[28] Isozymes of pyruvate kinase from the grassfrog

166

KINASES

[28]

The kinetic characterization of the pyruvate kinase of B. licheni]ormis, conducted with a constant assessment of the effects of reaction conditions on enzyme stability, reveals the following properties. 2 Catalysis by this enzyme proceeds optimally at pH 7.0-7.4. The initial rate of catalysis is modulated by substrate activation (both PEP and ADP), activation by AMP, and inhibition by ATP, Pi and carbamyl phosphate. Positive cooperatively is manifested by PEP, ADP, Pi and ATP in the presence of Mg(II) ions. AMP relieves the inhibition by ATP, yielding hyperbolic saturation curves for both substrates. The apparent substrate Km values for the fully activated enzyme, 0.2 mM for PEP, and 0.7 mM for ADP, in the presence of Mg(II) are independent of the concentration of the second substrate. In the presence of Mn(II) as the required divalent cation, the Km for PEP is 7-fold lower than that obtained with the Mg(II)-activated enzyme. Finally, the inhibition of catalysis by ATP in the presence of Mn(II) is not reversed by AMP and does not effect the hyperbolic nature of the PEP saturation curve observed under these conditions. Thus, the K-type 1° allosteric properties manifested in the presence of Mg(II) are obliterated when Mn(II) is employed as the activating divalent cation. loj. Monod, J. Wyman, and J.-P. Changeux, J. Mol. Biol. 12, 88 (1965).

[28] Isozymes of Pyruvate Kinase from the Grassfrog 1 B y L. E. FLANDERS, L. H. SCHLOEN, and H. J. SALLACH Phosphoeno/pyruvate + ADP ~ pyruvate + ATP

The occurrence of multiple forms of pyruvate kinase in different tissues of the rat was first reported by Tanaka et al. 2,3 These investigators detected from one to four isozymes in a given tissue as did Susor and Rutter. 4 Early work with human tissues demonstrated three different isozymes. ~ On the other hand, work in this laboratory 6 demonstrated that 1This work was supported by Research Contract No. AT(ll-1)-1631 from the U.S. Atomic Energy Commission and by National Institutes of Health Grant No. NS10287. T. Tanaka, Y. Harano, H. Morimura, and R. Mori, Bioehem. Biophys. Res. Commun. 21, 55 (1965). 8 T. Tanaka, Y. Harano, F. Sue, and H. Morimura, J. Biochem. (Tokyo) 62, 71 (1967). W. A. Susor and W. J. Rutter, Biochem. Biophys. Res. Commun. 30, 14 (1968). 5 R. H. Bibley, P. Stanzel, R. T. Jones, J. P. Campas, and R. ]5. Koler, Enzyme Biol. CIin. 9, 10 (1968). 8L. H. Schloen, J. R. Bamburg, and H. J. SaUach, Biochem. Biophys. Res. Commun. 36, 823 (1969).

[28]

AMPHIBIAN PYRUVATE KINASE ISOZYMES

167

five isozymes of pyruvate kinase are found in a given cell type, e.g., the unfertilized frog egg, as well as in certain tissues of the adult grassfrog, R a n a pipiens. Subsequent studies carried out in a number of laboratories have demonstrated multiple forms of pyruvate kinase in a variety of tissues from a number of different species (for further discussion and references, the reader is directed to recent articles 7-'* on this subject). On the basis of electrophoretic, kinetic, chromatographic, and immunological data, 7,s there appear to be three unique forms of pyruvate kinase in animal systems; using the nomenclature of Tanaka and associates, 7,8 these are type L (major liver form), type M1 (the only form in skeletal muscle), and type M2 (predominant fetal form but found in certain adult tissues). Unique kinetic properties that have been reported for the three isozymes include the following. Although there are quantitative differences, both types L and M~ show cooperativity with respect to phosphoenolpyruvate (PEP) concentration, inhibition by ATP or alanine, and activation by fructose 1,6-diphosphate (FDP) which lowers the apparent K,1 and shifts PEP kinetics from sigmoidal to hyperbolic. However, the two types differ in that inhibition of type L by either ATP or alanine is reversed by the addition of FDP whereas that of type M2 is not. Both types appear to exist in sensitive and desensitive forms with respect to the effects of modulators. On the other hand, type M1 displays hyperbolic kinetics with respect to PEP concentration either in the presence or in the absence of FDP. Each of the three isozymes can be distinguished on the basis of their electrophoretic mobilities. Type L has been shown to be under dietary and hormonal control.: With respect to the isozymes of pyruvate kinase in the grassfrog, present data 1°,11 indicate that the liver (major form), cardiac and skeletal muscle isozymes, the purification of which is described below, are similar in their properties to types L, M~, and M1 of the rat, respectively.

Assay Method Principle. The assay method is essentially that described by Biicher and Pfeiderer ~2 and the reader is referred to a discussion of the spectrophotometric assay in which the pyruvate kinase reaction is coupled with that of lactate dehydrogenase (Vol. 1 [66]).

7K. Imamura and T. Tanaka, J. Biochem. (Tokyo) 71, 1043 (1972). s K. Imamura, K. Taniuchi and T. Tanaka, J. Biochem. (Tokyo) 72, 1001 (1972). ~J. Osterman, P. J. Fritz, and T. Wuntch, J. Biol. Chem. 248, 1011 (1973). loL. E. Flanders, J. R. Bamburg, and H. J. Sallach, Biochim. Biophys. Acta 242, 566 (1971). 11L. I-I. Schloen and It. J. Sallach, unpublished observations. "0T. Bficher and G. Pfleiderer, this series, Vol. 1 [66].

168

KINASES

[28l

Reagents. For routine assays, the composition of the assay mixture is: 50 m M Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, p H 7.4; 0.46 m M A D P ; 0.77 m M P E P ; 0.15 m M N A D H ; 10 m M MgC12; 100 m M KC1; 5 m M dithiothreitol; and 5 units of lactate dehydrogenase. 13 When F D P is included in the incubation system, it is added as an aqueous solution of the tetracyclohexylammonium salt to a final concentration of 0.5 m M ; the same activation is achieved with the sodium salt. Procedure. The reagents are pipetted into a cuvette (1-cm light path) which is placed in the spectrophotometer for temperature equilibration at 15 ° . The t e m p e r a t u r e is very important for cardiac and liver enzymes and should not exceed 15°; the muscle enzyme can be assayed at room temperature. The reaction is initiated by the addition of p y r u v a t e kinase (25-90 u n i t s ) l ' and is followed by measuring the change in absorbaney at 340 nm with a B e c k m a n DU-Gilford 2000 recording spectrophotometer. T h e control cuvette contains all components except P E P . Definition o/ Unit and Specific Activity. One unit is defined as t h a t amount of enzyme t h a t produces a change in absorbancy of 0.001 per minute under the above conditions. Specific activity is defined as the number of units per milligram of protein as determined by the method of L o w r y et al. 1~ with bovine serum albumin as the standard or from the absorbancy at 280 nm according to the method of W a r b u r g and Christian. 1~

Purification Procedures The following general conditions are used unless stated otherwise. All steps are carried out at 0-4 °. Fractionations with a m m o n i u m sulfate are made b y the slow addition, with stirring, of the calculated amount of the solid salt. The resulting suspensions are equilibrated with stirring for 15 rain prior to eentrifugation. Centrifugations are carried out at 14,000 g for 30 rain. All a m m o n i u m sulfate residues are dissolved in the 1~The lactate dehydrogenase (Sigma, Type II) is in an ammonium sulfate suspension. A 1:250 dilution of the stock suspension into 25 mM tris(hydroxymethyl)aminomethane buffer, pH 7.4, prior to use in the assay is sufficient both to perform the coupling of pyruvate to lactate and dilute out the ammonium sulfate present. ~' The pyruvate kinase can be assayed directly from any of the t~urifieation steps except the ammonium sulfate fractionation; dialysis is used to eliminate the ammonium sulfate or dilution greater than 1:250 as with the lactate dehydrogenase. 1~O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). ~60. Warburg and W. Christian, Biochem. g. 310, 384 (1941). See also this series Vol. 3 [73].

[28]

AMPHIBIAN PYRUVATE KINASE ISOZYMES

169

appropriate buffer for the given purification step and are dialyzed against the same buffer until free of ammonium ions as measured by Nessler's reagent. CM- and DEAE-Sephadex ion exchange resins are used in column chromatography and are pretreated according to the directions supplied by Pharmacia. The final equilibration is carried out in the buffer to be used in the given chromatographic step. Protein in effluent solutions from columns is detected by absorbancy at 280 nm. Enzymic solutions are concentrated by ultrafiltration (Diaflo ultrafiltration cell, Amicon Corp., Lexington, Massachusetts). Grassfrogs, Rana pipiens, are purchased from Mogel-Ed Corp., Oshkosh, Wisconsin.

Purification o] Skeletal Muscle Pyruvate Kinase TM Step 1. Crude Extract. One gram of muscle from the hind legs is homogenized in a Waring Blendor for 1 rain in 5 ml of homogenizing buffer consisting of 0.25 M sucrose, 25 mM Tris.HC1, 2.5 mM EDTA, and 1 mM 2-mercaptoethanol (pH 7.5). Celhdar debris is removed by centrifugation and discarded. Step 2. First Ammonium Sulfate Fractionation. Ammonium sulfate (45 g/100 ml) is added to the supernatant solution from the above step. The 0-45% ammonium sulfate residue is removed by centrifugation and, to the resulting supernatant solution, ammonium sulfate (15 g/100 ml) is added. The 45-60% ammonium sulfate fraction is recovered by centrifugation and can be stored at --20 ° for 6 months with minimal loss in activity. Step 3. CM-Sephadex Chromatography. The ammonium sulfate precipitate from the above step is dissolved in 0.04 imidazole acetate buffer, pH 7.4, containing 1 mM EDTA and 10 mM 2-mercaptoethanol, using a volume of buffer which results in a protein concentration of 10 mg/ml. The dialyzed fraction is then applied to a CM-Sephadex column which is equilibrated with the above buffer; a bed volume of 1 liter of resin is used for each 0.8 g of protein. After the protein solution has run into the column bed and the latter is washed with 50 ml of buffer, a linear grad:ent of 0 to 0.15 M KC1, dissolved in column buffer, is applied. A total volume of 2 liters is used for a resin bed volume of 1 liter. The fractions comprising the peak of pyruvate kinase activity are pooled and concentrated by ultrafiltration to give a protein concentration of 2.5 mg/ml. Step 4. Second Ammonium Sulfate Fractionation. The concentrated enzyme solution from the above step is dialyzed for 48 hr against a solution of ammonium sulfate containing 52 g/100 ml to precipitate pyruvate

170

KINASES

TABLE

[28]

I

PURIFICATION DATA FOR CARDIAC AND SKELETAL MUSCLE PYRUVATE KINASES

Fraction Skeletal muscle a 1. Crude extract 2. First ammonium sulfate fraction 3. CM-Sephadex chromatographyb 4. Second ammonium sulfate fraction 5. DEAE-Sephadex chromatographyb Cardiac muscle ~ 1. Crude extract 2. Ammonium sulfate fraction 3. CM-Sephadex chromatography P-I P-II

Total units ( X l0 s)

Total protein (mg)

70 60 30 28 22

5000 800 45 32 17

0.525 0.210 0. 065 O. 077

100 21 1.65 1.70

Yield (%)

--

86 43 40 31

Specific activity ( X 104)

1.4 7.5 67.0 87.0 133.0

-40

0.52 1.0

12 15

4.0 4.6

Reproduced from L. E. Flanders, J. R. Bamburg, and H. J. Sallach, Biochim. Biophys. Acta, 242, 569 (1971) (Table I). b This step is quite variable with respect to yield. Recovery of 100% of the activity can occasionally be obtained. Data are for 16 g of frog heart. Values in step 3 are theoretical from the total of step 2; in practice, only 72,000 units from step 2 are run on a CM-Sephadex column (1.8 X 42 cm). The total yield for step 3 from step 2 is 68%.

kinase. A ratio of one volume of enzyme solution to 50 volumes of ammonium sulfate solution is used. (The resulting suspension is a convenient storage form for the enzyme; no loss in activity is observed for a period of over 1 month.) The precipitated protein is recovered by centrifugation, and the supernatant solution is discarded. Step 5. DEAE-Sephadex Chromatography. The ammonium sulfate residue from the above step is dissolved in 40 m M imidazole acetate buffer, pH 7.4, containing 1 m M E D T A , 10 m M 2-mercaptoethanol, and 0.5 m M F D P to give a protein concentration of 5-7 m g / m l (approximate dissolution volume is about two-thirds that of the volume of the suspension centrifuged in the above step). After dialysis against the same buffer, the enzyme solution is applied to a DEAE-Sephadex column (150 ml bed volume per 30 m~ of protein) equilibrated with the same buffer. The column is washed with 10 ml of buffer, then the enzyme is eluted with a linear gradient of 0 to 0.3 M KCI, dissolved in column buffer; a ~ t a l volume of 300 ml is used for a resin bed volume of 150 ml. The fractions containing pyruvate kinase are pooled and concentrated via ultrafiltration to give a final protein concentration of 4-8 mg/ml. This fraction

[28]

AMPHIBIAN PYRUVATE KINASE ISOZYMES

171

can be stored at 4 ° for several weeks with no loss in activity. This fraction is judged to be homogeneous as determined by sedimentation velocity, gel electrophoresis in 1% sodium dodecyl sulfate, double diffusion precipitin reaction and immunoelectrophoresis.1° The results of a typical purification are summarized in Table I.

Purification o] Cardiac Pyruvate Kinase ~° Step 1. Crude Extract. Pyruvate kinase in cardiac tissue is extracted by homogenization in the same buffer as that employed above for the skeletal muscle enzyme except that a tissue to buffer ratio of 1:2 (w/v) is used. Step 2. Ammonium Suljate Fractionation. Ammonium sulfate (18 g/100 ml) is added to the supernatant solution from the above step. The 0-18% ammonium sulfate precipitate is removed by centrifugation and, to the resulting supernatant solution, is added 20 g of ammonium sulfate per 100 ml of solution. The ammonium sulfate precipitate is recovered by centrifugation and is retained. Step 3. CM-Sephadex Chromatography. The ammonium sulfate fraction from the above step is dissolved in 40 mM imidazole acetate buffer, pH 7.4, containing 1 mM EDTA and 10 mM 2-mercaptoethanol and dialyzed against the same buffer until free of ammonium ions. For CM-Sephadex chromatography, the enzyme solution (100 mg of protein per 100 ml bed volume of resin) is applied to the column and is then eluted with a 1 liter linear gradient of 0 to 0.15 M KC1 (dissolved in column buffer). At the termination of the KC1 gradient, column buffer containing 0.15 M KC1 is passed over the column until all pyruvate kinase activity is eluted. The heart pyruvate kinase elutes as two peaks; the first (P-I) starts near the end of the gradient and the second (P-II) elutes with the 0.15 M KCl-column buffer wash applied at the end of the gradient. 17 (These two individual fractions can be rechromatographed separately on CM-Sephadex columns under conditions identical to those employed above. Pyruvate kinase in P-II upon rechromatography is eluted from the second column in its original position whereas 62% of that originally in P - I is eluted in the position of P-II when rechromatographed with the remainder of the activity eluting in its original position. It can be estimated that the separated fractions used for the second chromato" For a typical run, 72,000 units of fraction I I are placed on a column 1.8 × 42 cm. Flow rate is 25 ml/hr, and fraction volumes are 2.5 ml. Under these conditions, peak I begins eluting around fraction 100, ends at about fraction 130, at which point peak I I begins to elute and is eluted after fraction 170; the KC1 gradient ends at about fraction 125.

172

KINASES

[28]

graphic experiments contained no more than 10% of the other fraction. As outlined below, these two fractions have different kinetic properties.) The results of a typical purification are summarized in Table I.

Purification o.i Liver Pyruvate Kinase There are five different forms of pyruvate kinase in frog liver. The major isozyme is the most rapidly migrating anodal band, and the proeedure reported below is for the purification of this isozyme. The method 11 is a modification of the procedure of Staal et al2 s used for the purification of pyruvate kinase from human erythrocytes and utilizes the property of the enzyme to bind specifically to blue dextran. 1s,19 Step 1. Crude Extract. Liver (66 g) is homogenized with two volumes (w/v) of the 50 mM imidazole acetate buffer, pH 7.4, containing 80 mM KC1, 10 mM 2-mereaptoethanol, and 1 mM EDTA, in a Waring Blendor at the low setting for 2 rain. The homogenate is centrifuged at 14,000 g for 20 rain, and the resulting supernatant solution is then centrifuged at 100,000 g for 30 rain. The residue is discarded. Step 2. Ammonium Sulfate Fractionation. Ammonium sulfate (30 g/100 ml) is added to the supernatant solution from the above step. The precipitate is removed by centrifugation and discarded. To the resulting supernatant solution, 20 g of ammonium sulfate per 100 ml of original solution is added. The precipitated protein is recovered by centrifugation and this 30-50% ammonium sulfate residue is stored at --15 ° . Step 3. Gel Filtration of Enzyme-Blue Dextran Complex. One-third of the ammonium sulfate fraction from the above step is dissolved in 10 ml of 5 mM potassium phosphate buffer, pH 6.8, containing 5raM magnesium sulfate and 4 mM 2-mercaptoethanol. (The volume of buffer used is one-tenth of the volume from which the ammonium sulfate fraction was prepared.) The resulting solution is dialyzed against 1 liter of the same buffer, with 30-rain changes, until free of ammonium ions. After dialysis, any insoluble protein is removed by centrifugation and, to the resulting supernatant solution, Blue De×tran 200050 is added (final concentration = 0.5%). After equilibration for 10 rain, any insoluble material is removed by centrifugation. The clear supernatant solution (12-13 ml) is applied to a Sephadex G-200 column (6 X 45 cm) equilibrated with the same buffer as used in the dialysis. The column is ,8G. E. ft. Staal, J. F. Koster, H. Kamp, L. Van Milligen-Boersraa, and C, Veeger, Biochim. Biophys. Acta 227, 86 (1971). 1~R. Haeckel, B. I-Ie~, W. Lauterborn and K. H. Wiister, Hoppe-Seyler's Z. Physiol. Chem. 349, 699 (1968). ~oThe Blue Dextran 2000 (Pharmacia) is suspended in the same buffer (100 mg/ml) and is shaken at room temperature for several hours before use.

[28]

AMPHIBIAN PYRUVATE KINASE ISOZYMES

173

washed with the same buffer (flow rate = 35-40 ml/hr) and 10-ml fractions are collected. Pyruvate kinase is eluted together with the blue dextran. Fractions which are blue in color, not green, are pooled (110120 ml). Step 4. First DEAE-Sephadex Chromatography. The pooled fractions from the above step are applied to a DEAE-Sephadex column (5 X 8 cm) that is equilibrated with 0.2 M potassium phosphate buffer, pH 7.4, containing 50 mM 2-mercaptoethanol and 1 mM EDTA. The column is washed with the same buffer. Under these conditions, the complex is dissociated and the bulk of the blue dextran is adsorbed to the gel whereas the enzyme is not and elutes in the main protein fraction. The enzyme is precipitated from this fraction by the addition of 55 g of ammonium sulfate per 100 ml of solution. The 0-55% ammonium sulfate residue is recovered by centrifugation and retained. Step 5. Second Gel Filtration. The ammonium sulfate precipitate from the above step is dissolved in 6 ml of 0.2 M potassium phosphate buffer, pH 6.8, containing 4 mM 2-mercaptoethanol and 5% ammonium sulfate. Any insoluble material is removed by centrifugation prior to the application of the solution to a Sephadex G-200 column (4.7 X 90 cm) equilibrated with the above buffer. The column is washed with the same buffer (flow rate = 25-30 ml/hr) and 10 ml fractions are collected. Fractions with enzyme activity are pooled, concentrated by ultrafiltration (1-2 mg protein/ml) and enzyme activity is precipitated by the addition of 60 g of ammonium sulfate per 100 ml of solution. The 0-60% ammonium sulfate precipitate is recovered by centrifugation and is stored at --15 °. In processing all of 30-50% ammonium sulfate residues from step 2, steps 3-5 are repeated two additional times and the 0-60% ammonium sulfate fractions from step 5 are pooled prior to proceeding to the next step. Step 6. Chromatography on Hydroxyapatite. The ammonium sulfate fractions from the above step are dissolved in a minimal volume of 5 mM potassium phosphate buffer, pH 6.5, containing 10 mM 2-mercaptoethanol and 1 mM EDTA and dialyzed against the same buffer. The sample is then applied to a hydroxyapatite column (1 X 15 cm) which has been equilibrated with the same buffer. For elution of the enzyme, a two-chamber linear gradient apparatus containing 200 ml of solution per chamber is used. The mixing vessel contains the above buffer and the reservoir contains 0.1 M potassium phosphate buffer, pH 6.8, containing 10 mM 2-mercaptoethanol and 1 mM EDTA. The flow rate of the column is 45-50 ml/hr and 5-ml fractions are collected. Fractions containing enzyme activity are pooled. 21 2~Pyruvate kinase appears in the first protein eluting from the column; hence, enzyme in the first fractions has the highest specific activity. In practice, the last third to fourth of the activity peak is usually discarded.

174

[28]

KINASES TABLE II PURIFICATION DATA FOR LIVER PYRUVATE KINASE

Fraction 1. Crude extract 2. Ammonium sulfate fraction 3. Gel filtration of enzyme-blue dextran complex 4. First DEAE-Sephadex chromatography 5. Second gel filtration 6. Chromatography on hydroxyapatite 7. Second DEAE-Sephadex chromatography~

Total units (X 106)

Total protein (mg)

Yield (%)

Specific activity (X 104)

2.00 1.84 2.05

6670 1935 603

-91.8 102.'0

0.3 0.95 3.40

2.00

372

100.0

5.40

1.57 0.49 0.26

116 10.8 1.2

78.0 24.4 13.0

13.58 45.22 220.00

, Low recoveries at this stage are due to: (a) separation of other isozymes; and (b) lability of the enzyme to concentration by ultrafiltration at this point.

Step 7. Second DEAE-Sephadex Chromatography. The pooled fractions from the above step are dialyzed against 25 volumes of 93 m M imidazole acetate buffer, p H 7.4, containing 10 m M 2-mercaptoethanol and 1 m M E D T A , with hourly changes for 4 hr. The dialyzed solution is then applied to a D E A E - S e p h a d e x column (1..5 X 15 cm) t h a t has been equilibrated with the same buffer. The column is eluted with a linear gradient (150 ml of the above buffer in the mixing vessel and 150 ml of the same buffer containing 0.125 M KC1 in the reservoir), and fractions of 4 ml are collected. Under these conditions, the main peak of p y r u v a t e kinase activity usually elutes around fraction numbers 50-55. 22 Fractions containing activity are pooled and concentrated (1-2 mg protein/ml). The purified enzyme preparation is free of all of the other four isozymes found in liver as determined by zone electrophoresis2 The enzyme is sta. bilized by 50% glycerol at this point. The results of a typical preparation are summarized in Table II. Properties of the I s o z y m e s 6,10,11 The electrophoretic mobilities of cardiac, skeletal muscle, and liver (major form) isozymes of p y r u v a t e kinase are different. Immunological cross-reactivity of the antibody to the skeletal muscle enzyme with the ~2The conditions used in this chromatographic step resolve the chief isozyme from the four other isozymes found in liver. The main liver isozyme is the last to be eluted. Therefore, any pyruvate kinase activity eluting in earlier fractions, or as an ascending shoulder on the main peak, is discarded.

[28]

AMPHIBIAN PYRUVATE KINASE ISOZYMES

175

cardiac isozyme, but not with the purified liver isozyme, is observed. The antibody to the purified liver isozyme does not cross-react with either the cardiac or skeletal muscle isozymes. On the basis of gel filtration studies, all three isozymes appear to have approximately the same molecular weight. A detailed description of the kinetic and other properties of the cardiac and skeletal muscle enzymes can be found in Flanders et al. TM Briefly, the skeletal muscle enzyme exhibits Michaelis-Menten kinetics and is not affected by any of the modifiers demonstrated for regulatorytype pyruvate kinases from other sources. On the other hand, the cardiac isozyme is activated by PEP and FDP and is inhibited by L-alanine, but the extent to which the activity of the enzyme is altered by these modifiers is dependent upon the isolation procedure. Of the two fractions obtained upon CM-Sephadex chromatography, P-I and P - I I (see above), only the P - I I fraction exhibits allosteric kinetics indicative of a regulatory isozyme. With time (24 hr), the P-II fraction becomes desensitized to the effects of modifiers but can be regenerated by passage over CMSephadex as originally run. Although there are quantitative differences, the purified liver isozyme is similar kinetically to the cardiac isozyme in that it is subject to activation by FDP and PEP and is inhibited by L-alanine. In addition, two different fractions of liver pyruvate kinase, analogous to those obtained with the cardiac enzyme, can be obtained by CM-Sephadex chromatography. With respect to the effects of modulators on the amphibian cardiac and liver isozymes, the basis for the occurrence and/or interconversion of sensitive and insensitive forms is unknown. Other studies with hepatic pyruvate kinase from mammalian sources 2~--~ indicate that the amount of FDP bound to the enzyme can be one explanation although similar changes have been observed in the presence of organic solvents2 ~3B. Hess and C. Kutzbach, Hoppe Seyler's Z. Physiol. Chem. 352, 453 (1971). M. G. Irving and J. F. Williams, Biochem. J. 131, 287 (1973). ~ M. G. Irving and J. F. Williams, Biochem. J. 131, 303 (1973).