No abstract

The mitochondrial metabolism of 5-enoylCoAs, which are formed during the (3-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms, was studied with mitochondrial extracts and purified enzymes of (3-oxidation. Metabolites were identified spectrophotometrically and by high performance liquid chromatography. 5-cis-Octenoyl-CoA, a putative metabolite of linolenic acid, was efficiently dehydrogenated by mediumchain acyl-CoA dehydrogenase (EC 1.3.99.3) to 2-trans-5-cisoctadienoyl-CoA, which was isomerized to 3,5-octadienoylCoA either by mitochondrial A3,A2-enoyl-CoA isomerase (EC 5.3.3.8) or by peroxisomal trifunctional enzyme. Further isomerization of 3,5-octadienoyl-CoA to 2-trans-4-transoctadienoyl-CoA in the presence ofsoluble extracts of either rat liver or rat heart mitochondria was observed and attributed to a A3',542'4-dienoyl-CoA isomerase. Qualitatively similar results were obtained with 2-trans-5-trans-octadienoyl-CoA formed by dehydrogenation of 5-trans-octenoyl-CoA. 2-trans-4-trans-Octadienoyl-CoA was a substrate for NADPHdependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34). A soluble extract of rat liver mitochondria catalyzed the isomerization of 2-trans-5-cis-octadienoyl-CoA to 2-trans-4-trans-octadienoylCoA, which upon addition of NADPH, NAD+, and CoA was chain-shortened to hexanoyl-CoA, butyryl-CoA, and acetylCoA. Thus we conclude that odd-numbered double bonds, like even-numbered double bonds, can be reductively removed during the (3-oxidation of polyunsaturated fatty acids.The degradation of unsaturated fatty acids by 8-oxidation involves at least two auxiliary enzymes in addition to the enzymes required for the breakdown of saturated fatty acids (1). The auxiliary enzymes acting on double bonds are 2,4-dienoyl-CoA reductase or 4-enoyl-CoA reductase (EC 1.3.1.34) and A3,A2-enoyl-CoA isomerase (EC 5.3.3.8) (2).Chain shortening of unsaturated fatty acids with double bonds extending from even-numbered carbon atoms leads to the formation of4-enoyl-CoAs, which are dehydrogenated by acyl-CoA dehydrogenase (EC 1.3.99.3) to 2,4-dienoyl-CoAs. An NADPH-dependent 2,4-dienoyl-CoA reductase, originally described by Kunau and Dommes (3), catalyzes the reduction of 2,4-dienoyl-CoAs to 3-enoyl-CoAs, which, after isomerization by A3,A2-enoyl-CoA isomerase to 2-enoylCoAs, can be completely degraded via the P-oxidation spiral.Unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms are, according to Stoffel and Caesar (4), chain-shortened to 3-enoyl-CoAs, which, after isomerization to 2-enoyl-CoAs by A3,A2-enoyl-CoA isomerase, reenter the p-oxidation spiral. If so, 5-enoyl-CoAs are intermediates that would pass once more through the ,8-oxidation spiral before being acted upon by A3,A2-enoylCoA isomerase. This prediction, however, is contradicted by a recent observation of Tserng and Jin (5) who reported that the mitochondrial -oxidation of 5-enoyl-CoAs is dependent on NADPH. Their analysis of metabolites by gas chromatography/mass spectrometry...

Geraylgeranylated Rab proteins usually terminate In either Cys-Cys or Cys-Xaa-Cys, where Xaa is Ala, Ser, or Gly. In both cdases of proteins, the two cysteines are geranylgeranylated, but only the Cys-Xaa-Cys class has been shown to be carboxyl-methylated on the terminal cysteine in Wvo. In the current study, we used recombinant Rab geranylgeranyltransferase and a Rab escort protein (REP-1) to attach geranylgeranyl residues to the two cysteines at the carboxyl termius of Rab3A (Cys-Ala-Cys) and RablA (CysCys). The Many proteins in eukaryotic cells contain prenyl groups, either farnesyl (C15) or geranylgeranyl (C20), attached in thioether linkage to cysteine residues at or near the carboxyl terminus (1). Two broad classes of prenylated proteins exist: (i) those that terminate in CAAX boxes, where C is cysteine, A is an aliphatic amino acid, and X is typically methionine, serine, or leucine, and (ii) those that terminate in either CC or CXC, where C is cysteine and X is typically alanine or serine. After prenylation with either farnesyl or geranylgeranyl groups, the CAAX proteins are cleaved by a protease that removes the three carboxyl-terminal amino acids, after which a methyltransferase carboxyl-methylates the farnesylated or geranylgeranylated cysteine (2). These reactions render the carboxyl terminus hydrophobic, facilitating its interaction with membranes or with other proteins. The CAAX proteins include GTP-binding proteins, such as Ras, Rho, and Rac, as well as the y subunits of heterotrimeric guanine nucleotide binding proteins and nuclear lamins, among others.All known CC-or CXC-terminated proteins belong to the Rab family, and all are modified by geranylgeranyl groups (3-5). The Rab family contains more than 30 low molecular weight GTP-binding proteins, each of which is attached to a specific set of membranous organelles. Rab proteins are required for the budding and fusion process by which membrane vesicles move from one organelle to another (6).Only fragmentary information currently exists regarding the methylation of Rab proteins. YPT5, a Rab protein from the yeast Schizosaccharomyces pombe, terminates in CysAla-Cys (CAC) and was shown to be methylated (7,8). Two other Rab proteins from the same organism, YPT1 and YPT3, which terminate in CC, were not methylated (8). Rab3A, a protein of brain synaptic vesicles, terminates in CAC. In vivo, both cysteines are geranylgeranylated, and the terminal cysteine is carboxyl-methylated (4). Similarly, Rab4, which terminates in CGC, is carboxyl-methylated (9). In contrast, Rab2, which terminates in CC, is geranylgeranylated but not carboxyl-methylated (10).Amembrane-boundenzymethatcarboxyl-methylatesprenylated cysteine has been characterized biochemically in membranes from rat liver (11), bovine brain (12), rabbit brain (13), bovine retinal rod outer segments (14), and human neutrophils (15). The enzyme uses S-adenosyl-L-methionine (SAM) as a methyl donor, and it is inhibited by the end product S-adenosyl-L-homocysteine. It carboxyl-methylates either f...