Fat metabolism in higher plants. Further characterization of wheat germ acetyl coenzyme A carboxylase.

Wheat germ acetyl CoA carboxylase was purified 600-fold over the crude homogenate. The purified enzyme gave rise to complex electrophoretic patterns in dissociating gels. As isolated, the activity of wheat germ acetyl CoA carboxylase exhibited profound dependence on the composition of the reaction mixture. In addition to the substrates MgATP, HCO₃, and acetyl CoA, the enzyme required both free Mg²⁺ and K⁺ for optimal activity. The effects of the two ions were additive. At pH 8.5, Mg²⁺ activated the carboxylase by adding to the enzyme prior to the other reactants in an equilibrium ordered reaction mechanism.     

Autotrophic CO2 fixation in Acetobacterium woodii

Earlier labeling experiments have shown that autotrophically grown Acetobacterium woodii assimilates cell carbon via direct acetyl CoA formation from 2 CO₂, rather than via the Calvin cycle. Cell extracts contained the enzymes required for biosynthesis starting from acetyl CoA and CO₂. Notably, pyruvate synthase, pyruvate phosphate dikinase, and phosphoenolpyruvate carboxytransphosphorylase were present in sufficiently high activities. Ribulose-1,5-bisphosphate carboxylase activity could not be detected. The observed enzyme pattern was consistent with the postulated biosynthetic pathway as deduced from ¹⁴C-labeling experiments.     

Differential effects of metabolites on the active and inactive forms of hepatic acetyl CoA carboxylase

Partially purified acetyl CoA carboxylase was converted in vitro to its predominately phosphorylated (less active, b) or dephosphorylated (active, a) form. Studies of the properties of the two forms of carboxylase indicated that the a-form had a greater V than the b-form in the presence of different concentrations of citrate, pyruvate, MgATP^2?, MnATP^2?, acetyl CoA, and palmityl CoA. The concentration required for half-maximum stimulation of the a-form was less for citrate and the same as the b-form for MgATP^2?, MnATP^2?, and acetyl CoA. The concentration required for half-maximum inhibition of the a-form was higher for palmityl CoA, avidin, and ATP. The b-form was more strongly inhibited by palmityl CoA and avidin and this inhibition was partially reversed by citrate. These studies indicate that under normal physiological concentrations of metabolites, the b-form is virtually inactive. The physiological significance of the interconversion between the two forms of acetyl CoA carboxylase thus appears to lie in their differential response to the various metabolites which regulate the enzyme activity.     

Acetyl-CoA-carboxylase activity in normally developing wheat leaves

In order to investigate the role of acetyl CoA carboxylase (ACC) in the regulation of fatty-acid biosynthesis in chloroplasts, the activities and relative amounts of the enzyme have been measured in the tissue of wheat (Triticum aestivum L.) leaves undergoing development and cellular differentiation. The total activity in the first leaves of 5- to 7-d-old plants was similar but decreased to less than half in 9-d-old plants. The activity of ACC in the cells of the first leaf of 7-d-old plants doubled when cell age increased from 24 to 48 h, remained relatively constant for a further 24 h and then declined. The amount of ACC in cells increased 15-fold during the first 36 h of cell enlargement. Cells more than 36 h old contained about two-thirds the maximum amount of ACC found in younger cells. The most rapid phase of fatty-acyl accumulation in lipids was in cells aged between 60 and 84 h. Tenfold changes in the activity of ACC were observed when the assay conditions with respect to ATP, ADP, Mg²⁺ and pH were changed to correspond to the physiological conditions in chloroplasts during light/dark transitions. This observation and the magnitude of the changes in the optimum activity and amount of ACC in leaf cells undergoing development are consistent with a role for ACC in the regulation of the flow of carbon from acetyl CoA to fatty acids in chloroplasts.Abbreviation ACC acetyl CoA carboxylase     

Fatty acid synthesis in chick embryonic heart and liver during development.

Fatty acid synthesis by subcellular fractions of heart and liver of chick embryos at varying stages of development has been studied. Fatty acid synthetase activity is associated with the embryonic heart at early stages of development, as suggested by substrate requirement, Schmidt decarboxylation of synthesized fatty acids and gas liquid chromatographic identification of the products as palmitic and stearic acids. The fatty acid synthetase activity decreases in heart cytosol with age of the embryo and is absent in the newly hatched chick and in older chicken. The acetyl CoA carboxylase activity is negligible in embryonic and adult chicken heart. The fatty acid synthetase activity in liver is low, but measurable during the entire embryonic development. The activity increases by about three-fold on hatching and thereafter in fed, newly hatched chicks by about 35-fold, over the basal embryonic activity. The acetyl and malonyl transacylase activities in the heart and liver cytosols during development followed closely the fatty acid synthetase activities in heart and liver, respectively. A non-coordinate induction of fatty acid synthetase and acetyl CoA carboxylase activities in liver was observed during development. The microsomal chain elongation in liver and heart followed the pattern of fatty acid synthetase activity in liver and heart, respectively. The mitochondrial chain elongation in embryonic heart is initially low and increases with age; while this activity in liver is higher in early stages of embryonic development than in the older embryos and the chicks. Measurement of lipogenesis from acetate-1-¹⁴C by liver and heart slices from chick embryos and newly hatched chicks support the conclusions reached in the studies with the subcellular fractions. The results obtained indicate that the major system of fatty acid synthesis in embryonic and adult heart is the mitochondrial chain elongation. In embryonic liver, fatty acid synthesis proceeds by chain elongation, while the de novo system is the major contributor to the lipogenic capacity of the liver after hatching.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

Hydrogen-stimulated CO2 fixation and coordinate induction of hydrogenase and ribulosebiphosphate carboxylase in a H2-uptake positive strain of Rhizobium japonicum

H₂-uptake positive strains (122 DES and SR) and H₂-uptake negative strains SR2 and SR3 of Rhizobium japonicum were examined for ribulosebisphosphate (RuBP) carboxylase and H₂-uptake activities during growth conditions which induced formation of the hydrogenase system. The rate of ¹⁴CO₂ uptake by hydrogenase-derepressed cells was about 6-times greater in the presence than in the absence of H₂. RuBP carboxylase activity was observed in free-living R. japonicum strains 122 DES or SR only when the cells were derepressed for their hydrogenase system. Hydrogenase and RuBP carboxylase activities were coordinately induced by H₂ and both were repressed by added succinate. Hydrogenase-negative mutant strains SR2 and SR3 derived from R. japonicum SR showed no detecyable RuBP carboxylase activities under hydrogenase derepression conditions. No detectable RuBP carboxylase was observed in bacteroids formed by H₂-uptake positive strains R. japonicum 122 DES or SR. Propionyl CoA carboxylase activity was consistently observed in extracts of cells from free-living cultures of R. japonicum but activity was not appreciably influenced by the addition of H₂. Neither phosphoenolpyruvate carboxylase nor phosphoenolpyruvate carboxykinase activity was detected in extracts of R. japonicum.Abbreviations RuBP Ribulose 1,5-bisphosphate - (Na₂EDTA) (Ethylenedinitrilo)-tetraacetic acid, disodium salt - (propionyl CoA) Propionyl coenzyme A - (PEP) Phosphoenolpyruvate - (GSH) Reduced glutathione - (Tricine) N-tris(hydroxymethyl)-methylglycine     

Effect of iron on activity of soybean multi-subunit acetyl-coenzyme A carboxylase

Multi‐subunit acetyl‐coenzyme A carboxylase (MS‐ACCase; EC 6.4.1.2) isolated from soybean chloroplasts is a labile enzyme that loses activity during purification. We found that incubating the chloroplast stromal fraction under anaerobic conditions or in the presence of 5 mM FeSO₄ stimulated ACCase (acetyl‐CoA→malonyl‐CoA) and carboxyltransferase (malonyl‐CoA→acetyl‐CoA) activity. Fe‐stimulation of activity was associated with ⁵⁹Fe binding to a stromal protein fraction. ACCase and carboxyltransferase activities measured in the stromal protein fraction containing bound ⁵⁹Fe were 2‐fold and 6‐fold greater, respectively, than the control (stromal fraction not pretreated with FeSO₄). Superose 6 gel filtration chromatography indicated ⁵⁹Fe comigrated with stromal protein of approximately 180 kDa that exhibited carboxyltransferase activity, but lacked ACCase activity. Anion exchange (Mono‐Q) chromatography of the Superose 6 fraction yielded a protein peak that was enriched in carboxyltransferase activity and contained protein‐bound ⁵⁹Fe. Denaturing gels of the Mono‐Q fraction indicated that the 180‐kDa protein was composed of a 56‐kDa subunit that was bound by an antibody raised against a synthetic β‐carboxyltransferase (β‐CTase) peptide. Incubation of the Mono‐Q carboxyltransferase fraction with increasing concentrations of iron at a fixed substrate concentration resulted in increased initial velocities that fit well to a single rectangular three parameter hyperbola (v=v_o+V_max[FeSO₄]/K_m+[FeSO₄]) consistent with iron functioning as a bound activator of catalysis. UV/Vis spectroscopy of the partially purified fraction before and after iron incubation yielded spectra consistent with a protein‐bound metal cluster. These results suggest that the β‐CTase subunit of MS‐ACCase in soybean chloroplasts is an iron‐containing enzyme, which may in part explain its labile nature.     

Inhibition of pyruvate carboxylase by sequestration of coenzyme A with sodium benzoate

Pyruvate-dependent CO2 fixation by isolated mitochondria was strongly inhibited by sodium benzoate. Pyruvate carboxylase was identified as a site of inhibition by limiting flux measurements to assays of pyruvate carboxylase coupled with malate dehydrogenase. Benzoate reduced pyruvate-dependent incorporation of [14C]KHCO3 into malate and pyruvate-dependent malate accumulation by 74 and 72%, respectively. Aspartate-dependent malate accumulation was insensitive to benzoate, ruling out malate dehydrogenase as a site of action. Inhibition by benzoate was antagonized by glycine, which sharply accelerated conversion of benzoate to hippurate. Assays of coenzyme A and its acyl derivatives revealed inhibition to correlate with depletion of acetyl CoA and accumulation of benzoyl CoA. Depletion of acetyl CoA was sufficient to account for greater than 50% reduction in pyruvate carboxylase activity. Competition between acetyl CoA and benzoyl CoA for the activator site on pyruvate carboxylase was insignificant. Results support the interpretation that the observed inhibition of pyruvate carboxylase occurred primarily by depletion of the activator, acetyl CoA, through sequestration of coenzyme A during benzoate metabolism.     

Regulation of purified rat liver acetyl CoA carboxylase by phosphorylation

Acetyl CoA carboxylase was purified from liver of fasted-refed rats to near homogeneity, based on electrophoretic analysis and biotin content. These preparations contained an endogenous protein kinase that catalyzed the transfer of radioactive phosphate from [gamma-32P]ATP to acetyl CoA carboxylase, accompanied by a decrease in acetyl CoA carboxylase activity. Phosphate incorporated into acetyl CoA carboxylase was removed when the preparation was incubated with partially purified phosphorylase phosphatase catalytic subunit with regain of enzymatic activity. This endogenous protein kinase was shown not to be affected by either cyclic-AMP-dependent protein kinase inhibitor, EGTA, or trifluoperazine. The addition of either cyclic-AMP or purified cyclic-AMP-dependent protein kinase catalytic subunit to the purified acetyl CoA carboxylase preparation increased protein phosphorylation but had no further effect on acetyl CoA carboxylase activity. Purified acetyl CoA carboxylase was shown to act as an ATPase during the phosphorylation reaction.     

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.     

