The role of the cyclic AMP-dependent protein kinase in the glucagon-stimulated phosphorylation of ATP-citrate lyase

We have examined the mechanism whereby glucagon stimulates the phosphorylation of ATP-citrate lyase in intact rat hepatocytes. Purified ATP-citrate lyase is phosphorylated in vitro by the catalytic subunit of the cyclic AMP-dependent protein kinase, in a reaction wherein 2-3 mol phosphate/mol lyase are incorporated, at an initial rate that approaches that observed for mixed histone. This reaction is completely abolished by the protein kinase inhibitor protein. Limited tryptic digestion of ATP-citrate lyase phosphorylated in vitro by the cyclic AMP-dependent protein kinase yields a pattern of 32P-labeled peptides, indistinguishable from those observed in parallel digests of lyase isolated from 32P-labeled, glucagon-stimulated hepatocytes. Phosphorylase b kinase catalyzes the incorporation of 1 mol phosphate/mol lyase, albeit at less than 1/160 the rate observed for phosphorylase b. The phosphorylation of purified ATP-citrate lyase is also catalyzed by homogenates of hepatocytes. This reaction is stimulated by cyclic AMP. At 30 degrees C, in the presence of maximally stimulating concentrations of cyclic AMP, the addition of excess protein kinase inhibitor protein inhibits the phosphorylation of ATP-citrate lyase by 67%. Thus, hepatocytes contain both cyclic AMP-dependent and cyclic AMP-independent ATP-citrate lyase kinase activities. Pretreatment of hepatocytes with glucagon (10(-8) M for 2 min) prior to homogenization results in activation of an endogenous hepatocyte ATP-citrate lyase kinase, as well as histone kinase and phosphorylase b kinase; the glucagon-stimulated increment in lyase kinase (and histone kinase) is observed only when homogenates are assayed in the absence of added cyclic AMP, and is completely abolished by an excess of the protein kinase inhibitor protein. We conclude that the glucagon-stimulated phosphorylation of ATP-citrate lyase in intact hepatocytes is catalyzed directly by the cyclic AMP-dependent protein kinase.     

Characterization of the phosphorylation of rat mammary ATP-citrate lyase and acetyl-CoA carboxylase by Ca2+ and calmodulin-dependent multiprotein kinase and Ca2+ and phospholipid-dependent protein kinase

ATP-citrate lyase and acetyl-CoA carboxylase purified from lactating rat mammary gland are phosphorylated stoichiometrically by the calmodulin-dependent multiprotein kinase from rabbit skeletal muscle. The reactions are completely dependent on the presence of both Ca2+ and calmodulin. ATP-citrate lyase and acetyl-CoA carboxylase are also phosphorylated stoichiometrically by the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) purified from bovine brain. Phosphorylation of these substrates is stimulated 6-fold and 40-fold respectively by Ca2+ and phosphatidylserine. The calmodulin-dependent and phospholipid-dependent protein kinases phosphorylate the same serine residue on ATP-citrate lyase that is phosphorylated by cyclic-AMP-dependent protein kinase. The sequence of the tryptic peptide containing this site on the mammary enzyme is identical with the sequence of the peptide containing the site on ATP-citrate lyase that is phosphorylated in isolated hepatocytes in response to insulin and/or glucagon. The calmodulin-dependent, phospholipid-dependent and cyclic-AMP-dependent protein kinases phosphorylate distinct sites on acetyl-CoA carboxylase. However, one of the three phosphorylated tryptic peptides derived from enzyme treated with the phospholipid-dependent kinase is identical with the major phosphopeptide (T1) derived from enzyme treated with cyclic-AMP-dependent protein kinase. Phosphorylation of acetyl-CoA carboxylase by the phospholipid-dependent protein kinase inactivates acetyl-CoA carboxylase in a similar manner to cyclic-AMP-dependent protein kinase. With either protein kinase slightly greater phosphorylation and inactivation is seen after pretreatment of acetyl-CoA carboxylase with protein phosphatase-2A, but the effects of the protein phosphatase treatment are not completely reversed. Inactivation by the phospholipid-dependent protein kinase is Ca2+- and phospholipid-dependent, is reversed by protein phosphatase-2A, and correlates with the degree of phosphorylation. The relevance of these findings to insulin- and growth-factor-promoted phosphorylation of ATP-citrate lyase and acetyl-CoA carboxylase in intact cells is discussed.     

Insulin action rapidly decreases multifunctional protein kinase activity in rat adipose tissue

ATP-citrate lyase in vivo contains three phosphorylation sites on two tryptic peptides (peptides A and B). These phosphorylation sites are under hormonal control. Multifunctional protein kinase (MFPK) from rat liver phosphorylates peptide B on serine and threonine residues whereas cAMP-dependent protein kinase phosphorylates peptide A on a serine residue (Ramakrishna, S., and Benjamin, W. B. (1985) J. Biol. Chem. 260, 12280-12286). We now report that rat adipose tissue MFPK also phosphorylates serine and threonine residues of peptide B of ATP-citrate lyase. When the activity of MFPK was assayed using partially purified (by chromatography on phosphocellulose) cytosol fractions from insulin-treated adipose tissue, it was found that MFPK activity was decreased by over 55%. This decrease in MFPK activity occurs at physiological concentrations of insulin (EC50 = 1 x 10(-10) M). Its onset is rapid and almost maximal at 5 min after the addition of insulin. Even when new protein synthesis is inhibited by cycloheximide, extracts from insulin-treated fat pads have less MFPK activity compared to the control. The insulin effect is maintained after further chromatography on a gel filtration column suggesting that the decrease in MFPK activity is not due to a low molecular weight inhibitor. The insulin-induced decrease in MFPK activity is due to a decrease in Vmax whereas the affinity of this enzyme toward ATP-citrate lyase or ATP is unchanged.     

Cloning and expression of a human ATP-citrate lyase cDNA.

A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.     

The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary adipocytes

Protein kinase B (Akt) plays a central role in cellular regulation, although many of the physiologically relevant substrates for the kinase remain to be identified. In this study, we have isolated a protein from primary epididymal adipocytes with an apparent molecular weight of 125,000. This protein exhibited immunoreactivity, in an insulin-dependent manner, with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) as well as a phosphospecific antibody that recognizes serine 21/9 of GSK-3alpha/beta. MALDI-TOF mass spectrometry revealed the protein to be ATP-citrate lyase, suggesting that the two phosphospecific antibodies recognize phosphoserine 454, a previously reported insulin- and isoproterenol-stimulated ATP-citrate lyase phosphorylation site. Indeed, both insulin and isoproterenol stimulated the phosphorylation of this protein on the site recognized by the phosphospecific antibodies in a wortmannin-sensitive and -insensitive manner, respectively. In addition, transient expression of a constitutively active protein kinase B in primary adipocytes mimicked the effect of insulin on ATP-citrate lyase phosphorylation. Furthermore, ATP-citrate lyase was phosphorylated in vitro by recombinant protein kinase B on the same site. Taken together, these results demonstrate that serine 454 of ATP-citrate lyase is a novel and major in vivo substrate for protein kinase B.     

Phosphorylation of different sites of acetyl CoA carboxylase by ATP-citrate lyase kinase and cyclic AMP-dependent protein kinase

Native acetyl CoA carboxylase was phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase and ATP-citrate lyase kinase to 1 and 0.5 mol/subunit respectively. Both protein kinases added together increased acetyl CoA carboxylase phosphorylation additively. Partial proteolysis of 32P-acetyl CoA carboxylase followed by electrophoretic analysis showed that the 32P-phosphopeptides generated from acetyl CoA carboxylase phosphorylated with lyase kinase were different from the peptides obtained from the enzyme phosphorylated by cyclic AMP-dependent protein kinase. Mapping of tryptic 32P-phosphopeptides by high performance liquid chromatography showed that the major phosphopeptides phosphorylated by ATP-citrate lyase kinase were different from the major phosphopeptides phosphorylated by cyclic AMP-dependent protein kinase. The results suggest that at least one different site on acetyl CoA carboxylase is preferentially phosphorylated by each protein kinase.     

Purification and some properties of ATP-citrate lyase from rat brain

ATP-citrate lyase has been purified from rat brain by a new procedure which yields an enzyme of specific activity of 21 U/mg protein (37 °C) (2050-fold purification). Purity (by sodium dodecyl sulfate-gel electrophoresis) of the preparation was comparable to that of rat liver ATP-citrate lyase of similar specific activity. Both brain and liver ATP-citrate lyase have the same electrophoretic mobility, as well as the same immunoreactivity against specific rabbit anti-rat liver ATP-citrate lyase antibody. These data indicate that rat brain ATP-citrate lyase is similar or identical to that present in rat liver. Intraperitoneally injected ³²P_i was incorporated into the structural phosphate of ATP-citrate lyase in rat liver but not into the rat brain enzyme.     

Phosphorylation of sites 3 and 2 in rabbit skeletal muscle glycogen synthase by a multifunctional protein kinase (ATP-citrate lyase kinase)

A multifunctional protein kinase, purified from rat liver as ATP-citrate lyase kinase, has been identified as a glycogen synthase kinase. This kinase catalyzed incorporation of up to 1.5 mol of 32PO4/mol of synthase subunit associated with a decrease in the glycogen synthase activity ratio from 0.85 to a value of 0.15. Approximately 65-70% of the 32PO4 was incorporated into site 3 and 30-35% into site 2 as determined by reverse phase high performance liquid chromatography. Release of 32PO4 from the phosphopeptides during automated Edman degradation confirmed the site 3 and 2 assignment. Thermal stability studies established that the phosphorylations of sites 3 and 2 were catalyzed by the same kinase. This multifunctional kinase was distinguished from glycogen synthase kinase-3 on the basis of nucleotide (ATP versus GTP) and protein substrate (glycogen synthase, ATP-citrate lyase, and acetyl-CoA carboxylase) specificities. Since the phosphate contents in glycogen synthase of sites 3 and 2 are altered in diabetes and by insulin administration, the possible involvement of the multifunctional kinase was explored. Glycogen synthase purified from diabetic rabbits was phosphorylated in vitro by this multifunctional kinase at only 10% of the rate compared to synthase purified from control rabbits. Treatment of the diabetics with insulin restored the synthase to a form that was readily phosphorylated in vitro.     

Stoichiometry of phosphorylation of hepatic ATP-citrate lyase by protein kinase

Hepatic ATP-citrate lyase prepared with a fluoride-free step to allow endogenous phosphatases to dephosphorylate the enzyme was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase and [γ-³²P]ATP. After electrophoresis the radioactive phosphate was located predominantly in the gel slice containing the Coomassie blue stained protein corresponding to ATP-citrate lyase. The Stoichiometry of phosphorylation of hepatic ATP-citrate lyase in vitro by the catalytic subunit was such that 0.53 ± 0.02 molecules of phosphate were incorporated per subunit. The degree of phosphorylation was independent of the amount of ATP-citrate lyase present as substrate in the concentration range 1.2–6.4 μm. In the absence of catalytic subunit there was very little labeled phosphate incorporated into ATP-citrate lyase. Phosphorylation of ATP-citrate lyase by catalytic subunit was abolished by the specific protein inhibitor of cyclic AMP-dependent protein kinase. When ATP-citrate lyase was subjected to electrophoresis under nondenaturing conditions, lyase activity was recovered from the gel slice corresponding to the Coomassie blue staining phosphoprotein of a stained gel run in parallel.     

The reductive tricarboxylic acid cycle of carbon dioxide assimilation: initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum.

Carbon dioxide is fixed largely by the reductive tricarboxylic acid (RTCA) cycle in green sulfur bacteria. One of the key enzymes, ATP-citrate lyase, was purified to apparent homogeneity from the moderately thermophilic green sulfur bacterium Chlorobium tepidum. The molecular weight of the native enzyme was about 550,000, and the preponderance of evidence indicated that the protein is composed of identical subunits (Mr of approximately 135,000) which degraded to two major proteins with Mrs of approximately 65,000 and approximately 42,000. Western immunoblots and in vitro phosphorylation experiments indicated that these two species could have been the result of proteolysis by an endogenous protease, similar to what has been observed with mammalian, yeast, and mold ATP-citrate lyase. In addition to apparent structural similarities, the catalytic properties of C. tepidum ATP-citrate lyase showed marked similarities to the eukaryotic enzyme, with significant differences from other prokaryotic ATP-citrate lyases, including the enzyme from the closely related organism Chlorobium limicola. Phosphorylation of C. tepidum ATP-citrate lyase occurred, presumably on a histidine residue at the active site, similar to the case for the mammalian enzyme. In contrast to the situation observed for other prokaryotic ATP-citrate lyase enzymes, the C. tepidum enzyme was not able to replace ATP and GTP for activity or use Cu2+ to replace Mg2+ for enzyme activity. Given the apparent structural and catalytic similarities of the enzyme from C. tepidum and its eukaryotic counterpart, the C. tepidum system should serve as an excellent model for studies of the enzymology and regulation of this protein.     

Sequence of sites on ATP-citrate lyase and phosphatase inhibitor 2 phosphorylated by multifunctional protein kinase (a glycogen synthase kinase 3 like kinase)

Multifunctional protein kinase (MFPK) phosphorylates ATP-citrate lyase on peptide B on two sites, BT and BS, on threonine and serine, respectively, inhibitor 2 on a threonyl residue, and glycogen synthase at sites 2 and 3. The phosphorylation sites BT and BS of ATP-citrate lyase are dependent on prior phosphorylation at site A whereas site A phosphorylation is decreased by prior phosphorylation at sites BT and BS. To study the MFPK recognition sites and the site-site interactions, the amino acid sequences of ATP-citrate lyase peptide B and inhibitor 2 were determined and compared to each other and to glycogen synthase sites 3-5. The sequence of the tryptic peptide containing the two phosphorylation sites of peptide B is -Phe-Leu-Leu-Asn-Ala-Ser-Gly-Ser-Thr-Ser-Thr(P)-Pro-Ala-Pro-Ser(P)-Arg-, and the sequence of the MFPK phosphorylation site of inhibitor 2 is -Ile-Asp-Glu-Pro-Ser-Thr(P)-Pro-Tyr-. This inhibitor 2 site is identical with the site phosphorylated by glycogen synthase kinase 3/FA. These results suggest that at least some of the sites phosphorylated by MFPK (BT of ATP-citrate lyase, Thr 72 of inhibitor 2, and sites 3b and 4 of glycogen synthase) contain a Ser/Thr flanked by a carboxyl-terminal proline. However, as MFPK did not phosphorylate a series of peptides containing the -X-Thr/Ser-Pro-X- sequence, this minimum consensus sequence is not sufficient for phosphorylation by MFPK.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of mouse hepatic ATP-citrate lyase activity by dietary fat evidence for the presence of inactive enzyme

The induction of ATP-citrate lyase activity in mouse liver by dietary carbohydrate (glucose) is markedly reduced by including in the diet a source of polyunsaturated fatty acids. Within 72 h after changing from a standard mouse chow diet to a high carbohydrate diet containing 15% (w/w) of hydrogenated cottonseed oil (as a source of saturated fatty acids), the activity of mouse liver ATP-citrate lyase per milligram cytosolic protein was approx. 3-fold higher than that from mice fed a similar diet containing 15% (w/w) of corn oil. The rate of synthesis of ATP-citrate lyase relative to that for total protein and the rate of degradation of the enzyme were similar for both dietary groups. Elevated levels of enzyme activity in the hydrogenated cottonseed oil-fed livers were not accompanied by a similar increase in the amount of enzyme protein. To explain such findings, we propose that the activity of hepatic ATP-citrate lyase is regulated by dietary polyunsaturated fatty acids through a mechanism involving the conversion of a catalytically active form of the enzyme to a catalytically inactive form. A reversal of this conversion (inactive-active)_is evident within 72 h of removing the mice from the corn oil diet and placing them on the hydrogenated cottonseed oil diet. Futhermore, the conversion appears to be independent of the in vivo rate of synthesis of the enzyme.     

An insulin-stimulated (ribosomal S6) protein kinase from soluble extracts of H4 hepatoma cells

Insulin stimulates the phosphorylation of the 40 S ribosomal subunit protein, S6, in intact 32P-labeled H4IIE-C3 cells, a rat hepatoma line. Cell-free cytosolic extracts from H4 cells exhibit a 5- to 10-fold increase in S6 protein kinase activity (measured by transfer of 32P to exogenous 40 S rat liver ribosomal subunits) when prepared from cells exposed to insulin prior to homogenization. Stimulation of S6 phosphorylation in intact cells and activation of S6 protein kinase in cell-free extracts are both detectable within 2 min after insulin, and are maximally stimulated by 10 min. Half-maximal stimulation is observed at 10(-11) M insulin. The stimulated S6 kinase activity requires ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to be present during the kinase assay for full expression. Despite the presence of a 5- to 10-fold increase in S6 protein kinase activity, the extracts from insulin-treated cells exhibit no stimulated kinase activity toward casein, histone, or ATP-citrate lyase assayed under the conditions employed for S6. Thus, insulin mediates the rapid activation of protein kinase specific for ribosomal protein S6 by an as yet unidentified mechanism.     

Effects of phosphorylation on the kinetic properties of rat liver ATP-citrate lyase

Homogeneous rat liver ATP-citrate lyase (EC 4.1.3.8) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. In agreement with other workers, the maximum level of phosphorylation that we observed was approx. 2 mol/mol of tetramer. Phosphorylated and non-phosphorylated forms of ATP-citrate lyase were prepared. Their kinetic properties were examined using an assay system in which the concentrations of Mg.ATP, magnesium.citrate and CoA were varied systematically at a constant concentration of Mg2+. The phosphorylated form had a two-fold higher Km for Mg.ATP than did the non-phosphorylated form, but no other kinetic differences between the two forms were detected. When ATP-citrate lyase was assayed at a concentration of Mg.ATP well below Km, it was found that phosphorylation of the enzyme correlated well with a decrease of approx. 50% in its activity. This is the first demonstration that phosphorylation can affect the activity of ATP-citrate lyase.     

Long-term consumption of a diet with moderate medium chain triglyceride content does not inhibit the activity of enzymes involved in hepatic lipogenesis in the rat

The activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), malic enzyme (EC 1.1.1.40), ATP-citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase were lower (-25 to -60%) in liver of rats fed during 45 days with a moderate long-chain triglycerides (LCT) content diet (32% of metabolizable energy, ME), than in control rats fed with a low fat diet (LCT, 10% of ME). However, the fall in malic enzyme activity was not significant. In contrast, these activities were higher (+40 to +160%) in rats fed with a diet with a moderate medium-chain triglycerides (MCT) content (32% of ME), than in control rats. Nevertheless, the increase in activity of malic enzyme and ATP-citrate lyase was more important. Contrary to LCTs, MCTs had no inhibitory effect on the activity of enzymes involved in hepatic lipogenesis.     

Coordinate regulation of the biosynthesis of ATP-citrate lyase and malic enzyme during adipocyte differentiation. Studies on 3T3-L1 cells

The biosynthesis and degradation of two lipogenic enzymes were studied during the differentiation of 3T3-L1 preadipocytes into adipocytes. The activity and mass of malic enzyme, rose by an order of magnitude during adipocyte development and the enzyme accounted for 0.3% of the cytosol protein in mature fat cells. Similarly, the activity and amount of ATP-citrate lyase increased approximately 7-fold during the adipose conversion. The relative rates of synthesis of the two enzymes were less than or equal to 0.02% in preadipocytes, but increased sharply as the cells began to differentiate. Maximal steady state rates of malic enzyme and ATP-citrate lyase synthesis in 3T3-L1 adipocytes were 13- and 8-fold higher, respectively, than the basal rates in preadipocytes. In contrast, the half-lives of malic enzyme (67 h) and ATP-citrate lyase (47 h) were not altered during adipocyte development. Thus, accelerated rates of enzyme synthesis account for the differentiation-dependent accumulation of the two lipogenic enzymes. Increased rates of malic enzyme, ATP-citrate lyase, and fatty acid synthetase biosynthesis are expressed in a highly coordinated manner during adipocyte differentiation and are associated with parallel elevations in the levels of translatable mRNAs for these enzymes.     

Phosphorylation of the glucose transporter in rat adipocytes. Identification of the intracellular domain at the carboxyl terminus as a target for phosphorylation in intact-cells and in vitro

Phosphorylation of the insulin-regulatable glucose transporter (IRGT) is increased by incubating rat adipocytes with isoproterenol or by incubating microsomal membranes with cAMP-dependent protein kinase. To attempt to locate the sites of phosphorylation, the IRGT (apparent Mr = 46,000) was immunoprecipitated from 32P-labeled adipocytes and cleaved with CNBr or trypsin. Essentially all of the 32P could be recovered in a single CNBr fragment, denoted CB-T (Mr = 8,000), which bound a polyclonal antibody (R820) against a peptide having the sequence of the last 12 amino acids in the COOH terminus of the IRGT. 32P-Labeling of the IRGT was also confined to CB-T when membranes were incubated with [gamma-32P]ATP and cAMP-dependent protein kinase. Isoproterenol increased phosphorylation of CB-T, but insulin was without effect. To resolve phosphorylation sites further, IRGT from 32P-labeled cells was subjected to exhaustive proteolysis with trypsin. Samples were applied to a C-18 column, and 32P-labeled fragments were resolved into three peak fractions by elution with an increasing gradient of acetonitrile. [32P]Phosphoserine was the only phosphoamino acid detected in any of the peaks. Peak III contained approximately 80% of the 32P and was increased by isoproterenol. Almost all of the 32P introduced by cAMP-dependent protein kinase in vitro eluted in Peak III. In all cases, the 32P-labeled species in Peak III were quantitatively immunoprecipitated by R820. Digesting the peptide(s) in Peak III with V8 protease generated a single peak of 32P which eluted at lower acetonitrile than Peak III and contained 32P-labeled species that did not interact with R820. Automated Edman degradation indicated that the serine residue in Peak III phosphorylated by cAMP-dependent protein kinase was the 3rd or 4th residue from the NH2 terminus of the peptide. These findings indicate that phosphorylation of the IRGT is restricted to the presumed intracellular domain at the COOH terminus and that Ser488 is a major site phosphorylated both by cAMP-dependent protein kinase in vitro and in response to isoproterenol in vivo.     

