Binding of ATP citrate lyase to the microsomal fraction of rat liver.

Purified rat liver ATP citrate lyase is shown to bind to the microsomal fraction of rat liver. Under the same conditions the enzyme does not bind significantly to the mitochondrial fraction or to the outer membrane prepared from the mitochondrial fraction. The binding component of the microsomal fraction is further identified as the endoplasmic reticulum, and a protein component of the membrane is involved in binding. Binding decreases with increasing salt concentration. It requires more than 50 mM potassium phosphate or 60 mM potassium chloride to decrease binding significantly whereas complete inhibition of binding is observed in the presence of 0.01 mM CoA. The binding capacity of the microsomal fraction is shown to be high enough to allow most, if not all, the ATP citrate lyase present in rat liver to be bound to the microsomal fraction even when the enzyme has been induced over 10-fold by dietary manipulations. No significant difference has been found when comparing the binding of the phospho and dephospho forms of ATP citrate lyase.

Phosphorylation of dephospho-ATP citrate lyase by the catalytic subunit of cAMP-dependent protein kinase.

The native form of ATP citrate lyase (2 mol of phosphate/tetramer) and the dephospho-ATP citrate lyase (phosphate-free) purified to homogeneity from rat liver, are phosphorylated by ATP and by the catalytic subunit of cAMP-dependent protein kinase from rabbit muscle. A total of 2 mol of phosphate/tetramer were incorporated into native enzyme, while with the dephospho form, 4 mol of phosphate were incorporated. The phosphopeptides resulting from trypsin treatment which were isolated from phosphorylated forms of both native enzyme and the dephospho enzyme were similar. The ATP citrate lyase, phosphorylated to an extent of 4 mol of phosphate/tetramer, has the same Vmax as the native enzyme (2 mol of phosphate/tetramer). Native ATP citrate lyase, trypsin-treated to remove the phosphopeptide, could not be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase from rabbit muscle, suggesting a common trypsin-sensitive specific phosphorylation site. The phosphorylation rate varied with pH in potassium phosphate, imidazole/HCl, and Tris/HCl buffers. Divalent cations were essential for the activity of the protein kinase. The apparent Km value for ATP was found to be 50 microM.

Coenzyme A requirement for the termination reaction of rat liver fatty acid synthetase.

Highly purified rat liver fatty acid synthetase is completely inhibited when assayed in the presence of a coenzyme A-depleting system such as that catalyzed by phosphotransacetylase, acetyl-CoA synthetase, or ATP citrate lyase. The addition of free CoA causes a reversal of this inhibition. The requirement of free CoA is the same whether acetyl-CoA or butyryl-CoA serves as the primer for fatty acid synthetase. The CoA-depleted and thus inactive fatty acid synthetase system can be reactivated by the addition of a rat brain thioesterase or a rat mammary gland thioesterase II preparation. This reactivation appears to occur in the absence of free CoA. Long chain fatty acids (mainly palmitate) are formed by the thioesterase reactivated system. These results suggest that CoA is required for the termination of the fatty acid synthetase reaction. Possible mechanisms are discussed.

Coenzyme A is required for rat liver fatty acid synthetase activity.

Inhibition of highly purified rat liver fatty acid synthetase occurs when it is assayed in the presence of the ATP citrate lyase reaction components. Citrate, Mg2+, ATP, and ATP citrate lyase were all necessary for the inhibition to take place. Inhibition was prevented by hydroxycitrate, a competitive inhibitor for ATP citrate lyase. The length of time for the onset of inhibition to take place was proportional to the ratio of ATP citrate lyase activity to the fatty acid synthetase activity. The inhibition was reversed by the addition of coenzyme A. This indicates a reaction mechanism for fatty acid synthetase which involves free coenzyme A. Two possible roles for CoA are discussed, one as an allosteric activator and the other in the cleavage of palmitoyl enzyme in the last step of the reaction.

Binding of specific ATP citrate lyase and fatty acid synthetase antibodies to heavy populations of rat liver polysomes.

The polysome fractions involved in the synthesis of the rat-liver inducible lipogenic enzymes, ATP citrate lyase and fatty acid synthetase, were identified by their binding of radioiodinated specific antibodies to enzyme. Both of these populations of specific polysomes were shown to be markedly heavier than specific polysomes involved in albumin synthesis. The quanity of antibody bound to the lipogenic enzyme-related polysomes was markedly affected by the dietary status of the animal. A dietary regimen which induced ipogenesis resulted in a tenfold increase in the hepatic activities of these enzymes found in normally fed animals. The radioactivity bound to hepatic polysomes of induced rats was likewise greater than tenfole higher, presumably reflecting an increase in the number of polysomes active in enzyme synthesis. The fasting state resulted in lower hepatic enzyme activity than normal and correspondingly less binding of ATP citrate lyase and fatty acid synthetase antibodies to the heavy polysomes of the sucrose gradient.

Identification of ATP citrate lyase as a phosphoprotein.

ATP citrate lyase has been purified from rat liver by a new procedure which results in high yields of an intact and stable enzyme. The pure lyase (specific activity approximately equal to 10 at 25 degrees C) exhibits a single protein band upon sodium dodecyl sulfate (SDS)-gel electrophoresis (Mr = 110,000). This procedure minimizes protease degradation that usually occurs when the enzyme is isolated by previously described isolation methods. In addition, the lyase is shown to be a phosphoprotein. 32P-labeled lyase has been purified from rat liver following an intraperitoneal injection of inorganic [32P]phosphate into the animals. It has been demonstrated that this phosphate (structural phosphate) behaves as a serine phosphate and is not the same as the enzyme-bound phosphate (catalytic phosphate) that is derived from ATP during the lyase reaction. There are 2 structural phosphate residues for each enzyme tetramer molecule. Removal of the structural phosphate has been accomplished using a partially purified phosphatase derived from rat liver. The dephospholyase has the same Vmax as the native phosphoenzyme. Evidence indicates that the structural phosphate resides in a protease-sensitive region of the native enzyme.

Lipoamide dehydrogenase in serum: a preliminary report.

Lipoamide dehydrogenase was identified in serum and the optimal conditions for its assay at 30 degrees C were defined. The pH optimum in tris(hydroxymethyl)aminomethane buffer is 7.8, and activity is inhibited if buffer concentration exceeds 100 mmol/liter. Saturating concentrations of the substrates NAD+ and lipoamide are 3 mmol/liter and 5 mmol/liter, respectively. Activity is decreased eightfold when lipoic acid is substituted for lipoamide. Activity is linearly related to enzyme concentration up to limiting absorbance change of 0.300 at 340 nm, and both within-day and day-to-day precision are satisfactory. Data suggest a normal range (2 SD) of 3-19 kU/liter. The highest value measured in serum was 473 kU/liter. A correlation with direct bilirubin concentrations (r equals 0.435, P less than 0.01) was found.

Alpha-keto acid dehydrogenase complexes. XI. Comparative studies of regulatory properties of the pyruvate dehydrogenase complexes from kidney, heart, and liver mitochondria.

The activity of the multienzyme pyruvate dehydrogenase complexes, isolated from mitochondria of beef kidney, beef heart, and pork liver, is regulated by phosphorylation and dephosphorylation. Phosphorylation and concomitant inactivation of each of the three complexes are catalyzed by an ATP-specific kinase, and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase. The phosphatase has been separated from the other component enzymes of each pyruvate dehydrogenase complex, and the three phosphatases are functionally interchangeable. The kinase has been isolated from the beef kidney complex, and it is functional with the beef heart and pork liver complexes. ADP is competitive with ATP, and the ADP effect is more pronounced with the kidney kinase than with the liver and heart kinases. Pyruvate protects strongly the heart and liver pruvate dehydrogenase complexes and, to a lesser extent, the kidney complex against inactivation by ATP. Pyruvate apparently exerts its effect on the pyruvate dehydrogenase component of the complex, rather than on the kinase.

Alpha-keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation.

This paper reports the discovery that the activity of the multienzyme pyruvate dehydrogenase complex from beef kidney mitochondria is regulated by a phosphorylation-dephosphorylation reaction sequence. The site of this regulation is the pyruvate dehydrogenase component of the complex. Phosphorylation and concomitant inactivation of pyruvate dehydrogenase are catalyzed by an ATP-specific kinase (i.e., a pyruvate dehydrogenase kinase), and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase (i.e., a pyruvate dehydrogenase phosphatase). The kinase and the phosphatase appear to be regulatory subunits of the pyruvate dehydrogenase complex.