, the solubilized mitochondrial P-enolpyruvate carboxykinase of rat liver is not precipitated by the antibody to the cytosol enzyme. We conclude that the mitochrondrial and cytosol P-enolpyruvate carboxykinases are distinct enzymes, notwithstanding their uniform kinetic and physical properties.

Phosphoenolpyruvate carboxykinase occurs in both the cytosol and mitochrondria of liver, although the distribution of enzyme between these two cell compartments varies from one species to another. In livers from pigeons and chickens, P-enolpyruvate carboxykinase appears to be completely localized in the mito- chrondria (1, 2) whereas in the rat and mouse almost all the ac- tivity is found in the cytosol (2). Most other species that have

* This research was supported in part by Grants HD-02758, CA-07174, and AM-11279 from the National Institutes of Health, and Grant P202 from the American Cancer Society.

$ Present address, Commonwealth Scientific and Industrial Research Organization, Division of Nutritional Biochemistry, Adelaide, South Australia 5000.

been tested, such as guinea pigs, rabbits, sheep (2, 3), cows,’ and humans,’ have significant activities in both cellular fractions of liver. In those animals where the effects of diet and hormonal changes on hepatic P-enolpyruvate carboxykinase have been tested, only the cytosol enzyme is adaptive, and this act#ivity alters in the same direction as the changes in over-all gluconeo- genesis (1, 4, 5). Furthermore, fetal livers from both rats and guinea pigs, which do not carry out gluconeogenesis, contain only the mitochondrial P-enolpyruvate carboxykinase (6), while the cytosol P-enolpyruvate carboxykinase’ activity appears at birth simultaneously with the development of the gluconeogenic path- way (6). This finding suggested to us that the mitochrondrial and cytosol activities of P-enolpyruvate carboxykinase were separate enzymes. We report in this paper the isolation, in homogeneous form, of cytosol P-enolpyruvate carboxykinase from rat liver and the investigation of the immunochemical properties of antibodies prepared against this enzyme with re- spect to mitochondrial and cytosol P-enolpyruvate carboxy- kinases.

EXPERIMENTAL PROCEDURE

&fateriaZsP-enolpyruvate and malate dehydrogenase (EC 1.1.1.37) were obtained from Boehringer; dithiothreitol was from Calbiochem, and IDP and NADH were from P-L Bio- chemicals. Enzyme grade ammonium sulfate from Mann was used for all ammonium sulfate fractionations. Type 52 DEAE- cellulose was a product of Whatman and was equilibrated by washing several times and removing fines in 0.05 M Tris-chloride, pH 8.0. Sephadex G-100 and G-25 were obtained from Phar- macia. The G-100 gel was swollen for several days in 0.05 M

Tris-chloride, pH 8.0, and fines were removed by washing in the same buffer. Calcium phosphate gel obtained from Bio-Rad (Richmond, California) was used without treatment. Ampho- lines with pH ranges of 3 to 10,4 to 6, and 5 to 8 were purchased from LKB Corporation. 14C-Labeled sodium bicarbonate with low specific activity was obtained from New England Nuclear,

1 F. J. Ballard, unpublished observations.

5625

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5626 Cytosob Phosphoenolpyruvate Carboxyykinase Vol. 244, No. 20

Assay of P-enolpyruvate Carboxykinase-The assay used in all experiments was essentially that reported by Chang and Lane (7) for the mitochondrial P-enolpyruvate carboxykinase. The bicarbonate fixation mixture contains 100 mM imidazole-chloride, pH 6.6; 2 mu MnC12; 15 rnM P-enolpyruvate; 1.25 mu IDP; 50 mM KHCOa (containing 2 PC of NaH14COa) ; 2 units of malate dehydrogenase; 1 MM dithiothreitol; 2.5 mu NADH; and enzyme. The total volume was 1.0 ml. The final pH was between pH 6.9 and 7.2. The activity of P-enolpyruvate carboxykinase as measured by this assay procedure did not vary significantly across this pH range. Assays were routinely carried out for 10, 20, and 30 min at 37”, and the reactions were stopped by addi- tion of trichloracetic acid to a final concentration of 4%. After each reaction tube was gassed with COz for 3 min, the radioac- tivity was determined with Diotol as solvent (8) in a Nuclear- Chicago liquid scintillation spectrometer. Units of enzyme ac- tivity are defined as micromoles of bicarbonate fixed per mm, at 37”. Under the conditions used the assay showed propor- tionality with time and with enzyme concentration up to 0.003 unit of activity.

x .c- 2 t 0 v)

C c 3 E

PI antibody preparation

FIG. 1. Titration of cytosol P-enolpyruvate carboxykinase against control (wm) or test (O-O) antibody preparations. Experimental details are given in the text. Enzyme activity is expressed as millimicromoles of bicarbonate fixed at 37” per min.

TABLE I Purification of cytosol P-enolpyruvate carboxykinase

Further details of this purification from livers of 30 fasted rats are given in the text.

Stage -~

fw units units/?ng

1. 100,000 X g supernatant . . . . . . . 22,500 950 0.042 2. (NH&SO4 precipitate (4S65%) . 5,150 708 0.138 3. Sephadex G-100 chromatography.. 1,720 492 0.286 4. DEAE-cellulose chromatography.. 159 336 2.11

5. Cas(PO& gel adsorption and elution . . . . . . . . . . . . . . . 38 201 5.3

6. Electrofocusing pH 5-8.. . . . . . . . . . 6.4 102 15.9

Protein Measurements--Protein was measured by the ultra- violet method of Warburg and Christian (9).

Ouchterlony Double Difusion Patterns-Thin layer Ouch- terlony double diffusion precipitation analysis of P-enolpyruvate carboxykinase antibody was carried out on microscope slides. Approximately 5 ml of a solution containing 1% Nobles agar in 0.02 M KH2P04, pH 7.4, with 0.1 M KC1 were layered onto a standard microscope slide. A center well 4 mm in diameter was filled with approximately 0.02 ml of antibody to P-enolpyruvate carboxykinase. The outer wells contained 0.02 ml of either pure enzyme or other fractions to be tested. A 10% (w/v) homogenate of liver from normal rats and a 30% homogenate of adipose tissue from fasted rats were prepared with 0.25 M buffered sucrose (10). The supernatants obtained after cen- trifugation for 30 min at 105,000 x g were used undiluted for the Ouchterlony double diffusion analysis. The precipitation reaction was completed after 24 hours at room temperature in a humidified chamber.

Preparation of Antibodies against Cytosol P-enolpyruvate Car- boxykinase-Approximately 2 mg of purified enzyme were mixed with equal volumes of Freunds adjuvant and injected subcuta- neously into a rabbit. Additional injections of enzyme were made over a l-month period, after which blood was withdrawn by heart puncture. Serum was obtained and y-globulin was isolated by precipitation with 50% saturation of ammonium sulfate. This precipitate was washed with 50% saturated am- monium sulfate, dissolved in half the original volume of 0.15 M NaCl and dialyzed for 2 days against 0.15 M NaCl. y-Globulin from control serum was isolated from rabbits injected with Freunds adjuvant and 0.15 M NaCl but without enzyme.

These antibodies were titrated against a cytosol fraction of rat liver to determine potency (Fig. 1). Variable dilutions of anti- body were mixed with a constant amount of liver cytosol, incu- bated at 37” for 15 mm, and left overnight at 04”. This solu- tion was centrifuged at 700 x g for 15 min and the resulting supernatant was assayed for P-enolpyruvate carboxykinase activity. Since the antibody-antigen precipitate was enzymati- tally active, care was taken to avoid taking any of the precipi- tate for enzyme assays. It can be seen from Fig. 1 that about 97 y. of the P-enolpyruvate carboxykinase was precipitated under these conditions by the antibodies to the pure enzyme, while none was precipitated by control antibodies.

RESULTS

Purification of P-enolpyruvate Carboxykinase-All steps in the purification were carried out at O-4” as preliminary experiments had indicated that the enzyme was unstable when higher tem- peratures were used. The purification procedure as summarized in Table I lists recoveries obtained in a particular purification. Although the over-all recovery of enzyme was never greater than 15yo, most of the loss of activity could be accounted for in dis- carded fractions. Only in the final electrofocusing step was separation of activity into more than one peak noted.

Preparation of Liver Supernatant-Thirty Wiitar rats weighing about 400 g were starved overnight prior to killing. This treat- ment resulted in a 3-fold induction of the cytosol P-enolpyruvate carboxykmase activity without increasing the activity of the mitochondriil enzyme. Livers of animals fasted in this way contained 5 to 7 units of cytosol and 0.2 to 0.3 unit of mito- chondrial P-enolpyruvate carboxykinase activity per g of tissue.

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Issue of October 25, 1969 F. J. Ballard and R. W. Hanson 5627

Livers were homogenized in 2 volumes of 0.25 M buffered sucrose with a coaxial homogenizer and centrifuged at 100,000 X g for 1 hour to obtain 450 ml of clear supernatant.

Ammonium Sulfate Fractionation-Suflicient solid ammonium sulfate was added to the supernatant from the previous step to bring the solution to 45% saturation (at 25”). It was usually necessary to add some 1 M Tris during this fractionation to main- tain the pH of the enzyme solution at 7.0. After standing for 15 min the suspension was centrifuged at 5000 x g for 15 min, and the precipitate was discarded. Additional solid ammonium sulfate was added to increase the percentage saturation (at 25”) of the solution to 65%, and after standing for 15 min this suspen- sion was centrifuged as before. The precipitate was dissolved in 0.05 M T&-chloride, pH 8, to give a volume of approximately 75 ml.

Sephadex G-100 Chromatography-Seventy-five milliliters of enzyme from the ammonium sulfate fractionation were placed on a column of Sephadex G-100 (30 sq cm x 45 cm) that had previously been equilibrated with 0.05 M T&-chloride, pH 8. The enzyme was eluted with the same buffer at a flow rate of 4 ml per min.

DEAE-cellulose Chromatography-The fractions of highest specific activity from the Sephadex chromatography were com- bined, giving a total volume of about 130 ml, and applied to a DEAE-cellulose column (3 sq cm x 20 cm) previously equili- brated with 0.05 M T&-chloride, pH 8. The enzyme was eluted from the columns with 450 ml of linear gradient of 0 to 0.15 M

NaCl in 0.05 M Tris-chloride, pH 8, at a flow rate of 2 ml per min. Under these conditions P-enolpyruvate carboxykinase was eluted at a sodium chloride concentration of about 0.1 M. Although the degree of puri6cation was not significantly altered, more complete separation of P-enolpyruvate carboxykinase from hemoglobin could be obtained by carrying out this DEAE- cellulose chromatography at pH 7 rather than pH 8 since at the lower pH hemoglobin was not retained on the cellulose.

Calcium Phosphate Gel Titration-The combined fractions from DEAE-cellulose chromatography (60 ml; At&Q were treated with sufficient calcium phosphate gel to reduce the absorbance at 280 rnp to 35% of the initial value. This was accomplished by sequential addition of small amounts of gel, allowing the solution to stand for 5 min and centrifuging at 700 x g for 5 mm. Ap- proximately 15 ‘% of the starting P-enolpyruvate carboxykinase was not adsorbed by this treatment. The gel with adsorbed enzyme was washed 3 times with 0.025 M potassium phosphate, pH 6.6, resulting in the removal of a further 15% of the P-enol- pyruvate carboxykinase and at least 50% of the adsorbed protein. This enzyme was discarded. Three additional lo-ml elutions with 0.1 M potassium phosphate, pH 7.0, removed the remaining enzyme in a volume of 30 ml with a resulting specific activity of 5.0 to 6.0 units per mg of protein.

Akernatiue Ammonium Sulfate Extraction-In some experi- ments the fractions eluted from DEAE-cellulose were brought to 70% saturation with solid ammonium sulfate and extracted sequentially with 10 ml each of solutions that were 60, 58, 54, and 52% with respect to ammonium sulfate. The percentages refer to saturation at 25”. It was found that at least 80% of the P-enolpyruvate carboxykinase was obtained in the 58, 54, and 52% saturation solutions, and the specific activity attained was comparable to the calcium phosphate gel titration. Al- though this method was more rapid and resulted in a greater

yield of P-enolpyruvate carboxykinase, it was not found suitable if electrofocusing was to be the subsequent purification step since the large amount of ammonium sulfate was difEicult to remove.

EZectTojocusing-Thirty to forty milliliters of enzyme from the calcium phosphate or ammonium sulfate steps were deionized by passage through a 350-ml column of Sephadex G-25 which had been equilibrated with 0.02 M Tris-chloride, pH 7.5. All fractions containing protein were combined and applied to an LKB electrofocusing column (450 ml) which had been poured in g-ml fractions according to the manufacturer’s directions. The pH 5 to 8 gradient was stabiliied with a sucrose density gradient. The use of a mixer for preparation of the density gradient was not satisfactory as this resulted in considerable dilution of the enzyme and loss of activity of the P-enolpyruvate carboxykinase. The prepared column was cooled by passing water at 2” through the cooling compartments and the current set to give an initial amperage of 8 to 10 at 500 volts. After electrofocusing for 36 hours the amperage at constant voltage had dropped to 2. Frac- tions were drained from the column and analyzed for pH, protein, and enzyme activity (see Fig. 2). Those fractions of constant and high specific activity were combined and deionized by pas- sage through Sephadex G-25 that had been equilibrated with 0.05 ml of Tris-chloride, pH 8. This eluent was concentrated by adsorption on a Z-ml column of DEAE-cellulose followed by elution with 0.5 M NaCl in 0.05 M Tris-chloride, pH 8. In thii way the enzyme was obtained in a volume of about 1.5 ml at a concentration of 4 mg of protein per ml.

