PubMed · 7985227
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
J M Gunn. 1994. Many an error by the same example.. https://doi.org/10.1016/0968-0004(94)90110-4
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
The kinetic properties of the purified trifunctional beta-oxidation complex (TOC) from pig heart mitochondria were analyzed with the aim of elucidating the functional consequence of having three sequentially acting enzymes of beta-oxidation associated in one complex. The kinetic parameters of TOC and of the component enzymes of TOC, long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase, were determined with substrates having acyl chains with 16 carbon atoms. Quantification by high performance liquid chromatography of intermediates formed during the degradation of 2-trans-hexadecanoyl-CoA to myristoyl-CoA and acetyl-CoA by TOC revealed the accumulation of 3-hydroxyhexadecanoyl-CoA, whereas 3-ketohexadecanoyl-CoA was undetectable. The observed rates of NADH and acetyl-CoA formation were higher than the theoretical rates calculated by use of the kinetic parameters and measured concentrations of intermediates. When the sequence of reactions catalyzed by TOC was inhibited by acetyl-CoA, the steady-state concentration of the 3-hydroxyacyl-CoA intermediate was not affected, whereas a small amount of 3-ketohexadecanoyl-CoA was detected. The differences between observed and predicted reaction rates and between measured and expected concentrations of intermediates are best explained by the operation of a channeling mechanism. As a consequence of intermediate channeling between the active sites on the complex, more coenzyme A is available in the mitochondrial matrix and metabolites like 3-ketoacyl-CoA thioesters, which are strong inhibitors of several beta-oxidation enzymes, do not accumulate.
The synthesis of 4,7,10,13,16,19-docosahexaenoic acid (22:6(n-3)) requires that when 6,9,12,15,18,21-tetracosahexaenoic acid (24:6(n-3)) is produced in the endoplasmic reticulum, it preferentially moves to peroxisomes for one cycle of beta-oxidation rather than serving as a substrate for membrane lipid synthesis. Both 24:6(n-3) and its precursor, 9,12,15,18,21-tetracosapentaenoic acid (24:5(n-3)), were poor substrates for acylation into 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) by rat liver microsomes. When peroxisomes were incubated with 1-14C- or 3-14C-labeled 7,10,13,16,19-docosapentaenoic acid (22:5(n-3)), [1-14C]22:6(n-3), [3-14C]24:5(n-3), or [3-14C]24:6(n-3), only small amounts of acid-soluble radioactivity were produced when double bond removal at positions 4 and 5 was required. When microsomes and 1-acyl-GPC were included in incubations, the preferred metabolic fate of acids, with their first double bond at either positions 4 or 5, was to move out of peroxisomes for esterification into the acceptor rather than serving as substrates for continued beta-oxidation. When [1-14C]22:6(n-3) or [3-14C]24:6(n-3) was incubated with peroxisomes, 2-trans-4,7,10,13,16,19-22:7 accumulated. The first cycle of 20:5(n-3) beta-oxidation proceeds through 2-trans-4,8,11,14,17-20:6 and thus requires both Delta3,5,Delta2, 4-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase. The accumulation of the substrate for 2,4-dienoyl-CoA reductase, as generated from 22:6(n-3), but not from 20:5(n-3), suggests that this enzyme distinguishes between subtle structural differences. When 22:6(n-3) is produced from 24:6(n-3), its continued degradation is impaired because of low 2,4-dienoyl-CoA reductase activity. This slow reaction rate likely contributes to the transport of 22:6(n-3) out of peroxisomes for rapid acylation into 1-acyl-GPC by microsomes.
A cDNA clone was obtained from Arabidopsis thaliana that encodes a protein containing 92 amino acid residues with high sequence identity (57%) to bovine acyl-CoA-binding protein (ACBP). The coding sequence of this clone was expressed in Escherichia coli and the gene product (10.4 kDa) was purified. The recombinant A. thaliana ACBP (rAthACBP) was shown to bind acyl-CoA esters and protect acyl-CoAs from degradation by microsomal acyl-hydrolases. Antibodies that were raised to rAthACBP recognized the native Arabidopsis ACBP and also cross-reacted with a number of other plant ACBPs, including rapeseed (Brassica napus) ACBP. The pattern of expression and level of the gene product were examined in various tissues of Arabidopsis and Brassica using Western blotting. A. thaliana tissues contained between 3 and 143 micrograms AthACBP g(-1) FW depending on the tissue (0.4 to 14 nmol g(-1) FW). Developing B. napus seeds underwent a 12-fold increase in ACBP levels during seed maturation (20 to 250 micrograms ACBP g(-1) FW); the highest concentration occurring near the peak of triacylglycerol accumulation (26 nmol g(-1) FW.