Citrate Cleavage Research Articles

Influence of Fasting and Refeeding on Lipogenesis and Enzymatic Activity of Pig Adipose Tissue

Fatty acid synthesis and 14CO2 production from glucose-U-14C were virtually abolished in biopsy adipose tissue samples obtained from pigs subjected to a 4-day fast. Refeeding for 2 days fully restored lipogenesis to the prefasting level, and refeeding for 4 days was associated with a twofold overshoot in the capacity for glyceride-glycerol synthesis. No overshoot in fatty acid synthesis could be detected after 4, 6, or 12 days of refeeding. When pigs were refed diets high in protein or fat after a 4-day fast, the restoration of the lipogenic capacity of the adipose tissue was limited to about 50% of that observed in animals refed a high carbohydrate diet. The nonparallel behavior of citrate cleavage enzyme activity and lipogenesis discounts any regulatory role for this enzyme in fatty acid synthesis. Among the NADPH-generating dehydrogenase enzymes studied, malic enzyme appeared more adaptive than the pentose pathway enzymes. The rate of lipogenesis was more closely correlated with the activity of acetyl CoA carboxylase than with any other enzyme assayed. The regulatory implications of these findings in relation to lipogenic control in pig adipose tissue are discussed.

Citryl Phosphate and the Mode of Action of the Citrate Cleavage Enzyme

Abstract When the citrate cleavage reaction is conducted with 18O-citrate, 1 atom of citrate oxygen is incorporated into each molecule of orthophosphate stemming from ATP. This is consistent with the notion that citryl phosphate is an intermediate of the reaction. Chemically synthesized dl-citryl 1-phosphate is an active substrate of the citrate cleavage enzyme, undergoing stereospecific cleavage in the presence of coenzyme A to oxalacetate and acetyl coenzyme A. Citryl phosphate transfers its citryl group to the enzyme to form citryl enzyme, which is identical in its biochemical properties with the citryl enzyme formed from citrate plus ATP. Citryl phosphate can also transfer its phosphoryl group to the enzyme to form a phosphoprotein which differs in its properties from the phosphoenzyme formed from ATP.

Fluorometric micro determination of citrate in tissue extracts

An enzymic procedure has been developed for the determination of micro quantities of tissue citrate. The reactions involve the cleavage of citrate by citrate lyase to oxalacetate and its subsequent oxidation of NADH by malic dehydrogenase. The NAD + thus produced is cycled with lactate, α-ketoglutarate, and, ammonium ion, in the presence of the enzymes lactate dehydrogenase and glutamic dehydrogenase. Conversion of the cycling product (i.e., pyruvate) plus NADH to NAD + and lactate leads to a strongly fluorescent substance when heated in strong alkali. The fluorescence thus produced from NAD + can be equated by means of known standards, with the citrate originally present in tissue samples. Data obtained from several tissues are presented to demonstrate the potential applicability of the method to very small tissue samples and even to single cells.

NADP specific dehydrogenases and heaptic lipogenesis in the hibernator

1. 1. Glucose and acetate metabolism and the activity of several NAdP specific dehydrogenases were studied in hepatic tissue from the ground squirrel, Citellus tridecemlineatus. Tissues were obtained from hibernating and arousing squirrels in January and from normathermic squirrels in June and Sepatember. 2. 2. Glucose and acetate oxidation to CO 2 and incorporation into lipids and activities of NADP malic dehydrogenase, citrate cleavage enzyme and hexose monophosphate shunt dehydrogenases were lowest during hibernation, not changed during arousal, and attained macimum levels in June.

Nature of the phosphorylated residue in citrate cleavage enzyme

Citrate cleavage enzyme was phosphorylated with γ- 32P-ATP. A 32P-compound was isolated by Dowex chromatography of an alkaline hydrolysate of the 32P-phosphoenzyme. The isolated compound behaved chromatographically and electrophoretically like phosphohistidine.