Vanadate induces normolipidemia and a reduction in the levels of hepatic lipogenic enzymes in obese Zucker rat

The effects of vanadate administration on the plasma lipids and hepatic lipogenic enzymes were investigated in Zucker (fa/fa) rat, a model for obesity and non insulin-dependent diabetes. These animals were administered sodium orthovanadate through drinking water for a period of four months. The plasma levels of insulin, triacylglycerols and total cholesterol were significantly (p<0.001) elevated in untreated obese control rats as compared to the lean animals. In the livers of obese rats, the number of insulin receptors decreased by 60% and the activities of lipogenic enzymes acetyl-CoA carboxylase and ATP-citrate lyase increased by 4.7- and 5.6-folds, respectively. The messenger RNA for ATP-citrate lyase as measured by Northern blot analysis showed a parallel increase in obese control rats. Treatment of these rats with vanadate caused 56–77% decreases in the plasma levels of insulin, triacylglycerols and total cholesterol. The insulin receptor numbers in vanadate-treated obese rats increased (119%) compared to levels in untreated obese animals. The elevated activities of acetyl-CoA carboxylase and ATP-citrate lyase observed in livers of obese rats were significantly reduced by vanadate. The messenger RNA for ATP-citrate lyase also decreased in vanadate-treated obese rats back to the lean control levels. This study demonstrates that vanadate exerts potent actions on lipid metabolism in diabetic animals in addition to the recognized effects on glucose homeostasis.     

Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

Escherichia coli isocitrate lyase (EC 4.1.3.1.) can be phosphorylated in vitro by an ATP-dependent reaction. The enzyme becomes phosphorylated by an endogenous kinase when partially purified sonic extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with diethyl pyrocarbonate, a histidine-modifying reagent, blocked incorporation of [32P]phosphate from [gamma-32P]ATP. The isoelectric point of the enzyme was altered by treatment with phosphoramidate, a histidine phosphorylating agent, which suggests that isocitrate lyase can be phosphorylated at a histidine residue(s). Immunoprecipitated 32P-labeled isocitrate lyase was subjected to alkaline hydrolysis, mixed with chemically synthesized phosphohistidine standards, and analyzed by anion exchange chromatography. Characterization of the phosphoamino acid was based on the demonstration that the 32P-labeled product from alkali-hydrolyzed isocitrate lyase comigrated with synthetic 1-phosphohistidine. In addition, loss of catalytic activity after treatment with potato acid phosphatase indicates that catalytically active isocitrate lyase is the phosphorylated form of the enzyme.     

ATP-citrate lyase from rat liver. Characterisation of the citryl-enzyme complexes.

The mechanism of ATP-citrate lyase has been proposed to involve a citryl-enzyme intermediate. When the enzyme is incubated with its substrates ATP and [14C]citrate, but in the absence of the final acceptor, two distinct types of citrate-containing complex can be isolated. At early time points, a highly unstable complex can be isolated by gel filtration which has a half-life of 36 s at 25 degrees C. This complex reacts rapidly with CoA, but cannot be acid-precipitated; behaviour consistent with its identification as enzyme-citryl phosphate. However, ATP-citrate lyase is also capable of undergoing a slow time-dependent covalent incorporation of radiolabel from [14C]citrate. This modification is acid-stable, non-specific, and cannot be reversed by the addition of CoA. When cytochrome is included in the reaction mixture as a heterologous acceptor, it is also citrylated. These reactions require that when ATP-citrate lyase is incubated with all its substrates except for CoA, a freely diffusible citrylating species is generated within the active site. This evidence suggests that there is no requirement for the mechanism of ATP-citrate lyase to proceed via a covalent citryl-enzyme intermediate. By analogy with succinyl-CoA synthetase, an enzyme which has a high degree of sequence similarity with ATP-citrate lyase, a simple mechanism is proposed for the enzyme in which citryl-CoA is produced by direct nucleophilic attack on citryl phosphate.