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Turnover of succinyl-CoA:3-oxoacid CoA-transferase in glioma and neuroblastoma cells. Specific influence of acetoacetate in neuroblastoma cells.

ABSTRACT: The specific activity of succinyl-CoA:3-oxo-acid CoA-transferase (3-oxoacid CoA-transferase, EC 2.8.3.5) increases significantly during growth in culture in both mouse neuroblastoma N2a and rat glioma C6 cells. To investigate the mechanism(s) responsible for this, antibody specific for rat brain 3-oxoacid CoA-transferase was raised in rabbits. Immunotitrations of 3-oxoacid CoA-transferase from neuroblastoma and glioma cells on days 3 and 7 of growth after subculture showed that the ratio of 3-oxoacid CoA-transferase activity to immunoprecipitable enzyme protein remained constant, indicating that differences in specific activity of the enzyme at these times in both cell types reflect differences in concentration of enzyme protein. In glioma cells, the relative rate of 3-oxoacid CoA-transferase synthesis was about 0.04-0.05% throughout 9 days in culture. In contrast, the relative rate of synthesis of 3-oxo-acid CoA-transferase in neuroblastoma cells was about 0.07-0.08% on days 3, 5 and 7 after subculture, but fell to 0.052% on day 9. The degradation rates of total cellular protein (t1/2 = 28 h) and 3-oxoacid CoA-transferase (t1/2 = 46-50 h) were similar in both cell lines. The rise in specific activity of the enzyme in both cell lines from days 3 to 7 without a significant increase in the relative rate of synthesis reflects a slow approach to steady-state conditions for the enzyme secondary to its slow degradation. Differences in 3-oxoacid CoA-transferase specific activity between the two cell lines are apparently due to a difference of about 60% in relative rates of enzyme synthesis. The presence of 0.5 mM-acetoacetate in the medium significantly increased the specific activity of 3-oxoacid CoA-transferase in neuroblastoma cells during the early exponential growth phase. This treatment increased the relative rate of synthesis of 3-oxoacid CoA-transferase by 23% (P less than 0.025) in these cells on day 3, suggesting that substrate-mediated induction of enzyme synthesis is a mechanism of regulation of 3-oxoacid CoA-transferase.

SUBMITTER: Haney PM

PROVIDER: S-EPMC1144398 | biostudies-other | 1984 Nov

REPOSITORIES: biostudies-other

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Project description:The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act(TBEA6) (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3'-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn5::mob-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as the sole source of carbon and energy. The transposon Tn5::mob was inserted in a gene showing high homology to class III CoA-transferases. In the present study, analyses of the translation product clearly allocated Act(TBEA6) to this protein family. The predicted secondary structure indicates the lack of a C-terminal ?-helix. Act(TBEA6) was heterologously expressed in Escherichia coli Lemo21(DE3) and was then purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography. Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent V(max) of 44.6 ?mol min(-1) mg(-1) for succinyl-CoA, which corresponds to a turnover number of 36.0 s(-1) per subunit of Act(TBEA6). For 3SP, the apparent V(max) was determined as 46.8 ?mol min(-1) mg(-1), which corresponds to a turnover number of 37.7 s(-1) per subunit of Act(TBEA6). The apparent K(m) values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, the V. paradoxus ?act mutant did not reproduce the phenotype of the Tn5::mob-induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like the wild type. Complementation of the Tn5::mob-induced mutant with pBBR1MCS5::acdDPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn5::mob transposon on acd(TBEA6), located downstream of act(TBEA6).

Project description:Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ?40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to ?-methylmalyl-CoA. The cleavage of ?-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C4-dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica, succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii, and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as characteristic enzymes of this cycle.

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Turnover of succinyl-CoA:3-oxoacid CoA-transferase in glioma and neuroblastoma cells. Specific influence of acetoacetate in neuroblastoma cells.

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