scholarly journals Both Subunits of ATP-Citrate Lyase from Chlorobium tepidum Contribute to Catalytic Activity

2006 ◽  
Vol 188 (18) ◽  
pp. 6544-6552 ◽  
Author(s):  
Wonduck Kim ◽  
F. Robert Tabita
Keyword(s):  
Catalytic Mechanism ◽  
Specific Activity ◽  
High Specific Activity ◽  
Small Subunit ◽  
High Yield ◽  
Chlorobium Tepidum ◽  
Atp Citrate Lyase ◽  
Citrate Lyase ◽  
Green Sulfur

ABSTRACT ATP-citrate lyase (ACL) is an essential enzyme of the reductive tricarboxylic acid (RTCA) pathway of CO2 assimilation. The RTCA pathway occurs in several groups of autotrophic prokaryotes, including the green sulfur bacteria. ACL catalyzes the coenzyme A (CoA)-dependent and MgATP-dependent cleavage of citrate into oxaloacetate and acetyl-CoA, representing a key step in the RTCA pathway. To characterize this enzyme from the green sulfur bacterium Chlorobium tepidum and determine the role of its two distinct polypeptide chains, recombinant holo-ACL as well as its two individual subunit polypeptides were synthesized in Escherichia coli. The recombinant holoenzyme, prepared from coexpressed large and small ACL genes, and the individual large and small subunit polypeptides, prepared from singly expressed genes, were all purified to homogeneity to high yield. Purified recombinant holo-ACL was isolated at high specific activity, and its k cat was comparable to that of previously prepared native C. tepidum ACL. Moreover, the purified recombinant large and small subunit polypeptides were able to reconstitute the holo-ACL in vitro, with activity levels approaching that of recombinant holo-ACL prepared from coexpressed genes. Stoichiometric amounts of each subunit protein were required to maximize the activity and form the most stable structure of reconstituted holo-ACL. These results suggested that this reconstitution system could be used to discern the catalytic role of specific amino acid residues on each subunit. Reconstitution and mutagenesis studies together indicated that residues of each subunit contributed to different aspects of the catalytic mechanism, suggesting that both subunit proteins contribute to the active site of C. tepidum ACL.

The role of ATP citrate lyase in the transfer of acetyl groups in rat liver

1968 ◽  
Vol 158 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Yasushi Daikuhara ◽  
Takuo Tsunemi ◽  
Yoshiro Takeda
Keyword(s):  
Rat Liver ◽  
Atp Citrate Lyase ◽  
Citrate Lyase

Synthesis and N.M.R. Spectra of Substituted Aminoiodoacridines

1979 ◽  
Vol 32 (12) ◽  
pp. 2637 ◽  
Author(s):  
RF Martin ◽  
DP Kelly
Keyword(s):  
Electron Density ◽  
Electrophilic Substitution ◽  
Specific Activity ◽  
Diazonium Salt ◽  
High Specific Activity ◽  
High Yield ◽  
Model Compounds ◽  
Iodide Ion ◽  
Ion Substitution ◽  
Chloramine T

3-Amino-6-iodoacridine (10), 3,6-diiodoacridine (11) and 9-amino-2-ethoxy-6-iodoacridine (14) were prepared by iodide ion substitution of the corresponding diazonium salt whereas 3,6-diamino-4,5-diiodoacridine (12) and 6,9-diamino-2-ethoxy-5-iodoacridine (13) were prepared by direct iodination with iodide ion in the presence of chloramine-T. The latter reaction proceeded in relatively high yield and has been used for the synthesis of high specific activity 125I-labelled compounds (12), (13). The 1H and 13C N.M.R. spectra of (10)-(14) and model compounds indicate higher electron density at C4(C5) than at C2(C7) in 3(6)-amino-substituted acridines in agreement with the observed pattern of electrophilic substitution.

Preparation of 14C-Labeled Penicillic Acid with High Specific Activity and Yield

1977 ◽  
Vol 40 (2) ◽  
pp. 90-93 ◽  
Author(s):  
DOUGLAS L. PARK ◽  
PHILIP B. MISLIVEC ◽  
JAMES L. HEATH
Keyword(s):  
Acid Production ◽  
Specific Activity ◽  
High Specific Activity ◽  
High Yield ◽  
Liquid Media ◽  
Penicillic Acid ◽  
Stationary Culture ◽  
Radioactive Compound ◽  
Growth Substrates ◽  
Label Incorporation

14C-Labeled penicillic acid was produced by stationary culture incubation of Penicillium cyclopium (NRRL 1888) on a modified Raulin-Thom broth medium containing 14C-labeled acetate. Approximately 1.2 g of radioactive compound, with a specific activity of 23.0 μCi/mmole, was produced in 9 days in 1500 ml of the broth. Incorporation of the isotope into penicillic acid was 11. 9%. Production of the radiolabeled compound with high specific activity was achieved by correlating the monitoring of expired 14C-CO2 with production of penicillic acid during the fermentation. The effects of various growth substrates, pH, and incubation times on production of non-labeled penicillic acid also were investigated. Results show that sterile rice is an excellent substrate, that among liquid media examined, higher yields were obtained in stationary rather than in shake cultures, and that higher yields of penicillic acid were obtained at pH 3.5 or lower. Simultaneous monitoring of penicillic acid production and 14C-label incorporation is essential to detect and isolate a high yield of labeled compound with high specific activity.

Role of vacuoles and vesicles in extracellular enzyme secretion from yeast

1973 ◽  
Vol 19 (1) ◽  
pp. 113-117 ◽  
Author(s):  
R. A. Holley ◽  
D. K. Kidby
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Extracellular Enzyme ◽  
Invertase Activity ◽  
Preliminary Evidence ◽  
Acid Protease ◽  
Intracellular Enzyme ◽  
Per Se ◽  
Equilibrium Centrifugation

Preliminary evidence has been obtained which suggests that the intracellular invertase of Saccharomyces cerevisiae may not be localized in the vacuole per se. Alkaline phosphatase, an intracellular enzyme, and acid protease, a typically lysosomal enzyme, both showed high specific activity in the vacuole fraction prepared by equilibrium centrifugation of lysed sphaeroplasts in Ficoll gradients. Invertase activity has been found to be associated with vacuoles only when glucose-repressed cells are derepressed. Cells derepressed for invertase biosynthesis contained a population of vesicles which were virtually absent from the repressed cells. Evidence is presented which strongly suggests that these vesicles rather than the vacuoles are the vehicle by which invertase is secreted from the cell.

scholarly journals A biochemical explanation for the fatty liver and kidney syndrome of broilers: its alleviation by the short-term use of dietary fat

1977 ◽  
Vol 38 (3) ◽  
pp. 319-328 ◽  
Author(s):  
D. Balnave ◽  
R. B. Cumming ◽  
T. M. Sutherland
Keyword(s):  
Fatty Liver ◽  
Broiler Chickens ◽  
Specific Activity ◽  
Liver Lipid ◽  
Liver Weight ◽  
Atp Citrate Lyase ◽  
Commercial Diet ◽  
Meat Meal ◽  
Citrate Lyase ◽  
Liver And Kidney

1. Fatty liver and kidney syndrome (FLKS) was induced in young broiler chickens by giving them a diet composed principally of wheat and meat meal.2. FLKS resulted in reduced growth and increased liver weight; fasting for 18 h increased mortality, liver lipid and the specific activity of hepatic ATP-citrate lyase compared with birds fed on a commercial diet. The specific activities of hepatic fructose-l,6-diphosphate-l-phosphohydrolase and pyruvate carboxylase were reduced in birds suffering from FLKS and fasted for 18 h.3. Feeding of the FLKS-inducing diet supplemented with 150 g animal tallow/kg for 54 h considerably reduced mortality while restoring liver composition and enzyme activities towards those observed in birds fed a commercial diet. Investigations indicated that the glycerol component of the fat was not responsible for the observed responses.4. The present results suggest that in FLKS insufficiencies of biotin are induced in specific enzyme systems, but the syndrome may be alleviated without the use of supplementary biotin.5. The evidence indicates that, when stressed, birds affected by FLKS die from the hypoglycaemia occurring as a result of a reduced capacity for gluconeogenesis.