Graminicide resistance in a blackgrass (Alopecurus myosuroides) population correlates with insensitivity of acetyl-CoA carboxylase

The appearance of biotypes of the annual grass weed black‐grass (Alopecurus myosuroides L. Huds), which are resistant to certain graminicides, is the most significant example of acquired resistance to herbicides seen so far in European agriculture. An investigation was perfomed into the basis of the specific cross‐resistance to cyclohexanedione (CHD) and aryloxyphenoxypropionoic acid (AOPP) herbicides in the ‘Notts A1’ population of A. myosuroides, which survived treatment of fields with recommended rates of AOPP herbicides. In comparison with the wild‐type ‘Rothamsted’ population, the resistant biotype showed over 100‐fold resistance to these herbicides in a hydroponic growth system. Biosynthesis of fatty acids and activity of crude extracts of acetyl‐CoA carboxylase (ACCase) were commensurately less sensitive to these herbicides in Notts A1 compared with the Rothamsted biotype. These data are consistent with the hypothesis that the highly resistant population has arisen through selection of a mutant ACCase which is much less sensitive to the AOPP and CHD graminicides. Rapidly growing cell suspension cultures established from the Notts A1 population also showed high resistance indices for CHD or AOPP herbicides compared with cultures from the Rothamsted biotype. Fatty acid biosynthesis and ACCase activity in the cell suspensions were similarly sensitive towards the graminicides to those in the foliar tissue counterparts of the resistant and sensitive populations. Moreover, purification of the main (chloroplast) isoform of acetyl‐CoA carboxylase showed that this enzyme from the Notts A1 population was over 200‐fold less sensitive towards the AOPP herbicide, quizalofop, than the equivalent isoform from the Rothamsted population. These data again fully supported the proposal that resistance in the Notts biotype is due to an insensitive acetyl‐CoA carboxylase isoform. Overall, cell suspensions were also demonstrated to be excellent tools for further investigation of the molecular basis of the high level herbicide resistance which is prone to occur in A. myosuroides.     

Lipid Synthesis and Abundance of Acetyl CoA Carboxylase in Isochrysis galbana (Prymnesiophyceae) Following Nitrogen Starvation

Fatty acid content and the rate of lipid synthesis were measuredin the marine prymnesiophyte Isochrysis galbana grown undernitrogen starvation and in cultures recovering from nitrogendeprivation. Nitrogen starvation imposed a reduction in cellularsoluble protein content, variation in fatty acid compositionand reduction in the in vitro activity of the enzyme acetylCoA carboxylase. An increase in total fatty acid content isattributed to a differential reduction in cell division andthe rate of lipid synthesis. Recovery from nitrogen deprivationwas characterized by an increase in cellular soluble proteincontent and in the rate of lipid synthesis. Although the invitro activity of acetyl CoA carboxylase increased as the culturesrecovered from nitrogen starvation, the total cellular fattyacid content decreased, evidently due to an acceleration incell division. The relative cellular pool size of acetyl CoAcarboxylase determined by immunoblotting decreased under nitrogenstarvation conditions and increased as cells recovered fromit. Cellular accumulation of acetyl CoA carboxylase during recoveryfrom nitrogen starvation is ascribed to de novo synthesis ofthe enzyme that takes place in the cytoplasm. However, photosyntheticproteins such as ribulose bisphosphate carboxylase are synthesizedearlier than acetyl CoA carboxylase in the recovery process. (Received June 12, 1992; Accepted September 21, 1992)     

Short-term regulation of acetyl CoA carboxylase: is the key enzyme in long-chain fatty acid synthesis regulated by an existing physiological mechanism?

Acetyl CoA carboxylase, the rate-limiting enzyme in regulating fatty acid synthesis, is thought to be controlled by allosteric effectors, its state of aggregation, covalent modulation and protein inhibitors. It is still obscure whether citrate, a positive allosteric effector, and long-chain fatty acyl CoA esters, negative allosteric effectors, function physiologically to regulate acetyl CoA carboxylase activity. New evidence from several laboratories reveals that the covalent phosphorylation may not involve regulation of acetyl CoA carboxylase activity. Protein inhibitors from liver cytosol and a peptide from fat cells were found to regulate acetyl CoA carboxylase both in vivo and in vitro. Coenzyme A, guanosine 5-monophosphate and phosphatidylinositol 4,5-bisphosphate may have an indirect effect, but certainly no direct involvement, on carboxylase activity.     

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.     

Studies on wheat acetyl CoA carboxylase and the cloning of a partial cDNA

Wheat germ acetyl CoA carboxylase (ACCase) was purified by liquid chromatography and electroelution. During purification bovine serum albumin (BSA) was used to coat Amicon membranes used to concentrate partially pure ACCase. Despite further SDS-PAGE/electroelution and microbore HPLC steps BSA remained associated. This presented serious protein sequencing artefacts which may reflect the affinity of BSA for fatty acids bound to ACCase. To avoid these artefacts the enzyme was digested in gel with Endoproteinase LysC protease without the presence of BSA, and the resulting peptides blotted and sequenced.A partial cDNA (1.85 kb) encoding ACCase from a wheat embryo library was cloned, which hybridised to a 7.5 kb RNA species on northern blot of wheat leaf poly(A)⁺ RNA. The partial cDNA therefore represents about 0.25 of the full-length cDNA. The clone was authenticated by ACCase peptide sequencing and immuno cross-reactivity of the overexpressed clone. The derived amino acid sequence showed homology with both rat and yeast ACCase sequences (62%).Antibodies raised against wheat acetyl CoA carboxylase were specific for a 220 kDa protein from both wheat embryo and leaf. In addition, by using a novel quick assay for ACCase that utilised ¹²⁵I-streptavidin, we showed the major biotin containing protein to be 220 kDa in both leaf and germ. This is in marked contrast to the previously published molecular mass of 75 kDa allocated to wheat leaf ACCase.     

Detection of acetyl coenzyme A as an early CO2 assimilation intermediate in Methanobacterium

The pivotal role of acetyl coenzyme A in CO₂ assimilation by autotrophic methanogenic bacteria has been demonstrated by pulse-labelling of growing Methanobacterium thermoautotrophicum with ¹⁴CO₂. After very short incubation with ¹⁴CO₂ (1.5 s) approximately 1% of label incorporated into the soluble cell fraction was contained in acetyl coenzyme A. The percentage distribution of ¹⁴C within acetyl CoA markedly decreased with time, which is indicative for acetyl CoA being an immediate ¹⁴CO₂ fixation product. Label in the acetate molecule first appeared in the carboxyl carbon, but the methyl carbon became equally labelled within only 10 s. The acetyl CoA was compared with authentic material by various criterions and its cellular concentration was determined to be 52 M. This small cellular pool size of acetyl CoA as compared to e.g. alanine (6.4 mM) provides an explanation for the observed labelling kinetics. The data are fully consistent with autotrophic carbon assimilation via a total synthesis of acetyl coenzyme A from 2 CO₂.Dedicated to Professor Dr. Gerhart Drews on occasion of his 60th birthday     

Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation

Acetyl CoA carboxylase, in a partially purified preparation, was inactivated by ATP in a time- and temperature-dependent reaction. Adenosine 3′,5′-monophosphate did not affect the inactivation. Further purification separated the carboxylase from a protein fraction which could greatly enhance the inactivation of the enzyme.Inactivation of the enzyme with [γ-³²P]ATP resulted in the incorporation of ³²P which copurified with the enzyme. No label was incorporated when [U-¹⁴C]ATP was used. When carboxylase inactivated by exposure to [γ-³²P]ATP was precipitated with antibody, isotope incorporation into the precipitate paralleled enzyme inactivation. The phosphate was bound to serine and threonine residues by an ester linkage.Sodium fluoride completely inhibited the activation of partially purified enzyme by magnesium ions. Activation by magnesium, accompanied by the release of protein-bound ³²P, was antagonistic to inactivation of the enzyme by ATP.The data presented in this communication are consistent with a mechanism for controlling acetyl CoA carboxylase activity by interconversion between phosphorylated and dephosphorylated forms. Phosphorylation of the enzyme by a portein kinase decreases enzyme activity, whereas dephosphorylation by a protein phosphatase reactivates the enzyme.