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ABSTRACT: The present review addresses recent advances in research into a family of bifunctional enzymes that are responsible for the twofold task of synthesizing and hydrolyzing fructose-2,6-bisphosphate (Fru-2,6-P2), which in turn regulates the rate of glycolysis in most cells. The structure of the synthetic kinase, conjoined at its carboxyl-terminus to the phosphatase, is very highly conserved throughout evolution and differentiation, with isotypic expression arising from highly variable amino-terminal and carboxyl-terminal regulatory domains. These domains, which frequently contain protein-kinase-catalyzed phosphorylation motifs, are responsible for the widely divergent kinetics observed in various tissues and species, and for the hormonal modulation that alters intracellular levels of Fru-2,6-P2. The present review discusses recent advances in relating structure to function, and the identification of new pathways of transcriptional regulation of this important family of regulatory enzymes.
Current Opinion in Clinical Nutrition and Metabolic Care 10/2001; 4(5):411-8. · 4.38 Impact Factor
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ABSTRACT: To assess the role of glucocorticoid receptor antagonists and mediators released by Kupffer cells and other resident macrophages, we have used RU486 and gadolinium chloride to prevent the induction of glucose-6-phosphatase (Glu-6-Pase) gene expression in the liver following hemorrhagic shock (HS) and lactated Ringer's (LR) solution resuscitation. HS was induced in fasted, anesthetized, and cannulated rats by rapid phlebotomy to a mean arterial pressure of 40 mmHg and maintained for 30 min by withdrawal or infusion of blood. The LR solution group underwent induction and maintenance of HS for 30 min followed by LR resuscitation. Rats were injected with gadolinium chloride (7 mg/kg) to inhibit the phagocytic function of Kupffer cells, and with glucocorticoid receptor antagonist RU486 (20 mg/kg) prior to induction of HS. Arterial blood samples were obtained and livers were freeze clamped in liquid nitrogen and stored at -70 degrees C for subsequent analysis. Northern blot analysis indicated that Glu-6-Pase mRNA abundance increased 2-fold in HS rats and a further 2-fold with resuscitation. Gadolinium chloride administration had no significant effect on Glu-6-Pase mRNA abundance in HS or in LR solution. In contrast, RU486 pre-treatment reduced Glu-6-Pase mRNA by about one half in HS rats compared with control and that in LR solution to normal. This was associated with a normalization of Glu-6-Pase activity and plasma glucose toward pre-hemorrhage levels. These results suggest that gadolinium chloride inhibition of macrophage factor release has no effect on the induction of Glu-6-Pase mRNA during HS or in LR solution resuscitation. On the other hand, the suppression of Glu-6-Pase mRNA by RU486 suggests that glucocorticoids are responsible for the induction of the mRNA in HS and during LR resuscitation. KEYWORDS-Shock, hyperglycemia, corticosterone, gadolinium chloride, diltiazem, animal model, mRNA
Shock 12/2000; 14(5):578-81. · 2.85 Impact Factor
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ABSTRACT: The authors have recently demonstrated that increased gene expression of glucose-6-phosphatase (Glu-6-Pase) in hemorrhagic hypotension (HH) and following lactated Ringer's resuscitation (LR) is associated with a decrease in insulin and an increase in corticosterone concentrations.
To evaluate the in-vivo role of hormones the authors used insulin (IN), phentolamine and propranolol (PP) as an adrenergic blocker, and cyclic somatostatin (CS) as a glucagon blocker to prevent the induction of Glu-6-Pase gene expression in liver and kidney following HH and LR.
Hemorrhage was induced in fasted anesthetized rats, and the reduction of blood pressure to 40 mm Hg for a duration of 30 minutes was accomplished by withdrawal or infusion of shed blood. The resuscitated group underwent hemorrhage followed by fluid resuscitation with lactated Ringer's solution.
Neither PP nor CS treatment could block the induction of Glu-6-Pase messenger ribonucleic acid (mRNA) following either HH or LR. However, the administration of IN significantly prevented the increase of Glu-6-Pase mRNA level and activity in both liver and kidney following HH and LR. This was associated with a normalization of plasma glucose, corticosterone, and glucagon levels and glucose-6-phosphate concentrations in liver and kidney toward prehemorrhage levels.
These results indicate that in-vivo treatment with insulin during hemorrhagic hypotension and resuscitation is capable of preventing the increase in Glu-6-Pase gene expression in liver and kidney responsible for the observed hyperglycemia.
Academic Emergency Medicine 08/2000; 7(7):731-8. · 1.86 Impact Factor
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ABSTRACT: The influence of sepsis on the expression and activity of hepatic glucose-6-phosphatase (Glu-6-Pase) was examined during the early hyperglycemic phase and the later hypoglycemic phase.
Sepsis was induced in anesthetized, fasted rats by cecal ligation and puncture, and liver samples were taken at 0, 0.5, 1, 1.5, and 20 hours after cecal ligation and puncture.
The mRNA abundance of hepatic Glu-6-Pase increased fourfold at 0.5 hours over healthy control values, two-fold after 1 hour, and returned to normal after 1.5 hours. This finding was followed by a corresponding increase in Glu-6-Pase activity and was coincident with increased plasma glucose levels and decreased liver glucose-6-phosphate (Glu-6-P) at 0.5 and 1 hours. Plasma insulin and glucagon levels remained unchanged during this period, whereas corticosterone levels increased 2.5-fold over control values. At 20 hours cecal ligation and puncture, plasma glucose levels returned to normal, coincident with a 90% reduction in Glu-6-Pase mRNA abundance. Glu-6-Pase activity and Glu-6-P concentration returned to normal levels, while insulin, glucagon, and corticosterone levels increased significantly, i.e., 40-fold, 6.5-fold, and 6-fold, respectively.
The initial rise and subsequent decline in blood glucose correlate very well with a corticosterone-dependent induction of hepatic Glu-6-Pase, mRNA, and protein, followed by an insulin-dependent suppression of its expression.
The Journal of trauma 08/2000; 49(1):38-42. · 2.48 Impact Factor
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ABSTRACT: Liver and skeletal muscle isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-P(2)ase) isoenzymes are products of alternatively spliced first exons of the same gene, with common kinase and bisphosphatase domains. The muscle-specific exon-1 encodes nine unique amino acids, that lack the cAMP-dependent protein kinase (PK-A) phosphorylation site, and differ in sequence from those encoded by the liver-specific exon-1 (32 amino acids), contributing to its much lower affinity for fructose 6-phosphate (Fru-6-P). PK-A phosphorylation of the liver isoform at Ser(32) reduces the affinity of the kinase for Fru-6-P, and stimulates the bisphosphatase V(max). In the present study, we have defined the locus of interaction of the N-terminal residues with the N-terminal kinase and C-terminal domains by successive N- and C-terminal deletions. This study shows that: (1) residues Gly(5)-Glu(6)-Leu(7) of the liver isoform are responsible for increasing the affinity of 6PF2K for Fru-6-P, maintaining the inhibition of Fru-2,6-P(2)ase activity, and mediating the effects of PK-A phosphorylation on the two activities; (2) the loss of Fru-6-P inhibition of the bisphosphatase and the enhancement of its V(max), rather than the inhibition of the kinase, may be responsible for the behaviour of the muscle isoform primarily as a bisphosphatase; (3) the composition of residues 24-32 of the liver form appears to confer the enhanced kinase catalytic rate of this form over that of the muscle isoform. It is concluded that specific regions of the N-terminus of liver and skeletal muscle 6PF2K/Fru-2,6-P(2)ase have a role in adapting the two activities to work in the physiological range of pH and substrate concentrations found in each particular tissue.
Biochemical Journal 05/2000; 347(Pt 2):459-67. · 4.90 Impact Factor
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ABSTRACT: Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the betagamma subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-beta(2) (PLC-beta(2)), which is regulated by Gbetagamma, replaces the PH domain of PLC-delta(1) which binds to, but is not regulated by, Gbetagamma. We found that attachment of the PH domain of PLC-beta(2) onto PLC-delta(1) not only causes the membrane-binding properties of PLC-delta(1) to become similar to those of PLC-beta(2), but also results in a Gbetagamma-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.
Journal of Biological Chemistry 04/2000; 275(11):7466-9. · 4.77 Impact Factor
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ABSTRACT: Recombinantly expressed human ATP:citrate lyase was purified from E. coli, and its kinetic behavior was characterized before and after phosphorylation. Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein. Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits. Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic. The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity. Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction. Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation.
Biochemistry 03/2000; 39(5):1169-79. · 3.42 Impact Factor
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ABSTRACT: The mechanisms responsible for the glycemic changes associated with endotoxic shock are not fully understood, but are known to involve the ability of the liver to produce glucose. The purpose of the present study was to determine whether endotoxin (LPS) influences the expression and activity of glucose-6-phosphatase (Glu-6-Pase) during the early hyperglycemic phase and the later hypoglycemic phase. Rats were injected with a relatively large dose of LPS (20 mg/kg) or saline (control), and sacrificed at 1 or 5 h post-injection. Both the plasma glucose concentration and glucose production were elevated 1 h post-LPS (2-fold) and both decreased at 5 h postinjection (50%). Compared to time-matched control values, hepatic glucose-6-phosphate and fructose-6-phosphate levels were significantly decreased at both 1 and 5 h. Hepatic Glu-6-Pase activity and mRNA levels were moderately increased, 1 h after injection of LPS. At 5 h, an 88% decrease in mRNA abundance for Glu-6-Pase was associated with a 30% decrease in activity of this enzyme. Plasma insulin concentrations were not different 1 h after LPS and were elevated 2-fold from control values at 5 h. Circulating levels of glucagon and corticosterone were elevated at both time points following LPS. Our data indicate that the LPS-induced hypoglycemia and reduction in hepatic glucose production were accompanied by a depression in Glu-6-Pase activity and gene expression.
Molecular and Cellular Biochemistry 07/1999; 196(1-2):79-83. · 2.06 Impact Factor
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ABSTRACT: In a previous study [Nachaliel et al., 1993], we identified an RNA-binding protein (RBP) in FTO-2B rat hepatoma cells whose activity was stimulated upon the dissociation of a protein factor. We report in this article that the RBP is a complex protein of about 400 kDa, composed of RNA-binding subunit(s) (RBS), and regulatory subunit(s) (RS). We purified the RS to near-homogeneity (Mr approximately 25,000) and determined the amino acid sequence of a peptide derived from RS. On the basis of this sequence information, the cDNA for RS was obtained. Recombinant RS protein expressed in Escherichia coli had the capacity to bind RBS and inhibit its RNA-binding activity. The cDNA contains the complete coding sequence because the recombinant protein has the same electrophoretic mobility as that of the native RS in SDS-polyacrylamide gels. Sequence comparison showed that RS is almost identical to DJ-1, a recently discovered protein with an oncogenic potential, and CAP1, a rat sperm protein. However, the protein does not contain any known motifs that can provide a clue as to its exact function. Indirect immunofluorescence analyses showed that in addition to the cytoplasm, where RS is associated with microtubular filaments, the polypeptide is localized to the cell nucleus. The possible role of RS is discussed.
Journal of Cellular Biochemistry 04/1999; 72(3):435-44. · 2.87 Impact Factor
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ABSTRACT: The mRNA abundance of several hepatic glycolytic and gluconeogenic enzymes and blood hormone concentrations were determined in hemorrhagic hypotension-induced rats before and after resuscitation with lactated Ringer's. Northern blot analysis of total liver RNA after 30 min of hemorrhage showed control values for phospho-enolpyruvate carboxykinase and fructose-1,6-bisphosphatase mRNA, but significantly lower values for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/FBPase) as well as 2.5-fold increases in glucose-6-phosphatase (Glu-6-Pase) mRNA. The latter finding is in agreement with the greatly reduced intracellular levels of fructose-6-phosphate and glucose-6-phosphate, and the results are consistent with a rapid activation of hepatic gluconeogenesis by the concomitant decrease in 6PF2K/FBPase and increase in Glu-6-Pase. Blood insulin levels were decreased during hemorrhage and with resuscitation, whereas glucocorticoids were increased 1.5-fold in both cases. Glucagon was unchanged during hemorrhage, but was reduced with resuscitation. Lactated Ringer's resuscitation seemed to affect 6PF2K/FBPase only, which was restored to, and even exceeded, control values. In contrast, Glu-6-Pase mRNA was increased to fourfold control values. The increase in Glu-6-Pase and the decrease in 6PF2K/FBPase mRNA is probably at the level of altered transcriptional rates, because insulin, which plays a dominant role in the regulation of these genes, was decreased during hemorrhage. It remains to be determined what factors are causing further induction of Glu-6-Pase gene after lactated Ringer's resuscitation when hepatic glucose metabolism seems to have reverted to the glycolytic mode.
