Mitochondrial acetyl-CoA acetyltransferase in various organs from rat: form patterns and coenzyme-A-mediated modification

The mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9), which is involved in the biosynthesis or degradation of ketone bodies, was directly demonstrated in organ extracts applying a two-step chromatography-immunoelectrophoresis method. In liver, the enzyme can be shown in at least three forms: in an unmodified state, designated as AAT, and in the CoASH-modified forms A1 and A2, in amounts of 51.5 +/- 5.0%, 39.4 +/- 4.8% and 9.1 +/- 2.7% (areas of immunoprecipitation), respectively. This pattern, which could not be altered by a treatment with glutathione, resembles that of mitochondrial acetyl-CoA acetyltransferase in extrahepatic tissues. However, the proportion of the unmodified enzyme (AAT) is lower as compared to those in other tissues such as brain (81.5 +/- 4.4%). CoASH-modification and transformation into modified forms, which equal naturally occurring forms, can be demonstrated in vitro with acetyl-CoA acetyltransferase from both liver and brain. Thus CoASH-modification of mitochondrial acetyl-CoA acetyltransferase seems to be a process of general importance.     

Mitochondrial acetyl-CoA acetyltransferase in liver and extrahepatic tissues: role of modification by coenzyme A

The influence of clofibrate and di(2-ethylhexyl)phthalate on mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA: acetyl-CoA C-acetyltransferase, EC 2.3.1.9), the rate-limiting ketogenic enzyme, which can be modified and inactivated by CoA, was investigated. In fed rats, both compounds induced a doubling of ketone bodies in the blood and, moreover, an increase by about 13% in the hepatic relative amount of the unmodified, i.e., the most active form of the enzyme (immunoreactive protein). This shift would account for an elevation of overall enzyme activity by about 5% only. Thus, the CoA modification of mitochondrial acetyl-CoA acetyltransferase did not explain the entire augmentation of ketone bodies. However, clofibrate and di(2-ethylhexyl)phthalate also increased the immunospecific protein and enzyme activity by approx. 2- and 3-fold, respectively. These effects were observed in liver, but not in several extrahepatic tissues.     

Immunochemical aspects, molecular and kinetic properties of multiple forms of acetyl-CoA acetyltransferase from rat liver mitochondria.

Acetyl-CoA acetyltransferase (EC 2.3.1.9) from rat liver mitochondria, which catalyzes the first step in the biosynthesis of ketone bodies, exists in two forms, designated transferase A and transferase B. Both transferases showed immunochemical cross-reactivity, but are immunologically unrelated to cytosolic acetyl-CoA acetyltransferase activity and the mitochondrial acetyl-CoA acyltransferase from rat liver. The transferases A and B were estimated to have molecular weights of 151 000 in the absence and 40 000 in the presence of sodium dodecyl sulfate. They differ with respect to charge states and multiplicity of forms as indicated by isoelectric focusing. Transferase A appeared in two forms with isoelectric points of 8.4 and 9.1, whereas transferase B represents a stable protein state with an isoelectric point of 9.0. Kinetic analysis of the reactions leading to acetoacetyl-CoA synthesis revealed saturation curves with multiple intermediary plateaus, indicating a complex kinetic behaviour. The data presented are interpreted as representing a microheterogeneity of forms of the mitochondrial acetyl-CoA acetyltransferase. The kinetic properties exhibited suggest a role for this microheterogeneity in the regulation of ketogenesis.     

Modulation of lyso-platelet-activating factor: acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor: acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca2+ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca2+. The effect of Ca2+ was on the apparent Km of the enzyme for the substrate acetyl-CoA without showing any significant effect on the Vmax of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca2+ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca2+, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca2+ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.     

Modulation of lyso-platelet-activating factor:acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca²⁺ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca²⁺. The effect of Ca²⁺ was on the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the enzyme for the substrate acetyl-CoA without showing any significant effect on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca²⁺ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca²⁺, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca²⁺ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.     

Phosphorylation of partially purified 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP.     

Partial purification and characterization of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

The enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) was purified from rat spleen approx. 1500-fold in 1.6% yield. The specific activity of the purified enzyme was 0.317 +/- 0.089 mumol/min per mg of protein (mean +/- S.D., n = 6). The Km for the substrate acetyl-CoA was 137 +/- 13 microM and the pH optimum was about 8. Incubation of the purified enzyme was 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine followed by electrophoresis resulted in the incorporation of radioactivity into a protein of Mr 29,000. The enzyme was most active towards 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine as substrate, 1-palmitoyl-2-lyso-glycero-3-phosphocholine being a poor substrate. In addition, the enzyme preferred acetyl-CoA to palmitoyl-CoA or oleoyl-CoA as substrate.     

Quantitation of ketogenesis in periportal and pericentral regions of the liver lobule

A method has been devised to quantitate rates of ketogenesis (acetoacetate + beta-hydroxybutyrate production) in discrete regions of the liver lobule based on changes in NADH fluorescence. In perfused livers from fasted rats, ketogenesis was inhibited nearly completely with either 2-bromoctanoate (600 microM) or 2-tetradecylglycidic acid (25 microM). During inhibition of ketogenesis, a linear relationship (r = 0.90) was observed between decreases in NADH fluorescence detected from the liver surface and decreases in ketone body production. NADH fluorescence was monitored subsequently from individual regions of the liver lobule by placing microlight guides on periportal and pericentral regions of the liver lobule visible on the liver surface. Rates of ketogenesis in sublobular regions were calculated from regional decreases in NADH fluorescence and changes in the rate of ketone body formation by the whole liver during infusion of inhibitors. In the presence of bromoctanoate, ketogenesis was reduced 80% and local rates of ketogenesis were decreased 31 +/- 4 mumol/g/h in periportal areas and 28 +/- 3 mumol/g/h in pericentral regions. Similar results were observed with tetradecylglycidic acid. Therefore, it was concluded that submaximal rates of ketogenesis from endogenous, mainly long-chain fatty acids are nearly equal in periportal and pericentral regions of the liver lobule in liver from fasted rats. Rates of ketogenesis and NADH fluorescence were strongly correlated during fatty acid infusion. Infusion of 250 microM oleate increased NADH fluorescence maximally by 8 +/- 1% over basal values in periportal regions and 17 +/- 4% in pericentral areas. Local rates of ketogenesis, calculated from these changes in fluorescence, increased 35 +/- 6 mumol/g/h in periportal areas and 55 +/- 5 mumol/g/h in pericentral regions. Thus, oleate stimulated ketogenesis nearly 60% more in pericentral than in periportal regions of the liver lobule.     

Regulation of rat liver acetyl-CoA carboxylase. Stimulation of phosphorylation and subsequent inactivation of liver acetyl-CoA carboxylase by cyclic 3':5'-monophosphate and effect on the structure of the enzyme.

The effects of citrate and cyclic AMP on the rate and degree of phosphorylation and inactivation of rat liver acetyl-CoA carboxylase were examined. High citrate concentrations (10 to 20 mM), which are generally used to stabilize and activate the enzyme, inhibit phosphorylation and inactivation of carboxylase. At lower concentrations of citrate, the rate and degree of phosphorylation are increased. Furthermore, phosphorylation and enzyme inactivation are affected by cyclic AMP under these conditions. At high citrate concentrations, cyclic AMP has little or no effect on inactivation and phosphorylation of acetyl-CoA carboxylase. Phosphorlation and inactivation of carboxylase is accompanied by depolymerization of the polymeric form of the enzyme into intermediate and protomeric forms. Depolymerization of carboxylase requires the transfer of the gamma-phosphate group from ATP to carboxylase. Inactivation occurs in the absence of CO2, which indicates that phosphorylation of the enzyme is the cause of inactivation and depolymerization, i.e. carboxylation of the enzyme is not responsible for inactivation of the enzyme.     

Regulation of cytoplasmic acetyl-CoA and acetoacetyl-CoA synthetases by dexamethasone phosphate in rat liver and adipose tissue.

The activity of cytoplasmic acetyl-CoA synthetase and acetoacetyl-CoA synthetase in both liver and adipose tissue was measured in five groups of rats : fed, fasted, fasted and re-fed, fasted, re-fed and injected with dexamethasone during the re-feeding period, fed and injected with dexamethasone. Fasting was found to have a strong inhibitory effect on the activity of the two enzymes in both adipose tissue and liver, which could be abolished by re-feeding. In adipose tissue, dexamethasone prevented this re-establishment of normal enzyme activity but was without effect in the liver. It can therefore be concluded that dexamethasone inhibits the synthesis of acetyl-CoA and acetoacetyl-CoA synthetases in adipose tissue.     

Regulation of ketogenesis during the suckling-weanling transition in the rat. Studies with isolated hepatocytes.

