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S J Pilkis

Publications and source records attributed to S J Pilkis.

At least 55 records · Page 3Linked to original sources
PubMed1992

Arg-257 and Arg-307 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase bind the C-2 phospho group of fructose-2,6-bisphosphate in the fructose-2,6-bisphosphatase domain.

Rat liver fructose-2,6-bisphosphatase, which catalyzes its reaction via a phosphoenzyme intermediate, is evolutionarily related to the phosphoglycerate mutase enzyme family (Bazan, F., Fletterick, R., and Pilkis, S.J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). Arg-7 and Arg-59 of the yeast phosphoglycerate mutase have been postulated to be substrate-binding residues based on the x-ray crystal structure. The corresponding residues in rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, Arg-257 and Arg-307, were mutated to alanine. The Arg257Ala and Arg307Ala mutants and the wild-type enzyme were expressed in Escherichia coli and then purified to homogeneity. Both mutant enzymes had identical far and near UV circular dichroism spectra and 6-phosphofructo-2-kinase activities when compared with the wild-type enzyme. However, the Arg257Ala and Arg307Ala mutants had altered steady state fructose-2,6-bisphosphatase kinetic properties; the Km values for fructose-2,6-bisphosphate of the Arg257Ala and Arg307Ala mutants were increased by 12,500- and 760-fold, whereas the Ki values for inorganic phosphate were increased 7.4- and 147-fold, respectively, as compared with the wild-type values. However, the Ki values for the other product, fructose-6-phosphate, were unchanged for the mutant enzymes. Although both mutants exhibited parallel changes in kinetic parameters that reflect substrate/product binding, they had opposing effects on their respective maximal velocities; the maximal velocity of Arg257Ala was 11-fold higher, whereas that for Arg307Ala was 700-fold lower, than that of the wild-type enzyme. Pre-steady state kinetic studies demonstrated that the rate of phosphoenzyme formation for Arg307Ala was at least 4000-fold lower than that of the wild-type enzyme, whereas the rate for Arg257Ala was similar to the wild-type enzyme. Furthermore, consistent with the Vmax changes, the rate constant for phosphoenzyme breakdown for Arg257Ala was increased 9-fold, whereas that for Arg307Ala was decreased by a factor of 500-fold, as compared with the wild-type value. The results indicate that both Arg-257 and Arg-307 interact with the reactive C-2 phospho group of fructose 2,6-bisphosphate and that Arg-307 stabilizes this phospho group in the transition state during phosphoenzyme breakdown, whereas Arg-257 stabilizes the phospho group of the ground state phosphoenzyme intermediate.

Amino Acid Sequence↗
PubMed1992

Bovine brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Evidence for a neural-specific isozyme.

Bovine brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was purified to homogeneity and characterized. This bifunctional enzyme is a homodimer with a subunit molecular weight of 120,000, which is twice that of all other known bifunctional enzyme isozymes. The kinase/bisphosphatase activity ratio was 3.0. The Km values for fructose 6-phosphate and ATP of the 6-phosphofructo-2-kinase were 27 and 55 microM, respectively. The Km for fructose 2,6-bisphosphate and the Ki for fructose 6-phosphate for the bisphosphatase were 70 and 20 microM, respectively. Physiologic concentrations of citrate had reciprocal effects on the enzyme's activities, i.e. inhibiting the kinase (Ki of 35 microM) and activating the bisphosphatase (Ka of 16 microM). Phosphorylation of the brain enzyme was catalyzed by the cyclic AMP-dependent protein kinase with a stoichiometry of 0.9 mol of phosphate/mol of subunit and at a rate similar to that seen with the liver isozyme. In contrast to the liver isozyme, the kinetic properties of the brain enzyme were unaffected by cyclic AMP-dependent protein kinase phosphorylation, and also was not a substrate for protein kinase C. The brain isozyme formed a labeled phosphoenzyme intermediate and cross-reacted with antibodies raised against the liver isozyme. However, the NH2-terminal amino acid sequence of a peptide generated by cyanogen bromide cleavage of the enzyme had no identity with any known bifunctional enzyme sequences. These results indicate that a novel isozyme, which is related to other 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes, is expressed specifically in neural tissues.

Amino Acid Sequence↗
PubMed1992

Human glucokinase gene: isolation, characterization, and identification of two missense mutations linked to early-onset non-insulin-dependent (type 2) diabetes mellitus.

DNA polymorphisms in the glucokinase gene have recently been shown to be tightly linked to early-onset non-insulin-dependent diabetes mellitus in approximately 80% of French families with this form of diabetes. We previously identified a nonsense mutation in exon 7 in one of these families and showed that it was the likely cause of glucose intolerance in this dominantly inherited disorder. Here we report the isolation and partial sequence of the human glucokinase gene and the identification of two missense mutations in exon 7, Thr-228----Met and Gly-261----Arg, that cosegregate with early-onset non-insulin-dependent diabetes mellitus. To assess the molecular mechanism by which mutations at these two sites may affect glucokinase activity, the crystal structure of the related yeast hexokinase B was used as a simple model for human beta-cell glucokinase. Computer-assisted modeling suggests that mutation of Thr-228 affects affinity for ATP and mutation of Gly-261 may alter glucose binding. The identification of mutations in glucokinase, a protein that plays an important role in hepatic and beta-cell glucose metabolism, indicates that early-onset non-insulin-dependent diabetes mellitus may be primarily a disorder of carbohydrate metabolism.

Adult↗
PubMed1992

Lysine 356 is a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Lysine 356 has been implicated by protein modification studies as a fructose-2,6-bisphosphate binding site residue in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kitajima, S., Thomas, H., and Uyeda, K. (1985) J. Biol. Chem. 260, 13995-14002). However, Lys-356 is found in the fructose-2,6-bisphosphatase domain (Bazan, F., Fletterick, R., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). In order to ascertain whether Lys-356 is involved in fructose-2,6-bisphosphatase catalysis and/or domain/domain interactions of the bifunctional enzyme, Lys-356 was mutated to Ala, expressed in Escherichia coli, and then purified to homogeneity. Circular dichroism experiments indicated that the secondary structure of the Lys-356-Ala mutant was not significantly different from that of the wild-type enzyme. The Km for fructose 2,6-bisphosphate and the Ki for the noncompetitive inhibitor, fructose 6-phosphate, for the fructose-2,6-bisphosphatase of the Lys-356-Ala mutant were 2700- and 2200-fold higher, respectively, than those of the wild-type enzyme. However, the maximal velocity and the Ki for the competitive product inhibitor, inorganic phosphate, were unchanged compared to the corresponding values of the wild-type enzyme. Furthermore, in contrast to the wild-type enzyme, which exhibits substrate inhibition, there was no inhibition by substrate of the Lys-356-Ala mutant. In the presence of saturating substrate, inorganic phosphate, which acts by relieving fructose-6-phosphate and substrate inhibition, is an activator of the bisphosphatase. The Ka for inorganic phosphate of the Lys-356-Ala mutant was 1300-fold higher than that of the wild-type enzyme. The kinetic properties of the 6-phosphofructo-2-kinase of the Lys-356-Ala mutant were essentially identical with that of the wild-type enzyme. The results demonstrate that: 1) Lys-356 is a critical residue in fructose-2,6-bisphosphatase for binding the 6-phospho group of fructose 6-phosphate/fructose 2,6-bisphosphate; 2) the fructose 6-phosphate binding site is responsible for substrate inhibition; 3) Inorganic phosphate activates fructose-2,6-bisphosphatase by competing with fructose 6-phosphate for the same site; and 4) Lys-356 is not involved in 6-phosphofructo-2-kinase substrate/product binding or catalysis.

Amino Acid Sequence↗
PubMed1992

Regulation of gene expression of rat skeletal muscle/liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Isolation and characterization of a glucocorticoid response element in the first intron of the gene.

At least two genes encode isoenzymes of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Alternative splicing of one of these genes generates a skeletal muscle-specific transcript from an upstream promoter and a liver-specific transcript from a downstream promoter. A potent glucocorticoid response element was identified in the first intron of the gene, i.e. between liver exon I and exon II. The element is approximately 3.5 kilobase pairs (kb) downstream of the liver isoenzyme transcription start site and 13 kb upstream of exon II of the gene and confers dexamethasone-sensitive expression of chloramphenicol acetyltransferase (CAT) activity from a heterologous thymidine kinase promoter and from both homologous 5'-flanking regions of the gene. This glucocorticoid response element also exhibits androgen- but not estrogen-sensitive expression of CAT activity in HeLa cells cotransfected with the appropriate receptor expression vector. DNase footprint and sequence analysis revealed that the element is comprised minimally of two adjacent 15-mer glucocorticoid receptor dimer binding sites situated in opposite orientations. Glucocortcoid regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in liver and skeletal muscle is mediated by a single complex glucocorticoid response element located in the first intron of the skeletal muscle/liver gene.

Animals↗
PubMed1992

Cloning and sequencing of a cDNA encoding 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize. Possible relationship to the alkaline phosphatase family.

The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced from cDNAs isolated from maize cDNA libraries by screening with specific antibodies to the cofactor-independent enzyme and from a maize genomic clone. The genomic clone provided the 5'-nucleotide sequence encoding the N-terminal amino acids which could not be obtained from the cDNA. Confirmation that the nucleotide sequence was for the cofactor-independent phosphoglycerate mutase was obtained by sequencing the peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequence of a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which consists of 559 amino acids and is twice the molecular size of the mammalian cofactor-dependent enzyme subunit. Analysis of the cofactor-independent phosphoglycerate mutase amino acid sequence revealed no identity with the cofactor-dependent mutase types. Northern blot analysis confirmed this difference since the maize cofactor-independent phosphoglycerate mutase cDNA did not hybridize with mRNA of the cofactor-dependent mutase. The lack of amino acid identity between cofactor-dependent and -independent enzymes is consistent with their different catalytic mechanisms and suggests that both enzymes are unrelated evolutionarily and arose from two independent ancestral genes. However, a constellation of residues which are involved in metal ion binding in various alkaline phosphatases is conserved in the maize cofactor-independent phosphoglycerate mutase, which suggests that the enzyme is a member of the alkaline phosphatase family of enzymes.

Alkaline Phosphatase↗
PubMed1992

Transcriptional and posttranscriptional regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase during liver regeneration.

The control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2; EC 2.7.1.105/3.1.3.46) gene expression during liver regeneration was studied. The level of PFK-2/FBPase-2 mRNA decreased to about 5% of the control value 6 hr after partial hepatectomy. Thereafter the mRNA increased to a maximum at 48 hr and returned to normal levels by 96 hr. In sham-operated animals, only a small increase was observed during the first 4 hr. The mRNA was recognized by a 299-base-pair liver-specific cDNA probe but not by a muscle-specific probe. The time course of mRNA modulation was well correlated with PFK-2/FBPase-2 activity and with the amount of bifunctional enzyme protein determined by immunoblotting with an antibody raised against the N-terminal decapeptide of liver PFK-2/FBPase-2. No alteration in the degradation rate of PFK-2/FBPase-2 mRNA was noted after partial hepatectomy. The modulation of PFK-2/FBPase-2 gene expression during liver regeneration involved changes in the transcription rate. The rate decreased by 50% at 6 hr after liver resection. The rate increased thereafter to a maximum at 72 hr and then returned to control values by 96 hr. The transcription rate of albumin did not change, whereas that of phosphoenolpyruvate carboxykinase increased 12-fold at 6 hr. These results show that PFK-2/FBPase-2 gene transcription is specifically regulated and that this regulation is in part responsible for the alterations in hepatic metabolism seen in regenerating liver.

Animals↗
PubMed1992

Lysine 274 is essential for fructose 2,6-bisphosphate inhibition of fructose-1,6-bisphosphatase.

