Schistosoma mansoni phosphofructokinase: immunolocalization in the tegument and immunogenicity.

Schistosoma mansoni depends for its survival on glycolysis. Two glycolytic enzymes, glyceraldehyde-3P-dehydrogenase and triose-phosphate dehydrogenase, found in both the adult and schistosomular tegument, have been reported to confer partial protection against cercarial infection. This paper describes the immunogenic properties of phosphofructokinase (PFK), a rate-limiting enzyme of glycolysis, and its localization in the tegument and adjacent tissues. Recombinant schistosome PFK was used as antigen. A polyclonal antibody against purified PFK from Fasciola hepatica was affinity purified using recombinant PFK and used in combination with immunogold labelling to identify PFK by transmission electron microscopy in cryosections. In both adult worms and in schistosomula most immunogold label localized in the cytoplasmic syncytial region with less being found in the tegument. There was no significant PFK localization within or external to the outer membrane. Sera from mice immunized with recombinant S. mansoni PFK with Freund's adjuvant or alum plus rIL-12 demonstrated high titres of anti-PFK IgG, but no protection against cercarial infection. Sera from mice that were acutely or chronically infected or multiply exposed to irradiated cercariae did not recognize recombinant schistosome PFK in either Western blotting or ELISA. Similarly, sera from humans infected with S. mansoni did not recognize PFK. We conclude that in spite of the high immunogenicity of rPFK in mice, it is not a significant immunogen during the course of infection and does not confer protection from schistosomiasis. One main difference between PFK and the other 2 glycolytic enzymes seems to be the inaccessibility of PFK to the outside surface of the tegument.

Epitope mapping of a monoclonal antibody directed against the alpha-subunit of phosphofructokinase-1 from Saccharomyces cerevisiae by screening phage display libraries.

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of two types of subunits, alpha and beta. Subunit-specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, alpha-F3, binds to an epitope either at the C-terminal or at the N-terminal part of the alpha-polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D-A-F and D-S-F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody alpha-F3 to native phosphofructokinase-1. The peptide G-I-K-D-A-F-L inhibited the binding more strongly (IC50 = 1.5 microM) than the peptide A-P-W-H-D-S-F (IC50 = 33.3 microM). Sequence matching revealed the presence of the D-A-F motif in the polypeptide chain of phosphofructokinase-1 at amino acid position 172-174. As a control, the nonapeptide A-P-T-S-K-D-A-F-L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC50 = 0.3 microM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase-1 that is recognized by antibody alpha-F3.

Tissue distribution of placenta-type 6-phosphofructo- 2-kinase/fructose-2,6-bisphosphatase.

Several isozymes of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase have been characterized from mammalian tissues and, based on tissue origin, they are classified as liver, skeletal muscle, heart, testis, and placenta isozymes. In this paper, we examined the tissue distribution of placenta-type isozyme in rat tissues at the levels of transcription and translation. Analysis by Northern blotting showed that placenta, brain, testis, liver, kidney, and skeletal muscle expressed mRNA of placenta-type isozyme. Western blot analysis of fractions from POROS-HQ column chromatography of extracts from various rat tissues showed that proteins of placenta-type isozyme are expressed in placenta, brain, testis, liver, spleen, heart and lung, but not in kidney and skeletal muscle. An immunohistochemical study showed that, in liver, placenta-type isozyme is localized in Kupffer cells. These results indicate that isozymes of this particular enzyme may occur in particular cell types within each tissue.

Cellular distribution of 6-phosphofructo-1-kinase isoenzymes in rat brain.

In the brain, all three isoenzyme types [muscle (M), liver (L), and brain (C)] of 6-phosphofructo-1-kinase (PFK; EC 2.7.1.11) occur, forming a complex mixture of homo- and heterotetramers. The PFK isoenzyme pattern of the different brain cell types is yet unknown. In the present study, we investigated the distribution of the PFK isoenzyme subunits in primary and secondary cell cultures and in bulk-isolated cells of rat brain by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. All three PFK isoenzymes are present in all cell types but in different proportions. The cellular distribution of the PFK isoenzymes in situ was studied immunohistochemically with different polyclonal antisera against purified rat PFKs. The monospecific antibody against M-type PFK stained preferentially the perinuclear areas of neurons and glial cells. The antibodies that in immunoblots detected mainly the L-type PFK showed a characteristic staining in only the cytoplasma and the processes of cells, whereas the C-type antibodies almost homogeneously stained whole cell bodies as well as large dendrites. Because the PFK isoenzymes differ with respect to their allosteric properties, their differential distribution in different brain cells might be of importance for the regulation of brain glycolysis in the different cellular compartments of the brain.

Changes of activity and isozyme pattern of phosphofructokinase in the brains of patients with Alzheimer's disease.

A severe reduction of the in vivo cerebral glucose consumption rate is generally found in patients with Alzheimer's disease. In postmortem studies changes in the activities of key regulatory glycolytic enzymes, including 6-phosphofructokinase (PFK), have been reported in Alzheimer's disease brains, but the results obtained so far are inconsistent and controversial. We reevaluated the activity of PFK in brain tissue from clinically and neuropathologically confirmed cases of Alzheimer's disease using optimized tissue disintegration and assay methods and determined the PFK isozyme pattern. PFK activity in brains from patients with Alzheimer's disease was significantly increased in frontal and temporal cortex and unchanged in the other brain areas studied when compared with control brains. All three PFK isozymes were detected in each of the brain areas studied. In brains of Alzheimer's disease patients the level of the C-type PFK was slightly reduced at the expense of the M- and L-type subunits. The data presented do not support the results of other groups, which reported up to a 90% reduction of PFK activity in Alzheimer's disease. In contrast, the data presented clearly rule out the suggestion that changes of PFK activity might be one of the causes for the reduced glucose consumption in Alzheimer's disease brains.

Preparation of monoclonal antibodies against chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and their effects on enzyme activities.

The effects of the monoclonal antibodies (McAbs) directed against chicken liver 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) on the structure and function of the enzyme were studied. Using chicken liver 6PF-2-K/Fru-2, 6-P2ase as antigen, 7 clones of monoclonal antibodies specifically binding with the antigen were obtained. The epitopes of the antigen recognized by the 6 McAbs localized on the fructose-2, 6-bisphosphatase domain of chicken liver 6PF-2-K/Fru-2, 6-P2ase, and the other (H2) are on the 6-phosphofructo-2-kinase domain. All of the 7 McAbs could activate the kinase activity of the bifunctional enzyme by twofold and had a similar effect on the bisphosphatase activity of the bifunctional enzyme which resulted in a fourfold increase of the bisphosphatase activity of the bifunctional enzyme. However, the McAbs did not affect the activity of the separated fructose-2, 6-bisphosphatase domain. The results suggested that the Fru-2, 6-P2ases in the bifunctional enzyme and in the separated bisphosphatase domain were in two different conformation and activity states.

Interaction of murine monoclonal subunit-specific antibodies with phosphofructokinase-1 from Saccharomyces cerevisiae.

Murine monoclonal subunit-specific antibodies against heterooctameric phosphofructokinase-1 from Saccharomyces cerevisiae exhibiting no cross-reactivity were purified and characterized regarding complex formation with the native enzyme, immunological reactivity to the SDS-denatured subunits originating from native and proteolytically truncated phosphofructokinase, and protection against proteolytic degradation. Strong complex formation was found with one alpha-specific antibody. Western blotting employing different enzyme forms allowed to localize epitope sites of the alpha-polypeptide chain. Monoclonal antibodies protect phosphofructokinase-1 against chymotryptic degradation.

Glycogen synthase and phosphofructokinase protein and mRNA levels in skeletal muscle from insulin-resistant patients with non-insulin-dependent diabetes mellitus.

In patients with non-insulin-dependent diabetes mellitus (NIDDM) and matched control subjects we examined the interrelationships between in vivo nonoxidative glucose metabolism and glucose oxidation and the muscle activities, as well as the immunoreactive protein and mRNA levels of the rate-limiting enzymes in glycogen synthesis and glycolysis, glycogen synthase (GS) and phosphofructokinase (PFK), respectively. Analysis of biopsies of quadriceps muscle from 19 NIDDM patients and 19 control subjects showed in the basal state a 30% decrease (P < 0.005) in total GS activity and a 38% decrease (P < 0.001) in GS mRNA/microgram DNA in NIDDM patients, whereas the GS protein level was normal. The enzymatic activity and protein and mRNA levels of PFK were all normal in diabetic patients. In subgroups of NIDDM patients and control subjects an insulin-glucose clamp in combination with indirect calorimetry was performed. The rate of insulin-stimulated nonoxidative glucose metabolism was decreased by 47% (P < 0.005) in NIDDM patients, whereas the glucose oxidation rate was normal. The PFK activity, protein level, and mRNA/microgram DNA remained unchanged. The relative activation of GS by glucose-6-phosphate was 33% lower (P < 0.02), whereas GS mRNA/micrograms DNA was 37% lower (P < 0.05) in the diabetic patients after 4 h of hyperinsulinemia. Total GS immunoreactive mass remained normal. In conclusion, qualitative but not quantitative posttranslational abnormalities of the GS protein in muscle determine the reduced insulin-stimulated nonoxidative glucose metabolism in NIDDM.

Comparison of muscle phosphofructokinase from euthermic and hibernating Jaculus orientalis. Purification and determination of the quaternary structure.

1. The structural properties of skeletal muscle phosphofructokinase from euthermic and hibernating jerboa were compared. 2. The enzyme was purified by a rapid procedure; suspended in ammonium sulfate in the presence of ATP, it was found to be stable for three weeks. 3. A specific activity of 76 U/mg and at most 65 U/mg was obtained for the enzyme from the euthermic and hibernating jerboa, respectively. 4. The molecular weight was estimated to be 320 kDa for the oligomer and 80 kDa for the subunit. 5. A unique alanine residue was found at the C-terminal end, suggesting that the enzyme is a tetramer made of four identical subunits. 6. The tetrameric structure of phosphofructokinase was confirmed by using crosslinking with disuccinimidyl esters. 7. The kinetics of formation of the different crosslinked species were found to be in agreement with a model of the tetramer corresponding to a dihedral symmetry with isologuous contacts between protomers. 8. The same molecular characteristics and immunochemical properties were found for the enzyme extracted from the euthermic and hibernating animals.

[Immune response induced by phosphofructokinase from E. histolytica in hamsters]. / Respuesta inmune inducida por la fosfofructoquinasa de E. histolytica en hámsters.

The enzymatic activity of inorganic pyrophosphate (PPi) dependent phosphofructokinase became manifest in the supernatant obtained by centrifugation in a homogenate of E. histolytica strain HMI-IMSS at 700,000 g. Partial purification of the enzyme was achieved by column chromatography with Ultrogel AcA-34. Ten protein elution spikes were obtained: five showed enzymatic activity. Elution spikes I and II attained the highest values of specific enzymatic activity 6.45 and 6.98 U/mg of protein, respectively. Next were spikes X and III with similar values 2.55 and 2.63 U/mg of protein, and spike IV presented the lowest value of 0.86 U/mg of protein. The five spikes were used to immunize hamsters which were challenged intrahepatically, four weeks later, with 3 x 10(5) trophozoites of E. histolytica. A control group of animals not immunized underwent intrahepatic challenge with the same number of amebae. The proteins with enzymatic activity contained in elution spikes I and II conferred immunologic protection in 100% of the animals, while elution spikes X and III were protective in 50 to 63%, and spike IV gave the lowest value of 37%. It can be assumed that there is an antienzyme antibody responsible for the absence of hepatic abscesses in the immunized hamsters.

Antigenic probes locate binding sites for the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase on the actin monomer in microfilaments.

The topology of the interfaces between actin monomers in microfilaments and three glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase) was investigated using several specific antibodies directed against precisely located sequences in actin. A major contact area for glyceraldehyde-3-phosphate dehydrogenase was characterized in a region near residue 103. This interaction altered, by long-range conformational changes, the reactivity of antigenic epitopes in the C-terminal part of actin. The interface between actin and aldolase appeared to involve a sequence around residue 299 in the C-terminal region of actin. The interaction of phosphofructokinase, in contrast, modified the reactivity of all antibodies tested. Finally, the phosphagen kinases arginine kinase and creatine kinase showed no interaction with the microfilament.

Molecular characterization of four forms of phosphofructokinase purified from potato tuber.

Four forms of phosphofructokinase (PFK) have been purified to apparent homogeneity from tubers of potato (Solanum tuberosum cv. Record). Each had a final specific activity of about 200 mumol.min-1.mg-1 protein. Similar forms of PFK were found in partially purified extracts from tubers and leaves of other potato cultivars and related wild species. The relative molecular masses of three forms of PFK were about 200,000 whereas that of the fourth PFK was greater than 800,000. The four forms of PFK contained different proportions of four polypeptides which had apparent relative molecular masses of 46,300, 49,500, 50,000, and 53,000. These polypeptides gave different patterns of peptide fragments after chemical and proteolytic cleavage. Western blots and immunoprecipitation studies using antibodies raised against the individual polypeptides showed that all four are associated with PFK. Thus, potato tubers contain four distinct forms of PFK that differ in their subunit composition.

