Purification and properties of Escherichia coli coenzyme A-transferase.
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Acetoacetates↗
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Escherichia coli coenzyme A-transferase: kinetics, catalytic pathway and structure.
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Acetates↗
3-oxo acid coenzyme A-transferase in normal and diabetic rat muscle.
The amounts of succinyl-CoA--3-oxo acid CoA-transferase (EC 2.8.3.5) decrease progressively in skeletal muscle in streptozotocin-diabetic rats, reaching after 10 days about 50% of the value in normal rat muscle. Electrofocusing studies indicate the occurrence of partial proteolysis of the enzyme in diabetic muscle. However, several functional parameters relating to acetoacetate utilization, including substrate inhibition, are quite similar for muscle transferase preparations from normal and diseased rats. The development of pathological ketoacidosis is discussed in the light of these observations.
Acetoacetates↗
Purification and properties of succinyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney.
CoA-transferase (succinyl-CoA-3-oxo acid CoA-transferase, EC 2.8.3.5) isolated from sheep kidney was purified to homogeneity. The purified enzyme has a specific activity of approx. 200 units/mg. A mol.wt. of 110000 was obtained by gel filtration on Sephadex G-200, and a lower mol.wt. of 102000 was determined by analytical ultracentrifugation. A sedimentation coefficient of 5.6S was also determined. A subunit mol.wt. of 56000 was obtained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing of sheep kidney extracts indicated the presence of a single band of CoA-transferase activity with pI9.0. However, isoelectric focusing of purified CoA-transferase showed the presence of two peaks of CoA-transferase activity with pI values of 8.7 and 8.4, suggesting the presence of proteolytic activity during purification. Evidence for sheep kidney CoA-transferase being a dimer of two identical subunits has been obtained from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the amino acid composition, peptide 'mapping' and N-terminal analysis.
Acetoacetates↗
A new case of succinyl-CoA: acetoacetate transferase deficiency.
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Acidosis↗
Enzymes that metabolize acyl-coenzyme A in the monkey--their distribution, properties and roles in an alternative pathway for the excretion of nitrogen.
1. In various tissues from the monkey (Macaca fuscata), acyl-coenzyme A (CoA) hydrolase activities were found to be widely distributed within a 2-10 times range and present in liver cytosol having mol. wt of ca 60,000. 2. Acyl-CoA: amino acid N-acyltransferase activity were 4-250 times higher in liver and kidney than in other tissues, even no activity in heart, lung, and plasma. 3. The transferases abounded in liver mitochondria, being distributed evenly between the intracristate space, the inner membrane, and the matrix. 4. The partially purified transferases with benzoyl-CoA or phenylacetyl-CoA as substrates were shown to have mol. wt of ca 30,000 and reacted only with glycine or L-glutamine, respectively. 5. No amino acid tested had any effects on the enzyme as either inhibitors or activators. 6. These results suggest that the enzymes that metabolize acyl-CoA constitute an alternative pathway for the excretion of nitrogen.
Acyl Coenzyme A↗
Ketogenesis in vertebrate livers.
1. The hypothesis is advanced that a gluconeogenic organ such as the liver would evolve to oxidise fatty acids as its source of ATP for gluconeogenesis. It is also argued that such an organ might, in the light of current knowledge, be expected to be ketogenic. The animals investigated were lamprey, rainbow trout, eel, toad, axolotl, lizard and rat. 2. The respiratory quotients of liver slices from all animals was close to 0.74. Ketone bodies were produced from butyrate by all livers excepting the lamprey and ketone bodies were present in all blood samples examined. 3. There was no convincing evidence that direct deacylation of acetoacetyl CoA was important in any liver. HMGCoA synthase activity could not be found in the livers of the lamprey and eel. This enzyme was present in livers of the other animals. There was a large amount of acetoacetyl CoA-succinate transferase in the livers of the rainbow trout and eel, but only small amounts in the higher animals. 4. It is suggested that, initially the transferase was the important ketogenic pathway and the HMGCoA pathway evolved later.
Acyl Coenzyme A↗
The effects of dietary fiber feeding on cholesterol metabolism in rats.
