In vitro phosphorylation and inactivation of rat liver acetyl-CoA carboxylase purified by avidin affinity chromatography

Acetyl-CoA carboxylase (EC 6.4.1.2) has been isolated from rat liver by an avidin-affinity chromatography technique. This preparation has a specific activity of 1.17 +/- 0.06 U/mg and appears as a major (240,000 dalton) and minor (140,000 dalton) band on SDS-polyacrylamide gel electrophoresis. Enzyme isolated by this technique can incorporate 1.09 +/- 0.07 mol phosphate per mol enzyme (Mr = 480,000) when incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase at 30 degrees C for 1 h. The associated activity loss under these conditions is 57 +/- 4.0% when the enzyme is assayed in the presence of 2.0 mM citrate. Less inactivation is observed when the enzyme is assayed in the presence of 5.0 mM citrate. The specific protein inhibitor of the cyclic AMP-dependent protein kinase blocks both the protein kinase stimulated phosphorylation and inactivation of acetyl-CoA carboxylase. The phosphorylated, inactivated rat liver carboxylase can be partially dephosphorylated and reactivated by incubation with a partially purified protein phosphatase. Preparations of acetyl-CoA carboxylase also contained an endogenous protein kinase(s) which incorporated 0.26 +/- 0.11 mol phosphate per mol carboxylase (Mr = 480,000) accompanied by a 26 +/- 9% decline in activity. We have additionally confirmed that the rat mammary gland enzyme, also isolated by avidin affinity chromatography, can be both phosphorylated and inactivated upon incubation with the cyclic AMP-dependent kinase.     

Mechanism of glucagon inhibition of liver acetyl-CoA carboxylase. Interrelationship of the effects of phosphorylation, polymer-protomer transition, and citrate on enzyme activity

The short-term regulation of rat liver acetyl-CoA carboxylase by glucagon has been studied in hepatocytes from rats that had been fasted and refed a fat-free diet. Glucagon inhibition of the activity of this enzyme can be accounted for by a direct correlation between phosphorylation, polymer-protomer ratio, and activity. Glucagon rapidly inactivates acetyl-CoA carboxylase with an accompanying 4-fold increase in the phosphorylation of the enzyme and 3-fold increase in the protomer-polymer ratio of enzyme protein. Citrate, an allosteric activator of acetyl-CoA carboxylase required for enzyme activity, has no effect on these phenomena, indicating a mechanism that is independent of citrate concentration within the cell. The observation of these effects of glucagon on acetyl-CoA carboxylase activity is absolutely dependent upon the minimization of proteolytic degradation of the enzyme after cell lysis. Therefore, for the first time, an interrelationship has been demonstrated between phosphorylation, protomer-polymer ratio, and citrate for the inactivation of acetyl-CoA carboxylase by glucagon.     

Isolation of three cyclic-AMP-independent acetyl-CoA carboxylase kinases from lactating rat mammary gland and characterization of their effects on enzyme activity

Three cyclic AMP-independent acetyl-CoA carboxylase kinases (A, B1 and B2) have been isolated from lactating rat mammary gland, using phosphocellulose chromatography, high performance gel filtration, and affinity chromatography on casein-Sepharose and phosvitin-Sepharose. These protein kinases have been identified with previously described kinases by the following criteria. Kinase A phosphorylates the same sites on rabbit mammary acetyl-CoA carboxylase as acetyl-CoA carboxylase kinase 2, which was originally described as a contaminant of rabbit mammary acetyl-CoA carboxylase purified by the poly(ethylene glycol)procedure. Kinase A will henceforth be referred to as acetyl-CoA carboxylase kinase-2. Kinase B1 has been identified with casein kinase II by its heparin sensitivity, elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. Kinase B2 has been identified with casein kinase I by its elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. The three kinases phosphorylate distinct sites on acetyl-CoA carboxylase. Phosphorylation by either casein kinase I or II does not affect enzyme activity. However, acetyl-CoA carboxylase kinase 2 inactivates acetyl-CoA carboxylase reversibly, in an identical manner to cyclic-AMP-dependent protein kinase, and phosphorylates sites located on identical peptides. Acetyl-CoA carboxylase kinase-2 can, however, be distinguished from the free catalytic subunit of cyclic-AMP-dependent protein kinase by its molecular mass, its substrate specificity, its elution behaviour on phosphocellulose, and its complete lack of sensitivity to the protein inhibitor of cyclic-AMP-dependent protein kinase. We also present evidence that phosphorylation of acetyl-CoA carboxylase by cyclic-AMP-dependent protein kinase occurs directly and not via a bicyclic cascade system as proposed by other laboratories.     

Acetyl-coenzyme A carboxylase in maize leaves

Purified chloroplasts from mesophyll and bundle sheath cells of maize leaves have been shown to be the location of acetyl-CoA carboxylase. In disrupted chloroplasts the enzyme was recovered in the stromal fraction, along with protein-bound biotin; acetyl-CoA carboxylase activity did not require a membrane component. Mg²⁺ and ATP are required for activity and sulfhydryl protecting agents enhance stability of the enzyme. Acetyl-CoA carboxylase activity was independent of leaf development in cell-free extracts of maize. Comparison of acetyl-CoA carboxylase activity with [¹⁴C]acetate incorporation into lipids, in isolated chloroplasts from developing leaves of maize, indicate that acetyl-CoA carboxylase is not limiting fatty acid synthesis.     

Insulin and the regulation of adipose-tissue acetyl-coenzyme A carboxylase

Rat epididymal fat-pads were incubated for 30min with glucose (2mg/ml) in the presence or absence of insulin. A twofold or greater increase in acetyl-CoA carboxylase activity was observed in extracts from insulin-treated tissue provided that assays were performed rapidly after extraction. This effect of insulin was evident whether or not extracts were prepared with albumin, and was not noticeably diminished by the presence of citrate or albumin or both in the assay. Incubation of extracts before assay led to activation of acetyl-CoA carboxylase and a marked diminution in the insulin effect. The enzyme in extracts was very sensitive to reversible inhibition by palmitoyl-CoA even in the presence of albumin (10mg/ml); inhibition persisted on dilution of enzyme and inhibitor. It is suggested that the observed activation of acetyl-CoA carboxylase by insulin may reflect changes in enzyme activity in the fat-cell resulting from the reduction of long-chain fatty-acyl-CoA that occurs in the presence of insulin. Activation of the enzyme with loss of the insulin effect on incubation of the extracts may be due to the slow dissociation of long-chain fatty acyl-CoA from the enzyme.     

Affinity labelling of rat liver acetyl-CoA carboxylase by a 2',3'-dialdehyde derivative of ATP

The interaction of rat liver acetyl-CoA carboxylase with a 2',3'-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was proved to be the only substrate which protected the inactivation. Acetyl-CoA did not effect inactivation, while HCO3- accelerated the process. Ki values for oATP in the absence and presence of HCO3- were 0.35 +/- 0.04 and 0.5 +/- 0.06 mM, and those of the modification constant (kmod) were 0.11 and 0.26 min-1 respectively. oATP completely inhibited the [14C]ADP in equilibrium ATP exchange and did not effect the [14C]acetyl-CoA in equilibrium malonyl-CoA exchange. Incorporation of approximately 1 equivalent of [3H]oATP per acetyl-CoA carboxylase subunit has been shown. No recovery of the modified enzyme activity has been observed in Tris or beta-mercaptoethanol containing buffers, and treatment with NaB3H4 has not led to 3H incorporation. The modification elimination of the ATP triphosphate chain. The results indicated the affinity modification of acetyl-CoA carboxylase by oATP. It was shown that the reagent apparently interacted selectively with the epsilon-amino group of lysine in the ATP-binding site to form a morpholine-like structure.     

Kinetic characterization of yeast pyruvate carboxylase isozyme Pyc1 and the Pyc1 mutant, C249A

The yeast Pyc1 isoform of pyruvate carboxylase has been further characterized and shown to differ from the Pyc2 isoform in its K(a) for K(+) activation. Pyc1 differs from chicken liver pyruvate carboxylase in the lack of effect of acetyl-CoA on ADP phosphorylation by carbamoyl phosphate, which may be a result of differences in the loci of action of the effector between the two enzymes. Solvent D(2)O isotope effects have been measured with Pyc1 on the full pyruvate carboxylation reaction, the ATPase reaction in the absence of pyruvate, and the carbamoyl phosphate-ADP phosphorylation reaction for the first time for pyruvate carboxylase. Proton inventories indicate that the measured isotope effects are due to a single proton transfer step in the reaction. The inverse isotope effects observed in all reactions suggest that the proton transfer step converts the enzyme from an inactive to an active form. Kinetic measurements on the C249A mutant enzyme suggest that C249 is involved in the binding and action of enzyme activators K(+) and acetyl-CoA. C249 is not involved in ATP binding as was observed for the corresponding residue in the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase, nor is it directly responsible for the measured inverse (D)(k(cat)/K(m)) isotope effects. The size of the inverse isotope effects indicates that they may result from formation of a low-barrier hydrogen bond. Modification of the wild type and C249A mutant with o-phthalaldehyde suggests that C249 is involved in isoindole formation but that the modification of this residue is not directly responsible for the accompanying major loss of enzyme activity.     

Protein-serine kinase from rat epididymal adipose tissue which phosphorylates and activates acetyl-CoA carboxylase. Possible role in insulin action.

1. Most of the cyclic-nucleotide-independent acetyl-CoA carboxylase kinase activity in an extract of rat epididymal adipose tissue was evaluated from a Mono Q column by 0.175 M-NaCl at pH 7.4. The activity of the kinase in this fraction (fraction 1) was increased after exposure of intact tissue to insulin. 2. Incubation of purified adipose-tissue acetyl-CoA carboxylase with [gamma-32P]ATP and samples of fraction 1 led to the incorporation of up to 0.4 mol of 32P/mol of enzyme subunit. Most of the phosphorylation was on serine residues within a single tryptic peptide. This peptide, on the basis of two-dimensional t.l.c. analysis, h.p.l.c. and Superose 12 chromatography, appeared to be the same as the acetyl-CoA carboxylase peptide ('I'-peptide) which exhibits increased phosphorylation in insulin-treated tissue. 3. Phosphorylation of purified acetyl-CoA carboxylase by the kinase in fraction 1 was found to be associated with a parallel 4-fold increase in activity. However, increases in both phosphorylation and activity were much diminished if fraction 1 was treated by Centricon centrifugation to remove low-Mr components. Among these components was a potent inhibitor of acetyl-CoA carboxylase activity which appeared to be necessary for the kinase in fraction 1 to be fully active. 4. The inhibitor remains to be identified, but inhibition requires MgATP, although the inhibitor itself does not cause any phosphorylation of the carboxylase. No effects of insulin were observed on the activity of the inhibitor. 5. It is concluded that the kinase probably plays an important role in the mechanism whereby insulin brings about the well-established increases in phosphorylation and activation of acetyl-CoA carboxylase in adipose tissue.     

Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.     

Changes in mammary-gland acetyl-coenzyme A carboxylase associated with lactogenic differentiation

The process leading to the rise of acetyl-CoA carboxylase activity in rat mammary tissue after the onset of lactation was investigated. The kinetics of change in enzyme activity and enzyme immunotitratable with antibody against avian liver acetyl-CoA carboxylase were determined during the course of lactogenic differentiation. The antibody inactivates and specifically precipitates acetyl-CoA carboxylase from rat mammary tissue as well as that from chicken liver cytosol. Characterization of the immunoprecipitate of the mammary tissue carboxylase by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals a single biotin-containing polypeptide of about 230000mol.wt. This molecular weight is approximately twice that reported for the avian liver enzyme. However, chicken liver cytosol prepared in the presence of trypsin inhibitor and subjected to immunoprecipitation gives rise to a biotin-containing subunit of 230000mol.wt. as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; omission of proteinase inhibitor leads to a subunit(s) approximately one-half this size. Throughout gestation both carboxylase activity and amounts of immunotitratable enzyme remained low; however, after parturition both parameters rose concomitantly to values 30-40 times the initial values. Therefore the elevated concentration of acetyl-CoA carboxylase appears to result from an increased rate of synthesis of enzyme relative to degradation rather than to activation of a pre-existing form of the enzyme.     

Enzymatic activities of cytoplasmic and of microsomal acetyl-CoA carboxylase of rat epididymal adipose tissue; different regulatory effects of a short-term fasting and palmitoyl-CoA on these two enzymes

The existence of a microsomal acetyl-CoA carboxylase in the rat epididymal adipose tissue was demonstrated in vitro in the present study. Its specific activity was of the same order of magnitude as that of the cytoplasmic acetyl-CoA carboxylase. The effect of several experimental conditions on the enzymatic activities of both enzymes were tested; fasting for 24 hr strongly increased (2.5-4 times) the activity of the microsomal enzyme while the cytoplasmic enzyme remained unchanged. Palmitoyl-CoA (1 and 5 microM), an inhibitor of acetyl-CoA carboxylase, had a greater effect on the cytoplasmic (33 and 88% inhibition) than on the microsomal enzyme (0 and 37% inhibition).     

Acetyl-CoA-carboxylase: modification of ATP-binding site of the active center by 2',3'-dialdehyde derivative of ATP

The interaction of rat liver acetyl-CoA carboxylase with a 2',3'-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was the only reaction substrate which provided protection from inactivation. Acetyl-CoA did not affect inactivation, while HCO3- accelerated the process. Ki values for oATP in the absence and the presence of HCO3- were 0.35 +/- 0.04 and 0.5 +/- 0.06 mM, and those of the modification constant (k) were 0.11 and 0.26 min-1, respectively. oATP completely inhibited the reaction of [14C]ADP in equilibrium ATP exchange, whereas produced actually no effect on [14C]acetyl-CoA equilibrium with malonyl-CoA exchange. Incorporation of about one equivalent of [3H]oATP per acetyl-CoA carboxylase subunit has been shown. No restoration of the modified enzyme activity has been observed in Tris or beta-mercaptoethanol containing buffers, and treatment with NaB[3H]4 has not led to 3H incorporation. The modification process involves elimination of the triphosphate chain of oATP. The results obtained indicate the affinity character of oATP-mediated modification of acetyl-CoA carboxylase. The reagent apparently interacts selectively with the epsilon-amino group of lysine in the ATP-binding site to form a morpholine-like structure.     

Formation of malonyl coenzyme A in rat heart. Identification and purification of an isozyme of A carboxylase from rat heart

Acetyl-CoA carboxylase is thought to be absent in the heart since the latter is highly catabolic and nonlipogenic. It has been suggested that the high level of malonyl-CoA that is found in the heart is derived from mitochondrial propionyl-CoA carboxylase, which also uses acetyl-CoA. In the present study, acetyl-CoA carboxylase was identified and purified from homogenates of rat heart. The isolated enzyme had little activity in the absence of citrate (specific activity, less than 0.1 units/mg); however, citrate stimulated its activity (specific activity, 1.8 units/mg in the presence of 10 mM citrate). Avidin inhibited greater than 95% of activity, and addition of biotin reversed this inhibition. Further, malonyl-CoA (1 mM) and palmitoyl-CoA (100 microM) inhibited greater than 90% of carboxylase activity. Similar to acetyl-CoA carboxylase of lipogenic tissues, the heart enzyme could be activated greater than 6-fold by preincubation with liver (acetyl-CoA carboxylase)-phosphatase 2. The activation was accompanied by a decrease in the K0.5 for citrate to 0.68 mM. These observations suggest that the activity in preparations from heart is due to authentic acetyl-CoA carboxylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparation from heart showed the presence of one major protein band (Mr 280,000) and a minor band (Mr 265,000) while that from liver gave a major protein band (Mr 265,000). A Western blot probed with avidin-peroxidase suggested that both the 280- and 265-kDa species contained biotin. Antibodies to liver acetyl-CoA carboxylase, which inhibited greater than 95% of liver carboxylase activity, inhibited only 35% of heart enzyme activity. In an immunoblot (using antibodies to liver enzyme) the 265-kDa species, and not the major 280-kDa species, in the heart preparation was specifically stained. These observations suggest the presence of two isoenzymes of acetyl-CoA carboxylase that are immunologically distinct, the 265-kDa species being predominant in the liver and the 280-kDa species being predominant in the heart.     

Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.     

Diurnal variations of lipogenic enzymes, their substrate and effector levels, and lipogenesis from tritiated water in rat liver

The activities of glucose-6-phosphate dehydrogenase, malic enzyme, fatty acid synthetase and acetyl-CoA carboxylase (extracted with or without phosphatase inhibitor) in rat liver did not vary significantly during 24 h. The hepatic levels of glucose 6-phosphate and malate increased coordinately 3-6 h after the beginning (1900 h) of food intake and were high until morning, whereas the levels of acetyl-CoA and citrate peaked at 1900 h and then decreased. However, it is remarkable that the in vivo incorporation of 3H from tritiated water into fatty acids in liver increased with the level of malonyl-CoA after food intake. Comparing the substrate and effector levels with the Km and Ka values for the enzymes, the levels of acetyl-CoA, malonyl-CoA and citrate appear to limit the enzyme activities. It is suggested that, after food intake, the physiological activity of acetyl-CoA carboxylase was increased with the substrate increase and/or with the catalytic activation with citrate, and consequently, the fatty acid synthetase activity was also increased, whereas the enzyme activities measured under optimum conditions were not.     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

Phosphoinositide inhibition of acetyl-CoA carboxylase from rat liver

When purified acetyl-CoA carboxylase was incubated with various phospholipids, the effects on carboxylase activity were quite diverse. Phosphatidic acid, phosphatidylcholine, and phosphatidylinositol were slightly stimulatory, whereas carboxylase was inhibited by polyphosphoinositides in a time- and concentration-dependent manner. Phosphatidylinositol 4,5-bisphosphate (TPI) was the most effective inhibitor; carboxylase activity was inhibited 50% after incubation with 1.5 μm TPI for 30 min. Incubation of carboxylase with citrate reduced the susceptibility to inhibition by TPI. The inhibition was reversed by removal of TPI from the inhibited enzyme. Incubation of TPI with divalent metal cations removed its ability to inhibit carboxylase. Sedimentation studies showed that TPI treatment shifts carboxylase to a less-polymerized form. The K_m for ATP, 24 μm, was not affected by the inhibitor. However, the apparent K_m for acetyl-CoA was decreased from 44 to 11 μm following incubation with TPI. The possibility that polyphosphoinositides may play a role in acetyl-CoA carboxylase regulation is discussed.     

Studies on the assay, activity and sedimentation behaviour of acetyl-CoA carboxylase from isolated hepatocytes incubated with insulin or glucagon.

The activity of acetyl-CoA carboxylase, measured in various ways, was studied in 15000g extracts of rat liver hepatocytes and compared with the rate of fatty acid synthesis in intact hepatocytes incubated with insulin or glucagon. Hepatocyte extracts were prepared by disruption of cells with a Dounce homogenizer or by solubilization with 1.5% (v/v) Triton X-100. Sucrose-density-gradient centrifugation demonstrated that the sedimentation coefficient of acetyl-CoA carboxylase from cell extracts was 30-35S, regardless of the conditions of incubation or disruption of hepatocytes. Solubilization of cells with 1.5% Triton X-100 yielded twice as much enzyme activity (measured by [14C]bicarbonate fixation) in the sucrose-gradient fractions as did cell disruption by the Dounce homogenizer. Analysis by high-performance liquid chromatography of acetyl-CoA carboxylase reaction mixtures showed that [14C]malonyl-CoA accounted for 10-60% of the total acid-stable radioactivity, depending on the method for disrupting hepatocytes and on the preincubation of the 15000g extract, with or without citrate, before assay. Under conditions in which incubation of cells with insulin or glucagon caused an activation or inhibition, respectively, of acetyl-CoA carboxylase, only 25% of the acid-stable radioactivity was [14C]malonyl-CoA and enzyme activity was only 13% (control), 16% (insulin), and 57% (glucagon) of the rate of fatty acid synthesis. Under conditions when up to 60% of the acid-stable radioactivity was [14C]malonyl-CoA and acetyl-CoA carboxylase activity was comparable with the rate of fatty acid synthesis, there was no effect of insulin or glucagon on enzyme activity.     

Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation.

Autotrophic Archaea of the family Sulfolobaceae (Crenarchaeota) use a modified 3-hydroxypropionate cycle for carbon dioxide assimilation. In this cycle the ATP-dependent carboxylations of acetyl-CoA and propionyl-CoA to malonyl-CoA and methylmalonyl-CoA, respectively, represent the key CO2 fixation reactions. These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl-CoA and propionyl-CoA. The carboxylase was purified and characterized and the genes were cloned and sequenced. In contrast to the carboxylase of most other organisms, acetyl-CoA/propionyl-CoA carboxylase from M. sedula is active at 75 degrees C and is isolated as a stabile functional protein complex of 560 +/- 50 kDa. The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (alpha) and carboxytransferase (gamma), respectively, and a small 18.6 kDa biotin carrier protein (beta). These subunits probably form an (alpha beta gamma)4 holoenzyme. It has a catalytic number of 28 s-1 at 65 degrees C and at the optimal pH of 7.5. The apparent Km values were 0.06 mm for acetyl-CoA, 0.07 mm for propionyl-CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate. Acetyl-CoA/propionyl-CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes. Due to its stability the archaeal carboxylase may prove an ideal subject for further structural studies.     

Acetyl-coenzyme-A carboxylase of Candida Lipolytica. 1. Purification and properties of the enzyme.

Acetyl-coenzyme-A carboxylase has been isolated in homogeneous form from Candida lipolytica. The homogeneity of the enzyme preparation is evidenced by analytical ultracentrifugation, dodecyl-sulfate-polyacrylamide gel electrophoresis and Ouchterlony double-diffusion analysis. The purified enzyme exhibits a specific activity of 8.0 U/mg protein at 25 degrees C and contains 1 mol biotin/263000 g protein. The sedimentation coefficient (S20,W) of the enzyme is 18 S. It has been shown by dodecyl-sulfate-polyacrylamide gel electrophoresis that the enzyme possesses only one kind of subunit with a molecular weight of 230000. This finding, together with the biotin content, indicates that the C. lipolytica enzyme has a highly integrated subunit structure. The C. lipolytica enzyme is very labile, but is stabilized by glycerol. The enzyme is markedly activated by poly(ethyleneglycol), the activation being due principally to a decrease in the Km values for substrates. Even in the presence of this activator, the Km value for acetyl-CoA of the C. lipolytica enzyme is much higher than that of the enzyme from Saccharomyces cerevisiae and animal tissues. The C. lipolytica enzyme, unlike the enzyme from animal tissues, is not activated by citrate.