Specific antibodies to phenobarbital-induced cytochrome P-450 were prepared by affinity chromatography and coupled to ferritin with glutaraldehyde. The ferritin antibody conjugates with molecular ratio of approximately one were isolated by gel filtration and were used for immunochemical and immunoelectron-microscopic analyses of the distribution of cytochrome P-450 on microsomes from untreated, phenobarbital- and methylcholanthrene-treated rats.

Binding assay showed that at the saturation level of the antibodies, microsomes from untreated, phenobarbital- and methylcholanthrene-treated rats bind 0·25, 0·41 and 0·14 mol of the antibody per mol of cytochrome P-450, respectively. From these data, the maximum number of the ferritin particles which can bind with microsomes was calculated. This number was in good agreement with the average number of ferritin particles bound per microsome which was determined by electron-microscopic observations of the microsomes incubated with the antibody conjugates at saturation level. Electron-microscopic observations also indicated that smooth microsomes can bind more conjugates than rough microsomes and this finding was consistent with the biochemical data that, on the protein basis, smooth microsomes contain more cytochrome P-450 than rough microsomes, even after correction for ribosomal proteins. The number of ferritin particles bound per smooth microsome was proportional to the diameter and non-random distribution of the ferritin particles on the microsomal vesicles, which was deduced simply by inspection in the previous paper from this laboratory, was confirmed by statistical analyses of electron micrographs of the labelled microsomes.

Previous immunoelectron-microscopic studies from this laboratory revealed that in rat hepatocytes cytochrome P-450 is localized exclusively on almost all the microsomal vesicles derived from endoplasmic reticulum (ER) membrane and on outer nuclear envelopes where the cytochrome is distributed non-randomly, forming small clusters (Matsuura, Fujii-Kuriyama & Tashiro, 1978).

This communication will describe semiquantitative analyses on the distribution of cytochrome P-450 on the microsomal membranes. For this purpose specific antibodies to the cytochrome were coupled with ferritin and the antibody conjugates were used for immunocytochemical localization of cytochrome P-450 on the microsomal membranes.

First of all, from the binding assay of the labelled antibody conjugates to the microsomes, the maximum number of the antibody conjugates which can bind to microsomes at the saturation level was determined. This value was in good agreement with the number of ferritin particles per microsome determined by electron-microscopic observation, indicating that the present direct ferritin antibody technique could be used at least for a semiquantitative estimation of cytochrome P-450 on each microsomal vesicle.

Induction of the biosynthesis of cytochrome P-450 by treatment with phenobarbital increased the proportion of cytochrome P-450 which can bind with the conjugates, while induction with methylcholanthrene decreased it. This finding supports the possibility that the biosyntheses of cytochrome P-450 and cytochrome P-448 are independently regulated as suggested by previous authors (Bidleman & Mannering, 1970; Alvares & Kappas, 1978).

Distribution of the ferritin particles on the surface of the microsomal vesicles was analysed statistically by comparing it with the expected random (Poisson) distribution and shown to be non-random to a high degree of statistical significance.

Animals

Male Sprague-Dawley rats (∼ 200 g) were used. They were fed laboratory chow ad libitum and fasted 24 h before sacrifice. Liver microsomes were prepared from untreated, phenobarbital- or 3-methylcholanthrene-treated rats. For the latter purpose rats were given a single daily intraperitoneal injection of phenobarbital or 3-methylcholanthrene for 4 days (10 and 2-5 mg per too g body weight, respectively) and were fasted for 24 h before sacrifice.

Preparation of microsomes and other cell fractions

Total microsomes were prepared as described previously (Matsuura et al. 1978), and smooth and rough microsomes were prepared by discontinuous sucrose density gradient centrifugation according to Cardelli, Long & Pitot (1976). Golgi fraction was prepared by the procedures of Hino, Asano, Sato & Shimizu (1978). Mitochondria were prepared by conventional method as described by Brunner & Bygrave (1969) and plasma membrane was prepared by the procedure of Ray (1970).

Biochemical analyses

Schneider’s procedures (1961) were used for the extraction of RNA. The amount of RNA was determined by the orcinal reaction (Schneider, 1961) and protein was determined by the method of Lowry, Rosebrough, Farr & Randall (1951) using bovine serum albumin as the standard.

Cytochrome P-450 and P-448 were determined by the method of Omura & Sato (1964) from the CO-difference spectra of the reduced samples using an extinction coefficient of 91 mM−1 cm−1 for the difference in absorbance between 450 (or 448) and 490 nm. NADPH-cytochrome c reductase and cytochrome b6 were estimated as described by Omura & Takesue (1970). Glucose-6-phosphatase (G6Pase), galactosyl transferase, 5’-nucleotidase and succinatecytochrome c reductase were determined by the procedures of Leskes, Siekevitz & Palade (1971), Fleischer, Fleischer & Ozawa (1969), Emmelot, Bos, Benedetti & Rümke (1964), and Stotz (1955), respectively.

Immunochemical procedures

Cytochrome P-450 was prepared from liver microsomes of phenobarbital-treated rats and antiserum to the cytochrome was produced in rabbits as described previously (Matsuura et al. 1978). The immunoglobulin G (IgG) fraction to the antiserum was precipitated with ammonium sulphate and the specific antibody was purified by affinity chromatography using Sepharose 4B conjugated with the cytochrome as will be described in detail elsewhere (Fujii-Kuriyama, Mikawa, Matsuura & Tashiro, unpublished). Preparation of the ferritin-antibody conjugates, 125I-labelled antibodies and ferritin-125I-antibody conjugates were also described in the previous paper (Matsuura et al. 1978).

The antibody content of the specific antibody preparation and the ferritin-antibody conjugates were estimated from the binding assay to the immunoadsorbent gel as described by Matsuura et al. (1978). The value obtained was 80–90% for the former and ∼ 40% for the latter, respectively. Probably inactivation by glutaraldehyde in the course of the coupling procedures and the steric hindrance by the ferritin molecules may be responsible for the decrease in the antibody content of the ferritin-antibody conjugates.

Binding of the ferritin-antibody conjugates

Rough and smooth microsome fractions were resuspended in 0·25 M sucrose-TKM (0·05 M Tris-HCl, pH 7·4, 0·05 M KC1 and 0·005 M MgCl2). To aliquots of microsomal suspension containing 100 μg membrane proteins, 50, 100, 150, 200 and 300 μl each of the 125I-labelled antibody conjugate solution (∼ 1·3 mg IgG/ml) was added and incubated for 60 min at ∼ 4 °C. After incubation, the solutions were overlayered onto 0·44-1·15 M continuous sucrose density gradient containing TKM and centrifuged for 45 min at 40000 rev/min in a Hitachi RPS-65T rotor. Measurement of radioactivity indicates that unbound conjugates were around the top of the sucrose density gradient. While 94-95 % of the microsomal proteins was recovered as a pellet at the bottom of the centrifuge tube, the rest of the microsomal proteins (5-6 %) was recovered from the lower part of the gradient, where hardly any radioactivity was detected. This means that almost all the microsomal vesicles containing cytochrome P-450 were recovered in the pellet and the membrane structures remained in the lower part of the gradient do not contain the cytochrome and therefore are probably not derived from ER membranes.

Similar experiments were carried out using ferritin conjugated with control IgG (control conjugates). Binding of the microsomes with 125I-labelled control conjugates was 7-8% of the experiments in the case of microsomes from untreated rats and these values were subtracted from the experimental radioactivities.

Effect of glutaraldehyde fixation on the binding

Rat liver smooth microsomes prepared from untreated and phenobarbital-treated rats were fixed for 15 min at o °C with glutaraldehyde (final cone. 0·5 %) in 0·1 M triethanolamine buffer, pH 7·4 and centrifuged for 5 min at 1000 g. The pellet was resuspended in 0·1 M glycine in the same buffer and then enough ferritin 125I-labelled antibody conjugates were added to the suspension over the saturation level for the immunochemical reaction. After incubation, the bound conjugates were separated from the unbound conjugates by the sucrose density gradient centrifugation as described above and the radioactivities of the bound conjugates in the pellets were counted by a Packard 5330 autogamma spectrophotometer.

Electron microscopy

The thin pellets of the microsomal fractions were fixed in a mixture of glutaraldehyde and osmium tetroxide (Trump & Bulger, 1966; Hirsch & Fedorko, 1968), dehydrated, embedded, and sectioned as described previously (Matsuura et al. 1978). The membranous structure of the pellets was well preserved throughout the procedures for electron microscopy so that a systematic electron-microscopic survey from the top to the bottom of the pellets was easily performed.

Biochemical data

In Table 1 are listed some biochemical properties of the smooth and rough microsomes from untreated and phenobarbital-treated rats. To assess the purity of the isolated microsome fractions, we determined the activities of several marker enzymes in the preparation and compared them with those of the other subcellular fractions. The marker enzymes used for this purpose were galactosyltransferase for Golgi, 5–-nucleotidase for plasma membrane and succinate-cytochrome c reductase for mitochondria.

Table 1.

Biochemical properties of the microsomal preparations

Biochemical properties of the microsomal preparations
Biochemical properties of the microsomal preparations

From these data it is roughly estimated that the smooth microsome fraction is slightly contaminated with Golgi and plasma membranes, while the rough microsome fraction is slightly contaminated with mitochondria. More detailed analyses will be described in the Discussion.

Table 2 shows the content of cytochrome P-450 and P-448 in the rough and smooth microsomes from control, phenobarbital- and methylcholanthrene-treated rats. On the protein basis, smooth microsomes contain more P-450 than the rough microsomes, as reported previously (Orrenius, Ericsson & Ernster, 1965; Staeubli, Hess & Weibel, 1969; Ichikawa & Yamano, 1970; Fleischer, Fleischer, Azzi & Chance, 1971; Craft, Cooper, Shephard & Rabin, 1975; Matsuura et al. 1978). Table 2 also indicates that the phenobarbital and methylcholanthrene treatments induced a marked increase in the cytochrome content of the smooth microsomes.

Table 2.

Cytochrome P-450 (P-448) content of rough and smooth microsomes from untreated, phenobarbital- and methylcholanthrene-treated rats

Cytochrome P-450 (P-448) content of rough and smooth microsomes from untreated, phenobarbital- and methylcholanthrene-treated rats
Cytochrome P-450 (P-448) content of rough and smooth microsomes from untreated, phenobarbital- and methylcholanthrene-treated rats

In addition to the increase in the cytochrome content, membrane proteins of the smooth microsomes were also increased by these treatments (Table 3). Thus the total amount of cytochrome P-450 and P-448 of the smooth microsomes increased ∼ 6-fold and ∼ 4-fold after 4 days treatment with phenobarbital and methylcholanthrene, respectively, while that of the rough microsomes increased less than i-5-fold.

Table 3.

Percentage increase in amount of microsomal membrane proteins and total cytochrome P-450 (P-450 content times membrane protein) of rough and smooth microsomes after phenobarbital and methylcholanthrene treatment for 4 days

Percentage increase in amount of microsomal membrane proteins and total cytochrome P-450 (P-450 content times membrane protein) of rough and smooth microsomes after phenobarbital and methylcholanthrene treatment for 4 days
Percentage increase in amount of microsomal membrane proteins and total cytochrome P-450 (P-450 content times membrane protein) of rough and smooth microsomes after phenobarbital and methylcholanthrene treatment for 4 days

This finding is consistent with the previous electron-microscopic observations that treatment with phenobarbital and methylcholanthrene induced preferential proliferation of smooth ER (Orrenius et al. 1965; Staeubli et al. 1969; Matsuura et al. 1978).

Immunochemical analyses

Fig. 1 shows binding of the antibody conjugates with rat liver smooth microtomes, where the amount in μg of the antibody bound per μg of microsome protein (vertical axis) were plotted against mol of antibody added per mol of cytochrome P-450. It is evident that the amount of the antibody conjugates bound increased linearly with the increase in the amount of the conjugates added, reaching a plateau when the specific antibodies were added in more than 6-8 mol per mol of cytochrome P-450.

Fig. 1.

Binding of ferritin 125I-labelled antibody conjugates to liver smooth microsomes from untreated rat (•), and rat treated with phenobarbital (▪) and methylcholanthrene (▴). Binding to rough microsomes from untreated (○) and phenobarbital-(▫) treated rats are also plotted. Ordinate in Figs. 1 and 2 show μg of antibody bound per μg of microsomal protein and mol of antibody bound per mol of cytochrome P-450, respectively. Cytochrome P-450 content of each sample in nmol per mg protein as follows: smooth microsomes from untreated rats, 1·1; from phenobarbital-treated rats, 2·0; from 3-methylcholanthrene-treated rats, 2·6. Rough microsomes from untreated rats, 0·45; from phenobarbital-treated rats, 0·7.

Fig. 1.

Binding of ferritin 125I-labelled antibody conjugates to liver smooth microsomes from untreated rat (•), and rat treated with phenobarbital (▪) and methylcholanthrene (▴). Binding to rough microsomes from untreated (○) and phenobarbital-(▫) treated rats are also plotted. Ordinate in Figs. 1 and 2 show μg of antibody bound per μg of microsomal protein and mol of antibody bound per mol of cytochrome P-450, respectively. Cytochrome P-450 content of each sample in nmol per mg protein as follows: smooth microsomes from untreated rats, 1·1; from phenobarbital-treated rats, 2·0; from 3-methylcholanthrene-treated rats, 2·6. Rough microsomes from untreated rats, 0·45; from phenobarbital-treated rats, 0·7.

Phenobarbital treatment markedly increased the amount of the conjugates bound to the microsomes as shown in Fig. 1 (▪). In contrast, methylcholanthrene treatment did not facilitate binding of the antibody to the microsomes (▴), although cytochrome P-448 content of the smooth microsomes itself increased to about double after the treatment with methylcholanthrene, as shown in Table 2. This result is to be expected, because the antibody used for the present experiment is monospecific to phenobarbital-induced P-450 and cross-reacts very little with methylcholanthrene-induced P-448 (Mikawa, Fujii-Kuriyama & Tashiro, manuscript in preparation). Fig. 1 also shows that binding of the antibody conjugates to rough microsomes (▫) from phenobarbital-treated rats is less than one half of that with smooth microsomes (○). This result is in agreement with the biochemical data shown in Table 2.

In Fig. 2, ratio of mol of antibody bound per mol of cytochrome P-450 was plotted against that of mol of antibody added per mol of the cytochrome. Microsomes from phenobarbital-treated rats (▪) bound more conjugates than those from untreated rats (•), while microsomes from 3-methylcholanthrene-treated rats bound less conjugates (▴). This result is also to be expected because the proportion of phenobarbital-induced cytochrome P-450 in the total microsomal cytochrome P-450S increases in microsomes from phenobarbital-treated rats, while it decreases in those from methylcholanthrene-treated rats.

Fig. 2.

Binding of ferritin 125I-labelled antibody conjugates to liver smooth microsomes from untreated rat (•), and rat treated with phenobarbital (▪) and methylcholanthrene (▴). Binding to rough microsomes from untreated (○) and phenobarbital-(▫) treated rats are also plotted. Ordinate in Figs. 1 and 2 show μg of antibody bound per μg of microsomal protein and mol of antibody bound per mol of cytochrome P-450, respectively. Cytochrome P-450 content of each sample in nmol per mg protein as follows: smooth microsomes from untreated rats, 1·1; from phenobarbital-treated rats, 2·0; from 3-methylcholanthrene-treated rats, 2·6. Rough microsomes from untreated rats, 0·45; from phenobarbital-treated rats, 0·7.

Fig. 2.

Binding of ferritin 125I-labelled antibody conjugates to liver smooth microsomes from untreated rat (•), and rat treated with phenobarbital (▪) and methylcholanthrene (▴). Binding to rough microsomes from untreated (○) and phenobarbital-(▫) treated rats are also plotted. Ordinate in Figs. 1 and 2 show μg of antibody bound per μg of microsomal protein and mol of antibody bound per mol of cytochrome P-450, respectively. Cytochrome P-450 content of each sample in nmol per mg protein as follows: smooth microsomes from untreated rats, 1·1; from phenobarbital-treated rats, 2·0; from 3-methylcholanthrene-treated rats, 2·6. Rough microsomes from untreated rats, 0·45; from phenobarbital-treated rats, 0·7.

From Fig. 2, it is possible to estimate mol of antibody bound per mol of cytochrome at the saturation level. Table 4 shows the results of 2 experiments. On average, 0·25, 0·41 and 0·14 mol of antibody were bound per mol of cytochrome in the microsomes from untreated, phenobarbital-treated, and methylcholanthrene-treated rats, respectively.

Table 4.

