Influence of the growth substrate and the oxygen concentration in the medium on the cytochrome P-450 content in Candida guilliermondii

Summary The induction of cytochrome P-450 in Candida guilliermondii by different growth substrates was investigated. Hexadecane is a strong inducer, whereas its oxidation products induce only weakly. It could be shown that the oxygen concentration in the medium influences the cytochrome P-450 content in cells growing on hexadecane. At oxygen concentrations between 100% and 15% of saturation the cytochrome P-450 content reaches 25–35 nmol/g dry weight. Reducing the oxygen to lower concentrations increase the cytochrome P-450 content up to approximately 100 nmol/g dry weight.     

Cytochrome P-450 accumulation and loss as controlled by growth phase of Saccharomyces cerevisiae: relationship to oxygen, glucose and ethanol concentrations

Ethanol induced small amounts of cytochrome P-450 in Saccharomyces cerevisiae NCYC 754 under conditions in which it is not normally detectable. Moreover, in non-growing yeast the existing cytochrome P-450 content was increased by 50% at a limited range of glucose concentrations (8-12% in 0.1 M-potassium phosphate buffer, pH 7.0), in which ethanol is produced by fermentation, possibly at an optimum concentration for induction of cytochrome P-450. Added alkanols, other than ethanol, caused rapid degradation of cytochrome P-450 in non-growing yeast; the rate of loss was directly related to the lipid solubility of the alkanol. Ethanol therefore favoured the accumulation of cytochrome P-450 in yeast; this may be related to an important putative role of one of the isoenzymes in ethanol-tolerance of the yeast, by the oxidative removal of ethanol from the endoplasmic reticulum of the cell. It is the accumulation of dissolved oxygen, rather than ethanol, that occurs on cessation of yeast growth that is likely to trigger the rapid disappearance of cytochrome P-450 observed at this time.     

The alkane-hydroxylating enzyme system of the yeast Candida guilliermondii.

The growth of the investigated Candida guilliermondii strain on n-alkanes induces an alkane-hydroxylating enzyme system, which consists of a cytochrome P-450 and a NADPH-dependent reductase. The cytochrome P-450 was purified to 4 nmoles per mg protein. Long-chain alkanes, preferably hexadecane to octadecane, are hydroxylated to the corresponding primary alcohol by this enzyme system. The substrate induces a type I spectrum, other compounds checked type II spectra.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

Differential time course of induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by Aroclor 1254

The time course of induction of rat liver microsomal cytochromes P-450a, P-450b + P-450e, P-450c, and P-450d and epoxide hydrolase has been determined in immature male rats administered a single large dose [1500 mumol (500 mg)/kg body wt] of the polychlorinated biphenyl mixture Aroclor 1254. Differential regulation of these xenobiotic-metabolizing enzymes was indicated by their characteristic patterns of induction. The rate of induction of cytochrome P-450a and epoxide hydrolase was relatively slow, and steady-state levels of these enzymes were maintained from approximately Days 9 to 15 after Aroclor 1254 treatment. In contrast, cytochrome P-450c was maximally induced 2 days after Aroclor 1254 treatment and remained at a constant level through Day 15. Steady-state levels of cytochrome P-450d, beginning 1 week after Aroclor 1254 treatment, were preceded by a fairly rapid rate of induction and possibly by a small decline from maximal levels observed around Days 4 to 5. Like those of the other cytochrome P-450 isozymes and epoxide hydrolase, the levels of cytochromes P-450b + P-450e were constant from Day 9 to 15 after Aroclor 1254 treatment. However, an unexpected but reproducible decline (approximately 25%) in total cytochrome P-450 content observed between Days 4 and 9 after Aroclor 1254 treatment principally reflected a dramatic and totally unanticipated decrease (approximately 45%) in the level of cytochromes P-450b + P-450e. This transient decline in the level of cytochromes P-450b + P-450e was not due to an unusual effect of a mixture of polychlorinated biphenyls, since identical results were obtained with two individual congeners, namely 2,3,4,5,4'-penta- and 2,3,4,5,3',4'-hexachlorobiphenyl, that induced the same isozymes as Aroclor 1254. In contrast, when rats were treated with 2,4,5,2',4',5'-hexachlorobiphenyl, which induces cytochromes P-450a and P-450b + P-450e and epoxide hydrolase but not cytochromes P-450c or P-450d, maximal levels of cytochromes P-450b + P-450e were attained on Day 4 and no decrease was observed over the next 11 days. These results suggest that there may be an interaction in the regulation of induction of certain individual cytochrome P-450 isozymes.     

Evidence that 2-allyl-2-isopropylacetamide and phenobarbital induce the same cytochrome P-450 in cultured chick embryo hepatocytes

The induction of cytochrome P-450 in cultured chick embryo hepatocytes was studied using two structurally unrelated compounds, 2-allyl-2-isopropylacetamide and phenobarbital. Pulse-labeling of these cells showed enhanced de novo synthesis of cytochrome P-450. The cytochrome induced by 2-allyl-2-isopropylacetamide, as well as the one induced by phenobarbital, reacted immunologically with antibodies raised against the major hepatic phenobarbital-induced isozyme. Additional form of cytochrome P-450 is induced exclusively by phenobarbital. These results clearly demonstrate that these two drugs induce at least one form of cytochrome P-450 in common.     

Induction of cytochrome P-450 and ethanol oxidation in Yarrowia lipolytica

The study of the effect of different ethanol concentrations in the medium on the growth and the activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. But when the initial concentration of ethanol in the medium was 3 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

The cytochrome P-450 alkane monooxygenase system of the yeast Lodderomyces elongisporus: purification and some properties of the NADPH-cytochrome P-450 reductase

NADPH-cytochrome P-450 reductase has been purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis, from microsomal fraction of Lodderomyces elongisporus using an effective 2-step chromatography procedure. One mol enzyme contains 1 mol each of FAD and FMN and exhibits an apparent molecular weight of 79.000. Recombination of the NADPH-cytochrome P-450 reductase with highly purified cytochrome P-450 results in an active alkane monooxygenase system. The activity of the hexadecane hydroxylation was enhanced by the addition of non-ionic detergent.     

Maintenance and induction of cytochrome P-450 in cultured rat hepatocytes

Maintenance of microsomal cytochrome P-450 content by cultured rat hepatocytes has proven an elusive goal. It is reported here that exogenous heme maintains cytochrome P-450 content of cultured rat hepatocytes at high levels during the first 72 h of incubation. The maintenance studies have been expanded to demonstrate the in vitro induction of cytochrome P-450 by phenobarbital treatment. The induction of P-450 in vitro by phenobarbital required the trace element, selenium, in the presence of exogenous heme. The present findings suggest that selenium, and other trace elements, may have an essential role in the formation of holocytochrome P-450 in vitro.     

Inhibition of uroporphyrinogen decarboxylase by halogenated biphenyls in chick hepatocyte cultures. Essential role for induction of cytochrome P-448.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was assayed in cultures of chick-embryo hepatocytes by the changes in composition of porphyrins accumulated after addition of excess 5-aminolaevulinate. Control cells accumulated mainly protoporphyrin, whereas cells treated with 3,4,3',4'-tetrachlorobiphenyl or 2,4,5,3',4'-pentabromobiphenyl accumulated mainly uroporphyrin, indicating decreased activity of the decarboxylase. 3-Methylcholanthrene and other polycyclic-hydrocarbon inducers of the P-448 isoenzyme of cytochrome P-450, did not affect the decarboxylase in the absence of the biphenyls. Induction of P-448 was detected as an increase in ethoxyresorufin de-ethylase activity. Pretreatment of cells with methylcholanthrene decreased the time required for the halogenated biphenyls to inhibit the decarboxylase. The dose response of methylcholanthrene showed that less than 40% of the maximal induction of cytochrome P-448 was needed to produce the maximum biphenyl-mediated inhibition of the decarboxylase. In contrast, induction of the cytochrome P-450 isoenzyme by propylisopropylacetamide had no effect on the biphenyl-mediated decrease in decarboxylase activity. Use of inhibitors of the P-450 and P-448 isoenzymes (SKF-525A, piperonyl butoxide and ellipticine) supported the concept that only the P-448 isoenzyme is involved in the inhibition of the decarboxylase by the halogenated biphenyls. The effect of preinduction with methylcholanthrene to enhance inhibition of the decarboxylase was also shown by the increased rate at which porphyrin accumulated from endogenously synthesized 5-aminolaevulinate after treatment of cells with the combination of propylisopropylacetamide and the biphenyls. Antioxidants, chelators of iron, and chromate affected the decrease in decarboxylase activity only if they prevented the induced increase in cytochrome P-448. We conclude that the P-448 and not the P-450 isoenzyme of cytochrome P-450 plays an obligatory role in the inhibition of uroporphyrinogen decarboxylase caused by halogenated biphenyls.     

