Formation of glycine and aminoacetone from l-threonine by rat liver mitochondria

Threonine is a precursor of glycine in the rat, but the metabolic pathway involved is unclear. To elucidate this pathway, the biosynthesis of glycine, and of aminoacetone, from l-threonine were studied in rat liver mitochondrial preparations of differing integrities. In the absence of added cofactors, intact mitochondria formed glycine and aminoacetone in approximately equal amounts from 20 mM l-threonine, but exogenous NAD⁺ decreased and CoA increased the ratio of glycine to aminoacetone formed. In intact and freeze-thawed mitochondria, the ratio of glycine to aminoacetone formed was markedly sensitive to the concentration of l-threonine, glycine being the major product at low l-threonine concentrations. Disruption of mitochondrial integrity by sonication (1 min) decreased the ratio of glycine to aminoacetone formed, and in 20 000 × g supernatant fractions from sonicated (3 min) mitochondria, aminoacetone was the major product. The main non-nitogenous tow-carbon compound detected when intact mitochondria catabolized l-threonine to glycine was acetate, which was probably derived from deacylation of acetyl-CoA. These results suggest that glycine formation from l-threonine in rat liver mitochondria occured primarily by the coupled activities of threonine dehydrogenase and 2-amino-3-oxobutyrate CoA-ligase, the extent of coupling between the enzymes being dependent upon a close physical relationship and upon the flux through the dehydrogenase reaction. In vivo glycine synthesis would predominate, and aminoacetone would be a minor product.     

Interaction between L-threonine dehydrogenase and aminoacetone synthetase and mechanism of aminoacetone production

A mixture of threonine dehydrogenase and aminoacetone synthetase will catalyze the conversion of L-threonine to glycine. The overall reaction likely involves the conversion of L-threonine, NAD+, and CoA to glycine, NADH, and acetyl-CoA. Physical separation of L-threonine dehydrogenase from aminoacetone synthetase results in the formation of aminoacetone and CO2 from their substrates. A physical interaction between threonine dehydrogenase and aminoacetone synthetase has been demonstrated by gel permeation chromatography and fluorescence polarization. Polarization of fluorescence measurements of threonine dehydrogenase and aminoacetone synthetase labeled with fluorescein isothiocyanate indicated the formation of a soluble active complex, with an apparent dissociation constant (Kd) of 5-10 nM and an apparent stoichiometry of 2 aminoacetone synthetase dimers/1 threonine dehydrogenase tetramer. Chemical experiments have identified aminoacetone as the enzymatic product of L-threonine dehydrogenase acting on L-threonine. These experiments involved trapping pyrrole derivatives, [3H]NaBH4 reduction, and coupling with plasma amine oxidase. Kinetic experiments also showed NADH, CO2, and aminoacetone to inhibit threonine dehydrogenase in a manner consistent with an ordered Bi-Ter kinetic mechanism. NAD+ is the lead substrate followed by threonine, and the products are released in the order: CO2, aminoacetone, and NADH.     

Purification and properties of aminoacetone synthetase from beef liver mitochondria

Aminoacetone synthetase from beef liver mitochondria was purified to homogeneity and shown to be a member of the pyridoxal 5'-phosphate-dependent family of enzymes. This enzyme catalyzes the condensation of glycine and acetyl-CoA to produce CO2, CoA, and the stable product aminoacetone. Bovine aminoacetone synthetase is a dimer (Mr 56,000) of identical subunits and contains 2 mol of pyridoxal phosphate/mol of dimer. The holoenzyme was resolved by dialysis against cysteine and has a pI of 5.2. The holoenzyme shows an absorption maximum at 428 nm which undergoes a shift to 335 nm when reduced with sodium borohydride. The Km values of glycine and acetyl-CoA were 22 mM and 53 microM, respectively. Initial velocity studies indicate that the condensation reaction proceeds by an ordered mechanism. With the exception of aminomalonate, bovine aminoacetone synthetase acts specifically on glycine and acetyl-CoA. Coupled reactions of purified bovine aminoacetone synthetase and porcine L-threonine dehydrogenase demonstrated the interconversion of threonine and glycine.     

Bacterial catabolism of threonine. Threonine degradation initiated by L-threonine-NAD+ oxidoreductase.

