Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

Seidel, Sharon L.; Shawver, Laura K.; Shires, Thomas K.

doi:10.1016/0003-9861(84)90183-8

Cited by 12 publications

(6 citation statements)

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“…Solubilization and fractionation of microsomal protein and sample concentration and dialysis follow the procedures described previously (Seidel et al, 1984). All purification buffers contained 20% (w/v) glycerol.…”

Section: Purification Of Cytochrome P450 Isoenzymesmentioning

confidence: 99%

“…Elution of protein from the column with a 1000 ml linear 0-85 mM-KCl Vol. 235 Abbreviations used: MC or 3-MC, 3-methylcholanthrene; cytochrome P-450 PB, a major phenobarbital-inducible form of rat liver cytochrome P-450 previously purified in our laboratory (Seidel et al, 1984).…”

Section: Purification Of Cytochrome P450 Isoenzymesmentioning

confidence: 99%

“…After electro-transfer, non-specific proteinbinding sites on the papers were blocked with 10% (v/v) foetal calf serum and 1 % (w/v) bovine serum albumin in phosphate-buffered saline overnight at 0-4 'C. The blocked papers were then incubated in succession with the primary rabbit anti-(rat cytochrome P-450) IgG and peroxidase-conjugated goat anti-(rabbit IgG) F(ab')2 fragment (Cappel, Cochranville, PA, U.S.A.) as described previously (Seidel et al, 1984). Immuno-blots were developed by using 4-chloro-1-naphthol as the peroxidase substrate (Hawkes et al, 1982), and the reaction was stopped by displacement with distilled water.…”

Section: Immuno-blottingmentioning

confidence: 99%

“…Scheme 1 indicates the general scheme used in the separation of different forms of cytochrome P-450 from liver microsomes of 3-MC-pretreated rats. Solubilization of microsomes with cholate and poly(ethylene glycol) fractionation followed procedures previously described (Seidel et al, 1984). The first column contained DE-53 DEAE-cellulose in the lower half of the column and DE-52 DEAE-cellulose in the upper half.…”

Section: Purification Of Cytochrome P450 Isoenzymesmentioning

confidence: 99%

“…A polyclonal antibody against purified cytochrome P-450 MC-D was raised in rabbits. The IgG fraction from this antiserum was tested for cross-reaction with all of the forms of cytochrome P-450 purified from liver microsomes of 3-MC-pretreated rats and with a phenobarbital-inducible form (cytochrome P-450 PB) which was previously purified and characterized in our laboratory (Seidel et al, 1984). Fig.…”

Section: Immunological Characterizationmentioning

confidence: 99%

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Purification and characterization of a previously unreported form of cytochrome P-448 from the liver of 3-methylcholanthrene-pretreated rats

Seidel¹,

Shires²

1986

Biochemical Journal

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At least four hepatic isoenzymes of cytochrome P-450 were purified and characterized from rats treated with 3-methylcholanthrene. A monoclonal antibody developed against one of the forms (designated cytochrome P-450 MC-B) and polyclonal antibodies against others were used to demonstrate that form MC-B is immunologically distinct from other methylcholanthrene-inducible forms. Limited N-terminal amino acid sequencing showed that cytochrome P-450 MC-B has a primary structure that differs from the N-terminal sequences of other established rat isoenzymes. Cytochrome P-450 MC-B has a minimum Mr of 53,000, a CO-reduced spectral maximum at 448 nm, a Soret maximum of 417 nm in the absolute oxidized spectrum and a pattern of substrate preferences that differs from those of the other methylcholanthrene-induced forms. The other forms (MC-A, MC-C and MC-D) share characteristics with isoenzymes previously reported by other investigators.

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Section: Purification Of Cytochrome P450 Isoenzymesmentioning

confidence: 99%

Section: Purification Of Cytochrome P450 Isoenzymesmentioning

confidence: 99%