Luteinizing hormone and cyclic AMP-mediated induction of microsomal cytochrome P-450 enzymes in cultured mouse Leydig cells

Treatment of mouse Leydig cell cultures with luteinizing hormone (LH) or with 8-bromo-cAMP (8-Br-cAMP) for 5 days elicited a dose- and time-dependent increase in the microsomal cytochrome P-450 enzyme activities. 17 alpha-Hydroxylase and C17-20 lyase as well as a parallel increase in testosterone production. Reduction of the oxygen tension from 19 to 1% resulted in a greater increase in enzyme activity. Induction of microsomal cytochrome P-450 activities was 35 to 50% greater with 8-Br-cAMP than with LH and the increase in C17-20 lyase activity was 4-fold greater than that of 17 alpha-hydroxylase. Maximal induction of P-450 enzyme activities was observed between 3 and 5 days of continual treatment with 8-Br-cAMP or LH. Removal of 8-Br-cAMP from the culture medium inhibited any further increase in C17-20 lyase activity and testosterone production. The role of protein synthesis in the induction process was investigated by incubating Leydig cell cultures with and without cycloheximide between 24 and 48 h of treatment with 8-Br-cAMP. Cycloheximide completely inhibited the induction of C17-20 lyase activity and the increase in testosterone production. After removal of the inhibitor, cultures responded in a manner that paralleled induction in cultures that had not been treated with cycloheximide. In both cases, a 24-h lag period occurred prior to an increase in cytochrome P-450 activity. These data suggest that the increase in microsomal cytochrome P-450 activities represents an increase in enzyme synthesis and, furthermore, that reduction of oxygen tension decreases degradation of newly synthesized Leydig cell microsomal cytochrome P-450 activities as recently reported (Quinn, P.G., and Payne, A.H. (1984) J. Biol. Chem. 259, 4130-4135).     

The Induction of Cytochrome P-450 and Ethanol Oxidation in Yarrowia lipolytica Cells

The study of the effect of different ethanol concentrations in the medium on the growth and activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. However, when the initial concentration of ethanol in the medium was 4 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

Role of haem in the induction of cytochrome P-450 by phenobarbitone. Studies in chick embryos in ovo and in cultured chick embryo hepatocytes.

The role of haem synthesis during induction of hepatic cytochrome P-450 haemoproteins was studied in chick embryo in ovo and in chick embryos hepatocytes cultured under chemically defined conditions. 1. Phenobarbitone caused a prompt increase in the activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, and in the concentration of cytochrome P-450. This induction response occurred without measurable initial destruction of the haem moiety of cytochrome P-450. 2. When intracellular haem availability was enhanced by exogenous haem or 5-aminolaevulinate, phenobarbitone-medicated induction of cytochrome P-450 was not affected in spite of the well known repression of 5-aminolaevulinate synthase by haem. These data are consistent with the concept that haem does not regulate the synthesis of cytochrome P-450 haemoproteins. 3. Acetate inhibited haem biosynthesis at the level of 5-aminolaevulinate formation. When intracellular haem availability was diminished by treatment with acetate, phenobarbitone-medicated induction was decreased. 4. This inhibitory effect of acetate on cytochrome P-450 induction was reversed by exogenous haem or its precursor 5-aminolaevulinate. These data suggest that inhibition of haem biosynthesis does not decrease synthesis of apo-cytochrome P-450. Moreover, they indicate that exogenous haem can be incorporated into newly formed aop-cytochrome P-450.     

Phenobarbital induces cytochrome P-450- and cytochrome P-448-dependent monooxygenases in rat hepatoma cells

The induction by phenobarbital (PB) of aldrin epoxidase (AE) and aryl hydrocarbon hydroxylase (AHH), markers of cytochrome P-450- and cytochrome P-448-dependent monooxygenases, was studied in cell lines derived from Reuber H35 rat hepatoma which differ widely in their degree of differentiation. The following results were obtained: (1) PB induced AE 2-6-fold and AHH 2-4-fold in the differentiated clones, Fao, 2sFou, and C2Rev7 during an exposure period of 72 h. The barbiturate increased AHH but not AE in the dedifferentiated clone H5, the poorly differentiated line H4IIEC3/T, and in the well differentiated line H4IIEC3/G-. (2) Continuous presence of the barbiturate was required for maintaining the induction of the two monooxygenase activities in C2Rev7 cells. (3) Maximum induction of AE was observed at a PB concentration of 1.5-3.0 mM. (4) The effects of 7,8-benzoflavone on AHH-activities induced by phenobarbital in C2Rev7 and H5 cells suggested that they are mediated by cytochrome P-450- and cytochrome P-448-dependent monooxygenase forms, respectively. Thus, the flavonoid had only a slight inhibitory effect on PB-induced AHH in C2Rev7 cells, but strongly inhibited PB-induced AHH in H5 cells. The induction of AE and of 7,8-benzoflavone-inhibitable AHH in 2sFou cells indicated that PB is capable of inducing cytochromes P-450 and cytochrome P-448 in the same cell.     