INDUCTION BY GONADAL HORMONES OF HEPATIC LIPOGENIC ENZYME ACTIVITY IN IMMATURE PULLETS

1974 ◽  
Vol 61 (1) ◽  
pp. 29-43 ◽  
Author(s):  
D. BALNAVE ◽  
J. PEARCE
Keyword(s):  
Enzyme Activity ◽  
Corn Oil ◽  
Specific Activity ◽  
Liver Lipid ◽  
Lipogenic Enzyme ◽  
Atp Citrate Lyase ◽  
Liver Lipid Content ◽  
Hormone Administration ◽  
Citrate Lyase ◽  
Specific Activities

SUMMARY The effect of gonadal hormone administration on hepatic lipogenic enzyme activity, and some physiological parameters was investigated in immature pullets. Pullets (aged 4 weeks) were allocated to treatment groups and received intramuscular injections of oestradiol, testosterone, progesterone or oestradiol + testosterone (all in 0·3 ml corn oil) or corn oil alone (control group). There was no evidence of any hormone-induced changes in the specific activity of hepatic ATP-citrate lyase 1, 3, 6 and 12 h after hormone administration. NADP-malate dehydrogenase exhibited significant variations in specific activity over this period of time and it is probable that these changes reflected an increased requirement for NADPH for synthetic purposes in hormone-treated birds. The effect of 1, 2, 4 and 9 days of hormone administration was also investigated. In testosterone-treated birds there were significant increases in the specific activities of both lipogenic enzymes after 1 day of hormone treatment whereas for birds receiving oestradiol the maximum specific activities were found on the second day. Similarly, the liver lipid content of oestradioltreated birds showed a substantial increase on day 2. After 9 days of hormone administration no significant differences in the specific activity of ATP-citrate lyase were observed between treatments but the specific activity of NADP-malate dehydrogenase was significantly reduced in oestradiol- or mixed hormone-treated birds; it is possible that the reduced enzyme activity is associated with a reduced requirement for NADPH and in this connexion there were no further increases in liver lipid content or liver weight after 4 days of hormone administration. The liver RNA:DNA ratio tended to be greatest in birds receiving oestradiol or oestradiol + testosterone. Studies utilizing inhibitors of RNA and protein synthesis showed that such compounds abolished the increases in lipogenic enzyme activity following hormone administration suggesting that these increases were hormone-induced effects. These results are discussed in relation to the effects of the various hormones on liver lipid metabolism and also in relation to the situation in the mature laying hen.

Analysis of the Yarrowia lipolytica proteome reveals subtle variations in expression levels between lipogenic and non-lipogenic conditions

2021 ◽  
Vol 21 (2) ◽  
Author(s):  
Ryan Sestric ◽  
Vic Spicer ◽  
Oleg V Krokhin ◽  
Richard Sparling ◽  
David B Levin
Keyword(s):  
Yarrowia Lipolytica ◽  
Carbon Flux ◽  
Neutral Lipids ◽  
Pyruvate Decarboxylase ◽  
Atp Citrate Lyase ◽  
Expression Levels ◽  
Rich Media ◽  
Citrate Lyase ◽  
The Relationship

ABSTRACT Oleaginous yeasts have the ability to store greater than 20% of their mass as neutral lipids, in the form of triacylglycerides. The ATP citrate lyase is thought to play a key role in triacylglyceride synthesis, but the relationship between expression levels of this and other related enzymes is not well understood in the role of total lipid accumulation conferring the oleaginous phenotype. We conducted comparative proteomic analyses with the oleaginous yeast, Yarrowia lipolytica, grown in either nitrogen-sufficient rich media or nitrogen-limited minimal media. Total proteins extracted from cells collected during logarithmic and late stationary growth phases were analyzed by 1D liquid chromatography, followed by mass spectroscopy. The ATP citrate lyase enzyme was expressed at similar concentrations in both conditions, in both logarithmic and stationary phase, but many upstream and downstream enzymes showed drastically different expression levels. In non-lipogenic conditions, several pyruvate enzymes were expressed at higher concentration. These enzymes, especially the pyruvate decarboxylase and pyruvate dehydrogenase, may be regulating carbon flux away from central metabolism and reducing the amount of citrate being produced in the mitochondria. While crucial for the oleaginous phenotype, the constitutively expressed ATP citrate lyase appears to cleave citrate in response to carbon flux upstream from other enzymes creating the oleaginous phenotype.

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