Cloning, expression, and properties of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.
cDNA encoding the regulatory subunit of bovine mitochondrial pyruvate dehydrogenase phosphatase (PDPr) has been cloned. Overlapping cDNA fragments were generated by the polymerase chain reaction from bovine genomic DNA and from cDNA synthesized from bovine poly(A)+ RNA and total RNA. The complete cDNA (2885 base pairs) contains an open reading frame of 2634 nucleotides encoding a putative presequence of 31 amino acid residues and a mature protein of 847 residues with a calculated Mr of 95,656. This value is in agreement with the molecular mass of native PDPr (95,800 +/- 200 Da) determined by matrix-assisted laser desorption-ionization mass spectrometry. The mature form of PDPr was expressed in Escherichia coli as a maltose-binding protein fusion, and the recombinant protein was purified to near homogeneity. It exhibited properties characteristic of the native PDPr, including recognition by antibodies against native bovine PDPr, ability to decrease the sensitivity of the catalytic subunit to Mg2+, and reversal of this inhibitory effect by the polyamine spermine. A BLAST search of protein data bases revealed that PDPr is distantly related to the mitochondrial flavoprotein dimethylglycine dehydrogenase, which functions in choline degradation.
Lawson
J E
JE
Biochemical Institute and Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712, USA.
Park
S H
SH
Mattison
A R
AR
Yan
J
J
Reed
L J
LJ
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D050893:Dimethylglycine Dehydrogenase; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015870:Gene Expression; D008099:Liver / Q000201:enzymology; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry; D011487:Protein Conformation; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D010089:Oxidoreductases, N-Demethylating; D050893:Dimethylglycine Dehydrogenase; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase