Variations in the activity of several enzymes in the mammary glands of non-pregnant, pregnant and lactating rabbits

1. The enzymes phosphofructokinase (EC 2.7.1.11), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), ATP-citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) were assayed in rabbit mammary glands at various stages of the pregnancy-lactation cycle. 2. The activities of all enzymes were low during pregnancy and, with the exception of phosphofructokinase, in non-pregnant animals. Two- to ten-fold increases in enzyme activities occurred over the first 20 days of lactation. Although milk yield was considerably decreased, the enzyme activities remained elevated in late lactation (45 days after parturition). 3. These findings are discussed in relation to mammary-gland metabolism and compared with similar observations previously made on ruminants and other small mammals.     

Opposing effects of two osmolytes – trehalose and glycerol – on thermal inactivation of rabbit muscle 6-phosphofructo-1-kinase

Trehalose and glycerol are known as good stabilizers of function and structure of several macromolecules against stress conditions. We previously reported that they have comparable effectiveness on protecting two yeast cytosolic enzymes against thermal inactivation. However, enzyme protection has always been associated to a decrease in catalytic activity at the stabilizing conditions i.e., the presence of the protective molecule. In the present study we tested trehalose and glycerol on thermal protection of the mammalian cytosolic enzyme phosphofructokinase. Here we found that trehalose was able to protect phosphofructokinase against thermal inactivation as well as to promote an activation of its catalytic activity. The enzyme incubated in the presence of 1 M trehalose did not present any significant inactivation within 2 h of incubation at 50 C, contrasting to control experiments where the enzyme was fully inactivated during the same period exhibiting a t_0.5 for thermal inactivation of 56± 5 min. On the other hand, enzyme incubated in the presence of 37.5% (v/v) glycerol was not protected against incubation at 50 C. Indeed, when phosphofructokinase was incubated for 45 min at 50 C in the presence of lower concentrations of glycerol (7.5–25%, v/v), the remaining activity was 2–4 times lower than control. These data show that the compatibility of effects previously shown for trehalose and glycerol with some yeast cytosolic enzymes can not be extended to all globular enzyme system. In the case of phosphofructokinase, we believe that its property of shifting between several different complex oligomers configurations can be influenced by the physicochemical properties of the stabilizing molecules. (Mol Cell Biochem 269: 203)     

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.     

Changes in the enzyme pattern of the mammary gland of the lactating rat after hypophysectomy and weaning

1. The enzymes glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, UDP-glucose pyrophosphorylase, phosphofructokinase, ATP-citrate lyase and acetyl-CoA carboxylase have been assayed in rat mammary glands in various stages of involution after hypophysectomy and weaning. 2. After hypophysectomy all seven enzymes decline in activity over a 12–16hr. period but the extent of the decline varies, with acetyl-CoA carboxylase becoming almost totally inactive, ATP-citrate lyase and phosphofructokinase showing a large decrease, and the remaining enzymes a less marked decline. 3. Within 24hr. of removing the litter a change in the pattern of enzyme activity is found very similar to that after hypophysectomy. 4. The significance of these results is discussed in relation to the endocrine control of mammary gland metabolism and the mechanisms of involution.     

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.     

Rapid Purification of Enzymes of Fatty Acid Biosynthesis from Rat Adipose Tissue

Abstract

Acetyl CoA carboxylase, ATP-citrate lyase and fatty acid synthetase were purified to homogeneity by a simple procedure. The purification method consists of polymerization of acetyl CoA carboxylase with citrate followed by avidin-Sepharose affinity chromatography. ATP-citrate lyase and fatty acid synthetase were isolated as by-products of acetyl CoA carboxylase purification and are separated from each other by chromatography on DE-52. ATP-citrate lyase was further purified by CoA-agarose affinity chromatography and fatty acid synthetase was purified on Bio-Gel A-1.5m. Purified ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase had specific activities of 9.9, 2.8 and 1.8 U/mg respectively with an over all recovery of 30, 25 and 50% respectively. Using these purified enzymes, we found that ATP-citrate lyase and acetyl CoA carboxylase were phosphorylated in vitro by both cAMP-dependent protein kinase and ATP-citrate lyase kinase whereas fatty acid synthetase was not phosphorylated by these protien kinases.     

