Form Of acetyl-CoA Carboxylase Research Articles

Depletion and Repletion of Biotinyl Enzymes in Liver of Biotin-Deficient Rats: Evidence of a Biotin Storage System

The quantities of biotinyl proteins in liver of young rats were compared with age-matched controls at intervals during depletion and repletion of biotin. Growth rate and the concentrations of biotinyl proteins previously proposed as mitochondrial storage forms of acetyl CoA carboxylase rapidly decreased in response to biotin deprivation, whereas neither the concentration nor activity of cytosolic acetyl CoA carboxylase was affected. Concentrations of carboxylases active within mitochondria (pyruvate carboxylase, propionyl CoA carboxylase and 3-methyl crotonyl CoA carboxylase) decreased only after d 28. When biotin was injected into biotin-deficient rats, concentrations of the carboxylases active within mitochondria were restored to control levels within 3 h, whereas the concentrations of putative mitochondrial storage forms of acetyl CoA carboxylase reached normal levels only after 9 h, indicating that the injected biotin was preferentially used for the synthesis of the carboxylases active within mitochondria rather than acetyl CoA carboxylase. Mitochondrial acetyl CoA carboxylase may serve as a reservoir to maintain a normal concentration of cytosolic acetyl CoA carboxylase in liver of rats deprived of biotin and provide biotin, indirectly, to maintain essentially normal concentrations of the biotinyl enzymes active within mitochondria for several weeks after rats were fed a biotin-deficient diet.

Dual effects of fibroblast growth factor 21 on hepatic energy metabolism.

The aim of this study was to investigate the mechanisms by which fibroblast growth factor 21 (FGF21) affects hepatic integration of carbohydrate and fat metabolism in Siberian hamsters, a natural model of adiposity. Twelve aged matched adult male Siberian hamsters maintained in their long-day fat state since birth were randomly assigned to one of two treatment groups and were continuously infused with either vehicle (saline; n=6) or recombinant human FGF21 protein (1 mg/kg per day; n=6) for 14 days. FGF21 administration caused a 40% suppression (P<0.05) of hepatic pyruvate dehydrogenase complex (PDC), the rate-limiting step in glucose oxidation, a 34% decrease (P<0.05) in hepatic acetylcarnitine accumulation, an index of reduced PDC flux, a 35% increase (P<0.05) in long-chain acylcarnitine content (an index of flux through β-oxidation) and a 47% reduction (P<0.05) in hepatic lipid content. These effects were underpinned by increased protein abundance of PD kinase-4 (PDK4, a negative regulator of PDC), the phosphorylated (inhibited) form of acetyl-CoA carboxylase (ACC, a negative regulator of delivery of fatty acids into the mitochondria) and the transcriptional co-regulators of energy metabolism peroxisome proliferator activated receptor gamma co-activator alpha (PGC1α) and sirtuin-1. These findings provide novel mechanistic basis to support the notion that FGF21 exerts profound metabolic benefits in the liver by modulating nutrient flux through both carbohydrate (mediated by a PDK4-mediated suppression of PDC activity) and fat (mediated by deactivation of ACC) metabolism, and therefore may be an attractive target for protection from increased hepatic lipid content and insulin resistance that frequently accompany obesity and diabetes.

Sethoxydim affects lipid synthesis and acetyl-CoA carboxylase activity in soybean

With rare exceptions, dicot plastids have been reported to contain only a multisubunit (prokaryotic) form of acetyl-coA carboxylase (ACCase), the first committed step of lipid biosynthesis. The sensitivity of most monocots to cyclohexanediones (CHDs) such as sethoxydim, has been shown to be associated with the presence in their plastids of a multifunctional (eukaryotic) form of ACCase. Little is known about the effects of sethoxydim on lipid metabolism and ACCase activity in dicots. Here it is shown that foliar lipid biosynthesis is differentially affected by the herbicide treatment in two dicot species, Nicotiana sylvestris (wild tobacco) and Glycine max (soybean). In N. sylvestris, the total lipid content of neoformed leaves harvested 2 weeks after the sethoxydim treatment was unaffected by doses of up to 10(-3) M sethoxydim. In soybean, lipid content decreased by 45% when 10(-5) M sethoxydim was used, and this was associated with a 30% reduction in fatty acid synthesis activity. ACCase activity of soybean plastidial preparations was 60% reduced in the presence of sethoxydim, whereas that of N. sylvestris was unaffected. Finally, the presence of a biotinylated 220 kDa polypeptide, corresponding in size to multifunctional ACCase, was observed in soybean plastids. Possible relationships between sensitivity of plastidial soybean ACCase towards sethoxydim, plastidial protein content, and altered de novo lipid biosynthesis in herbicide-treated plants are discussed.

The pea chloroplast membrane-associated protein, IEP96, is a subunit of acetyl-CoA carboxylase.

Two forms of acetyl-CoA carboxylase (ACCase) have been characterized in pea (Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and beta-carboxyltransferase (CT) subunits of the plastidial-ACCase have recently been characterized and cloned. To further characterize the carboxyltransferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co-purifies with ACCase activity during gel permeation chromatography. However, upon anion-exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidial-ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the alpha-CT subunit of the MS-ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co-eluted during gel permeation chromatography with plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the beta-CT subunit. Furthermore, antibodies against beta-CT immunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE purification of ACCase protein demonstrated that the beta-CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the alpha-CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the alpha-CT subunit of pea chloroplast ACCase.

Localization and characterization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides

Young pea leaves contain two structurally different forms of acetyl-CoA carboxylase (EC 6.4.1.2; ACCase). A minor form, which accounted for about 20% of the total ACCase activity in the whole leaf, was detected in the epidermal tissue. This enzyme was soluble and was purified to homogeneity from young pea leaf extracts. It consisted of a dimer of two identical biotinyl subunits of molecular mass 220 kDa. In this respect, this multifunctional enzyme was comparable with that described in other plants and in other eukaryotes. A predominant form was present in both the epidermal and mesophyll tissues. In mesophyll protoplasts, ACCase was detected exclusively in the soluble phase of chloroplasts. This enzyme was partially purified from pea chloroplasts and consisted of a freely dissociating complex, the activity of which may be restored by combination of its separated constituents. The partially purified enzyme was composed of several subunits of molecular masses ranging from 32 to 79 kDa, for a native molecular mass > 600 kDa. One of these subunits, of molecular mass 38 kDa, was biotinylated. This complex subunit structure was comparable with that of microorganisms and was referred to as a 'prokaryotic' form of ACCase. Biochemical parameters were determined for both ACCase forms. Finally, both pea leaf ACCases exhibited different sensitivities towards the grass ACCase herbicide, diclofop. This compound had no effect on the 'prokaryotic' form of ACCase, while the 'eukaryotic' form was strongly inhibited.

Role of reversible phosphorylation of acetyl‐CoA carboxylase in long‐chain fatty acid synthesis 1

Acetyl-CoA carboxylase, the rate-limiting enzyme in the biogenesis of long-chain fatty acids, is regulated by phosphorylation and dephosphorylation. The major phosphorylation sites that affect carboxylase activity and the specific protein kinases responsible for phosphorylation of different sites have been identified. A form of acetyl-CoA carboxylase that is independent of citrate for activity occurs in vivo. This active form of carboxylase becomes citrate-dependent upon phosphorylation under conditions of reduced lipogenesis. Therefore, phosphorylation-dephosphorylation of acetyl-CoA carboxylase is the enzyme's primary short-term regulatory mechanism; this control mechanism together with cellular metabolites such as CoA, citrate, and palmitoyl-CoA serves to fine-tune the synthesis of long-chain fatty acids under different physiological conditions.

Acute Alloxan Diabetes Alters the Activity but Not the Total Quantity of Acetyl CoA Carboxylase in Rat Liver

Alloxan diabetes has repeatedly been shown to reduce lipogenesis in rat liver concomitant with decreased activity of acetyl CoA carboxylase. This and other observations led to the deduction that insulin is required for the synthesis of acetyl CoA carboxylase even though the actual amount of enzyme was not measured. We have developed methods to determine the quantity of acetyl CoA carboxylase in crude tissue extracts with which we have reexamined the role of insulin in regulating the amount of the enzyme in liver of acute (3-d) alloxan diabetic rats. The results show that although there was a decrease in the quantity of the active cytoplasmic form of acetyl CoA carboxylase in the liver of alloxan diabetic rats, there was a corresponding increase in the quantity of relatively inactive forms of the enzyme associated with mitochondria. Thus, the total amount of enzyme was minimally affected by the diabetic state. Instead, the results indicate that decreased acetyl CoA carboxylase activity in liver of the diabetic rats was attributable to a shift in the subcellular distribution of the enzyme from the active cytoplasmic to inactive mitochondrial forms. We have shown previously that subcellular distribution of the enzyme is dietary dependent. Results of this study implicate insulin in the mobilization and activation of mitochondrial acetyl CoA carboxylase.

Molecular weights of subunits of acetyl CoA carboxylase in rat liver cytoplasm

Monomeric [14C] methyl avidin was shown to bind to sodium dodecyl sulfate-denatured biotinyl proteins and remain bound through polyacrylamide gel electrophoresis which allowed their detection by fluorography. This method was used to show that purified rat liver acetyl CoA carboxylase contained two high molecular weight forms of the enzyme (MR = 241,000 and 252,000) while rapidly prepared, crude rat liver cytoplasm contained two larger molecular weight (MR = 257,000 and 270,000) forms. Thus, the enzyme had undergone substantial proteolysis during purification. The crude enzyme preparation also contained a smaller biotinyl protein (MR = 141,000) which is likely a proteolytic product of the larger forms of acetyl CoA carboxylase.

Acetyl-CoA carboxylase during development of plastids in wild-type and mutant barley seedlings.

A soluble acetyl-CoA carboxylase in homogenates of leaves from wild-type barley seedlings was studied. Centrifuging the homogenate at 150,000 X g did not reduce the total activity, but raised the specific activity. During chloroplast development in light-grown seedlings or during light-dependent greening of leaves grown in the dark, both the total activity of the carboxylase per plant and the specific activity per mg of protein in homogenates of the seedlings increased rapidly. The soluble leaf acetyl-CoA carboxylase was studied in a number of barley mutants with lesions in chloroplast development. In a group of three mutants light elicited an increase in acetyl-CoA carboxylase activity as in the wild-type. In two mutants light caused a decrease in activity. Dark-grown leaves of mutant albina-f17 contained levels of soluble acetyl-CoA carboxylase reached only in the light by the wild-type, whereas light-grown albina-f17 seedlings lacked carboxylase activities. The possibility is discussed that leaf cells contain two forms of acetyl-CoA carboxylase, one soluble with unknown location and a dissociable form located in the chloroplast.

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.

LIVER ACETYL COA CARBOXYLASE: THE DISSOCIATION-REASSOCIATION PROCESS AND ITS RELATION TO CATALYTIC ACTIVITY

In a previous paper' we reported that acetyl CoA carboxylase (E.C. 6.4.1.2) isolated in homogeneous form from chicken liver is an unusually large and asymmetric protein structure. The enzyme (in phosphate buffer, pH 7) has a molecular weight of about 8 million and a sedimentation coefficient of 53-58S. Its characteristic filamentous structure, revealed by electron microscopy, was easily dissociated into subunits with concomitant loss of enzymatic activity. Active filaments (70-100 A X up to 4000 A) were rapidly reconstituted by addition of isocitrate or other triand dicarboxylic acid anions. The present paper provides a more complete physicalchemical characterization of the protomeric and polymeric forms of acetyl CoA carboxylase, as well as an insight into the reversible dissociation-reassociation process and its relation to catalytic activity.