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S J Pilkis

Publications and source records attributed to S J Pilkis.

At least 73 records · Page 4Linked to original sources
PubMed1990

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Use of site-directed mutagenesis to evaluate the roles of His-258 and His-392 in catalysis.

The current model for hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase divides the protein into two functional domains: an N-terminal kinase domain and a carboxyl-terminal bisphosphatase domain. Site-directed mutagenesis was used to evaluate the role of two putative bisphosphatase active site histidyl residues in catalysis. His-258 has been implicated as a phosphoacceptor (Pilkis, S. J., Lively, M. O., and El-Maghrabi, M. R. (1987) J. Biol. Chem. 262, 12672-12675), and the importance of this residue was confirmed when it was mutated to alanine and neither bisphosphatase activity nor a phosphoenzyme intermediate could be detected. Mutation of His-392 to alanine produced an enzyme which had five percent of wild-type fructose 2,6-bisphosphatase activity, and the rate of phosphoenzyme formations was decreased from 4800 nmol/min/mg to 2.9 nmol/min/mg. Mutation of His-392 to phenylalanine, lysine, or aspartic acid also produced proteins that did not hydrolyze fructose 2,6-bisphosphate or form a phosphoenzyme intermediate. These results are consistent with an important role for His-392 in the bisphosphatase reaction, probably as a proton donor, and with its designation as an active site residue based on homology modeling (Bazan (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). H258A had the same Vmax for 6-phosphofructo-2-kinase as the wild-type enzyme, and the mutant's kinase was inhibited by cAMP-dependent phosphorylation. In addition, H392F and H392K did not catalyze the kinase reaction, although H392D had normal kinase activity which was also modulated by cAMP-dependent phosphorylation in the same manner as the wild-type enzyme. Thus, an active bisphosphatase domain is not a necessary condition for phosphorylation-induced changes in 6-phosphofructo-2-kinase activity. The results also suggest that structural and/or active site interactions exist between the two domains of the enzyme.

Amino Acid Sequence↗
PubMed1990

Multihormonal regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in primary cultures of rat hepatocytes.

In the absence of hormone, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA rapidly declined to undetectable levels in primary cultures of rat hepatocytes. Dexamethasone increased mRNA levels and this effect was blocked by actinomycin D or cycloheximide. Insulin or thyroxine had no effect but potentiated the dexamethasone induction. Dibutyryl cyclic AMP decreased the stimulatory effect of dexamethasone at early times but after 24 hours the cyclic nucleotide increased mRNA levels compared with dexamethasone alone. It was concluded that expression of the bifunctional enzyme gene in hepatocytes is under a complex multihormonal control. Glucocorticoids are the most important transcriptional factor and their effect requires ongoing protein synthesis.

Animals↗
PubMed1990

Expression of mammalian liver glycolytic/gluconeogenic enzymes in Escherichia coli: recovery of active enzyme is strain and temperature dependent.

A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, fructose-2,6-bisphosphatase, glucokinase, and fructose-1,6-bisphosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37 degrees C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22 degrees C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thio-beta-D-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose-1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37 degrees C and was essentially unchanged at an induction temperature of 22 degrees C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Fructose-2,6-bisphosphate in control of hepatic gluconeogenesis. From metabolites to molecular genetics.