Shock 12/1997; 8(5):385-8. · 2.85 Impact Factor
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ABSTRACT: 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been postulated to be a metabolic signaling enzyme, which acts as a switch between glycolysis and gluconeogenesis in mammalian liver by regulating the level of fructose 2,6-bisphosphate. The effect of overexpressing the bifunctional enzyme was studied in FAO cells transduced with recombinant adenoviral constructs of either the wild-type enzyme or a double mutant that has no bisphosphatase activity or protein kinase phosphorylation site. With both constructs, the mRNA and protein were overexpressed by 150- and 40-fold, respectively. Addition of cAMP to cells overexpressing the wild-type enzyme increased the S0.5 for fructose 6-phosphate of the kinase by 1.5-fold but had no effect on the overexpressed double mutant. When the wild-type enzyme was overexpressed, there was a decrease in fructose 2,6-bisphosphate levels, even though 6-phosphofructo-2-kinase maximal activity increased more than 22-fold and was in excess of fructose-2,6-bisphosphatase maximal activity. The kinase:bisphosphatase maximal activity ratio was decreased, indicating that the overexpressed enzyme was phosphorylated by cAMP-dependent protein kinase. Overexpression of the double mutant resulted in a 28-fold increase in kinase maximal activity and a 3-4-fold increase in fructose 2,6-bisphosphate levels. Overexpression of this form inhibited the rate of glucose production from dihydroxyacetone by 90% and stimulated the rate of lactate plus pyruvate production by 200%. In contrast, overexpression of the wild-type enzyme enhanced glucose production and inhibited lactate plus pyruvate production. These results provide direct support for fructose 2,6-bisphosphate as a regulator of gluconeogenic/glycolytic pathway flux and suggest that regulation of bifunctional enzyme activities by covalent modification is more important than the amount of the protein.
Journal of Biological Chemistry 11/1995; 270(41):24229-36. · 4.77 Impact Factor
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ABSTRACT: Fructose-1,6-bisphosphatase (EC 3.1.3.11) is a key regulatory enzyme of gluconeogenesis that catalyzes the hydrolysis of fructose-1,6-bisphosphate to generate fructose-6-phosphate and inorganic phosphate. Deficiency of fructose-1,6-bisphosphatase is associated with fasting hypoglycemia and metabolic acidosis because of impaired gluconeogenesis. We have cloned and characterized the human liver fructose-1,6-bisphosphatase gene (FBP1). FBP1, localized to chromosome bands 9q22.2-q22.3 by fluorescence in situ hybridization, consists of seven exons that span > 31 kb, and the six introns are in the same position as in the rat gene. FBP1 was screened for mutations in two subjects with fructose-1,6-bisphosphatase deficiency. Four nucleotide substitutions were identified, two of which were silent mutations in the codons for Ala-216 (GCT-->GCC) and Gly-319 (GGG-->GGA). The other substitutions were in intron 3, a C-->T substitution 7 nucleotides downstream from the splice donor site, and in the promoter region, an A-->T substitution 188 nucleotides upstream from the start of transcription. These nucleotide substitutions were also found in normal unaffected subjects and thus are not the cause of fructose-1,6-bisphosphatase deficiency in the two subjects studied. The molecular basis of hepatic fructose-1,6-bisphosphatase deficiency in these subjects remains undetermined but could result from unidentified mutations in the promoter that decrease expression or from mutations in another gene that indirectly lead to decreased fructose-1,6-bisphosphatase activity.
Genomics 07/1995; 27(3):520-5. · 3.02 Impact Factor
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ABSTRACT: A cDNA encoding the catalytic core of a novel brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoenzyme was isolated from a lambda gt10 bovine brain library. This brain cDNA begins and ends in an open reading frame encoding a peptide of 476 amino acids. This peptide contains both the catalytic kinase and bisphosphatase domains and has an overall 65% and 67% indentity with the bovine heart and liver isozymes, respectively, whereas the NH2 and COOH-termini are divergent. An active catalytic core brain bifunctional enzyme was expressed in E. coli using a T7 RNA polymerase-based expression system. These results support the presence of a distinct gene coding for the protein in bovine brain.
Biochemical and Biophysical Research Communications 05/1995; 209(3):1140-8. · 2.48 Impact Factor
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ABSTRACT: Decreased hepatic fructose 2,6-bisphosphate levels were observed in the early phase of hemorrhagic shock. The lower sugar bisphosphatae level was a result of increased phosphoenolpyruvate levels and decreased glucose-6-phosphate and fructose-6-phosphate levels. The decreased glucose-6-phosphate levels correlated with increased activity of liver glucose-6-phosphatase and a concomitant 2.5-fold increase in glucose-6-phosphatase mRNA abundance. In addition, protein-free filtrate from hemorrhagic shock rats, but not from control rats, increased glucose-6-phosphatase activity. However, when control and hemorrhagic shock protein-free filtrates were heated, they both increased the glucose-6-phosphatase activity of the respective microsomes to the same extent. It is concluded that the early hyperglycemic phase of hemorrhagic shock is due to enhanced glucose-6-phosphatase gene expression and activity and the generation of a heat sensitive activator of the enzyme.
