Richard E. Dugan

Regulation of the diurnal rhythm of rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity by insulin, glucagon, cyclic AMP and hydrocortisone☆

Abstract Rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity and the amplitude of the diurnal variation of this enzyme are progressively reduced to very low levels within 1 week after the onset of diabetes induced by streptozotocin. Daily insulin therapy to 7-day diabetic rats restores the activity and the amplitude of this diurnal variation in enzyme activity to near-normal levels within 4 days. Insulin also produces a rapid 2-hr stimulation of the reductase activity in diabetic rats to the level found in normal animals at that time of day regardless of the duration of diabetes. Hence, insulin is required for the diurnal rise of reductase activity in rat liver. Glucagon, dibutyryl cyclic AMP, and hydrocortisone, in contrast, markedly inhibit the diurnal rise of reductase activity in normal rats. Therefore, the relative concentrations of insulin, glucagon, and glucocorticoids are important in the regulation of the diurnal variation of hepatic reductase activity.

Factors affecting the diurnal variation in the level of β-hydroxy-β-methylglutaryl coenzyme A reductase and cholesterol-synthesizing activity in rat liver☆

Abstract It has been demonstrated through kinetic analysis that the diurnal variation in β-hydroxy-β-methylglutaryl coenzyme A reductase activity in rat liver microsomes is produced by changes in the rate of enzyme formation. The rate constant for enzyme breakdown is essentially the same for the periods of diurnal rise and diurnal fall in enzyme activity; whereas the rate constant for enzyme formation for the period of diurnal rise is 7- to 10-fold that for the period of diurnal fall. The location of the rise and fall of enzyme activity in the daily cycle is feeding and not light dependent. However, the diurnal rise in enzyme activity can be elicited without feeding at the time of day that it ordinarily occurs. The diurnal variation in cholesterol synthesis from acetate, when animals are fed, may be controlled by the quantity of an enzyme of the cholesterol pathway that acts prior to the reductase. Under fasting conditions when β-hydroxy-β-methylglutaryl coenzyme A reductase activity increases similar to the increase under feeding conditions, this enzyme is not rate limiting in the synthesis of cholesterol from acetate.

Comparative effects of dietary regimens on the levels of enzymes regulating the synthesis of fatty acids and cholesterol in rat liver.

Abstract The activities, in subcellular fractions of rat liver, for acetyl-CoA carboxylation, fatty acid synthesis, and β-hydroxy-β-methylglutaryl-CoA reduction and for the overall conversion of acetate to cholesterol were determined for animals in different nutritional states. Acetyl-CoA carboxylase and fatty acid synthetase activities increased or decreased together whenever the nutritional state was changed. A similar relationship was observed between β-hydroxy-β-methylglutaryl-CoA reductase activity and the synthesis of cholesterol from acetate. Feeding a fat-free diet after either a normal or fasted regimen increased the activity of acetyl-CoA carboxylase and fatty acid synthetase but suppressed the activities for β-hydroxy-β-methylglutaryl CoA reduction and cholesterol synthesis. Thus, the quantity of fat in the diet apparently regulates the levels of activity of the key enzymes of two divergent pathways in an opposite manner. The levels of these enzymes in turn control the proportion of acetyl-CoA used by each pathway. When a diet containing 2% cholesterol is fed, the levels of β-hydroxy-β-methylglutaryl-CoA reductase activity and cholesterol synthesis in liver are markedly depressed. In contrast, the levels of acetyl-CoA carboxylase and fatty acid synthetase activities are unaffected. These results suggest that cholesterol and fat exert their effects on the cholesterol-synthesizing and fatty acid-synthesizing pathways by different mechanisms.

Regulation of hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase by the interplay of hormones☆

We have previously established that insulin causes a marked and rapid stimulation of hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase activity in normal and diabetic rats [Biochem. Biophys. Res. Commun.50, 504 (1973)], whereas l-triiodothyronine stimulates the reductase activity to supranormal levels in hypophysectomized rats two days after administration [Proc. Nat. Acad. Sci. (1974) In press]. In the present investigation it is demonstrated that the stimulation of the reductase activity in hypophysectomized-diabetic rats requires the mediation of both insulin and l-triiodothyronine. Neither hormone alone is effective. The rapid stimulation of reductase activity by insulin and the delayed stimulation elicited by l-triiodothyronine are both inhibited by either glucagon or hydrocortisone. Thus, an interplay of hormones regulates reductase activity and consequently cholesterol biosynthesis.

High-performance liquid chromatography of coenzyme A esters formed by transesterification of short-chain acylcarnitines: diagnosis of acidemias by urinary analysis

A protocol for the identification and estimation of short-chain esters of carnitine is described; it is useful for the diagnosis of acidemias. By this method, carnitine esters in urine are converted to coenzyme A esters enzymatically with carnitine acetyltransferase (CAT): short-chain acylcarnitine + CoA cat in equilibrium short-chain acyl-CoA + carnitine. The coenzyme A esters are separated by high-performance liquid chromatography using a radial compression system with a C8 Radial-Pak cartridge and a mobile phase containing 0.025 M tetraethylammonium phosphate in a linear gradient of 1 to 50% methanol. Coenzyme A esters are quantitated by integrator determination of the area under the 254-nm absorption peaks. Enzymatic conversion approaches 100% for acetyl and propionyl esters except in the presence of high levels of free carnitine, which lowers the proportion of ester as acyl-CoA at equilibrium. However, since acidemia patients produce urine low in free carnitine, this problem is minimized. The method is rapid and simple and identifies propionic, methylmalonic, and isovaleric acidemias.

