Biochemistry of acyl-CoA ester utilization in liver mitochondria and endoplasmic reticulum

Liver mitochondria, confirmed as intact by complete suppression of succinate uptake and oxidation, possess a carnitine palmitoyltransferase I that is totally inhibited by malonyl CoA. Succinate dehydrogenase activity in mitochondria, which were isolated by centrifuging partially-purified mitochondria through 1.315 M sucrose, was completely suppressed when [14C]-succinate uptake was abolished by prior incubation of the mitochondria with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and valinomycin. The conclusion that these mitochondria were intact was confirmed by the fact that, when these mitochondria were broken by a freeze-thaw cycle followed by sonication, such inhibition was totally abolished. The yield of mitochondria, microsomes and peroxisomes from the initial homogenate was 17.8%, < 0.1% and zero, respectively, indicating that the mitochondria were not only intact but also essentially free of contamination from microsomes and peroxisomes. The overt form of carnitine palmitoyltransferase (CPT I) in these intact and pure mitochondria was totally inhibited by malonyl CoA, indicating that previous reports of incomplete inhibition in mitochondrial preparations resulted from interference from CPT activity in the inner mitochondrial membrane (CPT II), microsomes or peroxisomes. The complementary roles of microsomal lipase, carnitine acyltransferase and latent diacylglycerol acyltransferases in the synthesis of triacylglycerol within microsomes. It has been previously documented that cytosolic triacylglycerol (TAG) cannot be incorporated en bloc into very-low-density-lipoproteins (VLDL). In order to identify the pathway for synthesis of VLDL TAG within the lumen of the endoplasmic reticulum, the microsomes had to be preconditioned by depleting their endogenous substrates and then fusing them with biotinylated phosphatidylserine liposomes containing CoASH and Mg2+. Incubating these fused microsomes with tri[3H]oleoylglycerol and [14C]oleoyl-CoA yielded intraluminal triacylglycerol with a [3H]:[14C] ratio close to 2:1. This suggests that the precursor tri[3H]oleoylglycerol was hydrolysed by microsomal lipase to di[3H]oleoylglycerol, a significant proportion of which was then internalised into the microsomes and re-esterified with [14C]oleoyl-CoA by the activity of latent diacylglycerol acyltransferase. Since the pathway of intraluminal synthesis of triacylglycerol was carnitine-dependent, and glybenclamide, a potent inhibitor of carnitine acyltransferase activity, markedly reduced the extent of intraluminal triacylglycerol synthesis, the participation of microsomal carnitine acyltransferases in the trafficking of the [14C]oleoyl-CoA into the microsomal lumen for subsequent incorporation into newly synthesised triacylglycerol is established. This study provides the first direct demonstration of the enzymatic processes involved in the intraluminal synthesis of triacylglycerol, which is a major component of VLDL. Fluorescent and thermodynamic techniques to determine the equilibrium binding constants of acyl-CoA binding protein for different acyl-CoAs: A comparative study. Long-chain acyl-CoA esters serve as substrates for carnitine acyltransferases, which catalyze the interconversion of acyl-CoAs to their corresponding carnitine esters and hence facilitate their entry, via carnitine translocase, to the cellular sites of β-oxidation and TAG biosynthesis. However, there is strong evidence that cytosolic acyl-CoA binding protein (ACBP) plays a key role in acyl-CoA sequestration, transport and utilization within cells. Thus, an understanding of the relationship between the concentrations of free and ACBP-bound acyl-CoA is likely to assist in determining whether CAT and CPT I recognize free and/or protein-bound acyl-CoA. The present study demonstrates that, in the presence of ACBP, the free concentration of acyl-CoA is too small to account for the activities measured for these two enzymes. The binding affinity of recombinant rat ACBP, cloned in E. coli, towards different acyl-CoAs was determined using two alternative approaches which have the advantage of not requiring the physical separation of bound from free ligand for determining the dissociation constants. These involved analysing the results of both microcalorimetric thermodynamic and fluorescent displacement studies. For the latter, the choice of the fluorescent probe has been shown to be critical for generating reliable data. A mathematical linearization of the fluorescent displacement curve was developed to facilitate the determination of the dissociation constant (Kd). The affinities of ACBP towards each of the acyl-CoAs used in this study were similar. The Kd values for 16:0-CoA, 16:1-CoA, 18:1-CoA and 22:6-CoA in a buffer identical to the one used in the CPT I assay were 14.2 ± 3.9, 21.5 ± 6.6, 14.1 ± 0.8 and 16.2 ± 4.0 nM, respectively. Determination of the kinetic parameters which control the partitioning of acyl- CoAs between hepatic mitochondrial and microsomal carnitine acyltransferases. Mitochondrial carnitine palmitoyltransferase I (CPT I) and microsomal carnitine acyltransferase (CAT I) regulate the entry of fatty acyl units into their respective organelles. Thus, CPT I and CAT I occupy prominent positions in energy generation by mitochondria and the assembly of VLDLs by microsomes, respectively. The present study has determined the extent to which acyl-CoAs are partitioned between CAT I and CPT I by determining the dissociation constant (Kd) and the turnover number of these two enzymes towards the CoA esters of oleic acid (18:1) and docosahexaenoic acid (22:6), together with the total functional enzyme (Et) values expressed as pmoles of enzyme per mg organelle protein. The latter parameter was experimentally obtained by specific radiolabeling of the enzyme active site. A model of the partitioning of acyl-CoAs between the two enzymes is proposed in this study based on the kinetic parameters derived by mathematical analysis of the raw data