THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 2, Issue of January 25, pp, 726-730.1985 Printed in U.S.A.

Isolation and Characterization of Phosphofructokinase C from Rabbit Brain*

(Received for publication, May 25, 1984)

Lawrence G . Foe and Robert G . KempS From the Department of Biological Chemistry and Structure, University of Health ScienceslThe Chicago Medical School, North Chicago, Zllinois 60064

Phosphofructokinase from rabbit brain consists of hybrids of the A, B, and C isozymes. Phosphofructo- kinase C was isolated from a purified mixture of such hybrids in a 2-step procedure. In the first step, phos- phofructokinase B was removed by chromatography on DEAE-Sephadex. In the second step, subunits of phosphofructokinases A and C were separated by dis- sociation at pH 5.0 followed by chromatography on carboxymethylcellulose. The separated isozymes were then reassociated by neutralization. Phosphofructoki- nase C was structurally distinct from phosphofructo- kinases A (obtained from muscle or brain) and B (ob- tained from liver) as shown by one-dimensional chy- motryptic and staphylococcal VS protease fingerprints of all three isozymes. In addition, phosphofructokinase C cross-reacted weakly or not at all with antisera raised against phosphofructokinase B or phosphofruc- tokinase A. Phosphofructokinase C was also kineti- cally distinct from the A and B isozymes. The C isozyme was more sensitive than the A isozyme but less sensi- tive than the B isozyme to inhibition by ATP, was less sensitive than the A isozyme but more sensitive than the B isozyme to inhibition by citrate, and was less sensitive than either of the other two isozymes to ac- tivation by inorganic phosphate, AMP, and fructose 2,6-bisphosphate. The self-association properties of phosphofructokinase C differed from those of the A and B isozymes in that at pH 8.0, the C isozyme did not form oligomers larger than a tetramer under condi- tions where the other two isozymes did. Thus the prop- erties of phosphofructokinase C are in general quite distinct from those of the other two phosphofructoki- nase isozymes.

Three isozymes of phosphofructokinase (ATP:D-fructose 6- phosphate 1-phosphotransferase, EC 2.7.1.11), designated A, B, and C, are present in rabbits (1). The A and B isozymes can be isolated in essentially homogeneous form from rabbit skeletal muscle and liver, respectively (2, 3). The C isozyme, however, has not previously been isolated in pure form. This is because phosphofructokinase C is not expressed alone but rather occurs only in hybrid mixtures with phosphofructoki- nase A and/or B (1, 4). The similarities and differences between the enzymatic properties of the C isozyme and those of the A and B isozymes have not been described.

* This work was supported by United States Public Health Service Grant AM19912. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

In a previous paper (4) we reported that phosphofructoki- nase purified from rabbit brain consists of a hybrid mixture of all three isozyme forms. The proportion of phosphofructo- kinase C in these hybrids is about 50%. The present paper describes the isolation of phosphofructokinase C from this hybrid mixture purified from rabbit brain and examines some of the properties of the homogeneous C isozyme. It will be shown that the properties of phosphofructokinase C are, in general, distinctly different from those of the other two phos- phofructokinase isozymes.

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION’

Self-association Properties of Phosphofructokinase Iso- zymes-A general property of mammalian phosphofructoki- nase is the tendency to self-associate to oligomeric forms higher than the active tetramer (13). It is generally found that liver phosphofructokinase (corresponding to phosphofructo- kinase B) self-associates to a greater extent than muscle phosphofructokinase (corresponding to phosphofructokinase A) under comparable conditions (14, 15). The self-associate behavior of the C isozyme has not been previously examined. When the A, B, and C isozymes were centrifuged on sucrose gradients at pH 8.0 as described under “Experimental Proce- dures” (initial protein concentrations were 0.1 mg/ml for each isozyme) the sedimentation coefficients of phosphofructoki- nases C, A, and B were found to be 13, 18, and 38 S, respec- tively. A sedimentation coefficient of 13 S corresponds to a tetramer (12); hence under the experimental conditions phos- phofructokinases A and B self-associated to oligomers higher than tetramers, but phosphofructokinase C did not.

One-dimensional Peptide Mapping and Immunoprecipita- tion of Phosphofructokinase Isozymes-Partial digestion of phosphofructokinase isozymes A, B, and C with chymotrypsin or staphylococcal V8 protease followed by SDS-PAGE’ (Figs. 4 and 5) established two facts. First, the identical cleavage patterns given by phosphofructokinase A from muscle and phosphofructokinase A from brain proved the identity of these isozymes. Second, the fact that the patterns given by phos-

Portions of this paper (including “Experimental Procedures,” part of “Results and Discussion,” Figs. 1-8, and Tables I and 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-1574, cite the authors, and include a check or money order for $4.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; Tris, tris(hydroxymethy1)- aminomethane.

726

Phosphofructokinase C from Brain 727

phofructokinase C were very different from those given by isozymes A and B was a strong indication that phosphofruc- tokinase C was not simply a modified form (e.g. a “proenzyme” with a short amino-terminal or carboxyl-terminal extension) of the A or B isozymes. Although relatively minor differences in amino acid sequences could give rise to markedly different digestion patterns in this peptide-mapping procedure, the results do show that the three isozymes must have some sequence differences. Immunoprecipitation of phosphofruc- tokinase isozymes with antisera raised against purified muscle and liver phosphofructokinases supported the results of the one-dimensional peptide-mapping experiments. As shown in Fig. 6, phosphofructokinase A isolated from brain was com- pletely precipitated by antiserum against phosphofructoki- nase from muscle, again indicating the identity of these phos- phofructokinases. Phosphofructokinase C was not precipi- tated by antiserum against muscle phosphofructokinase or by an amount of antiserum raised against liver phosphofructo- kinase just sufficient to completely precipitate an equivalent amount of phosphofructokinase B. However, phosphofructo- kinase C could be precipitated by antiserum against liver phosphofructokinase if larger amounts of antiserum were employed (Fig. 6). This could mean that phosphofructokinase C has some antigenic determinants in common with phos- phofructokinase B. However, since the liver enzyme injected into the guinea pigs contained traces of phosphofructokinases A and C, the weak cross-reactivity of phosphofructokinase C to anti-liver phosphofructokinase antiserum may merely re- flect the presence of small amounts of specific anti-phospho- fructokinase C antibodies in this antiserum.

Regulatory Kinetic Properties of Phosphofructokinase Iso- zymes-Fig. 7 shows the activities of phosphofructokinase isozymes at pH 7.2 as a function of increasing concentrations of substrates. In the experiments illustrated here, the source of the A isozyme was brain; essentially the same results were obtained when phosphofructokinase A from muscle was em- ployed instead. As can be seen, all three isozymes exhibited a sigmoidal response to increasing fructose 6-phosphate concen- trations and an inhibition by increasing concentrations of ATP. Phosphofructokinase C was inhibited by ATP to an extent intermediate between that of the A and B isozymes. As shown in Fig. 7A and more explicitly in Fig. 8, it was noted that the “true” maximal velocity for phosphofructokinase C was not achieved with a saturating concentration of fructose 6-phosphate; an activator such as inorganic phosphate, AMP, or fructose 2,6-bisphosphate was also necessary. In other words, in contrast to the situation with the A and B isozymes, allosteric activators of phosphofructokinase C not only in- creased apparent fructose 6-phosphate affinity via reversal of inhibition by ATP but also increased the apparent maximal reaction velocity about 50-100% (Fig. 8). Addition of activa- tors to the pH 8.0 assay solution, however, did not result in an increase in the specific activity of phosphofructokinase C.

The responses of phosphofructokinase isozymes to allo- steric effectors are summarized in Table 11. A consistent pattern was seen, namely that phosphofructokinase C was less sensitive to allosteric activators than were phosphofruc- tokinases A or B. The C isozyme was 2- to 4-fold less sensitive to inorganic phosphate, 10-fold less sensitive to AMP, and almost 100-fold less sensitive to fructose 2,6-bisphosphate than phosphofructokinases A and B. On the other hand, the sensitivity of phosphofructokinase C to inhibition by citrate or 3-phosphoglycerate was intermediate between phospho- fructokinases A and B.

Human phosphofructokinase also has been shown to exist in three isozyme forms. These were designated as M, L, and

P by Vora (16). The P isozyme was referred to as F by Kahn et al. (17); whatever its designation, this isozyme probably corresponds with the rabbit phosphofructokinase C based on its similar tissue distribution (1, 17), while the human M and L isozymes correspond to the rabbit A and B isozymes. The human F isozyme was purified from platelets (la), and some of its regulatory kinetic properties were described in a prelim- inary communication (19). The F isozyme was reported to be less sensitive to inhibition by ATP or citrate than either the M or L isozymes and was also reported to be completely refractory to activation by either glucose 1,6-bisphosphate or AMP. In contrast, the rabbit phosphofructokinase C is inter- mediate in sensitivity to inhibition by ATP or citrate relative to phosphofructokinases A and B, and while it is relatively insensitive to allosteric activators, especially fructose 2,6- bisphosphate, it will respond to sufficiently high concentra- tions. Indeed, the presence of activators is required for the achievement of maximum specific activity of phosphofructo- kinase C at pH 7.2.

The separate kinetic properties of phosphofructokinases A, B, and C do not appear to predict the kinetic properties of hybridized mixtures of these isozymes. For example, rabbit brain phosphofructokinase was previously reported to be less inhibited by ATP than the isolated A isozyme (20), although since brain phosphofructokinase consists of hybrids of phos- phofructokinase A with the more “inhibitable” B and C iso- zymes, the opposite property would have been expected. Sim- ilarly, brain phosphofructokinase showed similar or higher affinity for AMP and inorganic phosphate as activators com- pared to phosphofructokinases A and B, a result that would not be expected considering the high proportion of the rela- tively insensitive C isozyme present in brain phosphofructo- kinase. This would suggest that the regulatory kinetic prop- erties of phosphofructokinase hybrids are influenced by inter- actions between the different subunits as well as by properties intrinsic to the individual subunits themselves. A similar conclusion was reached previously in experiments with phos- phofructokinases A, B, and the A2B2 phosphofructokinase hybrid (21).

General Comment-As described herein, the purification of phosphofructokinase C from rabbit has made the structural and kinetic characterization of this isozyme possible. Since pure C is now available as an antigen, it should be possible to obtain antibodies specific for phosphofructokinase C, which will enable us to identify phosphofructokinase C in crude tissue or cell extracts. Since expression of phosphofructoki- nase isozymes has been reported to change during develop- ment or malignant transformation (16, 17, 22-25), the ability to identify phosphofructokinase C as well as phosphofructo- kinases A and B in crude extracts by immunological means could be useful in the characterization of these cellular proc- esses.

REFERENCES 1. Tsai, M. Y., and Kemp, R. G. (1973) J. Bid. Chem. 248, 785-

2. Kemp, R. G. (1975) Methods Enzymol. 42, 71-77 3. Kemp, R. G. (1975) Methods Enzymol. 42, 67-71 4. Foe, L. G., and Kemp, R. G. (1984) Arch. Biochern. Biophys. 228,

5. Kawahara, K., and Tanford, C. (1966) Biochemistry 5,1578-1591 6. Warner, R. C. (1958) Arch. Biochern. Biophys. 78,494-496 7. Kemp, R. G., Foe, L. G., Latshaw, S. P., Poorman, R. A., and

Heinrikson, R. L. (1981) J. Bid. Chem. 256, 7282-7286 8. Morrissey, J. H. (1981) Anal. Biochem. 117,307-310 9. Foe, L. G., and Kemp, R. G. (1982) J. Biol. Chem. 257, 6368-

792

503-511

6372 10. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254

728 Phosphofructokinase C from Brain 11. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, 19.

12. Hesterberg, L. K., and Lee, J. C. (1981) Biochemistry 20, 2974- 20.

13. Uyeda, K. (1979) Adu. Enzymol. Relat. Areas Mol. Biol. 48, 193- 21.

14. Reinhart, G. D., and Lardy, H. A. (1980) Biochemistry 19, 1484- 22,

15. Trujillo, J. L., and Deal, W. C. (1977) Biochemistry 16, 3098- 23.

16. Vora, S. (1981) Blood 57, 724-732 17. Kahn, A,, Meienhofer, M. C., Cottreau, D., Lagrange, J. L., and 24.

18. Kahn, A., Cottreau, D., and Meienhofer, M. C. (1980) Biochim. 25.

U. K. (1977) J. Biol. Chem. 252,1102-1106

2980

244

1490

3104

Dreyfus, J. C. (1979) Hum. Genet. 48,93-108

Bi0ph.y~. Acta 611, 114-126

Meienhofer, M. C., Cottreau, D., Dreyfus, J. C., and Kahn, A,

Tsai, M. Y., and Kemp, R. G. (1974) J. Biol. Chem. 249, 6590-

Gonzalez, F., and Kemp, R. G . (1978) J. Biol. Chem. 253, 1493-

Thrasher, J. R., Cooper, M. D., and Dunaway, G. A. (1981) J.

Davidson, M., Collins, M., Byrne, J., and Vora, S. (1983) Biochem.

De Faria, J. B., El-Dorry, H. A., and Bacila, M. (1978) Deu. Biol.

Kahn, A. Cottreau, D., and Dreyfus, J. C. (1980) Pediatr. Res.

(1980) FEBS Lett. 110, 219-222

6596

1497

Biol. Chem. 256,7844-7848

J. 214,703-710

65,547-552

14, 1162-1167

Phosphofruc :tokinme C from Brain 729

QLAE-Srphanex I8 Z . ? 2 7 5 ! ? 5

CM-52

- 2pca - P - AYP -?

A 100 hN 103 .M 80, M 10 M I1.835 Y

B ,2000 >2000 zoo 10 0.35

C 750 1600 350 75 4.5

s 1 2 3 4 s ;.- --".n.-c..- . . . . " I C . ."" "

22a

730 Phosphofructokinase C from Brain

10

10 20 30 40 50

0.2 - 1 E E H

0.1 f

Fractm Hmber

19

1

4

2 3 4 5 s -. " "

- 29

!1

14

.. 1 2 3 4 5 2 0

J

A B

0.6 1

V

V -

1 .o

0.8

0.6

0.4

0.2

1 2 3 4 5 1 0

mM Fructose 6-P