Electrochemical investigations on the oxygen activation by cytochrome P-450.

The application of cytochrome P-450 in substrate conversion is complicated both due to the limited stability and the cofactor regeneration problems. To overcome the disadvantages of NADPH consumption the transfer of the reduction equivalents from an electrode into the cytochrome P-450-system was studied: 1. NADPH was cathodically reduced at a mercury pool electrode. By immobilization of NADP on dialdehyde Sephadex the reductive recycling was possible. 2. Different forms of reduced oxygen were produced by the cathode: a) The reaction of O2- with deoxycorticosterone yields a carboxylic acid derivative. In contrast the cytochrome P-450 catalyzed NADPH-dependent reaction with the same substrate gives corticosterone, O2- represents only an intermediate in the activation of oxygen and is not the "activated oxygen" species. b) Molecular oxygen was reduced to HO2- and H2O2, respectively. The interaction of adsorbed cytochrome P-450 on the electrode surface with the reduced oxygen species in the absence of NADPH was studied. The electrochemically generated peroxide seems to be more active than added H2O2. 3. In a model of electro-enzyme-reactor several substrates were hydroxylated by microsomal cytochrome P-450 with cathodically reduced oxygen which substitutes NADPH.     

Metabolic conditions determining the composition and catalytic activity of cytochrome P-450 monooxygenases in Candida tropicalis.

In the microsomal fraction of Candida tropicalis cells, two distinct monooxygenases were detected, depending on the growth conditions. The distinction of the two monooxygenases was evident from: (i) the absorption maxima in the reduced CO difference spectra of the terminal oxidases (cytochromes P-450 and P-448); (ii) the contents of the monooxygenase components (cytochromes P-450/P-448, NADPH-cytochrome c (P-450) reductase, and cytochrome b5) and (iii) the catalytic activity of the complete system (aliphatic hydroxylation and N-demethylation activity). The occurrence of the respective monooxygenases could be related to the carbon source (n-alkanes or glucose). Oxygen limitation led to a significant increase of cytochrome P-450/P-448 content, independent of the carbon source utilized by the cells. An improved method for the isolation of microsomes enabled us to demonstrate the presence of cytochrome P-448 in glucose-grown cells.     

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

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Fatty acid hydroxylation in rat kidney cortex microsomes

Rat kidney microsomes have been found to catalyze the hydroxylation of medium-chained fatty acids to the omega- and (omego-1)-hydroxy derivatives. This reaction, which requires NADPH and molecular oxygen, is a function of monooxygenase system present in the kidney microsomes, containing NADPH-cytochrome c reductase and cytochrome P-450K. NADH is about half as effective as an electron donor as NADPH and there is an additive effect in the presence of both nucleotides. Cytochrome P-450K absorbs light maximally at 452-3 nm, when it is reduced and bound to carbon monoxide. The extinction coefficient of this complex is 91 mM(-1) cm(-1). Electrons from NADPH are transferred to cytochrome P-450K via the NADPH-cytochrome c reductase. The reduction rate of cytochrome P-450K is stimulated by added fatty acids and the reduction kinetics reveal the presence of endogenous substrates bound to cytochrome P-450K. Both cytochrome P-450K concentration and fatty acid hydroxylation activity in kidney microsomes are increased by starvation. On the other hand, phenobarbital treatment of the rats has no effect on either the hemoprotein or the overall hydroxylation reaction and 3,4-benzpyrene administration induces a new species of cytochrome P-450K not involved in fatty acid hydroxylation. Cytochrome P-450K shows, in contrast to liver P-450, high substrate specificity. The only substances forming enzyme-substrate complexes with cytochrome P-450K are the medium-chained fatty acids and certain derivatives of these acids. The chemical requirements for substrate binding include a carbon chain of medium length and at the end of the chain a carbonyl group and a free electron pair on a neighbouring atom. The distance between the binding site for the carbonyl group and the active oxygen is suggested to be in the order of 16 A. This distance fixes the ratio of omega- and (omega-1)-hydroxylated products formed from a certain fatty acid by the single species of cytochrome P-450K involved. The membrane microenvironment seems also to be of importance for the substrate specificity of cytochrome P-450K, since removal of the cytochrome from the membrane lowers its binding specificity to some extent. A comparison between the liver and kidney cytochrome P-450 systems suggests that the kidney cytochrome P-450K system is specialized for fatty acid hydroxylation.