Hormonal regulation of hepatic gluconeogenic pathway flux is brought about by phosphorylation/dephosphorylation and control of gene expression of several key regulatory enzymes. Regulation by cAMP-dependent phosphorylation occurs at the level of pyruvate kinase and 6-phosphofructo-2-kinase (6PF-1-K)/fructose-2,6-bisphosphatase (Fru-2,6-P2ase). The latter is a unique bifunctional enzyme that catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P2), which is an activator of 6PF-1-K and an inhibitor of Fru-1,6-P2ase. The bifunctional enzyme is a homodimer whose activities are regulated by cAMP-dependent protein kinase-catalyzed phosphorylation at a single NH2-terminal seryl residue/subunit, which results in activation of the Fru-2,6-P2ase and inhibition of the PF-1-K reactions. Hormone-mediated changes in the phosphorylation state of the bifunctional enzyme are responsible for acute regulation of Fru-2,6-P2 levels. 6PF-2-K/Fru-2,6-P2ase thus provides a switching mechanism between glycolysis and gluconeogenesis in mammalian liver. Pyruvate kinase is regulated by both phosphorylation and allosteric effectors. Fru-1,6-P2, an allosteric activator, also inhibits cAMP-dependent enzyme phosphorylation, and its steady-state concentration is indirectly determined by the level of Fru-2,6-P2. Therefore, acute regulation of both pyruvate kinase and the bifunctional enzyme provide coordinated control at both the pyruvate/phosphoenolpyruvate and Fru-6-P/Fru-1,6-P2 substrate cycles. The Fru-2,6-P2 system is also subject to complex multihormonal long-term control through regulation of 6 PF-2-K/Fru-2,6-P2ase gene expression. Glucocorticoids are the major factor in turning on this gene in liver, but insulin is also a positive effector. cAMP prevents the effects of glucocorticoids and insulin. Although Fru-2,6-P2 plays a key role in the regulation of carbon flux in the gluconeogenic pathway, the regulation of this flux depends on several factors and regulation of other key enzymes whose importance varies depending on the dietary and hormonal status of the animal. Molecular cloning of the cDNA encoding PF-2-K/Fru-2,6-P2ase has elucidated its structure and permitted analysis of its evolutionary origin as well as its tissue distribution and control of its gene expression. The rat liver and skeletal muscle isoforms arose by alternative splicing of a single gene. The muscle form differs from the liver form only at the NH2-terminal and does not have a cAMP-dependent protein kinase phosphorylation site. The hepatic enzyme subunit consists of 470 amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

Expression of rat hepatic glucokinase in Escherichia coli.

Rat liver glucokinase was expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The expressed protein starts with the predicted initiator methionine residue and ends at the appropriate carboxyl terminal residue. It was partially purified by ammonium sulfate precipitation and gel filtration and had kinetic and physical properties similar to the purified rat liver enzyme. The efficient expression of this low abundance hepatic protein in bacteria provides a system for in vitro analysis of mutations of the enzyme.

Base Sequence↗
PubMed1989

Sequence of the 5'-flanking region of the rat 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase gene: regulation by glucocorticoids.

Dexamethasone addition to cultured hepatocytes caused a 90-fold increase in mRNA for 6-phosphofructo 2-kinase/fructose-2,6-bisphosphatase. Glucocorticoid administration in vivo also increased the enzyme's mRNA in skeletal muscle by 3-4-fold. The sequence of the 5'-flanking region of the enzyme's gene revealed at least one consensus glucocorticoid response element. The amino acid sequence derived from a partial cDNA clone for the rat skeletal muscle bifunctional enzyme was identical to that of the liver isozyme except for an undetermined amount of N-terminal sequence. It is concluded that the rat muscle and liver isozymes, which are postulated to be identical except for the N-terminal region, are both regulated by glucocorticoids.

Animals↗
PubMed1989

Glucocorticoid regulation of hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression.

The effect of adrenalectomy and triamcinolone treatment on mRNA encoding rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was studied. Adrenalectomy decreased both the kinase and the bisphosphatase activities of the bifunctional enzyme to about 30% of the values in livers of normal rats. Triamcinolone treatment restored both activities to normal by 24 h. These changes were caused by alterations in the concentration of the enzyme as determined by immunoblotting and by an assay that measures phosphoenzyme formation. Messenger RNA for liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was markedly decreased by adrenalectomy and was increased 15-fold by triamcinolone administration for 8 h. The rate of transcription of the bifunctional enzyme gene, measured in rat liver nuclei, was also decreased in adrenalectomy, and triamcinolone treatment increased this rate 5-fold within 8 h. Similarly, liver nuclear precursors of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA were decreased by adrenalectomy to 25% of the level in nuclei from normal rats. Triamcinolone treatment restored heterogeneous values by 2 h, while treatment for 30 h increased it 12-fold over the adrenalectomized levels. It was concluded that glucocorticoids regulate the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, at least in part, by modulating the transcription rate of the gene.

Adrenalectomy↗
PubMed1989

The amino acid sequence of rat liver glucokinase deduced from cloned cDNA.

Rat liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified to homogeneity, cleaved, and subjected to amino acid sequence analysis. Forty-five percent of the protein sequence was obtained, and this information was used to design oligonucleotide probes to screen a rat liver cDNA library. A 1601-base pair cDNA (GK1) contained an open reading frame that encoded the amino acid sequences found in the peptides used to generate the oligonucleotide probes. A second cDNA was subsequently identified (GK.Z2), which is 2346 base pairs long and corresponds to nearly the entire glucokinase mRNA. Blot transfer analysis of hepatic RNA showed that glucokinase mRNA exists as a single species of about 2400 nucleotides. Four hours of insulin treatment of diabetic rats resulted in a 30-fold induction of this mRNA. GK.Z2 has a long open reading frame which, with the known partial peptide sequence, allowed us to deduce the primary structure of glucokinase. The enzyme is composed of 465 amino acids and has a mass of 51,924 daltons. Glucokinase has 53 and 33% amino acid sequence identities with the carboxyl-terminal domains of rat brain hexokinase I and yeast hexokinase, respectively. If conservative amino acid replacements are also considered, glucokinase is similar to these two enzymes at 75 and 63% of positions, respectively. The putative glucose- and ATP-binding domains of glucokinase were identified, and these regions appear to be highly conserved in the hexokinase family of enzymes.

Amino Acid Sequence↗
PubMed1989

Expression of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its kinase domain in Escherichia coli.

The rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) and its separate kinase domain were expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The bifunctional enzyme (470 residues per subunit) was efficiently expressed as a protein that starts with the initiator methionine residue and ends at the carboxyl-terminal tyrosine residue. The expressed protein was purified to homogeneity by anion exchange and Blue Sepharose chromatography and had kinetic and physical properties similar to the purified rat liver enzyme, including its behavior as a dimer during gel filtration, activation of the kinase by phosphate and inhibition by alpha-glycerol phosphate, and mediation of the bisphosphatase reaction by a phosphoenzyme intermediate. The expressed 6-phosphofructo-2-kinase also started with the initiator methionine but ended at residue 257. The partially purified kinase domain was catalytically active, had reduced affinities for ATP and fructose 6-phosphate compared with the kinase of the bifunctional enzyme, and had no fructose-2,6-bisphosphatase activity. The kinase domain also behaved as an oligomeric protein during gel filtration. The expression of an active kinase domain and the previous demonstration of an actively expressed bisphosphatase domain provide strong support for the hypothesis that the hepatic enzyme consists of two independent catalytic domains encoded by a fused gene.

Animals↗
PubMed1989

Evolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The bifunctional rat liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) is constructed of two independent catalytic domains. We present evidence that the kinase and bisphosphatase halves of the bifunctional enzyme are, respectively, structurally similar to the glycolytic enzymes 6-phosphofructo-1-kinase and phosphoglycerate mutase. Computer-assisted modeling of the C-terminal bisphosphatase domain reveals a hydrophobic core and active site residue constellation equivalent to the yeast mutase structure; structural differences map to length-variable, surface-located loops. Sequence patterns derived from the structural alignment of mutases and the bisphosphatase further detect a significant similarity to a family of acid phosphatases. The N-terminal kinase domain, in turn, is predicted to form a nucleotide-binding fold that is analogous to a segment of 6-phosphofructo-1-kinase, suggesting that these unrelated enzymes bind fructose 6-phosphate and ATP substrates in a similar geometry. This analysis indicates that the bifunctional enzyme is the likely product of gene fusion of kinase and mutase/phosphatase catalytic units.

Acid Phosphatase↗
PubMed1989

Indirect versus direct routes of hepatic glycogen synthesis.

During refeeding after a brief period of starvation, glucose carbon is deposited into hepatic glycogen by both a direct and an indirect route. In the indirect route glucose is first metabolized to 3-carbon precursors, which then transverse the gluconeogenic pathway before being deposited into glycogen. Recent studies have yielded widely different estimates of the percentage of glucose carbon that follows the indirect route. Work summarized here demonstrates that the relative contributions of glucose carbon to hepatic glycogen formation by the indirect and direct pathways are greatly dependent on experimental design, and at least in vitro, are possibly dependent on the extent of glycogen/glucose 1-P recycling. Under physiological refeeding conditions in vivo, both pathways are used, each contributing approximately 50% of the amount of carbon appearing in glycogen. The level of glucokinase activity does not appear to be responsible for poor glucose utilization in liver. Poor glucose utilization in isolated liver preparations may result from the absence of a neurophysiological feedback loop that senses the arterial/portal glucose gradient and then regulates whole liver glucose uptake.

Animals↗
PubMed1988

Induction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA by refeeding and insulin.

The effects of fasting/refeeding and untreated or insulin-treated diabetes on the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its mRNA in rat liver were determined. Both enzymatic activities fell to 20% of control values with fasting or streptozotocin-induced diabetes and were coordinately restored to normal within 48 h of refeeding or 24 h of insulin administration. These alterations in enzymatic activities were always mirrored by corresponding changes in amount of enzyme as determined by phosphoenzyme formation and immunoblotting. In contrast, mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase did not decrease during starvation or in diabetes, but there was a 3-6-fold increase upon refeeding a high carbohydrate diet to starved rats or insulin treatment of diabetic rats. The decrease of the enzyme in starved or diabetic rats without associated changes in mRNA levels suggests a decrease in the rate of mRNA translation, an increase in enzyme degradation, or both. The rise in enzyme amount and mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with refeeding and insulin treatment suggests an insulin-dependent stimulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression. Northern blots of RNA from heart, brain, kidney, and skeletal muscle probed with restriction fragments of a full-length cDNA from liver showed that only skeletal muscle contained an RNA species that hybridized to any of the probes. Skeletal muscle mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was 2.0 kilobase pairs but in contrast to the liver message (2.2 kilobase pairs) was not regulated by refeeding.

Animals↗
PubMed1988

The stereochemical course of phospho group transfer catalyzed by rat liver 6-phosphofructo-2-kinase.

Adenosine [gamma-(S)-16O,17O,18O]triphosphate was used as substrate in the phosphokinase reaction catalyzed by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The product D-fructose 2,6-[2-16O,17O,18O]bisphosphate was then used as substrate in the alkaline phosphatase-mediated transfer of the phospho groups to (S)-butane-1,3-diol, where the configuration at phosphorus has been determined. Although there was approximately equal transfer by alkaline phosphatase of the labeled 2- and the unlabeled 6-phospho groups, the subsequent assignment of configuration of the chiral phospho group from the 2-position was unambiguous. It was found that 6-phosphofructo-2-kinase proceeds by a pathway that results in net inversion of the configuration at phosphorus. The simplest interpretation of this result is that the phospho group is transferred directly between substrates in a ternary complex by an "in-line" mechanism not involving a phosphoenzyme intermediate. This conclusion is consistent with the fact that the enzyme cannot be labeled by [gamma-32P]ATP and with steady-state kinetic data that suggest an ordered sequential mechanism. This finding also indicates that the adenine nucleotide and sugar phosphate isotope exchange reactions catalyzed by the enzyme are not relevant to the normal catalytic pathway of the kinase.

