ABSTRACT
The catalytic activity and the state of aggregation of the dihydrolipoyl transacetylase-lipoamide dehydrogenase binding protein (E2-E3BP) subcomplex of the bovine heart pyruvate dehydrogenase multienzyme complex were investigated. Treatment of E2-E3BP with the chaotropic salts GndnCl or KSCN led to a rapid decrease in transacetylase activity which was accompanied by a loss of the native quaternary structure, as indicated by changes in the sedimentation properties of the E2-E3BP subcomplex. Reassembly or refolding of dissociated E2-E3BP was achieved for the GndnCl-treated subcomplex using a defined protocol. This reassembly procedure effectively excluded all E3BP from the reassembled oligomeric transacetylase. The reassembled oligomeric E2, free of E3BP, was unable to reconstitute the overall activity of the complex following incubation with pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3). In binding studies using radiolabeled components it was demonstrated that the reassembled transacetylase, while retaining its capacity for reductive acetylation and its ability to bind E1, lost its ability to bind E3. The evidence presented in this study indicates that the strong association of E3BP with E2 facilitates the binding of E3, the lipoamide dehydrogenase component, and therefore may have an important role in the assembly and ultimately the catalytic activity of the pyruvate dehydrogenase multienzyme complex.
Subject(s)
Acetyltransferases/metabolism , Myocardium/enzymology , Pyruvate Dehydrogenase Complex/metabolism , Animals , Cattle , Dihydrolipoyllysine-Residue Acetyltransferase , Enzyme ActivationABSTRACT
Pyruvate dehydrogenase complex (PDC) has a key role in the regulation of hepatic lipogenesis by dietary factors. We have investigated the effects of dietary carbohydrate and fat on hepatic PDC. Sucrose-based or starch-based diets were administered for 15 days. A positive correlation between PDC activity and the lipogenic potential of the diet was found. A high-sucrose, fat-free diet caused a 3-fold increase in total activity whereas a high-starch, fat-free diet caused a 1.5-fold increase, as compared with chow-fed rats. Dietary polyunsaturated fat (PUF) caused a marked inhibitory effect on total and active PDC; fish oil being more effective than corn oil. Dietary saturated fat (butter) failed to inhibit the sucrose-induced elevation in total activity, but was almost as effective as fish oil in depressing percent active enzyme. Changes in total PDC activity closely correlated with modifications in the content of enzyme quantitated by immunoblotting, indicating that increased enzyme content and not activation is the predominant mechanism underlying the adaptive response to high-sucrose feeding. This response is suppressed by dietary PUF. Inhibition of hepatic lipogenesis by PUF involves a reduction of PDC content as well as that of several lipogenic enzymes. The relevant mechanisms remain to be established.
Subject(s)
Dietary Fats, Unsaturated/pharmacology , Liver/drug effects , Pyruvate Dehydrogenase Complex/metabolism , Sucrose/antagonists & inhibitors , Animals , Dietary Carbohydrates/administration & dosage , Dietary Fats/administration & dosage , Dietary Fats, Unsaturated/administration & dosage , Lipids/biosynthesis , Liver/enzymology , Male , Rats , Rats, Sprague-Dawley , Sucrose/administration & dosageABSTRACT
1. Pyruvate dehydrogenase complex (PDC) activity was measured in several tissues of rats fed for 7 or 15 days on control, or high-sucrose or high-fat diets. 2. Total activity in adipose tissue increased in the three groups 3-4 fold as compared with chow-fed animals in the first week. Total activity was 60% lower in rats fed the diet containing 22% corn oil for 2 weeks. 3. Hepatic total and PDCa activities were 50-80% higher in rats fed the sucrose diet for 7 or 15 days and decreased 30-40% in those fed on the high-fat diet for 2 weeks.
Subject(s)
Adaptation, Physiological/physiology , Adipose Tissue/enzymology , Dietary Fats/pharmacology , Pyruvate Dehydrogenase Complex/metabolism , Sucrose/pharmacology , Adipose Tissue/drug effects , Animals , Diet , Liver/drug effects , Liver/metabolism , Male , Rats , Rats, Sprague-DawleyABSTRACT
The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.