title: Sequence of rat mitochondrial glycerol-3-phosphate dehydrogenase cDNA. Evidence for EF-hand calcium-binding domains.
abstract: The FAD-dependent, mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) is an essential component of the glycerol phosphate shuttle and is abundant in the pancreatic insulin cell, skeletal muscle, and brain. Although DNA clones for this enzyme and its homologues have been isolated from bacteria and yeast, it has never been cloned from a higher eukaryote. We have cloned and sequenced cDNAs encoding the rat mitochondrial glycerol-3-phosphate dehydrogenase. The longest cDNA (2337 base pairs) encodes a deduced protein of 727 amino acids that shows strong homology to the yeast and bacterial FAD-dependent glycerol phosphate dehydrogenases. The amino terminus of the purified mature protein was sequenced and shows identity with the deduced amino acid sequence beginning with residue 43. The 42 preceding amino acids are consistent with a mitochondrial leader peptide. A highly conserved FAD-binding domain and conserved regions possibly involved with glycerol phosphate binding are present. An unexpected finding was the homology of the deduced protein to calmodulin. Analysis of the deduced protein sequence shows a region near the carboxyl terminus containing two sequences homologous to "EF-hand" calcium-binding domains that are not present in the shorter yeast and bacterial homologues. The second of these domains appears to have features compatible with considerable affinity for calcium, whereas the first does not. The finding of a potential calcium-binding region is consistent with the known enhancement by calcium of the mammalian enzyme activity at low substrate concentrations and the lack of a requirement for calmodulin. This is the first report of EF-hands in a metabolic enzyme or in a mitochondrial protein.
authors.affiliation: University of Wisconsin Childrens Diabetes Center, Madison 53706.
authors.forename: L J
authors.initials: LJ
authors.lastname: Brown
authors.forename: M J
authors.initials: MJ
authors.lastname: MacDonald
authors.forename: D A
authors.initials: DA
authors.lastname: Lehn
authors.forename: S M
authors.initials: SM
authors.lastname: Moran
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000737:chemistry; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000201:enzymology
chemical_list: D018076:DNA, Complementary; D005993:Glycerolphosphate Dehydrogenase; D002118:Calcium