Adenosine Triphosphate↗
PubMed1988

Complete amino acid sequence of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The complete amino acid sequence of 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was determined by direct analysis of the S-carboxamidomethyl protein. A complete set of nonoverlapping peptides was produced by cleavage with a combination of cyanogen bromide and specific proteolytic enzymes. The active enzyme is a dimer of two identical polypeptide chains composed of 470 amino acids each. The NH2-terminal amino acid residue of the polypeptide chain was shown to be N-acetylserine by fast atom bombardment mass spectrometry of the purified N-terminal tetradecapeptide isolated after cleavage of the intact S-carboxamidomethylated protein with lysyl endoproteinase (Achromobacter protease I). Alignment of the set of unique peptides was accomplished by the analysis of selected overlapping peptides generated by proteolytic cleavage of the intact protein and the larger purified cyanogen bromide peptides with trypsin, Staphylococcus aureus V8 protease, and lysyl endoproteinase. Four nonoverlapping peptides were aligned by comparison with the amino acid sequence predicted from a partial cDNA clone encoding amino acid positions 166-470 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Colosia, A.D., Lively, M., El-Maghrabi, M. R., and Pilkis, S. J. (1987) Biochem. Biophys. Res. Commun. 143, 1092-1098). The nucleotide sequence of the cDNA corroborated the peptide sequence determined by direct methods. A search of the Protein Identification Resource protein sequence database revealed that the overall amino acid sequence appears to be unique since no obviously homologous sequences were identified. However, a 100-residue segment of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (residues 250-349), including the active site histidine residue of the bisphosphatase domain, was found to be homologous to the active site regions of yeast phosphoglycerate mutase and human bisphosphoglycerate mutase.

Amino Acid Sequence↗
PubMed1988

Expression of the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli.

The fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (EC 2.7.105/EC 3.1.3.46) was expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The protein was efficiently expressed (i) as a fusion protein that starts at the T7 major capsid protein initiation site in a pET expression vector and (ii) as a protein that starts within the bisphosphatase sequence by translation reinitiation. Both proteins have similar properties. The protein was purified to homogeneity by anion-exchange chromatography and gel filtration. The purified fructose-2,6-bisphosphatase domain was active and no 6-phosphofructo-2-kinase activity was found associated with it. In contrast to the dimeric bifunctional enzyme, the fructose-2,6-bisphosphatase domain behaved as a monomer of 30 kDa. The turnover number and kinetic properties of the separate bisphosphatase domain were similar to those of the bisphosphatase of the bifunctional enzyme, including the ability to form a phosphoenzyme intermediate. These results support the hypothesis that the rat liver enzyme consists of two independent domains and is a member of a class of enzymes formed by gene fusion.

Animals↗
PubMed1988

cDNA sequence of rat liver fructose-1,6-bisphosphatase and evidence for down-regulation of its mRNA by insulin.

A coding-length clone of rat liver fructose-1,6-bisphosphatase (EC 3.1.3.11) was isolated by immunological screening of a cDNA library in lambda gt11. Its identity was verified by comparing the deduced amino acid sequence with that obtained by direct sequencing of a complete set of CNBr and proteolytic peptides from the purified protein. The enzyme subunit is composed of 362 amino acids and has N-acetylvaline as the amino-terminal residue. The cDNA, 1255 base pairs (bp) long, consisted of 1086 bp of coding region, 15 bp of 5' untranslated sequence, and 154 bp at the 3' untranslated end. The 3' untranslated sequence contained a polyadenylylation signal (AATAAA) followed after 30 bp by a stretch of 7 adenines at the end of the clone. The deduced amino acid sequence was identical to the primary sequence of the protein and confirmed the alignment of five nonoverlapping peptides. It also confirmed the 27-residue extension, unique to the rat liver subunit, ending with a carboxyl-terminal phenylalanine. RNA blot analyses using the radiolabeled liver cDNA as a probe revealed a single band of fructose-1,6-bisphosphatase mRNA, 1.4 kilobases long, in liver and kidney but not in nongluconeogenic tissues. Fructose-1,6-bisphosphatase mRNA was increased 10-fold in livers from diabetic rats and was reduced to control levels after 24 hr of insulin treatment, suggesting that the changes in enzyme activity observed in diabetes and after insulin treatment are due to alterations in mRNA abundance.

Amino Acid Sequence↗