Lysine 274 is conserved in all known fructose-1,6-bisphosphatase sequences. It has been implicated in substrate binding and/or catalysis on the basis of reactivity with pyridoxal phosphate as well as by x-ray crystallographic analysis. Lys274 of rat liver fructose-1,6-bisphosphatase was mutated to alanine by the polymerase chain reaction, and the T7-RNA polymerase-transcribed construct containing the mutant sequence was expressed in Escherichia coli. The mutant and wild-type forms of the enzyme were purified to homogeneity, and their specific activity, substrate dependence, and inhibition by fructose 2,6-bisphosphate and AMP were compared. While the mutant exhibited no change in maximal velocity, its Km for fructose 1,6-bisphosphate was 20-fold higher than that of the wild-type, and its Ki for fructose 2,6-bisphosphate was increased 1000-fold. Consistent with the unaltered maximal velocity, there were no apparent difference between the secondary structure of the wild-type and mutant enzyme forms, as measured by circular dichroism and ultraviolet difference spectroscopy. The Ki for the allosteric inhibitor AMP was only slightly increased, indicating that Lys274 is not directly involved in AMP inhibition. Fructose 2,6-bisphosphate potentiated AMP inhibition of both forms, but 500-fold higher concentrations of fructose 2,6-bisphosphate were needed to reduce the Ki for AMP for the mutant compared to the wild-type. However, potentiation of AMP inhibition of the Lys274----Ala mutant was evident at fructose 2,6-bisphosphate concentrations (approximately 100 microM) well below those that inhibited the enzyme, which suggests that fructose 2,6-bisphosphate interacts either with the AMP site directly or with other residues involved in the active site-AMP synergy. The results also demonstrate that although Lys274 is an important binding site determinant for sugar bisphosphates, it plays a more significant role in binding fructose 2,6-bisphosphate than fructose 1,6-bisphosphate, probably because it binds the 2-phospho group of the former while other residues bind the 1-phospho group of the substrate. It is concluded that the enzyme utilizes Lys274 to discriminate between its substrate and fructose 2,6-bisphosphate.

Animals↗
PubMed1992

Glu327 is part of a catalytic triad in rat liver fructose-2,6-bisphosphatase.

The fructose-2,6-bisphosphatase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to be structurally and functionally homologous to phosphoglycerate mutase. Both enzymes catalyze their reactions via phosphoenzyme intermediates which utilize an active site histidine as a nucleophilic phosphoacceptor and another histidine as a proton donor to the leaving group. Glu327 in the bisphosphatase domain of the rat liver bifunctional enzyme is conserved in all phosphoglycerate mutase structures and is postulated, by modelling studies, to be located in the active site. Glu327 was mutated to Ala, Gln, or Asp. The mutant and wild-type enzymes were expressed in Escherichia coli with a T-7 RNA polymerase-based expression system and purified to homogeneity by substrate elution from phosphocellulose. The Glu327 mutants had apparent molecular weights of 110,000 by gel filtration and had unaltered 6-phosphofructo-2-kinase activity. Circular dichroism showed that the secondary structure of the Glu327 mutant enzyme forms was the same as the wild-type enzyme. The maximal velocity of the fructose-2,6-bisphosphatase of the Glu327----Ala, Glu327----Gln, and Glu327----Asp mutants was 4, 2, and 20%, respectively, that of the wild-type enzyme, but the rate of phosphoenzyme formation of the mutants was reduced by at least a factor of 1000. In addition, the rate constants of phosphoenzyme hydrolysis for the Glu372----Ala and Glu327----Gln mutants were 2.7 and 1.3%, respectively, of the wild type, whereas the rate constant for the Glu327----Asp mutant was 60% of the wild-type value. Glu327 was not a substrate or product binding site determinant since the Km for fructose-2,6-bisphosphate and Ki for fructose-6-phosphate of the mutants were not appreciably changed. The results implicate Glu327 as part of a catalytic triad in fructose-2,6-bisphosphatase and suggest that it influences the protonation state of the active site histidine residues during phosphoenzyme formation and/or acts as a base catalyst to enhance the nucleophilic attack of water on the phosphoenzyme intermediate.

Animals↗
PubMed1992

Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site affects phosphate activation.

To identify those residues involved in fructose 6-phosphate binding to the kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase site-directed mutations were engineered at Lys194, Arg195, Arg230, and Arg238. The mutant enzymes were purified to homogeneity by anion exchange and Blue-Sepharose chromatography and/or substrate elution from phosphocellulose columns. Circular dichroism experiments demonstrated that all of the single amino acid mutations had no effect on the secondary structure of the protein. In addition, when fructose-2,6-bisphosphatase activity was measured, all mutants had Km values for fructose 2,6-bisphosphate, Ki values for fructose 6-phosphate, and maximal velocities similar to that of the wild-type enzyme. Mutation of Arg195----Ala, or His, had little or no effect on the maximal velocity of the kinase but increased the Km for fructose 6-phosphate greater than 3,000-fold. Furthermore, the Ka for phosphate for Arg195Ala was increased 100-fold compared with the wild-type enzyme. Mutation of Lys194----Ala had no effect on maximal velocity or the Km for fructose 6-phosphate. Mutation of either Arg230 or Arg238----Ala increased the maximal velocity and the Km for fructose-6 phosphate of the kinase by 2-3-fold but had no effect on fructose-2,6-bisphosphatase. However, the Km values for ATP of the Arg230Ala and Arg238Ala mutants were 30-40-fold higher than that for the wild-type enzyme. Mutation of Gly48----Ala resulted in a form with no kinase activity, but fructose-2,6-bisphosphatase activity was identical to that of the wild-type enzyme. The results indicate that: 1) Arg195 is a critical residue for the binding of fructose 6-phosphate to the 6-phospho-fructo-2-kinase domain, and that interaction of the sugar phosphate with Arg195 is highly specific since mutation of the adjacent Lys194----Ala had no effect on fructose 6-phosphate binding; 2) Arg195 also play an important role in the binding of inorganic phosphate; and 3) Gly48 is an important residue in the nucleotide binding fold of 6-phosphofructo-2-kinase and that both Arg230 and Arg238 are also involved in ATP binding; and 4) the bifunctional enzyme has two separate and independent fructose 6-phosphate binding sites.

Animals↗
PubMed1992

Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho- forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis.

The mechanism by which cAMP-dependent protein kinase-catalyzed phosphorylation modulates the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was examined after site-specific mutation of the cAMP-dependent phosphorylation site (Ser32) to aspartic acid or alanine. The mutant and wild-type enzymes were overexpressed in Escherichia coli in a rich medium to levels as high as 30 mg/liter and were then purified to homogeneity. The kinetic properties of the Ser32-Ala mutant were identical with the dephosphorylated wild-type bifunctional enzyme. Mutation of Ser32 to aspartic acid mimicked several effects of cAMP-dependent phosphorylation: there was an increase in the Km for fructose 6-phosphate for 6-phosphofructo-2-kinase and an increase in the maximal velocity of fructose-2,6-bisphosphatase. Fructose-2,6-bisphosphatase activity of the Ser32-Asp mutant was 75% that of the phosphorylated wild-type enzyme, the mutant's kinase reaction had an identical dependence on fructose 6-phosphate, while its maximum velocity was only 60% that of the phosphorylated wild-type enzyme over a wide pH range. Furthermore, catalytic subunit-catalyzed in vitro phosphorylation of the Ser32-Ala mutant on Ser33 increased the Km for fructose 6-phosphate by 4-fold for the 6-phosphofructo-2-kinase. The results support the hypothesis that Ser32 is an important residue in the regulation of the activities of the bifunctional enzyme and that phosphorylation of Ser32 can be functionally substituted by aspartic acid. The results suggest a role for negative charge in the effect of phosphorylation.

Animals↗
PubMed1992

Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis.