Mol of specific antibodies bound per mol of cytochrome -P-450 on the microsomes

Mol of specific antibodies bound per mol of cytochrome -P-450 on the microsomes
Mol of specific antibodies bound per mol of cytochrome -P-450 on the microsomes

Morphological observations

Figs. 3,4, 5 show high-magnification views of ferritin immuno-electron micrographs of liver microsomes from untreated rats and rats treated with phenobarbital and methylcholanthrene, respectively. As has been reported recently (Matsuura et al. 1978), ferritin particles labelled almost all the microsomal vesicles from untreated rats where they exist non-randomly, sometimes forming micro-clusters as shown by the arrowheads in Fig. 3, while almost all the surface of the vesicles from phenobarbital-treated rats are completely covered by the particles, as shown in Fig. 4. Even in the latter case heterogeneous distribution of the particles is evident on some of the vesicles (arrowheads). When liver microsomes from rats treated with 3-methylcholanthrene were incubated with the antibody conjugates, labelling of the microsomes was similar to those from untreated rats as shown in Fig. 5.

Fig. 3.

Electron micrographs of microsomes incubated with ferritin-antibody conjugates. Liver microsomes were prepared from untreated rats (Fig. 3), and from rats treated with phenobarbital (Fig. 4), and methylcholanthrene (Fig. 5), respectively. In Fig. 3, arrowheads show clustering of ferritin particles and the small arrows in a cross-sectional microsomal profile at the left side of the photograph indicate how the profile was divided into 50-nm sections, starting from the most upper arrow marked with an asterisk. In Fig. 4, arrowheads indicate heterogeneous distribution of ferritin particles on the microsome from phenobarbital-treated rat. Bar, too nm, × 112500.

Fig. 3.

Electron micrographs of microsomes incubated with ferritin-antibody conjugates. Liver microsomes were prepared from untreated rats (Fig. 3), and from rats treated with phenobarbital (Fig. 4), and methylcholanthrene (Fig. 5), respectively. In Fig. 3, arrowheads show clustering of ferritin particles and the small arrows in a cross-sectional microsomal profile at the left side of the photograph indicate how the profile was divided into 50-nm sections, starting from the most upper arrow marked with an asterisk. In Fig. 4, arrowheads indicate heterogeneous distribution of ferritin particles on the microsome from phenobarbital-treated rat. Bar, too nm, × 112500.

Fig. 4.

Electron micrographs of microsomes incubated with ferritin-antibody conjugates. Liver microsomes were prepared from untreated rats (Fig. 3), and from rats treated with phenobarbital (Fig. 4), and methylcholanthrene (Fig. 5), respectively. In Fig. 3, arrowheads show clustering of ferritin particles and the small arrows in a cross-sectional microsomal profile at the left side of the photograph indicate how the profile was divided into 50-nm sections, starting from the most upper arrow marked with an asterisk. In Fig. 4, arrowheads indicate heterogeneous distribution of ferritin particles on the microsome from phenobarbital-treated rat. Bar, too nm, × 112500.

Fig. 4.

Electron micrographs of microsomes incubated with ferritin-antibody conjugates. Liver microsomes were prepared from untreated rats (Fig. 3), and from rats treated with phenobarbital (Fig. 4), and methylcholanthrene (Fig. 5), respectively. In Fig. 3, arrowheads show clustering of ferritin particles and the small arrows in a cross-sectional microsomal profile at the left side of the photograph indicate how the profile was divided into 50-nm sections, starting from the most upper arrow marked with an asterisk. In Fig. 4, arrowheads indicate heterogeneous distribution of ferritin particles on the microsome from phenobarbital-treated rat. Bar, too nm, × 112500.

Fig. 5.

Electron micrographs of microsomes incubated with ferritin-antibody conjugates. Liver microsomes were prepared from untreated rats (Fig. 3), and from rats treated with phenobarbital (Fig. 4), and methylcholanthrene (Fig. 5), respectively. In Fig. 3, arrowheads show clustering of ferritin particles and the small arrows in a cross-sectional microsomal profile at the left side of the photograph indicate how the profile was divided into 50-nm sections, starting from the most upper arrow marked with an asterisk. In Fig. 4, arrowheads indicate heterogeneous distribution of ferritin particles on the microsome from phenobarbital-treated rat. Bar, too nm, × 112500.

Fig. 5.

Electron micrographs of microsomes incubated with ferritin-antibody conjugates. Liver microsomes were prepared from untreated rats (Fig. 3), and from rats treated with phenobarbital (Fig. 4), and methylcholanthrene (Fig. 5), respectively. In Fig. 3, arrowheads show clustering of ferritin particles and the small arrows in a cross-sectional microsomal profile at the left side of the photograph indicate how the profile was divided into 50-nm sections, starting from the most upper arrow marked with an asterisk. In Fig. 4, arrowheads indicate heterogeneous distribution of ferritin particles on the microsome from phenobarbital-treated rat. Bar, too nm, × 112500.

In order to analyse the microsomal profiles more quantitatively, they are classified according to their diameter and the direction of sectioning (either cross-sectional or tangential) as shown in Table 5. Since most of the smooth vesicles were labelled with ferritin as reported previously (Matsuura et al. 1978), the non-labelled vesicles were not classified as were the labelled vesicles. It is evident, however, that the unlabelled vesicles are rather large in diameter and sometimes show open membrane structures, suggesting that they are probably derived either from plasma membranes or Golgi membranes. This problem will be discussed later.

Table 5.

Distribution of profiles of liver microsomes from untreated, phenobarbital-, and methylcholanthrene-treated rats*

Distribution of profiles of liver microsomes from untreated, phenobarbital-, and methylcholanthrene-treated rats*
Distribution of profiles of liver microsomes from untreated, phenobarbital-, and methylcholanthrene-treated rats*

The other vesicles are labelled with various numbers of ferritin particles and their cross-sectional diameter ranged from 60 to 200 nm, with mean diameter of ∼ 140, 120 and 130 nm for microsomes from untreated, phenobarbital- and methylcholanthrene-treated rats, respectively. The former diameter is in good agreement with the corresponding value (135·4 nm) reported by Wibo, Amar-Costesec, Berthet & Beaufay (1971).

Diameter of the microsomal vesicles and the number of bound ferritin particles

We tried to find correlation between the diameter of microsome section and the number of conjugates bound to microsomes. For this purpose microsome profiles were classified first into tangential and cross-sectional ones, and then according to their diameter, and the ferritin particles on each microsomal profile were counted. These analyses were carried out on a number of smooth microsomes and frequency distributions of the number of ferritin particles per profile were plotted on microsomes from untreated rats (Fig. 6A), and rats treated with phenobarbital (Fig. 6B) and methylcholanthrene (Fig. 6 c), respectively. Then the average numbers of ferritin particles bound per microsome were plotted against the mean diameters of microsomes. As shown in Fig. 7, it is apparent that a linear relationship exists on microsomes from untreated, phenobarbital- and methylcholanthrene-treated rats, respectively.

Fig. 6.

Distribution of number of profiles in relation to the ferritin load on the microsomes from untreated rats (A), and rats treated with phenobarbital (B) and methylcholanthrene (C) treated rats, respectively. Profiles were grouped according to their direction of sectioning (either tangential, I, or cross-sectional, II) and their diameter (numerals at right-hand side), and the numbers of profiles bearing n ferritin images were determined. The analysis was performed on 500 profiles for crosssectional and tangential views, respectively. The number of the particles in the control experiments was so small that the control values were neglected.

Fig. 6.

Distribution of number of profiles in relation to the ferritin load on the microsomes from untreated rats (A), and rats treated with phenobarbital (B) and methylcholanthrene (C) treated rats, respectively. Profiles were grouped according to their direction of sectioning (either tangential, I, or cross-sectional, II) and their diameter (numerals at right-hand side), and the numbers of profiles bearing n ferritin images were determined. The analysis was performed on 500 profiles for crosssectional and tangential views, respectively. The number of the particles in the control experiments was so small that the control values were neglected.

Fig. 7.

Relationships between the mean number of ferritin particles bound per microsomal profile and the diameter of smooth microsomes from untreated rats (○, cross sectional; •, tangential), and rats treated with phenobarbital (▫, cross sectional; ▪, tangential) and methylcholanthrene (▵, cross sectional; ▴ tangential), respectively. •, cross sectional from untreated rats, fixed with 0·5 % glutaraldehyde.

Fig. 7.

Relationships between the mean number of ferritin particles bound per microsomal profile and the diameter of smooth microsomes from untreated rats (○, cross sectional; •, tangential), and rats treated with phenobarbital (▫, cross sectional; ▪, tangential) and methylcholanthrene (▵, cross sectional; ▴ tangential), respectively. •, cross sectional from untreated rats, fixed with 0·5 % glutaraldehyde.

This analysis clearly indicates that cytochrome P-450 molecules are distributed on almost all the microsomal vesicles in proportion to the perimeter of each vesicle.

Statistical analysis of ferritin particles on each microsomal vesicle

In the previous paper (Matsuura et al. 1978) we reported that the distribution of ferritin particles on each microsomal vesicle is usually heterogeneous, indicating clustering of cytochrome P-450. It is difficult, however, to determine by simple inspection whether an array of ferritin particles is ordered in some way or truly random. We have used statistical analysis to compare the distribution pattern of ferritin particles observed with an expected random pattern. The method is based on the fact that if one samples small unit areas from a large array of elements and scores the number of elements occurring in each area, the probability Px of finding x elements in an area is given by the formula of the Poisson distribution,
formula
where m is the mean number of elements per unit area sampled. This distribution is valid when the elements are arranged randomly, i.e. when each is independent of the others and each has an equal probability of occurring in any sample area. This kind of analysis has been applied for determination of two-dimensional distribution of surface immunoglobulin on murine lymphocytes by Abbas, Ault, Kamovsky & Uanue (1975).

In order to apply this method to sectioned specimens, 2 methods appear to be possible. One is to use tangential or grazing profiles of the vesicles and another is to use cross-sectional profiles. Difficulties associated with the former approach are that the boundary of the microsomal vesicles is not always clear and it is not always easy to observe a number of large tangential profiles of microsomes, while difficulty with the latter approach is the problem of overlapping of the ferritin particles. We analysed the cross-sectional profiles, neglecting for a while overlapping of the ferritin particles. Since it is difficult to determine precisely the thickness of the thin sections used in electron microscopy, thin sections with an interference colour of grayish tone were exclusively employed.

We observed a number of photographs of microsomes from untreated rats which showed ferritin particles clearly and enlarged them to a final magnification of 100000 times or more as shown in Fig. 3. Then we selected smooth microsomal vesicles with strictly cross-sectional profile and scored them from the top of the vesicles (the arrow with star in Fig. 3) along the perimeter at intervals of 50 nm as shown by a series of arrows in Fig. 3.

We counted successively the number of ferritin particles on each 50-nm section which corresponds to the number of the particles in a small rectangle, 2125 nm2 in area, if the thickness of the sections is assumed to be 42-5 nm as is described later. The total number of 50-nm sections (n) and the total number of ferritin particles were counted to determine m, the mean number of ferritin per section. Then each section was examined and a tally was kept of the number of sections containing 0, 1, 2, …, etc. ferritin particles. It was then possible to draw a bar graph of the number of sections containing x ferritin versus each value of x. At the same time, the value of Px was calculated and the products of Px times n were plotted. Fig. 8 shows comparison of the bar graph of the actual distribution of ferritin particles on a number of liver microsomes from untreated rats and the calculated values expected if the same number of ferritin particles were randomly distributed through the same sections.

Fig. 8.

Statistical analyses of distribution of ferritin particles on smooth microsomes from untreated rats. Bars represent the observed distribution and the curve represents the corresponding expected random (Poisson) distribution in terms of the numbers of 50-nm sections (f) containing x ferritin particles.

Fig. 8.

Statistical analyses of distribution of ferritin particles on smooth microsomes from untreated rats. Bars represent the observed distribution and the curve represents the corresponding expected random (Poisson) distribution in terms of the numbers of 50-nm sections (f) containing x ferritin particles.

There were considerably more sections observed to contain no particles (x = 0) than would be expected. Thus, the observed pattern deviates from random in such a way that there is too much unoccupied area, which implies that the ferritin particles tend to be clustered together more than would be expected on the bases of random distribution.

The first row of Table 6 gives the basic data for a number of cross-sectional profiles of microsomes from untreated rats we have tested. For true random (Poisson) distribution, the variance of the distribution σ2 is equal to the mean, and if it deviates toward clustering, the variance will be greater than the mean (Abbas et al. 1975). It can be seen that the variance is greater than the mean, confirming the tendency toward clustering of the particles.

Table 6.

Statistical analysis of the distribution of cytochrome P-450 on the liver microsomal vesicles prepared from untreated and methylcholanthrene-treated rats

Statistical analysis of the distribution of cytochrome P-450 on the liver microsomal vesicles prepared from untreated and methylcholanthrene-treated rats
Statistical analysis of the distribution of cytochrome P-450 on the liver microsomal vesicles prepared from untreated and methylcholanthrene-treated rats

In order to compare the observed distribution quantitatively with the expected random distribution, a ‘goodness of fit’ was performed as used by Abbas et al. (1975). To do this we used a Chi-square test in which the Chi-square value is calculated for each value of x and these are summed to obtain a Chi-square value which reflects the magnitude of the difference between the two distributions. One can then obtain the probability of the null hypothesis (‘P’ value) that the distribution are in fact the same. As shown in Table 6, the observed pattern was convincingly non-random as judged by this method (P < 0·005).

Phenobarbital treatment resulted in almost complete covering of the microsomal vesicles with ferritin as shown in Fig. 4. Statistical analyses on the microsomes from rats treated with 3-methylcholanthrene showed that heterogeneous labelling was similar to that on microsomes from untreated rats (see the second row of Table 6).

Effect of glutaraldehyde fixation

Since the present immunocytochemical reactions were carried out using ferritinantibody conjugates containing divalent antibodies, it could be argued that the heterogeneous distribution was created artificially by lateral diffusion and artificial aggregation of the cytochrome during immunocytochemical procedures. In order to avoid such argument, microsomes were first fixed with glutaraldehyde and then used for the binding assay. Qualitative observation has been reported in detail in the previous paper (Matsuura et al. 1978), and quantitative analyses are described in this paper.

First, the effects of glutaraldehyde fixation on the binding of the antibody conjugates to microsomes were investigated as described in Materials and methods. After fixation for 15 min at o °C with 0 · 5% glutaraldehyde, the binding capacity of microsomes from untreated and phenobarbital-treated rats decreased to 51 and 66% of that from the untreated rats (average of 2 experiments), respectively.

Morphological observations confirmed this immunochemical result. That is, after fixation with glutaraldehyde, the number of ferritin particles bound to smooth microsomal vesicles decreased to ∼ 55% of the value for unfixed microsomes, shown by a small closed circle in Fig. 7.

Statistical analyses of the distribution of ferritin particles also confirmed the nonrandom distribution of cytochrome P-450 on the fixed microsomal vesicles (Table 6, the third row).

Composition of the microsomal fractions

By assuming the exclusive localization of galactosyltransferase in Golgi fraction, 5’-nucleotidase in plasma membrane and succinate-cytochrome c reductase in mitochondria, it is possible to obtain rough estimates for the degree of contamination of these organelles to the smooth and rough microsomes fraction as shown in Table 7. In the present experiment, Golgi fraction was prepared according to the procedures of Hino et al. (1978), who reported that the fraction is contaminated on the protein basis with 13–17% of microsomes, 2–4% of lysosomes and 4–6% of plasma membrane. After correction for these contaminations, the specific activity of galactosyltransferase in pure Golgi apparatus is calculated to be 805·8. Using this value, it is estimated that ∼ 5·0% of the total proteins in the smooth microsomes fraction are of Golgi origin.

Table 7.

Contamination of various cell organelles to smooth and rough microsome fraction

Contamination of various cell organelles to smooth and rough microsome fraction
Contamination of various cell organelles to smooth and rough microsome fraction

According to Table 1, the specific activity of 5’-nucleotidase of plasma membrane purified by the procedures of Ray (1970) was 0·537μmol of inorganic phosphate liberated per min per mg protein. It can be calculated, therefore, that 4·7% of the total protein in the smooth microsomes fraction was plasma membrane in origin. Similarly specific activity of succinate-cytochrome c reductase of mitochondria was 0·11 μmol of cytochrome c reduced per min per mg protein, and 1-4 and 6-9% of the total protein in the smooth and the rough microsome fraction, respectively, were estimated to be of mitochondrial origin.

Marker enzymes of the other cell organelles were not determined. According to Beaufay et al. (1974), however, the microsomal fraction prepared by their procedures contains, on the protein basis, 3 % of outer mitochondrial membranes, 1 % of lysosomes and 1 % of peroxisomes. Thus the present smooth microsome elements deriving from ER may account for ∼ 85 % of microsomal proteins.

