Activation of acetyl-CoA carboxylase. Purification and properties of a Mn2+-dependent phosphatase

Acetyl-CoA carboxylase was isolated from rat liver by polyethylene glycol precipitation and avidin affinity chromatography. Sodium dodecyl sulfate electrophoresis of the enzyme gives one protein band (Mr 250,000). Phosphate analysis of the carboxylase showed the presence of 8.3 mol of phosphate/mol of subunit (Mr 250,000). The purified carboxylase has low activity in the absence of citrate (specific activity = 0.3 units/mg). However, addition of 10 mM citrate activates the carboxylase 10-fold, with half-maximal activation observed at 2 mM citrate, well above the physiological citrate level. Using this carboxylase as a substrate, we have isolated from rat liver a protein that activates the enzyme about 10-fold. This protein has been purified to near homogeneity (Mr 90,000). Incubation of this protein with 32P-labeled acetyl-CoA carboxylase results in a time-dependent activation of carboxylase with concomitant release of 32Pi, indicating that this protein is a phosphoprotein phosphatase. Both activation and dephosphorylation are dependent on Mn2+, but not citrate. This phosphatase does not hydrolyze p-nitrophenyl phosphate but does show high affinity for acetyl-CoA carboxylase (Km = 0.2 microM) as compared to its action on phosphorylase a (Km = 5.5 microM) and phosphohistone (Km = 20 microM). Activated acetyl-CoA carboxylase was isolated after dephosphorylation by the phosphatase. Such preparations contain about 5 mol of phosphate/mol of subunit and have specific activities of 2.6-3.0 units/mg in the absence of citrate. These activities are comparable to those of the phosphorylated carboxylase in the presence of 10 mM citrate. Thus, dephosphorylation by the Mn2+-dependent phosphatase renders the carboxylase citrate-independent, as compared to the phosphorylated form, which is citrate-dependent. To our knowledge this is the first report of a preparation of animal acetyl-CoA carboxylase that has substantial catalytic activity independent of citrate.     

Acute hormonal control of acetyl-CoA carboxylase. The roles of insulin, glucagon, and epinephrine

Acetyl-CoA carboxylase, purified from rapidly freeze-clamped livers of rats maintained on a normal laboratory diet and given 0-5 units of insulin shortly before death, gives a major protein band (Mr 265,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase from untreated rats has relatively low activity (0.8 unit/mg protein when assayed in the absence of citrate) and high phosphate content (8.5 mol of Pi/mol of subunit), while the enzyme from livers of rats that received 5 units of insulin has higher activity (2.0 units/mg protein) and lower phosphate content (7.0 mol of Pi/mol of subunit). Addition of citrate activates both preparations with half-maximal activation (K0.5) at 1.0 and 0.6 mM citrate, respectively. The enzyme from rats that did not receive insulin is mainly in the octameric state (Mr approximately 2 x 10(6)), while that from rats that received insulin is mainly in the polymeric state (Mr approximately 10 x 10(6)). Thus, short-term administration of insulin results in activation of acetyl-CoA carboxylase, lowering of its citrate requirement, and dephosphorylation and polymerization of the protein. The insulin-induced changes in the carboxylase are probably due to dephosphorylation of the protein since similar changes are observed when the enzyme from rats that did not receive insulin is dephosphorylated by the Mn2(+)-dependent [acetyl-CoA carboxylase]-phosphatase 2. The effect of glucagon or epinephrine administration on acetyl-CoA carboxylase was also investigated. The carboxylase from fasted/refed rats has a relatively high specific activity (3.4 units/mg protein in the absence of citrate), lower phosphate content (4.9 mol of Pi/mol of subunit), and is present mainly in the polymeric state (Mr approximately 10 x 10(6)). Addition of citrate activates the enzyme with K0.5 = 0.2 mM citrate. Glucagon or epinephrine injection of fasted/refed rats yielded carboxylase with lower specific activity (1.4 or 1.9 units/mg, respectively, in the absence of citrate), higher phosphate content (6.4 or 6.7 mol of Pi/mol of subunit, respectively), and mainly in the octameric state (Mr approximately 2 x 10(6)). Treatment of these preparations with [acetyl-CoA carboxylase]-phosphatase 2 reactivated the enzyme (specific activity approximately 8 units/mg protein in the absence of citrate) and polymerized the protein (Mr approximately 10 x 10(6]. These observations indicate that insulin and glucagon, by altering the phosphorylation state of the acetyl-CoA carboxylase, play antagonistic roles in the acetyl-control of its activity and therefore in the regulation of fatty acid synthesis.     

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.     

Regulation of acetyl-coenzyme A carboxylase. I. Purification and properties of two forms of acetyl-coenzyme A carboxylase from rat liver

Acetyl-CoA carboxylase of animal tissues is known to be dependent on citrate for its activity. The observation that dephosphorylation abolishes its citrate dependence (Thampy, K. G., and Wakil, S. J. (1985) J. Biol. Chem. 260, 6318-6323) suggested that the citrate-independent form might exist in vivo. We have purified such a form from rapidly freeze-clamped livers of rats. Sodium dodecyl sulfate gel electrophoresis of the enzyme gave one protein band (Mr 250,000). The preparation has high specific activity (3.5 units/mg in the absence of citrate) and low phosphate content (5.0 mol of Pi/mol of subunit). The enzyme isolated from unfrozen liver or liver kept in ice-cold sucrose solution for 10 min and then freeze-clamped has low activity (0.3 unit/mg) and high phosphate content (7-8 mol of Pi/mol of subunit). Citrate activated such preparations with half-maximal activation at greater than 1.6 mM, well above physiological range. The low activity may be due to its high phosphate content because dephosphorylation by [acetyl-CoA carboxylase]-phosphatase 2 activates the enzyme and reduces its dependence on citrate. Since freeze-clamping the liver yields enzyme with lower phosphate content and higher activity, it is suggested that the carboxylase undergoes rapid phosphorylation and consequent inactivation after the excision of the liver. The carboxylase is made up of two polymeric forms of Mr greater than or equal to 10 million and 2 million based on gel filtration on Superose 6. The former, which predominates in preparations from freeze-clamped liver, has higher activity and lower phosphate content (5.3 units/mg and 4.0 mol of Pi/mol of subunit, respectively) than the latter (2.0 units/mg and 6.0 mol of Pi/mol of subunit, respectively). The latter, which predominates in preparations from unfrozen liver, is converted to the active polymer (Mr greater than or equal to 10 million) by dephosphorylation. Thus, the two polymeric forms are interconvertible by phosphorylation/dephosphorylation and may be important in the physiological regulation of acetyl-CoA carboxylase.     

Regulation of acetyl-coenzyme A carboxylase. II. Effect of fasting and refeeding on the activity, phosphate content, and aggregation state of the enzyme

Acetyl-CoA carboxylase isolated from freeze-clamped livers of fed rats has relatively low phosphate content (5.0 mol of Pi/mol of subunit) and high specific activity (3.5 units/mg in the absence of citrate). The enzyme from rats fasted for 12, 18, 24, and 48 h exhibited decreasing specific activities of 2.75, 1.85, 1.7, and 0.9 units/mg, respectively. Citrate activated all preparations of carboxylase, with most activation observed with the least active preparation. There was no significant change in the sensitivity of the enzyme to citrate since half-maximal activation was observed at 0.2 mM for carboxylase from fed as well as fasted rats. With the decrease in activity as a function of fasting, there was a concomitant increase in the phosphate content of carboxylase, with values of 5.3, 5.6, 6.7, and 7.6 mol of Pi/mol of subunit obtained for preparations from rats fasted for 12, 18, 24, and 48 h, respectively. Refeeding the fasted rats resulted in increased specific activity of carboxylase (3.4 units/mg) and decreased phosphate content (5.1 mol of Pi/mol of subunit). Moreover, dephosphorylation by [acetyl-CoA carboxylase]-phosphatase 2 activated the carboxylase from 48-h fasted rats to a value of 2.9 units/mg, assayed in the absence of citrate, indicating that the low activity of carboxylase from fasted rats was due to its increased phosphate content. Superose 6 chromatography showed that the enzyme exists in two polymeric forms, a highly active polymer of greater than or equal to 40 subunits and less active octamer. The former predominates in livers of fed rats, whereas the latter predominates in livers of fasted rats. The octamer could be converted to the highly active polymer by dephosphorylation. These observations indicate that fasting/refeeding results in phosphorylation/dephosphorylation of acetyl-CoA carboxylase with concomitant depolymerization/polymerization of the protein and ultimately decreasing or increasing its specific activity.     