Understanding the regulation of hepatic glucose metabolism had its foundation in the elucidation of several pathways, but recent advances have come from the application of molecular genetics. Five years ago little was known about the primary structure of the key regulatory enzymes. Since then, the primary sequence of liver GK, 6-PF-1-K, Fru-1,6-P2ase, PK, PEPCK, and 6-PF-2-K/Fru-2,6-P2ase have been derived from cDNA sequences and/or determined by direct protein sequencing. This has provided new insights into the molecular mechanisms of catalysis and the regulation of these enzymes by covalent modification. Isolation of the cDNAs for these enzymes also has allowed for the quantitation of specific mRNAs and permitted analysis of hormonal control of specific gene expression. The genes for these enzymes have been isolated and sequenced, and their promoter regions are being identified and characterized. Hormone response elements have been delineated in several of the promoters. The promoter regions for 6-PF-2-K/Fru-2,6-P2ase and Fru-1,6-P2ase have also been identified, and future research will focus on the elucidation of the mechanisms whereby hormones regulate the expression of these genes. A number of generalizations can be made about the regulation of gene expression of glycolytic/gluconeogenic enzymes. First, there is coordinate hormonal regulation of gene expression and these effects are consonant with their physiologic actions. Insulin induces the mRNAs that encode glycolytic enzymes and represses the mRNAs that encode gluconeogenic enzymes; cAMP has opposite effects. Both can increase or decrease transcription. Whereas insulin and cAMP affect all of these mRNAs, glucocorticoids appear to have a more restricted action. Second, transcriptional and posttranscriptional regulatory mechanisms are involved. The synthesis of all of the mRNAs discussed is regulated by hormones. Relatively little is known about how mRNA stability is regulated in general, but it is clear that PEPCK mRNA is stabilized by agents that increase the rate of transcription of the gene. Under appropriate metabolic signals this dual control of mRNA synthesis and stability provides for a long-term increase in PEPCK mRNA and protein. Studies with PK mRNA are less direct, but suggest a similar dual mechanism. It will be interesting to see whether multilevel regulation is restricted to these two mRNAs, both of which are involved in the same substrate cycle, or whether the stability of other mRNAs involved in hepatic glucose metabolism is also affected. Third, glucose appears to be important in the regulation of these hepatic genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Expression and site-directed mutagenesis of hepatic glucokinase.

Soluble rat liver glucokinase was expressed at high levels at 22 degrees C in the BL21(DE3)pLysS strain of Escherichia coli. Aspartate-211 of yeast hexokinase has been implicated as a catalytic residue from crystallographic data. The corresponding residue in rat liver glucokinase, aspartate-205, was mutated to alanine and the expressed mutant had 1/500th of the activity of the wild type, with no change in the Km values for glucose or ATP. The results support a role for this residue as a base catalyst in the glucokinase reaction and, most probably, a similar role in the reactions of all members of the hexokinase family.

Animals↗
PubMed1991

Expression of rat liver fructose-1,6-bisphosphatase in Escherichia coli.

Rat liver fructose-1,6-bisphosphatase was expressed in Escherichia coli using a T7 RNA polymerase-transcribed expression system. Maximum yields of soluble active enzyme were obtained when the bacterial host cell, BL21(DE3), carrying the expression plasmid was grown and transcription induced in LB medium at 37 degrees C. Approximately 20mg of fructose-1,6-bisphosphatase are synthesized per liter of culture after 4hr, of which about 10mg are soluble and enzymatically active. Expressed fructose-1,6-bisphosphatase, purified to homogeneity by substrate elution from a carboxymethyl Sephadex column, was indistinguishable from that purified from rat liver in terms of subunit size and kinetic properties. The in vitro expression of fructose-1,6-bisphosphatase in an heterologous system is a necessary preliminary step for future studies on site-directed mutant enzyme forms.

Animals↗
PubMed1991

The rat fructose-1,6-bisphosphatase gene. Structure and regulation of expression.

