Cloning of a rat brain succinic semialdehyde reductase involved in the synthesis of the neuromodulator gamma-hydroxybutyrate.
The gamma-hydroxybutyrate biosynthetic enzyme succinic semialdehyde reductase (SSR) was purified to homogeneity from rat brain. Peptides were generated by tryptic cleavage and sequenced. PCR primers were designed from the amino acid sequences of two of the peptides showing a similarity (75-85%) to a mitochondrial aldehyde dehydrogenase. A PCR-amplified DNA fragment was generated from recombinant plasmids prepared by a mass excision procedure from a rat hippocampal cDNA library and used as a probe to screen this cDNA library. One cDNA of 1341 bp had an open reading frame encoding a protein of 447 residues with a deduced molecular mass of 47967 Da. The enzyme was expressed in Escherichia coli. Immunoblotting analysis revealed the existence of a protein with the same electrophoretic mobility as the SSR purified from rat brain and with an estimated molecular mass of 45 kDa. Northern blot experiments showed that this enzyme was not expressed in the kidney or in the liver. In the brain tissue, a single but rather broad band was labelled under high stringency conditions, suggesting the presence of more than one messenger species coding for SSR. Hybridization in situ performed on brain tissue slices showed specific labelling of the hippocampus, the upper cortex layer, the thalamus, the substantia nigra, the cerebellum, the pons medulla and the olfactory tract. The recombinant enzyme showed catalytic properties similar to those of the SSR purified from rat brain, particularly in regard to its substrate affinities and Ki for inhibition by phthalaldehydic acid. Valproic acid did not inhibit the cloned SSR. This enzyme had 20-35% identity in highly conserved regions involved in NADPH binding with four other proteins belonging to the aldo-oxo reductase family.
Laboratoire de Neurobiologie Moléculaire des Interactions Cellulaires, UPR 416 CNRS, Centre de Neurochimie, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France.
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C
Andriamampandry
J C
JC
Siffert
M
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Schmitt
J M
JM
Garnier
A
A
Staub
C
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Muller
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Gobaille
J
J
Mark
M
M
Maitre
D000449:Aldehyde Reductase / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D005260:Female; D015723:Gene Library; D006624:Hippocampus / Q000201:enzymology; D006801:Humans; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012978:Sodium Oxybate / Q000378:metabolism*; D014158:Transcription, Genetic
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012978:Sodium Oxybate; D000449:Aldehyde Reductase; D006884:Hydroxybutyrate Dehydrogenase; C019416:4-hydroxybutyrate dehydrogenase