Identification of the AMP binding sites of rabbit phosphofructo-1-kinase isozymes B and C.

Rabbit liver phosphofructo-1-kinase, designated isozyme B, and rabbit brain phosphofructokinase, which contains all three isozymes as heteropolymers, have been modified by [14C]fluorosulfonylbenzoyladenosine (FSBAdo). Several lines of evidence supported modification at the binding site for AMP. The modification proceeded to the extent of 2 to 4 mol of reagent incorporated per mol of tetramer, and AMP protected against the reaction. The kinetic properties of modified isozymes A and B and of modified brain phosphofructokinase were examined and compared to their unmodified forms. It was observed that modification greatly diminished ATP inhibition of all of the isozymes. Furthermore, equilibrium binding studies of modified phosphofructokinase B showed a greatly diminished capacity and affinity for cyclic AMP. Cyclic AMP had little or no influence on the properties of modified A isozyme or brain phosphofructokinase, but was capable of further deinhibiting modified B isozyme, apparently at sites remaining unmodified by FSBAdo. Phosphofructokinase B, modified by radiolabeled FSBAdo, was digested by trypsin, and the digest separated by high-pressure liquid chromatography. The labeled peptide was isolated and sequenced to provide the sequence: Asn-Tyr-Gly-Thr-Lys-Leu-Gly-Val-Lys, with the lysine in the fifth position being the site of modification. To isolate isozyme C, a monoclonal antibody to this isozyme was produced by injecting purified rabbit brain phosphofructokinase into mice, and subsequently selecting for those clones that recognized brain phosphofructokinase but not purified phosphofructokinases A and B. The selected monoclonal was specific for native rabbit isozyme C and would not recognize mouse or rat brain phosphofructokinases. Linking the antibody to an inert phase provided an efficient means of purifying rabbit isozyme C from rabbit brain. The enzyme so recovered retained little of its original activity, but the method provided a simple technique for the preparation of enzyme for protein chemistry studies. The modified C isozyme was isolated on the immuno-affinity column and digested with trypsin. A tryptic peptide bearing the label was isolated and sequenced to provide the structure: Asn-Phe-Gly-Thr-Lys-Ile-Ser-Ala-Arg, with position 5 being the site of modification. The sequences of isozymes B and C are homologous to the site of modification of the A isozyme by FSBAdo.

Immunological and biochemical studies on the relationship between two actin-binding proteins, phosphofructokinase and gelsolin.

Phosphofructokinase and gelsolin-like proteins coexist in many muscle and non-muscle tissues. They are both actin-binding proteins, and some of their biochemical parameters are remarkably similar. In a previous report [Füchtbauer, A., Jockusch, B. M., Leberer, E. & Pette, D. (1986) Proc. Natl Acad. Sci. USA 83, 9502-9506] it was shown that phosphofructokinase preparations contained actin-filament-severin activities characteristic for gelsolin. Therefore, we investigated a possible relationship between these proteins with respect to their actin-binding properties. Immunoblotting experiments with specific and non-cross-reacting antibodies to both proteins revealed two distinct polypeptides with slightly different molecular mass in SDS-PAGE of crude extracts from rabbit skeletal muscle, indicating that phosphofructokinase and gelsolin are not identical. An actin-filament-severing activity as well as the component detected by anti-gelsolin were found to copurify with phosphofructokinase during its preparation. However, the presumptive gelsolin was completely eliminated after a heat-denaturation step leaving the phosphofructokinase activity unaffected. Purified phosphofructokinase had no effects on the polymer state of preformed actin filaments. Unlike gelsolin, phosphofructokinase did not promote nucleation of actin polymerization but delayed the nucleation step. We therefore conclude that phosphofructokinase and gelsolin are functionally and structurally distinct proteins.

Analysis of the phosphofructokinase subunits and isoenzymes in human tissues.

The 6-phosphofructo-1-kinase (PFK) subunits and isoenzymes were studied in human muscle, heart, brain, liver, platelets, fibroblasts, erythrocytes, placenta and umbilical cord. In each tissue, the subunit types in the native isoenzymes were characterized by immunological titration with subunit-specific antibodies and by column chromatography on QAE (quaternary aminoethyl)-Sephadex. Further, the subunits of the partially purified native isoenzymes were resolved by SDS/polyacrylamide-gel electrophoresis, identified by immunoblotting, and quantified by scanning gel densitometry of silver-stained gels and immunoblots. Depending on the type of tissue, one to three subunits were detected. The Mr values of the L, M and C subunits regardless of tissue were 76,700 +/- 1400, 82,500 +/- 1640 and 86,500 +/- 1620. Of the tissues studied, only the muscle PFK isoenzymes exhibited one subunit, which was the M-type subunit. Of the other tissues studied, the PFK isoenzymes contained various amounts of all three subunits. Considering the properties of the native PFK isoenzymes, it is clear that, in human tissues, they are not simply various combinations of two or three homotetrameric isoenzymes, but complex mixtures of homotetramers and heterotetramers. The kinetic/regulatory properties of the various isoenzyme pools were found to be dependent on subunit composition.

Phosphofructokinase from Dirofilaria immitis. Stimulation of activity by phosphorylation with cyclic AMP-dependent protein kinase.

Phosphofructokinase has been partially purified from the filariid helminth, Dirofilaria immitis, using ion exchange and affinity chromatography. The D. immitis phosphofructokinase cross-reacted with antibodies prepared against the phosphofructokinase from Ascaris suum. These antibodies had been bound to agarose beads. The enzyme was eluted from the immobilized antigen-antibody complex by denaturing agents, and the subunit molecular weight determined by sodium dodecyl sulfate gel electrophoresis was identical to that of the ascarid enzyme, 90,000. At pH 6.8, substrate saturation curves of the filarial phosphofructokinase with ATP revealed that the enzyme was inhibited by ATP. The fructose-6-P saturation curve was sigmoid at all ATP levels tested. Phosphorylation of the D. immitis phosphofructokinase by the catalytic subunit of beef heart cyclic AMP-dependent protein kinase resulted in incorporation of 0.8 mol of phosphate/mol of subunit and in a 3-4-fold increase in catalytic activity when measured at pH 6.8 at inhibitory levels of ATP. Additional kinetic studies revealed that the phosphorylated enzyme was less susceptible to ATP inhibition than was the nonphosphorylated form. It is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of carbohydrate metabolism in the filarial as well as the intestinal-dwelling nematodes.

A novel type of phosphofructokinase from plants.

A phosphofructokinase (PFK) has been purified to homogeneity from carrot roots as a large aggregated form (molecular weight greater than 5 million). The purified plant PFK, seemingly the cytosolic form, differed from its mammalian counterpart in a lower subunit molecular weight (60,000 verses 80,000), in being only sluggishly activated by fructose-2,6-bisphosphate, and in immunological properties. Similar to liver PFK, the purified carrot PFK could be dissociated by addition of 5 mM ATP to small and intermediate forms (respective molecular mass values of 2.4 X 10(5) and 6 X 10(5) Da). These small and intermediate forms could partially reassociate to the original large form in the presence of 5 mM Fru-6-P. Alkaline pH also effected the dissociation of the large and intermediate forms to the small form of PFK. All forms were present in significant amounts in freshly prepared carrot root extracts. The different forms of PFK showed characteristic pH activity profiles with pH optima of 8.6 (small form), 5.5 and 9.0 (intermediate form), and 7.0 and 8.5 (large forms). As alkaline pH (greater than or equal to approximately 8.5) dissociated the large and intermediate enzyme forms to yield the small form, it was concluded the "true" pH optima of the intermediate and large forms are pH 5.5 and 7.0, respectively. The pH optimum displayed by the intermediate and large forms in the alkaline region (pH 8.5-9.0) was considered to be due to their dissociation during assay. The different forms of PFK also had dissimilar regulatory properties, each showing a characteristic response to ATP, citrate, and Pi, but all were sensitive to inhibition by phosphoenolpyruvate and NADPH. Leaf cytosolic PFK, partially purified from spinach, showed similar properties. The results suggest that metabolite-dependent aggregation-disaggregation is a mechanism whereby plants regulate the activity of cytosolic PFK and the accompanying rate of glycolytic carbon flux.

Localization of binding sites of F-protein (phosphofructokinase) on the myosin molecule.

To determine the location of F-protein binding sites on myosin, the interaction of F-protein with myosin and its proteolytic fragments in 0.1 M KCl, 10 mM potassium phosphate, pH 6.5, has been investigated using sedimentation, electron microscopy and optical diffraction methods. Sedimentation experiments show that F-protein can bind to myosin and myosin rod rather than to light meromyosin or subfragment-1. The F-protein binding to myosin and rod is of a similar character. The calculated values of the constants of F-protein binding to myosin and rod are 2.6 X 10(5) M-1 and 2.1 X 10(5) M-1, respectively. The binding sites are probably located on the subfragment-2 portion of the myosin molecule. The number of the F-protein binding sites calculated per chain weight of 80,000 is 5 +/- 1. Electron microscopic observations confirm the sedimentation results. F-protein does not bind to light meromyosin paracrystals, but decorates myosin and rod filaments with the interval of 14.3 nm regardless of whether F-protein is added prior to or after filamentogenesis. The comparison of optical diffraction patterns obtained from myosin and rod filaments with those from decorated ones reveals the marked enhancement of meridional reflection at (14.3 nm)-1 in the latter case. Neither the increase in ionic strength from 0.1 to 0.15 and pH from 6.5 to 7.3 nor substitution of potassium phosphate buffer by imidazole-HCl buffer, or Tris-HCl influences F-protein binding to myosin and rod filaments as visualized by electron microscopy. The possible significance of F-protein location in the thick filament structure is discussed.

Actin-severing activity copurifies with phosphofructokinase.

Microinjection of muscle 6-phosphofructokinase (PFK; EC 2.7.1.11) into tissue culture cells led to a reversible disintegration of microfilament bundles (stress fibers). The mode of disruption as well as of recovery of stress fibers was very similar to that found previously in experiments performed with the actin-severing protein brevin, an extracellular variant of gelsolin. PFK, like brevin, was also capable of disrupting stress fibers in detergent-extracted cells and in ethanol-fixed cells, in a Ca2+-dependent manner. When compared with heart muscle gelsolin, PFK comigrated with the 85- to 90-kDa band. Antibodies against PFK crossreacted with gelsolin from the same species. These results point to a tight association between polypeptides with similar biochemical and immunological parameters present in both preparations. They suggest hitherto unexpected cellular control mechanisms for both microfilament functions and glycolysis.

Isoenzymes of phosphofructokinase in the rat. Demonstration of the three non-identical subunits by biochemical, immunochemical and kinetic studies.

In man and the rabbit, 6-phosphofructokinase (PFK, EC 2.7.1.11) exists in tetrameric isoenzymic forms composed of muscle (M or A), liver (L or B) and platelet or brain (P or C) subunits, which are under separate genetic control. In contrast, the genetic control of the rat PFK has not yet been conclusively established; it is unclear whether the P-type or C-type subunit exists in this species. To resolve this question, we investigated the enzyme from the skeletal muscle, liver and brain of rats of Wag/Rij strain. Our studies demonstrate that the rat PFK is also under the control of three structural loci and that the homotetramers M4, P4 and L4 exhibit unique chromatographic, immunological and kinetic-regulatory properties. Skeletal-muscle and brain PFKs consist of isolated M4 and P4 homotetramers respectively. Although liver PFK consists predominantly of L4 homotetramer, it also contains small amounts of PL3 and P2L2 species. All three PFKs exhibit allosteric properties: co-operativity with fructose 6-phosphate and inhibition by ATP decrease in the order P4 greater than L4 greater than M4. P4 and M4 tetramers are the most sensitive to citrate inhibition, whereas L4 tetramer is the least sensitive. More importantly, P4 and L4 isoenzymes are the most sensitive to activation by fructose 2,6-bisphosphate, whereas M4 isoenzyme is the least sensitive. These results indicate that the brain PFK in this strain of rat is a unique tetramer, P4, which also exhibits allosteric kinetics, as do the well-studied M4 and L4 isoenzymes. The reported differences in the number and nature of isoenzymes present in the rat brain and liver most probably reflect the differences in the strains studied by previous investigators. Since the nature of the rat PFK isoenzymes and nomenclatures reported by previous investigators have been now reconciled, it is proposed that, for the sake of uniformity, only well-established nomenclatures used for the rabbit or human PFK isoenzymes be used for the rat isoenzymes.