Role of ferredoxin in the activation of sedoheptulose diphosphatase in isolated chloroplasts

Sedoheptulose 1,7-diphosphatase activity of isolated spinach chloroplasts shows a requirement for (i) reduced ferredoxin and (ii) a protein factor. Activation by ferredoxin, reduced photochemically by chloroplast fragments, was optimal at pH 7.8 and at a Mg²⁺ concentration of 5 mM. The protein factor needed for activation appears to be the same as that required by the chloroplast fructose-1,6-diphosphatase that is activated by reduced ferredoxin. The results indicate that sedoheptulose-1,7-diphosphatase, like fructose-1,6-diphosphatase, is a regulatory enzyme whose activity in chloroplasts is controlled via ferredoxin by light.     

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.     

In vitro phosphorylation of fructose-1,6-bisphosphatase from rabbit and pig liver with cyclic AMP-dependent protein kinase

Homogeneous preparations of fructose-1,6-bisphosphatase from mouse, man, rabbit, pig, and rat were tested as substrates for cyclic AMP-dependent protein kinase. Up to 1 mol of [32P]phosphate per mole enzyme subunit was incorporated into fructose-1,6-bisphosphatase from pig and rabbit liver, which should be compared with 2.6 mol of phosphate per mole enzyme subunit in the case of the rat liver enzyme. The phosphorylation of fructose-1,6-bisphosphatase from the livers of man and mouse was negligible. Phosphorylation of pig and rabbit fructose-1,6-bisphosphatase decreased the apparent Km for fructose-1,6-bisphosphate, but in contrast to the case of the rat liver enzyme it did not change the inhibition constants for AMP and fructose-2,6-bisphosphate. The phosphorylation sites in rabbit and pig liver fructose-1,6-bisphosphatase were located close to the carboxyterminal of the polypeptide chains, since trypsin treatment of the phosphorylated enzyme quantitatively removed all of the protein-bound radioactivity without significantly altering the subunit molecular weight and with a maintained neutral pH optimum.     

Purification and properties of mouse liver fructose 1,6-bisphosphatase.

A simple procedure has been developed for the purification of mouse liver and kidney fructose-1,6-bisphosphatase. In addition to the conventional method, including substrate elution from phosphocellulose, Blue Sepharose column chromatography made the purification procedure highly reproducible. The enzyme from rabbit liver was also purified by this method with a small modification. The isolated preparation was electrophoretically homogeneous. The mouse liver enzyme was identical with the kidney enzyme, and different from the rabbit liver enzyme electrophoretically. The structural properties and the amino acid composition were similar to those of this enzyme from other mammalian livers; the molecular weight was 143,000, subunit size was 37,500, S20, w was 7.0, and partial specific volume was 0.74. Cysteine and methionine residues amounted to 5-6 mol per subunit. Tryptophan was not detected. The Km value for fructose-1,6-bisphosphate was 1.3 microM. The Ki value for AMP was 19 microM. EDTA strongly activated the activity of the mouse liver enzyme at neutral pH. A partial proteolytic digestion of the mouse liver enzyme decreased the activity at neutral pH, and increased it at alkaline pH.     

Fructose-1, 6-diphosphatase and acid hexose phosphatase of Escherichia coli

Fraenkel, D. G. (Albert Einstein College of Medicine, New York, N.Y.), and B. L. Horecker. Fructose-1,6-diphosphatase and acid hexose phosphatase of Escherichia coli. J. Bacteriol. 90:837-842. 1965.-The conversion of fructose-1,6-diphosphate to fructose-6-phosphate (fructose-1,6-diphosphatase activity) is essential for growth of Escherichia coli on glycerol, acetate, or succinate, but is unnecessary for growth on hexoses or pentoses. It has sometimes been assumed that fructose-1,6-diphosphatase activity is due to a nonspecific acid hexose phosphatase. We have now obtained a number of one-step mutants which have lost the ability to grow on glycerol, succinate, or acetate, but which grow normally on hexoses; these mutants are deficient in a fructose-1,6-diphosphatase which can be assayed spectrophotometrically in the presence of Mg(++) and low concentrations of substrate. These mutants still possess the nonspecific acid hexose phosphatase, which does not require Mg(++) and is active only at much higher concentrations of fructose-1,6-diphosphate. Evidence is presented to support the hypothesis that the newly described activity is the physiological fructose-1,6-diphosphatase. The acid hexose phosphatase is a different enzyme whose function remains unknown.     