The gene encoding rat fructose-1,6-bisphosphatase was isolated from a rat genomic Charon 4A library by screening with a cDNA to the rat liver enzyme. Southern blotting of rat genomic DNA showed that there is a single copy of the fructose-1,6-bisphosphatase gene. It extends over 23 kilobases and is composed of seven exons and six introns that range in size from 93 to 267 base pairs and from 1,400 to 11,300 base pairs, respectively. The intron/exon boundary sequences conform to consensus acceptor (GTn) and donor (nAG) sequences, and the exons in the gene appear to code for functional protein domains. The transcription start site, determined by 5'-extension sequencing of mRNA, was assigned to a guanine 119 bases 5' to the translation initiation AUG. The sequence of the gene upstream to the cap site contains characteristic RNA polymerase II promoter-binding sites: a putative TATA box at position -29 and a Sp 1 binding site (GGGGCGGAGA) at position -48. A 1,300-base pair fragment of 5'-flanking sequence containing these elements, ligated upstream from a firefly luciferase reporter gene and transfected into cultured normal rat kidney cells, demonstrated strong promoter activity. The accumulation of fructose-1,6-bisphosphatase mRNA in hepatocytes incubated with cAMP suggests that the gene may be cAMP-responsive, which is consistent with the presence of three consensus cAMP regulatory elements at positions -169, -282, and -698 in the 5'-flanking region of the gene. Expression of the fructose-1,6-bisphosphatase promoter-driven luciferaes gene was 2-3-fold activated by the cyclic nucleotide, suggesting that one or more of these elements may be functional. On the other hand, insulin decreased the expression of the endogenous gene in hepatocytes. Thus, expression of the fructose-1,6-bisphosphatase gene is regulated independently by both cAMP and insulin.

Amino Acid Sequence↗
PubMed1991

Hormonal control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in rat hepatoma cells.

The hormonal control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression was studied in the rat hepatoma cells, FTO-2B. In contrast to another hepatoma cell line (HTC), the enzyme in FTO-2B cells displays both kinase and bisphosphatase activities. As in rat liver, the mRNA in FTO-2B cells is 2.2-kilobases in length. However, the 5' region of the mRNA differs from the mRNA in the liver in that it contains sequences unique to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA from skeletal muscle. These results suggest that the mRNA in FTO-2B cells may represent an additional alternative splicing product of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene. Exposure of FTO-2B cells to media containing either insulin (10(-7) M) or dexamethasone (10(-6) M) induced about a 10-fold increase in the level of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA within 6-10 h of hormone treatment. The concentrations of insulin or dexamethasone giving half-maximal stimulation were 10(-9) M and 2 x 10(-8) M, respectively, and dibutyryl cyclic AMP (5 x 10(-7) M) completely prevented the increase in enzyme mRNA induced by these hormones. Exposure of cells to glucose-free medium abolished the insulin-mediated enhancement in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA, but not that induced by dexamethasone. No alteration in the degradation rate of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA was noted when cells were treated with insulin. Run-on transcription assays with isolated nuclei showed an increase in the relative transcription rate of the gene in cells treated with either insulin or dexamethasone. The time course of transcription activation preceded the increase in the level of the mRNA, indicating that the main mechanism for the induction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expression by insulin and dexamethasone is mediated by stimulation of gene transcription.

Animals↗
PubMed1991

Isolation of bovine liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase cDNA: bovine liver and heart forms of the enzyme are separate gene products.

In order to ascertain whether the heart and liver forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were products of two different genes or arose via alternative splicing of a single gene, the bovine liver cDNA of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a lambda gt10 phage library and its sequence compared with that of bovine heart cDNA. The deduced amino acid sequence of the bovine liver cDNA was also compared with the amino acid sequence of the human and rat liver phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme. The bovine liver cDNA codes for a protein that has 81.6% amino acid identity with the bovine heart form and 97.0 and 98.3% identity with the rat and human liver forms of the enzyme, respectively. Comparison of the nucleotide sequences of the two bovine cDNAs and their deduced amino acid sequences demonstrates that while there is conservation of the active sites of liver/muscle and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases they are encoded by different genes.

Amino Acid Sequence↗