1. Isolates representing seven bacterial genera capable of growth on L-threonine medium, and possessing high L-threonine 3-dehydrogenase activity, were examined to elucidate the catabolic route. 2. The results of growth, manometric and enzymic experiments indicated the catabolism of L-threonine by cleavage to acetyl-CoA plus glycine, the glycine being further metabolized via L-serine to pyruvate, in all cases. No evidence was obtained of a role for aminoacetone in threonine catabolism or for the metabolism of glycine by the glycerate pathway. 3. The properties of a number of key enzymes in L-threonine catabolism were investigated. The inducibly formed L-threonine 3-dehydrogenase, purified from Corynebacterium sp. B6 to a specific activity of about 30-35 mumol of product formed/min per mg of protein, exhibited a sigmoid kinetic response to substrate concentration. The half-saturating concentration of substrate, [S]0.5, was 20mM and the Hill constant (h) was 1.50. The Km for NAD+ was 0.8mM. The properties of the enzyme were studied in cell-free extracts of other bacteria. 4. New assays for 2-amino-3-oxobutyrate-CoA ligase were devised. The Km for CoA was determined for the first time and found to be 0.14mM at pH8, for the enzyme from Corynebacterium sp. B6. Evidence was obtained for the efficient linkage of the dehydrogenase and ligase enzymes. Cell-free extracts all possessed high activities of the inducibly formed ligase. 5. L-Serine hydroxymethyltransferase was formed constitutively by all isolates, whereas formation of the 'glycine-cleavage system' was generally induced by growth on L-threonine or glycine. The coenzyme requirements of both enzymes were established, and their linked activity in the production of L-serine from glycine was demonstrated by using extracts of Corynebacterium sp. B6. 6. L-Serine dehydratase, purified from Corynebacterium sp. B6 to a specific activity of about 4mumol of product formed/min per mg of protein, was found to exhibit sigmoid kinetics with an [S]0.5 of about 20mM and h identical to 1.4. Similar results were obtained with enzyme preparations from all isolates. The enzyme required Mg2+ for maximum activity, was different from the L-threonine dehydratase also detectable in extracts, and was induced by growth on L-threonine or glycine.     

Catabolism of threonine in mammals by coupling of l-threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase

There is doubt about the l-threonine 3-dehydrogenase (EC 1.1.1.103) and threonine aldolase (EC 2.1.2.1) catabolic pathways of l-threonine in mammals which are believed to produce aminoacetone and glycine plus acetaldehyde, respectively. l-Threonine 3-dehydrogenase in disrupted guinea-pig liver mitochondria was investigated in a reaction mixture containing l-threonine without and with CoA and oxaloacetate; l-[U-¹⁴C]threonine was included in four similar experiments for autoradiograms. Threonine aldolase was examined in similar mitochondria from liver and kidney. CoA reduced the aminoacetone formed from l-threonine to 10–14% and CoA plus oxaloacetate produced citrate (from CoASAc) in approximately equal amounts to the decrease in aminoacetone. Autoradiograms confirmed the decrease in aminoacetone with the simultaneous appearance of citrate and glycine. No evidence was obtained that threonine aldolase catabolised l-threonine at the concentration used to assay the dehydrogenase. It is concluded that 2-amino-3-oxobutyrate (precursor of aminoacetone), which is produced from l-threonine by l-threonine 3-dehydrogenase, undergoes CoA-dependent cleavage to glycine and CoASAc by 2-amino-3-oxobutyrate-CoA ligase. The results suggest that the coupling of these enzymes provides a new pathway for the catabolism of threonine in mammals.     

Catabolism of threonine in mammals by coupling of l-threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase

There is doubt about the l-threonine 3-dehydrogenase (EC 1.1.1.103) and threonine aldolase (EC 2.1.2.1) catabolic pathways of l-threonine in mammals which are believed to produce aminoacetone and glycine plus acetaldehyde, respectively. l-Threonine 3-dehydrogenase in disrupted guinea-pig liver mitochondria was investigated in a reaction mixture containing l-threonine without and with CoA and oxaloacetate; l-[U-¹⁴C]threonine was included in four similar experiments for autoradiograms. Threonine aldolase was examined in similar mitochondria from liver and kidney. CoA reduced the aminoacetone formed from l-threonine to 10–14% and CoA plus oxaloacetate produced citrate (from CoASAc) in approximately equal amounts to the decrease in aminoacetone. Autoradiograms confirmed the decrease in aminoacetone with the simultaneous appearance of citrate and glycine. No evidence was obtained that threonine aldolase catabolised l-threonine at the concentration used to assay the dehydrogenase. It is concluded that 2-amino-3-oxobutyrate (precursor of aminoacetone), which is produced from l-threonine by l-threonine 3-dehydrogenase, undergoes CoA-dependent cleavage to glycine and CoASAc by 2-amino-3-oxobutyrate-CoA ligase. The results suggest that the coupling of these enzymes provides a new pathway for the catabolism of threonine in mammals.     

Inhibition and regulation of rat liver L-threonine dehydrogenase by different fatty acids and their derivatives.

Rat liver L-threonine dehydrogenase is a mitochondrial enzyme which transforms L-threonine either into aminoacetone or into acetyl-CoA. We show that it is inhibited by several fatty acids and their derivatives: short chain fatty acids, L-2-hydroxybutyrate and D-3-hydroxybutyrate, long chain fatty acids, such as lauric acid, myristic acid, palmitic and stearic acids, bicarboxylic acids such as malonic acid and its derivatives methyl- and hydroxymalonic acids. The inhibition occurs at low and physiological concentrations of such compounds, which are normally present and metabolized in mitochondria. It presumably plays a role in the physiology of acetyl-CoA-dependent formation of fatty acids and ketobodies, in L-threonine-dependent gluconeogenesis, and in the regulation of L-threonine metabolism by L-threonine dehydrogenase and L-threonine deaminase.     

Inhibition and regulation of rat liver L-threonine dehydrogenase by different fatty acids and their derivatives

Rat liver L-threonine dehydrogenase is a mitochondrial enzyme which transforms L-threonine either into aminoacetone or into acetyl-CoA. We show that it is inhibited by several fatty acids and their derivatives: short chain fatty acids, L-2-hydroxybutyrate and D-3-hydroxybutyrate, long chain fatty acids, such as lauric acid, myristic acid, palmitic and stearic acids, bicarboxylic acids such as malonic acid and its derivatives methyl- and hydroxymalonic acids. The inhibition occurs at low and physiological concentrations of such compounds, which are normally present and metabolized in mitochondria. It presumably plays a role in the physiology of acetyl-CoA-dependent formation of fatty acids and ketobodies, in L-threonine-dependent gluconeogenesis, and in the regulation of L-threonine metabolism by L-threonine dehydrogenase and L-threonine deaminase.     

Growth, enzyme levels, and some metabolic properties of an Escherichia coli mutant grown on L-threonine as the sole carbon source.

A mutant of Escherichia coli (designated E. coli SBD-76) that utilizes L-threonine as the sole carbon source was isolated. In contrast with levels in extracts of wild-type cells, the levels of threonine dehydrogenase in extracts of this mutant were 100-fold higher than levels of threonine aldolase or degradative threonine dehydratase. Catabolite repression of threonine dehydrogenase was manifested in wild-type, but not SBD-76, cells. For purposes of isolating enzymes, large quantities of SBD-76 cells with the elevated threonine dehydrogenase level could be grown in a fermentor in modified Fraser medium containing 1% glycerol, rather than in the 0.2% L-threonine minimal medium used to isolate the mutant. SBD-76 cells grown on L-threonine excreted glycine and aminoacetone into the medium, and extracts of the mutant strain catalyzed a quantitative conversion of L-threonine to glycine and aminoacetone.     

Metabolism of 2-oxoaldehydes in yeasts. Possible role of glycolytic bypath as a detoxification system in L-threonine catabolism by Saccharomyces cerevisiae

L-Threonine catabolism by Saccharomyces cerevisiae was studied to determine the role of glycolytic bypath as a detoxyfication system of 2-oxoaldehyde (methylglyoxal) formed from L-threonine catabolism. During the growth on L-threonine as a sole source of nitrogen, a large amount of aminoacetone was accumulated in the culture. The enzymatic analyses indicated that L-threonine was converted into either acetaldehyde and glycine by threonine aldolase or 2-aminoacetoacetate by NAD-dependent threonine dehydrogenase. Glycine formed was condensed with acetyl-CoA by aminoacetone synthase to form 2-aminoacetoacetate, a labile compound spontaneously decarboxylated into aminoacetone. The enzyme activities of the glycolytic bypath of the cells grown on L-threonine were considerably higher than those of the cells grown on ammonium sulfate as a nitrogen source. The result indicated the possible role of glycolytic bypath as a detoxification system of methylglyoxal formed from L-threonine catabolism.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

L-threonine aldolase is not a genuine enzyme in rat liver.

