Changes in subcellular localization of fructose 1,6-bisphosphatase during differentiation of isolated muscle satellite cells.

Subcellular localization of FBPase, a regulatory enzyme of glyconeogenesis, was examined inside dividing and differentiating satellite cells from rat muscle. In dividing myoblasts, FBPase was located in cytosol and nuclei. When divisions ceased, FBPase became restricted to the cytosolic compartment and finally was found to associate with the Z-lines, as in adult muscle. Moreover, a 12-fold decrease was observed in the number of FBPase-positive nuclei associated with muscle fibres of adult rat, as compared with young muscle, possibly reflecting the reduction in number of active satellite cells during muscle maturation. The data might suggest that FBPase participates in some nuclear processes during development and regeneration of skeletal muscle.

Subcellular localization of muscle FBPase in carp (Cyprinus carpio) tissues.

Subcellular localization of muscle FBPase-a regulatory enzyme of glyconeogenesis-was investigated in carp using immunohistochemistry and protein exchange method. Results of the experiments revealed that, in striated muscles, FBPase associates with alpha-actinin of the Z-line and co-localizes with aldolase. Additionally, in cardiac and smooth muscle cells FBPase is present inside the nuclei. In the light of findings on mammalian muscle FBPase, the data presented here indicates that interaction of the enzyme with specific cellular partners and nuclear presence of FBPase is a general phenomenon in contemporary vertebrates.

The origin of the high sensitivity of muscle fructose 1,6-bisphosphatase towards AMP.

Adenosine 5'-monophosphate (AMP) inhibits muscle fructose 1,6-bisphosphatase (FBPase) about 44 times stronger than the liver isozyme. The key role in strong AMP binding to muscle isozyme play K20, T177 and Q179. Muscle FBPase which has been mutated towards the liver enzyme (K20E/T177M/Q179C) is inhibited by AMP about 26 times weaker than the wild-type muscle enzyme, but it binds the fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), similarly to the wild-type liver enzyme. The reverse mutation of liver FBPase towards the muscle isozyme significantly increases the affinity of the mutant to TNP-AMP. High affinity to the inhibitor but low sensitivity to AMP of the liver triple mutant suggest differences between the isozymes in the mechanism of allosteric signal transmission.

Nuclear localization of fructose 1,6-bisphosphatase in smooth muscle cells.

Fructose 1,6-bisphosphatase (FBPase)--a key enzyme of gluconeogenesis--for a long time was regarded to be soluble, and freely diffused in the cytoplasm. Our recent investigation revealed however, that in skeletal muscles of mammals, FBPase is located on both sides of the Z-line and, in cardiomyocytes, it is also present inside the cells' nuclei. In the current paper we demonstrate that, in smooth muscle cells, FBPase is located in the cytoplasm and the nucleus, and that the presence of the enzyme in the nucleus is almost completely restricted to the heterochromatin area. In search for additional evidence for the nuclear localization of FBPase and for a possible explanation of its role in the nucleus, we have analyzed the primary structures of muscle FBPases, finding on their molecular surface a number of domains specific for proteins transported into the nucleus.

Calcium inhibits muscle FBPase and affects its intracellular localization in cardiomyocytes.

As our recent investigation revealed, in mammalian heart muscle, fructose 1,6-bisphosphatase (FBPase)--a key enzyme of glyconeogenesis--is located around the Z-line, inside cells' nuclei and, as we demonstrate here for the first time, it associates with intercalated discs. Since the degree of association of numerous enzymes with subcellular structures depends on the metabolic state of the cell, we studied the effect of elevated Ca2+ concentration on localization of FBPase in cardiomyocytes. In such conditions, FBPase dissociated from the Z-line, but no visible effect on FBPase associated with intercalated discs or on the nuclear localization of the enzyme was observed. Additionally, Ca2+ appeared to be a strong inhibitor of muscle FBPase.

Nuclear localization of aldolase A in pig cardiomyocytes.

The subcellular localization of the muscle aldolase (aldolase A) in cardiomyocytes was determined immunocytochemically by light and electron microscopy. The enzyme was localized in the cytoplasm and also in cardiomyocyte nuclei. Inside the nuclei it was preferentially localized in the heterochromatin region. The nuclear localization was confirmed by the measurement of aldolase activity in subcellular fractions of a heart muscle, and in isolated nuclei of cardiomyocytes. There was no detectable aldolase activity in isolated cardiomyocyte nuclei fractions if the fraction was not preincubated with a solution containing Triton X-100 and KCl. The calculated concentration of aldolase in the nucleus was about 0.6 micro M. This paper is the first report on the localization of aldolase A inside cardiomyocyte nuclei.

Colocalization of muscle FBPase and muscle aldolase on both sides of the Z-line.

Previously we have reported that in vitro muscle aldolase binds to muscle FBPase [Biochem. Biophys. Res. Commun. 275 (2000) 611-616] which results in the changes of regulatory properties of the latter enzyme. In the present paper, the evidence that aldolase binds to FBPase in living cell is presented. The colocalization experiment, in which aldolase was diffused into skinned fibres that had been pre-incubated with FBPase, has shown that aldolase in the presence of FBPase binds predominantly to the Z-line. The existence of a triple aldolase-FBPase-alpha-actinin complex was confirmed through a real-time interaction analysis using the BIAcore biosensor. The colocalization of FBPase and aldolase on alpha-actinin of the Z-line indicates the existence of glyconeogenic metabolon in vertebrates' myocytes.

Muscle FBPase in a complex with muscle aldolase is insensitive to AMP inhibition.

Real-time interaction analysis, using the BIAcore biosensor, of rabbit muscle FBPase-aldolase complex revealed apparent binding constant [K(Aapp)] values of about 4.4x10(8) M(-1). The stability of the complex was down-regulated by the glycolytic intermediates dihydroxyacetone phosphate and fructose 6-phosphate, and by the regulator of glycolysis and glyconeogenesis--fructose 2,6-bisphosphate. FBPase in a complex with aldolase was entirely insensitive to inhibition by physiological concentrations of AMP (I(0.5) was 1.35 mM) and the cooperativity of the inhibition was not observed. The existence of an FBPase-aldolase complex that is insensitive to AMP inhibition explains the possibility of glycogen synthesis from carbohydrate precursors in vertebrates' myocytes.

FBPase is in the nuclei of cardiomyocytes.

Intracellular localization of FBPase in the cardiac muscle of the pig was studied by immunohistochemistry. In contrast to data from skeletal muscle [Gizak, A., Rakus, D. and Dzugaj, A. (2003) Histol. Histopathol. 18, 135-142], in cardiomyocytes FBPase was present not only in the cytoplasm, but surprisingly, also in the nucleus. Results of the microscopic investigation were confirmed by immunoblotting, measurement of FBPase activity in isolated cardiomyocyte nuclei and by determination of the nuclear FBPase I(0.5) toward adenosine monophosphate (AMP), which was the same as for the purified enzyme.

Immunohistochemical localization of human fructose-1,6-bisphosphatase in subcellular structures of myocytes.

The localization of fructose-1,6-bisphosphatase (FBPase) in human skeletal muscle was determined immunohistochemically using polyclonal antibodies. Light microscopy analysis, confirmed with the use of confocal microscopy, indicated that the enzyme is localized on both sides of the Z line of myocytes. The immunohistochemical investigation was confirmed by a co-sedimentation experiment which revealed that muscle FBPase binds strongly to alpha-actinin--a major structural protein of the Z line. This is the first report on localization of FBPase in skeletal muscle tissue.

Hematological effects of high dose of cortisol on the carp (Cyprinus carpio L.): cortisol effect on the carp blood.

The level of circulating cortisol and peripheral blood parameters were determined in carp age 2 years (K(2)) 24, 72, and 216 h after a single intraperitoneal injection of a high dose (200 mg x kg(-1) body wt) of hydrocortisone. The most striking effect of cortisol was manifest as a significant change in the percentage composition of leukocytes, whose number per unit volume of blood remained relatively constant. A profound lymphopenia and eosinopenia were compensated for in the general balance by an increased number of circulating promyelocytes and myelocytes as well as metamyelocytes and mature polymorphonuclear neutrophilic granulocytes. The results and their possible reasons are discussed on the background of literature data.

Human lung fructose-1,6-bisphosphatase is localized in pneumocytes II.

The localization of fructose-1,6-bisphosphatase (Fru-1,6-Pase EC 3.1.3.11) in human alveolar epithelium was determined immunohistochemically using a polyclonal antibody raised against the enzyme purified from human liver. The immunohistochemical analysis revealed that the Fru-1,6-Pase was localized in pneumocytes II and was absent in pneumocytes I. Hypothetically Fru-1,6-Pase participating in glucose-6-phosphate synthesis from noncarbohydrate precursors increases NADPH level which is used for surfactant synthesis and for glutathione redox cycle.

Muscle aldolase decreases muscle FBPase sensitivity toward AMP inhibition.

Muscle aldolase bound to muscle FBPase (K(d) = 8.7 microM) decreases the latter's sensitivity towards AMP inhibition. I(0.5) of muscle FBPase was increased from 0.06 microM to 0.65 microM when determined in the presence of 10 microM of muscle aldolase. In the presence of 10 microM of liver aldolase I(0.5) of liver FBPase was increased only twofold, from 11.0 microM to 21.7 microM. The effect of muscle aldolase on liver FBPase and liver aldolase on muscle FBPase is rather negligible. Aldolase slightly affected interaction of FBPase with magnesium ions decreasing K(a) and Hill constant (n). No effect of aldolase on FBPase pH optimum was observed.

Kinetic properties of pig (Sus scrofa domestica) and bovine (Bos taurus) D-fructose-1,6-bisphosphate 1-phosphohydrolase (F1,6BPase): liver-like isozymes in mammalian lung tissue.

F1,6BPases from porcine and bovine lung were isolated and their kinetic properties were determined. Ks, Kis and beta were determined assuming partial-noncompetitive inhibition (simple intersecting hyperbolic noncompetitive inhibition) of the enzyme by the substrate. Values for Ks were 4.1 and 4.4 microM for porcine and bovine F1,6BPase, respectively and values for 1 were close to 0.55 in both cases. Kis were 9 and 15 microM for porcine and bovine F1,6BPase, respectively. I0.5 for AMP were determined as 7 microM for pig enzyme and 14 microM for F1,6BPase from bovine lung. The enzymes were inhibited by F2,6BP with Ki's of 0.19 and 0.21 microM for porcine and bovine enzymes, respectively. In the presence of AMP concentration equal to I0.5, the Ki values for pig and bovine enzymes were 0.07 and 0.09 microM, respectively. The levels of F2,6BP, AMP and antioxidant enzymes activities in pig and bovine lung tissues were also determined. The cDNA coding sequence of pig lung F1,6BPase1 showed a high homology with pig liver enzyme, differing only in four positions (G/C-63, T/A-808, G/C-884 and T/A-1005) resulting in a single amino acid substitution (Gly-295 for Ala-295). It is hypothesized that the lung F1,6BPase participates in gluconeogenesis, surfactant synthesis and antioxidant reactions.

Regulatory properties of Rana esculenta liver D-fructose-1,6-bisphosphate 1-phosphohydrolase and their comparison with properties of other vertebrate liver isoenzymes.

