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K Uyeda

Publications and source records attributed to K Uyeda.

16 recordsLinked to original sources
PubMed1992

Oscillation in fructose 2,6-bisphosphate levels and in the phosphorylation states of fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase in ischemic rat liver.

In order to determine the role of fructose (Fru) 2,6-P2 in stimulation of phosphofructokinase in ischemic liver, tissue contents of Fru-2,6-P2, hexose-Ps, adenine nucleotides, and Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated during the first few minutes of ischemia. The Fru-2,6-P2 concentration in the liver changed in an oscillatory manner. Within 7 s after the initiation of ischemia, Fru-2,6-P2 increased from 6 to 21 nmol/g liver and decreased to 5 nmol/g liver within 30 s. Subsequently, it reached the maximum value at 50, 80, and 100 s and decreased to the basal concentration at 60, 90, and 120 s. Oscillatory patterns were also observed with Glc-6-P and Fru-6-P, but the ATP/ADP ratio decreased monotonically. Determination of Fru-6-P,2-kinase activity and the phosphorylation states of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase demonstrated that at 7 and 50 s, where Fru-2,6-P2 was the highest, the enzyme was activated and mostly in a dephosphorylated form. On the other hand, at 0, 30, and 300 s, the enzyme was predominantly in the phosphorylated form. The concentration of cAMP in the liver also changed in an oscillatory manner between 0.5 to 1.3 nmol/g with varying frequency of 10 to 40 s. These results indicated that: (a) Fru-2,6-P2 was important in rapid activation of phosphofructokinase in the first few seconds and up to 2-3 min, and (b) the oscillation of Fru-2,6-P2 concentration was the result of activation and inhibition of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase, which was caused by changes in the phosphorylation state of the enzyme.

Animals↗
PubMed1992

Endotoxin stimulation of liver parenchymal cell phosphofructokinase activity requires nonparenchymal cells.

The rate of carbohydrate flux through phosphofructokinase (measured as the rate of [3-3H]glucose detritiation) was increased fourfold in rat liver parenchymal cells incubated with conditioned medium from lipopolysaccharide-stimulated adherent liver non-parenchymal cells. The rate was not affected in parenchymal cells incubated either with lipopolysaccharide directly or with conditioned medium from non-stimulated non-parenchymal cells. The stimulation of carbohydrate flux through phosphofructokinase by conditioned medium was not duplicated by peptide cytokines known to be released by lipopolysaccharide-activated liver non-parenchymal cells (interleukin-1, interleukin-6, tumor necrosis factor-alpha, and transforming growth factor-beta) or platelet activating factor. Furthermore, formation of the active conditioned medium was not prevented by inclusion of cycloheximide or dexamethasone to inhibit cytokine synthesis, or indomethacin or BW755c to inhibit arachidonic acid metabolism, during lipopolysaccharide-stimulation of the non-parenchymal cells. The results indicate that intercellular communication between lipopolysaccharide-stimulated liver non-parenchymal cells and parenchymal cells by soluble mediators is responsible for the stimulation of liver phosphofructokinase activity during endotoxin-induced shock. Studies to isolate and identify the factor(s) in the conditioned medium are currently in progress.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1991

Characterization of two isozymic forms of heart fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Occurrence of two isozymic forms of fructose 6-P, 2-kinase: fructose 2,6-bisphosphatase in bovine heart was investigated by transcribing mRNAs and amplifying the cDNAs with polymerase chain reactions. Analysis of the PCR products revealed 1.7 Kb and 1.5 Kb DNAs, and the determination of their nucleotide sequences showed that these DNAs are identical except for the lack of 180 base pairs near the 3' of the bovine heart enzyme DNA previously reported (6). This missing nucleotide sequence encodes Asn451-Gln510 and contains the phosphorylation sites for cAMP dependent protein kinase and protein kinase C.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase.

We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA fragment. A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a Mr of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced Mr of 54,023. Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same Mr 55,000 was also present in rat testis extract. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. Various kinetic constants of the expressed fructose 6-P,2-kinase were KmFru 6-P = 85 microM and KmATP = 270 microM, and those of fructose 2,6-bisphosphatase were KmFru 2,6-P2 = 21 microM and KiFru 6-P = 3.4 microM. The enzyme was phosphorylated by Fru-2,6[2-32P]P2 and also by protein kinase C, but not by cAMP-dependent protein kinase, which is in contrast to the liver and heart isozymes.

Amino Acid Sequence↗
PubMed1979

Hormone-stimulated phosphorylation of liver phosphofructokinase in vivo.

