Pyruvate dehydrogenase E1 alpha isoform in rat testis: cDNA cloning, characterization, and biochemical comparison of the recombinant testis and liver enzymes.
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 alpha 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 alpha and E1 beta 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.
Jeng
J
J
Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD 20852, USA.
Kallarakal
A T
AT
Kim
S F
SF
Popov
K M
KM
Song
B J
BJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013737:Testis / Q000201:enzymology*
D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases