| The mammalian pyruvate dehydrogenase complex (PDH) is the pivotal enzyme of carbohydrate metabolism. Starvation, diabetes, sepsis, Alzheimer disease as well as primary PDH mutations can affect PDH activity. The detection of PDH deficiencies is complicated due to the complexity of expression and regulation of the PDH complex. This dissertation reports the development of novel methods (i) to measure PDH activity, (ii) to characterize mosaic expression, and (iii) to study of the assembly of the mammalian PDH complex.; By using monoclonal antibodies against four subunits of the PDH complex we have shown that the deficiencies of various subunits can be detected by quantitative Western blot in most patients. Also we have demonstrated that by labeling two different subunits of PDH by an immunocytochemical technique, we can detect mosaicism in cell cultures from female patients. This approach will be particularly useful in identifying females with PDH E1α subunit deficiency as well as for prenatal diagnostics.; The monoclonal anti-PDH antibodies were also used to study PDH complex assembly, activity and regulation based on immunoprecipitation of PDH complex from bovine, human and rat heart mitochondria as well as from human fibroblast cells. The purified complex was used for development of an immunocapture PDH activity assay and for characterization of the assembly of the complex. Two specific pyruvate dehydrogenase kinases (PDK), i.e. PDK1 and PDK2 were found to coimmunopurify with PDH in rat heart mitochondria.; The structure and assembly of immunopurified PDH complex, has been further investigated by 2-D gel electrophoresis. Addition of specific PDH inhibitors and activators was found to alter the charge of E1α subunit and can be used to study phosphorylation levels of this subunit. Such an analysis might be important in examining patients with diabetes, starvation and PDH deficient patients, when it is thought that altered kinetics of the enzyme results from E1α PDH mutations.; To conclude, methods have been developed that will complement those already available to the clinicians and researchers for the analysis of PDH expression, activity and regulation. This dissertation includes my previously published and coauthored materials. |