D-Fructose-1,6-bisphosphate 1-phosphohydrolase [EC 3.1.3.11] (Fru-1,6P2ase), a regulatory enzyme of gluconeogenesis, was isolated from Rana esculenta liver in homogeneous from with approximately 30% yield. Basic kinetic properties of the enzyme and its subunit molecular weight were determined. Km is 1.72 microM. Like other vertebrate Fru-1,6P2ase, the frog liver enzyme is inhibited by fructose-2,6-bisphosphate (Fru-2,6P2) competitively, Ki is 78 nM and by AMP allosterically, I0.5 is 10.9 microM. Both inhibitors (Fru-2,6P2 and AMP) act synergistically on liver Fru-1,6-P2ase. Ki for Fru-2,6P2 determined in the presence of 1-10 microM of AMP were 35-2 nM, respectively. Maximum activity was found at pH 7.5. Like other Fru-1,6P2ases, the frog enzyme requires magnesium ions for its activity and is activated by potassium ions; the Ka for Mg2+ is 267 microM, Ka for K+ is 77 mM. The subunit molecular weight of the frog liver Fru-1,6P2ase was 37,300 Da. A great similarity between regulatory properties of frog liver Fru-1,6P2ase and liver enzymes of other vertebrates, suggests a similar regulation of gluconeogenesis in amphibia and other vertebrates.

cDNA sequence and kinetic properties of human lung fructose(1, 6)bisphosphatase.

A cDNA encoding fructose(1,6)bisphosphatase was isolated from total human lung RNA. The cDNA contained an open reading frame encoding 337 amino acids. The determined nucleotide sequence of the lung cDNA was significantly different from muscle cDNA and slightly differed from human liver cDNA in a single mutation (Gly-336 for Ala-336) and a T for C substitution in position 648. The human lung fructose(1, 6)bisphosphatase [Fru(1,6)Pase] was isolated and its kinetic parameters were compared with liver and muscle isoenzymes. Values of kcat for the lung Fru(1,6)Pase were lower than for the liver and muscle enzyme. Like the liver isoenzyme, lung Fru(1,6)Pase is significantly less inhibited by AMP than the muscle enzyme. The values of I0.5 were 9.5, 9.8, and 0.3 microM for the liver, lung, and muscle enzyme, respectively. The lung enzyme was slightly more sensitive to fructose(2,6)bisphosphate [Fru(2,6)P2] inhibition than the liver enzyme. Ki was 75 microM for the lung and 96 microM for the liver enzyme. The synergistic effect of AMP and Fru(2,6)P2 on the lung and liver Fru(1,6)Pase was also observed. In the presence of AMP the corresponding values of Ki for Fru(2,6)P2 were 16 microM for the lung and 10 microM for the liver enzyme.

Rana esculenta L. liver Fru-1,6-P2ase and G-6-Pase activity and Fru-2,6-P2 concentration after acclimation at 5 and 25 degrees C.

The activities of Fru-1,6-P2ase and G-6-Pase in liver and kidney of frogs acclimated at 5 and 25 degrees C and Fru-2,6-P2 level in liver were investigated. The aim of this study was to examine the effect of thermal acclimation on regulatory enzymes of gluconeogenesis and on concentration of gluconeogenesis regulator. Fru-1,6-P2ase activity in liver of frogs acclimated at 5 degrees C was 6.16 +/- 0.77 and 4.46 +/- 0.46 U/g wt in those acclimated at 25 degrees C; the respective values for G-6-Pase were 0.46 +/- 0.04 and 0.25 +/- 0.02 U/g wt. Fru-1,6-P2ase activity in kidney was 3.2 +/- 0.48 U/g wt at 5 degrees C and 2.64 +/- 0.23 U/g wt at 25 degrees C; the respective values for G-6-Pase were 0.2 +/- 0.05 and 0.17 +/- 0.05 U/g wt. K(m) of frog liver Fru-1,6-P2ase determined after acclimation at 5 degrees C and to 25 degrees C was 1.36 and 1.41 microM, respectively. Frog liver Fru-1,6-P2ase was allosterically inhibited by AMP. I0.5 determined after acclimation at 5 degrees C was 10.55 microM and after acclimation at 25 degrees C was 10.88 microM. Liver Fru-2,6-P2 concentration after acclimation at 5 degrees C was 0.44 +/- 0.13 nmol/g wt in comparison with 0.58 +/- 0.19 nmol/g wt after acclimation at 25 degrees C. In conclusion, cold exposure increased hepatic gluconeogenic capacity of Rana esculenta.

