6-Phosphofructo-1-kinase isoenzymes of the jejunal mucosa of rabbit, rat and mouse

1. 6-Phosphofructo-1-kinase (PFK) isoenzymes were studied in the jejunal mucosa of rabbit, rat and mouse. 2. The rat mucosal enzyme was found to be very similar to, although not identical with, the mouse mucosal enzyme, as the physical and regulatory properties of these two enzymes were nearly similar except that the immunological studies were dissimilar. 3. PFK prepared from rabbit mucosa showed different and distinct properties from the rat and mouse mucosal PFK when studied by (NH4)2SO4-precipitation, polyacrylamide gel electrophoresis, immunological cross-reactivity and regulatory properties. 4. The difference between the rabbit enzyme and the rat or mouse enzymes is suggested to be due to the lower rate of glycolysis observed in the rabbit jejunal mucosa as the total enzyme activities of the rabbit were found to be less than half of those activities of the rat and mouse mucosa. 5. The dissimilarities among the species in mucosal isoenzymes obtained in the present study are rather expected since the term isoenzyme is now properly reserved for forms that have been shown to be genetically distinct as shown for different tissues in the same species. Such multigenic control does not appear to have been established for the same tissue in different species.     

Phosphofructokinase from the epithelial cells of rat small intestine. Comparison of regulatory properties with those of skeletal muscle, liver and brain phosphofructokinase

The regulatory properties of phosphofructokinase from rat mucosa, liver, brain and muscle were investigated. Mucosal phosphofructokinase displayed cooperativity with respect to fructose 6-phosphate at pH 7.0 and so did the muscle, brain and liver isoenzymes. All these four isoenzymes were inhibited by ATP, the mucosal isoenzyme being the least inhibited. They were also inhibited by citrate and creatine phosphate. AMP, ADP, glucose 1,6-diphosphate, fructose 2,6-bisphosphate and inorganic phosphate were all strong activators for the mucosal, brain, liver and muscle phosphofructokinase, but the mucosal isoenzyme was found to be more activated than the others, accounting for the higher rates of glycolysis observed in mucosa. The results suggest that mucosal phosphofructokinase is unique and different from all the other isoenzymes.     

The purification, characterization and regulatory properties of liver phosphofructokinase in the Arabian one-humped camel (Camelus dromedarius)

1. Phosphofructokinase from camel liver was purified to homogeneity more than 3600-fold, and the yield of the preparation was 46%. 2.The sodium dodecyl sulphate-treated purified enzyme migrated as a single band in 10% polyacrylamide gel. 3. The enzyme is a tetramer, with a monomer Mr 90,000. 4. The regulatory properties of the purified enzyme from camel liver were studied at pH 7.0. 5. The enzyme displayed cooperativity with respect to fructose 6-phosphate and was inhibited by high concentrations of ATP. 6. The enzyme was also inhibited by citrate, phosphocreatine and 2,3-bisphosphoglycerate. 7. On the other hand, ADP, AMP, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate were all found to be strong activators for camel liver phosphofructokinase.     

A Rapid Purification Procedure for Camel Kidney Glutathione S-Transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37, 000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 μ;mol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100.

The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29, 000 D and 26, 000 D to give a native molecular weight of 55, 000 D.

The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. l-chloro-2, 4-dinltrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse.

Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

A rapid purification procedure for camel kidney glutathione S-transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37,000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 mumol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100. The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29,000 D and 26,000 D to give a native molecular weight of 55,000 D. The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. 1-chloro-2,4-dinitrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse. Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Tissue Specific Expression of Glutathione S-transferases, Glutathione Content and Lipid Peroxidation in Camel Tissues

Differential expression of glutathione S-transferase (GST) enzyme activity in various tissues of the camel was observed with a maximum activity in the liver. Compared with the rat and human livers, GST activity in camel liver was 50% lower than that of rat liver and similar to that of human liver. Extrahepatic tissues in camel have a comparable GST activity with those of similar tissues in the rat. Assay of GST activity using ethacrynic acid as substrate demonstrated maximum activity in the camel brain followed by intestine, liver and kidney. Microsomal GST activity in camel tissues was expressed in the order of liver > testis > intestine ≈ kidney ≈ brain. Phenotyping of GST was performed in camel hepatic and extrahepatic tissues using human specific antibodies to class α, μ, and π cytosolic GST isoenzymes and rat specific antibody to the microsomal GST. Western immunoblot and immunohistochemical analyses showed an abundant expression of GST α and μ in the camel liver, while π was very poorly expressed. Camel extrahepatic tissues however, had a significant expression of GST π. The camel GST isoenzymes were found to be predominantly expressed in the hepatocytes around the central vein with a gradual decrease in expression in the hepatocytes located toward the periphery. Kidney cortex exhibited a greater expression of the enzyme protein in the proximal tubules as compared to the glomeruli. Glutathione (GSH) concentration in rat tissues, except in the brain, was about 2-fold higher than that of camel tissues. Rate of NADPH-dependent microsomal lipid peroxidation was comparable both in the rat and camel tissues with the highest activity in the brain and lowest activity in the intestine. The differential expression of GST isoenzymes in different organs of the camel, GSH concentration and the rate of lipid peroxidation in different tissues may be important factors in determining the differential susceptibility of camel tissues to the toxic effects of xenobiotics.     

Multiple forms of phosphofructokinase in developing rabbit and guinea pig tissues.

Multiple forms of phosphofructokinase in striated muscle and cardiac muscle of developing rabbit (Oryctolagus cuniculus) undergo changes with development, but not in brain and liver. The cardiac muscle of the 1-day-old rabbit contains phosphofructokinase A₄ together with the four hybrid forms which were tentatively called A₃C, A₂C₂, AC₃, and C₄. In older animals, phosphofructokinase C₄ disappears first, followed by the hybrid forms, and only phosphofructokinase A₄ persists in the adult animal. Both phosphofructokinase A₄ and phosphofructokinase C₄, as well as their hybrid forms, are present in developing embryonic brain and also in the brains of adult animals. Developing rabbit liver contains a single form of phosphofructokinase, but two isoenzymes are consistently seen in guinea pig liver. In striated muscle from fetal and 1-day-old rabbit, two isoenzymes are found, tentatively identified as A₄ and the A₃C hybrid. The results suggest that fetal phosphofructokinase A₄ and phosphofructokinase C₄, and their hybrids, might be present in striated muscle. Guinea pig tissues show a pattern of phosphofructokinase isoenzymes different from that in rabbit tissues.     

Phosphofructokinase D from the epithelial cells of rat small intestine.

Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.     

The A2B2 hybrid isozyme of phosphofructokinase.

The hybrid isozyme of phosphofructokinase, A2B2, was formed by incubation of rabbit muscle enzyme. A4, and rabbit liver enzyme, B4, in the presence of sodium citrate at neutral pH. The enzyme composition of the resulting mixture of A2B2 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-P2, fructose-6-P, and high concentrations of MgATP. The A2B2 isozyme was separated from A4 and B4 by ion exchange chromatography. The kinetic regulatory properties of A2B2 were compared with those of A4, B4, and a 1:1 mixture of A4 and B4. ATP inhibition of A2B2 was intermediate between that observed with A4 and B4 and was clearly not identical to a simple summing of the effects of A and B subunits. Similar comparisons were made using other phosphofructokinase inhibitors, citrate, 2,3-P2-glycerate, and P-creatine. In each case the observed inhibition was intermediate between the observed with A4 and B4. The existence in a number of tissues of phosphofructokinase A2B2 provides added diversity to the regulatory mechanisms of glycolysis.     

Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.     

Fructose 2,6-bisphosphate. A new activator of phosphofructokinase

A new activator of rat liver phosphofructokinase was partially purified from rat hepatocyte extracts by DEAE-Sephadex chromatography. The activator, which eluted in the sugar diphosphate region, was sensitive to acid treatment but resistant to heating in alkali. Mild acid hydrolysis resulted in the appearance of a sugar monophosphate which was identified as fructose 6-phosphate by gas chromatography/mass spectroscopy. These observations suggest that the activator is fructose 2,6-bisphosphate. This compound was synthesized by first reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide and then treating the cyclic intermediate with alkali. The structure of the synthetic compound was definitively identified as fructose 2,6-bisphosphate by 13C NMR spectroscopy. Fructose 2,6-bisphosphate had properties identical with those of the activator purified from hepatocyte extracts. It activated both the rat liver and rabbit skeletal muscle enzyme in the 0.1 microM range and was several orders of magnitude more effective than fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate was not a substrate for aldolase or fructose 1,6-bisphosphatase. It is likely that this new activator is an important physiologic factor of phosphofructokinase in vivo.     

A comparative study of the rate-limiting enzymes of cholesterol synthesis, esterification and catabolism in the rat and rabbit

1. The rat and rabbit are amongst the animal models most widely used in the study of human atherosclerosis, a disease state correlating with disturbances in cholesterol metabolism. 2. In order to relate the key regulatory enzymes of cholesterol synthesis, esterification and catabolism in the rat and rabbit to their differing degree of susceptibility to atherosclerosis, enzyme levels and their properties were determined in liver and intestine of both species. 3. Hepatic HMG CoA reductase and cholesterol 7 alpha-hydroxylase levels were significantly higher in the rat than in the rabbit, while intestinal HMG CoA reductase activity in the two species was comparable. Conversely, the capacity to esterify cholesterol as measured by ACAT activities was considerably greater in both sites in the rabbit compared to the rat. 4. The data suggest that differences in the key regulatory enzymes of cholesterol metabolism in both liver and intestine may reflect different methods of cholesterol utilization in the two species.     

Modulation of lactate dehydrogenase activity by enzyme-protein interaction

Some lactate dehydrogenase modulator proteins have been isolated from the lactate dehydrogenase-free crude mitochondrial fraction of rabbit muscle, beef liver and chicken liver. It was shown that beef and chicken liver mitochondrial extracts exhibited activatory capacity in contrast to the inhibitory capacity of rabbit muscle mitochondrial extracts. All modulators can be precipitated by 80% ammonium sulphate saturation and show high anodic electrophoretic mobility and heat stability. Modulators have higher affinity for alkaline pI lactate dehydrogenase isoenzymes, independent of whether the M and H subunits are predominant. The inhibitor and the activator molecules compete for lactate dehydrogenase since their modulatory capacity was nullified when similar relative amounts were used. This study shows the existence of analogous proteins with an acidic pI in the different mitochondrial fractions which modify lactate dehydrogenase activity.     

Rat thyroid phosphofructokinase. Comparison of the regulatory and molecular properties with those of rat muscle enzyme.

The kinetic and molecular properties of rat thyroid phosphofructokinase (specific activity 134 units/mg) were compared with those of rat muscle phosphofructokinase (specific activity 135 units/mg). Thyroid and muscle phosphofructokinase showed similar sedimentation patterns in sucrose density gradients; their affinity for DEAE-cellulose was similar but not identical. A comparison of the kinetic properties revealed differences in the pH optima. Striking differences in the kinetic properties were shown below pH 7.4; the thyroid enzyme was less inhibited by ATP or citrate and more sensitive to activation by cyclic 3':5'-AMP than the muscle enzyme. A study of the effects of some cyclic as well as linear mononucleotides, such as cyclic AMP, cyclic IMP, cyclic GMP, cyclic CMP, cyclic UMP, 5'-AMP, and 3'-AMP on thyroid phosphofructokinase showed that at concentrations as low as 1 micrometer only cyclic AMP and cyclic IMP were able to activate thyroid enzyme in the presence of low fructose-6-P and high ATP concentrations.     

Differences in phosphofructokinase regulation in normal and tumor rat thyroid cells.

The kinetic and molecular properties of a phosphofructokinase derived from a transplantable rat thyroid tumor lacking regulatory control on the glycolytic pathway were studied. The properties of the near-purified enzyme (specific activity 140 units/mg) were compared with those of phosphofructokinase from normal rat thyroid (specific activity 134 units/mg). The electrophoretic mobilities and gel elution behavior of these two enzymes were almost similar. The thyroid tumor phosphofructokinase showed, however, a greater degree of size and/or shape heterogeneity in the presence of ATP than the normal thyroid enzyme, as determined by gel filtration and sucrose density gradient centrifugation. Kinetic studies below pH 7.4 showed a sigmoid response curve for both enzymes when the velocity was determined at 1 mM ATP with varying levels of fructose-6-P. The interaction coefficient, however, was 4.2 and 2.6 for normal and tumor thyroid phosphofructokinase, respectively. Ammonium sulfate decreased the cooperative interactions with the substrate fructose-6-P in both enzymes. The thyroid tumor enzyme, however, was less sensitive to the inhibition by ATP and by citrate. The reversal of citrate inhibition by cyclic 3':5'-adenosine monophosphate was also less effective with the thyroid tumor phosphofructokinase, while the protective effect of fructose-6-P was stronger. The difference in citrate inhibition between tumor and normal thyroid enzyme was not strongly affected by varying the MgCl2 concentration up to 10 mM. It is concluded that the complex allosteric regulation typical of the normal thyroid phosphofructokinase is still present in the enzyme isolated from the thyroid tumor tissue. The latter, however, is more loosely controlled by its physiological effectors, such as ATP, citrate, and cyclic AMP.     

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.     

Phosphorylation of phosphofructokinase by protein kinase C changes the allosteric properties of the enzyme

The Ca2+- and phospholipid-dependent protein kinase C from rat brain phosphorylates rabbit muscle phosphofructokinase at the same trypsin-labile site as cyclic AMP-dependent protein kinase. However, protein kinase C also effectively phosphorylates one or more separate sites. Incubation of phosphofructokinase in the presence of protein kinase C, phospholipids, Ca2+, and ATP appears to affect the allosteric properties of phosphofructokinase by shifting the fructose 6-phosphate saturation curve to lower substrate concentrations in a time-dependent manner and decreasing cooperativity of the enzyme.     

Regulation of rat liver phosphofructokinase by glucagon-induced phosphorylation

In perfused rat liver, the effects of various hormones on the stimulation of phosphorylation and allosteric properties of purified phosphorfructokinase were investigated. Rat livers were perfused with [³²P]phosphate followed with various hormones or cyclicAMP, and ³²P-labeled phosphofructokinase was isolated. ³²P incorporation into the enzyme and enzyme inhibition by ATP or citrate were determined. Only glucagon increased the ³²P incorporation into phosphofructokinase and this increase was approximately threefold. The cyclicAMP level was increased simultaneously approximately four- to fivefold compared to the control perfused liver. Similar results were obtained by perfusing the liver with cyclicAMP (0.1 mm). The phosphorylated phosphofructokinase showed a decrease in the K_i values for ATP (from 0.4 to 0.2 mm) and citrate (from 2 to 0.6 mm). Neither epinephrine nor insulin affected the extent of phosphorylation or the allosteric properties of the enzyme. The half-maximal concentration of glucagon required for phosphorylation of phosphofructokinase and modification of its allosteric properties was approximately 6 × 10^?11m. It is concluded that glucagon increases the inhibition of liver phosphofructokinase by ATP and citrate through phosphorylation of the enzyme involving a β-receptor-mediated cyclicAMP-dependent mechanism.     

Relationship between fructose 2,6-bisphosphate activation and MgATP inhibition of rat liver phosphofructokinase at high pH. Kinetic evidence for individual binding sites linked by finite couplings

The concentration of fructose 6-phosphate required to produce half-maximal velocity of rat liver phosphofructokinase at pH 9 (Ka) has been measured at 110 different combinations of MgATP and fructose 2,6-bisphosphate (Fru-2,6-BP) concentrations spanning the range 0.1-100 mM and 0.003-100 microM, respectively. The data have been evaluated by nonlinear regression to an equation resulting from a linked-function analysis of an enzyme capable of binding three ligands simultaneously at separate sites. In addition, the data have been fit to equations, derived from the linked-function expression, that would result if various combinations of antagonistic ligands were unable to bind to the enzyme simultaneously, even at high concentration, either because they compete for a single binding site or because they bind exclusively to different conformational forms of the enzyme. The complete linked-function equation is able to predict the Ka for rat liver phosphofructokinase as a function of any Fru-2,6-BP and/or MgATP concentration significantly better than any of the alternatives examined, particularly at high concentrations of one or both modifier ligands. The free energy couplings between all three possible pairs of ligands are of quite moderate magnitude, especially when the multiplicity of binding sites for each ligand that actually exists on the functional enzyme is considered. Therefore, we conclude that any explanation of the action of Fru-2,6-BP and MgATP by a model more elaborate than the simple linked-function case considered herein cannot be simplified by assuming that the properties of rat liver phosphofructokinase result from an equilibrium of limiting conformational states that exhibit exclusive binding properties.     

Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate.

Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.