Identification of human cytochromes P-450 analogous to forms induced by phenobarbital and 3-methylcholanthrene in the rat

Abstract

Antibodies to four rat liver forms of cytochrome P-450, two phenobarbital-inducible (PB1 and PB2) and two 3-methylcholanthrene-inducible (MC1 and MC2) proteins, have been used to make a structural and functional comparison of rat and human cytochromes P-450. Proteins from both species were identified on Western blots by their reaction with these antibodies. In the human liver preparations, structurally related proteins to PB1 and to PB2 were identified in all the samples tested with apparent Mr values of 51 800 and 54 800 for PB1 and 53 600 and 57 200 for PB2. Considerable variation in the content of the lower-Mr proteins was measured between samples and, as with the rat enzymes, samples which reacted well with anti-PB1 also reacted with anti-PB2, indicating that these proteins are regulated at least to some degree, co-ordinately. The apparent Mr values of the major human proteins identified with anti-MC1 and anti-MC2 were 54 400 and 57 000 respectively. Only six (of 31) human samples contained significant amounts of these proteins. The same six samples which reacted with anti-MC1 also reacted with anti-MC2, again indicating co-ordinate regulation of these two proteins. Antibody inhibition of microsomal 7-ethoxycoumarin and 7-ethoxyresorufin metabolism demonstrated a degree of conservation of substrate specificity related to specific P-450 isoenzymes between the species. However, the contributions of the different P-450 isoenzymes to the human microsomal activity were not always related to the rat enzyme with the highest activity towards these substrates.

Multiple forms of cytochrome P-450 related to forms induced marginally by phenobarbital. Differences in structure and in the metabolism of alkoxyresorufins

“…2(a) The proteins (1 ,ug) were a run on SDS/9% -polyacrylamide gels and transferred to nitrocellulose and developed by using the 'Western blot' procedure as described by Adams et al [14]. The rabbit antisera were used at a dilution of 1:1000 and the bands revealed by using peroxidase-labelled anti-(rabbit IgG) and 4-chloro-l-naphthol as peroxidase substrate.…”

“…The filters were repeatedly washed with TBST containing 0.025% SDS until no more radioactivity was eluted, and then dried before exposure to X-ray film. Western blots were carried out as described previously [14].…”

“…Interestingly, PB1 also appears to be elevated at early time points in preneoplastic foci in rat liver [13]. Proteins structurally related to PB1 and PB2 are expressed in high levels in human liver, but with significant inter-individual variation [14]. In order to identify structurally related forms of PB1 and PB2 we have isolated several cytochrome P-450 forms from rat liver and have compared their properties.…”

Potentiation and suppression of mouse liver cytochrome P‐450 isozymes during the acute‐phase response induced by bacterial endotoxin

“…SDS/polyacrylamide gel electrophoresis and transfer to nitrocellulose were carried out as described previously [27]. Antibody-reactive proteins were visualised by peroxidase staining [27], or by incubation with '251-labelled Staphylococcus aureus protein A (0.1 pCi/ml for 45 min at room temperature) followed by autoradiography.…”

“…Antibody-reactive proteins were visualised by peroxidase staining [27], or by incubation with '251-labelled Staphylococcus aureus protein A (0.1 pCi/ml for 45 min at room temperature) followed by autoradiography. Band intensities were estimated by densitometric scanning of either photopositives (peroxidase blots) or autoradiographs (1251 blots) using a Joyce-Loebl Chromoscan-3 densitometer.…”