The flux through the sterol biosynthetic pathway was studied in hepatocytes isolated from male Sprague-Dawley rats fed diets containing one of four fiber sources: cellulose, pectin, oat bran and wheat bran. Sterol synthesis measured by the incorporation of tritiated water or [2-14C]mevalonic acid was not inhibited in hepatocytes isolated from animals fed diets containing cellulose, pectin, oat bran or wheat bran when compared to animals fed a fiber-free diet. Based on these results, it is concluded that the intake of fiber has no inhibitory effect on endogenous sterol synthesis. In fact, in comparison to that in fiber-free controls, sterol synthesis was markedly elevated in pectin- and wheat bran-fed animals. In the case of the pectin-treated animals, the higher synthetic rate corresponded to an increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.
Animals↗
Effect of microgravity on metabolic enzymes of individual muscle fibers.
Eleven enzymes were measured in individual fibers of soleus and tibialis anterior (TA) muscles from two flight and two control (synchronous) animals. There were five enzymes of glycogenolytic metabolism: phosphorylase, glucose-6-phosphate isomerase, glycerol-3-phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase (group GLY); five of oxidative metabolism: citrate synthase, malate dehydrogenase, beta-hydroxyacyl-CoA dehydrogenase, 3-ketoacid CoA-transferase, and mitochondrial thiolase (group OX); and hexokinase, subserving both groups. Fiber size (dry weight per unit length) was reduced about 35% in both muscles. On a dry weight basis, hexokinase levels were increased 100% or more in flight fibers from both soleus and TA. Group OX enzymes increased 56-193% in TA without significant change in soleus. Group GLY enzymes increased an average of 28% in soleus fibers but underwent, if anything, a modest decrease (20%) in TA fibers. These changes in composition of TA fibers were those anticipated for a conversion of about half of the originally predominant fast glycolytic fibers into fast oxidative glycolytic fibers. Calculation on the basis of fiber length, rather than dry weight, gave an estimate of absolute enzyme changes: hexokinase was still calculated to have increased in both soleus and TA fibers, but only by 50 and 25%, respectively. Three of the OX enzymes were, on this basis, unchanged in TA fibers, but 3-ketoacid CoA-transferase and thiolase had still nearly doubled, whereas TA GLY enzymes had fallen about 40%. In soleus fibers, absolute levels of OX enzymes had decreased an average of 25% and GLY enzymes were marginally decreased.
3-Hydroxyacyl CoA Dehydrogenases↗
The oxidation of 3-hydroxybutyrate in developing rat jejunum.
Ketone bodies and glutamine are the primary oxidative substrates in the intestine of fasting adult rats. Because suckling rats consume the majority of their calories in the form of lipid and as a result have elevated blood ketone concentrations similar to fasting adult rats, we examined the role of ketone oxidation as a source of energy in the intestine of suckling rats. In suckling rats the rate of [3-14C]DL-3-hydroxybutyrate oxidation in intestinal tissue slices, enterocytes, and intestinal mitochondria was 0.15 +/- 0.01 nmol/h/mg wet wt, 8.6 +/- 1.1 nmol/h/mg protein and 8.3 +/- 1.3 nmol/h/mg protein, respectively. In suckling rats the rate of intestinal oxidation of [3-14C]DL-3-hydroxybutyrate was 75% lower than the rate in weaned rats in intestinal tissue slices, 55% lower in enterocytes, and 50% lower in intestinal mitochondria. The activity of two enzymes involved in ketone oxidation, 3-hydroxybutyrate dehydrogenase, and acetoacetyl-CoA thiolase was 38% and 55% higher in intestinal homogenates of suckling rats compared with adult rats. In contrast, 3-oxoacid CoA-transferase activity, an enzyme also involved in 3-hydroxybutyrate oxidation, was one third that of adults. These studies indicate that intestinal oxidation of 3-HB occurs in suckling rat pups, but is lower than the rate of oxidation in the intestine of weaned rats.
3-Hydroxybutyric Acid↗
3-Oxo acid coenzyme A transferase activity in brain and tumors of the nervous system.
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Animals↗
Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum.
A new coenzyme A (CoA)-transferase from the anaerobe Clostridium aminobutyricum catalyzing the formation of 4-hydroxybutyryl-CoA from 4-hydroxybutyrate and acetyl-CoA is described. The enzyme was purified to homogeneity by standard techniques, including fast protein liquid chromatography under aerobic conditions. Its molecular mass was determined to be 110 kDa, and that of the only subunit was determined to be 54 kDa, indicating a homodimeric structure. Besides acetate and acetyl-CoA, the following substrates were detected (in order of decreasing kcat/Km): 4-hydroxybutyryl-CoA, butyryl-CoA and propionyl-CoA, vinyl-acetyl-CoA (3-butenoyl-CoA), and 5-hydroxyvaleryl-CoA. In an indirect assay the corresponding acids were also found to be substrates; however, DL-lactate, DL-2-hydroxybutyrate, DL-3-hydroxybutyrate, crotonate, and various dicarboxylates were not.
Carbon-Carbon Double Bond Isomerases↗
Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida.
beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified.
Adipates↗
Ketone body utilization and its metabolic effect in resting muscles of normal and streptozotocin-diabetic rats.
By using an in situ rat hindquarter perfusion, we evaluated ketone body utilization and its metabolic effects in the resting muscle of 24 h fasted normal and streptozotocin (STZ)-diabetic rats. Under the perfusion with ketone body-supplementation (1 mM each of acetoacetic acid (AcAc) and 3-hydroxybutyric acid (3-OHB], the AcAc and 3-OHB uptake of STZ-diabetic rats was significantly (P less than 0.05) smaller than that of normal rats. This might be explained by the low enzyme activity of 3-oxoacid CoA transferase demonstrated in the hindlimb muscles of STZ-diabetic rats and this reduced ketone body uptake would be one of the causes of the development of diabetic ketoacidosis. The glucose uptake and the phosphofructokinase (PFK) activity of normal rats were significantly (P less than 0.05) higher than those of STZ-diabetic rats. In both normal and STZ-diabetic rats, the glucose utilization and PFK activity of the muscles in the ketone body-supplemented condition were significantly (P less than 0.05) lower than those in the non-supplemented condition. This inhibition of glucose utilization by ketone bodies should be due to the mechanism by which the oxidation of ketone bodies inhibits PFK in the muscle.
Animals↗
Properties of 5-hydroxyvalerate CoA-transferase from Clostridium aminovalericum.
5-Hydroxyvalerate CoA-transferase from Clostridium aminovalericum, strain T2-7, was purified approximately 100-fold to homogeneity. The molecular mass of the native enzyme was determined by three different methods to be 178 +/- 11 kDa; that of the subunit was 47 kDa, indicating a homotetrameric structure. The following CoA esters acted as substrates (decreasing specificity, V/Km): 5-hydroxyvaleryl-CoA greater than propionyl-CoA greater than acetyl-CoA greater than (Z)-5-hydroxy-2-pentenoyl-CoA greater than butyryl-CoA greater than valeryl-CoA. 4-Pentenoate and 3-pentenoate were also activated by acetyl-CoA to the corresponding CoA esters, whereas crotonate, (E)-5-hydroxy-2-pentenoate, (E)-2-pentenoate and 2,4-pentadienoate were not attacked. 5-Hydroxyvalerate CoA-transferase showed ping-pong kinetics and was inactivated by sodium boranate only in the presence of a CoA substrate. This indicated the formation of a thiolester between a specific carboxyl group of the enzyme and CoASH during the course of the reaction. The CoA-transferase was inhibited by ATP and CTP, slightly by ADP, GTP and UTP, but not by AMP. The inhibition by ATP was competitive towards CoA esters and noncompetitive towards acetate.
Clostridium↗
Synthesis and properties of (R)-2-hydroxyglutaryl-1-CoA. (R)-2-hydroxyglutaryl-5-CoA, an erroneous product of glutaconate CoA-transferase.
