The intensification of absorbance changes in leaves by light-dispersion

In dispersive samples, like leaves, the absorbance of pigments is intensified. The intensification is due to a longer optical path through the dispersive sample. However, in chloroplast suspensions the optical path is not much longer than in clear solutions. The factor of intensification (=the lengthening of the optical path) is calculated by comparing the absorbance of leaves and the absorbance of chloroplast suspensions with equal pigment-content. This method also includes the influence of possible sieve effects which could decrease absorbance. The measurements are carried out with high- and low-light leaves of different thickness and pigment content. The intensification of absorbance was 2–2.5 fold. In highlight leaves it was somewhat less than in low-light leaves. The factor is better correlated to the pigment content than to the thickness of the leaves. The plot of absorbance versus the pigment content of the leaves shows that decreases with increasing pigment content. In contrast, chloroplast suspensions show a linear dependence as expected from Lambert-Beer's law. Thus, in leaves with very low pigment content the absorbance is intensified up to 6 fold while the intensification decreases with increasing absorbance. These results are in good agreement with measurements of Tsel'niker (1975) and with the theoretical predictions of Butler's formula (1960). Absorbance changes due to photooxidation of P-700 and cytochrome f in intact leaves are measured, and is used to calculate the amount of the oxidized components. Without correction for the values would be much greater than the amount actually present. The corrected data show that between 70 and 90% of the present P-700 and cytochrome f can be photooxidized in the intact leaf.Abbreviations A absorbance - factor of intensification=lengthening of the optical path - Chl chlorophyll a+b content - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FW fresh weight - HL high-light - LA leaf area - LL low-light - PhAR photosynthetically active radiation     

Measurements of manganese in thylakoids of Sinapis alba grown under high-light and low-light conditions

The manganese content of thylakoids and tissues was measured in leaves grown under high- and low-light conditions. Especially when grown in a nutrient medium enriched in manganese (20 M), the thylakoids contained large amounts of manganese, which could be removed by EDTA washing without impairment of the Hill reaction. The unremovable content of manganese was almost the same in thylakoids from plants grown in nutrient media of normal (2 M) and reduced (0.2 M) manganese content. Up to this limit of manganese content, Hill activity did not seem to be impaired. 1.2 atoms Mn per 100 molecules chlorophyll were found in low-light thylakoids and 1.6 atoms Mn in high-light thylakoids. This is similar to the behaviour of other electron transport components, the number of which is also decreased under low-light conditions. However, the decrease in the manganese content is not as striking as the decrease in, for example, the cytochrome f and ferredoxin content. This may be attributed to an invariable pool of manganese which is not involved in the oxygen evolving system. Alternatively, if all of our measured manganese is involved in electron transport to PS II, this could indicate that in low-light chloroplasts the ratio of PS II/PS I components may be somewhat increased.

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Zusammenfassung Der Mangangehalt von Thylakoiden und Gewebe aus Starklicht- und Schwachlichtblättern wurde untersucht. Besonders bei Pflanzen, welche unter erhöhtem Manganangebot (20 M) angezogen wurden, besaßen die Thylakoide sehr viel Mangan, welches durch Waschen mit EDTA entfernt werden konnte, ohne die Hill-Aktivität zu beeinträchtigen. In Thylakoiden aus Pflanzen, welche unter normalem (2 M) und reduziertem (0,2 M) Manganangebot gewachsen waren, unterschied sich der nicht entfernbare Mangangehalt nicht sehr. Dies scheint die untere Grenze des Mangangehalts zu sein, bis zu welchem die Hill-Aktivität noch nicht beeinträchtigt wird. Schwachlicht-Thylakoide besitzen 1,2 Atome Mn pro 100 Chlorophyllmoleküle, während Starklicht-Thylakoide 1,6 Atome Mn pro 100 Chlorophyllmoleküle enthalten. Dies gleicht dem Verhalten anderer Komponenten des Elektronentransports, welche ebenfalls im Starklicht vermehrt vorkommen. Die Unterschiede im Mangangehalt sind jedoch geringer als die Unterschiede im Gehalt von z.B. Cytochrom f und Ferredoxin. Dies könnte auf einen konstanten Anteil von Mangan zurückzuführen sein, welcher nicht am wasserspaltenden System beteiligt ist. Wenn jedoch das gesamte gemessene Mangan am Elektronentransport zum PS II beteiligt ist, könnte dies ein Hinweis sein, daß in Schwachlicht-Chloroplasten sich das Verhältnis der PS II-/PS I-Komponenten etwas vergrößert.

     

The concentration of Cytochrome f and P700 in chlorophyll-deficient mutants of Chlorella fusca

The ratio of Chlorophyll: Cytochrome f and of Chlorophyll: P700 (reaction center pigment in photosystem I) is essentially lower in chlorophyll-deficient mutants than in the normal green strain. On a dry weight basis, the mutants have the same or a higher content of redox enzymes than the normal form. The size of the photosynthetic unit of the mutants is 4 to 7 times smaller than that of the normal strains, due mainly to a deficiency of the light-harvesting chlorophyll-protein complex.Abbreviations Chl chlorophyll - Cyt f Cytochrome f - P700 reaction center pigment in photosystem I - PS photosystem - LH light-harvesting     

The effect of ethylenediamine chemical modification of plastocyanin on the rate of cytochrome f oxidation and P-700 reduction

Chemical modification of plastocyanin was carried out using ethylenediamine plus a water-soluble carbodiimide, which has the effect of replacing a negatively charged carboxylate group with a positively charged amino group at pH 6–8. The conditions were adjusted to produce a series of singly and doubly modified forms of plastocyanin. Differences in charge configuration allowed separation of these forms on a Pharmacia fast protein liquid chromatograph using a Mono Q anion exchange column. These forms were used to study the interaction of plastocyanin with its reaction partner cytochrome f. The rate of cytochrome f oxidation was progressively inhibited upon incorporation of increasing numbers of ethylenediamine moieties indicating a positively charged binding site on cytochrome f. However, differential inhibition was obtained for the various singly modified forms allowing mapping of the binding site on plastocyanin. The greatest inhibition was found for forms modified at negatively charged residues Nos. 42–45 and Nos. 59–61 which comprise a negative patch surrounding Tyr-83. In contrast, the form modified at residue No. 68, on the opposite side of the globular plastocyanin molecule, showed the least inhibition. It can be concluded that the binding site for cytochrome f is located in the vicinity of residues Nos. 42–45 and Nos. 59–61. Modification of plastocyanin at residues Nos. 42–45 showed no effect on the rate of P-700⁺ reduction, suggesting that these residues are not involved in the binding of Photosystem I. However, an increase in the rate of P-700⁺ reduction was observed for plastocyanins modified at residue No. 68 or Nos. 59–61, which is consistent with the idea that the reaction domain of Photosystem I is negatively charged and Photosystem I binds at the top of the molecule and accepts electrons via His-87 in plastocyanin. These results raise the possibility that plastocyanin can bind both cytochrome f and Photosystem I simultaneously. The effect of ethylenediamine modification on the formal potential of plastocyanin was also examined. The formal potential of control plastocyanin was found to be +372 ± 5 mV vs. normal hydrogen electrode at pH 7. All modified forms showed a positive shift in formal potential. Singly modified forms showed increases in formal potentials between +8 and +18 mV with the largest increases being observed for plastocyanins modified at residues Nos. 42–45 or Nos. 59–61.     

Cyclic and noncyclic photophosphorylation during the ontogenesis of high-light and low-light leaves of Sinapis alba