Troglitazone stimulates acetyl-CoA carboxylase activity through a post-translational mechanism

Troglitazone, a thiazolidinedione, is known to act as an insulin sensitizer. The various effects of the drug include stimulation of glucose utilization and inhibition of gluconeogenesis and fatty acid oxidation. We studied the effect of troglitazone treatment on rat liver acetyl-CoA carboxylase (ACC), the key enzyme that catalyzes the formation of malonyl-CoA, the rate-limiting step in the synthesis of long chain fatty acids. Treatment of rats with troglitazone for 18 days resulted in more than 200% increase in the activity of hepatic acetyl-CoA carboxylase (1.01+/-0.14 and 2.33+/-0.28 mU/mg supernatant protein for control and troglitazone-treated rats, respectively) (p<0.001). The expression of acetyl-CoA carboxylase mRNA, as studied by RNAse protection assay, was not significantly different between the two groups of animals. The ACC from control and troglitazone-treated groups was purified by avidin-affinity chromatography. The purified enzyme migrated as a major protein band (Mr 262,000) on SDS-polyacrylamide gels. Troglitazone treatment was associated with increased citrate sensitivity of ACC. The specific activity of the purified preparation in troglitazone-treated rats was increased by 67% (2.5 vs. 1.5 U/mg). Quantitation of alkali-labile phosphate content of the purified preparation revealed 5.66+/-0.17 and 6.29+/-0.13 mol Pi/mol subunit of 262 Kda for control and troglitazone-treated rats, respectively (P<0.01). The subtle increase in phosphate content does not explain the observed activation of the enzyme. It is possible that additional mechanisms such as troglitazone related rearrangement of the occupancy of select phosphate binding sites or altered binding of the biotin cofactor may also contribute to the observed activation of ACC.     

Evidence that insulin activates fat-cell acetyl-CoA carboxylase by increased phosphorylation at a specific site.

1. A new rapid method for the purification of fat-cell acetyl-CoA carboxylase is described; the key step is sedimentation after specific polymerization by citrate. 2. Incubation of epididymal fat-pads or isolated fat-cells with insulin or adrenaline leads to a rapid increase or decrease respectively in the activity of acetyl-CoA carboxylase measured in fresh tissue extracts. The persistence of the effect of insulin through high dilution of tissue extracts and through purification involving precipitation with (NH4)2SO4 suggests that the enzyme undergoes a covalent modification after exposure of intact tissue to the hormone. The opposed effects of insulin and adrenaline are not adequately explained through modification of a common site on acetyl-CoA carboxylase, since these hormones bring about qualitatively different alterations in the kinetic properties of the enzyme measured in tissue extracts. 3. The state of phosphorylation of acetyl-CoA carboxylase within intact fat-cells exposed to insulin was determined, and results indicate a small but consistent rise in overall phosphorylation of the Mr-230000 subunit after insulin treatment. 4. Acetyl-CoA carboxylase from fat-cells previously incubated in medium containing [32P]phosphate was purified by immunoprecipitation and then digested with performic acid and trypsin before separation of the released phosphopeptides by two-dimensional analysis. Results obtained show that the exposure of fat-cells to insulin leads to a 5-fold increase in incorporation of 32P into a peptide which is different from those most markedly affected after exposure of fat-cells to adrenaline. 5. These studies indicate that the activation of acetyl-CoA carboxylase in cells incubated with insulin is brought about by the increased phosphorylation of a specific site on the enzyme, possibly catalysed by the membrane-associated cyclic AMP-independent protein kinase described by Brownsey, Belsham & Denton [(1981) FEBS Lett. 124, 145-150].     

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.     

The role of acetyl-CoA carboxylase phosphorylation in the control of mammary gland fatty acid synthesis during the starvation and re-feeding of lactating rats.

Activation of acetyl-CoA carboxylase during incubation of crude extracts of lactating rat mammary gland with Mg2+ and citrate can be blocked by NaF, suggesting that it represents a dephosphorylation of the enzyme. The greater extent of activation in extracts from 24 h-starved rats (200%) compared with fed controls (70%) implies that the decrease in acetyl-CoA carboxylase activity in response to 24 h starvation may involve increased phosphorylation of the enzyme. Acetyl-CoA carboxylase was purified from the mammary glands of lactating rats in the presence of protein phosphatase inhibitors by avidin-Sepharose chromatography. Starvation of the rats for 24 h increased the concentration of citrate giving half-maximal activation by 75%, and decreased the Vmax. of the purified enzyme by 73%. This was associated with an increase in the alkali-labile phosphate content from 3.3 +/- 0.2 to 4.5 +/- 0.4 mol/mol of enzyme subunit. Starvation of lactating rats for 6 h, or short-term insulin deficiency induced by streptozotocin injection, did not effect the kinetic parameters or the phosphate content of acetyl-CoA carboxylase purified from mammary glands. The effects of 24 h starvation on the kinetic parameters and phosphate content of the purified enzyme were completely reversed by re-feeding for only 2.5 h. This effect was blocked if the animals were injected with streptozotocin before re-feeding, suggesting that the increase in plasma insulin that occurs on re-feeding was responsible for the activation of the enzyme. The effects of re-feeding 24 h-starved rats on the kinetic parameters and phosphate content of acetyl-CoA carboxylase could be mimicked by treating enzyme purified from 24 h-starved rats with protein phosphatase-2A in vitro. Our results suggest that, in mammary glands of 24 h-starved lactating rats, insulin brings about a dephosphorylation of acetyl-CoA carboxylase in vivo, which may be at least partly responsible for the reactivation of mammary lipogenesis in response to re-feeding.     

The low activity of acetyl-CoA carboxylase in basal and glucagon-stimulated hepatocytes is due to phosphorylation by the AMP-activated protein kinase and not cyclic AMP-dependent protein kinase

Acetyl-CoA carboxylase purified from isolated hepatocytes is activated dramatically by protein phosphatase treatment, concomitant with a reduction of the phosphate content from 3.7 to 1.1 mol/subunit. Glucagon treatment of the cells produces a further inactivation of the enzyme that is totally reversed by phosphatase treatment, and is associated with an increase in phosphate content of 0.8 mol/subunit, distributed in two peptides which contain the sites phosphorylated in vitro by the cyclic AMP-dependent and AMP-activated protein kinases. Sequencing of these peptides shows that the low activity of acetyl-CoA carboxylase is due to phosphorylation by the AMP-activated protein kinase, and not cyclic AMP-dependent protein kinase, even after glucagon treatment.     

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.     

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.     

Comparison of acetyl-CoA carboxylases from parsley cell cultures and wheat germ

The activity of acetyl-CoA carboxylase of suspension-cultured cells of parsley (Petroselinum hortense Hoffm.) is greatly stimulated by light soon after transferring cells to new culture medium. Parsley acetyl-CoA carboxylase has been purified from frozen cells by treatment of the crude protein extract with Dowex 1 × 2 and polyethyleneimine, precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose and blue Sepharose CL-6B, and gel filtration on Sepharose 6B. A recovery of about 8% has been achieved with a 300-fold increase in specific activity. Wheat germ acetyl-CoA carboxylase has been purified 2180-fold by a similar procedure. The two carboxylases have the following characteristics: Molecular weights of 840,000 for the parsley carboxylase and 700,000 for the wheat germ carboxylase have been estimated from the elution volumes of a calibrated Sepharose 6B column. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the carboxylases from parsley and wheat each are composed of one large subunit (M_r = 210,000 and 240,000, respectively) and possibly one smaller polypeptide component (M_r = 105,000 and 98,000, respectively). Avidin-binding experiments demonstrated that the 240,000 — M_r component of wheat germ carboxylase is the biotin-containing subunit of this enzyme. No isoenzymes of the parsley carboxylase could be demonstrated.     

Acetyl-coenzyme-A carboxylase from rat liver. Subunit structure and proteolytic modification.

The subunit structure of rat liver acetyl-coenzyme-A carboxylase has been studied by polyacrylamide gel electrophoresis in the presence of dodecylsulfate. A number of individual preparations of the enzyme purified by the same procedures exhibited three different types of electrophoretic patterns as follows: first, a single slow-moving protein bands (Mr 230000); secondly, two adjacent fast-moving protein band (M4 124000 and 118 000); finally, all three protein bands. With the use of the [14C]biotin-labelled enzyme, the biotinyl prosthetic group was shown to be associated with the polypeptide of 230000 Mr as well as with that of 124000 Mr, but not with the polypeptide of 118000 Mr. Studies were next made with the labelled enzyme to examine the possibility that the two light polypeptides might have been formed by proteolytic modification of the heavy polypeptide during the procedures used for the purification of the enzyme. Treatment of the enzyme with trypsin or chymotrypsin resulted in cleavage of the heavy polypeptide into two nonidentical polypeptides with molecular weights of approximately 120000. Incubation of the enzyme with proteases derived from rat liver converted the heavy polypeptide into lighter polypeptides of 80000-130000 Mr. Acetyl-CoA carboxylase isolated from crude rat liver extracts by means of immunoprecipitation with specific antibody invariably showed only the heavy polypeptide. The biotin content of the enzyme was found to be 1 mol per 237000 g protein. These results indicate that rat liver acetyl-CoA carboxylase, unlike bacterial and plant biotin enzymes, has only one kind of subunit, which has a molecular weight of 230000 and contains one molecular of biotin. Thus, the mammalian enzyme exhibits a highly integrated subunit structure.     

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.     

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.     

Regulation of rat liver fructose 2,6-bisphosphatase

An enzyme activity that catalyzes the hydrolysis of phosphate from the C-2 position of fructose 2,6-bisphosphate has been detected in rat liver cytoplasm. The S0.5 for fructose 2,6-bisphosphate was about 15 microM and the enzyme was inhibited by fructose 6-phosphate (Ki 40 microM) and activated by Pi (KA 1 mM). Fructose 2,6-bisphosphatase activity was purified to homogeneity by specific elution from phosphocellulose with fructose by specific elution from phosphocellulose with fructose 6-phosphate and had an apparent molecular weight of about 100,000, 6-phosphofructo 2-kinase activity copurified with fructose 2,6-bisphosphatase activity at each step of the purification scheme. Incubation of the purified protein with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in the incorporation of 1 mol of 32P/mol of enzyme subunit (Mr = 50,000). Concomitant with this phosphorylation was an activation of the fructose 2,6-bisphosphatase and an inhibition of the 6-phosphofructo 2-kinase activity. Glucagon addition to isolated hepatocytes also resulted in an inhibition of 6-phosphofructo 2-kinase and activation of fructose 2,6-bisphosphatase measured in cell extracts, suggesting that the hormone regulates the level of fructose 2,6-bisphosphate by affecting both synthesis and degradation of the compound. These findings suggest that this enzyme has both phosphohydrolase and phosphotransferase activities i.e. that it is bifunctional, and that both activities can be regulated by cAMP-dependent phosphorylation.     

Analysis of sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Comparison with sites phosphorylated by casein kinase-2 and the calmodulin-dependent multiprotein kinase

We have examined the sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Two tryptic peptides derived from the enzyme become more radioactive after treatment of 32P-labelled cells with insulin. One of these (T4a) accounts for a large part of the total increase in phosphate observed after insulin treatment, and comigrates with the peptide containing the sites phosphorylated in vitro by casein kinase-2. The other may correspond to the 'I' site peptide originally described by Brownsey and Denton in 1982: labelling of this peptide is stimulated at least threefold by insulin treatment, but it is a minor phosphopeptide and, even after insulin treatment, accounts for only about 2.5% of the enzyme-bound phosphate (equivalent to less than 0.1 mol phosphate/mol 240-kDa subunit). Two other major tryptic phosphopeptides (T1 and T4b) labelled in adipocytes do not change significantly in response to insulin, and comigrate with peptides containing sites phosphorylated in vitro by cyclic-AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase respectively. We have sequenced peptides T4a and T4b from acetyl-CoA carboxylase derived from control and insulin-treated adipocytes, and also after phosphorylation in vitro with casein kinase-2 and the calmodulin-dependent multiprotein kinase. The results show that T4a and T4b are forms of the same peptide containing phosphate groups on different serine residues: Phe-Ile-Ile-Gly-Ser4-Val-Ser5-Gln-Asp-Asn-Ser6-Glu-Asp -Glu-Ile-Ser-Asn-Leu-. Site 5 was phosphorylated by the calmodulin-dependent protein kinase and site 6 by casein kinase-2. Migration in the T4a position was exclusively associated with phosphorylation in site 6, irrespective of the presence of phosphate in sites 4 and 5. Sites 5 and 6 were partially phosphorylated in control adipocytes, and there were also small amounts of phosphate in site 4. On stimulation with insulin, phosphorylation appeared to occur primarily at site 6, thus accounting for the increase in 32P-labelling of T4a. We were unable to isolate sufficient quantities of the other insulin-sensitive peptide to determine its sequence. Our results are consistent with the idea that insulin activates either casein kinase-2, or a protein kinase which has the same specificity as casein kinase-2. The function of this modification is not clear, since phosphorylation by casein kinase-2 has no direct effect on acetyl-CoA carboxylase activity.     

Multiple phosphorylation of acetyl-CoA carboxylase in chick liver cells. A cyclic AMP-independent process.

When chick liver cells in monolayer culture were incubated with 32Pi in the presence of insulin, acetyl-CoA carboxylase became extensively labeled with 32Pi reaching a stoichiometry of 9 to 10 mol of phosphoryl group per mol of 240,000-dalton enzyme subunit. The covalently bound phosphate was found to be metabolically labile, turning over with a t1/2 of approximately 2 h (enzyme t1/2 approximately equal to 24 h). Addition of Bt2cAMP altered neither the rate nor extent of phosphorylation. Contrary to other reports, the fully phosphorylated acetyl-CoA carboxylase appears to be catalytically active.     

Evidence that activation of acetyl-CoA carboxylase by insulin in adipocytes is mediated by a low-Mr effector and not by increased phosphorylation.

The activation of acetyl-CoA carboxylase (measured in a crude supernatant fraction) caused by insulin treatment of adipocytes was completely unaffected by the addition of a large amount of highly purified protein phosphatase to the supernatant fraction. Under the same conditions the inhibition of acetyl-CoA carboxylase by adrenaline was totally reversed. Experiments with 32P-labelled adipocytes showed that insulin increased the total phosphorylation of acetyl-CoA carboxylase from 2.7 to 3.5 molecules of phosphate/240 kDa subunit, and confirmed that this increase was partially accounted for by phosphorylation within a specific peptide (the 'I-site' peptide). Protein phosphatase treatment of the crude supernatant fractions removed over 80% of the 32P radioactivity from the enzyme and removed all detectable radioactivity from the I-site peptide. The effect of insulin on acetyl-CoA carboxylase activity, but not the effect on phosphorylation, was lost on purification of the enzyme on avidin-Sepharose. The effect on enzyme activity was also lost if crude supernatant fractions were subjected to rapid gel filtration after treatment under conditions of high ionic strength, similar to those used in the avidin-Sepharose procedure. These results show that, although insulin does increase the phosphorylation of acetyl-CoA carboxylase at a specific site, this does not cause enzyme activation. They suggest instead that activation of the enzyme by insulin is mediated by a tightly bound low-Mr effector which dissociates from the enzyme at high ionic strength.