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.     

Metabolic effects of tumour necrosis factor-α on rat brown adipose tissue

Intravenous administration of a single dose (100 g/kg bw) of recombinant tumour necrosis factor- (TNF, cachectin) to rats increased the rate ofin vitro fatty acid synthesis in interscapular brown adipose tissue (IBAT) from both glucose and alanine, without changes in the oxidation of these substrates to¹⁴CO₂. Lactate production and glycerol release were also unaffected by treatment with the cytokine. Additionally, the presence of TNF in the incubation media did not affect fatty acid synthesis, suggesting an indirect effect of the cytokine. The activities of different enzymes of glucose and alanine metabolism such as hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase and alanine transaminase, did not suffer changes as a consequence of TNF administration. The same applied to the enzymatic activities involved in fatty acid synthesis such as fatty acid synthase, acetyl-CoA carboxylase and ATP-citrate lyase. Conversely, citrate levels in IBAT were increased in animals treated with TNF, suggesting that it could be the cause for the increased fatty acid synthesis in this tissue.     

Opposing effects of two osmolytes--trehalose and glycerol--on thermal inactivation of rabbit muscle 6-phosphofructo-1-kinase

Trehalose and glycerol are known as good stabilizers of function and structure of several macromolecules against stress conditions. We previously reported that they have comparable effectiveness on protecting two yeast cytosolic enzymes against thermal inactivation. However, enzyme protection has always been associated to a decrease in catalytic activity at the stabilizing conditions i.e., the presence of the protective molecule. In the present study we tested trehalose and glycerol on thermal protection of the mammalian cytosolic enzyme phosphofructokinase. Here we found that trehalose was able to protect phosphofructokinase against thermal inactivation as well as to promote an activation of its catalytic activity. The enzyme incubated in the presence of 1 M trehalose did not present any significant inactivation within 2 h of incubation at 50 degrees C, contrasting to control experiments where the enzyme was fully inactivated during the same period exhibiting a t0.5 for thermal inactivation of 56+/-5 min. On the other hand, enzyme incubated in the presence of 37.5% (v/v) glycerol was not protected against incubation at 50 degrees C. Indeed, when phosphofructokinase was incubated for 45 min at 50 degrees C in the presence of lower concentrations of glycerol (7.5-25%, v/v), the remaining activity was 2-4 times lower than control. These data show that the compatibility of effects previously shown for trehalose and glycerol with some yeast cytosolic enzymes can not be extended to all globular enzyme system. In the case of phosphofructokinase, we believe that its property of shifting between several different complex oligomers configurations can be influenced by the physicochemical properties of the stabilizing molecules.     

Regional and Subcellular Distribution of ATP-Citrate Lyase and Other Enzymes of Acetyl-CoA Metabolism in Rat Brain

The activities of ATP-citrate lyase in frog, guinea pig, mouse, rat, and human brain vary from 18 to 30 μmol/h/g of tissue, being several times higher than choline acetyltransferase activity. Activities of pyruvate dehydrogenase and acetyl coenzyme A synthetase in rat brain are 206 and 18.4 μmol/h/g of tissue, respectively. Over 70% of the activities of both choline acetyltransferase and ATP-citrate lyase in secondary fractions are found in synaptosomes. Their preferential localization in synaptosomes and synaptoplasm is supported by RSA values above 2. Acetyl CoA synthetase activity is located mainly in whole brain mitochondria (RSA, 2.33) and its activity in synaptoplasm is low (RSA, 0.25). The activities of pyruvate dehydrogenase, citrate synthase, and carnitine acetyltransferase are present mainly in fractions C and B_p. No pyruvate dehydrogenase activity is found in synaptoplasm. Striatum, cerebral cortex, and cerebellum contain similar activities of pyruvate dehydrogenase, citrate synthase, carnitine acetyltransferase, fatty acid synthetase, and acetyl-CoA hydrolase. Activities of acetyl CoA synthetase, choline acetyltransferase and ATP-citrate lyase in cerebellum are about 10 and 4 times lower, respectively, than in other parts of the brain. These data indicate preferential localization of ATP-citrate lyase in cholinergic nerve endings, and indicate that this enzyme is not a rate limiting step in the synthesis of the acetyl moiety of ACh in brain.     

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.     

Purification of Leuconostoc mesenteroides Citrate Lyase and Cloning and Characterization of the citCDEFG Gene Cluster

A citrate lyase (EC 4.1.3.6) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the β subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the α subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris. This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD, citE, and citF, encoding, respectively, the three citrate lyase subunits γ (acyl carrier protein [ACP]), β (citryl-S-ACP lyase; EC 4.1.3.34), and α (citrate:acetyl-ACP transferase; EC 2.8.3.10). The gene (citC) encoding the citrate lyase ligase (EC 6.2.1.22) was localized in the region upstream of citD. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of Klebsiella pneumoniae and Haemophilus influenzae. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the citCDEFG gene cluster in Escherichia coli led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in E. coli is not complete or not linked to the protein.Lactic acid bacteria of the genus Leuconostoc play important roles in the dairy industry because of their ability to produce carbon dioxide and C₄ aroma compounds through lactose heterofermentation and citrate utilization. The carbon dioxide produced is responsible for eye formation in certain types of cheese. Citrate utilization by these bacteria leads to the production of diacetyl, which is considered a main flavor compound of a range of fermented dairy products such as cultured butter, buttermilk, and cottage cheese.The citrate utilization by lactic acid bacteria requires specifically three enzymes involved in the conversion of citrate to pyruvate: a citrate permease, a citrate lyase, and an oxaloacetate decarboxylase. The energetic role of citrate metabolism in Leuconostoc mesenteroides has been recently described (24, 25). The citrate permease catalyzes an electrogenic exchange of divalent anionic citrate and monovalent lactate, resulting in the generation of a membrane potential (Fig. ​(Fig.1,1, reaction 1) (24, 25). The intracellular citrate is cleaved by a citrate lyase (EC 4.1.3.6), yielding acetate and oxaloacetate (Fig. ​(Fig.1,1, reactions 2 and 3). The oxaloacetate is decarboxylated into carbon dioxide and pyruvate in a reaction catalyzed by the enzyme oxaloacetate decarboxylase (Fig. ​(Fig.1,1, reaction 4). Open in a separate windowFIG. 1Citrate fermentation pathway in L. mesenteroides and role of the different subunits in the reaction catalyzed by citrate lyase (EC 4.1.3.6). The proteins involved are citrate permease (1), citrate lyase α subunit citrate:acetyl-ACP transferase (EC 2.8.3.10) (2), citrate lyase β subunit citryl–S-ACP lyase (EC 4.1.3.34) (3) oxaloacetate decarboxylase (4), acetate:SH-CL ligase (EC 6.2.1.22) (5), and lactate dehydrogenase (6). ACP, γ subunit of ACP; R, prosthetic group. Acetic anhydride is used for chemical specific acetylation of the prosthetic group. Acetic anhydride is an analog of the mixed anhydride of citric and acetic acids which corresponds probably to an intermediate analog in the acyl-exchange reaction (7a, 14a).Understanding of the molecular genetics of these lactic acid bacteria is not far advanced, and the genes encoding the enzymes citrate lyase and oxaloacetate decarboxylase are unknown.On the basis of previous studies (22, 33), the citrate lyase of Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc can be considered a functional complex (M_r, 585,000) composed of three proteins: α, β, and γ subunits in a stoichiometric relationship of 6:6:6. The structure and the mechanism of action are similar to those of the citrate lyase of Klebsiella pneumoniae, which has been extensively studied (1, 15, 16, 34, 36). The citrate lyase is active only if the thioester residue of the prosthetic group linked to its acyl carrier protein (ACP) (γ subunit) is acetylated. This activation is catalyzed by an acetate:SH-citrate lyase ligase (CL ligase) (EC 6.2.1.22), which converts HS-ACP with ATP and acetate into the acetyl-S-ACP (Fig. ​(Fig.1,1, reaction 5) (32). The α subunit replaces the acyl group with a citryl group to form the citryl-S-ACP (Fig. ​(Fig.1,1, reaction 2) (16). At last, the β subunit cleaves citryl-S-ACP into oxaloacetate and regenerates the acyl-S-ACP (Fig. ​(Fig.1,1, reaction 3) (16).Different mechanisms of regulation of citrate lyase have been reported, such as configurational changes, reversible covalent modification by acetylation-deacetylation, and phosphorylation-dephosphorylation (1, 2). In microorganisms like Klebsiella, in which the reactions of the tricarboxylic acid cycle are operative and therefore contain citrate synthase, a strict regulation of citrate lyase activity is necessary to avoid a futile cycle between citrate fermentation and the l-glutamate biosynthetic pathway. After citrate depletion from the growth medium or upon transfer from an anaerobic citrate medium to an aerobic glucose medium, the synthesis of l-glutamate from oxaloacetate and acetyl coenzyme A (CoA) via citrate can be ensured only if the citrate fermentation pathway is turned off. The intracellular l-glutamate concentration controls these pathways by modulating the activity of the citrate lyase complex (1, 2).An induction of citrate lyase activity has been observed in Leuconostoc but never in all Lactococcus strains tested (21, 26). In L. mesenteroides, the citrate lyase activity is induced by citrate and rapidly repressed after the citrate consumption in the medium. However, the regulation mechanisms remain unknown. In this paper, we report the purification of L. mesenteroides citrate lyase and an approach based on reverse genetics that yielded the full-length sequence of CL ligase and citrate lyase genes encoding the α, β, and γ subunits. The citrate lyase and CL ligase genes were sequenced and expressed in Escherichia coli.     

ATP citrate lyase in cholinergic nerve endings

The activity of ATP-citrate lyase in homogenates of five selected rat brain regions varied from 2.93 to 6.90 nmol/min/mg of protein in the following order: cerebellum < hippocampus < parietal cortex < striatum < medulla oblongata and that of the choline acetyltransferase from 0.15 to 2.08 nmol/min/mg of protein in cerebellum < parietal cortex < hippocampus=medulla oblongata < striatum. No substantial differences were found in regional activities of lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase or acetyl-CoA synthase. High values of relative specific activities for both choline acetyltransferase and ATP-citrate lyase were found in synaptosomal and synaptoplasmic fractions from regions with a high content of cholinergic nerve endings. There are significant correlations between these two enzyme activities in general cytocol (S₃), synaptosomal (B) and synaptoplasmic (B_s) fractions from the different regions (r=0.92–0.99). These data indicate that activity of ATP-citrate lyase in cholinergic neurons is several times higher than that present in glial and noncholinergic neuronal cells.     

Studies on pituitary follitropin. III. Functional role of the amino groups in subunit and receptor interactions of the ovine hormone.

The effectiveness of Glc, mannose, 2-deoxyglucose, fructose, galactose, arabinose, and N-acetylglucosamine at protecting rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) from inactivation by chymotrypsin, glutaraldehyde, heat, and Ellman's reagent have been compared. The relative effectiveness at protecting against these inactivating agents decreases in the order Glc > mannose > 2-deoxyglucose > fructose, galactose, and arabinose, the last three providing no significant protection at all. The nonphosphorylatable substrate analog, N-acetylglucosamine, provides substantial protection against heat inactivation, but is ineffective against inactivation by the other agents. Similar inactivation studies were conducted using several hexose 6-phosphates. Glc-6-P and 1,5-anhydroglucitol-6-P provided substantial protection while 2-deoxyglucose-6-P, fructose-6-P, mannose-6-P, and galactose-6-P were all relatively ineffective at protecting hexokinase activity. The protective effect of these ligands is taken as an indication of ligand-induced conformational changes in brain hexokinase. The results are interpreted in terms of, and considered to support, a recently proposed model (J. E. Wilson, 1978, Arch. Biochem. Biophys.185, 88–99) in which the suitability of a carbohydrate as a substrate depends directly on its ability to induce specific conformational changes prerequisite for subsequent catalytic events while the inhibitory effectiveness of a hexose 6-phosphate is likewise related to its ability to evoke appropriate conformational change in the enzyme. Synergistic interactions between hexose and hexose-6-P binding sites, first reported for Glc and Glc-6-P by Ellison et al. (1975, J. Biol. Chem.250, 1864–1871), have been confirmed. Thus, although fructose and galactose were themselves quite ineffective at providing protection against inactivation of hexokinase by chymotrypsin or glutaraldehyde, they greatly increased the protection afforded by suboptimal (with respect to degree of protection in the absence of added hexose) levels of Glc-6-P. Conversely, the 6-phosphates of fructose, galactose, mannose, and 2-deoxyglucose, which were themselves ineffective at protecting the enzyme activity, markedly enhanced the protection provided by suboptimal levels of Glc or mannose. Based on the relationship between this enhancement of Glc-dependent protection and the hexose-6-P concentration, the dissociation constants for the complexes of hexokinase with 2-deoxyglucose-6-P and mannose-6-P were estimated to be ?0.5 mm.     

Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation in Desulfobacter postgatei growing on acetate and sulfate

Desulfobacter postgatei is an acetate-oxidizing, sulfate-reducing bacterium that metabolizes acetate via the citric acid cycle. The organism has been reported to contain a si-citrate synthase (EC 4.1.3.7) which is activated by AMP and inorganic phosphate. It is show now, that the enzyme mediating citrate formation is an ATP-citrate lyase (EC 4.1.3.8) rather than a citrate synthase. Cell extracts (160,000xg supernatant) catalyzed the conversion of oxaloacetate (apparent K _m=0.2 mM), acetyl-CoA (app. K _m=0.1 mM), ADP (app. K _m=0.06 mM) and phosphate (app. K _m=0.7 mM) to citrate, CoA and ATP with a specific activity of 0.3 mol·min^-1·mg^-1 protein. Per mol citrate formed 1 mol of ATP was generated. Cleavage of citrate (app. K _m=0.05 mM; V _max=1.2 mol · min^-1 · mg^-1 protein) was dependent on ATP (app. K _m=0.4 mM) and CoA (app. K _m=0.05 mM) and yielded oxaloacetate, acetyl-CoA, ADP, and phosphate as products in a stoichiometry of citrate:CoA:oxaloacetate:ADP=1:1:1:1. The use of an ATP-citrate lyase in the citric acid cycle enables D. postgatei to couple the oxidation of acetate to 2 CO₂ with the net synthesis of ATP via substrate level phosphorylation.     

The fluctuation of various enzyme activities due to myo-inositol deficiency in Saccharomyces carlsbergensis

The neutral lipid accumulation in myo-inositol deficient Saccharomyces carlsbergensis results at least partly from an enhancement of acetyl CoA carboxylase activity due to the high level of fructose 1,6-bisphosphate which activates acetyl CoA carboxylase, and due to the low level of citrate which counteracts the activation [4].In an attempt to explore the effect of myo-inositol deficiency on the metabolic fluxes, various enzyme activities were compared between the myo-inositol supplemented and deficient cells. The activities of phosphofructokinase and ATP-citrate lyase increased by 74 and 83%, respectively, in the deficient cell, whereas those of aldolase and citrate synthase decreased by 65 and 27%, respectively. The activity of glucose-6-phosphate dehydrogenase was unchanged. Unlike acetyl CoA carboxylase, elimination of low molecular effectors had no influence on their activities.The thermostability of phosphofructokinase (at 53°C) increased, while that of aldolase (at 48°C) greatly decreased due to the deficiency. The thermostability of glucose-6-phosphate dehydrogenase (at 52°C) was also unchanged.     

