Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.
Female; Animals; Rats; Amino Acid Sequence; *Gene Expression Regulation; Kinetics; Base Sequence; Immunoblotting; Molecular Sequence Data; Steroid Hydroxylases/*genetics; Circadian Rhythm; DNA/genetics/isolation & purification; Restriction Mapping; Enzyme Induction; Liver/drug effects/*enzymology; Cell Fractionation; Cholesterol 7-alpha-Hydroxylase/biosynthesis/*genetics/immunology; Cholestyramine Resin/pharmacology; Cytochrome P-450 Enzyme System/genetics; Epitopes/analysis; Polyribosomes/metabolism/ultrastructure; Inbred Strains; RNA; Enzymologic; Sequence Homology; Cloning; Nucleic Acid; Messenger/*genetics; Centrifugation; Density Gradient; Molecular/methods
Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.
Li Y C; Wang D P; Chiang J Y
The Journal of biological chemistry
1990
1990-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Cloning and characterization of two major 3-methylcholanthrene inducible hamster liver cytochrome P450s.
Female; Humans; Male; Animals; Rats; Amino Acid Sequence; Reference Values; Base Sequence; Liver/enzymology; Immunoblotting; Molecular Sequence Data; Cricetinae; Mesocricetus; Phenobarbital/pharmacology; Restriction Mapping; Nucleic Acid Hybridization; Cytochrome P-450 Enzyme System/biosynthesis/*genetics; Enzyme Induction; Liver/drug effects/*enzymology; Methylcholanthrene/*pharmacology; Inbred Strains; Sequence Homology; Cloning; Molecular; Nucleic Acid; Microsomes
We have studied the immunochemical properties of two major 3-methylcholanthrene inducible hamster liver cytochrome P450 isozymes, P450 MC1 and P450 MC4. Immunoblots using specific antibodies against P450 MC1 and P450 MC4 demonstrated that these two P450s were present in very low levels in control hamster livers and were greatly induced by 3-methylcholanthrene treatment. P450 MC1 was immunochemically different from P450 MC4, rat P450c and P450d, and rabbit LM4. The immunorelated polypeptide to P450 MC1 was not present in the control or the
Lai T S; Chiang J Y
Archives of biochemistry and biophysics
1990
1990-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/0003-9861(90)90664-k" target="_blank" rel="noreferrer noopener">10.1016/0003-9861(90)90664-k</a>
Aflatoxin B1 metabolism by 3-methylcholanthrene-induced hamster hepatic cytochrome P-450s.
Animals; Rats; Gene Expression Regulation; Liver/enzymology; Antibodies/immunology; Cricetinae; Enzyme Induction/drug effects; Mesocricetus; Aflatoxin B1; Liver/drug effects/*enzymology; Methylcholanthrene/*pharmacology; Aflatoxins/immunology/*metabolism; Cytochrome P-450 Enzyme System/biosynthesis/isolation & purification/*metabolism; Mutagenicity Tests; Salmonella typhimurium/enzymology/genetics; Subcellular Fractions/drug effects/enzymology; Inbred Strains; Enzymologic; Microsomes
We have studied the activation of aflatoxin B1 by hamster liver microsomes and purified hamster cytochrome P-450 isozymes using a umu mutagen test. The hamster liver microsomes or S-9 fractions were much more active than rat liver microsomes or S-9 fractions in the activation of umu gene expression by aflatoxin B1 metabolites. 3-Methyl-cholanthrene treatment increased aflatoxin B1 activation by hamster liver microsomes. Two major 3-methylcholanthrene-inducible cytochrome
Lai T S; Chiang J Y
Journal of biochemical toxicology
1990
1905-06
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/jbt.2570050303" target="_blank" rel="noreferrer noopener">10.1002/jbt.2570050303</a>
Regulation of cholesterol 7 alpha-hydroxylase in the liver. Purification of cholesterol 7 alpha-hydroxylase and the immunochemical evidence for the induction of cholesterol 7 alpha-hydroxylase by cholestyramine and circadian rhythm.
Animals; Rats; Organ Specificity; Kinetics; *Circadian Rhythm; Enzyme Induction; Liver/drug effects/*enzymology; Cholesterol 7-alpha-Hydroxylase/*biosynthesis/isolation & purification/metabolism; Cholestyramine Resin/*pharmacology; Chromatography; Durapatite; Hydroxyapatites; Isoenzymes/*biosynthesis/isolation & purification; Obesity/enzymology; Polyethylene Glycols; Steroid Hydroxylases/*biosynthesis; Inbred Strains; Zucker; Microsomes; Ion Exchange
Two cholesterol 7 alpha-hydroxylase isozymes were purified from liver microsomes of cholestyramine-treated female rats by using anion exchange high performance liquid chromatography. These two cytochrome P-450 isozymes were similar in electrophoretic mobility, immunocross-reactivity, and Vmax but differed in Km for cholesterol, turnover number, and charges. Antibody against the major isozyme was raised in rabbit. This antibody specifically inhibited microsomal cholesterol 7 alpha-hydroxylase activity. Immunoblot of microsomal polypeptides indicated that microsomal cholesterol 7 alpha-hydroxylase enzyme levels were increased in parallel with cholesterol 7 alpha-hydroxylase activity upon the treatment of rats with diet supplemented with cholestyramine. Both cholesterol 7 alpha-hydroxylase activity and enzyme levels were drastically reduced immediately after the removal of cholestyramine from the diet. Cholesterol 7 alpha-hydroxylase activity was also detected in the microsomes of kidney, heart, and lung in about 7-27% of the level found in the liver. 3-Methylcholanthrene treatment induced cholesterol 7 alpha-hydroxylase activity and enzyme level. In contrast, pregnenolone-16 alpha-carbonitrile or dexamethasone treatment greatly depressed enzyme and activity in rats. Cholesterol 7 alpha-hydroxylase enzyme level was 2-3-fold higher in liver microsomes of rats maintained under the reversed light cycle than under the normal light cycle. In genetically obese Zucker rats, cholesterol 7 alpha-hydroxylase activity and enzyme level did not respond to the change in the light cycle, however, were induced to the same levels as in the lean rats by cholestyramine treatment. This study provided the first direct evidence that the bile acid feedback regulation and circadian rhythm of microsomal cholesterol 7 alpha-hydroxylase activity involved the induction of cholesterol 7 alpha-hydroxylase enzyme level.
Chiang J Y; Miller W F; Lin G M
The Journal of biological chemistry
1990
1990-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).