1) (R)-2-Hydroxyglutaryl-1-CoA was synthesised starting from (R)-5-oxotetrahydrofuran-2-carboxylic acid (gamma-lactone of (R)-2-hydroxyglutarate) which was converted to the acylchloride and condensed with N-capryloylcysteamine. The lactone ring of the resulting thiolester was opened by acid hydrolysis and the CoA derivative was obtained by transesterification. 2) Pure glutaconate CoA-transferase from Acidaminococcus fermentans catalysed the formation of the 1- and the 5-isomer of (R)-2-hydroxyglutaryl-CoA from acetyl-CoA and (R)-2-hydroxyglutarate. The isomers were separated by HPLC and characterised by their reaction with acetate under the catalysis of the CoA-transferase. V/Km for the 1-isomer was 80 times higher than that for the 5-isomer. 3) Studies with cell-free extracts from A. fermentans showed that only (R)-2-hydroxyglutaryl-1-CoA but not its 5-isomer was dehydrated to glutaconyl-1-CoA. The data indicate that (R)-2-hydroxyglutaryl-5-CoA is an erroneous product of glutaconate CoA-transferase which only occurs in vitro.
Acyl Coenzyme A↗
Growth and development of brain of artificially reared hypoketonemic rat pups.
Three groups of rats were reared: mother-reared controls; artificially reared controls (AR-c), which were fed a milk substitute with the same composition of macro-nutrients as natural rat's milk; and an artificially reared test group (AR-h), which was fed a milk substitute identical to that fed AR-c pups except that the component of fat containing medium chain length fatty acids was omitted (medium chain triglyceride deficient) and replaced on an isocaloric basis with carbohydrate. The AR rats were fed the milk substitute from postnatal Day 5 until Day 17 by fitting them with gastric cannulas through which the milk could be infused automatically. The nutritional impact of the milk substitutes on growth and the integrity of the brain was assessed by a comparison of morphologic and biochemical markers. Pups in the AR-h group were hypoketonemic. Animals in all groups attained the same body weight by Day 17 and there was no difference in the morphologic markers among the groups with one exception: the vibrissal "barrel fields" of the somatosensory cortex of rat pups in both AR groups were reduced in size but not in number of distribution from those of the mother-reared groups. Furthermore, the brains of the rat pups in the AR groups were not different in weight, but they weighed less than brains of mother-reared controls. Our data show that although there are many similarities in the status of AR rat pups when compared with mother-reared controls, distinctive differences associated with artificial rearing are evident. We conclude that medium chain fatty acids in milk fat and the circulating ketone bodies are not mandatory substrates for growth and the development of the brain. Mechanisms must exist whereby alternative substrates are used to compensate when these metabolites are diminished in supply.
Animal Nutritional Physiological Phenomena↗
Gender differences in the response of hepatic fatty acids and cytosolic fatty acid-binding capacity to alcohol consumption in rats.
To investigate possible gender differences in the response of hepatic fatty acids and cytosolic fatty acid-binding capacity to ethanol consumption, both female and male rats (41 days of age) were pair fed liquid diets (with a littermate of the same sex) for 28 days. The diets contained 36% of energy either as ethanol or as additional carbohydrate. After ethanol feeding, the hepatic concentration of fatty acids increased 155% in females (P less than 0.01), whereas there was only a trend for an increase (22%) in males. This was associated with a much smaller increase of cytosolic fatty acid-binding capacity in females (58%) than in males (161%). Whereas the ethanol-induced increase in fatty acid-binding capacity provided an ample excess of binding sites for the fatty acids in males, the increase in females was barely sufficient for the binding of the large increase of fatty acids produced by ethanol in the females. The cytosolic protein responsible for this binding, the liver fatty acid-binding protein of the cytosol (L-FABPc), also promotes esterification of the fatty acids. In keeping with the postulated role of this protein, the ethanol-induced increases in hepatic triacylglycerols, phospholipids, and cholesterol esters were smaller in females than in males. The gender difference in cholesterol esters was associated with parallel changes in acyl-CoA transferase activity. A possible implication of the relatively small and most likely inadequate increase in liver fatty acid-binding capacity and fatty acid esterification during alcohol consumption in the females is that under these circumstances the risk for development of a potentially deleterious accumulation of fatty acids in the liver is increased, thereby contributing to the enhanced vulnerability of females to alcohol-induced hepatotoxicity.
Alcohol Drinking↗