Cloning and functional expression of the cDNA encoding rat lanosterol 14-alpha demethylase.
Lanosterol 14 alpha-demethylase (LDM) is a cytochrome P-450 enzyme in the biosynthetic pathway of cholesterol. As such, it represents a target for cholesterol-lowering drugs. Rat LDM (rLDM) has been purified from the livers of rats treated with cholestyramine. The purified protein was used to generate tryptic fragments which were then sequenced. The amino acid (aa) sequences were used to design oligodeoxyribonucleotide primers and a DNA fragment was generated by RT-PCR to probe a phagemid library. A clone encoding rLDM was isolated from the livers of cholestyramine-treated rats. The clone contains an open reading frame encoding a polypeptide of 486 aa and a predicted molecular mass of 55 045 Da. The deduced aa sequence shows a high degree of identity to the yeast LDM sequences, as well as sequences which match typical P-450 sequence motifs. When produced in a baculovirus/insect cell culture system, LDM activity was detected and inhibited by the specific inhibitor azalanstat with an IC50 value of less than 2 nM. The isolation of this full-length coding sequence should facilitate research into understanding the direct and indirect effects of LDM in the regulation of cholesterol biosynthesis and the search for cholesterol-lowering drugs.
Sloane
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DL
Syntex Discovery Research, Palo Alto, CA 94303, USA.
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O Y
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Leung
R
R
Scarafia
L E
LE
Saldou
N
N
Jarnagin
K
K
Swinney
D C
DC
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002784:Cholesterol / Q000096:biosynthesis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D058886:Sterol 14-Demethylase; D015003:Yeasts / Q000201:enzymology / Q000235:genetics
C548417:Cyp51 protein, rat; D017931:DNA Primers; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase