Sten Orrenius

The presence of a monooxygenase system in human fetal liver microsomes

Abstract Human fetal liver microsomes were shown to contain the components of the NADPH- and NADH-linked electron transport systems. The NADPH-linked system was capable of hydroxylating testosterone and laurate, but unable to oxidize exogenous substrates such as 3, 4-benzpyrene and aminopyrine. The spectral change associated with the binding of the substrate to cytochrome P-450 was of the type I variety with testosterone and laurate and type II with aminopyrine.

A Comparative study on the effects of phenobarbital and 3,4-benzpyrene on the hydroxylating enzyme system of rat-liver microsomes

Abstract The effects of phenobarbital and 3,4-benzpyrene on the hydroxylating enzyme system of rat-liver microsomes have been compared. Repeated injections of phenobarbital into rats caused a marked increase in the concentration of cytochrome P-450, and in the activities of NADPH-cytochrome P-450 reductase and NADPH-cytochrome b 5 reductase, as well as an enhancement of the rate of aminopyrine demethylation, whereas 3,4-benzpyrene hydroxylation was only moderately stimulated. The latter activity, however, was enhanced several-fold by the injection of a single dose of 3,4-benzpyrene. Associated herewith there was a formation of cytochrome P-448 but there was no obvious increase in the rate of reduction of the hemoprotein by NADPH. When added to the assay system in vitro , aminopyrine and to a minor extent 3,4-benzpyrene stimulated the initial rate of NADPH-cytochrome P-450 reduction catalyzed by liver microsomes from controls. Using liver microsomes from 3,4-benzpy-rene-treated rats, 3,4-benzpyrene was more potent than aminopyrine in stimulating the rate of NADPH-cytochrome P-448 reduction. The concentration of the polycyclic hydrocarbon needed to obtain maximum stimulation of the rate of reduction of the cytochrome by NADPH was considerably lower with liver microsomes from 3,4-benz-pyrene-treated rats than from controls. Furthermore, aminopyrine, which markedly inhibited 3,4-benzpyrene hydroxylation catalyzed by control liver microsomes, had considerably less effect on this activity when microsomes from 3,4-benzpyrene-treated rats were used as the source of enzyme. The results support the hypothesis that in contrast to phenobarbital, 3,4-benzpyrene yields a modified cytochrome (P-448) in the liver microsomes with an increased affinity for 3,4-benzpyrene as well as a decreased affinity for aminopyrine. Possible mechanisms for cytochrome P-448 formation are discussed.

Cytochrome P-450K of rat kidney cortex microsomes: Further studies on its interaction with fatty acids

Abstract The cytochrome P-450K containing monooxygenase system of rat kidney cortex microsomes catalyzes the hydroxylation of various saturated fatty acids of medium chain length to the corresponding ω- and (ω-1)-hydroxy derivatives. The hydroxylation activity, as well as the ratio between the two hydroxylated products, vary with the carbon chain length of the fatty acid. Optimal hydroxylation activity is observed with myristic acid which yields the 13- and 14-hydroxylated products at a ratio of about 1. The ω/(ω-1)-hydroxylation ratio decreases with increasing carbon chain length of the fatty acid. On the other hand, with lauric acid as a substrate the ratio between ω- and (ω-1)-hydroxylation does not change significantly with varying time of incubation or substrate concentration, or incubation in a medium containing D2O or after induction of enhanced hydroxylation activity by starvation of the animals. Furthermore, 12-hydroxylauric acid and capric acid—which is almost exclusively ω-hydroxylated by rat kidney cortex microsomes—inhibit both 11- and 12-hydroxylation of lauric acid to a similar extent whereas 11-hydroxylauric acid does not seem to inhibit either 11- or 12-hydroxylation. C10-C16 fatty acids produce the type I spectral change upon addition to rat kidney cortex microsomes and seem to interact with similar amounts of the cytochrome P-450K present in these particles. In agreement with the metabolic studies, 12-hydroxylauric acid interacts with cytochrome P-450K giving rise to a reverse type I spectral change, whereas 11-hydroxylauric acid does not produce an observable spectral change. Finally, results of binding experiments with a series of derivatives of dodecane suggest that type I binding to cytochrome P-450K requires, besides a proper chain length, the presence of a carbonyl group together with an electron pair on a neighboring atom at the end of the carbon chain. A chain length of 14 carbon atoms seems to be optimal and it is suggested that this chain length may correspond to the distance between a possible binding site and the catalytic site of cytochrome P-450K

Spectral Studies on the Interaction of Imipramine and some of its Oxidized Metabolites with Rat Liver Microsomes

1. Imipramine and its oxidized metabolites 2-hydroxyimipramine, desmethylimipramine (DMI), 2-hydroxydesmethylimipramine (2-OH-DMI), didesmethylimipramine (DDMI) and iminodibenzyl (IDB) all interact with cytochrome P-450 to give rise to the type I spectral change when added to suspensions of rat liver microsomes.2. Imipramine, DMI and the more polar 2-OH-DMI, all exhibit high affinities for binding to the cytochrome.3. The magnitude of the type I spectral change produced by imipramine is not enhanced by the further addition of DMI, neither is the DMI-induced type I spectral change increased by 2-OH-DMI. Thus it appears that imipramine and its oxidized metabolites interact with a common cytochrome P-450 species and that the inhibitory effect of DMI and 2-OH-DMI on the oxidation of imipramine and DMI, respectively, may be due to competition for binding to cytochrome P-450.4. At low concentrations DDMI elicits a type I spectral change with microsomes but with increased concentration produces a type II spectra...

