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Department ofPharmacology, School ofMedicine,
In the past decade, efforts made by severallaboratories have created much progress in
The most meaningful information obtained is that there are multipleforms of
cytochrome P-450 in liver microsomes. Thus, the pharmacological significance of
the multiple forms ofcytochrome P-450 will be discussed.
Multiple forms of cytochrome P-450 in experimental animals
There has been a great number of reports suggesting the presence of at least two
forms of drug metabolizing enzymes in liver microsomes of experimental animals.
For example, treatment of rats with phenobarbitone induces a wide variety of drug
metabolizing activities while treatment with polycyclic hydrocarbons, such as
3-methylcholanthrene, induces only limited activities. The difference in the drug
metabolizing activities were later proved to be caused by the presence of different
forms of cytochrome P-450 in these induced microsomes (Kuntzman, Lu, West,
Jacobson & Conney, 1971; Lu & Levin, 1974; Ryan, Lu, Kawelek, West & Levin,
1975). The purified preparations showed substrate specificities for the oxidations of
benzo[a]pyrene, benzphetamine and other drugs, Thereafter, the presence ofmultiple
forms of cytochrome P-450, which are distinguishable in their catalytic activities, in
molecular weight, in physical property during purification, in amino acid sequence
and in immunological cross reactivity between each form of purified cytochrome
P-450 and the antibody, were suspected. Up to date , the presence of eight forms of
cytochrome P-450 in rabbits and at least four forms of cytochrome P-450 in rat liver
microsomes have been postulated and some of these forms were purified and
partially characterized (Levin, 1977; Guengerich, 1979). The substrate specificity of
the purified cytochrome P-450 is in contrast to the classical concept of the drug
metabolizing enzyme , since a drug metabolizing enzyme having a quite broad
substrate specificity had been believed to be involved in metabolizing a wide variety
ofdrugs consisting ofeven newly synthesized drugs.
PURIFICATION OF HEPA TIC CYTOCHROME P-450
Multiple forms of cytochrome P-450 in human Iiver microsomes
Mark (1969). As reviewed by Pelkonen, Haltiala, Larmi & Karki (1974), the specific
content of cytochrome P-450 in human liver microsomes has been reported to vary
from one individual to another, probably depending upon the freshness of the
sampie, cause of death, intake of drugs, and so on. The purification of cytochrome
protein-I and it was demonstrated that the human cytochrome P-450 was capable of
catalyzing ethylmorphine N-demethylation, requiring a phospholipid for the
activity. Human cytochrome P-450 was purified in this laboratory, to a specific
content of 10.6 nmol mg protein-', the purified preparation apparently showed a
single band on SDS-polyacrylamide gel electrophoresis, with a molecular weight of
To examine further the properties of human cytochrome P-450, Wang, Mason &
Guengerich (1980) purified, from autopsy sampies, a form of cytochrome P-450 to
homogeneity with a specific content ofl4 nmol mg proteirr",
As mentioned above, the presence of multiple forms of cytochrome P-450 was
confirrned in experimental animals. Thus, it is reasonable to assurne that multiple
forms are also present in human microsomes and that they oxidize numerous
endogenous and exogenous compounds. The difficulty in purification ofthe multiple
forms from human liver was due mainly to the type of sample available. When an
autopsy sampie, wh ich was most readily available, was used, a minor or unstable
form ofcytochrome P-450 might have been degraded before starting purification, To
minimize such degradation of the cytochrome after death, Beaune, Remers &
Dansette (1980) employed livers from kidney transplantation donors immediately
after death, to separate three fractions containing cytochrome P-450. The purified
preparations were distinguishable in their catalytic activities. Although furt her
characterization is needed, their fmding was the first evidence showing that there are
multiple forms of cytochrome P-450 in human livers. To further support this, two
forms of cytochrome P-450, from autopsy sampies were recently purified in our
laboratory. Human microsomes were solubilized by sodium cholate, then
cytochrome P-450 was precipitated by polyethylene glycol 6,000. The polyethylene
glycol precipitates were applied to a w-amino-n-octyl Sepharose 4B column.
Cytochrome P-450 was eluted from the column using Emulgen 911 as an inhibitor.
The fractions containing cytochrome P-450 were passed through a DEAE-cellulose
(DE-52) column and then applied to a CM-Sephadex (C-50) column. A majority of
cytochrome P-450 which did not adsorb on the CM-Sephadex column was applied
to a hydroxylapatite column. The cytochrome P-450 was eluted from the column by
increasing the phosphate concentration. This cytochrome P-450 preparation was
designated as Form 1.Cytochrome P-450 adsorbed on the CM -Sephadex column was
eluted by 500 mM potassium phosphate (pH 7.25) containing 20% glyceroI. This
cytochrome P-450 preparation was designated as Form ll . The specific contents of
Form 1 and Form Il were 15.2-16.8 and 6.8-9.0 nmol mg protein-I, respectively.
These two forms still contained a protein as a contaminant but were distinguishable
in their molecular weights (Figure I) and in their catalytic activities (Table I). These
results were then compared with those ofBeaune et a/. (1980) and those obtained by
Wang et a/. (1980). A form ofcytochrome P-450 present in the B2 fraction ofBeaune
et a/. (1980) preparation can be assumed to be unstable, since the cytochrome P-450
preparation by Wang et al. (1980) and the present two cytochrome P-450
preparations, from autopsy sampies, did not show such high activities, in the
oxidation ofethoxycoumarin and benzo[a]pyrene, as did the B2 fraction.
Figure 1 SDS-polyacrylamide gel electrophoresis of partially purified preparations of
protein and in smoke as the result oftryptophan pyrolysis. Trp-P-I and Trp-P-2, as
weil as benzo(a)pyrene, show potent mutagenic activity after undergoing metabolie
activation by cytochrome P-450 of experimental animals (Ishii , Ando, Kamataki &
Kato, 1980; Yamazoe, Ishii, Kamataki, Kato & Sugimura, 1980). The present work
showed that in contrast to rat liver microsomes, 7,8-benzoflavone stimulated
metabolie activation of Trp-P-2 in human liver microsomes. To confirm the ability
of human cytochrome P-450 to activate Trp-P-I and Trp-P-2, cytochrome P-450
was fractionated into four fractions. Human microsomes, from normal portions ofa
Iiver isolated by operation for hepatic cancer, were treated with cholic acid, and
cytochrome P-450 was fractionated by means of to-amino-n-octyl Sepharose 4B,
Table 1 Comparison ofthe activitiesof cytochrome P-450 purified from human and rat liver
microsomes. Activitiesexpressed, for human liver, as nmol substrate nmol P-450-1 mirr" and,
forrat liver,as nmol substratenmol P-450-1or P-448-1mirr" .
• CytochromeP-450 purifiedfrom phenobarbitone-treatedrats.
•• CytochromeP-448 purifiedfrom3-methylcholanthrene-treated rats. ••• Not determined.
PURIFICATIO N OF HEPA TIC CYTOCHROME P-450 83
hydroxylapatite and CM-Sephadex(C-50) columns. Fractions land 11 were
separated by the hydroxylapatite column and subfractions, I-a , I-b, II-a and II-b ,
were separated by CM -Sephadex columns. As can be seen in Table 2, Fraction I-b
showed relatively high mutagen-producing activity.
Table 2 Metabolie activation oftryptophan pyrolysis products by cytochrome P-450 purified
(TA 98 revertants plate" , 0.2 nmol P-450)
(nmol mg proteinrl) 5.3 1.2 16.3
* Subtracted by the number ofcontrol revertants, 27.
Pharmacological implications of multiple forms of cytochrome P-450
There are marked differences in the activities of drug metabolizing enzymes between
species and strains of experimental an imals. If human microsomes metabolize a
particular drug, with a similar rate to microsomes from an experimental animal, it
does not necessarily mean that the m icrosomes from man and the experimental
animal contain the same forms ofcytochrome P-450 and at the same level. Thus, the
nature of human drug metabolizing enzymes can not be estimated without using
human sampIes. In addition, racial differences, probably due to genetic and
nutritional differences, have also been noted. Thus, these differences can, at least in
part, be accounted for by the amount and characteristics of one or more forms of
studies have indicated significant changes in drug metabolizing activities caused by
endogenous and exogenous factors, such as, disease states and drug inta ke. Thus, the
next challenge must be to determine relative changes in the amounts of multiple
forms of cytochrome P-450 by these numerous factors, and implication of the
the cytochrome P-450 forms might have been degraded. On the other hand, it is
impossible to obtain large amounts of human liver sampies to determine the forms of
cytochrome P-450. With progress in the purification procedure, it will be possible to
extract minor forms of cytochrome P-450 remaining in the autopsy sampIes, if the
sampIes can be treated within one hour after death. Thus, the practical approach may
Table 3 Purposes and benefltsin the puriflcation of cytochrome P-450 from human liver.
I Determination of multiplicity of cytochrome P-450 and preparation ofthe antibody.
2 Quantification ofvarious formsof cytochrome P-450 from small sizeof'biopsy sampies using
3 Resolution ofsome important problems.
i Alterations ofcytochrome P-450 under pathological and nonphysiologicalstates.
ii EtTects ofdrugs, diets and environmental chemicals.
iii Quantitative speciesditTerences in cytochrome P-450.
be to use an antibody method whereby even small amounts of human liver may be
used for the determination of the cytochromes. As mentioned above, there are
marked racial differences in drug metabolism. It is hoped that it will be possible to
exchange antibodies internationally for studies on racial differences of cytochrome
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