The effect of glucagon on the phosphorylation and the enzymic activity of phosphofructokinase in rat liver in vivo was investigated. Glucagon stimulated the phosphorylation of liver phosphofructokinase approximately 3- to 5-fold and increased cAMP levels 5-fold and blood glucose levels 2-fold over the values obtained for control animals. The specific radioactivity of ATP isolated from liver was the same in both control and hormone-treated animals. During the purification of the 32P-labeled enzyme from both animals, no difference was observed in the total or specific enzyme activities of the enzymes from the various fractions. Thus, phosphofructokinase appears to be phosphorylated in vivo by a cyclic AMP-dependent protein kinase. Although phosphorylation does not affect the maximum catalytic activity of the enzyme, it does render the enzyme significantly more sensitive to ATP inhibition. Thus, at a given concentration of ATP, the phosphorylated phosphofructokinase exhibits considerably lower activity than the unphosphorylated enzyme. The possible relationship between our observations and glucagon-mediated control of glycolysis is discussed.

Adenosine Triphosphate↗
PubMed1979

Phosphofructokinase.

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Allosteric Regulation↗
PubMed1977

Changes in allosteric properties of phosphofructokinase bound to erythrocyte membranes.

Human and rabbit erythrocyte membranes prepared by hypotonic hemolysis contained 5 to 15% of the phosphofructokinase in the erythrocytes. The membrane-bound phosphofructokinase can be eluted by a saline wash. Human erythrocyte and rabbit muscle phosphofructokinase bind to the saline-washed membranes. This binding is specific for the inner surface of the membrane. The amount of phosphofructokinase bound is dependent on pH; at pH 7, 6 times more enzyme is bound than at pH 7.5. Unlike free phosphofructokinase, the membrane-bound phosphofructokinase is not inhibited by ATP or 2,3-diphosphoglycerate, and its fructose-6-P saturation curve is nonsigmoidal.

Adenosine Triphosphate↗
PubMed1977

Erythrocyte adenosine triphosphate and 2,3-diphosphoglycerate after human renal transplantation: dissociation from hypophosphataemia.

1. Erythrocyte 2,3-diphosphoglycerate (2,3-DPG) and adenosine triphosphate (ATP) were determined in normal individuals, uraemic patients on chronic haemodialysis and patients who underwent renal transplantation, and correlated with plasma phosphate and arterial blood pH. 2. Significant increases in the 2,3-DPG and ATP content were found in the uraemic patients and these persisted after transplantation in spite of marked hypophosphataemia. 3. No correlation was established with plasma phosphate for either of the compounds but 2,3-DPG had a significant correlation with arterial blood pH. 4. Normal values for ATP and 2,3-DPG were observed in post-transplant patients with normal haematological values. The high amounts of erythrocyte 2,3-DPG and ATP in the early post-transplant period are independent of the circulating concentration of inorganic phosphate, and might represent the response of erythrocyte glycolysis to changing arterial blood pH.

Adenosine Triphosphate↗
PubMed1976

Studies on the fatty acid inactivation of phosphofructokinase.

Investigation of phosphofructokinase in normal and regenerating livers led to the discovery of an inactivating factor in the extracts of these livers. The inactivating factor was found to be a mixture of free fatty acids. The fatty acid compositions of the normal and regenerating livers are the same, but the concentrations of most of the fatty acids are at least 3 to 4 times higher in the latter. Inactivation of phosphofructokinase by palmitate and oleate was investigated using purified rabbit muscle enzyme. Incubation of the enzyme with palmitate (250 muM) or oleate (50 muM) resulted in rapid inactivation of the enzyme with biphasic curves. The concentrations of oleate and palmitate required to produce 50% inactivation of the enzyme were 35 muM and 75 muM, respectively. Fructose-6-P (0.5 mM), MgATP, (1 mM), fructose-1,6-P2 (1 mM), AMP (1 mM), and cyclic adenosine 3':5'-monophosphate (20 muM) protected the enzyme against inactivation when these metabolites were incubated with the enzyme before the addition of fatty acid. Bovine serum albumin (100 muM) and beta-cyclodextrin (0.25 mM) also protected the enzyme against the inactivation. However, if the enzyme was inactivated by fatty acid, subsequent addition of the above metabolites or bovine serum albumin did not reactivate the enzyme. Binding studies with [3H]oleate revealed at least three types of binding sites. The first site binds 2 to 4 mol of oleate/mol of enzyme. Oleate binding to this site did not seem to affect the enzyme activity. The second binding site binds 5 to 15 mol of oleate/mol of enzyme resulting in complete loss of the activity. This is followed by an increase in oleate binding to the third site of the enzyme. Sucrose density gradient centrifugation of oleate-inactivated enzyme indicated that the enzyme dissociated to the dimeric form. Similarly, centrifugation of [3H]oleate-treated enzyme revealed that all polymeric forms of phosphofructokinase bound approximately 6 to 8 mol of oleate/mol of enzyme. In the presence of fructose-6-P, oleate is bound to the polymers to a lesser degree and therefore protects against the fatty acid inactivation. Various polymers which are cross-linked with dimethylsuberimidate are also inhibited by oleate.

Adenosine Monophosphate↗