Biochemical and Biophysical Research Communications 11/1994; 204(2):716-24. · 2.48 Impact Factor
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ABSTRACT: cDNA clones coding for the catalytic subunit of rat liver glucose-6-phosphatase (EC 3.1.3.9) were isolated from a rat liver cDNA library in lambda gt11 phage. The sequence of the cDNA and the amino acid sequence derived from it were greater than 90% identical to the corresponding sequences for the mouse and human forms of liver glucose-6-phosphatase. Northern blot analysis of RNA from FAO hepatoma cells revealed that dexamethasone induced the glucose-6-phosphatase mRNA while insulin suppressed its expression. When both hormones were added together insulin completely suppressed the effect of glucocorticoid. cAMP addition alone decreased the abundance of glucose-6-phosphatase mRNA. The results demonstrate multihormonal regulation of gene expression of hepatic glucose-6-phosphatase and support a dominant role for insulin.
Biochemical and Biophysical Research Communications 06/1994; 201(1):302-9. · 2.48 Impact Factor
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ABSTRACT: Six novel alkaloids that contain a fused tetracyclic pyrido[2,3,4-kl]acridine ring system were purified recently from the Red Sea purple tunicate Eudistoma sp. Evaluation of the effects of these alkaloids on cultured neuroblastoma and fibroblast cells revealed that they possess potent growth regulatory properties, and affect cell shape and adhesion. In mouse neuroblastoma cells, the Eudistoma alkaloids inhibited cell proliferation and induced a process of differentiation during which the cells flattened onto the surface, increased considerably in size, and extended long neurites. In hamster fibroblasts the alkaloids slowed down cell multiplication, and caused an exceptional cell flattening or elongation. In a virus-transformed derivative of the hamster fibroblasts the alkaloids restored many aspects of normal cell growth and morphology. In addition, several of the alkaloids mimicked the effects of cAMP analogs on two well-characterized cAMP-mediated processes involved in hepatic glucose metabolism--inhibition of pyruvate kinase (PK) activity and induction of mRNA for phosphoenolpyruvate carboxykinase (PEPCK). All these effects suggest that the Eudistoma alkaloids may act on the cAMP signaling system. However, a single application of these compounds was sufficient to completely block cell multiplication and to induce and sustain differentiation and "reverse transformation". Furthermore, these effects were not readily reversible following removal of the drugs. In contrast, a single application of agents that mimic or elevate cAMP induced a transient response that waned with time in culture, and the effects induced by constant elevation of cAMP reverse rapidly following drug removal. We propose that the Eudistoma alkaloids cause growth inhibition, differentiation, and reverse transformation by modifying the activity state of proteins that are involved in the regulation of cell shape and adhesion and serve as a target for the cAMP and/or other second messenger systems.
Journal of Cellular Physiology 01/1994; 157(3):481-92. · 3.87 Impact Factor
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ABSTRACT: The role of Cys-138 in the catalysis of the skeletal muscle 6-phosphofructo-2-kinase reaction was investigated by mutating this residue to serine, glutamine and alanine, expressing the mutants in E. coli with a T7 RNA polymerase-based expression system, and analyzing their kinetic properties. The Cys138Ala mutant had greatly diminished activity, while the Cys138Ser and Cys138Gln mutants had maximal velocities 2-3 fold higher than the wild-type enzyme. It was concluded that Cys-138 does not act as a base catalyst in the kinase reaction, but that it plays a significant structural role in the enzyme's active site.
Biochemical and Biophysical Research Communications 09/1993; 195(1):229-36. · 2.48 Impact Factor
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ABSTRACT: A cDNA for an adipocyte membrane protein, implicated in the transport of long-chain fatty acids, was isolated by screening with a synthetic oligonucleotide derived from the amino terminal sequence of the protein. The 88-kDa adipocyte membrane protein was previously identified by covalent labeling with N-sulfosuccinimidyl esters of long-chain fatty acids which irreversibly inhibited fatty acid transport by 75% (Harmon, C. M., and Abumrad, N.A. (1993) J. Membr. Biol. 124, 261-268). The cDNA (FAT, 2432 base pairs (bp)) contained 70 bp of 5'-untranslated sequence, an open reading frame encoding a 472-amino acid protein with a predicted molecular mass of 52466, and 940 bp of 3'-untranslated sequence with two polyadenylation signal sequences but with no polyadenylation tail. The deduced protein sequence predicted two transmembrane segments and 10 potential N-linked glycosylation sites. Extensive glycosylation most likely explains why the molecular mass of the isolated protein (88 kDa) is different from that deduced from the cDNA sequence (53 kDa). The sequence of FAT is 85% homologous with that of glycoprotein IV (CD36) identified in human platelets and in lactating mammary epithelium. Consistent with this, a polyclonal antibody against CD36 reacted with adipocyte plasma membranes and detected a single band at 88 kDa. Northern blot analysis of RNA obtained from rat adipose tissue and probed with the cDNA identified two major transcripts of 4.8 and 2.9 kilobases which were abundant in heart, intestine, fat, muscle, and testis. The mRNAs were not detectable in cultured adipose cell lines (Ob1771, 3T3F442A) at the fibroblastic stage but were strongly induced during the differentiation process and by treatment of preadipocytes with dexamethasone, conditions that were also associated with an increase in oleate transport. In contrast, the fibroblastic cell lines 3T3-C2 and L929, which do not differentiate, did not express the mRNAs at all stages of culture. The data suggest that FAT and CD36 belong to a family of proteins that bind/transport long-chain fatty acids or function as regulators of these processes.
Journal of Biological Chemistry 09/1993; 268(24):17665-8. · 4.77 Impact Factor
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ABSTRACT: The role of the NH2-terminal region of the liver and skeletal muscle 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases was investigated, as well that of a mutant of the liver isoform lacking the first 22 amino acids, by the overexpression of these enzymes in Escherichia coli and the comparison of their kinetic properties. The muscle isoform and the deletion mutant had Km values for fructose 6-phosphate which were 50- and 20-fold higher, respectively, than that of the liver isoform, and the bisphosphatase maximal velocity of the liver deletion mutant was 4-fold higher than that of the native liver isoform. Phosphorylation of the liver isoform increased bisphosphatase activity by 2-3-fold and the Km for fructose 6-phosphate of the 6-phosphofructo-2-kinase by 10-15-fold, but these kinetic effects were greatly diminished for the deletion mutant despite equivalent phosphorylation by cAMP-dependent protein kinase. Arg-173 of the skeletal muscle isoform was found to be functionally equivalent to the residue corresponding to the essential fructose 6-phosphate binding residue of the liver kinase domain, Arg-195. The results suggest that 1) the NH2-terminal regions of the liver and skeletal muscle isoforms are important determinants of fructose 6-phosphate affinity, and 2) the initial 22 amino acids of the liver isoform exert an inhibitory influence on the bisphosphatase and mediate, at least in part, the response of both activities of the enzyme to cAMP-dependent phosphorylation.
Journal of Biological Chemistry 08/1993; 268(19):14056-64. · 4.77 Impact Factor
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ABSTRACT: The hormonal regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression was studied in the rat hepatoma cell line FAO-1. Both 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities were detected in FAO-1 cells, at 68% of the levels found in rat liver. Northern blot analysis showed that FAO-1 cells, like rat liver, contained a predominant species of bifunctional enzyme mRNA, which is 2.2 kb in size. A sensitive RNAase protection assay revealed the presence in FAO-1 cells of an additional mRNA species, which is generated when transcription is initiated from the skeletal muscle promoter of the rat liver/skeletal muscle gene. The liver/skeletal muscle mRNA ratio in FAO-1 cells was 10:1, which is similar to that observed in rat liver. In contrast, in another rat hepatoma cell line, FTO-2B, only the skeletal muscle mRNA was detected. Insulin and dexamethasone induced the liver bifunctional enzyme mRNA in FAO-1 cells by 2-4-fold and 10-20-fold respectively in a concentration- and time-dependent manner, and their effects were antagonized by cyclic AMP. Transcription of the gene in FAO-1 cells, measured by nuclear run-on assays, was also enhanced by dexamethasone and insulin. It is concluded that the FAO-1 cell line is similar to liver with respect to both the preferential use of the liver promoter of the gene and its regulation by hormones, and is therefore an excellent model for the study of the hepatic expression of this gene.
Biochemical Journal 08/1993; 293 ( Pt 1):173-9. · 4.90 Impact Factor