These biochemical data do not agree with our immunoelectron-microscopic observations. Our previous and present observations showed that only ∼ 6% of the smooth vesicles are not labelled with the anti-P-450 antibody conjugates. This discrepancy may be explained mainly by the fact that 5–6% of the microsomal proteins are not recovered in the pellets but are found in the sucrose density gradient. They were not labelled with the conjugates and so probably derived from smooth vesicles other than ER membranes. This loss might correspond to more than 10% loss of the smooth microsomal proteins, and in addition, mitochondria were eliminated from the morphological counting of the microsomal vesicles as reported in the previous paper (Matsuura et al. 1978).

Electron-microscopic observation revealed that 98–99% of the rough microsomal vesicles are labelled with the antibody conjugates. This result is to be expected because the major contaminants to this fraction are mitochondria which were eliminated from counting of the microsomal vesicles as described above.

This proportion of the microsomal elements which are determined biochemically as derived from ER membranes agrees well with the proportion of the vesicles which are labelled with the ferritin-antibody conjugates. This means that practically all the microsomal vesicles derived from ER membrane contain cytochrome P-450.

Correlation between immunochemical and morphological data

As our next problem, the number of antibody conjugates bound per microsome was calculated from the immunochemical data, and was then compared with the average number of ferritin particles bound per microsomal vesicle as determined by morphological observation.

According to Ito (1974) and Morimoto et al. (1976), the amount of protein per microsomal vesicle, ∼ 120 nm in average diameter, is 2·88 × 10−13 and 2·16 × 10−13 mg, respectively. The former value was calculated from the data of Ito (1974) assuming that the amount of membrane protein of microsomal vesicle is proportional to the square of the diameter.

According to Wibo et al. (1971), the average radius and number of microsomal vesicles per 10−12 mg protein were 67·7 nm and 2·37, respectively. From the latter value the amount of protein per vesicle of average diameter 135·4 nm is calculated to be 3·3 × 10−13 mg, which corresponds to 2·59 × 10−13 mg for a vesicle with an average diameter of 120 nm. We will use this last value because it is nearest to the average of the 3 values.

Cytochrome P-450 content of the smooth microsomes from untreated rats is ∼ 1·0 nmol per mg microsomal protein (Table 2). As the amount of protein per vesicle is assumed to be 2·59 × 10−13 mg, the number of cytochrome P-450 molecules per vesicle will be 156. Since the molar ratio of the antibody conjugates bound per mol of cytochrome P-450 at saturation level is 0·25, the average number of conjugates expected to bind to a microsome of ∼ 120 nm average diameter is calculated to be 39. If we assume that the average thickness of the grey sections is 42·5 nm according to Wibo et al. (1971) and the microsomal vesicles are spherical bodies, ∼ 120 nm in average diameter, and the thin sections are made across the spheres, the proportion of the surface area of the sectioned vesicle to the total surface of the spherical body is 35·4%. So the expected number of the ferritin particles on a sectioned microsome will be 13·8. This value is in good agreement with the experimental value of 12·3, the number of ferritin particles counted as bound per microsome in cross-sectional profile (Fig. 7). It is to be pointed out here that the latter value is definitely smaller than the corresponding number in tangential profile (15·5). This is probably due to overlapping of ferritin particles in cross-sectional profiles, because the surface areas of the 2 profiles are always equal provided the sections are cut through the surface of a sphere. Comparing the mean number of the ferritin particles in both profiles (Fig. 7), it is estimated that the overlapping of the ferritin particles on cross-sectional profiles of the microsomes from untreated rat is ∼ 20%.

We could roughly estimate, therefore, the amount of cytochrome P-450 molecules from the number of ferritin particles on the microsomal membrane. That is, under the present experimental conditions each ferritin particle represents ∼ 4 molecules of cytochrome P-450 on microsomes from untreated rats.

It is to be noted here that ratio of mol of specific antibody bound per mol of cytochrome P-450 of rat liver microsome depends on the composition of the molecular forms of cytochrome P-450S as discussed in detail in the following section. For quantitative immunoelectron-microscopic analyses, therefore, the ratio should be evaluated experimentally in each case. The thickness of thin sections should be always constant or measured precisely to allow comparison of electron micrographs from different sections.

Multiplicity of cytochrome P-450 and binding of the conjugates with microsomes

Cytochrome P-450 purified from the hepatic microsomes of phenobarbital-treated male rats was electrophoretically homogeneous and ∼ 45000 in molecular weight. It cross-reacts little with the antibody against cytochrome P-448 as reported by Thomas et al. (1976), Dean & Coon (1977), and Mikawa, Fujii-Kuriyama & Tashiro (manuscript in preparation).

When ferritin-antibody conjugates were incubated with smooth microsomes from phenobarbital-treated rats, the amount of conjugates bound per μg of microsomal protein was much higher than when incubated with those from untreated rats. This result is consistent with the electron-microscopic observation that the number of ferritin particles bound to the microsomal membrane was markedly increased by phenobarbital treatment.

When the conjugates were incubated with the microsomes from methylcholanthrene-treated rats, however, neither immunochemical nor morphological observations showed any increase in the amount or in the number of bound conjugates, respectively, despite the fact that methylcholanthrene induced marked increase in the cytochrome P-448 content. This indicates that methylcholanthrene treatment did not induce any increase in the amount of phenobarbital-induced cytochrome P-450 and the biosyntheses of P-450 and P-448 are independently regulated as has been suggested previously (Bidleman & Mannering, 1970; Alvares & Kappas, 1978).

Table 4 shows that the maximum numbers of antibody molecules which can bind with each cytochrome P-450 molecule on microsomal membranes prepared from untreated, phenobarbital- and methylcholanthrene-treated rats were ∼ 0·25, 0·43 and 0·10 mol per mol of cytochrome P-450, respectively.