Activity of L-threonine aldolase in rat liver cytosolic extract was not affected by the omission of alcohol dehydrogenase in a previously established NADPH-linked alcohol dehydrogenase-coupled assay. The liver extract was able to catalyse the dehydrogenation of NADPH with either acetaldehyde (a product of L-threonine aldolase action) or 2-oxobutyrate (a product of L-threonine dehydratase action). When the liver extract was chromatographed on a Sephacryl S-200 column, no threonine aldolase activity was detected in the eluate. However, activity of threonine aldolase re-appeared when the fractions with highest activity of lactate dehydrogenase and threonine dehydratase were mixed. Activity of threonine aldolase could also be abolished by removing threonine dehydratase from the liver extract with a specific antibody. Hence L-threonine aldolase should not be a genuine enzyme in the rat liver, and the apparent enzyme activity may result from a combined effect of threonine dehydratase and lactate dehydrogenase (or an oxo acid-linked NADPH dehydrogenase) in the liver cytosolic extract.     

Cell-free synthesis of a putative precursor to the rat liver mitochondrial glycerol-3-phosphate dehydrogenase

Antibodies to purified glycerol-3-phosphate dehydrogenase were raised in rabbits and purified from serum by affinity chromatography on enzyme-bound Sepharose columns. RNA from membrane-free polyribosomes, or poly(A)+ RNA (total cellular RNA) of rat liver, was translated in a rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, and the glycerol-3-phosphate dehydrogenase synthesized was isolated by immunoprecipitation using the antibody. The in vitro product moved on sodium dodecyl sulfate-polyacrylamide gels as a polypeptide that was about 5,000 daltons larger than the subunit of the mature enzyme (74,000 daltons). Digestion of both the mature and the in vitro newly synthesized forms of the enzyme yielded respective sets of peptide fragments which had similar patterns upon sodium dodecyl sulfate-gel electrophoresis. When the presumptive precursor that had been synthesized in vitro was incubated with isolated intact rat liver mitochondria, it was converted to "mature" subunits that were no longer susceptible to externally added proteases. Import of the presumptive precursor is dependent upon an electrochemical potential across the inner mitochondrial membranes. The mature form of the protein is assembled in its native location (the outer surface of the inner mitochondrial membrane).     

A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver.

The total activity of pyruvate dehydrogenase in mitochondria isolated from rat brain and liver was 53.5 and 14.2nmol/min per mg of protein respectively. Pyruvate dehydrogenase in liver mitochondria incubated for 4 min at 37 degrees C with no additions was 30% in the active form and this activity increased with longer incubations until it was completely in the active form after 20 min. Brain mitochondrial pyruvate dehydrogenase activity was initially high and did not increase with addition of Mg2+ plus Ca2+ or partially purified pyruvate dehydrogenase phosphatase or with longer incubations. The proportion of pyruvate dehydrogenase in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge, whereas total pyruvate dehydrogenase did not change. The chelators citrate, isocitrate, EDTA, ethanedioxybis(ethylamine)tetra-acetic acid and Ruthenium Red each lowered pyruvate dehydrogenase activity in brain mitochondria, but only citrate and isocitrate did so in liver mitochondria. These chelators did not affect the energy charge of the mitochondria. Mg2+ plus Ca2+ reversed the pyruvate dehydrogenase inactivation in liver, but not brain, mitochondria. The regulation of the activation-inactivation of pyruvate dehydrogenase in mitochondria from rat brain and liver with respect to energy charge is similar and may be at least partially regulated by this parameter, and the effects of chelators differ in the two types of mitochondria.     

L-Threonine dehydrogenase from goat liver. Feedback inhibition by methylglyoxal

L-Threonine dehydrogenase, which forms aminoacetone from L-threonine and NAD, has been extensively purified from goat liver. A feedback inhibition of this enzyme has been observed with methylglyoxal. Kinetic data and other experiments indicate that methylglyoxal acts at a site other than the active site of the enzyme. The enzyme contains a single subunit of Mr 89,000. The apparent Km values of the enzyme for L-threonine and NAD were found to be 5.5 and 1 mM, respectively.     

Transport of proteins into mitochondrial matrix. Evidence suggesting a common pathway for 3-ketoacyl-CoA thiolase and enzymes having presequences

Rat liver 3-ketoacyl-CoA thiolase, a mitochondrial matrix enzyme which catalyzes a step of fatty acid beta-oxidation, was synthesized in a rabbit reticulocyte lysate cell-free system. The in vitro product was apparently the same in molecular size and charge as the subunit of the mature enzyme. The enzyme synthesized in vitro was transported into isolated rat liver mitochondria in an energy-dependent manner. In pulse experiments with isolated rat hepatocytes at 37 degrees C, the radioactivity of the newly synthesized enzyme in the cytosolic fraction remained essentially unchanged during 5-20 min of incubation, whereas that of the enzyme in the particulate fraction increased with time during the incubation. The pulse-labeled enzyme disappeared with an apparent half-life of less than 3 min from the cytosolic fraction, in pulse-chase experiments. Purified 3-ketoacyl-CoA thiolase inhibited the mitochondrial uptake and processing of the precursors of the other matrix enzymes, ornithine carbamoyltransferase, medium-chain acyl-CoA dehydrogenase and acetoacetyl-CoA thiolase. These results indicate that 3-ketoacyl-CoA thiolase has an internal signal which is recognized by the mitochondria and suggest that this enzyme and the three others are transported into the mitochondria by a common pathway.     

Threonine degradation by Serratia marcescens.

The wild strain of Serratia marcescens rapidly degraded threonine and formed aminoacetone in a medium containing glucose and urea. Extracts of this strain showed high threonine dehydrogenase and "biosynthetic" threonine deaminase activities, but no threonine aldolase activity. Threonine dehydrogenase-deficient strain Mu-910 was selected among mutants unable to grow on threonine as the carbon source. This strain did not form aminoacetone from threonine, but it slowly degraded threonine. Strain D-60, deficient in both threonine dehydrogenase and threonine deaminase, was derived from strain Mu-910 and barely degraded threonine. A glycine-requiring strain derived from the wild strain grew in minimal medium containing threonine as the glycine source, whereas a glycine-requiring strain derived from strain Mu-910 did not grow. This indicates that threonine dehydrogenase participates in glycine formation from threonine (via alpha-amino-beta-ketobutyrate) as well as in threonine degradation to aminoacetone.     

Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.     

Calcium sensitive isocitrate and 2-oxoglutarate dehydrogenase activities in rat liver and AS-30D hepatoma mitochondria

NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase in extracts of mitochondria from the highly malignant AS-30D rat hepatoma cell line demonstrate Ca2+ sensitivities and affinities for substrates similar to those of normal liver mitochondria. However, the maximal activities of NAD+- and NADP+-dependent isocitrate dehydrogenase were found to be 8 and 3.5 fold higher in hepatoma mitochondrial extracts than those of liver mitochondria, whereas maximal activities of succinate and 2-oxoglutarate dehydrogenases were similar in the two tissues. At pyridine nucleotide concentrations giving the lowest physiological NADH/NAD+ ratio, NAD+-isocitrate dehydrogenase activity in hepatoma mitochondrial extracts was completely inhibited at subsaturating concentrations of Ca2+, substrate, and NAD+, in contrast to rat liver mitochondrial extracts which retained significant activity.     

Studies on the metabolism of glycolyl-CoA

Glycolyl-CoA can be formed during the course of the beta-oxidation by rat liver mitochondria of 4-hydroxybutyrate. The existence of this beta-oxidation has been previously supported by the occurrence of 4-hydroxybutyrate and its beta-oxidation catabolites in urine from patients with 4-hydroxybutyric aciduria, an inborn error of gamma-aminobutyric acid metabolism due to the deficiency of succinic semialdehyde dehydrogenase. The characteristics of the mitochondrial beta-oxidation of 4-hydroxybutyrate were, in rat liver, compared with those of the mitochondrial beta-oxidation of butyrate. The inhibition by malonate of the oxidation of 4-hydroxybutyrate was about twofold weaker than that of oxidation of butyrate, whereas both oxidations were abolished by preincubating the mitochondria with 1 mM valproic acid, a known inhibitor of mitochondrial beta-oxidation. Mitochondria from rat kidney cortex were demonstrated to catalyse, as previously shown for hepatic mitochondria, the carnitine-dependent oxidation of 12-hydroxylauroyl-CoA-omega-Hydroxymonocarboxylyl-CoAs are thus concluded to be precursors of glycolyl-CoA also in rat kidney cortex. In addition, 3-hydroxypyruvate was found to be a precursor of glycolyl-CoA, since it was oxidized by bovine heart pyruvate dehydrogenase with a cofactor requirement similar to that of pyruvate oxidation. Glycolyl-CoA was a substrate of carnitine acetyltransferase (pigeon breast muscle). Pig heart citrate synthase was capable of catalyzing the condensation of glycolyl-CoA with oxaloacetate. The product of this reaction induced low NADH production rates dependent on the addition of porcine heart aconitase and isocitrate dehydrogenase.