Hormonal regulation of key gluconeogenic enzymes and glucose release in cultured hepatocytes: effects of dexamethasone and gastrointestinal hormones on glucagon action

Hormonal regulation of key gluconeogenic enzymes and glucose release by glucagon, dexamethasone, secretin and somatostatin was evaluated in maintenance cultured rat hepatocytes. (i) Phosphoenolpyruvate (PEP)-carboxykinase activity declined rapidly during the first 24 h in serum- and hormone-free culture with a further slight decay during the following 2 days. Dexamethasone and glucagon independently increased PEP-carboxykinase and acted synergistically when added in combination. Glucose-6-phosphatase activity declining linearly during hormone-free culture was stimulated by glucagon. Dexamethasone itself was without significant effects but completely abolished glucagon action. Fructose-1,6-diphosphatase was maintained at its initial level during the first day under control conditions and declined thereafter. Neither glucagon nor dexamethasone affected total activity or substrate (fructose-1,6-diphosphate) affinity of this enzyme. In short-term experiments on cells cultured under control conditions, protein synthesis-dependent stimulation of PEP-carboxykinase by glucagon and the permissive action of dexamethasone was demonstrated. Glucose-6-phosphatase and fructose-1,6-diphosphatase were not altered by hormones within this period. (ii) Stimulation by glucagon of gluconeogenesis was independent of its action on PEP-carboxykinase. Dexamethasone inhibited glycogenolysis but maintained glucose release at control levels probably by stimulation of gluconeogenesis. When added in combination, the glycogen-preserving action of dexamethasone acutely reduced the glucose release in response to glucagon. Glucagon sensitivity remained unchanged. (iii) The gastrointestinal hormones secretin and somatostatin were ineffective in modulating basal or glucagon-stimulated glucose release and gluconeogenic key enzymes. They are therefore unlikely to play a physiological role in hepatic glucose metabolism.     

Dietary and hormonal regulation of some enzyme activities associated with gluconeogenesis in rabbit liver.

1. Starvation increases the activity of cytosolic P-enolpyruvate carboxkinase in rabbit liver some 4-5 fold but does not alter the activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase or glucose-6-phosphatase.2. Alloxan-induced diabetes increases the activities of cytosolic P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase approx. 6-, 2- and 2-fold, respectively. Again the activity of mitochondrial P-enolpyruvate carboxykinase is not altered. 3. Administration of mannoheptulose rapidly increases blood glucose levels and also causes a significant increase in cytosolic P-enolpyruvate carboyxkinase activity within 4 h. The activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase are not affected. 4. Administration of hydrocortisone also increases blood glucose levels and the activities of cytosolic P-enolpyruvate carboxykinase and glucose-6-phosphatase are significantly increased within 12h. Again, mitochondrial P-enolpyruvate carboxykinase and fructose-1,6-diphosphatase activities remain unaffected. 5. The observations that (A) the activity of cytosolic P-enolpyruvate carboxykinase responds to more situations conducive to gluconeogenesis than do the activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase, and (B) cytosolic P-enolpyruvate carboxykinase activity is rapidly adaptive under appropriate circumstances, suggests that this particular enzyme's activity plays an important role in the regulation of gluconeogenesis in rabbits.     

The effect of fructose 2,6-bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase from rat liver

Rat liver fructose-1,6-bisphosphatase was partially phosphorylated in vitro and separated into unphosphorylated and fully phosphorylated enzyme. The effects of fructose 2,6-bisphosphate and AMP on these two enzyme forms were examined. Unphosphorylated fructose-1,6-bisphosphatase was more easily inhibited by both effectors. Fructose 2,6-bisphosphate affected both K0.5 and Vmax, while the main effect of AMP was to lower Vmax. Fructose 2,6-bisphosphate and AMP together acted synergistically to decrease the activity of fructose-1,6-bisphosphatase, and since unphosphorylated and phosphorylated enzyme forms are affected differently, this might be a way to amplify the effect of phosphorylation.     

Accumulation of neutral lipids in Saccharomyces carlsbergensis by myo-inositol deficiency and its mechanism. Reciprocal regulation of yeast acetyl-CoA carboxylase by fructose bisphosphate and citrate.