FATTY ACID HYDROXYLATION IN RAT KIDNEY CORTEX MICROSOMES

SummaryRat kidney microsomes have been found to catalyze the hydroxylation of medium-chained fatty acids to theω- and (ω-1)-hydroxy derivatives. This reaction, which requires NADPH and molecular oxygen, is a function of a monooxygenase system present in the kidney microsomes, containing NADPH-cytochromec 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 91mm−1 cm−1. Electrons from NADPH are transferred to cytochrome P-450K via the NADPH-cytochromec 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 Å. This distance fixes the ratio ofω- and (ω-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.

Substrate binding to cytochrome P-450 of liver and adrenal microsomes

The binding of various substrates of the microsomal mono-oxygenase system to its terminal oxidase, cytchrome P450, is associated with characteristic absorbance changes in the difference spectrum of the microsomes [l-3] . Thus, hexobarbital, aminopyrine, testosterone and laurate, as well as a number of other substances, elicit spectral changes characterized by a trough in the 420 nm region and a peak in the 390 nm region when added to a suspension of rat liver microsomes. This spectral change has been termed type I. The type II spectral change, on the other hand, has a peak at about 430 mn and trough at 390-400 nm and is likewise produced by a variety of substances, among them aniline. In a recent paper we have suggested that the competitive ’ :‘-?i;n that certain drugs, steroids and fatty acids exert on each other’s oxidative metabolism in the liver microsomes may be related to the competition for binding to a common cytochrome P-450 species [4]. The substances used in that study, hexobarbital, aminopyrine, testosterone and laurate, were all potent inducers of type I spectra. However, it was not possible to decide whether all of these substrates may interact at a common or at different site(s) of the cytochrome P-450 molecule. Furthermore, aminopyrine and hexobarbital, though slowly metabolized by guinea-pig adrenal microsomes,

The Role of Cytochrome P-450 in Microsomal Mixed Function Oxidation Reactions

The central role of oxygen in many metabolic reactions of the cell is the general theme of this colloquium. Prof. Lubbers [1] has described the role of the hemoproteins, hemoglobin and myoglobin in oxygen transfer reactions whereas Prof. Chance [2] has presented his elegant experiments on the reduction of oxygen by the mitochondrial respiratory chain. The present discussion will center on a different type of reaction in which oxygen participates in many mammalian cells. These reactions are those which have been termed [3] mixed function oxidation reactions — reactions which are catalyzed [4] by a unique hemoprotein called cytochrome P-450.

A study of the modified type II spectral change produced by the interaction of agroclavine with cytochrome P-450

Abstract Previous work in this laboratory has demonstrated that agroclavine is metabolized by liver microsomes and that cytochrome P-450 is involved as the terminal oxidase in the reaction. Addition of agroclavine to suspensions of liver microsomes in the same concentration as used in the metabolic studies produced a modified Type II spectral change with peak near 420 nm and trough near 390 nm. Using modifiers such as hexobarbital, this spectrum has now been shown to contain a Type 1 component. Furthermore, when sufficiently low concentrations of agroclavine were added to rat liver microsomes, a Type I spectral change appeared, which gradually turned into the modified Type II variety upon increase in agroclavine concentration. Moreover, tryptophan, which also elicits a modified Type II spectral change in rat liver microsomes at certain concentrations, was shown to be capable of producing both a Type I and a Type II difference spectrum, dependent on the concentration in which this substance was added to the microsomes. It is suggested that compounds which are metabolized by cytochrome P-450 interact at a “Type I binding-site”, but that the optical manifestation of this interaction may sometimes be hidden by a second interaction whose spectral manifestations are appreciably larger.

Preparation of antisera against cytochrome b5 and NADPH-cytochrome c reductase from rat liver microsomes

Abstract Antisera were prepared in rabbits against purified rat liver cytochrome b 5 and NADH-cytochrome c reductase, respectively. With the antiserum against cytochrome b 5 , the cytochrome could be precipitated in immunoelectrophoresis from rough and smooth liver microsomal membranes, kidney microsomes and outer mitochondrial membranes. The cytochrome was precipitable from the rough membranes on the 2nd prenatal day, from the smooth membranes on the day of birth and from the kidney microsomes on the 2nd postnatal day. The NADPH-cytochrome c reductase could be precipitated from the rough and smooth membranes and from kidney microsomes by the specific antiserum. It was precipitable from the rough and smooth membranes at birth and from kidney microsomes on the 2nd postnatal day. No NADPH-cytochrome c reductase was found in the mitochondrial membranes. None of the two antisera contained inhibiting antibodies against the catalytic action of the enzyme against which it was directed.

Conversion of agroclavine by mammalian cytochrome P-450

Abstract 1. 1. Agroclavine has been converted to noragroclavine and elymoclavine in a mammalian microsomal system. 2. 2. Conversion has been shown to be dependent upon cytochrome P-450. 3. 3. Spectra of the binding of agroclavine to the hemoprotein are of a modified Type II with microsomes from rat liver and guinea pig adrenal. The spectrum is of Type I with rabbit-liver and guinea pig-liver microsomes.