Xylitol prevents NEFA-induced insulin resistance in rats.

AIMS/HYPOTHESIS: Increased NEFA levels, characteristic of type 2 diabetes mellitus, contribute to skeletal muscle insulin resistance. While NEFA-induced insulin resistance was formerly attributed to decreased glycolysis, it is likely that glucose transport is the rate-limiting defect. Recently, the plant-derived sugar alcohol xylitol has been shown to have favourable metabolic effects in various animal models. Furthermore, its derivative xylulose 5-phosphate may prevent NEFA-induced suppression of glycolysis. We therefore examined whether and how xylitol might prevent NEFA-induced insulin resistance. METHODS: We examined the ability of xylitol to prevent NEFA-induced insulin resistance. Sustained ~1.5-fold elevations in NEFA levels were induced with Intralipid/heparin infusions during 5 h euglycaemic-hyperinsulinaemic clamp studies in 24 conscious non-diabetic Sprague-Dawley rats, with or without infusion of xylitol. RESULTS: Intralipid infusion reduced peripheral glucose uptake by ~25%, predominantly through suppression of glycogen synthesis. Co-infusion of xylitol prevented the NEFA-induced decreases in both glucose uptake and glycogen synthesis. Although glycolysis was increased by xylitol infusion alone, there was minimal NEFA-induced suppression of glycolysis, which was not affected by co-infusion of xylitol. CONCLUSIONS/INTERPRETATION: We conclude that xylitol prevented NEFA-induced insulin resistance, with favourable effects on glycogen synthesis accompanying the improved insulin-mediated glucose uptake. This suggests that this pentose sweetener has beneficial insulin-sensitising effects.

MinK-related peptide 1: A beta subunit for the HCN ion channel subunit family enhances expression and speeds activation.

The HCN family of ion channel subunits underlies the currents I(f) in heart and I(h) and I(q) in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a beta subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a beta subunit for the cardiac delayed rectifier I(Kr), these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.

Metabolism of lipoprotein acylglycerols by liver parenchymal cells.

We investigated the metabolism by hepatocyte suspensions of the acylglycerols in lipoprotein remnants as well as those associated with albumin and low or high density lipoproteins. Remnants, albumin and plasma lipoproteins, rich in monoacylglycerol were prepared by short-term incubations of radio-labeled chylomicra or very low density lipoproteins with extrahepatic lipoprotein lipase in the presence of albumin and low and high density lipoproteins. We demonstrated that liver parenchymal cells contain an active monoacylglycerol acyltransferase that is located on the extracellular surface of the cell plasma membrane. Further, the enzyme is capable of degrading the monoacylglycerol in all the above forms. Triacylglycerol in intact chylomicra and very low density lipoproteins were not metabolized by the cells to any appreciable degree. The degradation of the remnant triacylglycerol appeared to depend solely on the activity of the lipoprotein lipase bound to the lipoprotein remnants. Little uptake of intact lipoprotein acylglycerols by the hepatocytes was observed; instead, hydrolysis of the substrates in the medium always preceded the uptake of the products. The products were then utilized for the synthesis of triacylglycerol and phospholipid within the cells.

Effect of diabetes, insulin, starvation, and refeeding on the level of rat hepatic fructose 2,6-bisphosphate.

The influence of alloxan diabetes and starvation for 72 h on the level of rat hepatic fructose 2,6-biphosphate was investigated. Both diabetes and starvation decreased the level to 10% of the value found in livers of normal, fed rats (10 nmol/g liver). The activity of the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, 6-phosphofructo 2-kinase, was also decreased in livers of diabetic rats. Insulin administration for 24 h to diabetic rats restored the level of fructose 2,6-bisphosphate to normal. Refeeding a high carbohydrate diet for 24 h to starved rats resulted in fructose, 2,6-biphosphate levels that were 2.5-fold higher than that in livers of fed rats. The level of fructose 2,6-bisphosphate in diabetes and starvation, and after refeeding correlates as well with the rate of glycolysis and gluconeogenesis in these states and thereby provides further support for its role in regulating hepatic carbohydrate metabolism.

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)

Alterations in hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glucose-6-phosphatase gene expression after hemorrhagic hypotension and resuscitation.

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.

Effects of RU486 on Glu-6-pase gene expression in hemorrhage and resuscitation.

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

Rat hepatic 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a unique bifunctional enzyme.

Fructose 2,6-bisphosphate is a potent allosteric activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. It potentiates the effect of AMP on both enzymes. A great deal of compelling evidence supports the hypothesis that fructose 2,6-bisphosphate plays a key role in the hormonal and substrate regulation of substrate cycling at the fructose 6-phosphate/fructose 1,6-bisphosphate level in liver. This regulation is exerted at the level of the enzyme activities responsible for the synthesis and degradation of fructose 2,6-bisphosphate. Synthesis of the compound is catalyzed by a unique enzyme which transfers the gamma-phosphate of ATP to the C2 position of fructose 6-phosphate (ATP:D fructose 6-phosphate 2-phosphotransferase) while degradation is catalyzed by a phosphohydrolase activity which is specific for the C-2 position of fructose 2,6-bisphosphate (D-fructose 2,6-bisphosphate 2-phosphohydrolase). These activities are distinct from the classical 6-phosphofructo 1-kinase and fructose 1,6-bisphosphatase with regard to molecular weight, interaction with ligands, and the efficiency with which phosphoryl transfer occurs. Both activities have been purified to homogeneity and have been shown to be present in a single enzyme protein, i.e. the enzyme is bifunctional. Incubation of the 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase with cAMP-dependent protein kinase and ATP leads to phosphorylation of the enzyme resulting in inactivation of the phosphotransferase activity and stimulation of the phosphohydrolase activity. Since fructose 2,6-bisphosphate is not further metabolized and can only be recycled to fructose 6-phosphate, simultaneous modulation of the synthesis and degradation of the compound by covalent modification of a single protein provides a very efficient and sensitive regulatory mechanism. The bifunctional enzyme was also shown to possess an ATPase activity which was nearly equal to the activity of the kinase reaction. However, in the presence of fructose 6-phosphate the enzyme did not transfer phosphate to water but rather to the C-2 position of the phosphorylated sugar. The ability of the enzyme to catalyze a partial reaction at a rate nearly equal to that of the forward reaction suggested that the reaction mechanism of the kinase proceeds by a two step transfer, i.e. via a phosphoryl enzyme intermediate.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of liver and kidney glucose-6-phosphatase gene expression in hemorrhage and resuscitation.

UNLABELLED: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a review of relationships between the two activities of the enzyme.

Both the synthesis and the degradation of Fru-2,6-P2 are catalyzed by a single enzyme protein; ie, the enzyme is bifunctional. This protein, which we have designated 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase is an important enzyme in the regulation of hepatic carbohydrate metabolism since its activity determines the steady-state concentration of fructose 2,6-P2, an activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. Regulation of the bifunctional enzyme in intact cells is a complex function of both covalent modification via phosphorylation/dephosphorylation and the influence of substrates and low molecular weight effectors. Recent evidence suggests that both reactions may proceed by two-step transfer mechanisms with different phosphoenzyme intermediates. The enzyme catalyzes exchange reactions between ADP and ATP and between fructose 6-P and fructose 2,6-P2. A labeled phosphoenzyme is formed rapidly during incubation with [2-32P]Fru-2,6-P2. The labeled residue has been identified as 3-phosphohistidine. However, it was not possible to demonstrate significant labeling of the enzyme directly from [gamma-32P]ATP. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a fructose 2,6-bisphosphatase site which is readily phosphorylated by fructose 2,6-P2. Additional evidence in support of two active sites include: limited proteolysis with thermolysin results in loss of 6-phosphofructo 2-kinase activity and activation of fructose 2,6-bisphosphatase, mixed function oxidation results in inactivation of the 6-phosphofructo 2-kinase but no affect on the fructose 2,6-bisphosphatase, N-ethylmaleimide treatment also inactivates the kinase but does not affect the bisphosphatase, and p-chloromercuribenzoate immediately inactivates the fructose 2,6-bisphosphatase but not the 6-phosphofructo 2-kinase. Our findings indicate that the bifunctional enzyme is a rather complicated enzyme; a dimer, probably with two catalytic sites reacting with sugar phosphate, and with an unknown number of regulatory sites for most of its substrates and products. Three enzymes from Escherichia coli, isocitric dehydrogenase kinase/phosphatase, glutamine-synthetase adenylyltransferase, and the uridylyltransferase for the regulatory protein PII in the glutamine synthetase cascade system also catalyze opposing reactions probably at two discrete sites. All four enzymes are important in the regulation of metabolism and may represent a distinct class of regulatory enzymes.

Effect of pentobarbital on fructose 2,6-bisphosphate metabolism in isolated rat hepatocytes.

Addition of the commonly used anesthetic pentobarbital to hepatocytes from fed rats resulted in a dose-dependent decrease in the level of fructose 2,6-bisphosphate. At a concentration of pentobarbital (0.4 mM) that lowered fructose 2,6-bisphosphate by 60%, there was no significant change in the level of fructose 6-phosphate, ATP, or L-glycerol 3-phosphate. Higher concentrations of pentobarbital (2 mM) enhanced both glycolysis and glycogenolysis and fructose 2,6-bisphosphate levels were reduced to less than 10% of the control. Concomitant with these changes there was a decrease in ATP, glucose 6-phosphate, and fructose 6-phosphate and a two- and fivefold increase in ADP and AMP, respectively. In hepatocytes from starved rats pentobarbital also lowered ATP levels and inhibited gluconeogenesis but had no effect on either lactate production or the already low level of sugar diphosphate. However, in the fasted case pentobarbital completely prevented the 10-fold elevation of fructose 2,6-bisphosphate brought about by 30 mM glucose. The anesthetic had no effect on cAMP-dependent protein kinase activity or on pyruvate kinase activity in hepatocytes from fed or starved rats but caused reciprocal changes in the activities of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Kinase activity was decreased and bisphosphatase activity was increased. These results suggest that the effects of pentobarbital on gluconeogenesis and glycolysis are due to inhibition of energy metabolism with elevated AMP levels causing activation of 6-phosphofructo-1-kinase and inhibition of fructose 1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of capillary electrophoresis and indirect detection to quantitate in-capillary enzyme-catalyzed microreactions.

The use of capillary electrophoresis and indirect detection to quantify reaction products of in-capillary enzyme-catalyzed microreactions is described. Migrating in a capillary under conditions of electrophoresis, plugs of enzyme and substrate are injected and allowed to react. Capillary electrophoresis is subsequently used to measure the extent of reaction. This technique is demonstrated using two model systems: the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by fructose-biphosphate aldolase (ALD, EC 4.1.2.13), and the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate by fructose-1,6-bisphospatase (FBPase, EC 3.1.3.11). These procedures expand the use of the capillary as a microreactor and offer a new approach to analyzing enzyme-mediated reactions.

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.

N- and C-termini modulate the effects of pH and phosphorylation on hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

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.

Functional homology of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, phosphoglycerate mutase, and 2,3-bisphosphoglycerate mutase.

The bisphosphatase domain of the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to exhibit a structural similarity to yeast phosphoglycerate mutase and human red blood cell 2,3-bisphosphoglycerate mutase including very similar active site sequences with a histidyl residue being involved in phospho group transfer. The liver bifunctional enzyme was found to catalyze the hydrolysis of glycerate 1,3-bisphosphate to glycerate 3-phosphate and inorganic phosphate. The Km for glycerate 1,3-bisphosphate was 320 microM and the Vmax was 11.5 milliunits/mg. Incubation of the rat liver enzyme with [1-32P]glycerate 1,3-bisphosphate resulted in the formation of a phosphoenzyme intermediate, and the labeled amino acid was identified as 3-phosphohistidine. Tryptic and endoproteinase Lys-C peptide maps of the 32P-phosphoenzyme labeled either with [2-32P]fructose 2,6-bisphosphate or [1-32P]glycerate 1,3-bisphosphate revealed that 32P-radioactivity was found in the same peptide, proving that the same histidyl group accepts phosphate from both substrates. Fructose 2,6-bisphosphate inhibited competitively the formation of phosphoenzyme from [1-32P]glycerate 1,3-bisphosphate. Effectors of fructose-2,6-bisphosphatase also inhibited phosphoenzyme formation. Substrates and products of phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase also modulated the activities of the bifunctional enzyme. These results demonstrate that, in addition to a structural homology, the bisphosphatase domain of the bifunctional enzyme has a functional similarity to phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase and support the concept of an evolutionary relationship between the three enzyme activities.

Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity. Allosteric activation of ATP:citrate lyase by phosphorylated sugars.

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.

Phosphorylation of liver pyruvate kinase by Ca++/calmodulin-dependent protein kinase: characterization of two phosphorylation sites.

Rat liver pyruvate kinase is phosphorylated by calcium/calmodulin-dependent protein kinase II at serine and threonine residues in a 3-4 kDa CNBr fragment located near the amino terminus. The two sites of phosphorylation were separated by reverse-phase HPLC of a thermolysin digest. Sequence analysis established the sites of phosphorylation as follows: Leu-Arg-Arg-Ala-Ser(PO4)-Val-Ala-Gln-Leu-Thr(PO4)-Gln-Glu.