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Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its amino-acid sequence is more similar to pyrophosphate (PPi)-dependent PFKs than to other ATP-dependent PFKs. a phylogenetic analysis suggests that the enzyme must have been derived from a PPi-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the chage in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary hitory, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs. [ABSTRACT FROM AUTHOR]

The paper describes a simple kinetic model of an open monosubstrate enzyme reaction with substrate inhibition and product activation. A comparison between the model and the phosphofructokinase reaction shows a close resemblance between their dynamical properties. This makes it possible to explain qualitatively most experimental data on single-frequency oscillations in glycolysis. A mathematical analysis of the model has shown the following. In the model, at a definite relationship between the parameters, self-oscillations arise. The condition of self-excitation is satisfied more readily with a lower source rate, larger product sink rate constants, lower product-enzyme affinity and higher enzyme activity. Self-oscillations exist only in a certain range of values of the parameter determining the degree of substrate inhibition. This range increases with decreasing source rate. Too strong or, conversely, too weak substrate inhibition leads to damped oscillations. The period of self-oscillations depends on the degree of substrate inhibition, the source rate, the sink rate constant, the enzyme activity, the affinity of the substrate and the product for the enzyme; it decreases with an increase in these values. With an increase in the relative sink rate constant the steady state amplitude of self-oscillations initially increases until a definite maximum is reached and then drops to zero. A self-oscillatory state in the phosphofructokinase reaction exists only when the maximum rate of this reaction is essentially higher than the source rate, and lower than the maximum rate of the reactions controlling the sink of the products. An experimental investigation of self-oscillations in the phosphofructokinase reaction may be considerably simplified by using a reconstituted system consisting of a small number of reactions with an irreversible sink of the products and artificial substrate supply. In this case the above relationship (section 6) should hold. [ABSTRACT FROM AUTHOR]

Reports on the purification and characterization of phosphofructokinase 2 and fructose 2,6-bisphosphatase extracted from chicken liver of pigeon muscle. Revelation of two homgenous proteins acting as dimers; Effect of chicken liver enzyme with the protein kinase subunit on mol enzyme incorporation; Phosphorylation of pigeon muscle; Absence of effect of hydrogen-ion changes on avian liver phosphofructokinase 2.

The characterization of the gene encoding Leishmania donovani phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported. L. donovani has a single PFK gene copy per haploid genome that encodes a polypeptide with a deduced molecular mass of 53 988 and a pI of 9.26. The predicted amino acid sequence contains a C-terminal tripeptide that conforms to an established signal for glycosome targeting. L. donovani PFK showed most sequence similarity to inorganic pyrophosphate (PPi )-dependent PFKs, despite being ATP-dependent. It thereby resembles PFKs from other Kinetoplastida such as Trypanosoma brucei , Trypanoplasma borreli (characterized in this study), and a PFK found in Entamoeba histolytica . It exhibited hyperbolic kinetics with respect to ATP whereas the binding of the other substrate, fructose 6-phosphate, showed slight positive cooperativity. PPi , even at high concentrations, did not have any effect. AMP acted as an activator of PFK, shifting its kinetics for fructose 6-phosphate from slightly sigmoid to hyperbolic, and increasing considerably the affinity for this substrate, whereas GDP did not have any effect. Modelling studies and site-directed mutagenesis were employed to shed light on the structural basis for the AMP effector specificity and on ATP/PPi specificity among PFKs. [ABSTRACT FROM AUTHOR]

Treatment of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with the arginine- specific reagent, phenylglyoxal, irreversibly inactivated both 6-phospbofructo-2-kinase and fructose- 6-bisphosphatase in a time-dependent and dose-dependent manner. Fructose 6-phosphate protected against 2,6-phosphofructo-2-kinase inactivation, whereas MgGTP protected against fructose-2,6- bisphosphatase inactivation. Semi-logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non-linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 tool/tool enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic.AMP-dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic-AMP-dependent protein kinase interacts with a modified arginine residue. Chymotryptic cleavage of the modified protein and microsequencing showed that Arg225, in the 6-phosphofructo-2-kinase domain, was one of the residues modified by phenylglyoxal. The protection by fructose 6-phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in fructose 6-phosphate binding in the 6-phosphofructo-2-kinase domain of the bifunctional enzyme. [ABSTRACT FROM AUTHOR]

We describe here the genetic and biochemical analyses of two classes of mutations in the soluble phosphofructokinase (PFK I) of Saccharomyces cerevisiae: those leading to the loss of activity and those giving rise to a kinetically altered enzyme. Complementation and allele-testing between these two classes of mutants show that loss of enzyme activity in vitro can come about not only by mutations in the catalytic subunit but also in the regulatory subunit. Also, a mutation in the catalytic subunit can give rise to an enzyme altered in its kinetic properties in a manner phenomenologically similar to that caused by a mutation in the regulatory subunit. The results of the complementation studies in diploids suggest that, in spite of their distinct functions, both the subunits are essential for activity to be detected in vitro. This is confirmed by the reconstitution of an active PFK I enzyme by mixing cell-free extracts of two complementing parents, each of which lacks the enzyme activity. PFK activity appears in the mixture, reaching a maximum value of 60-100% of that of the diploid in 15-30 min at 24° C. Unlike the catalytic subunit which exists in various multimeric states in cell-free extracts of the mutant bearing only this subunit, the regulatory subunit exists largely as a monomer in a mutant devoid of the catalytic subunit. The reconstituted enzyme, however, is indistinguishable from that of the wild type, as analysed by sedimentation studies and Western blot analysis, demonstrating that only the heteromeric complex of the two subunits is active, while neither of the individual subunits displays activity in vitro. [ABSTRACT FROM AUTHOR]

A 5S-rRNA-containing ribonucleoprotein was purified to homogeneity from a rabbit muscle extract through its affinity to phosphofructokinase-1 and then stucturally characterized. This RNP was compared to the 5S- rRNA-containing ribonucleoprotein extracted from rabbit liver ribosomal 60S subunits with EDTA. Analytical gel filtration revealed a molecular mass of 70-80 kDa for both complexes. Gel electrophoresis of the ribosomal complex revealed three protein components, one migrating as a band of 35 kDa and two other small polypeptides of apparently 16.5 kDa and 17.5 kDa. In the sarcoplasmic RNP these small polypeptides were absent. However. besides a major component of 35 kDa, up to five slightly larger and smaller species of 31.5-36.5 kDa were detected. Despite this heterogeneity, only one N-terminal amino acid sequence was obtained for the isolated sarcoplasmic protein, suggesting a C-terminal heterogeneity of one single polypeptide. Within the first 46 amino acid residues no difference between the sequences of the isolated 35-kDa components of sarcoplasmic and ribosomal complexes was found. Homology criteria indicated that this component belongs to the ribosomal protein L5 family. The RNA was identified by complete enzymatic sequencing as 5S rRNA; it was also identical in both complexes and is strongly homologous to 5S rRNA of man. Both L5 - 5S-RNA complexes could be resolved by hydroxyapatite chromatography into three species still consisting of both protein and RNA. 5'-Terminal dephosphorylation experiments showed that this heterogeneity is exclusively due to the differing number (1 -3) of 5-terminal phosphates. The two additional low-molecular-mass proteins were stably associated to the ribosomal RNP at high salt concentrations in a stoichiometry of about 2:1. They were identified as the acidic phosphoproteins P2/β by N-terminal sequencing. High phosphate concentrations facilitated their dissociation from the L5 - 5S-RNA complex. For the sarcoplasmic L5 - 5S-RNA complex a hitherto unknown interaction with phosphofructokinase-1, affecting the enzymatic properties, was demonstrated. [ABSTRACT FROM AUTHOR]

Reports on the reaction of monoclonal antibodies with phosphofructokinase 2/fructose 2,6-bisphosphatase. Derivation of monoclonal antibodies from immunized mice; Discovery of clones that secrete antibodies that react with immunabsorbent assay antigens; Use of subcloned clones for analyzing antibody reaction to bifunctional enzymes; Evidence of bifunctionality by the enzyme through the similar activities of the cloned antibodies.

Reports on the development of a method for the simultaneous purification of hexokinase, glucosephosphate isomerase, phosphofructokinase, fructose-1,6-biphosphate adolase, triosephosphate isomerase, D-glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase and glycerol kinase from Trypanosoma brucei. Preparation of crude glycosomes by differential centrifugation of cell homogenates; Features of the glycosomal glycolytic enzymes.

The initial kinetics of yeast phosphofructokinase was studied by stopped-flow measurements over an enzyme concentration range from 0.5 mg/ml to 0.01 mg/ml. Before attaining the steady state the reaction showed a lag phase in the product formation, the duration of which was found to decrease with increasing enzyme concentration. The lag phase disappeared after preincubation of the enzyme for at least five minutes with either fructose 6-phosphate, fructose 1,6-bisphosphate or fructose 2,6-bisphosphate. Preincubation of the enzyme with cither AMP or ADP resulted in a reduction of this phase, while ATP was without effect. Simultaneous addition of fructose 1,6-bisphosphate to the reaction mixture of the enzyme causes a significant shortening of the transient phase, whereas micromolar concentrations of fructose 2,6-bisphosphate are capable of abolishing the lag phase completely. The occurrence of an initial transient phase suggests that the enzyme after starting the reaction converts from a state of low activity to one of high activity. This conversion mainly depends on the concentration of fructose 1,6-bisphosphate generated in the course of the reaction. In addition an association reaction of the enzyme seems to be involved in the process of conversion of the phosphofructokinase during the initial transient phase. [ABSTRACT FROM AUTHOR]

Phosphofructokinase from muscle has been shown to be a calmodulin-binding protein [Mayr, G. W. and Heilmeyer, L. M. G., Jr (1983) FEBS Lett. 159, 51–57]. Details of the influence of calmodulin on the aggregation state, the conformation and the catalytic properties of phosphofructokinase have been studied by enzymatic and light-scattering analyses. Calmodulin acts as a Ca2+-dependent hysteretic inhibitor of the highly active enzyme. At least one mole of calmodulin binds to each protomer of the enzyme, induces a shift from the highly active tetrameric towards an inactive dimeric state and slowly changes the conformation of the dimers. Dissociation of calmodulin from conformationally changed dimers by removal of Ca2+ stops the inactivation. Without a significant regain of catalytic activity large polymers are rapidly formed. For a reactivation of the inactivated enzyme, calmodulin has to remain associated and the incubation conditions must be changed in a way to allow for a back isomerization and reassociation of dimers. The isomerization reaction is promoted by Mg · ATP, the reassociation reaction most effectively by fructose bisphosphate. A model for the calmodulin-phosphofructokinase interaction is proposed. [ABSTRACT FROM AUTHOR]

The activity of rabbit muscle phosphofructokinase (EC 2.7.1.11) has been followed as a function of time under conditions where the enzyme is separated from the bulk solution by an inert membrane. An enzymatic coupling assay allows continuous measurement of the variations in NADH concentration, which is directly related to the enzyme catalytic activity. For given concentrations of substrates (ATP and Fru6P) in the outside reservoir and a given ratio between diffusion coefficients of both substrates, the activity of phosphofructokinase exhibits an oscillatory behavior during a period of about 5 h. The phenomenon is explained in terms of coupling between diffusion of metabolites and non-linear enzyme reaction. [ABSTRACT FROM AUTHOR]

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkBl mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e.. ‘gluconeogenic growthrsquo;). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1∗. We show here that strains with pfkBl∗ contain an altered form of phosphofructokinase 2, called phosphofructokinase 2∗, which has been purified. Phosphofructokinase 2∗ is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1.6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 × 10-5 M) in both phosphofructokinase 2 and phosphofructokinase 2∗. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2∗ instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal m phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2∗ strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a ‘non-allosteric’ protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 11 7,. 569–574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1.6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment. [ABSTRACT FROM AUTHOR]

The effect of glucose on the phosphorylation of phosphofructokinase has been investigated in hepatocytes isolated from fed and 24-h-starved rats by measuring the 32P radioactivity associated with the enzyme, (n addition, the effects of gluconeogenic precursors and of glucagon or Bt2cAMP were followed under these conditions. The basal incorporation of phosphate into phosphofructokinase in hepatocytes from starved and fed rats was 0.2 ± 0.1 and 0.6 ± 0.2 mol phosphate/mol phosphofructokinase (tetramer) respectively, whereas the maximal incorporation obtained was 1.03 ± 0.2 mol phosphate/mol phosphofructokinase. With 16 mM L-lactate + 4 mM pyruvate or with 20 mM L-alanine, the phosphorylation of the enzyme was lower than the basal incorporation found in hepatocytes from fed and starved rats. Glucose (20 mM) addition to isolated hepatocytes from fed rats resulted in an increase in the incorporation of phosphate into phosphofructokinase to almost maximal value. In hepatocytes isolated from starved rats, glucose led to a 2.5-fold increase of phosphorylation as compared to the basal incorporation. L-Alanine (20 mM) abolished the glucose-induced phosphorylation in hepatocytes from starved and from fed animals. Glucagon (0.1 μM) did not significantly enhance the phosphorylation of phosphofructokinase in the presence of L-lactate + pyruvate in hepatocytes from starved rats, but stimulated the phosphorylation to maximal value when cells had been preincubated in the presence of glucose. In contrast, in liver cells isolated from fed rats, glucagon had no additional effect on phosphofructokinase phosphorylation in the presence of glucose, but did increase the phosphorylation to maximal value when L-lactate + pyruvate served as substrates. L-Alanine inhibited the glucose-induced phosphorylation of phosphofructokinase also in the presence of glucagon. In contrast, glucagon stimulated the phosphorylation of L-type pyruvate kinase independently from exogenous precursors and from the nutritional slate of the animals. Bt2cAMP (0.1 mM) as glucagons, did not significantly enhance the phosphorylation of phosphofructokinase above the level obtained with glucose alone in hepatocytes from fed rats, but did increase the velocity of the phosphate incorporation in hepatocytes from starved rats, thus leading under both conditions to the maximal incorporation. It is concluded that the degree of phosphorylation of rat liver phosphofructokinase is affected by the metabolic state, and that glucagon (or cAMP) acts indirectly, probably by changing glucose metabolism due to increased glycogenolysis rather than by an activation of a cAMP-dependent protein kinase. [ABSTRACT FROM AUTHOR]

The self-stabilization of the energy charge and of ATP was investigated in an open reconstituted enzyme system containing phosphofructokinase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase. The experiments were performed in a stirred flow-through reactor containing gel-entrapped enzymes. The dynamics of the system were analyzed theoretically by a model based on the kinetic properties of the individual enzymes. The energy charge was identified as one of the essential variables of the system According to the theoretical prediction, homoeostasis of the energy charge was observed experimentally when either the maximal activity of phosphofructokinase, the energy charge of the influx solution or the flow rate through the react ion chamber was varied. It is shown that the efficiency of stabilization of the energy charge is related to the occurrence of alternative stationary states. [ABSTRACT FROM AUTHOR]

It is known from theoretical work that very simple enzyme systems may exhibit non-linear dynamic properties like those found in vivo. To realize such a system experimentally, an ATP-producing reaction (pyruvate kinase) was coupled with an ATP-consuming reaction (phosphofructokinase). The substrate-stat technique has been adapted to characterize each single enzyme as well as to follow up one enzyme in the coupled system. If the plots of both pyruvate kinase activity versus [ADP] and phosphofructokinase activity versus [ATP] are hyperbolic, the coupled system approaches a unique steady state as predicted from the single characteristics. Under assay conditions where phosphofurctokinase is inhibited by ATP, its characteristics as found in a single assay and in the coupled system are different. For a system with ATP-inhibited phosphofructokinase, a paradoxical behaviour is predicted and is demonstrated experimentally. If the total pyruvate kinase activity is increased over a certain limit, the system is switched from a low [ATP], high-activity steady state into a high [ATP] lowactivity steady state. [ABSTRACT FROM AUTHOR]

The properties of proteolytically non-modified phosphofructokinase from baker's yeast were investigated by means of hydrodynamic, electrophoretic, and chemical methods. The sedimentation coefficient shows a linear dependence on the protein concentration down to 0.01 mg/ml. The sedimentation coefficient has been determined to be s200.w = 20.81 S. At concentrations of the enzyme at less than 0.01 mg/ml a significant decrease of this value was found. The partial specific volume of the enzyme as determined by three different methods at 20 °C is v2 = 0.742 ml/g. From equilibrium sedimentation a molecular weight of 835000 + 32000 was calculated for the native enzyme. Amino acid analysis of the enzyme was performed and the number of half-cystine residues determined as 11⁄100000g is in good agreement with the number of titratable -SH groups after denaturation of the enzyme. The subunit molecular weight was determined by applying equilibrium sedimentation in the presence of sodium dodecylsulphate. Taking into account that 0.37 g of dodecylsulphate is bound per g of the enzyme protein as found by equilibrium dialysis, a subunit molecular weight of 104000 could be calculated. This value is about 15°/, smaller than that obtained by gel electrophoresis in the presence of sodium dodecylsulphate. Evidently, yeast phosphofructokinase seems to be octameric. Limited proteolysis in the presence of proteinase B from yeast was investigated by following the alterations of the sedimentation coefficient, the molecular weight, and the subunit pattern of the enzyme. During partial proteolytic degradation the molecular weight of the native enzyme decreases by about 230000. This diminution is in accordance with the difference in the molecular weight per subunit if an octameric composition is considered. [ABSTRACT FROM AUTHOR]

