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PubMed1992

The regulatory protein of liver glucokinase.

Fructose, sorbitol and D-glyceraldehyde stimulate the rate of glucose phosphorylation in isolated hepatocytes. This effect is mediated by fructose 1-phosphate, which releases the inhibition exerted by a regulatory protein on liver glucokinase. In the presence of fructose 6-phosphate, the regulatory protein binds to, and inhibits, liver glucokinase. Fructose 1-phosphate antagonizes this inhibition by causing dissociation of the glucokinase-regulatory protein complex. Both phosphate esters act by binding to the regulatory protein, and by presumably causing changes in its conformation. The regulatory protein behaves as a fully competitive inhibitor. It inhibits liver glucokinase from various species, and rat islet glucokinase, but has no effect on hexokinases from mammalian tissues or from yeast, or on glucokinase from microorganisms. Kinetic studies indicate that the regulatory protein binds to glucokinase at a site distinct from the catalytic site. Several phosphate esters, mainly polyol-phosphates, were found to mimick the effect of fructose 6-phosphate. The most potent is sorbitol 6-phosphate, suggesting that fructose 6-phosphate is recognized by the regulatory protein in its open-chain configuration. Other phosphate esters and Pi have a fructose 1-phosphate-like effect. The stimulatory effect of fructose on glucose phosphorylation is observed not only in isolated hepatocytes but also in the livers of anesthetized rats. This suggests that fructose could be a nutritional signal causing an increase in the hepatic glucose uptake.

Allosteric Regulation↗
PubMed1975

Glycogenolysis and control of anaphylactic histamine release by cyclic adenosine monophosphate-related agents. II. Modification of histamine release by glycogenolytic metabolites.

D-glucose-6-phosphate (less than or equal to 5 x 10(-3) M), pyruvate, lactate (less than or equal to 1 X 10(-2) M), and dibutyryl cyclic AMP (less than 5 X 10(-3) M) were capable of inhibiting anapylactic histamine release in vitro from chopped guinea pig lung. In lower concentrations, pyruvic acid and lactate, as well as dibutyryl cyclic AMP, enhanced the release. Significant synergism was observed betweenpyruvate (5 X 10(-3) M) and isoproterenol (1 X 10(-8) M) in the inhibition of histamine release. The inhibitory actions of isoproterenol, glucose-6-phosphate, and pyruvate were influenced by calcium ion concentration. However, beta blockade, which diminished the isoproterenol effect, was without efect on pyruvate (1 X 10(-2) M) to the release system. Glucose-6-phosphate and isoproterenol did not have this effect. The results, together with a prevouus study, suggest that glycogenolysis may possess a role in the anaphylactic istamine release in vitro from sensitized lung fragments...

Anaphylaxis↗
PubMed1992

Evidence for conservation of peripheral lymphocyte homing receptors between the bovine, murine and human species.

In rodents and humans, lymphocytes circulate throughout the body and return preferentially to their tissues of origin via a process termed homing. The specificity of homing is controlled by the binding of tissue-specific receptors on lymphocytes to ligands on specialized high-walled endothelial venules (HEV) found in lymphoid tissue. The murine and human peripheral lymphocyte homing receptors (PLHR) have been characterized and shown to be similar to each other. We present evidence for a similar receptor in the bovine. Bovine peripheral blood lymphocytes (PBL) bind to the HEV of murine peripheral lymph node tissue in vitro. The same sugars that have been shown to decrease the binding of murine or human lymphocytes to murine HEV also decrease the binding of bovine PBL to murine HEV. Neuraminidase treatment affects lymphocyte binding in a similar manner in the bovine, murine and human species. Phorbol myristate acetate (PMA) stimulation, which has been shown to reduce the expression of murine and human PLHR, also reduces the binding of bovine PBL to murine HEV. These data suggest conservation of PLHR between these species.

Animals↗
PubMed1988

A kinetic model of phosphofructokinase from Plasmodium berghei. Influence of ATP and fructose-6-phosphate.

Phosphofructokinase (PFK) from the malarial parasite Plasmodium berghei shows the following kinetic features: the more the pH is decreased, the more the enzyme is inhibited by ATP; in contrast to PFK from erythrocytes, this inhibition is less potent by two orders of magnitude; as in the red cell, fructose-6-phosphate (F6P) is a positive effector. Kinetic modelling of PFK from P. berghei has been performed by taking the pH-dependence of activity into regard, implicitly by the estimation of pH-dependent kinetic parameters for the inhibition by ATP and the activation by F6P and explicitly by the assumption of protonation-steps involved in allosteric regulation. By means of a novel procedure of model discrimination [D. Buckwitz and H.-G. Holzhütter: A new method to discriminate between enzyme-kinetic models. In: Application of Computational Methods in Medicine (Györi, I., ed.), Akademai, Budapest, in press] we have selected among several kinetic models the best rate equation which provides an adequate quantitative description of the kinetic behaviour of the enzyme in the relevant ranges of substrate concentrations and pH (5.8-7.6). It thus becomes clear how the highly increased glycolytic flux in malaria-infected cells could be affected through PFK.

Adenosine Triphosphate↗
PubMed1989

Presence of a fructose-2,6-bisphosphate-insensitive pyrophosphate: fructose-6-phosphate phosphotransferase in the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis and Isotricha prostoma.

Extracts of the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis and Isotricha prostoma contained a high activity (0.5-1 mumol min-1 (mg protein)-1) of pyrophosphate:fructose-6-phosphate phosphotransferase (PPi-PFK), but no detectable ATP: fructose-6-phosphate phosphotransferase. PPi-PFK from I. prostoma was purified close to homogeneity by adsorption on phospho-Ultrogel and elution with fructose-1,6-bisphosphate, and subsequent anion-exchange chromatography. The enzyme had an Mr of 95,000 as determined by gel filtration and consisted of subunits of Mr 48,000. PPi-PFK from I. prostoma and from T. foetus displayed hyperbolic kinetics with respect to their substrates and were not affected by fructose-2,6-bisphosphate. In sharp contrast with what has been found in other eukaryotes, no evidence could be found for the presence of fructose-2,6-bisphosphate in the two trichomonads, in I. prostoma and in Entamoeba histolytica.

Animals↗
PubMed1991

Changes in intracellular levels of fructose 2,6-bisphosphate and several glycolytic intermediates in Leishmania major promastigotes as a function of pO2.

Leishmania major promastigotes were grown to late log phase, washed and resuspended in Hanks' balanced salt solution, and incubated with glucose at various pO2s in the presence of 5% CO2. Samples were taken at times from 0-40 min and assayed for fructose 2,6-bisphosphate (Fru(2,6)P2), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), phospho(enol)pyruvate (PEP), and ATP. At 95% O2 ATP remained constant throughout the incubation. It did not decrease significantly at 10% O2, but decreased by about 20% and 30% at 6% and 0% O2, respectively. At 95% O2, Fru(2,6)P2 increased about 15-fold within 5 min after the addition of glucose and remained at this high level. At 10%, 6%, and 0% O2 Fru(2,6)P2 rose about 5-fold within 5 min and then declined slightly during the remainder of the incubation. G6P increased from about 0.5 to 12 nmol (mg protein)-1 at 5 min in cells incubated under 95% O2 and then declined to about 5 nmol (mg protein)-1. It increased to about 8 nmol (mg protein)-1 at 5 min and then declined slightly in cells incubated under 10% O2. F6P levels were approximately one-eighth of G6P levels under all conditions, suggesting that phosphohexoseisomerase was not subject to regulation. PEP levels were initially high, but at 95% O2 there was a 50% drop in PEP at 5 min, while at 10%, 6%, and 0% O2 there was less of a decline. The observation that the rise in Fru(2,6)P2 levels at 10%, 6%, or 0% O2 is the same at 5 min and less than the rise at 95% O2 supports the presence of a low affinity oxygen sensor. The different time course of changes in G6P, F6P, and PEP levels suggests that in addition to an activation of pyruvate kinase by Fru(2,6)P2, other regulatory events are also operative at low pO2.

Adenosine Triphosphate↗
PubMed1995

Kinetic mechanism of pyrophosphate-dependent phosphofructokinase from Giardia lamblia.

The steady-state kinetics of the reaction catalyzed by inorganic-pyrophosphate-dependent D-fructose-6-phosphate 1-phosphotransferase from Giardia lamblia have been investigated. The reactants for the forward and reverse reactions were the Mg-chelated complexes of pyrophosphate (PPi) and Pi. Uncomplexed ligands were not substrates. In the direction of phosphorylation of fructose-6-phosphate (F6P), initial velocity double-reciprocal plots for both PPi and F6P were intersecting suggesting sequential addition of substrates. Similarly, intersecting patterns were observed in the reverse reaction with either Pi or fructose-1,6-bisphosphate (FBP) as the variable substrate. Although the catalytic constants for the forward and reverse reactions were found to be identical (83 s-1), the kcat/Km for PPi is about two orders of magnitude higher than the kcat/Km for Pi, indicating that PPi is utilized much more efficiently than Pi. Product inhibition of Pi is competitive vs. PPi and noncompetitive vs. F6P, when the fixed substrate is subsaturating. Product inhibition by FBP was found to be noncompetitive with either Pi or F6P as the variable substrate. These results are consistent with a sequential ordered Bi Bi mechanism with PPi adding first and Pi dissociating last. In the reverse reaction, however, PPi and F6P were found to be noncompetitive with either Pi or FBP. Dead-end inhibition analysis with fructose 2,6-bisphosphate, a competitive substrate analog of FBP, gave uncompetitive inhibition with respect to Pi, indicating that fructose 2,6-bisphosphate (and hence FBP) binds after Pi. This kinetic mechanism is different from that observed with the enzyme from Propionibacterium freudenreichii, Entamoeba histolytica or Mung bean, which were concluded to be rapid equilibrium random mechanism.

Animals↗
PubMed1982

Modifications in the allosteric properties of phosphofructokinase in rat erythrocytes and reticulocytes cross-linked with dimethyl suberimidate and 3,3'-dithiobispropionimidate.

The kinetic behaviour of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been studied in situ, by using rat erythrocytes and reticulocytes treated with dimethyl suberimidate and 3,3'-dithiobispropionimidate as cross-linking reagents and with digitonin as the delipidating agent. Comparison of the ATP and fructose-6-P saturation curves of phosphofructokinase in dimethyl suberimidate-permeabilized cells with those obtained in haemolysates showed the enzyme to have reduced allosteric properties under in situ conditions, although it still responded to cyclic AMP (300 microM) added as allosteric effector. Non-sigmoidal fructose-6-P saturation curves were also observed using 3,3'-dithiobispropionimidate-permeabilized erythrocytes, either in the absence or in the presence of cyclic AMP. A hyperbolic behaviour was shown after cross-linking reversal of 3,3'-dithiobispropionimidate-permeabilized erythrocytes by treatment with dithiothreitol. Specific activity values of phosphofructokinase were always lower in permeabilized cells than in haemolysates. A significant inhibition of phosphofructokinase specific activity, without any effect on its allosteric behaviour, is exerted by reaction of dimethyl suberimidate or 3,3'-dithiobispropionimidate with erythrocyte lysates in the presence of an inhibitory concentration of ATP. These results suggest that penetration of the cross-linking reagent and its subsequent reaction with intracellular phosphofructokinase will have a direct effect upon the results obtained using this in situ approach.U

Adenosine Triphosphate↗
PubMed1988

Reactivity of the thiol groups of Escherichia coli phosphofructo-1-kinase.

Modification of Escherichia coli phosphofructo-1-kinase (6-phosphofructokinase; EC 2.7.1.11) with several thiol modifying reagents led to a pseudo-first-order loss of activity that was associated with the modification of a single cysteine residue, identified as the cysteine at position 119 in the protein sequence. This cysteine was protected from reaction with vinyl pyridine, bromopyruvate, and dithionitrobenzoic acid by the substrate, fructose-6-P. In the crystal structure of the highly homologous phosphofructokinase from Bacillus stearothermophilus, cysteine 119 is sufficiently distant from the catalytic site to exclude a direct steric inhibition of the binding of substrate as a mechanism of inactivation for the modification. Thus, the inhibition is unlikely to be a direct one but to be the result of interference with the conformational change that is associated with fructose-6-P binding. A second thiol, position 283, was shown to be protected from reaction when the enzyme was in its native conformation. In contrast to the previously published sequence for the E. coli enzyme six cysteines as opposed to seven have been found both in enzyme from strain LE392 and in enzyme produced by a plasmid that was derived from pLC 16-4. The position in question, 75, was identified as phenylalanine.

Chemical Phenomena↗
PubMed1989

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates; diastereotopic specificity, isotopic discrimination and intramolecular hydrogen transfer.

When D-[1-3H]fructose 6-phosphate generated from D-[2-3H]glucose 6-phosphate is converted, in a monodirectional manner to D-glucose 6-phosphate and then 6-phospho-D-gluconate, about 42% of the radioactivity is transferred from the C1 of the ketohexose ester to the C2 of the aldohexose phosphate, whereas the remaining 58% are produced as 3H2O. The velocity of the reaction catalyzed by phosphoglucoisomerase represents, in the case of the tritiated substrate, only 43% of that recorded with D-[U-14C]fructose 6-phosphate, such an isotopic discrimination being attributable mainly to a difference in maximal velocity rather than affinity. The phenomena of both intramolecular hydrogen transfer and isotopic discrimination were less pronounced than when D-[2-3H]glucose 6-phosphate is converted, in a monodirectional manner, to D-fructose 6-phosphate and then D-fructose 1,6-bisphosphate. In contrast, when either D-[1-3H]glucose 6-phosphate or D-[1-3H]fructose 6-phosphate prepared from D-[1-3H]glucose were tested, no 3H2O was formed, all radioactivity being recovered, respectively, in tritiated D-fructose 1,6-bisphosphate or NADP3H. Nevertheless, phosphoglucoisomerase was also found to discriminate between D-[U-14C]glucose 6-phosphate and D-[1-3H]glucose 6-phosphate or between D-[U-14C]fructose 6-phosphate and D-[1-3H]fructose 6-phosphate prepared from D-[1-3H]glucose. The reaction velocity with the tritiated esters averaged 78-83% of those recorded with the 14C-labelled esters. Such an isotopic discrimination was again attributable mainly to a difference in maximal velocity rather than affinity. These findings indicate that the mode of preparation of D-[1-3H]fructose cannot be ignored in considering the fate of this tritiated hexose, as ruled by the intrinsic properties, and especially the diastereotopic specificity of phosphoglucoisomerase.

Carbon Radioisotopes↗
PubMed1989

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates; comparison with phosphomannoisomerase.

The isotopic discrimination, diastereotopic specificity and intramolecular hydrogen transfer characterizing the reaction catalyzed by phosphomannoisomerase are examined. During the monodirectional conversion of D-[2-3H]mannose 6-phosphate to D-fructose 6-phosphate and D-fructose 1,6-bisphosphate, the reaction velocity is one order of magnitude lower than with D-[U-14C]mannose 6-phosphate and little tritium (less than 6%) is transferred intramolecularly. Inorganic phosphate decreases the reaction velocity but favours the intramolecular transfer of tritium. Likewise, when D-[1-3H]fructose 6-phosphate prepared from D-[1-3H]glucose is exposed solely to phosphomannoisomerase, the generation of tritiated metabolites is virtually restricted to 3H2O and occurs at a much lower rate than the production of D-[U-14C]mannose 6-phosphate from D-[U-14C]fructose 6-phosphate. However, no 3H2O is formed when D-[1-3H]fructose 6-phosphate generated from D-[2-3H]glucose is exposed to phosphomannoisomerase, indicating that the diastereotopic specificity of the latter enzyme represents a mirror image of that of phosphoglucoisomerase. Advantage is taken of such a contrasting enzymic behaviour to assess the back-and-forth flow through the reaction catalyzed by phosphomannoisomerase in intact cells exposed to D-[1-3H]glucose, D-[5-3H]glucose or D-[6-3H]glucose. Relative to the rate of glycolysis, this back-and-forth flow amounted to approx. 4% in human erythrocytes and rat parotid cells, 9% in tumoral cells of the RINm5F line and 47% in rat pancreatic islets.

Animals↗
PubMed1990

Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action.

The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.

Animals↗
PubMed1991

6-Phosphofructo-1-kinase of rat placenta.

6-Phosphofructo-1-kinase (PFK) of rat placenta was purified to homogeneity with a recovery of 56% of the enzyme activity in the original extract. The purified enzyme is a tetramer and the Mr value of the subunit is 85,000 +/- 1500 as shown by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Considering the properties of the native rat placental PFK isoenzyme, it is clear that this tissue is a complex mixture of homotetramer and heterotetramer. Purified placenta PFK displayed little cooperativity at pH 7.0 with respect to fructose 6-phosphate and was markedly inhibited with high concentrations of ATP. The affinity of the enzyme for fructose 6-phosphate was increased by fructose 2,6-biphosphate. The purified enzyme was highly inhibited by citrate, whereas it was only slightly inhibited by phosphoenol pyruvate. ADP, AMP and fructose 2,6-bisphosphate showed little stimulation towards placental PFK. The present study suggests that the placental PFK is a relatively active enzymic form and it is also probably characterized with a high rate of glycolysis possibly because this tissue requires a high energy production for the development and maintenance of the fetus as the placenta tends to be a semipermeable membrane through which substances are exchanged between mother and fetus.

Adenosine Triphosphate↗
PubMed1991

Modulation of the interaction between aldolase and glycerol-phosphate dehydrogenase by fructose phosphates.

Kinetics of fructose-1,6-disphosphate aldolase (EC 4.1.2.13) catalyzed conversion of fructose phosphates was analyzed by coupling the aldolase reactions to the metabolically sequential enzyme, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), which interacts with aldolase. At low enzyme concentration poly(ethylene glycol) was added to promote complex formation of aldolase and glycerol-phosphate dehydrogenase resulting in a 3-fold increase in KM of fructose-1,6-bisphosphate and no change in Vmax. Kinetic parameters for fructose-1-phosphate conversion changed inversely upon complex formation: Vmax increased while KM remained unchanged. Gel penetration and ion-exchange chromatographic experiments showed positive modulation of the interaction of aldolase and dehydrogenase by fructose-1,6-bisphosphate. The dissociation constant of the heterologous enzyme complex decreased 10-fold in the presence of this substrate. Fructose-1-phosphate or dihydroxyacetone phosphate had no effect on the dissociation constant of the aldolase-dehydrogenase complex. In addition, titration of fluorescein-labelled glycerol-phosphate dehydrogenase with aldolase indicated that both fructose-1,6-bisphosphate and fructose-2,6-biphosphate enhanced the affinity of aldolase to glycerol-phosphate dehydrogenase. The results of the kinetic and binding experiments suggest that binding of the C-6 phosphate group of fructose-1,6-bisphosphate to aldolase complexed with dehydrogenase is sterically impeded while saturation of the C-6 phosphate group site increases the affinity of aldolase for dehydrogenase. The possible molecular mechanism of the fructose-1,6-bisphosphate modulated interaction is discussed.

Animals↗
PubMed1992

An exploration of the binding site of aldolase using alkyl glycolamido phosphoric esters and alkyl monoglycolate phosphoric esters.

Alkyl glycolamido phosphoric esters (P-O-CH2-CO-NH-(CH2)n-CH3) and alkyl monoglycolate phosphoric esters (P-O-CH2-CO-O-(CH2)n-CH3), which are analogs of the aldolase substrate fructose-1-phosphate, were synthesized and use for probing the active site of rabbit muscle aldolase. The inhibition constants (Ki) were affected by the length of the alkyl groups of these compounds and a maximum value of Ki was observed between the number of methylene groups 2 and 4, depending on the type of compound. In the previous investigation, N-(omega-hydroxyalkyl)-glycolamido bisphosphoric esters (P-O-CH2-CO-NH-(CH2)n-O-P) and alkanediol monoglyclolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P) have a minimum Ki value between the number of methylene groups 1 and 4. The difference spectra of aldolase caused by binding of alkyl glycoamido phosphoric esters or alkyl monophosphates resembled that of their analogous bisphosphoric esters, but the intensity of absorbance was smaller than that of the bisphosphoric ester analogs. These results suggest that rabbit muscle aldolase has two binding sites for the phosphate groups on the entrance end of the active site cavity, the singly wound beta-barrel of the parallel alpha/beta class structure. The distance between the phosphate binding site Lys-107 in the beta-sheet structure (c) and Arg-148 in the beta-sheet structure (d) may possibly be expanded or contracted by the forms of the bending structure of the biphosphate compounds. Also, the change of distance between the beta-sheet structure (c) and (d) containing Trp-147, may have an effect on the environment of the tryptophan and cause a change of the absorbance of aldolase especially at 295-299 nm. On the other hand, the synthetic monophosphate compounds bind at only one of the two phosphate binding sites and have very little effect on the absorbance of Trp-147, in a similar manner as orthophosphate. The alkyl groups of monophosphate may be repelled by the ionic amino acid side chains, Asp-33, Lys-146, Glu-187 and/or Lys-229 in the middle of the active site cavity. However, the end of the long alkyl group of some monophosphates may possibly contact the hydrophobic bottom of the active site cavity without effect on the environment of Trp-147.

Binding Sites↗
PubMed1989

Regulation of hepatic inorganic phosphate and ATP in response to fructose loading: an in vivo 31P-NMR study.

Fructose loading results in hepatic accumulation of fructose 1-phosphate (Fru1 P). The goals of the present experiments were: first, to distinguish between ATP, intracellular inorganic phosphate (Pi), and extracellular Pi as sources of phosphate for the phosphorylation of fructose, and second, to examine the influence of ATP and Fru1 P on movement of phosphate into and out of these three pools. To achieve these goals, 31P-NMR was used to monitor the response of hepatic ATP, Pi and Fru1 P to two consecutive injections of fructose. The first was administered with ATP at the control level, and the second, 1 h after the first, with ATP at 65% of the control level. Changes in intra- and extracellular Pi were distinguished by correlating measurements of total NMR-detectable phosphorus and NMR-detectable Pi with measurements of plasma Pi. The initial fructose injection resulted in rapid accumulation of Fru1 P, small decreases in plasma and NMR-detectable Pi and a dramatic decrease in ATP. Total NMR-detectable phosphorus did not change, suggesting that phosphate did not enter or leave the liver. Therefore, accumulation of Fru1 P was initially balanced by an equivalent decrease in ATP, without large changes in Pi. Following the second injection, when ATP was at 65% of control. Fru1 P accumulated at approximately the same rate and to the same level as achieved following the first injection. There was little further change in ATP and a marked decrease in NMR-detectable Pi, while plasma Pi was higher than after the first injection. Therefore the greater decrease in NMR-detectable Pi following the second injection represented a significant decrease in intracellular Pi. Return of Fru1 P to control coincided with a dramatic increase in plasma Pi, and a decrease in total NMR-detectable phosphate. This suggests that phosphate released from Fru1 P entered the extracellular space. These data suggest the mechanisms by which intracellular Pi is regulated. When sufficient ATP is available, ATP hydrolysis supplies phosphate for the synthesis of Fru1 P, and prevents a significant decrease in intracellular Pi. When ATP is reduced, accumulation of Fru1 P depletes intracellular Pi. Therefore, decreased availability of ATP correlates with increased utilization of intracellular Pi. When Fru1 P returns to control, the increase in intracellular Pi is limited by release of Pi into the plasma.

Adenosine Triphosphate↗