Changes in the activities of the enzymes of hepatic fatty acid oxidation during development of the rat.

1. Changes in the activities of several enzymes involved in mitochondrial fatty acid oxidation were measured in livers of developing rats between late foetal life and maturity. The enzymes studied are medium- and long-chain ATP-dependent acyl-CoA synthetases of the outer mitochondrial membrane and matrix, GTP-dependent acyl-CoA synthetase, carnitine acyltransferase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, general 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase.     

Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very long-chain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal beta-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low Mr were seen in the patients. With anti-(oxoacyl-CoA thiolase), high Mr cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal beta-oxidation enzymes.     

The acetoacetyl-coenzyme A thiolases of rat brain and their relative activities during postnatal development

1. The apparent 3-oxoacyl-CoA thiolase activity of rat brain extracts is due to two different acetoacetyl-CoA thiolases, one cytoplasmic and the other mitochondrial. By the methods developed in the preceding paper (Middleton, 1973), the changes in activities of these two enzymes were determined during postnatal development. 2. Although the total brain acetoacetyl-CoA thiolase activity changes not more than 2-fold from birth to adulthood this masks large changes in the relative proportions of the two types of thiolase present. 3. Cytoplasmic acetoacetyl-CoA thiolase activity declines slowly from 4 units/g fresh wt. at birth to an adult value of 1.3 units/g fresh wt. 4. The mitochondrial acetoacetyl-CoA thiolase (activated by K(+)) rises rapidly in activity from 1 unit/g fresh wt. at birth to a peak value of 5 units/g fresh wt. at 20 days. After weaning the activity declines to 2.3 units/g fresh wt. in the adult. 5. These different developmental patterns are discussed in terms of the probable metabolic roles of the two brain acetoacetyl-CoA thiolases.     

Some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified from ox liver, and obtained essentially free from 3-oxoacyl-CoA thiolases. The kinetic behaviour, like that of the synthases from chicken liver and yeast, is compatible with a reaction pathway involving condensation of an acetyl-enzyme with acetoacetyl-CoA. The Km for acetoacetyl-CoA, less than 1 micronM at pH 7.8, may possibly be low enough to permit rapid ketogenesis under physiological conditions without the need for a binary complex between the synthase and oxoacyl-CoA thiolase.     

Regulation of thiolases from pig heart. Control of fatty acid oxidation in heart

The effects of various mitochondrial coenzymes and metabolities on the activities of 3-oxoacyl-CoA thiolase (EC 2.3.1.16) and acetoacetyl-CoA thiolase (EC 2.3.1.9) from pig heart were investigated with the aim of elucidating the possible regulation of these two enzymes. Of the compounds tested, acetyl-CoA was the most effective inhibitor of both thiolases. However, 3-oxoacyl-CoA thiolase was more severly inhibited by acetyl-CoA than was acetoacetyl-CoA thiolase. 3-Oxoacyl-CoA thiolase was also significantly inhibited by decanoyl-CoA while acetoacetyl-CoA thiolase was inhibited by 3-hydroxybutyryl-CoA as strongly as it was by acetyl-CoA. All other compounds either did not affect the thiolase activities or only at unphysiologically high concentrations. The inhibition of acetoacetyl-CoA thiolase by acetyl-CoA was linear and apparently noncompetitive with respect to CoASH (Ki = 125 microM) whereas that of 3-oxoacyl-CoA thiolase was nonlinear. However at low concentrations of acetyl-CoA the inhibition of 3-oxoacyl-CoA thiolase was linear competitive with respect to CoASH (Ki = 3.9 microM). It is concluded that 3-oxoacyl-CoA thiolase, but not acetoacetyl-CoA thiolase, will be completely inhibited by acetyl-CoA at concentrations of CoASH and acetyl-CoA which are assumed to exist intramitochondrially at state-4 respiration. It is suggested that fatty acid oxidation in heart muscle at sufficiently high concentrations of plasma free fatty acids is controlled via the regulation of 3-oxoacyl-CoA thiolase by the acetyl-CoA/CoASH ratio which is determined by the rate of the citric acid cycle and consequently by the energy demand of the tissue.     

Glyoxysomal acetoacetyl-CoA thiolase and 3-oxoacyl-CoA thiolase from sunflower cotyledons

Following chromatography on hydroxyapatite, the elution profile of the thiolase activity of the glyoxysomal fraction from sunflower (Helianthus annuus L.) cotyledons exhibited two peaks when the enzyme activity was assayed with acetoacetyl-CoA as substrate. Only one of these two activity peaks was detectable when a long-chain thiolase substrate was used in the activity assay. The proteins (thiolase I and thiolase II) underlying the two activity peaks detected with acetoacetyl-CoA were of glyoxysomal origin. They were purified using glyoxysomal matrices as starting material, and biochemically characterized. Thiolase I is an acetoacetyl-CoA thiolase (EC 2.3.1.9) exhibiting activity only towards acetoacetyl-CoA (Km = 11 microM). Its contribution to the total glyoxysomal thiolytic activity towards acetoacetyl-CoA amounted to about 15%. Thiolase II is a 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The activity of the enzyme towards 3-oxoacyl-CoAs increased with increasing chain length of the substrate. Thiolase II exhibited a Km value of 27 microM with acetoacetyl-CoA as substrate. and Km values between 3 and 7 microM with substrates having a carbon chain length from 6 to 16 carbon atoms. The thiolase activity of the glyoxysomes towards acetoacetyl-CoA and 3-oxopalmitoyl-CoA exceeded the glyoxysomal butyryl-CoA and palmitoyl-CoA beta-oxidation rates, respectively, by about 10-fold at all substrate concentrations employed (1-15 microM).     

Cloning, expression, and purification of glyoxysomal 3-oxoacyl-CoA thiolase from sunflower cotyledons

The glyoxysomal beta-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the coexistence of two different thiolase isoforms, thiolase I and II. So far, this phenomenon has only been described for glyoxysomes from sunflower cotyledons. Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9) recognizes acetoacetyl-CoA only, while thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16) exhibits a more broad substrate specificity towards 3-oxoacyl-CoA esters of different chain length. Here, we report on the cloning of thiolase II from sunflower cotyledons. The known DNA sequence of Cucumis sativus 3-oxoacyl-CoA thiolase was used to generate primers for cloning the corresponding thiolase from sunflower cotyledons. RT-PCR was then used to generate an internal fragment of the sunflower thiolase gene and the termini were isolated using 5'- and 3'-RACE. Full-length cDNA was generated using RT-PCR with sunflower thiolase-specific primers flanking the coding region. The resultant gene encodes a thiolase sharing at least 80% identity with other plant thiolases at the amino acid level. The recombinant sunflower thiolase II was expressed in a bacterial system in an active form and purified to apparent homogeneity in a single step using Ni-NTA agarose chromatography. The enzyme was purified 53.4-fold and had a specific activity of 235 nkat/mg protein. Pooled fractions from the Ni-NTA column resulted in an 83% yield of active enzyme to be used for further characterization.     

Comparison of the stability and substrate specificity of purified peroxisomal 3-oxoacyl-CoA thiolases A and B from rat liver

The specific activities and substrate specificities of 3-oxoacyl-CoA thiolase A (thiolase A) purified from normal rat liver peroxisomes and 3-oxoacyl-CoA thiolase B (thiolase B) isolated from livers of rats treated with the peroxisome proliferator clofibrate were virtually identical. The enzymes could be distinguished by their N-terminal amino acid sequences, their isoelectric points and their stability, the latter being higher for thiolase A. Contrary to thiolase B, which showed a marked cold lability in the presence of KCl by dissociating into monomers with poor activity, thiolase A retained its full activity and its homodimeric structure under these conditions.     

Mitochondrial acetoacetyl-CoA thiolase deficiency

A patient with severe progressive neuropathy and growth retardation who showed a persistent ketosis despite normal blood glucose levels is described. A liver biopsy was analyzed for 3-oxoacyl-CoA thiolase activity. One of the mitochondrial 3-oxoacyl-CoA thiolases which in normal control liver could be activated by K+ was virtually absent in the patient's liver. An intensive search for 3-methylhydroxybutyric acid and 3-methylacetoacetic acid by gas chromatography/mass spectroscopy in the patient's urine failed to show the presence of these acids, demonstrating that the 3-methylacetoacetyl-CoA thiolase is functioning in this patient. It is therefore concluded that the persistent ketosis is due to a deficiency of the mitochondrial acetoacetyl-CoA specific thiolase.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation

Catalase activity was inhibited by aminotriazole administration to rats in order to evaluate the influence of catalase on the peroxisomal fatty acyl-CoA beta-oxidation system. 2 h after the administration of aminotriazole, peroxisomes were prepared from rat liver, and the activities of catalase, the beta-oxidation system and individual enzymes of beta-oxidation (fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase) were determined. Catalase activity was decreased to about 2% of the control. Among the individual enzymes of the beta-oxidation system, thiolase activity was decreased to 67%, but the activities of fatty acyl-CoA oxidase, crotonase and beta-hydroxybutyryl-CoA dehydrogenase were almost unchanged. The activity of the peroxisomal beta-oxidation system was assayed by measuring palmitoyl-CoA-dependent NADH formation, and the activity of the purified peroxisome preparation was found to be almost unaffected by the administration of aminotriazole. The activity of the system in the aminotriazole-treated preparation was, however, significantly decreased to 55% by addition of 0.1 mM H2O2 to the incubation mixture. Hydrogen peroxide (0.1 mM) reduced the thiolase activity of the aminotriazole-treated peroxisomes to approx. 40%, but did not affect the other activities of the system. Thiolase activity of the control preparation was decreased to 70% by addition of hydrogen peroxide (0.1 mM). The half-life of 0.1 mM H2O2 added to the thiolase assay mixture was 2.8 min in the case of aminotriazole-treated peroxisomes, and 4 s in control peroxisomes. The ultraviolet spectrum of acetoacetyl-CoA (substrate of thiolase) was clearly changed by addition of 0.1 mM H2O2 to the thiolase assay mixture without the enzyme preparation; the absorption bands at around 233 nm (possibly due to the thioester bond of acetoacetyl-CoA) and at around 303 nm (due to formation of the enolate ion) were both significantly decreased. These results suggest that H2O2 accumulated in peroxisomes after aminotriazole treatment may modify both thiolase and its substrate, and consequently suppress the fatty acyl-CoA beta-oxidation. Therefore, catalase may protect thiolase and its substrate, 3-ketoacyl-CoA, by removing H2O2, which is abundantly produced during peroxisomal enzyme reactions.     

Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzme A cycle enzymes in liver. Separate cytoplasmic and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A generating systems for cholesterogenesis and ketogenesis.

Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme synthase which comprise the 3-hydroxy-3-methylglutaryl-CoA-generating system(s) for hepatic cholesterogenesis and ketogenesis exhibit dual mitochondrial and cytoplasmic localization. Twenty to forty per cent of the thiolase and synthase of avian and rat liver are localized in the cytoplasmic compartment, the remainder residing in the mitochondria. In contrast, 3-hydroxy-3 methylglutaryl-CoA lyase, an enzyme unique to the "3-hydroxy-3-methylglutaryl-CoA cycle" of ketogenesis, appears to be localized in the mitochondrion. The small proportion, 4 to 8 percent, of this enzyme found in the cytoplasmic fraction appears to arise via leakage from the mitochondria during cell fractionation in that its properties, pI and stability, are identical to those of the mitochondrial lyase. These results are consistent with the view that ketogenesis which involves all three enzymes, acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA lyase, occurs exclusively in the mitochondrion, whereas cholesterogenesis, a pathway which involves only the 3-hydroxy-3-methylglutaryl-CoA synthesizing enzymes, is restricted to the cytoplasm. Further fractionation of isolated mitochondria from chicken and rat liver showed that all three of the 3-hydroxy-3-methylglutaryl-CoA cycle enzymes are soluble and are localized within the matrix compartment of the mitochondrion. Likewise, cytoplasmic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase are soluble cytosolic enzymes, no thiolase or synthase activity being detectable in the microsomal fraction. Chicken liver mitochondrial 3-hydroxy-3methylglutaryl-CoA synthase activity consists of a single enzymic species with a pI of 7.2, whereas the cytoplasmic activity is composed of at least two species with pI values of 4.8 and 6.7. Thus it is evident that the mitochondrial and cytoplasmic species are molecularly distinct as has been shown to be the case for the mitochondrial and cytoplasmic acetoacetyl-CoA thiolases from avian liver (Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, W. D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275). Substantial mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase activity is present in all tissues surveyed, while only liver and kidney possess significant mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Therefore, it is proposed that tissues other than liver and kidney are unable to generate acetoacetate because they lack the mitochondrial synthase.     

S-(4-Bromo-2,3-dioxobutyl)CoA: an affinity label for certain enzymes than bind acetyl-CoA.

S-(4-Bromo-2,3-dioxobutyl)-CoA, a potential affinity label for enzymes possessing a receptor site(s) for short-chain acyl-CoA, was synthesized by condensing CoA and 1,4-dibromo-2,3-butanedione in acidified methanol. The new reagent was tested as an active site-directed irreversible inhibitor with four enzymes that accept a short-chain acyl-CoA as substrate. With citrate synthase (pig heart) and acetyl-CoA hydrolase (beef kidney) irreversible inhibition was observed, and the rate of inactivation obeyed first-order kinetics. Benzoyl-CoA, a reversible competitive inhibitor versus acetyl-CoA with both citrate synthase and acetyl-CoA hydrolase, protected the active site of both enzymes against the irreversible inhibitor. The new reagent was an exceptionally potent irreversible inhibitor of acetoacetyl-CoA thiolase (beef liver). Relatively low concentrations of the reagent (≥1 μm) completely inhibited the thiolase in less than 2 min. Preincubation of thiolase with acetoacetyl-CoA protected the enzyme against inhibition by S-(4-bromo-2,3-dioxobutyl)-CoA. In contrast, irreversible inhibition of l-3-hydroxyacyl-CoA dehydrogenase (pig heart) was not observed. Instead, the new reagent appeared to be a weak alternate substrate for this dehydrogenase. In all cases, the new reagent exhibited tight reversible binding at the active site since the measured K_i's (and K_m) were in the range, 30 to 120 μm. It is anticipated that the new reagent will be suitable for investigating a number of acyl-CoA using enzymes by affinity labeling techniques.     

Biosynthesis of enzymes of rat-liver mitochondrial beta-oxidation

The biogenesis of seven enzymes involved in the mitochondrial fatty acid beta-oxidation of rat liver was studied. Hepatic RNA was translated in vitro in a rabbit reticulocyte lysate cell-free system and the translation products were immunoprecipitated, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. The translation products obtained in vitro of medium-chain and/or long-chain acyl-CoA dehydrogenase (these enzymes were immunochemically cross-reactive), enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and acetoacetyl-CoA thiolase and probably also short-chain acyl-CoA dehydrogenase were larger than the subunits of the corresponding mature enzymes by 2-4.5 kDa, whereas the 3-oxoacyl-CoA thiolase obtained in vitro was approximately the same size as the mature subunit. The free polysome fraction of rat liver was 4.3-9.0-times more active than the membrane-bound polysome fraction in the synthesis of these seven enzymes. The enzyme activities were increased after administration of di(2-ethylhexyl)phthalate; the extent of the increase varied from one enzyme to another. The increase in the cell-free translation activity of total hepatic RNA for these enzymes after administration of the chemical was markedly different among individual enzymes and higher than that in the rates of synthesis of the corresponding enzymes which were determined by the experiment in vivo.     

The NH2-terminal 14-16 amino acids of mitochondrial and bacterial thiolases can direct mature ornithine carbamoyltransferase into mitochondria

Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein. This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence. A hybrid gene encoding the NH2-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed. Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria. These results indicate that the NH2-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. The fusion protein containing the NH2-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria. On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP) of the peroxisomal thiolase appeared not to be localized to the mitochondria. These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH2-terminal portion of the ancestral thiolase. The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NH2-terminal portion of the enzyme.     

Molecular and catalytic properties of the acetoacetyl-coenzyme A thiolase of Escherichia coli.

The acetoacetyl-CoA-thiolase, a product of the acetoacetate degradation operon (ato) was purified to homogeneity as judged by polyacrylamide-gel electrophoresis at pH 4.5, 7.0, and 8.3. The enzyme has a molecular weight of 166,000 and is composed of four identical subunits. The subunit molecular weight is 41,500. Histidine was the sole N-terminal amino acid detected by dansylation. The thiolase contains eight free sulhydryl residues and four intrachain disulfide bonds per mole. The ato thiolase catalyzes the CoA- dependent cleavage of acetoacetyl-CoA and the acetylation of acetyl-CoA to form acetoacetyl-CoA. The maximal velocity in the direction of acetoacetyl-CoA cleavage was 840 nmol min^? (enzyme unit)^?1 and the maximal velocity in the direction of acetoacetyl CoA formation was 38 nmol min^?1 (enzyme unit)^?1. Like other thiolases, the ato thiolase was inactivated by sulfhydryl reagents. The enzyme was protected from inactivation by sulfhydryl reagents in the presence of the acyl-CoA substrates, acetyl-CoA and acetoacetyl-CoA; however, no protection was obtained when the enzyme was incubated with the acetyl-CoA analog, acetylaminodesthio-CoA. Consistent with these results was the demonstration of an acetyl-enzyme compound when the thiolase was incubated with [1-¹⁴C]acetyl-CoA. The sensitivity of the acetyl-enzyme bond to borohydride reduction and the protection afforded by acyl-CoA substrates against enzyme inactivation by sulfhydryl reagents indicated that acetyl groups are bound to the enzyme by a thiolester bond.     

Mitochondrial metabolism of 3-mercaptopropionic acid. Chemical synthesis of 3-mercaptopropionyl coenzyme A and some of its S-acyl derivatives

The metabolism of 3-mercaptopropionic acid in mitochondria was studied by use of purified mitochondrial enzymes and rat heart mitochondria. Metabolites of 3-mercaptopropionic acid were separated by high performance liquid chromatography and identified by comparing them with chemically synthesized derivatives of 3-mercaptopropionic acid. The initial step in the metabolism of 3-mercaptopropionic acid is its conversion to a CoA thioester, most likely catalyzed by medium-chain acyl-CoA synthetase. The resulting 3-mercaptopropionyl-CoA is a poor substrate of acyl-CoA dehydrogenase but substitutes effectively for CoASH in reactions catalyzed by 3-ketoacyl-CoA thiolase and acetoacetyl-CoA thiolase. S-Acyl-3-mercaptopropionyl-CoA thioesters formed in the thiolase-catalyzed reactions are not at all or only poorly acted upon by acyl-CoA dehydrogenases. However, they are hydrolyzed by thioesterase(s) to CoASH and S-acyl-3-mercaptopropionic acid. The hydrolysis of S-acyl-3-mercaptopropionyl-CoA thioesters proceeds more rapidly than the hydrolysis of fatty acyl-CoA thioesters of comparable chain lengths. Free CoASH is also regenerated from S-acetyl-3-mercaptopropionyl-CoA and more rapidly from 3-mercaptopropionyl-CoA as a result of their reactions with carnitine catalyzed by carnitine acetyltransferase. These findings lead to the suggestion that the major mitochondrial CoA-containing metabolites of 3-mercaptopropionic acid are S-acyl-3-mercaptopropionyl-CoA thioesters.     

Phylogenetic Analysis of Eukaryotic Thiolases Suggests Multiple Proteobacterial Origins

Eukaryotic thiolases are essential enzymes located in three different compartments (peroxisome, mitochondrion, and cytosol) that can display catabolic or anabolic functions. They are responsible for the thiolytic cleavage of oxidized acyl-CoA (thiolase I; EC 2.3.1.16) and the synthesis or degradation of acetoacetyl-CoA (thiolase II; EC 2.3.1.9). Phylogenetic analysis of eukaryotic thiolase sequences showed that they form six distinct clusters, one of them highly divergent, which are in good correlation with their class and subcellular location. When analyzed together with a representative sample of prokaryotic thiolases, all eukaryotic thiolase groups emerged close to proteobacterial sequences. Metazoan cytosolic thiolase II was related to α-proteobacterial sequences, suggesting a mitochondrial origin. Unexpectedly, cytosolic thiolases from green plants and fungi as well as at least one member of all eukaryotic peroxisomal and mitochondrial thiolases had δ-proteobacteria as closest relatives. Our analysis suggests that these eukaryotic peroxisomal and mitochondrial thiolases may have been acquired from δ-proteobacteria prior to the ancestor of all known eukaryotes.     

Enzymes involved in acetoacetate formation in various bovine tissues

1. The activities of acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase and lyase and acetoacetyl-CoA deacylase were measured in homogenates of samples of liver, rumen epithelium (long papillae), kidney and lactating mammary gland derived from slaughtered cows. 2. The activities of the four enzymes in bovine liver were similar to the activities previously reported for the corresponding enzymes in rat liver. 3. Acetoacetyl-CoA thiolase and hydroxymethylglutaryl-CoA synthase and lyase were present in rumen epithelium. The activities of the enzymes were all lower on a wet weight basis than in liver. Only very slight deacylase activity was detected. 4. Kidney contained acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA lyase and acetoacetyl-CoA deacylase, but only trace amounts of hydroxymethylglutaryl-CoA synthase. 5. Mammary gland contained acetoacetyl-CoA thiolase and some hydroxymethylglutaryl-CoA lyase, but virtually no hydroxymethylglutaryl-CoA synthase or acetoacetyl-CoA deacylase. 6. Since physiologically significant ketogenesis probably occurs solely via the hydroxymethylglutaryl-CoA pathway, it is evident that, of the four tissues examined, such ketogenesis must be restricted to the liver and the rumen epithelium. 7. All the enzymes except hydroxymethylglutaryl-CoA lyase were also assayed in the four tissues derived from cows suffering from bovine lactational ketosis. Ketosis did not cause a statistically significant change in the activity of any of the enzymes measured. 8. Hepatic hydroxymethylglutaryl-CoA synthase and lyase were found to be associated mainly with the particulate fraction, as in the rat. A considerably greater proportion of these enzymes was found to be present in the cytoplasmic fraction from rumen epithelium, although it was not excluded that this was due to mitochondrial damage during homogenization. 9. Appreciable hydroxymethylglutaryl-CoA synthase was also present in epithelium from the dorsal region of the rumen, from the reticulum and from the omasum, but not from the abomasum.     

Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata — a complementation study

Summary The genetic relationship between 10 patients with clinical manifestations of rhizomelic chondrodysplasia punctata (RCDP) was studied by complementation analysis after somatic cell fusion. Biochemically, 9 out of the 10 patients were characterized by a partial deficiency of acyl-CoA: dihydroxyacetone phosphate acyltransferase (DHAP-AT) and an impairment of plasmalogen biosynthesis, phytanate catabolism and the maturation of peroxisomal 3-oxoacyl-CoA thiolase; 3-oxoacyl-CoA thiolase was strongly reduced in the peroxisomes of these patients. Fusion of fibroblasts from these 9 patients with Zellweger fibroblasts resulted in complementation as indicated by the restoration of DHAP-AT activity, plasmalogen biosynthesis, and punctate fluorescence after staining with a monoclonal antibody to peroxisomal thiolase. No complementation was observed after fusion of different combinations of the 9 RCDP cell lines, suggesting that they belong to a single complementation group. The tenth patient was characterized biochemically by a deficiency of DHAP-AT and an impairment of plasmalogen biosynthesis. However, maturation and localization of peroxisomal thiolase were normal. Fusion of fibroblasts from this patient with fibroblasts from the other 9 patients resulted in complementation as indicated by the restoration of plasmalogen biosynthesis. We conclude that mutations in at least two different genes can lead to the clinical phenotype of RCDP.     

The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver

1. Cytoplasmic acetoacetyl-CoA thiolase was highly purified in good yield from rat liver extracts. 2. Mg(2+) inhibits the rate of acetoacetyl-CoA thiolysis but not the rate of synthesis of acetoacetyl-CoA. Measurement of the velocity of thiolysis at varying Mg(2+) but fixed acetoacetyl-CoA concentrations gave evidence that the keto form of acetoacetyl-CoA is the true substrate. 3. Linear reciprocal plots of velocity of acetoacetyl-CoA synthesis against acetyl-CoA concentration in the presence or absence of desulpho-CoA (a competitive inhibitor) indicate that the kinetic mechanism is of the Ping Pong (Cleland, 1963) type involving an acetyl-enzyme covalent intermediate. In the presence of CoA the reciprocal plots are non-linear, becoming second order in acetyl-CoA (the Hill plot shows a slope of 1.7), but here this does not imply co-operative phenomena. 4. In the direction of acetoacetyl-CoA thiolysis CoA is a substrate inhibitor, competing with acetoacetyl-CoA, with a K(i) of 67mum. Linear reciprocal plots of initial velocity against concentration of mixtures of acetoacetyl-CoA plus CoA confirmed the Ping Pong mechanism for acetoacetyl-CoA thiolysis. This method of investigation also enabled the determination of all the kinetic constants without complication by substrate inhibition. When saturated with substrate the rate of acetoacetyl-CoA synthesis is 0.055 times the rate of acetoacetyl-CoA thiolysis. 5. Acetoacetyl-CoA thiolase was extremely susceptible to inhibition by an excess of iodoacetamide, but this inhibition was completely abolished after preincubation of the enzyme with a molar excess of acetoacetyl-CoA. This result was in keeping with the existence of an acetyl-enzyme. Acetyl-CoA, in whose presence the overall reaction could proceed, gave poor protection, presumably because of the continuous turnover of acetyl-enzyme in this case. 6. The kinetic mechanism of cytoplasmic thiolase is discussed in terms of its proposed role in steroid biosynthesis.