Evidence for catalytic site cysteine and histidine by chemical modification of β-hydroxy-β-methylglutaryl-coenzyme A reductase

Summary S-(4-Bromo-2,3-dioxobutyl)-coenzyme A inactivates both yeast and rat liver β-hydroxy-β-methylglutaryl-coenzyme A reductase. The inactivation is irreversible, complete in 15 s, and proportional to the concentration of the reagent. β-Hydroxy-β-methylglutaryl-CoA provides protection against inactivation, whereas NADPH does not. Inactivation is attributed to reaction with an essential cysteine at the β-hydroxy-β-methylglutaryl-CoA binding site. Experiments with other active site-directed reagents confirm the involvement of a cysteine and support the presence of an active-site histidine, but rule out the participation of arginine or serine.

Hog liver squalene synthetase: The partial purification of the particulate enzyme and kinetic analysis of the reaction☆

Abstract Hog liver squalene synthetase has been partially purified, without solubilization, by sonication of microsomes and subsequent gel filtration of the sonicated protein. A decrease in microsomal fragment size and an increase in soluble protein were observed as sonication time and power increased. The specific activity of squalene synthetase in the microsomal subfragments increased as the smaller particles and enzymatically inactive soluble protein were produced by sonication. Kinetic analysis with sonicated microsomal protein suspensions indicates that the reaction mechanism for the formation of squalene from farnesyl pyrophosphate is ping-pong. An irreversible step separates the addition of the first from the addition of the second molecule of farnesyl pyrophosphate to the enzyme. Another irreversible step separates the addition of farnesyl pyrophosphate from the addition of reduced nicotinamide adenine dinucleotide phosphate to the enzyme. From the results of kinetic analyses a partial scheme is proposed for the order of binding of substrates and release of products from the enzyme forms of squalene synthetase.

Affinity purification of anti-pigeon liver fatty acid synthetase immunoglobulin and comparative immunoreactivity of the catalytic reactions☆

Abstract Rabbit anti-pigeon liver fatty acid synthetase antibody was prepared by affinity chromatography on Sepharose-fatty acid synthetase to near monospecificity (98% or more) as shown by immunodiffusion plates and rocket immunoelectrophoresis. Immunotitrations of the highly purified monospecific antibody against the overall activity and partial activities of fatty acid synthetase were then carried out. Only 6 mol of antibody/mol of enzyme was required to inactivate overall fatty acid synthetase activity and the condensation reaction, while 12 to 18 mol were required to partially inactivate the β-ketoacyl reductase and the malonyl- and acetyl-CoA transferases. Palmitoyl-CoA thioesterase (deacylase) activity was not inhibited by the antibody. The degree of inactivation of the partial reactions by antibody was not affected by dissociation of the fatty acid synthetase. Immunoprecipitation of the enzyme indicated that there are approximately 35 immunoreactive sites on the fatty acid synthetase molecule. The possible implications of these results to an understanding of the structural organization of pigeon liver fatty acid synthetase and its antigenic determinants are discussed.

A malonyl-CoA-binding protein from liver

A soluble protein that binds malonyl-CoA without requiring cofactors has been purified from rat liver. Until saturated, it competes with fatty acid synthetase for free malonyl-CoA, temporarily reducing the rate of fatty acid synthesis at low levels of malonyl-CoA, as in fatty acid synthetase--coupled assays for acetyl-CoA carboxylase. These assays yield low estimates for carboxylase activity with crude and partially purified homogenates containing the malonyl-CoA-binding protein. The protein does not inhibit assays for carboxylase activity that measure nonvolatile radioactivity incorporated from bicarbonate or NADH oxidation coupled to ADP formation. It has an Mr of 180,000 and a subunit of 90,000. It has a lower affinity for ATP, ADP, and acetyl-CoA and none for CO2 or fatty acid synthetase. No enzymatic function has been identified. The protein may regulate malonyl-CoA-binding enzymes.

Membrane-Bound Enzymes of Sterol Metabolism

This chapter will be limited to enzymes of animal origin, and it will not, therefore, include those that make plant steroids, such as ergosterol or the vitamin D family of compounds. Steroids of animal origin are either precursors of cholesterol or have cholesterol as a common progenitor. Hence we will be concerned with the membrane-bound enzymes that catalyze specific reaction steps in the biosynthesis of cholesterol or the transformation of cholesterol to bile acids or steroid hormones. Microsomal enzymes that participate in sterol esterification or the hydrolysis of sterol esters will not be reviewed. The subject of cholesterol esterification has been reviewed previously (Norum, 1974). Studies on esterases, transferases, and hydrolases of sterol ester metabolism are cited in recent publications by Stokke (1974) and Sakamoto et al. (1974).