Fatty Acid Synthesis in Human Adipose Tissue

The concentration of enzymes required for fatty acid synthesis was considerably less in extracts prepared from human omental adipose tissue than from rat epididymal fat. Overall rates of synthesis as measured by the incorporation of radioactive precursors into fatty acids were also much lower in human adipose tissue. The most striking difference was found with citrate-1,5-14C, human adipose tissue incorporating the compound at a rate less than one-tenth that of rat epididymal fat. This finding was consistent with the virtual absence of the citrate cleavage enzyme in human adipose tissue. By contrast, the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase, two enzymes necessary for the synthesis of glyceride glycerol from pyruvate, and the long chain fatty acid activating enzyme (or enzymes) were essentially similar in both human and rat adipose tissues. The results are consistent with the hypothesis that synthesis de novo of fatty acids is not an important physiological function of human adipose tissue.

Partial purification and some properties of the (R)-citrate synthase from Clostridium acidi-urici.

The enzyme (R)‐citrate synthase which catalyzes essentially the same biochemical reaction as the citrate synthase but differs in its stereospecificity, has been studied. From cell‐free extracts of Clostridium acidi‐urici the enzyme has been purified 80‐fold by isoelectric precipitation and by chromatography on DEAE‐cellulose and DEAE‐Sephadex. The purification is made difficult by the instability of the enzyme. With the aid of the stereospecific cleavage of citrate by citrate lyase it was demonstrated that the purified (R)‐citrate synthase is not contaminated by the usual citrate synthase.The (R)‐citrate synthase differs in some properties from the usual citrate synthase: it is inactivated by exposure to oxygen, anaerobic conditions and a sulfhydryl compound being required for maximum activity. (R)‐citrate synthase has a specific requirement for manganese ions, the enzyme is inhibited by EDTA, and this inhibition can be reversed by manganese ions but not by magnesium ions.

The sulfhydryl groups of citrate cleavage enzyme

Chicken liver citrate cleavage enzyme has an apparent optimum activity at pH 8.7, similar to the enzyme isolated from rat liver. Early results indicating a lower pH optimum were due to a pH dependent disulfide inhibition of the enzyme. Both chicken liver and rat liver citrate cleavage enzymes are inhibited by oxidized glutathione, oxidized BAL, lipoate, and arsenite. Studies with the purified rat liver enzyme indicate the presence of 6–8 moles of very reactive SH groups per mole of enzyme which are essential for enzyme activity. These groups can be protected by Mg-citrate but not by Mg-ATP. The loss of enzyme activity observed upon storage and its subsequent reactivation by dithiothreitol does not result from changes in aggregation and dissociation of the enzyme as judged by gel electrophoresis and ultracentrifugation.

Tricarballylate and hydroxycitrate: Substrate and inhibitor of ATP: Citrate oxaloacetate lyase

Citrate cleavage enzyme shows atypical kinetics at low chloride concentrations, but normal kinetics at high chloride concentrations. Tricarballylate is a substrate for citrate cleavage enzyme. The reaction with tricarballylate can be demonstrated both by the disappearance of CoA and, in the presence of hydroxylamine, by the appearance of a hydroxamate. This indicates that tricarballylyl-CoA is formed in the reaction. When hydroxamate formation is used to assay activity, the apparent K m for tricarballylate is about three times greater than that for citrate, while the maximum reaction velocity with tricarballylate is about 90% of that observed with citrate. One of the stereoisomers of hydroxycitrate is a powerful inhibitor of citrate cleavage enzyme.

The role of hormones in glucose repression in rat liver

Previous studies from this laboratory have demonstrated that the admin istration of amino acid mixtures to rats previously fed a 0% protein diet will markedly induce the synthesis of several hepatic enzymes. In particular, serine dehydratase is markedly induced under these conditions. The induc tion is characterized by an initial period of RNA synthesis, but thereafter appears to continue independent of RNA synthesis. That the increases seen are the result of actual changes in the rate of synthesis of the enzyme has been demonstrated by immunochemical methods. Relatively low doses of glucose (150 mg) effectively repress enzyme synthesis under these conditions. Glucagon also induces serine dehydratase, this induction being repressed by glucose. Insulin given to animals partially repressed with glucose has no further effect. However, rats made diabetic with alloxan show marked increases in the level of serine dehydratase, this high level being unaffected by glucose, fructose, or insulin administration. Similar findings may be seen with the mitochondrial enzyme, ornithine transaminase. The enzymes of carbohyd rate metabolism, glucokinase, citrate cleavage enzyme and glucose 6-phos phate dehydrogenase, may be induced by protein and glucose in the intact animal, but appear to require glucose and insulin in the diabetic animal. Animals fed a high protein diet show marked increases in serine dehydratase and ornithine transaminase which is not affected by the chronic administra tion of insulin. These studies demonstrate that glucose repression is a complex phenomenon, probably requiring the interaction of hormones and substrates. The data seen in this paper, however, do not in any way support a primary role for insulin in the mechanism of glucose repression in rat liver.

The metabolic fate of the products of citrate cleavage. Adenosine triphosphate-citrate lyase and nicotinamide-adenine dinucleotide phosphate-linked malate dehydrogenase in foetal and adult liver from ruminants and non-ruminants.

1. Foetal rat liver slices incorporate the C-3 of aspartate and C-2 of glutamate into fatty acids at rates equal to those observed with adult rat liver slices. Incorporation of either of these labelled carbon atoms into fatty acids would require a functioning citrate-cleavage pathway which consists of the enzymes ATP-citrate lyase, NAD-malate dehydrogenase and NADP-malate dehydrogenase. However, NADP-malate dehydrogenase is present in foetal rat liver at only 5% of the activity detectable in adult rat liver. 2. From these findings and the effect of cofactors on the formation of (14)CO(2) from [1,5-(14)C(2)]citrate in liver supernatant fractions (100000g), it is suggested that NADP-malate dehydrogenase limits the citrate-cleavage sequence. 3. Measurement of the citrate-cleavage pathway by incorporation studies with [3-(14)C]aspartate and [U-(14)C]glucose and by determining the activities of ATP-citrate lyase and NADP-malate dehydrogenase have shown that this sequence of reactions is present in the liver of the bovine foetus but not in the adult. However, C-2 of glutamate is not incorporated into fatty acids or non-saponifiable lipid by bovine liver slices. This finding as well as those presented above for the adult and foetal rat liver are interpreted on the basis of a competition between phosphoenolpyruvate carboxykinase and NAD-malate dehydrogenase for oxaloacetate produced by the cleavage of citrate in the cytosol.

The stereospecificity of the citrate synthase in sulfate-reducing and photosynthetic bacteria.

The stereospecificity of the citrate synthase of a number of sulfate-reducing and photosynthetic bacteria was determined by the following method: radioactive citrate was synthesized from [4-14C]oxaloacetate and acetylcoenzyme A by cell-free extracts. The resulting citrate was then cleaved to acetate and malate by citrate lyase plus malate dehydrogenase. In the case that the usual citrate synthase was present in the cell-free extracts, the citrate cleavage yielded radioactive malate and unlabeled acetate. The presence of the Clostridium kluyveri type of citrate synthase led to the formation of radioactive acetate and unlabeled malate. It was found that four species of the Athiorhodaceae, Desultfovibrio gigas, D. sale xigens and Desulfotomaculum ruminis contain the usual citrate synthase. Two strains of D. desulfuricans and two strains of D. vulgaris contain the C. kluyveri type of citrate synthase, which is now referred to as (R)-citrate synthase. The presence of the (R)-citrate synthase in D. desulfuricans and D. vulgaris was confirmed by an in-vivo experiment. When grown on pyruvate and radioactive carbon dioxide, the protein of these two species contains [5-14C]glutamate, whereas the protein of D. gigas contains [1-14C]-glutamate. The presence of the (R)-citrate synthase in certain strictly anaerobic bacteria is discussed from an evolutionary point of view.

Systematic Oscillations in Tyrosine Transaminase and Other Metabolic Functions in Liver of Normal and Adrenalectomized Rats on Controlled Feeding Schedules

In an attempt to clarify the regulatory mechanism of synthesis and degradation of enzymes in mammalian cells in vivo, rats were fed 12, 30 and 60% protein diets and were adapted to 8 hours' feeding in either a 24-hour cycle or a 48-hour cycle under controlled lighting conditions. The rats were killed at various times in the cycle to determine any oscillatory changes in the activity of tyrosine transaminase, serine dehydratase, glucose 6-phosphate dehydrogenase and citrate cleavage enzyme in liver, compared with changes of glycogen deposition in liver and corticosterone level in plasma. Tyrosine transaminase shows increased activity 6 hours after the onset of food intake, and the increased activities are in proportion to the protein content of the diets. Direct proportionality of activity to protein level in the diet was also found for serine dehydratase and inverse proportionality for glucose 6-phosphate dehydrogenase and citrate cleavage enzyme. A secondary rise in tyrosine transaminase activity observed 16 hours into the fasting periods in rats adapted to 8 hours’ feeding in 48-hour cycle is clearly dependent upon the protein content of the diet previously ingested, and is also dependent upon the availability of adrenal hormone. Glucose 6-phosphate dehydrogenase shows higher activity in rats adapted to 8 hours' feeding in a 48-hour cycle than in rats in a 24-hour cycle, and, on the contrary, serine dehydratase presents higher activity in the latter group of rats.

Acetyl transport mechanisms in the nervous system. The oxoglutarate shunt and fatty acid synthesis in the developing rat brain.

Abstract Radioactive acetyl groups and lipids are produced from dl‐[5‐14C]glutamate. Degradation studies indicate that approximately 90 per cent of the radioactivity is localized in the original carboxyl groups of the two carbon unit. Since these results are shown not to be due to a 14CO2 fixation, it is concluded that the oxoglutarate shunt as an acetyl group transport system is functional in brain.The highest ratio of fatty’acid activity/CO2 activity in this pathway is found in the newborn rat brain and steadily decreases with development. This pattern is observed with incubations of brain slices with labelled glutamate or citrate and is similar to the changes observed in the activity of the citrate cleavage enzyme with brain maturation.In contrast to the previous studies with liver preparations, the conversion of [2‐14C]‐ and [5‐14C]glutamate to fatty acids is relatively small. This is particularly true during the period of maximal lipid synthesis.

Citrate Cleavage Enzyme and Fatty Acid Synthesis

A series of experiments was performed to test the proposal of others that citrate cleavage enzyme (EC 4.1.3.8) regulates the rate of fatty acid synthesis in rat liver. The results show that, in recovery from fasting, fatty acid synthesis increased markedly without change in citrate cleavage enzyme activity. After the administration of alloxan, fatty acid synthesis decreased prior to any change in citrate cleavage enzyme. Substitution of a high carbohydrate, low fat diet resulted in large increases in citrate cleavage enzyme activity without concomitant change in lipogenesis. The addition of purified citrate cleavage enzyme from rat liver to the fatty acid-synthesizing system from fasted animals did not restore fatty acid synthesis. It was concluded that citrate cleavage enzyme does not play a primary regulatory role in fatty acid synthesis.

Lipogenesis in human adipose tissue

The pathways and some critical enzymes involved in lipogenesis in adipose tissue from 82 patients have been studied. Of the glucose-(14)C metabolized to lipid by isolated adipose cells, approximately 0.6% was recovered in fatty acids and the rest in glyceride-glycerol. Palmitate-1-(14)C was readily incorporated into neutral lipid. Homogenates of human adipose tissue contained an active alpha-glycerophosphate dehydrogenase which was approximately twice as active as malate dehydrogenase. Mitochondria of human adipose tissue contained an NAD-independent alpha-glycerophosphate dehydrogenase; the reaction product, di-hydroxyacetone phosphate, was recovered extramitochondrially. Homogenates of human adipose tissue also contained an active fatty acyl CoA synthetase which required ATP, CoA, and Mg(++) for maximal activity. The activity of acyl CoA synthetase varied greatly in a group of 40 patients. By contrast, the range of activity of malate dehydrogenase assayed in the same group of patients was much smaller. When palmitate or palmitoyl CoA was used as substrate, no difference was found in the rate of incorporation of alpha-glycerol-1,3-(14)C phosphate into neutral lipid. If time was allowed for activation of palmitate, the incorporation of alpha-glycerol-1,3-(14)C phosphate into lipid was 3.5 times greater than for unactivated palmitate. Palmitate (200 mM) stimulated lipogenesis in homogenates of human adipose tissue and then caused a severe inhibition at 700 mM. Arachidate over the same concentration range did not depress lipogenesis below initial values.

Acetyl group transfer in lipogenesis: II. Fatty acid synthesis from intra- and extramitochondrial acetyl CoA

In order to evaluate the role of citrate as an acetyl carrier from the mitochondria to the cytosol, we compared pathways of fatty acid synthesis from acetyl CoA, doubly labeled with 14C and T in the acetyl moiety, generated in the mitochondria (by pyruvate decarboxylation) and in the cytosol (by the citrate cleavage enzyme). Epididymal fat pad segments were incubated with glucose-6- 14C-6-T, a precursor of mitochondrial acetyl CoA, and glutamate-5- 14C-4-T, a precursor of extramitochondrial acetyl CoA. The use of glutamate-5- 14C-4-T in conjunction with glutamate-5- 14C-3-T permits the calculation of the tritium loss in the conversion of extramitochondrial acetyl CoA to fatty acids. Acetate produced by the degradation of lactate from glucose-6- 14C-6-T permits the calculation of the tritium loss in the conversion of intramitochondrial acetyl CoA to fatty acids. It was found that the loss of tritium in the conversion of extramitochondrial acetyl CoA to fatty acids was about 20% less than the loss of tritium in the conversion of intramitochondrial acetyl CoA to fatty acids. The additional loss of tritium in the latter pathway corresponds closely to the loss of tritium in the conversion of T-acetyl CoA to T-citrate in the citrate synthase reaction, suggesting that citrate is involved in the transfer of the acetyl group of acetyl CoA.

Citryl 1-Phosphate and the Citrate Cleavage Reaction

Abstract Citryl 1-phosphate has been synthesized chemically. It is an active substrate of ATP:citrate lyase (EC 4.1.3.8). With coenzyme A present, the enzyme catalyzes the cleavage of citryl-1-P into oxaloacetate and acetyl-CoA. Starting with radioactive citryl-1-P, the products of cleavage have been isolated from incubation mixtures in the form of convenient derivatives which have been purified to constant specific activity. The identity of oxaloacetate as a cleavage product has been further confirmed through its specific oxidation of NADH in the presence of malate dehydrogenase (EC 1.1.1.37).

The citrate cleavage enzyme activity in chick embryo and chicken liver during development.

La relazione tra le misure sperimentali dei livelli di attivita del «Citrate cleavage enzyme» (E.C. 4.1.3.6.) e lo andamento dei processi neoglicogenetico e lipogenetico nel fegato di embrione di pollo e di pollo a vari tempi di sviluppo esclude che la scissione enzimatica del citrato partecipi alla neoglicogenesi e conferma il ruolo di questa reazione nella lipogenesi.

Purification and Kinetic Studies of the Citrate Cleavage Enzyme

Abstract The citrate cleavage enzyme (EC 4.1.3.8) was isolated from rat liver and purified. Reciprocal plots with coenzyme A or MgATP as variable substrates were linear, but when citrate was varied, the plot was curved, convex upward. The reciprocal plot became linear in the presence of 0.25 m chloride, and under these conditions Mg-citrate appeared to be the active substrate. Complete initial velocity studies run in the presence of chloride at pH 8.1 indicated a sequential reaction pathway and suggested that MgATP added to the enzyme first followed by a rapid equilibrium random addition of Mg-citrate and CoA. The associated Michaelis constants and inhibition constants have been determined. Apparent Km values for the reverse reaction and an approximate equilibrium constant for the reaction have also been measured. Isotopic exchange experiments indicated the presence of a ping-pong sequence in which MgADP could be released before the addition of the other two reactants. However, MgATP remains as the first substrate to add to the enzyme, and the ping-pong pathway is apparently an alternate sequence to the sequential pathway. A phosphorylated enzyme was formed either from MgATP-γ-32P or from phosphate-32P, acetyl-CoA, and oxaloacetate; however, the two phosphoenzymes were not equivalent. The one formed with MgATP could react with MgADP to give MgATP or with CoA and Mg-citrate to give free phosphate. The apparent phosphoenzyme formed from phosphate, acetyl-CoA, and oxaloacetate was unreactive with either MgADP or Mg-citrate and CoA.