Noncyclic electron transport to ferricyanide and photophosphorylation as well as the methylviologen mediated aerobic and anaerobic photophosphorylation with dichlorophenolindophenol-ascorbate as the electron donor of photosystem I were measured during the development of high-light and low-light adapted leaves of Sinapis alba. Anaerobic methylviologen-catalyzed phosphorylation is more than twice as high as aerobic phosphorylation. The difference between the rates of aerobic and anaerobic phosphorylation is sensitive to dibromothymoquinone. Thus, under anaerobic conditions, methylviologen mediates a cyclic phosphorylation including plastoquinone. All photochemical activities of high-light chloroplasts are about twice as high as that of low-light chloroplasts and show a permanent decline with increasing plant age. The lower activities of low-light chloroplasts correlate with a decrease of electron transport components, such as cytochrome f. This indicates that the number of electron transport chains is decreased under low-light conditions and more chlorophyll molecules interact with one electrontransport chain.Abbreviations Asc ascorbate - Chl chlorophyll a+b - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(dichlorophenyl)-1,1-dimethylurea - DCPIP dichlorophenolindophenol - HL high light - LL low light - MV methylviologen - PhAR photosynthetically active radiation - PS photosystem     

Effect of high-light treatments in inducing photoinhibition of photosynthesis in intact leaves of low-light grown Phaseolus vulgaris and Lastreopsis microsora

Photoinhibition studies, using gas-exchange techniques, were conducted with leaflets of Phaseolus vulgaris L. plants that were grown under low photonfluence rates. Comparative measurements were made on attached, intact leaflets and in subsequently isolated chloroplasts. Photoinhibition studies were also conducted with attached fronds of the deep-shade fern Lastreopsis microsora (Endl.) Tindale. Leaflets of lowlight-grown Phaseolus vulgaris and fronds of the shade fern were found to be subject to similar photoinhibition when exposed to photon-fluence rates in excess of those at which they were grown. Photoinhibition following exposure to a photon fluence-rate approximating full sunlight is manifested as a reduction in the capacity for both light-saturated and light-limited carbon uptake and is reflected at the chloroplast level as substantial inhibition of electron flow through photosystem (PS) II, with little effect on PS I. The extent of photoinhibition is markedly dependent on the length of exposure to a high-light regime and on the actual photon-fluence rate maintained during treatment. A greater degree of photoinhibition is evident if carbon metabolism is prevented by the removal of CO₂ than when maximum rates of CO₂ uptake prevail throughout the exposure to a high photonfluence rate. Apparently a certain level of CO₂ turnover is beneficial in providing a sink for photochemically generated energy. When leaf material is exposed to photon-fluence rates well in excess of the rate present during growth apparently the potentials of the various biophysical and photochemical means of dissipating excitation energy are exceeded and photoinhibition of photosynthesis results.Abbreviation PFR photon fluence rate     

The use of light-induced absorbance changes at 820 nm to monitor the oxidation state of P-700 in leaves

Abstract The use of the light-induced absorbance change at 820 nm (ΔA 820) to monitor the oxidation and reduction of P-700 in irradiated leaves is examined. Results obtained from leaves irradiated with a range of wavelengths of light, poisoned with DCMU, or lacking PS I, are consistent with the proposition that the light-induced ΔA 820 can be used to monitor P-700 oxidation in leaves.     

Transient kinetics of the electron transfer between P-700, plastocyanin and cytochrome f in chloroplasts suspended in fluid media at sub-zero temperatures

The kinetics of redox changes of P-700, plastocyanin and cytochrome f in chloroplasts suspended in a fluid medium at sub-zero temperatures have been studied following excitation of the chloroplasts with either a single-turnover flash, a series of flashes or continuous light. The results show that: (1) The kinetics of reduction of P-700⁺ and those of oxidation of plastocyanin are consistent with a bimolecular reaction between these two components as previously suggested (Olsen, L.F., Cox, R.P. and Barber, J. (1980) FEBS Lett. 122, 13–16). (2) Cytochrome f shows heterogeneity with respect to its kinetics of oxidation by Photosystem I. (3) In contrast to the situation when plastoquinol is the electron donor, reduction of cytochrome f by electrons derived from diaminodurene occurs with sigmoidal kinetics that shows a good fit to an apparent equilibrium constant of 12 between the cytochrome and P-700. (4) The rate of electron transfer from plastoquinol to Photosystem I depends on the redox state of the plastoquinone pool. (5) In relation to current ideas about the lateral heterogeneity of Photosystem I and Photosystem II in the thylakoid membrane, the results are consistent with the function of plastocyanin as a mobile carrier of electrons in the intrathylakoid space.     