The abnormal accumulation of lipids due to myo-inositol deficiency in Saccharomyces carlsbergensis, and the mechanism involved was investigated. The deficient cells contained much more neutral lipids with a greater ratio of unsaturated fatty acids compared to the supplemented cells, whereas there was no significant change in their phospholipid contents. The biosynthesis of fatty acids and sterols from acetate, and of triacylglycerols and sterol esters from palmitate was markedly augmented in the deficient cells. Acetyl-CoA carboxylase activity of the deficient supernatant was 2- to 5-fold higher than that of the supplemented. However, the activity from both sources was not significantly different after Sephadex G-25 gel filtration of the supernatant, suggesting the presence of low molecular effector(s) in the deficient supernatant. There was a great increase in acid-soluble glycogen, trehalose, and fructose-1,6-P2, as well as a drastic decrease in citrate in the deficient cells. Their intracellular levels were calculated so that their effects on acetyl-CoA carboxylase was examined over the range of physiological concentration. Citrate strongly inhibited the enzyme activity of the supernatant, but it had no effect on the preparation after gel filtration. On the other hand, fructose-1,6-P2 stimulated the enzyme activity both before and after gel filtration. The acetyl-CoA carboxylase activity in the gel filtrate was measured as a function of citrate concentration at several fixed concentrations of fructose-1,6-P2. Citrate counteracted the activation by fructose-1,6-P2 in a dose-dependent manner. Citrate lacked the inhibitory effect in the absence of fructose-1,6-P2. It was concluded from these results that neutral lipid accumulation in the deficient cells reflected an increase in the synthesis of fatty acids, at least partly based on an enhancement of acetyl-CoA carboxylase activity, and that the operation of a reciprocal regulation of the enzyme by fructose-1,6-P2 and citrate caused a marked elevation of the enzyme activity in the deficient cells with a high fructose-1,6-P2 level and a low citrate level.     

Modification of the kinetics and regulatory properties of bovine hepatic fructose 1,6-diphosphatase with pyridoxal 5'-phosphate

Treatment of purified fructose 1,6-diphosphatase from bovine liver (which is maximally active at neutral pH) with pyridoxal 5'-phosphate produces changes in the spectral, catalytic, and allosteric properties of the enzyme. After modification the Michaelis constants for fructose-1,6-diphosphate and Mg²⁺ are increased, and inhibition by AMP is decreased. Substrate inhibition is decreased, but not abolished; the curve is merely shifted toward higher substrate concentration. Fructose-1, 6-diphosphate protects against the increases in the K_m for fructose-1, 6-diphosphate and the K_m for Mg²⁺, and against the changes in substrate inhibition, but not against the changes in AMP inhibition. AMP protects against the changes in AMP inhibition and the increase in the K_m for magnesium, but does not prevent the changes in substrate inhibition or the increase in the K_m for fructose-1, 6-diphosphate. The pH curves in the modified enzyme are altered at high, but not at low, substrate concentration.     

Fructose-1-phosphate-6-sulfate as an alternative substrate for aldolase and fructose-1,6-diphosphatase.

Fructose-1-phosphate-6-sulfate was prepared by direct sulfurylation of fructose, and selective phosphorylation of the 6-sulfuryl isomer by phosphofructokinase. The ketose derivative was used as a substrate for aldolase and fructose-1,6-diphosphatase. Kinetic studies with aldolase showed that the alternative substrate binds one third as well as fructose-1,6-P₂ yet 900 fold greater than fructose-1-P. The V_m was intermediate between the two ketose phosphates. From kinetic studies with skeletal muscle fructose-1,6-diphosphatase at pH 7.5 a K_m of 8 μM and a V_m approximately 6% that for fructose-1,6-P₂ was obtained.     

Liver peptide stabilizing factor protects phosphofructokinase against inactivation by fructose-1,6-bisphosphatase.

Rabbit liver fructose-1,6-bisphosphatase (FDPase) can reversibly inactivate both rabbit muscle and rat liver phosphofructokinases (PFK) under appropriate conditions. The peptide factor which stabilizes rat liver PFK-L₂ against thermal inactivation has now been found to protect both PFKs from inactivation by FDPase. Assay at high ATP (ca. 3 mM) is necessary to demonstrate these reversible changes. In addition, the activation of FDPase by liver cytosol, by oleate plus cytosol, or by oleate plus muscle PFK is lowere about 50% in the presence of peptide factor. These observations suggest an active participation of the peptide factor in regulation of liver glycolysis and gluconeogenesis.     

Effect of calcitonin on fructose 1,6-diphosphatase activity in rat liver: role of cytosolic calcium concentration

The effect of calcitonin (CT) on fructose 1,6-diphosphatase activity in the hepatic cytosol was investigated after a single subcutaneous administration of the hormone to rats. Administration of CT (synthetic [Asu1,7] eel CT; 80 MRC mU/100 g body weight) produced significant increase in fructose 1,6-diphosphatase activity and calcium content in the hepatic cytosol of intact and thyroparathyroidectomized rats. Those alterations were also observed with the dose of CT at physiological level. The binding of calcium by 10 microM EGTA in the hepatic cytosol caused a clear reduction of the increase in fructose 1,6-diphosphatase activity produced by CT administration. The enzyme activity of CT-treated rats was markedly reduced by W-7 (100 microM), calmodulin inhibitor, while that of control rats was not significantly altered by the drug. Meanwhile, fructose 1,6-diphosphatase activity in the hepatic cytosol obtained from normal rats was significantly enhanced by addition of calcium ion (0.1-5.0 microM). This increase was also clearly inhibited by W-7. These results suggest that CT increases fructose 1,6-diphosphatase activity in the hepatic cytosol of rats, and that this hormonal regulation may depend on calmodulin, mediated through calcium increased in the cytosol.     

Kinetic studies on the mechanism and regulation of rabbit liver fructose-1,6-bisphosphatase

The interaction of Mg2+, AMP, and fructose 2,6-bisphosphate with respect to rabbit liver fructose-1,6-bisphosphatase was investigated by studying initial-rate kinetics of the system at pH 9.5. A rapid-equilibrium Random Bi Bi mechanism is suggested for the rabbit liver enzyme from the kinetic data. Our kinetic findings indicate that Mg2+ and the inhibitor AMP are mutually exclusive in their binding to fructose-1,6-bisphosphatase. This probably is the mechanism for AMP regulation of fructose-1,6-bisphosphatase and thus, to some extent, gluconeogenesis. A kinetic model for the interaction of these ligands with respect to rabbit liver fructose-1,6-bisphosphatase is presented.     

A fluorescence study of the effects of pH and Mg2+ on the conformation of fructose 1,6-diphosphatase from spinach chloroplasts.

2-p-Toluidino-naphthalene-6-sulfonate is a sensitive fluorescent reporter group which can be used for the detection of the conformation of fructose 1,6-diphosphatase from spinach chloroplasts. When fructose 1,6-diphosphatase was added to a dilute solution of 2-p-toluidino-naphthalene-6-sulfonate at pH 9.0, the fluorescence intensity gradually increased. At this pH, the enzyme activity decreased at the same rate. However, at neutral pH (7.5), this time-dependent fluorescence change was not observed. In the presence of Mg2+, which is an activator of the enzyme, the fluorescence intensity was increased instantly and did not change for 30 min in the pH range 8.0--9.0. From the concentration dependence of the fluorescence intensity, the dissociation constant for Mg2+ was determined, Kdis = 3 mM. The effects of pH and Mg2+ on the conformation and activity of chloroplast fructose 1,6-diphosphatase are discussed.     

Activities of malate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and fructose-1,6-diphosphatase with regard to metabolic subpopulations of fast- and slow-twitch fibres in rabbit muscles

Summary Activities of malate dehydrogenase (MDH), 3-hydroxyacyl-CoA dehydrogenase (HAD) and fructose-1,6-diphosphatase (FDPase) were determined in single fibres dissected from freeze-dried rabbit psoas and soleus muscles. Slow-twitch fibres as determined by qualitative ATPase reaction represent a rather uniform population with regard to HAD and MDH activities. In these fibres the two enzymes are in constant proportions. FDPase is found at extremely low activities in slow-twitch fibres and because of its relatively high activity in fast-twitch fibres of soleus and psoas muscle it might be used as a marker enzyme. Fast-twitch fibres in psoas muscle represent a heterogeneous population with regard to activities of MDH as well as of HAD. The two enzyme activities are not proportional in fasttwitch psoas fibres. These findings suggest the existence of metabolic sub-populations of fast-twitch fibres having a wide range of aerobic oxidative capacities and having differences in their capacity to oxidizing fatty acids.     

Biochemical compartmentation of fish tissues. Distribution of gluconeogenic enzymes in the brain

1. Specific glucose-6-phosphatase and fructose-1,6-diphosphatase activity were found to be biochemically compartmentalized in four parts of the brain in nine nutritionally important fishes. 2. Glucose-6-phosphatase and fructose-1,6-diphosphatase activity were highest in the cerebrum and lowest in the cerebellum. 3. Piscivorous fishes had the highest gluconeogenic enzyme content, followed by catfishes and major carps. 4. After the liver and muscles, the various parts of the brain play an important role in carbohydrate metabolism. 5. A direct relationship between the stage of evolution and elevation of gluconeogenic enzyme levels was observed. 6. It is evident from the results and the discussion that evolution modifies the biochemical organization of fishes in general and of their brain in particular.     

