Effect of insulin on association of acetyl CoA carboxylase phosphatase and acetyl CoA carboxylase

Insulin promotes an association between acetyl CoA carboxylase and acetyl CoA carboxylase phosphatase. The association between rat epididymal fat tissue carboxylase and the phosphatase occurs in both a tissue culture system and in vivo and is accompanied by an increase in acetyl CoA carboxylase activity.     

Inhibition of rat liver acetyl CoA carboxylase by chloride

The activity of acetyl CoA carboxylase in both crude and purified rat liver preparations was reduced in the presence of sodium or potassium chloride and increased in the presence of potassium acetate. The chloride inhibition was not competitive with bicarbonate. The use of Trischloride buffer did not alter the apparent pH optimum of the enzyme when compared with Tris-acetate buffer.     

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.     

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.     

Biotin subunits of acetyl CoA carboxylase and pyruvate carboxylase from a thermophilic Bacillus.

Two biotin-containing polypeptides of molecular weights 140,000 and 22,000 have been identified by gel electrophoresis in a sodium dodecyl sulfate-denatured extract of cells of a thermophilic Bacillus. These polypeptides can be separated from each other by either gel filtration through Sepharose 6B or affinity chromatography on a Sepharose-avidin column. The larger polypeptide is renatured under appropriate conditions to yield enzymically active pyruvate carboxylase. Enzyme reconstitution experiments show that the smaller polypeptide is a component of acetyl CoA carboxylase. The biotin subunits of these two carboxylases are thus distinct from, and dissimilar to, each other. The demonstration that a pyruvate carboxylase-deficient mutant of the Bacillus contains the smaller, but not the larger, polypeptide corroborates this conclusion.     

Cloning of acetyl CoA carboxylase cDNA and the effects of spirodiclofen on the expression of acetyl CoA carboxylase mRNA in Panonychus citri

The citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), is an important pest worldwide. Previous studies showed that acetyl CoA carboxylase (ACCase) may be a critical target of spirodiclofen, a recently introduced insecticide. Therefore, we cloned the PcACCase gene and obtained a full‐length cDNA (Genbank accession number KJ675439 ). The open reading frame was 6 972 bp, coding for 2 323 amino acids. The PcACCase protein had a theoretical molecular weight of 262.82 kDa and an isoelectric point of 6.31, and it contained six conserved domains and one low‐complexity region. Quantitative real‐time PCR showed that spirodiclofen can up‐regulate the expression levels of ACCase mRNA during all four developmental stages.     

Regulation of the structure and activity of pyruvate carboxylase by acetyl CoA

In this review we examine the effects of the allosteric activator, acetyl CoA on both the structure and catalytic activities of pyruvate carboxylase. We describe how the binding of acetyl CoA produces gross changes to the quaternary and tertiary structures of the enzyme that are visible in the electron microscope. These changes serve to stabilize the tetrameric structure of the enzyme. The main locus of activation of the enzyme by acetyl CoA is the biotin carboxylation domain of the enzyme where ATP-cleavage and carboxylation of the biotin prosthetic group occur. As well as enhancing reaction rates, acetyl CoA also enhances the binding of some substrates, especially HCO3-, and there is also a complex interaction with the binding of the cofactor Mg2. The activation of pyruvate carboxylase by acetyl CoA is generally a cooperative processes, although there is a large degree of variability in the degree of cooperativity exhibited by the enzyme from different organisms. The X-ray crystallographic holoenzyme structures of pyruvate carboxylases from Rhizobium etli and Staphylococcus aureus have shown the allosteric acetyl CoA binding domain to be located at the interfaces of the biotin carboxylation and carboxyl transfer and the carboxyl transfer and biotin carboxyl carrier protein domains.     

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.     

Hormonal regulation of acetyl CoA carboxylase effect of insulin and epinephrine

Acetyl CoA carboxylase in the isolated epididymal fat tissue is activated by insulin and inactivated by epinephrine and dibutyryl cAMP. Insulin activation of the enzyme occurs in the presence of inhibitors of protein synthesis and can be observed as early as 15 min. A time dependent inactivation of the enzyme occurs in the presence of ATP and Mg⁺⁺$\text{in vitro}$.     

Putative mediators of insulin action regulate hepatic acetyl CoA carboxylase activity

Incubation of rat liver plasma membranes with insulin enhances the production of small molecular weight substances which regulate the activity of liver acetyl CoA carboxylase. While low concentrations of insulin cause the release of a carboxylase stimulator from membranes, concentrations greater than 10^?9 M generate less stimulating activity. This biphasic concentration curve for insulin can be resolved by differential alcohol extraction into two fractions which have antagonistic activity. The production of both substances is enhanced by insulin. Chemical and chromatographic evidence suggest that these substances are identical to the previously described “mediators” which regulate both pyruvate dehydrogenase and adenylate cyclase activities.