The data from our laboratory indicated that 2,3-butanediol, one of the unusual metabolites found in human alcoholics, could be generated by the side reaction of pyruvate dehydrogenase (PDH) located in brain and testis. Based on this hypothesis, we have started to study the genes for the PDH multienzyme complex. All the subunits of the PDH complex were purified to near homogeneity from bovine kidney and heart. The purified proteins were subsequently used for antibody generation. We have then cloned and characterized full-length cDNAs for PDH E1a, E1b, and E3 subunits whose genes are located in chromosome X, 3, and 7, respectively. The nucleotide sequence of human brain PDH E1a clone was identical with that of liver clone indicating that the differences in the production of 2,3-butanediol in brain and liver may not be due to a structural difference in PDH E1a but rather due to alteration in metabolic regulators or tissue specific regulation of PDH differently modulated by PDH-specific kinase and phosphatase. Our clones, however, were different from other cDNA clones isolated from foreskin and fetal liver. Two different types of cDNA clones for PDH E1b subunits were also identified and completely characterized. The sequences of our clones were exactly identical, but they are different from that isolated from foreskin in several regions. The correct sequences of our clones were verified by a method of polymerase chain reaction and DNA sequencing. The differential regulation of PDH complex was also studied in cultured human fibroblasts from patients of lactic acidosis. In two cell lines of Leigh syndrome patients, no abnormality of PDH complex was observed while a major problem in PDH complex was observed in a TCA-cycle defective cell line. The defect in these cells was determined to be related to problems of PDH protein translation and processing. PDH-specific protein kinase and phosphatase were also purified and their N-terminal amino acid sequences were determined. 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.
