The effects of pent-4-enoate and methylenecyclopropylacetate on some enzymes of beta-oxidation in extracts of liver mitochondria.

Pent4enoate is the simplest hypoglycaemic analogue of methylencyclopropylacetate, an active metabolite of the toxic amino acid hypoglycin (see Sherratt et al., 1971). These unusual fatty acids inhibit 8-oxidation (von Holt et al., 1966; Senior et al., 1968); it has been assumed that they do so by the same mechanism because of the requirement for the CH,=C-C-C-CO,H group for hypoglycaemic activity in this series of compounds (Anderson et al., 1958) [see, however, Senior et al. (1968)l. Pent4enoate is oxidized to various extents by different liver preparations (Holland & Sherratt, 1973 ; Williamson et al., 1970). Holland & Sherratt (1973) showed that a metabolite specifically inhibits /I-oxidation in rat liver mitochondria and that purified pig liver acetoacetyl-CoA thiolase (EC 2.3.1.9) was inhibited by one of these, penta-2,4-dienoyl-CoA. Other products of the 8-oxidation of pent4enoate, 3-oxopent4enoyl-CoA and 3-hydroxypent-4-enoyl-CoA, could not be prepared chemically for study. There has been relatively little work on the inhibitory effects of methylenecyclopropylacetate (von Holt et al., 1966), although a metabolite inactivates butyryl-CoA dehydrogenase (EC 1.3.99.2) in mitochondria (Billington et al., 1974). Stewart et al. (1973) described the quantitative oxidation of some acyl-CoA derivatives to acetyl-CoA by an extract of liver mitochondria supplemented with 0.2m~-CoA, 0.3 mM-NAD+ and 125pg of catalase which could be followed polarographically when coupled to oxygen with 0.03 % phenazine methosulphate and 0.001 % Methylene Blue as artificial electron carriers. This system has been used to investigate the mechanism of inhibition of 8-oxidation by pent-4-enoate and by methylenecyclopropylacetate. Acetone-dried rat or ox liver mitochondria (2g) were extracted with l0mM-potassium phosphate, pH7.2, and the extract was centrifuged at 150000ga,. for 60min at 2°C. The extract was passed through a column of Sephadex G-25 pre-equilibrated with 10mMpotassium phosphate buffer, pH7.2, to give a final protein concentration of about 5mg/ ml. This extract rapidly and completely oxidized O.Sm~-butyryl-CoA to acetyl-CoA at 30°C (270ng-atoms/min per mg of protein). It also completely oxidized 0.1 m ~ p e n t 4 enoyl-CoA and 0.1 mM-n-pentanoybCoA at about one-third of the rate of the oxidation of butyryl-CoA, presumably to acetyl-CoA and acryloyl-CoA or propionyl-CoA respectively. This is in contrast with the self-limiting oxidation of pent4enoate in intact mitochondria (Holland & Sherratt, 1973). There was no decrease in the rate of oxidation of butyryl-CoA or hexanoyl-CoA added after the completion of the oxidation of 0.1 mMpent-4-enoyl-CoA. Any inhibition of acetoacetylCoA thiolase by endogenously generated penta-2,4-dienoyl-CoA would be distal in the 8-oxidation sequence to the dehydrogenases coupled to 0, with dyes, and would not therefore be reflected as a decreased rate of O2 uptake. However, the lack of inhibition of the oxidation of hexanoyl-CoA indicates that, in this system, 2-oxoacyl-CoA thiolase was not inhibited sufficiently to be rate-limiting in 8-oxidation. When 0.1 m-pent4enoyl-CoA was incubated with mitochondria1 extracts with the addition of electron acceptors and absence of cofactors, the O2 uptake was consistent with its conversion into penta-2,4-dienoyl-CoA by butyryl-CoA dehydrogenase. The extracts were then passed again through a column of Sephadex G-25 and, acetoacetylCoA thiolase and butyryl-CoA thiolase activities of the eluate were assayed directly (Holland & Sherratt, 1973). A similar incubation with 0.1 mM-n-pentanoyl-CoA, which was oxidized to pent-2enoyl-CoA, was done as a control. With these conditions, acetoacetyl-CoA thiolase was inhibited by 60-70 % in preparations from ox liver and by 30% in those from rat liver which had been incubated with pent-4-enoyl-CoA, but it was not inhibited in control incubations. There was no inhibition of butyryl-CoA dehydrogenase. Similar results were obtained if 0.1 m~-pent-4-enoate or 0.1 mM-n-pentanoate