Inhibition of Coumarin 7-Hydroxylase Activity in Human Liver Microsomes

1. We have constructed a full-length human liver cytochrome P450IIA cDNA from a partial-length clone by oligonucleotide-directed mutagenesis, and subcloned it into the monkey kidney (COS-7) cell expression vector, pSVL. 2. The cDNA encodes a 49 kDa protein with coumarin 7-hydroxylase (COH) activity which cross-reacts with antisera to the mouse cytochrome P-450 isoenzyme responsible for COH activity and comigrates with a human liver microsomal protein. 3. Western blot analysis of a panel of human livers indicates that the level of the 49 kDa protein, detected using antisera to either the mouse COH P-450 or rat P450IIA1 protein, correlates very highly with COH activity. 4. Antisera to the rat P450IIA1 protein can inhibit COH activity in human liver microsomes. Taken together, these data indicate that a member of the P450IIA subfamily is responsible for most, if not all, of the COH activity in human liver.

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Lauric acid hydroxylase activity and cytochrome P450 IV family proteins in human liver microsomes

Biochemical Pharmacology ◽

10.1016/0006-2952(91)90524-9 ◽

1991 ◽

Vol 42 (9) ◽

pp. 1841-1844 ◽

Cited By ~ 11

Author(s):

H.A.A.M. Dlrven ◽

J.G.P. Peters ◽

G.Gordon Gibson ◽

W.H.M. Peters ◽

F.J. Jongeneelen

Keyword(s):

Cytochrome P450 ◽

Lauric Acid ◽

Human Liver ◽

Liver Microsomes ◽

Human Liver Microsomes ◽

Hydroxylase Activity

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A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4′-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6β-hydroxylation

Chemico-Biological Interactions ◽

10.1016/s0009-2797(01)00159-4 ◽

2001 ◽

Vol 134 (3) ◽

pp. 243-249 ◽

Cited By ~ 47

Author(s):

Judy Easterbrook ◽

Drew Fackett ◽

Albert P. Li

Keyword(s):

Rat Liver ◽

Human Liver ◽

Liver Microsomes ◽

Human Liver Microsomes ◽

Rat Liver Microsomes ◽

Aroclor 1254 ◽

Coumarin 7

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A radiometric assay for debrisoquine 4-hydroxylase in human liver microsomes

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y84-013 ◽

1984 ◽

Vol 62 (1) ◽

pp. 84-88 ◽

Cited By ~ 5

Author(s):

M. Nakano ◽

T. Inaba

Keyword(s):

Human Liver ◽

High Performance ◽

Liquid Scintillation ◽

Liver Microsomes ◽

Microsomal Protein ◽

Scintillation Counting ◽

Human Liver Microsomes ◽

Hydroxylase Activity ◽

Radiometric Assay ◽

Layer Chromatography

A radiometric method to assay debrisoquine 4-hydroxylase activity in human liver microsomes was established. Following incubation with 14C-labelled debrisoquine, unreacted debrisoquine was extracted with chloroform; 4-hydroxydcbrisoquine was derivatized with hexafluoroacetylacetone, extracted, and subjected to high performance thin-layer chromatography (HP-TLC) followed by liquid scintillation counting. The Km values for debrisoquine 4-hydroxylase were 70 and 120 μM and the Vmax values were 8 and 24 pmol per milligram microsomal protein per minute. By application of this assay, it was possible to show that 4-hydroxydebrisoquine formation was competitively inhibited by sparteine. Antipyrine up to a concentration of 4 mM had no effect.

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