A preparation of phosphofructokinase from rabbit skeletal muscle is described which exploits the association-dissociation properties of the enzyme. Phosphofructokinase so prepared is partially phosphorylated and may be fractionated into three distinct species with sedimentation coefficients of 30 S, 18 S and 13 S by chromatography on agarose gels, hydroxyapatite or DEAE-cellulose. Measurements of alkali-labile phosphate content (phosphoserine and/or phosphothreonine) show that fractions consisting almost exclusively of 30-S species and fractions consisting predominantly of 18-S and 13-S species contain approximately 0.15 and 0.29 mol of phosphate per phosphofructokinase monomer (Mr = 80 000) respectively. The results are interpreted in terms of at least two 13-S components which differ in their phosphate contents and also in their self-association properties. The possible significance of phosphorylation is discussed. [ABSTRACT FROM AUTHOR]

The interaction of phosphofructokinase with NH4+, AMP, ATP, citrate, MgATP or fructose 6-phosphate, and in part with their mixtures forming either binary or ternary complexes has been studied by means of ultraviolet difference spectroscopy and circular dichroism spectroscopy in the wavelength range 265–300 nm with the aim of characterizing the conformational corollaries of the ligand effects on phosphofructokinase. The positive as well as the negative effectors change phosphofructokinase conformation in different ways, not easily interpretable in terms of one active and one inactive enzyme conformation. The spectroscopic equivalents of phosphofructokinase conformation changes resulting from catalytic activity are similar to those produced by the reaction products. The ligand concentration-dependent changes of absorption differences in the tryptophyl, tyrosyl and phenylalanyl region parallel each other, i.e. the interactions of the ligands with phosphofructokinase are not confined to specific aromatic side chains, but involve conformation changes of the large domains of the protein. ATP affinity to the enzyme shows temperature-dependent biphasic changes so that ATP binding appears to be either an entropy-driven or enthalpy-driven process. The dissociation constants of the ligands derived from spectroscopic titration of binary complex formation are comparable to those calculated from kinetic experiments, MgATP and fructose 6-phosphate each alone change phosphofructokinase conformation by binary complex formation in keeping with a random order of reaction sequence. [ABSTRACT FROM AUTHOR]

Phosphofructokinase has been purified from Escherichia coli strain K-12 grown in a glucose-limited chemostat, both aerobically and anaerobically. The enzymes migrated together in polyacrylamide gel electrophoresis, had the same subunit size in denaturing (dodecylsulfate) gels Mr approx. 34000) and the same kinetic characteristics as described earlier for E. coli phosphofructokinase [e.g. Blangy et al. (1968) J. Mol. Biol. 31, 13–35]: a sigmoid curve of velocity vs. fructose 6-phosphate concentration, activation by ADP, and inhibition by phosphoenolpyruvate. Findings [e.g. Doelle (1975) Eur. J. Biochem. 50, 335–342] of quite different enzymes in aerobic and anaerobic cells were not confirmed. [ABSTRACT FROM AUTHOR]

The influence of the positive effectors AMP, sulphate, glucose 1,6-bisphosphate and the negative effector 2,3-bisphosphoglycerate on rat erythrocyte phosphofructokinase has been investigated. The kinetic data have been fitted to the Monod-Wyman-Changeux model as well as to a model based on a closed association-dissociation equilibrium. The application of the fitting procedure yields for both models a good correspondence between theoretical and experimental data and equal results with respect to the action of the effectors on the enzyme. The corresponding dissociation constants for the binding of the positive effectors to the active state are: AMP 35 µM, sulphate 0.43 mM and glucose 1,6-bisphosphate 15 µM. 2,3-Bisphosphoglycerate as an inhibitor stabilizes the inactive state (dissociation constant: 1.4 mM). A preliminary discrimination between the Monod-Wyman-Changeux model and the association-dissociation model has been attempted. [ABSTRACT FROM AUTHOR]

The levels of several enzymes have been studied during sporulation of Saccharomyces cerevisia. The specific activities of ribonuclease and aminopeptidase I raised several-fold after transfer of the cells to sporulation medium, whereas the specific activities of phosphofructokinase. glucose-6-phosphate dehydrogenase, tryptophan synthase and pyruvate decarboxylase were not significantly altered. The specific activities of NAD-dependent glutamate dehydrogenase, isocitrate lyase, malate dehydrogenase and fructose bisphosphatase all decreased from the onset of sporulation. The inactivation of these latter enzymes was inhibited by cycloheximide and by inhibitors of energy metabolism. Hexokinase, alcohol dehydrogenase and glutamate oxaloacetate transaminase were partially lost from the cells during the period of ascus maturation. None of the enzymic changes observed proved to be ‘sporulation-specific’ in that it occurred exclusively in sporulating diploid yeast cells. Therefore it is postulated that the meiotic events and the metabolic changes required for ascospore formation are under separate genetic control in this organism. During sporulation, the cellular content of cytochromes b, c, and aa3 was reduced to 20% or less of that present in vegetative derepressed cells. Since the relative percentage of total to cycloheximide-insensitive mitochondrial protein synthesis was not significantly altered throughout sporulation, and the pattern of mitochondrially synthesized polypeptides was rather similar both in vegetative and in sporulating cells, it appeared that not not only degradation but also synthesis and therefore turnover of the mitochondrially coded polypeptides of cytochromes b and aa3 took place during sporulation. The activity ratio of cytochrome c oxidase to F1-ATPase in submitochondrial particles isolated from vegetative cells and from purified asci was almost identical. This indicates that the loss of membrane-bound mitochondrial cytochromes during sporulation is probably due to a nonselective degradation of inner mitochondrial membrane proteins. [ABSTRACT FROM AUTHOR]

Rabbit muscle phosphofructokinase uniformly carboxymethylated with iodo[2-14C]acetate consists of subunits with a molecular weight of 80000 ± 5000. The subunit polypeptide chain contains 16 and 52 residues respectively of cysteine and arginine and, contrary to previous results, peptide mapping experiments gave no indication that phosphofructokinase chains yield fewer than the expected numbers of cysteine and arginine containing peptides. To test further for the possible occurrence of repeat sequences within a single subunit chain, cysteine-containing peptides were isolated and sequenced from tryptic and thermolytic digests of s-[2-14C]carboxymethylated phosphofructokinase. In all, 15 different cysteine sequences (comprising a total of 104 residues) were identified, showing that not more than one of an expected 16 cysteine-containing sequences is repeated, and that the subunits in phosphofructokinase are of unique sequence along their entire length. The near quantitative isolation of several cysteine-containing peptides shows further that all subunits are of similar if not identical sequence. [ABSTRACT FROM AUTHOR]

A procedure is presented for the purification of 3 g of homogeneous methionyl-tRNA synthetase and 1 g of homogeneous tyrosyl-tRNA synthetase from 50 kg batches of Escherichia coli. The procedure permits the isolation of many enzymes simultaneously and the elution positions of seven other aminoacyl-tRNA synthetases, catalase, rhodanese, phosphofructokinase, elongation factor Tu and cytochrome b-562 are indicated. The problems of extraction work on this scale are discussed. [ABSTRACT FROM AUTHOR]

Yeast phosphofructokinase having a molecular weight of 750000-800000 (20 S) has been subjected to limited proteolysis by subtilisin and yeast proteases. Two steps of proteolytic degradation could be distinguished: in the first step, which is accompanied by an increase in molecular activity, the subunits α and β (Mr 120 000) are converted to α' and β' (Mr approximately 90 000), and in the second step, accompanied by a decrease in enzyme activity, α' is converted to α' (Mr 80 000) and two further fragments having Mr 45 000 and 35 000 become detectable. In the course of this conversion the sedimentation value of the undissociated enzyme drops from 20 S to about 17 S. The two substrates fructose 6-phosphate and ATP exhibit characteristic protective effects on enzyme activity and on subunit degradation. Whereas the first step is not strongly influenced by the substrates, fructose, 6-phosphate inhibits significantly the degradation of α' and β', whereas ATP prevents only degradation of β'. When in presence of ATP α' is degraded to α'', the quaternary structure of the 17-S enzyme is no longer stable and a dissociation of this molecule occurs to a 12-S form which is enzymically active and ATP-sensitive and in which the ratio of α'' to β' is one-to-one. [ABSTRACT FROM AUTHOR]

The purification and kinetic characteristics of two phosphofructokinases are described. Aerobic cultures of Escherichia coil exhibit two types of phosphofructokinase. Both types are dimers of mol. wt 150000 (subunit mol. wt 73000), whereas the anaerobic culture of E. coil revealed only one type, which is a tetramer of mol. wt 350000 (subunit mol. wt 90000). Type I of the aerobic enzyme, representing approximately 70 % of the total enzyme activity, is ATP-insensitive, whereas type II and the anaerobic enzyme are ATP-sensitive. The addition of AMP stimulates the tetramer, relieving ATP inhibition, and also the type II dimer, which is, however, inhibited at concentrations higher than 0.5 mM AMP. No effect was observed on the type I dimer of the aerobic preparation. ADP stimulates the tetramer and inhibits type I more strongly than type II of the aerobic dimer. The kinetic characteristics together with the effect of metabolites on these phosphofructokinase types are described and discussed in the light of their importance for the regulatory mechanism of the Pasteur effect. [ABSTRACT FROM AUTHOR]

The phosphofructokinase of the red cells of rats is exquisitely sensitive to inhibition by ATP. The apparent Ki for ATP is decreased with lower pH values and fructose-6-phosphate concentrations. 2,3-Bisphophoglycerate, which is present in the red cells of man and many mammals, inhibits the phosphofructokinase in a synergistic manner with ATP. The inhibitory effect of phosphoenolpyruvate is biologically insignificant. It may be estimated that the enzyme would be practically completely inactive under cellular conditions without the action of positive effectors. There exist a multitude of positive effectors. Significant activations under cellular conditions are exercised by AMP, glucose 1,6-bisphosphate and inorganic phosphate. Fructose 1,6-bisphosphate activates to a limited extent and ADP only at alkaline pH values. All positive effectors produce a decreased inhibition by ATP and increased affinity of the enzyme for fructose 6-phosphate. AMP is the activator with the highest affinity, while glucose 1,6-bisphosphate may be considered as a baseline activator owing to its sluggish turnover. The data may be fitted in a qualitative manner to the model of Monod, Wyman and Changeux. It is possible that the negative anionic effectors act on the same site. [ABSTRACT FROM AUTHOR]

By applying analytical gel chromatography technique the association behaviour of human erythrocyte phosphofructokinase has been studied. The molecular weight distribution of the polymerized forms of this enzyme covers a range of 380000 to about six millions. The degree of polymerization has been found to depend on the protein concentration, on the presence of various substrates and effectors and on the pH-value. ATP favours the aggregation of the enzyme with increasing pH, whereas in presence of fructose 6-phosphate or fructose 1,6-bisphosphate respectively the degree of association appears to be independent of the pH value between pH 6 and 9. In the microgram range the specific activity of the enzyme decreases with an increase of the enzyme concentration. [ABSTRACT FROM AUTHOR]

A new and efficient method for preparation of pure yeast phosphofructokinase is described, leading rapidly to an enzyme with minimum proteolytic alterations. Essential is an affinity chromatography step on the reactive dye Cibacron blue F3G-A® which has been covalently bound to Sephadex G-100. The enzyme prepared by this method has a significantly higher sedimentation constant (20.5 S) than those forms of yeast phosphofructokinase purified by procedures previously described (17.8 S) and is composed of two different kinds of subunits having molecular weights of 130000 and 96000, respectively. By gel filtration various enzyme fractions have been obtained, containing either mainly subunits of the 130000 or of the 96000 type, respectively. Samples of phosphofructokinase which have been prepared by the original and by the new method were compared by means of analytical gel chromatography and dodecylsulphate elcetrophoresis. In the course of the preparation procedure described previously, the enzyme is evidently proteolytically converted to the 570000 molecule by degradation of the subunits having 130000 molecular weight to those of 96000. [ABSTRACT FROM AUTHOR]