The rates of ketogenesis from endogenous substrates, butyrate or oleate, have been measured in isolated hepatocytes from suckling and weanling rats. Ketogenesis from endogenous substrate and from oleate decreased on weaning, whereas the rate from butyrate remained unchanged. It is concluded that the major site of regulation of ketogenesis during this period of development involves the disposal of long-chain fatty acyl-CoA between the esterification and beta-oxidation pathways. Modulators of lipogenesis [dihydroxyacetone and 5-(tetradecyloxy)-2-furoic acid] did not alter the rate of ketogenesis in hepatocytes from suckling rats, and it is suggested that this is due to the low rate of lipogenesis in these cells. Hepatocytes from fed weanling rats have a high rate of lipogenesis and evidence is presented for a reciprocal relationship between ketogenesis and lipogenesis, and ketogenesis, and esterification in these cells. Dibutyryl cyclic AMP stimulated ketogenesis from oleate in hepatocytes from fed weanling rats, even in the presence of an inhibitor of lipogenesis [5-(tetradecyloxy)-2-furoic acid], but not in cells from suckling rats. It is suggested that cyclic AMP may act via inhibition of esterification and that in hepatocytes from suckling rats ketogenesis is already maximally stimulated by the high basal concentrations of cyclic AMP [Beaudry, Chiasson & Exton (1977) Am. J. Physiol. 233, E175--E180].     

Heat activation of rat liver acetyl-CoA carboxylase in vitro.

Acetyl-CoA carboxylase in rat liver homogenates was activated in vitro in a time- and temperature-dependent manner. The activity of acetyl-CoA carboxylase in rat liver preparations was determined in a 1-min assay to preclude the possibility of citrate activation of the enzyme during the assay period. Activation of the enzyme occurred more rapidly in liver preparations continuously maintained at ambient or greater temperatures than in homogenates of liver which had been chilled. High speed supernatant (105,000 X g, 60 min) did not heat-activate, and reconstitution of the heat-activatable 27,000 X g, 20-min, fraction by recombining the high speed pellet with the high speed supernatant only partially restored the heat activatability. Elution of the 105,000 X g supernatant from Sephadex G-25 resulted in an enzyme preparation which was heat-activatable. Addition of boiled 105,000 X g supernatant to the Sephadex G-25-treated enzyme again prevented heat activation. Dilution of the enzyme 5-fold did not prevent heat activation.     

Modulation of lyso-platelet activating factor:acetyl-CoA acetyltransferase from rat splenic microsomes. The role of cyclic AMP-dependent protein kinase

Incubation of rat splenic microsomes with the catalytic subunit of cyclic AMP-dependent protein kinase in the presence of Mg-ATP stimulated 2-3-fold lyso-platelet-activating factor:acetyltransferase activity. This activation was due to an increase in the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the acetylation reaction, whereas the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for acetyl-CoA was not affected. The ATP derivative, AMPPNP, could not replace ATP and preincubation of the microsomes with the heat-stable inhibitor of protein kinase prevented the activation by Mg-ATP obtained in the presence of the protein kinase. Activation of the acetylation reaction by the protein kinase was reversible. Evidence is provided that the reversal of activation is due to dephosphorylation of the enzyme. These data provide evidence that in vitro lyso-platelet-activating factor:acetyltransferase from splenic microsomes is regulated by phosphorylation.     

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.     

Mitochondrial acetyl-CoA carboxylase. Time course of mobilization/activation in liver of refed rats.

Fasted (48 h) rats were killed at 0, 2, 4, 6, 8, 12, 16, 20 and 24 h after they were refed on a high-carbohydrate diet. An increase in the maximal activity and quantity of cystolic acetyl-CoA carboxylase was found in liver of refed rats after a lag time of about 8 h. The increased quantity of cytosolic enzyme was attributable primarily to mobilization of mitochondrial storage forms and not to substantial increase in the rate of synthesis of acetyl-CoA carboxylase.     

Periportal zonation of the cytosolic acetyl-CoA synthetase of male rat liver.

Several important metabolic functions of the mammalian liver have been shown to be located in zones with respect to the complex microcirculation of the organ. The zonal distribution of the cytosolic component of the acetyl-CoA synthetase activity has been investigated using the dual-digitonin-pulse-perfusion technique, which allows highly zone-selective sampling of cytosol from the periportal and perivenous zone of rat liver. Approximately 80% of the cytosolic enzymes are eluted from the hepatocytes in the periportal and perivenous sub-zones affected by digitonin, while less than 1% of the glutamate dehydrogenase activity (a marker enzyme of the mitochondrial compartment) is eluted. A twofold higher activity of the cytosolic form of acetyl-CoA synthetase is found in the periportal zone compared to the perivenous zone in fed male rats. Following a fasting/refeeding transition, this activity gradient is abolished in a manner similar to that observed for the enzyme acetyl-CoA carboxylase. Since the latter enzyme is utilizing the product of acetyl-CoA synthetase, acetyl-CoA, the similarity in the observed regulation suggests a functional coupling between cytosolic acetate activation and fatty-acid synthesis.     

Induction of carnitine acetyltransferase by clofibrate in rat liver.

Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine O-acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold.