Showing papers on "ATP citrate lyase published in 1986"

Autotrophic growth and CO2 fixation of Chloroflexus aurantiacus

Abstract: Chlorofluexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as the electron source. The lowest doubling time observed was 26 h. The mechanism of CO2 fixation in autotrophically grown cells was studied. The presence of ribulose-1,5-bis-phosphate carboxylase and phosphoribulokinase could not be demonstrated. Carbon isotope fractionation (δ13C) was small, and alanine and aspartate but not 3-phosphoglycerate were the major labelled compounds in short term 14CO2 labelling. Thus CO2 is not fixed by the Calvin cycle. Fluoroacetate (FAc) completely inhibited protein synthesis in cultures and caused a slight citrate accumulation. However, CO2 fixation continued and increased polyglucose formation occurred. Under these conditions added acetate was metabolized to polyglucose, as were glycine, serine, glyoxylate and succinate, but to a lesser extent; little or no formate or CO was utilised. Glyoxylate inhibited CO2 fixation in vivo, indicating that pyruvate is formed from acetyl-CoA and CO2 by pyruvate synthase. Two key enzymes of the reductive TCA cycle, citrate lyase and α-ketoglutarate synthase were not detected in cell free extracts, but pyruvate synthase and phosphoenolpyruvate carboxylase were demonstrated. It is concluded that acetyl-CoA is a central intermediate in the CO2 fixation process, but the mechanism of its synthesis is not clear.

Citrate transport in Klebsiella pneumoniae.

Abstract: Sodium ions were specifically required for citrate degradation by suspensions of K. pneumoniae cells which had been grown anaerobically on citrate. The rate of citrate degradation was considerably lower than the activities of the citrate fermentation enzymes citrate lyase and oxaloacetate decarboxylase, indicating that citrate transport is rate limiting. Uptake of citrate into cells was also Na+ -dependent and was accompanied by its rapid metabolism so that the tricarboxylic acid was not accumulated in the cells to significant levels. The transport could be stimulated less efficiently by LiCl. Li+ ions were cotransported with citrate into the cells. Transport and degradation of citrate were abolished with the uncoupler [4-(trifluoromethoxy)phenylhydrazono]propanedinitrile (CCFP). After releasing outer membrane components and periplasmic binding proteins by cold osmotic shock treatment, citrate degradation became also sensitive towards monensin and valinomycin. The shock procedure had no effect on the rate of citrate degradation indicating that the transport is not dependent on a binding protein. Citrate degradation and transport were independent of Na+ ions in K. pneumoniae grown aerobically on citrate and in E. coli grown anaerobically on citrate plus glucose. An E. coli cit+ clone obtained by transformation of K. pneumoniae genes coding for citrate transport required Na specifically for aerobic growth on citrate indicating that the Na-dependent citrate transport system is operating. Na+ and Li+ were equally effective in stimulating citrate degradation by cell suspensions of E. coli cit+. Citrate transport in membrane vesicles of E. coli cit+ was also Na+ dependent and was energized by the proton motive force (delta micro H+). Dissipation of delta micro H+ or its components delta pH or delta psi by ionophores either totally abolished or greatly inhibited citrate uptake. It is suggested that the systems energizing citrate transport under anaerobic conditions are provided by the outwardly directed cotransport of metabolic endproducts with protons yielding delta pH and by the decarboxylation of oxaloacetate yielding delta pNa+ and delta psi. In citrate-fermenting K. pneumoniae an ATPase which is activated by Na+ was not found. The cells contain however a proton translocating ATPase and a Na+/H+ antiporter in their membrane.

Extramitochondrial citrate synthase activity in bakers' yeast.

Abstract: We isolated the gene for citrate synthase (citrate oxaloacetate lyase; EC 4.1.3.7) from Saccharomyces cerevisiae and ablated it by inserting the yeast LEU2 gene within its reading frame. This revealed a second, nonmitochondrial citrate synthase. Like the mitochondrial enzyme, this enzyme was sensitive to glucose repression. It did not react with antibodies against mitochondrial citrate synthase. Haploid cells lacking a gene for mitochondrial citrate synthase grew somewhat slower than wild-type yeast cells, but exhibited no auxotrophic growth requirements.

Steady-state kinetic analysis of isocitrate lyase from Lupinus seeds: considerations on a possible catalytic mechanism of isocitrate lyase from plants.

Abstract: Isocitrate lyase catalyzes the reversible cleavage of isocitrate into glyoxylate and succinate. The kinetic mechanism of bacterial isocitrate lyase has been reported to be ordered uni-bi. Moreover, it has been proposed that isocitrate lyase in higher plants may be switched on and off by a succinylation/desuccinylation mechanism. Similarly to bacterial citrate lyase, in which an acetylation/deacetylation mechanism is operative, succinylation might also play a role in the catalytic mechanism of plant isocitrate lyase. We have investigated the kinetic mechanism of isocitrate lyase from Lupinus seeds. The results reported in this paper show that the system follows a preferentially ordered uni-bi pathway in which the succinate is released first. On the basis of our results and some other recently reported data, we conclude that it is unlikely that bacterial and plant isocitrate lyases have different catalytic mechanisms.

The Hypolipidemic Activity of a Series of 2,3-Dihydrophthalazine-l,4-dione Derivatives in Rodents.

Abstract: A series of substituted 2,3-dihydrophthalazine-l,4-dione derivatives as well as the corresponding N,N-diaminophthalamides were prepared and were demonstrated to have potent hypolipidemic activity, lowering both serum triglyceride and cholesterol levels significantly at 20 mg/kg/day after 16 days of dosing in CF1 male mice. The parent compound, 2,3-dihydrophthalazine-l,4-dione, lowered serum cholesterol 51% and serum triglyceride 43%. 2-(2-Carboxyethyl)-2,3-dihydrophthalazine-l,4-dione demonstrated the best hypocholesterolemic activity, with a 66% reduction after 16 days. The 2-(p-chlorophenyl) derivative demonstrated good activity (>40% reduction) in both screens, as did the 6-methyl-2,3-dihydrophthalazine-l,4-dione derivative. Of the amides, 4-methyk N,N-diaminophthalamide demonstrated the best hypolipidemic activity, affording a greater than 40% reduction. 2,3-Dihydrophthalazine-l,4-dione was found to inhibit the enzyme activity of acetyl CoA synthetase, ATP-dependent citrate lyase, sn-glycerol-3-phosphate acyl transferase, phosphatidylate phosphohydrolase, and mitochondrial citrate exchange of liver. In mice after 16 days of dosing, there was a reduction of cholesterol, triglycerides, neutral lipids, and phospholipids in the liver. Cholesterol and neutral lipids were reduced in rat chylomicrons, very low-density lipoproteins, and low-density lipoproteins. The cholesterol content of the high-density lipoprotein fraction was slightly elevated, but reductions in the triglycerides and phospholipids were observed in this lipoprotein fraction. 3H-Cholesterol distribution studies showed a lower concentration in the major organs and plasma, with a higher 3H-cholesterol content in the stomach and large intestine.

Hypolipidemic activity of 3- and 4-phenyl-piperidine-2,6-diones and selected N-substituted derivatives

Abstract: Three- and 4-phenyl-piperidine-2,6-dione derivatives were investigated for hypolipidemic activity at 20 mg/kg/day intraperitoneally in rodents. The 3-phenyl compound afforded the best activity and effectiveness in both normal and hyperlipidemia-induced mice. The agent lowered lipids by blocking the de novo hepatic synthesis of cholesterol and triglycerides, specifically at the sites of ATP-dependent citrate lyase, acetyl CoA synthetase,sn-glycerol-3-phosphate acyl transferase and phosphatidylate phosphohydrolase. The agent caused a more rapid clearance of cholesterol by the fecal route. Cholesterol levels of the chylomicrons, very low density lipoprotein and low density lipoprotein (LDL) were reduced, whereas high density lipoprotein cholesterol was significantly elevated after drug administration. Triglyceride content was lowered in the chylomicron and LDL fractions. These modulations of lipid content of serum lipoproteins by the drug suggest a favorable situation for treatment of hyperlipidemic states.

Lipid metabolism and enzyme activities in hormone-dependent and hormone-independent mammary adenocarcinoma in GR mice.

Abstract: Lipid metabolism in hormone-dependent (HD) GR mouse mammary tumors was compared to that in hormone-independent (HI) tumors and normal mammary tissues. HD tumors, like normal mammary tissue but unlike HI tumors, synthesized medium-chain-length fatty acids (MCFA). However, when treated with hormones (estrone and progesterone), the HI tumors were induced to produce MCFA. The activity of thioesterase II correlated positively with the synthesis of MCFA and was influenced by the hormones administered. The activities of NADP+-linked malate dehydrogenase, citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase, although lower in tumors than in normal glands, were not different in HD as compared to HI tumors. Whereas the predominating lipids synthesized in normal glands were triglycerides, phospholipids comprised about half of the lipid synthesized in the tumors, with no difference between HD and HI tumors. The conversion of D-[U-14C]glucose to 14CO2 was higher in HD tumors than in HI tumors but increased in HI tumors treated with hormones in vivo. By a comparison of the 14CO2 produced from D-[1-14C]glucose and from D-[6-14C]glucose in the presence and absence of an electron acceptor (methylene blue), it was demonstrated that regeneration of NADP+ from NADPH was a rate-limiting step for the pentose phosphate pathway in the tumors. Hence, while differences in the lipid metabolism can be demonstrated between HD and HI GR mouse mammary tumors, some of the changes are due to the hormone treatment rather than to a specific alteration in the tumor itself.

Apparent inhibition of ATP citrate lyase by L-glutamate in vitro is due to the presence of glutamine synthetase.

Abstract: Preincubation in assay mixture for 30 min at 37 degrees C of ATP citrate lyase from rat brain and liver results in 65-70% inhibition in the presence of 10 mM L-glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate-suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol-precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor L-methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elsewhere, L-glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.

Calcitonin action on ATP citrate lyase activity in the hepatic cytosol of fed rats and its calcium-calmodulin dependency.

Abstract: The effect of calcitonin (CT) on ATP citrate lyase activity in the hepatic cytosol was investigated after a single subcutaneous administration of the hormone to fed rats. Administration of CT (synthetic [Asu107] eel CT; 80 MRC mU/100 g body weight) produced significant increases in ATP citrate lyase activity and calcium content in the hepatic cytosol of intact and thyroparathyroidectomized rats. Those alterations were also observed with the dose of CT at physiological level. The increased cytosolic ATP citrate lyase activity resulting from CT administration was prevented by treatment with 10 microM EGTA. This enzyme activity was restored by addition of calcium ion (2.5-10 microM). The rise in enzyme activity of CT-treated rats was markedly reduced by the presence of W-7 (10 and 100 microM), a calmodulin inhibitor, in the enzyme assay system, while that of control rats was not significantly altered by the drug. These results suggest that CT increases ATP citrate lyase activity in the hepatic cytosol of fed rats, and that this hormonal regulation may depend on calmodulin, and be mediated through raised calcium in the cytosol.