These data are to be expected, because the molecular forms of cytochrome P-450S are multiple and the proportion of the phenobarbital-induced form on microsomes from phenobarbital-treated, untreated, and methylcholanthrene-treated rats may decrease in this order.

Number of ferritin-antibody conjugates bound at saturation level

As discussed above, mol of antibody conjugates which can bind per mol of cytochrome P-450 on microsomes was less than one. According to Dean & Coon (1977), about 10 Fab– fragments from goat bind per molecule of cytochrome P-450 from rabbit liver. It is certain, therefore, that there is a marked difference in the number of antibody molecules that can bind with free cytochrome P-450 and the number of conjugates that can bind with cytochrome on the microsomal vesicles. Several factors appear to be responsible for this difference.

First of all, multiplicity of the cytochrome P-450 haemoproteins (including, of course, cytochrome P-448) in rat liver microsomes is to be taken into consideration as discussed above.

The second factor is a topographical one. In order to bind to cytochrome P-450 on to microsomal vesicles, the antibody molecules in the conjugates should be accessible to the immunoreactive sites of the P-450 molecules there. It is possible that many of the antibody-binding sites of the cytochrome are buried in the membrane and thus not accessible to the antibodies.

The third factor is steric hindrance due to the size of the ferritin-antibody conjugates. The conjugates are so large that if a conjugate binds with some P-450 molecule on the microsomes, further binding of the conjugates to the neighbouring P-450 molecules on the microsomal membrane may be inhibited. This effect will be accentuated when cytochrome P-450 molecules on microsomal vesicles are distributed non-randomly, forming small clusters.

Cytochrome P-450 content of rough and smooth microsomes

In the previous paper (Matsuura et al. 1978), it was revealed that the regions of the rough microsomal surface studded with ribosomes are much less labelled with the antibody conjugates than the smooth regions. As the proportion of the rough regions to the total surface area of the rough microsomal vesicles is considerable, it is natural that we found less ferritin particles on rough microsomes than on smooth microsomes. The ratios of ferritin particles bound with the smooth to those bound with the rough microsomes from untreated and phenobarbital-treated rats were 1·7 and 2·1, respectively. It is suggested that the cytochrome does exist on the rough regions of rough ER but that these regions are less labelled with the conjugates simply because of the occupation of the regions by the membrane-bound ribosomes.

Biochemical analyses also clearly indicate that, on the protein basis, smooth microsomes contain ∼ 2·3 times more P-450 than rough counter part (Table 2). This value is consistent with previous data reported by Matsuura et al. (2·3 times (1978)) from this laboratory and by Ichikawa & Yamano (i-6 times (1970)), Fleischer et al. (1·6 times (1971)) and Craft et al. (2-0 times (1975)), from other laboratories.

As a matter of course, rough microsomes contain ribosomes and the above values should be corrected for the ribosomal proteins. If we assumed that RNA content of ribosomes is ∼ 50% (Tashiro & Siekevitz 1965), and subtract the amount of the ribosomal proteins from that of the total microsomal proteins using Table 1, the corrected ratio of the amounts of cytochrome P-450 in smooth to rough microsomes from untreated and phenobarbital-treated rats will be 1·6 and 2·5 times, respectively.

From these analyses, we could conclude that cytochrome P-450 is at least partially excluded from the rough regions on the microsomes. This conclusion is also supported by the fact that the ratio of mol of the antibody bound per mol of cytochrome P-450 for rough microsomes is similar to that of smooth microsomes as shown in Fig. 2. Thus steric hindrance of the ribosomes to the binding of the conjugates to rough microsomes does not appear to be significant.

The probable scarcity of cytochrome P-450 and other electron-transport enzymes on the ribosome-rich regions of the outer surface of rough microsomes may explain why the cytochrome (and other microsomal electron-transport enzymes as well) show characteristic heterogeneous distribution patterns of group b enzymes of Beaufay et al. (1974) when analysed by isopycnic centrifugation in sucrose density gradient, and why this heterogeneous distribution is converted to homogeneous distribution after detachment of ribosomes by treatment with pyrophosphate or EDTA (Amar-Costesec ei al. (1974)).

It is to be noted here that our conclusion is apparently contradictory to the hypothesis that cytochrome P-450 is a binding protein for ribosomes (Ohlsson & Jergil, 1977; Takagi, 1977).

Distribution of cytochrome P-450 on the microsomes

We have demonstrated in this and in the previous paper (Matsuura et al. 1978) that cytochrome P-450 molecules are present essentially in all the microsomal vesicles derived from ER, whether rough or smooth. A similar conclusion has been reported by Morimoto et al. (1976) on the intermicrosomal distribution of NADPH-cytochrome c reductase and by Remacle et al. (1976) on cytochrome b5, respectively. Thus the microsomal electron-transport enzymes so far studied appear to be distributed on any microsomal vesicle derived from ER.

In the previous paper we reported by inspection that the distribution of cytochrome P-450 molecules on each microsome vesicle is non-random. In this paper the distribution was analysed statistically by comparing it with the expected random distribution and shown to be non-random to a high degree of statistical significance. Non-random distribution of the cytochrome was evident even after fixation with glutaraldehyde as described in detail in the text.

It is to be noted here that the present statistical analyses were carried out on the cross-sectional profiles neglecting overlapping of the ferritin particles. Such overlapping may weaken the apparent clustering of the particles and so the above conclusion is valid even if the overlapping effect was taken into consideration.

Thus on ER membranes of hepatocytes not only NADPH-cytochrome c reductase (Morimoto et al. (1976)) but also cytochrome P-450 appear to be distributed heterogeneously, forming small clusters.

We thank Miss Keiko Miki for assistance with the manuscript. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, fapan and by a grant from the Naito Research Fund.

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