Pyruvate dehydrogenase E1 α isoform in rat testis: cDNA cloning, characterization, and biochemical comparison of the recombinant testis and liver enzymes

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology Vol. 120; no. 1; pp. 205 - 216
• Main Authors: Jeng, Jiingjau, Kallarakal, Abraham T., Kim, Sungmin F., Popov, Kirill M., Song, Byoung J.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.05.1998
• Subjects: Amino Acid Sequence; Animals; Base Sequence; cDNA cloning; Cloning, Molecular; Gene expression; Gene Expression Regulation, Enzymologic - genetics; Kinetics; Liver - enzymology; Male; Mitochondria - enzymology; Molecular Sequence Data; Phosphorylation; Protein Kinases - metabolism; Protein-Serine-Threonine Kinases; Pyruvate dehydrogenase; Pyruvate Dehydrogenase Complex - chemistry; Rat testis; Rats; Recombinant protein characterization; Recombinant Proteins - chemistry; RNA, Messenger - metabolism; Sequence Analysis, DNA; Sequence variation; Testis - enzymology; Tissue-specific isozyme; FPLC, fast protein liquid chromatography; Phosphorylation; 2,6-DCPIP, 2,6-dichlorophenolindophenol; Recombinant protein characterization; Tissue-specific isozyme; SDS, sodium dodecyl sulfate; Rat testis; Gene expression; DTT, dithiothreitol; TPP, thiamine pyrophosphate; cDNA cloning; PVDF, polyvinylidene difluoride; Sequence variation; PAGE, polyacrylamide gel electrophoresis; PDH, pyruvate dehydrogenase; PDC, pyruvate dehydrogenase complex; Pyruvate dehydrogenase; RT-PCR, reverse transcription followed by polymerase chain reaction; PMS, phenazine methosulphate; IPTG, isopropyl β- d-thiogalactopyranoside; GAPDH, glyceraldehyde-3-phosphate dehydrogenase
• ISSN: 1096-4959; 0305-0491; 1879-1107
• DOI: 10.1016/S0305-0491(98)10010-X

Previous data indicated a tissue-specific regulation of mitochondrial pyruvate dehydrogenase (PDH) complex, especially in the brain and testis. The lack of biochemical data on the rat testis PDH limits comparative analysis between testis and liver enzymes. Therefore, we have isolated a cDNA clone encoding rat testis PDH E1 α isoform, determined its nucleotide sequence, studied the tissue-specific expression, and characterized the recombinant protein produced in bacteria, compared to the liver counterpart. Our cDNA clone (2.2 kb) contained the identical open reading frame (from nt 974 to 2149) with that previously reported (Cullingford et al., 1993 Biochim Biophys Acta 1216:149–153) but contained a long 5′ untranslated region, which has little identity to the other clone. Northern blot confirmed testis-specific expression of this isoform. Genomic DNA analyses by PCR amplification suggested this clone is a gene product distinct from its X-linked somatic counterpart. Our biochemical and kinetic analyses revealed that the purified recombinant rat testis PDH E1 (containing both E1 α and E1 β subunits) was enzymatically active and phosphorylated in vitro by purified PDH-kinase p48 or p45, similar to the recombinant human liver enzyme. Our current data thus indicate that the differential regulation of testis PDH observed in the animal model may result from differential modulation of PDH-kinase or -phosphatase in this tissue rather than the presence of functionally different PDH E1 subunit.