THE JOURNAL OF B~OLOGKXL CHEMISTRY Vol. 253, No 5, Issue of March 10, pp. 1493-1497, 1978

Printed in U.S.A

The A,B, Hybrid Isozyme of Phosphofructokinase* (Received for publication, July 5, 19’77)

FRESIA GONZALEZ AND ROBERT G. KEMPS \

From the Departments of Biochemistry, The Medical College of Wisconsin, Milwaukee, Wisconsin, 53233 and The University of Health Sciences/The Chicago Medical School, Chicago, Illinois 60612

The hybrid isozyme of phosphofructokinase, A2B2, was formed by incubation of rabbit muscle enzyme, A,, and rabbit liver enzyme, Bq, in the presence of sodium citrate at neutral pH. The enzyme composition of the resulting mix- ture of AzB2 and the homoprotomeric forms was identical to that found in rabbit adipose tissue extracts. Hybrid formation, which apparently proceeds by way of dimers, can be blocked by fructose-1,6-P,, fructose-6-P, and high concentrations of MgATP. The A,B, isozyme was separated from A, and B, by ion exchange chromatography. The kinetic regulatory properties of A,B, were compared with those of A,, B,, and a 1:l mixture of A, and B,. ATP inhibition of A,B, was intermediate between that observed with A, and B, and was clearly not identical to a simple summing of the effects of A and B subunits. Similar com- parisons were made using other phosphofructokinase inhib- itors, citrate, 2,3-P,-glycerate, and P-creatine. In each case the observed inhibition was intermediate between that ob- served with A, and B,. The existence in a number of tissues of phosphofructokinase A2BZ provides added diversity to the regulatory mechanisms of glycolysis.

Phosphofructokinase, the enzyme catalyzing the principal regulatory step in glycolysis, is subject to regulatory control by a great variety of metabolites. Diversity in the control of glycolysis among various tissues is achieved by differences in effector concentrations in those tissues and by the presence of tissue-specific isozymes with differing sensitivities to the metabolite effecters (l-3). In the rabbit, the A isozyme of skeletal muscle and heart is very sensitive to inhibition by citrate and creatine-P, whereas the B isozyme of liver and erythrocytes is more sensitive to inhibition by ATP and 2,3- P,-glycerate (1). A number of tissues of the rabbit show hybrid patterns of the A and B isozymes (4), yielding a total of five electrophoretically distinct species. Hybrids may also be produced in vitro by dissociating the A and B isozymes at low pH followed by recombination at neutral pH (5). The distribution of such in vitro hybrids varies slightly from that

* This work was supported by United States Public Health Service Grant AM 19912 and a grant-in-aid from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. Present address, Department of Biochemistry, University of Health Sciences/The Chicago Medical School, 2020 West Ogden Ave., Chicago, Ill. 60612.

observed in tissue extracts. The in vitro hybrids represent a pattern that would be produced by the random association of monomers, whereas extracts of tissues such as adipose tissue show greater proportions of Aq, Bq, and AZB2, with A3B and AB3 in only low concentrations (4, 5).

In the present communication, conditions are described for the in vitro generation of phosphotiuctokinase isozyme pat- terns similar to, those seen in adipose tissue. The major isozyme species, A2B2, is isolated and its kinetic regulatory properties are compared with those of the parental forms, A, and B,.

MATERIALS AND METHODS

All nucleotides were obtained from P-L Biochemicals. Dithiothrei- tol, glycylglycine, Tris, Tes,’ a-glycero-P dehydrogenase, triose-P isomerase, all phosphorylated sugar derivatives, 2,3-P,-glyceric acid, and P-creatine were obtained from Sigma Chemical Co. Frozen rabbit livers used for the preparation of liver phosphofructokinase and frozen rabbit skeletal muscle used for the preparation of muscle phosphofructokinase, glyceraldehyde-3-P dehydrogenase, and aldol- ase were from young (8 to 12 weeks) New Zealand white rabbits of mixed sex purchased from Pel-Freez Biologicals, Rogers, Ark.

