PubMed · 27067
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J M Bourre, S Pollet, M Paturneau-Jouas, N Baumann. 1978. Fatty acid biosynthesis during brain development.. https://doi.org/10.1007/978-1-4615-9071-2_2
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Acetyl-CoA acetyltransferase (EC 2.3.1.9) from rat liver mitochondria, which catalyzes the first step in the biosynthesis of ketone bodies, exists in two forms, designated transferase A and transferase B. Both transferases showed immunochemical cross-reactivity, but are immunologically unrelated to cytosolic acetyl-CoA acetyltransferase activity and the mitochondrial acetyl-CoA acyltransferase from rat liver. The transferases A and B were estimated to have molecular weights of 151 000 in the absence and 40 000 in the presence of sodium dodecyl sulfate. They differ with respect to charge states and multiplicity of forms as indicated by isoelectric focusing. Transferase A appeared in two forms with isoelectric points of 8.4 and 9.1, whereas transferase B represents a stable protein state with an isoelectric point of 9.0. Kinetic analysis of the reactions leading to acetoacetyl-CoA synthesis revealed saturation curves with multiple intermediary plateaus, indicating a complex kinetic behaviour. The data presented are interpreted as representing a microheterogeneity of forms of the mitochondrial acetyl-CoA acetyltransferase. The kinetic properties exhibited suggest a role for this microheterogeneity in the regulation of ketogenesis.
The alpha and beta subunits of the acetyl-CoA:acetoacetate-CoA transferase were purified by isoelectric focusing of the enzyme in the presence of 6 M urea. The purified beta subunit, in which the active center of the enzyme is located, exhibits low catalytic activity (2% of the specific activity of the native enzyme) which is stimulated 5-6-fold in the presence of an equimolar concentration of alpha subunit. The presence of the substrate,acetoacetyl-CoA, is required to recover the catalytic activity of the beta subunit and mixtures containing purified alpha and beta subunits. When the enzyme is dissociation in the presence of 6 M urea and the subunits are not fractioned, removal of the urea by dialysis results in the recovery of 88-98% of enzymic activity and the native alpha2beta2 subunit structure. However, analysis of this renatured enzyme by immunochemical techniques shows that the enzyme does not refold to a completely native conformation. This renatured enzyme exhibits an immunological reactivity more closely resembling the isolated alpha subunit. The results indicate that the alpha subunit serves as a structural subunit, or possible a maturation subunit, imposing a conformation on the beta subunit that is catalytically more competent.
A partial inhibition of acylcarnitine oxidation by arsenite in rat liver mitochondria has been studied. This inhibition is confined to the thiolase(s). The inhibition was observed also in the presence of malate, indicating no selective effect on ketogenesis. Ketogenesis from acetyl-CoA was inhibited by arsenite. Mitochondrial CoA was acylated by acylcarnitine nearly as rapidly in the presence of arsenite as in its absence. Thus, arsenite did not interfere with the availibility of CoA in the mitochondria. No effect of arsenite on enzymes of beta-oxidation other than the thiolase(s) was observed. When arsenite and acylcarnitine were added simultaneously to mitochondria, there was a delay before maximal inhibition of oxygen uptake occurred. When the mitochondria were preincubated with arsenite before addition of acylcarnitine, the inhibitory effect on oxygen utilization was initially large, but then partially repealed. Similar time delays were observed in the activity of acetoacetyl-CoA thiolase of disrupted mitochondria depending on the sequence of arsenite and acetoacetyl-CoA addition. It is suggested that substrate and arsenite complete for the reactive sulfhydryl group at the active site of the thiolase(s).