Reaction kinetics of P-700, cytochrome c-553 and cytochrome f in the cyanobacterium, Synechococcus sp

Absorption changes invoked by short flashes in the Soret band region were measured in the thermophilic cyanobacterium Synechococcus sp. and photoresponses of P-700, cytochrome c-553 and cytochrome f were resolved with the aid of a microcomputer. Cytochrome c-553 was oxidized very rapidly with a half-time of less than 20 μs, while the half oxidation time of cytochrome f was 35–45 μs. The two cytochromes were reduced monophasically with half-time of 2 ms after a lag lasting a few milliseconds. The reduction kinetics of P-700 showed three exponential phases with half-times of 40 μs, 200 μs and 2 ms, which are ascribed to electron donation from cytochrome f, the Rieske iron-sulfur protein and plastoquinone, respectively. The results support the following sequence and rates of linear electron transport at the physiological temperature of the cyanobacterium: P-700

cytochrome c-553

cytochrome f

Rieske protein

plastoquinone.     

Development of the photosynthetic apparatus during light-dependent greening of a mutant of Chlorella fusca

The formation of chlorophyll, cytochrome f, P-700, ribulose bisphosphate carboxylase as well as photosynthesis and Hill reaction activities were tested during the light-dependent greening process of the Chlorella fusca mutant G 10. Neither chlorophyll nor protochlorophyllide was detected in the darkgrown cells. When transferred to light the mutant cells developed chlorophyll and established its photosynthetic capacity after a short lag phase. In the in vivo absorption spectra a spectral shift of the red absorption peak position from 674 to 680 nm was indicated during the first 3 h of greening. Cytochrome f was already present in the dark-grown cells, but during the greening phase a threefold increase in the cytochrome f content could be seen. At the early stages of greening a characteristic primary oscillation in the content of cytochrome f was observed. P-700 was lacking in the dark and during the first 30 min of illumination. From the first to the second h of light a forced synthesis of P-700 took place and the time-course curve for the ratios of P-700/chlorophyll rose to a sharp maximum. The synthesis of P-700 started together with photosystem I activity and showed similar kinetics. We found the simultaneous appearance of photosystem II, photosystem I, and photosynthetic activities 30 min after the beginning of the illumination. Based on chlorophyll content they attained maximum activity after 2 h of light, but at this time photosystem I capacity proved to be remarkably higher than photosynthetic and photosystem II activities. Highest carboxylase activity existed in darkgrown cells. During the greening process the activity of the enzyme decreased continuously. After 2 h of illumination chlorophyll synthesis partially served to increase the size of the photosynthetic unit, which consequently led to a decrease in the light energy needed to saturate photosynthesis and also to a decrease of photosynthetic rate based on chlorophyll content.Abbreviations Chl chlorophyll - Cyt f cytochrome f - DPIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - GSH glutathione - LH light-harvesting - PS photosystem - RuBP ribulose bisphosphate     

The kinetics of P-700+ reduction in leaves: a novel in situ probe of thylakoid functioning

Abstract. The half time (t_1/2) of the reduction of P-700⁺ in the millisecond time frame is known to be limited by the reaction between plastoquinol and the cytochrome cytb₆f complex. This is considered to be the rate limiting reaction of thylakoid electron transport and measurements of it provide a means of analysing how thylakoid election transport is regulated in vivo. The half time for the reduction of photochemically oxidized P-700 has been measured in vivo using absorbance changes around 820 nm. The results showed that t_1/2 is independent of irradiance and decreases as photosynthetic induction progresses. Even with a constant t_1/2 the quantum efficiency of PSI declined as irradiance increased. The significance of the concept of photosynthetic control of electron transport is discussed in the light of these observations.     

Some aspects of the role of cytochrome P-450 isozymes in the n-oxidative transformation of secondary and tertiary amine compounds

Indirect evidence of the participation of cytochrome P-450 (P-450) in the microsomal N-oxygenation of secondary and tertiary nitrogen functions is presented by studies employing diagnostic modifiers of the hemoprotein system as well as antibodies directed toward the diverse P-450 isoforms and NADPH-cytochrome P-450 reductase. Experiments with recombinant hemoproteins or P-450 isozymes directly purified from the tissues of various animal species support the results obtained by the inhibitor assays. Although the intermediacy of aminium radicals is thought to be restrictive to P-450-catalyzed N-oxygenation of secondary and tertiary amine groups bearing accessible hydrogens on the α-carbon, numerous exceptions to this rule are documented. It is proposed that aminium radicals partition between oxygen rebound and α-hydrogen abstraction to yield a finite level of N-oxygenated product in all P-450-mediated amine oxidations, the partition ratio depending on the amine structure and particular P-450 isozyme operative. In some instances, N-oxygenation appears to proceed by peroxidatic mechanisms. The relative contribution of P-450 to the N-oxygenation of secondary and tertiary amines in crude preparations or live animals, where competition with the flavin-containing monooxygenase (FMO) occurs, seems to be a function of the relative amounts and catalytic capacities of the two enzyme systems. Both parameters are species and tissue dependent. Accordingly, the extent to which P-450 contributes to total N-oxidative turnover of the amine substrates varies from minor to major. © 1996 John Wiley & Sons, Inc.     

NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Function and regulation of cytochrome P-450 in alkane-assimilating yeast

Transition of n-hexadecane utilizing cultures of Candida maltosa to oxygen-limited growth caused an up to 6-fold increase of the cellular cytochrome P-450 content. Enhanced cytochrome P-450 formation required protein de novo synthesis and was not due to a change of the apo/holo-enzyme ratio as demonstrated by cycloheximide inhibition and immunological quantitation. The effect of low oxygen concentration (pO₂=3–5%) was simulated by selective inhibition of alkane hydroxylation with carbon monoxide (at a pO₂ of 70–75%). Enhanced cytochrome P-450 formation occurred even when a constant growth rate was maintained through utilization of a second non-repressive growth substrate. However, the presence of n-alkanes was an essential precondition. It was concluded, that the cytochrome P-450 formation was mainly regulated by the intracellular inducer concentration which depends on the relative rates of alkane transport into the cell and the actual alkane hydroxylating activity of the enzyme system.Abbreviation cyt cytochrome     

Identification and chromosomal locations of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Nectria haematococca

Summary The ability to detoxify the phytoalexin, pisatin, an antimicrobial compound produced by pea (Pisum sativum L.), is one requirement for pathogenicity of the fungus Nectria haematococca on this plant. Detoxification is mediated by a cytochrome P-450, pisatin demethylase, encoded by any one of six Pda genes, which differ with respect to the inducibility and level of pisatin demethylase activity they confer, and which are associated with different levels of virulence on pea. A previously cloned Pda gene (PdaT9) was used in this study to characterize further the known genes and to identify additional members of the Pda family in this fungus by Southern analysis. DNA from all isolates which demethylate pisatin (Pda⁺ isolates) hybridized to PdaT9, while only one Pda^– isolate possessed DNA homologous to the probe. Hybridization intensity and, in some cases, restriction fragment size, were correlated with enzyme inducibility. XhoI/BamHI restricted DNA from reference strains with a single active Pda allele had only one fragment with homology to PdaT9; no homology attributable to alleles associated with the Pda^– phenotype was found. Homology to this probe was also limited to one or two restriction fragments in most of the 31 field isolates examined. Some unusual progeny from laboratory crosses that failed to inherit demethylase activity also lost the single restriction fragment homologous to PdaT9. At the chromosome level, N. haematococca is highly variable, each isolate having a unique electrophoretic karyotype. In most instances, PdaT9 hybridized to one or two chromosomes containing 1.6–2 million bases of DNA, while many Pda- isolates lacked chromosomes in this size class. The results from this study of the Pda family support the hypothesis that deletion of large amounts of genomic DNA is one mechanism that reduces the frequency of Pda genes in N. haematococca, while simultaneously increasing its karyotypic variation.     

Immunohistochemical study of temporal variations in cytochrome P-450 isozymes in rat testis and their modifications by the inductive effects of cadinenes

Temporal variations in cytochrome P-450 isozymes of rat testis, PB-P-450 (forms of cytochrome P-450 strongly induced by phenobarbital) and MC-P-448 (forms of cytochrome P-450 strongly induced by 3-methylcholanthrene), were investigated immunohistochemically by the avidin-biotin-complex method using specific antibodies against PB-P-450 and MC-P-448 isozymes. Immunoreactivity to both PB-P-450 and MC-P-448 isozymes was observed in Leydig cells. The number of PB-P-450 positive Leydig cells was found to undergo significant time-of-day variation with a peak time of 0000 hours (light phase from 0800 to 2000 hours). Injection of cadinenes (300 mg/kg per day intraperitoneally at 48 and 96 h before sacrifice) induced PB-P-450 isozyme but did not induce MC-P-448 isozyme. The induction of PB-P-450 isozyme by cadinenes was time dependent, and the early dark phase (2000 and 0000 hours) was most sensitive. These results suggest that temporal variation of cytochrome P-450 isozymes is one of the important physiological variations in detoxification and activation of various xenobiotics and chemicals in the testis.     

Redox states of photosystems I and II in irradiated leaves of wheat seedlings grown under different conditions of nitrogen nutrition

Changes in the redox states of photosystem I (PSI) and PSII in irradiated wheat leaves were studied after growing seedlings on a nitrogen-free medium or media containing either nitrate or ammonium. The content of P700, the primary electron donor of PSI was quantified using the maximum magnitude of absorbance changes at 830 nm induced by saturating white light. The highest content of P700 in leaves was found for seedlings grown on the ammonium-containing medium, whereas its lowest content was observed on seedlings grown in the presence of nitrate. At all irradiances of actinic light, the smallest accumulation of reduced Q_A was observed in leaves of ammonium-grown plants. Despite variations in light-response curves of P700 photooxidation and Q_A photoreduction, the leaves of all plants exposed to different treatments demonstrated similar relationships between steady-state levels of P700⁺ and Q_A ^–. The accumulation of oxidized P700 up to 40% of total P700 content was not accompanied by significant Q_A photoreduction. At higher extents of P700 photooxidation, a linear relationship was found between the steady-state levels of P700⁺ and Q_A ^–. The leaves of all treatments demonstrated biphasic patterns of the kinetics of P700⁺ dark reduction after irradiation by far-red light exciting specifically PSI. The halftimes of corresponding kinetic components were found to be 2.6–4 s (fast component) and 17–22 s (slow component). The two components of P700⁺ dark reduction were related to the existence of two PSI populations with different rates of electron input from stromal reductants. The magnitudes of these components differed for plants grown in the presence of nitrate, on the one hand, and plants grown either in the presence of ammonium or in the absence of nitrogen, on the other hand. This indicates the possible influence of nitrogen nutrition on synthesis of different populations of PSI in wheat leaves. The decrease in far-red light irradiance reduced the relative contribution of the fast component to P700⁺ reduction. The fast component completely disappeared at low irradiances. This finding indicates that the saturating far-red light must be applied to determine correctly the relative content of each PSI population in wheat leaves.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 165–171.Original Russian Text Copyright © 2005 by Dzhibladze, Polesskaya, Alekhina, Egorova, Bukhov.This revised version was published online in April 2005 with a corrected cover date.     

Localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats An immunohistochemical study

Summary Immunohistochemical localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats was studied by indirect fluorescent antibody staining technique. A polyclonal antibody for cytochrome P-450_MC purified from hepatic microsomes of 3-methylcholanthrene-pretreated rats was used for this experiment. A strong immunofluorescence was found to be localized in the cytoplasm of the surface epithelium of the mucosa in the colon of 3-methylcholanthrene-pretreated rats. A faint immunofluorescence was also observed in the epithelium of untreated rats. 7-Ethoxycoumarin O-deethylase activity of colonic microsomes was significantly enhanced by 3-methylcholanthrene-pretreatment in parallel with an increase in the intensity of immunostaining for cytochrome P-450_MC in Western blotting analysis. This is the first report on the localization of cytochrome P-450 in the colonic mucosa.