Crystalline rabbit muscle phosphofructokinase was prepared from frozen muscle as previously described (6). Rabbit liver phospho- fructokinase was prepared from frozen liver through Step 4 of the procedure of Kemp (7). The liver enzyme preparations used in these studies had specific activities of 6 to 25 unitslmg. Aldolase was prepared and crysta!lized twice by the method of Taylor (8). The enzyme was recrystallized two additional times at pH 6.2 to remove a trace of contamination by phosphofructokinase. Glyceraldehyde-3- P dehydrogenase was prepared from frozen rabbit skeletal muscle by the procedure of Velick (9).

Rabbit adipose tissue was obtained from New Zealand white rabbits killed by an overdose of Diabutal and bled by cutting vessels in the neck. The adipose tissue was removed from areas surrounding the kidneys, minced with scissors, homogenized in a Waring Blendor with 2 volumes of an ice-cold buffer consisting of 30 mM potassium fluoride, 4 mM EDTA, and 1 mM dithiothreitol, pH 7.5. The homog- enate was centrifuged at 18,000 x g and at 4” for 20 min, and the pink infranatant solution was removed with a Pasteur pipette. The extract was concentrated employing a Sartorius collodion bag vac- uum concentrator and was then dialyzed overnight against 1 liter of 50 mM Tris/HCl (pH 8.0) buffer containing 0.1 M (NH&SO,, 1 rnM ATP, 2 mM dithiothreitol, 1 mM EDTA. This buffer was described by Tarui et al. (10) as a means of protecting against the aggregation of phosphofructokinase.

Enzymic activity was determined at 30” and pH 8.2 in 3 ml of medium containing 25 rnM glycylglycine, 25 mM P-glycerol-P, 1 mM EDTA, 6 rnM MgCl,, 0.2 rnM NADH, 3 mM (NH&SO,, 0.1 rnM dithiothreitol, 0.6 unit of aldolase, 0.2 unit of triose-P isomerase, 0.3 unit of a-glycerol-P dehydrogenase, 1 mM ATP, and 1 rnM fructose-6-P. For studies of regulatory properties of the enzyme, the assay medium was buffered by 50 rnM Tes titrated with KOH to a

1 The abbreviation used is: Tes, N-[Tris(hydroxymethyl)methyl- 2-aminolethanesulfonic acid.

1493

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1494 A&l2 Isozyme of Phosphofructokinase

pH of 7.3. KC1 was then added to make the final concentration of potassium ion 150 mM. Other components of the assay in a total volume of 3 ml included 1 mM EDTA, 0.2 mM NADH, 1 mM dithiothreitol, 0.2 unit of triose-P isomerase, 0.6 unit of aldolase, 0.3 unit of a-glycerol-l? dehydrogenase, and ATP and fructose-6-P at the indicated concentrations. MgCl, was always present at 5 mM in excess of the concentration of ATP. Before their use in kinetic studies, the phosphofructokinase and the auxiliary enzymes were dialyzed overnight to remove ammonium sulfate.

Protein determinations were performed by the calorimetric method of Lowry et al. (11). Zone electrophoresis was performed at 4” at 4 to 6 mA and 300 V for 2l/z h on cellulose acetate strips (50 X 200 mm) (Shandon) in a buffer system at pH 8.1 that consisted of 50 mM glycylglycine, 0.1 mM EDTA, 1 mM ATP, and 0.25 mM dithiothreitol. For most studies, strips that were 50 mm wide were employed, which allowed the placing of two samples on the same strips. Following electrophoresis, enzyme activity on the strips was de- tected by an adaptation (1) of the method of Penhoet et al. (12) as follows. A gel was prepared that consisted of 0.3% Ionagar 2 (Colab), 50 rnM Tris/HCl, 10 mM Na*HAsO,, 2 rnM EDTA, 1 mM fructose-6-P, 1 mM ATP, 4 mM MgCl,, 1 mM NAD, 0.24 mg/ml of glyceraldehyde- 3-P dehydrogenase, 0.1 mg/ml of aldolase, 0.7 @g/ml of triose phosphate isomerase, 0.024 mg/ml of phenazine methosulfate, and 0.4 mg/ml of nitroblue tetrazolium (Nutritional Biochemical& The final pH was 8.3. The cellulose acetate strips were placed face down on a thin layer of the gel. After development by incubation in the dark at 37” for 10 to 20 min, the intensely blue activity bands of the reduced tetrazolium dye were observed on the inverted glass plate and subsequently photographed.

RESULTS

Studies of Hybrid Formation-In a previous study from this laboratory (5), it was shown that phosphofructokinases from rabbit muscle (A isozyme) and rabbit liver (B isozyme) could be hybridized by dissociation at pH 5.2 followed by recombination at neutrality. In that study, it was observed that three new electrophoretic species could be generated by dissociation-recombination to produce a live-band pattern sim- ilar to that seen with the well characterized cases of aldolase and lactic acid dehydrogenase. In Fig. 1 are shown electropho- retie analyses of isozyme A (Track 11, isozyme B (Track 31, and the pattern produced by low pH hybridization (Track 5). In that earlier study (4, 51, it was also noted that, whereas the electrophoretic pattern of phosphofructokinase in adipose

I

2

FIG. 1. Electrophoretic patterns of phosphofructokinase. Electro- phoresis conditions and activity stain are described under “Materials and Methods.” Origin is indicated at the lefi by a dotted line. Track 1, muscle phosphofructokinase; Truck 2, hybridized mixture of A, and B1; Track 3, liver phosphofrnctokinase; Tracks 4 and 6, adipose tissue extracts; Track 5, mixture of hybrids produced by exposure of A, and B, to pH 5.2.

tissue extracts bore a resemblance to that of in vitro hybrid patterns which produced a five-band set, the second and fourth bands from the origin were often very faint or missing, indicating a preferential recombination of dimers. These re- sults are confirmed by Tracks 4 and 6 of Fig. 1 which show the electrophoretic analysis of adipose tissue extracts of two different rabbits. The second and fourth activity bands of the five-band set are faint in one case and missing in the second. It was noted that the extract analyzed in Track 4 had a greater proportion of isozyme Bq, although there was no apparent difference between the two rabbits in age or nutri- tional status. At present, we do not know what factors contribute to these minor distribution differences.

Lad et al. (13) concluded from frontal gel chromatography studies that citrate stabilizes dissociated states of muscle phosphofructokinase while fructose-6-P and fructose-1,6-P sta- bilize a tetramer. This suggested that hybridization of phos- phofructokinases may occur more readily at neutral pH in the presence of citrate. To study this possibility, liver and muscle phosphofiuctokinases were dialyzed separately at pro- tein concentrations greater than 0.5 mg/ml in the presence of 50 mM Tes (pH 6.8) containing 0.1 mM ATP, 0.1 mM EDTA, and 0.05 mM dithiothreitol. The enzymes were mixed 1:l with a final activity of 10 units/ml in the presence of various effecters. These solutions were kept at 4” and analyzed for retention of activity as measured at pH 8.2 and for hybrid formation by electrophoresis at intervals for several days. Between one- and two-thirds of the initial activity was lost within 46 h in the absence of added ligand, in the presence of 1 mM MgCl*, or in the presence of low concentrations of citrate (0.1 mM1. On the other hand, activity losses were small in the presence of 6 mM citrate, or 6 mM MgATP, of 1 mM fructose-1,6-P,, or of combinations of these effecters. As predicted, hybrids were detected in the presence of citrate, but not when the incubation contained fructose-1,6-P, or 6 mM MgATP. Either fructose-1,6-P, or fructose-6-P at 1 mM could block hybrid formation in the presence of 6 mM citrate. The hybrid pattern produced in incubations containing citrate contained A2B2 as the major component with practically no AB, or A,B. This is shown in Track 2 of Fig. 1. The similarity of this pattern with that of adipose tissue extracts (Tracks 4 and 6) is striking. Hybrids have also been produced in the presence of 6 mM citrate at 23 and 37” but activity losses were greater under these conditions. Hybrids appeared more slowly at enzyme concentrations in excess of 40 units/ml.

Preparation and Purification of Hybrid Form of Phospho- fructokinase - Initial attempts to separate the hybrid A,B, from the parental forms using ion exchange chromatography procedures similar to those used in the purification of liver and brain phosphofiuctokinase were unsuccessful. The A,B, was not completely resolved from the A4 component and heavy losses in activity occurred. The procedure that was finally developed had some unusual features; the column was run at room temperature, and the operation was carried out rapidly by pumping the sample and elution buffer upward through the column bed with a total development time of less than 6 h. In further contrast to the previously published procedures for phosphofructokinases (2, 71, DEAE-Sephadex A-25 was used instead of A-50. The details of the procedure, which has been successfully repeated eight times, are as follows. The data are those of a typical preparation. Liver and muscle phosphofructokinases with specific activities of 25 and 160 units/ml, respectively, were dialyzed overnight at 4” against a 50 mM Tes buffer (pH 6.8 to 6.9) containing 0.1 mM

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AaBz Isozyme of Phosphofructokinase 1495

Comparison of Regulatory Properties of Aa2, A4, and B4, ATP, 0.1 mM EDTA, and 0.5 mM dithiothreitol. After dialysis, the activity was determined and a mixture of 350 units each of muscle and liver enzyme based on units of activity was adjusted to a final total phosphofructokinase concentration of 10 units/ml. Sodium citrate (pH 6.9) was added to a final concentration of 6 mM and the mixture was allowed to equili- brate for 3 days at 7-8” with continuous stirring. The forma- tion of A2BZ hybrid was followed by electrophoresis of samples on cellulose acetate. After the incubation period the mixture was dialyzed overnight at 4” against 50 mM TrislHCl, pH 7.5, containing 0.1 mM ATP, 0.1 mM fructose-1,6-P,, 2 mM EDTA, 0.5 mM dithiothreitol, 6 mM citrate, and 50 mM (NH,),SO+ A column with a 2.5~cm diameter was packed to a height of 30 cm with DEAE-Sephadex A-25 previously equilibrated at room temperature with a buffer of the same composition and pH as the dialysis buffer. The dialyzed fraction from the previous step was pumped to the column from the bottom with the outlet at the top. The column was washed with approximately one-third of its volume with the equilibration buffer (40 ml). A gradient from 0.05 to 0.40 M ammonium sulfate in the same buffer described above (250 ml in each gradient bottle) was used to elute the enzyme. This entire chromatographic procedure was performed at room tempera- ture.

ATP Inhibition -The activity of phosphofructokinase is known to be sensitive to inhibition by ATP, and to be further regulated by a large variety of metabolic effecters, including inorganic phosphate, citrate, creatine phosphate, adenine nucleotides, and several glycolytic intermediates (14). The sensitivities of isozymes A, and B, to ATP inhibition and to the regulation by other effecters have been shown to differ (1). Because of these differences in the parent A, and B4 isozymes, the properties of AzB2 are of interest from the standpoint of what we can learn about subunit communica- tion, as well as for the information it provides about glycolytic regulation in adipose tissue and in other tissues where A,B, represents the major isozymic species.

Electrophoretic evaluation of the various fractions indicated that the first peak of activity corresponded to Aq, the second, A,B,, and the third peak was B,. The elution profile and the electrophoretic characterization of several of the fractions are shown in Fig. 2. In several analyses, including the one shown in the figure, another small peak was observed at Fractions 105 to 115. This component could be identified as ABB. The A,B, hybrid was concentrated by ultrafiltration, then dialyzed against 50 mM Tris/HCl, pH 8.0, buffer containing 0.1 mM ATP, 2 mM EDTA, 0.5 mM dithiothreitol, 0.1 mM fructose- 1,6-P,. The enzyme could be stored in this buffer for several weeks with little or no loss of activity. Approximately 280 units of A,B, with a specific activity of 110 unitslmg were recovered in the procedure described above and in Fig. 2.

Fig. 3 describes the ATP inhibition curves at pH 7.3 and 1 mM fructose-6-P for At, Bq, AeB2, and 1:l mixture of A, and B,. ATP inhibition of phosphofructokinase decreases as pH is increased and as the concentration of fructose-6-P is increased. At the relatively high concentration of fructose-6-P and at the slightly alkaline pH used in Fig. 3, inhibition of A, (lower half of Fig. 3) is weak and requires approximately 11 mM ATP to achieve 50% inhibition. The much more sensitive B4 isozyme requires only 1 mM ATP for 50% inhibition. The ATP inhibition curve for A,B, is a smooth curve, giving an inhibi- tion pattern between that of A, and B,. Of greater interest is a comparison of A2B2 with a mixture of A4 and B, that was prepared by adding together equal amounts of each enzyme based on the activity at pH 8.2. This is shown in the upper part of Fig. 3. The curve given by the mixture appears biphasic and is virtually identical to that which can be calculated as the theoretical sum of the two activities at pH 7.3, if one assumes equivalent activities of the two enzymes at pH 6.2. It is obvious that in A,B, the intrinsic binding affinities of one subunit type has a profound influence on the binding of the second type.

Other Inhibitors, Citrate, and 2,3-Pzglycerate - It is difti- cult to readily assess the relative potency of other inhibitors with respect to phosphotiuctokinase isozymes because of the

20 40 60 80 100 120 140

Fraction Number FIG. 2. DEAE-Sephadex-A-25 chromatography of the hybrid mix-

ture. The column was 30 cm long and 2.5 cm in diameter, equili- brated with 50 mM Tris/HCl, pH 7.5, containing 2 mM EDTA, 6 mM citrate, 50 mM (NH&SO,, 0.1 mM dithiothreitol, 0.1 mM ATP, and 0.1 mM fructose-1,6-P,. The column was eluted with a linear gradient obtained by employing 250 ml of the above buffer in an open mixing chamber connected to a reservoir containing 250 ml of this buffer but with (NH&SO, concentration of 0.40 M. Activities were deter- mined at pH 8.2. The inset shows the electrophoresis pattern obtained from aliquots of fractions from the elution profile.

0.5

0.25

0.25

mM ATP FIG. 3. ATP inhibition of phosphofructokinase. Detailed proce-

dures are described under “Materials and Methods.” V, is the velocity at optimal conditions of pH 8.2 with 1 mM ATP and 1 rnM fructose-6-P. Data for velocities were obtained from assays at pH 7.3 in the Tes buffer system described in the text. Fructose-6-P was present at 1.0 mM. 0, muscle enzyme (A,); 0, A2B2; A, liver enzyme (B,); n , 1:l mixture of A4 and B4 based on assays at V,.

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1496 AJ3, Isozyme of Phosphofructokinase

differing sensitivities of the isozymes to ATP inhibition and because ATP acts synergistically with the other inhibitors (Xi), that is, an increase in ATP concentration results in an even greater sensitivity (lower K,) to the second inhibitor. To minimize this problem we (1, 2) have made comparisons of inhibitor actions at optimal ATP concentrations. In the in- stance of citrate and 2,3-P,-glycerate, inhibition was deter- mined at ATP concentrations of 2 mM for Ad, 1 mM for A2B2, and 0.4 mM for B,. Citrate inhibition is described in Fig. 4. As noted previously (l), the liver enzyme, Bq, is much less sensitive to citrate inhibition than is the A, isozyme. The inhibition curve of the hybrid falls between that of the two parental isozymes, but the data are not simple arithmetic sums of those two activities. The relative sensitivities of A4 and B, to 2,3-P,-glycerate inhibition is opposite that seen for citrate inhibition (1). As shown in Fig. 5, B, is sensitive to 2,3-P,-glycerate while the muscle isozyme is not. Although A,B, can be inhibited by this effector, the inhibition is much less than that of B,.

P-creatine Inhibition - P-creatine also acts synergistically with ATP but at a site distinct from the citrate site (15). P- creatine inhibition is readily reversed by high fructose-6-P concentrations and is thus not detectable at 1 mM fructose-6- P, the concentration used in Figs. 4 and 5. To define appropri- ate conditions for the assessment of P-creatine inhibition, sensitivity to ATP inhibition curves of Ad, A,B,, and B, were compared at several concentrations of fructose-6-P. In the case of B,, the enzyme was extremely sensitive to ATP inhibition at concentrations of fructose-6-P less than 1 mM. For example, at 0.75 mM fructose-g-p, maximal activity of B, was achieved at only 0.1 mM ATP. This condition was used in the results of Fig. 6. Under a number of conditions in which ATP and fructose-6-P were varied, no creatine-P inhibition of B, could be observed. On the other hand, A,B, was readily inhibited by P-creatine if the fructose-6-P concentration was less than 1 mM, but the concentration of P-creatine necessary for inhibition was greater than that necessary for inhibition of A, (Fig. 6).

DISCUSSION

In the presence of citrate, phosphofiuctokinase AzB2 could be produced in high yield from mixtures of A, and B,. It would appear that the role of citrate is to provide stability to the dissociated state of phosphofi-uctokinase. The enzyme is noto-

Citrate (mM)

riously unstable in dilute solution and loss of activity is presumed to be accompanied by dissociation. This phenome- non has been studied in detail by Bock and Frieden (16, 17), who showed that muscle phosphofi-uctokinase (A,) can exist in either protonated or unprotonated tetrameric and dimer forms, and that inactivation resulted from an isomerization of the tetramer followed by dissociation to the dimer. They concluded also that citrate was preferentially bound to proton- ated forms (17). In the present study, citrate may be prevent- ing the formation of an additional dimeric state that is or becomes an essentially irreversibly inactivated form.

The formation of A,B, at the virtual exclusion of hybrids containing either one A or B subunit indicates the absence of a significant amount of monomer in the hybridization incu- bation as a result of the greater stability of the dimeric form. Our earlier studies of hybridization under acidic conditions wherein one obtained A3B and AB, (5) suggested that even under low pH conditions the equilibrium favored dimer. In those experiments, if the period ‘of recombination of subunits was shortened by adjusting the pH from 5.3 to 7.0 quickly after the two isozymes were united, only recombinants of dimers could be demonstrated. This procedure, however, was not useful for the production of A,B, because of the low yields of active enzyme. It would appear that the dissociation of the tetrameric enzyme to dimer proceeds asymmetrically. I f not, then re-exposure of A,B, to hybridization conditions would be expected to produce AB, and A,B in addition to the other forms due to dissociation of hybrid along a different axis resulting in AB dimers. This does not occur, and is consistent with the electron microscopic study of Telford et al. (18) which showed that the two axes of tetramer of muscle enzyme are not identical.

An analysis of the kinetics of the A,B, hybrid reveals properties that were not what one would expect from equiva- lent mixtures of A, and B,. The affinities of individual subunits are obviously influenced by the nature of the remain- ing subunits of the tetramer. This influence of one subunit type on the affinity of the second is seen for ATP, citrate, creatine-P, and 2,3-P,-glycerate. For example, the B subunit’s aflinity for creatine-P is not detectable in the B, complex, but the subunit must have some capacity to bind the ligand as shown by the inhibition curve of A2B2, wherein 100% inhibi- tion can be demonstrated. In the complex, A subunits would appear to have reduced affinity for the ligand while B subunits

1

1 2 3 4 5

2.3 di F$ glycemte knM)

4 0 12

P-Creatme (mM)

FIG. 4 (left). Inhibition of phosphofructokinases by citrate. De- the presence of 1 rn~ fructose-6-P. 0, A,, at 2 rn~ ATP; W, A,B,, at tailed procedures were described under “Materials and Methods.” 1 rn~ ATP; A, B,, at 0.40 rn~ ATP. Data were obtained from assays at pH 7.3, in the presence of 1 rn~ FIG. 6 (right). Inhibition of phosphofructokinases by creatine fructose-6-P. 0, A,, at 2 rn~ ATP; n , AIBI. at 1 rn~ ATP: A, B1. at phosphate. Detailed Drocedures were described under “Materials 0.40 rn~ ATP. V, is defined in legend to Fig. 3. and &Iethods.” Conditions of the assay were the following at pH 7.3.

FIG. 5 (center). Inhibition of phosphofructokinases by 2,3-diphos- 0, A,, 1 rn~ ATP, 0.25 rn~ fructose-6-P; W, A2B2. 0.4 mM ATP, 0.50 phoglyceric acid. Detailed procedures were described under “Mate- mhl fructose-6-P, A, B,, 0.1 rn~ ATP, 0.75 IIIM fructose-6-P. V, is rials and Methods.” Data were obtained from assays at pH 7.3 in defined in the legend to Fig. 3.

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Aaz Isozyme of Phosphofructokinase 1497

have increased affinity. A less likely explanation for such an ences in effector responses among the various parental forms observation is that subunits retain their intrinsic binding and their hybrids. properties giving biphasic binding isotherms for the hybrid, but that the conformational and thus kinetic consequences of Acknowledgment-We are indebted to Pei-Yung Hsu for

the binding is monophasic. Such a possibility could be exam- excellent technical assistance.

ined by direct binding studies. The data for the behavior of phosphofructokinase A,B, relative to A, and B, are similar to that seen with pyruvate kinase hybrid isozyme (19, 20). Skeletal muscle pyruvate kinase shows hyperbolic kinetics and is unaffected by fructose-1,6-P,, while the L,M hybrid displays sigmoidal kinetics in addition to being activated by the sugar diphosphate (19).

REFERENCES

The kinetic properties of phosphofiuctokinase A,B, de- scribed here probably account for the minor isozyme compo- nent of rat liver described as L, by Dunaway and Weber (21). The L, enzyme was less susceptible to ATP inhibition than the major isozyme (L,) as one would predict on the assumption that L, represents B, and L, represents A,BZ. An earlier study from this laboratory (22) indicated the presence of a minor electrophoretic component in rat liver extracts that was assumed to be either A,B, or AB,.

The existence of hybrid phosphofiuctokinases with regula- tory properties distinct from those of the homoprotomeric enzymes provides additional regulatory diversity to tissues. In the case of adipose tissue where adaptive changes of phosphofructokinase in response to dietary and hormonal influences have been reported (23), the formation of hybrids may be of additional adaptive value. Although our prelimi- nary studies reported here showed all three forms present, under some conditions it is possible that one form is present to the exclusion of the other. A switch in a hormonally responsive tissue from a phosphofructokinase that is highly sensitive to ATP to one that is relatively insensitive would not require the complete replacement of isozyme B, by A, but only the synthesis of A subunits and their hybridization with pre-existing B subunits. Previous studies of adaptive changes of energy metabolism in response to hormones or diet have usually included phosphofructokinase in view of its role as a pacemaker, but future work should also include potential isozymic shifts of this enzyme because of the striking differ-

1. 2.

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Tsai, M. Y., Gonzalez, F., and Kemp, R. G. (1975) Isozyme II (Marker% C. L., ed) pp. 819-835, Academic Press, New York

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Lad, P. M., Doyle, E. H., and Hammes, G. G. (1973) Biochemis- try 12, 4303-4309

Bloxham, D. P., and Lardy, H. A. (1973) The Enzymes (Boyer, P. D., ed) 3rd Ed, Vol. 9, pp. 239-278, Academic Press, New York

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Colombo, G., Tate, P. W., Girotti, A. W., and Kemp, R. G. (1975) J. Biol. Chem. 250, 9404-9412

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Bock, P. E., and Frieden, C. (1976) J. Biol. Chem. 251, 5637- 5643

Telford, J. N., Lad, P. M., and Hammes, G. G. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3054-3056

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Hubbard, D. R., and Cardenas, J. M. (1975) J. Biol. Chem. 250, 4931-4936

Dunaway, G. A., Jr., and Weber, G. (1974) Arch. Biochem. Biophys. 162, 620-628

Gonzalez, F., Tsai, M. Y., and Kemp, R. G. (1975) Camp. Biochem. Physiol. 52B, 315-319

Orevi, M., Gorin, E., and Shafrir, E. (1972) Eur. J. Biochem. 30, 418-426

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F Gonzalez and R G KempThe A2B2 hybrid isozyme of phosphofructokinase.

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