As a continuing project, all the subunits of the pyruvate dehydrogenase (PDH) multienzyme complex were purified to near homogeneity from bovine kidney and heart. The purified proteins were subsequently used for further purification of PDH-kinase and PDH-phosphatase and generation of antibodies against each subunit. Using these antibodies and oligodeoxynucleotides, we have also cloned and determined the nucleotide structures of full-length cDNAs for PDH E1`, E1~, and E3 subunits. We also demonstrated differential expression of these PDH genes in cell- and tissue-specific manners. Particularly an immunologically distinct isoform of PDH E1` with different molecular mass was identified in rat testis. This PDH E1` isoform is only detected by anti-peptide antibodies and immunoprecipitated by monospecific antibodies against PDH E1~ subunit, common to PDH complex in all tissues examined but not by those against PDH E1` subunit of kidney. This specific PDH E1` isotype appeared to be present in testis and brain which may support the potential role of PDH isozyme in acetoin, thus butanediol production found in alcoholic individuals as well as in animal models. The coordinate, transcriptional activation of the subunits of the PDH complex during adipocyte differentiation was also demonstrated. PDH-specific protein kinase and phosphatase were also purified to homogeneity and their N-terminal amino acid sequences were determined. Another protein with PDH-kinase activity (M/r 68,000) was also purified: the peptide sequence analysis revealed that this protein is identical with calelectrin. Based on the partial amino acid sequences, several oligodeoxynucleotides were synthesized and used to clone the genes coding for PDH-specific kinase as well as PDH-phosphatase, whose structures and regulation were not characterized yet.