Kinetic properties of D-fructose-1,6-bisphosphate 1-phosphohydrolase isolated from human muscle.

D-Fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11) [Fru(1,6)Pase] was isolated from human muscle in an electrophoretically homogeneous form, free of aldolase contamination. The enzyme is inhibited by the substrate [fructose (1,6)-bisphosphate]. Km is 0.77 microM; Kis is 90 microM. The fructose-2,6-bisphosphate [Fru(2,6)P2], a regulator of gluconeogenesis, inhibits human muscle Fru(1,6)Pase with Ki = 0.13 microM. To determine Km, Kis and Ki the integrated method was used. AMP is an allosteric inhibitor of Fru(1,6)Pase. As with other mammalian isoenzymes, the human muscle enzyme is more strongly inhibited by AMP than is the liver isoenzyme [Dzugaj and Kochman (1980) Biochim. Biophys. Acta 614, 407-412]. Both of the inhibitors [AMP and Fru(2,6)P2] act synergistically on human muscle Fru(1,6)Pase. Ki for Fru(2,6)P2 determined in the presence of 0.4 microM AMP was 0.028 microM. The human muscle enzyme, like other mammalian Fru(1,6)Pases, requires Mg2+ for its activity. The Ka for magnesium was 232 microM, and h (Hill coefficient) = 2.0.

Kinetic parameters of human and rabbit liver D-fructose 1,6-diphosphate 1-phosphohydrolase determined at 25 and 37 degrees C.

1. Kinetic parameters of human and rabbit liver D-fructose 1,6-diphosphate 1-phosphohydrolase (EC 3.1.3.11) (FDP-ase) at 25 and 37 degrees C have been determined. 2. Km determined at 25 degrees C were 1.4 microM for human and 1.6 microM for rabbit enzyme; at 37 degrees C, corresponding values were 1.7 and 1.8 microM. 3. Both enzymes are allosterically inhibited by AMP. Respective values of I0.5 were 7.2 microM for human and 13.2 microM for rabbit at 25 degrees C, and 16.6 microM for human and 27.3 microM for rabbit at 37 degrees C. 4. Fructose 2,6-diphosphate, a potent regulator of gluconeogenesis, is more effective at 25 than at 37 degrees C. Ki determined at 25 degrees C was 0.07 microM for human and 0.035 microM for rabbit in comparison with 0.17 microM for human and 0.09 microM for rabbit at 37 degrees C. 5. Affinity of FDP-ase for magnesium is also dependent on temperature. For the human enzyme, Km at 25 degrees C was 226 microM and at 37 degrees C, 176 microM. For the rabbit enzyme, corresponding values were 256 and 240 microM. 6. Both enzymes are activated by KCl. Determined values of A0.5 were 91 mM for human, and 50 mM for rabbit enzyme at 25 degrees C, and 129 mM for human and 100 mM for rabbit enzyme at 37 degrees C.

Fluorescence investigations on the interaction of rabbit liver D-fructose-1,6-bisphosphate 1-phosphohydrolase with bovine serum albumin.

The interaction of D-fructose-1,6-bisphosphate 1-phosphohydrolase (Fru-P2-ase, EC 3.1.3.11), with bovine serum albumin (BSA) results in the fluorescence quenching of BSA. BSA increases fluorescence anisotropy of Fru-P2-ase modified with o-phthaldialdehyde. A program in Fortran, to simulate the experimental titration curves of BSA with Fru-P2-ase and o-phthaldialdehyde modified Fru-P2-ase with BSA, was written. For fluorescence quenching experiments the best fit was obtained for a model where one subunit of native Fru-P2-ase binds up two molecules of BSA. The determined dissociation constants at 5, 15, 25 and 35 degrees C were 2.2, 1.6, 0.83 and 0.03 microM, respectively.