Fructose-1,6-bisphosphatase from rat liver. A comparison of the kinetics of the unphosphorylated enzyme and the enzyme phosphorylated by cyclic AMP-dependent protein kinase

A purification procedure for rat hepatic fructose-1,6-bisphosphatase, described earlier, has been improved, resulting in an enzyme preparation with a neutral pH optimum and with both phosphorylatable serine residues present. The subunit Mr was 40,000. Phosphorylation in vitro with cyclic AMP-dependent protein kinase resulted in the incorporation of 1.4 mol of phosphate/mol of subunit and led to an almost 2-fold decrease in apparent Km for fructose-1,6-bisphosphate. In contrast to yeast fructose-1,6-bisphosphatase, fructose-2,6-bisphosphate had no effect on the rate of phosphorylation or dephosphorylation of the intact enzyme. The effects of the composition of the assay medium, with regard to buffering substance and Mg2+ concentration, on the apparent Km values of phosphorylated and unphosphorylated enzyme were investigated. The kinetics of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase were studied with special reference to the inhibitory effects of adenine nucleotides and fructose-2,6-bisphosphate. Unphosphorylated fructose-1,6-bisphosphatase was more susceptible to inhibition by both AMP and fructose 2,6-bisphosphate than phosphorylated enzyme, at high and low substrate concentrations. Both ATP and ADP had a similar effect on the two enzyme forms, ADP being the more potent inhibitor. Finally, the combined effect of several inhibitors at physiological concentrations was studied. Under conditions resembling the gluconeogenic state, phosphorylated fructose-1,6-bisphosphatase was found to have twice the activity of the unphosphorylated enzyme.     

Fructose-1,6-diphosphatase: a cellular site of hyperbaric oxygen toxicity.

The growth-inhibitory effect of 4.2 atm of hyperbaric oxygen for Escherichia coli was strongly influenced by available nutrients. A pattern of protection was achieved with various carbohydrate intermediates which was consistent with oxygen-induced poisoning of fructose-1,6-diphosphatase and of enzymes required in the pentose shunt and for converting galactose into glucose. Two of these sites have not been investigated further, but direct evidence was obtained that purified fructose-1,6-diphosphatase was inactivated in vitro by superoxide anion, but not by molecular oxygen at hyperbaric pressure (4.2 atm). Poisioning of fructose-1,6-diphosphatase by metabolically generated oxygen radicals, such as superoxide ion, would have deleterious effects for E. coli in media where synthesis of glucose by reverse glycolysis is required, and presumably for cells of higher organisms, including man.     

Relationship between the inhibition of phosphatidic acid phosphohydrolase-1 by oleate and oleoyl-CoA ester and its apparent translocation

Phosphatidic acid phosphohydrolase-1 (PAP-1) activity is reversibly inhibited by fatty acids and their acyl-CoA esters and it appears paradoxical that these effectors have been reported to increase the liver's esterification capacity by translocating the rate-limiting enzyme PAP-1 from cytosol to the endoplasmic reticulum. Therefore, we have examined the effect of oleate, oleoyl-CoA, and spermine on the activation and translocation of PAP-1 of rat liver. PAP-1 activity is directly inhibited by oleic acid and oleoyl-CoA ester in an allosteric manner, resulting in the formation of inactive PAP-1-fatty acid (or -acyl-CoA) complex, even in the absence of any subcellular structures. Such association/aggregation of PAP-1 can be easily collected by centrifugation and may explain the apparent translocation phenomenon of this enzyme to a particular structure in the presence of fatty acids or acyl-CoA esters as reported in many works. Indeed, incubation of cytosol fraction alone with oleate or oleoyl-CoA at 37 degrees C, followed by centrifugation, induces a significant increase (sevenfold) in PAP-1 activity in the pellet fraction. This displacement is accompanied by an increase in the specific activity of PAP-1 in the pellet fraction. Spermine is less effective than oleate in inducing the displacement of PAP-1 activity from cytosol to the pellet fraction in the absence of any membrane structures. This apparent translocation of PAP-1 is also promoted when homogenate fraction was incubated with oleate prior to the preparation of cytosol and microsomal fraction. Thus, many of the announced factors, including fatty acids, would promote the in vitro association/aggregation of PAP-1 enzyme rather than its translocation, and therefore, re-evaluation of the reported effects on PAP-1 translocation phenomenon is required. It is proposed that fatty acids and their esters would favour beta-oxidation over esterification by promoting the forming of inactive associated PAP-1 in situations such as starvation and metabolic stress in which there is an increased supply of fatty acids to the liver.