Rapid purification of enzymes of fatty acid biosynthesis from rat adipose tissue

Acetyl CoA carboxylase, ATP-citrate lyase and fatty acid synthetase were purified to homogeneity by a simple procedure. The purification method consists of polymerization of acetyl CoA carboxylase with citrate followed by avidin-Sepharose affinity chromatography. ATP-citrate lyase and fatty acid synthetase were isolated as by-products of acetyl CoA carboxylase purification and are separated from each other by chromatography on DE-52. ATP-citrate lyase was further purified by CoA-agarose affinity chromatography and fatty acid synthetase was purified on Bio-Gel A-1.5m. Purified ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase had specific activities of 9.9, 2.8 and 1.8 U/mg respectively with an over all recovery of 30, 25 and 50% respectively. Using these purified enzymes, we found that ATP-citrate lyase and acetyl CoA carboxylase were phosphorylated in vitro by both cAMP-dependent protein kinase and ATP-citrate lyase kinase whereas fatty acid synthetase was not phosphorylated by these protein kinases.     

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.     

Metabolic fate of initiation factors after inhibition of protein synthesis in Escherichia coli

Summary The metabolic fate of translation initiation factor after inhibition of protein synthesis by different means has been investigated. We have found a decay in initiation factor activity when protein synthesis is blocked by chloramphenicol but not during arginine starvation of PA1 (Rel^–) or PA2 (Rel⁺) strains or during puromycin incubation. These results suggest that inactivation of certain initiation factors occurs when the regeneration of ribosomal subunits from polysomes is inhibited in the cells.Complementation experiments indicate that IF₃ factor activity is preferentially affected during chloramphenicol treatment.Same preferential inhibition of IF₃ activity seems to occur during in vitro incubation of crude IF. 70S ribosomes or 30S subunits protect this factor against the inactivation. Preliminary results seem tosuggest that ATP is implicated in this in vitro inactivation process.     

Properties of the purified APS-kinase from Escherichia coli and Saccharomyces cerevisiae

Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3-phosphotransferase) has been isolated from Escherichia coli and from Saccharomyces cerevisiae. As major steps of purification, affinity chromatography on Sepharose CL 6B (blue or red) and chromatofocusing on polybuffer PBE 94^tm were employed. The proteins were obtained in nearly homogeneous state after five chromatographic steps.The isolated enzymes from both sources appeared predominantly to exist as dimers. Upon reduction of the protein with dithiothreitol, it desintegrated into assumingly identical smaller subunits (E. coli rom M_r 90-85000 to 45-40000 and s. cerevisiae from 52-49500 to 28-29500). Both forms, dimer and monomer were found catalytically active.Preincubation of the isolated enzyme from either source in the presence of thioredoxin plus DTT, reduced glutathione or DTT increased the activity significantly. Treatment of the enzyme with SH-blocking reagents inactivated the enzyme irreversibly as compared to the inactivation caused by oxidants (2,6-dichlorophenol-indophenol, ferricyanide or oxydized glutathione). This oxidant induced inactivation was less pronounced for the fungal enzyme than for the bacterial protein. The enzyme from E. coli required thioredoxin in order to alleviate the GSSG-induced inactivation.Abbreviations APS adenylylsulphate - APS kinase - ATP adenylylsulphate 3-phosphotransferase - DCPIP 2,6-dichlorophenol indophenol - DTT dithiothreitol - GSH reduced glutathione - GSSG oxidized glutathione - HPLC high performance liquid chromatography - -MSH -mercaptoethanol - PAPS 3-phosphoadenylylsulphate - TNBS 2,4,6 tri-nitrobenzenesulphonic acid