Tests of Homogeneity-P-enolpyruvate carboxykinase obtained by the above purification procedure was found to have constant specific activity in all protein-containing fractions upon chroma- tography on a Sephadex G-100 column. Polyacrylamide disc gel electrophoresis at pH 8.6 indicated a single protein band,

fraction number

FIG. 2. Elution pattern of P-enolpyruvate carboxykinase from a pH 5 to 8 electrofocusing column. Enzyme activity is given in units per ml and specific activity in micromoles of bicarbonate fixed at 37” per min per mg of protein, after subtracting a slight blank absorbance at 280 rnN from the indicated absorbance. This absorbance, due to ampholytes, had been determined in a separate electrofocusing experiment when protein was omitted.

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5628 Cytosol Phosphoemlpyruvate Carboxykinase Vol. 244, No. 20

although slight contamination by low molecular weight and highly charged material was always seen. As this material was only obtained after electrofocusing, it is assumed that it consists of peptides from the ampholine column. Attempts to elute the protein and measure enzyme activity from sliced, unstained disc gels were only partially successful since only 10% or less of the P-enolpyruvate carboxykinase activity was recovered. The position of this activity on the disc gel coincided with the protein band.

As can be seen from Fig. 2, a second peak of P-enolpyruvate carboxykinase was focused at slightly higher pH. We believe that this second peak is due to the binding of peptides by the enzyme. Substantially greater proportions of enzyme were found in this second peak if a gradient mixer was used to pre- pare the electrofocusing column (enzyme in the second of the two compartments in the mixer) or if the pH 4 to 6 ampholyte solu- tion was used. When a greater fraction of enzyme was .ob- tained in this second peak, the total recovery of enzyme from the column was lower than the 80% found by the technique re-

TABLE II

Properties of puri$.ed P-enolpyruvate carboxykinase The molecular weight was determined by chromatography on

Sephadex G-100 columns (11). Enzyme stability studies were carried out as described in the text. The isoelectric point was determined by the electrofocusing technique. Values are the means of the number of determinations in parentheses. Standard errors are given for the molecular weight and isoelectric point.

Molecular weight, Sephadex G-100 74,000 f 4,000 (6) Enzyme stability at 2”, percentage after

1 month 97 (3) 2 months 90 (3) 3 months 86 (3)

Isoelectric point, cytosol enzyme 5.04 f 0.05 (8) Isoelectric point, mitochondrial enzyme 4.9, 5.1

40- / 8 / .

.’ V

. /O

20- do A .

, /O

too d ova WV, ‘A I

20 40 60

m units added

FIG. 3. Titration of an antibody preparation against P-enol- pyruvate carboxykinase from different sources. l , mitochondria from fetal liver; 0, mitochondria from livers of fasted, refed adult animals; n , cytosol from normal liver; 0, cytosol from epididymal adipose tissue of normal animals; A., cytosol from livers of fasted, refed animals; v, cytosol from epididymal adipose tissue of fasted animals; v, purified cytosol liver enzyme. Activity is ex- pressed as milliunits of bicarbonate fixed at 37” per min. Anti- body, 30 ~1, was used for each experiment. Other details are given in the text.

ported in the procedure for enzyme purification. Additional evidence that this second activity peak is due to binding of ampholines rather than an isozyme of P-enolpyruvate carboxy- kinase was obtained by mixing the two activity peaks from a purification in which the second activity peak had a similar high specific activity to the main peak. When this mixture was applied to polyacrylamide gel electrophoresis, a single protein band was formed.

Properties of Puri$ed EnzymeThe purified enzyme was stable for several months at 2” when present as a suspension in 70% saturated ammonium sulfate so long as EDTA and dithiothreitol were present (Table II). Purified cytosol P-enolpyruvate car- boxykinase had a molecular size of 74,000 when determined on Sephadex G-100. The proteins used to standardize the Sephadex G-100 column were bovine thyroglobulin, chicken liver lactate dehydrogenase, yeast hexokinase, liver alcohol dehydrogenase bovine hemoglobin, and horse heart cytochrome c. The respec- tive elution volumes relative to the void volume of the column (V,/Vo) were 1.0, 1.12, 1.24, 1.35, 1.48, and 2.42. The V,/VO for P-enolpyruvate carboxykinase was 1.40. The enzyme had an isoelectric point of 5.04.

In order to check whether the mitochondrial P-enolpyruvate carboxykinase from rat liver had an obvious difference in iso- electric point from the cytosol enzyme, two purifjcations of mitochondrial P-enolpyruvate carboxykinase were attempted. Freeze-drying was used to solubilize the enzyme, and ammonium sulfate and Sephadex G-100 steps were carried out as described in the purification procedure for the cytosol enzyme. Following these steps the enzyme was placed on the electrofocusing column with the pH 5 to 8 ampholines. As shown in Table II, the mito- chondrial enzyme also had an isoelectric point of about pH 5.

Experiments with Antibodies to Cytosol P-enolpyruvate Car- boxykinase-Constant amounts of antibody were incubated, as described previously, with a portion of cytosol from normal liver and livers from rats that had been fasted for 1 day and refed for 2 days. A constant activity of P-enolpyruvate carboxykinase was precipitated by the antibody, and beyond this amount of enzyme the P-enolpyruvate carboxykinase appeared quantita- tively in the supernatant (Fig. 3). P-enolpyruvate carboxyki- nase in cytosol fractions of adipose tissue from either fasted or normal rats as well as pure enzyme also showed a similar titra- tion curve.

Washed mitochondria of fetal liver or livers from fasted-refed adults were prepared and freeze-dried. The freeze-dried mito- chondria were suspended in water and centrifuged at 100,000 X g for 30 min, and the supernatant was obtained. To test that mitochondrial P-enolpyruvate carboxykinase was solubilized by these procedures, we passed a portion of the extracts through Sephadex G-100 columns. In all cases the eluted activity ap- peared as a symmetrical peak with a molecular weight in the range of 65,000 to 80,000. The mitochondrial extracts were tested against the antibody to the cytosol P-enolpyruvate car- boxykinase; clearly, no enzyme was precipitated (Fig. 3).

Ouchterlony double diffusion experiments were carried out against cytosol P-enolpyruvate carboxykinase from fasted rat liver. Since a nonspecific precipitation line was evident in most cases, the antibody was fractionated by mixing it (1 :l) with cytosol prepared from a 10% homogenate of liver from a rat that had been fasted for 1 day and refed for 2 days. This sus- pension was incubated at 37” for 15 min, allowed to stand over- night, and centrifuged at 700 x g for 20 min. The supernatant

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Issue of October 25, 1969 F. J. Ballard and R. W. Hanson 5629

FIG. 4. Ouchterlony double diffusion analysis of P-enolpyru- vate carboxykinase antibody of rat liver cytosol. The center well cont,ained 0.02 ml of P-enolpyruvate carboxykinase antibody (Ab) and the outer wells contained 0.02 ml of the following: A, 10% liver cytosol; B, 30y0 adipose tissue cytosol; C, 10% liver cytosol; D, 30yo adipose t,issue cytosol; E, pure P-enolpyruvate carboxy- kinase from rat liver cytosol; F, 10% liver cytosol. The prepara- tion of the tissue extracts and treatment of the antibody are out- lined under “Experimental Procedure.” The patterns were allowed to develop for 24 hours prior to photography. The edges of t,he Ouchterlonywells are the outer white margins.

obtained in this manner had only slightly less potency when titrated against P-enolpyruvate carboxykinase, since very little cytosol P-enolpyruvate carboxykinase is present in livers of fasted-refed animals (12). The Ouchterlony double diffusion patterns obtained with this antibody preparation show a single continuous precipitation line against the cytosol P-enolpyruvate carboxykinase from liver, adipose tissue, and the pure enzyme (Fig, 4).

DISCUSSION

A comparison between the kinetic properties of P-enolpyru- vate carboxykinase in cytosol and mitochondrial fractions of guinea pig liver made by Holten and Nordlie (13) indicated only slight differences between the enzyme from these two fractions. The enzymes were found to have similar Michaelis constants for oxalacetate and ITP when assayed in the direction of P-enolpy- ruvate formation, and similar Michaelis constants for P-enol- pyruvate and IDP when the carboxylation assay was used: Both enzymes showed absolute specificity for Mn* rather than Mg* for the carboxylation of P-enolpyruvate. However, comparative inhibition studies of purified mitochondrial and cytosol P-enol- pyruvate carboxykinase of guinea pig liver made by Holten and Nordlie did indicate difference between the enzymes. With either ITP or GTP as phosphoryl donor, 1.33 mM AMP caused an inhibition of the mitochondrial but not the cytosol enzyme. Another difference noted was a variation in the relative levels of stimulation by Mn2f and Mg* of P-enolpyruvate production

catalyzed by the mitochondrial and cytosol enzymes. But, because of the marked similarities in fundamental catalytic properties, it was suggested that the two enzymes in guinea pig liver were variants of the same enzyme protein.

In an earlier study of the developmental pattern of P-enolpy- ruvate carboxykinase in rat liver we noted that, in the 17-day fetus, 90% of the activity of the enzyme is mitochondrial (6). The cytosol enzyme increases, from an almost undetectable level of activity, to 2.6 units per g within the first 24 hours after birth. During the same period the activity of mitochondrial P-enol- pyruvates carboxykinase drops from 0.38 unit per g to 0.25 unit per g. Although these developmental patterns of the two en- zymes were very different, we found no differences in the Michae- lis constant for P-enolpyruvate, the pH optimum, the molecular weight, or relative velocities with IDP and GDP. We were also unable to detect any difference in isoelectric point between the two enzymes. A comparison between the purified P-enol- pyruvate carboxykinase from guinea pig liver mitochondria (7) and rat liver cytosol indicates a close similarity in final specific activity and molecular size. Chang and Lane (7) reported a final specific activity of about 9 compared to 14 to 16 in this report, while the molecular sizes were 73,300 and 74,000, re- spectively.

Despite the similarities in properties of the P-enolpyruvate carboxykinases from mitochondria and cytosol of both rat and guinea pig livers, the control of the enzymes in the two cell compartments must be different since only the cytosol enzyme is adapative to hormonal and dietary alterations (1, 4, 5). The present antibody experiments indicate that the antigenic prop- erties of the cytosol enzyme are qualitatively different from those of the mitochondrial enzyme. This was true both for the mito- chondrial enzyme from fetal rat liver, where the negligible ac- tivity of cytosol P-enolpyruvate carboxykinase (6) makes contamination of the mitochondrial fraction impossible, and for the mitochondrial enzyme from the livers of fasted-refed adult rats. The lack of any precipitation of mitochondrial P-enol- pyruvate carboxykinase by antibody specific to the purified cytosol enzyme is compelling evidence that the two enzymes are immunologically distinct proteins.

Previous work has established the existence of a cytosol P-enol- pyruvate carboxykinase in adipose tissue (14, 15). The activity of this enzyme increases 5-fold after a 24-hour fast, and this increase can be blocked by injection of actinomycin D. Pre- liminary studies have shown that rat adipose tissue P-enolpyru- vate carboxykinase activity is elevated in diabetes and by adre- nalectomy and that the activity is reduced to near normal values by injection of insulin or triamcinolone (16, 17). This adaptive response of the enzyme to dietary and hormonal treatments is similar to those noted in liver by Lardy and his co-workers (4, 18). It is of interest, therefore, that antibody titration ex- periments indicate that P-enolpyruvate carboxykinases from cytosol of rat liver and adipose tissue are immunochemically similar proteins.

These findings of two different forms of P-enolpyruvate car- boxykinase in liver imply that both enzymes have important functions in the intermediary metabolism of the liver cell. While it is certain that in pigeon liver the mitochondrial enzyme is involved in gluconeogenesis and in rat liver the cytosol enzyme is involved, we are unable to decide on the respective functions of the cytosol and mitochondrial P-enolpyruvate carboxykinase in tissues in which both enzymes are present in large amounts.

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5630 Cytosol Phosphoemlpyruvat Carboxykinase Vol. 244, No. 20

A&nowtedgments--We wish to thank Miss Joann Varga and Mrs. Linda Grengle for technical assistance. We are indebted to Mr. Ronald Wieder for taking the photographs used in this paper, and to Dr. N. Yamamoto for his invaluable advice and assistance with the immunological studies.

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F. J. Ballard and R. W. HansonThis Enzyme and the Mitochondrial Phosphoenolpyruvate Carboxykinase

of Rat Liver and the Immunochemical Demonstration of Differences between Purification of Phosphoenolpyruvate Carboxykinase from the Cytosol Fraction

1969, 244:5625-5630.J. Biol. Chem. 

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