Microsomal enzymes of cholesterol biosynthesis from lanosterol. Purification and characterization of delta 7-sterol 5-desaturase of rat liver microsomes

Microsomal delta 7-sterol 5-desaturase of cholesterol biosynthesis is a multienzyme system which catalyzes the introduction of the delta 5-bond into delta 7-cholestenol to form 7-dehydrocholesterol. The detergent-solubilized 5-desaturase has been purified more than 70-fold and resolved from electron carriers and other rat liver microsomal enzymes of sterol biosynthesis by chromatography on DEAE-Sephacel, CM-Sepharose, and immobilized cytochrome b5; the 5-desaturase had not been fully resolved from cytochrom b5 reductase in earlier work. A functional electron transport system for the 5-desaturase has been reconstituted by combining the purified 5-desaturase and electron carriers with egg phosphatidylcholine liposomes. Optimizations of conditions for reconstitution have been obtained; both cytochrome b5 and NADH-cytochrome b5 reductase serve as electron carriers. A pyridine nucleotide-dependent flavoprotein is required and the requirement can be satisfied with either purified cytochrome b5 reductase or cytochrome P-450 reductase. Cyanide and iron-chelators strikingly inhibit the 5-desaturase activity, thus suggesting that 5-desaturase is a metalloenzyme as are other well-characterized cytochrome b5-dependent oxidases. 5-Desaturase is resolved from 4-methyl sterol oxidase activity of cholesterol biosynthesis by chromatography on the immobilized cytochrome b5. This resolution of the two oxidases not only indicates that introduction of the delta 5-bond and oxidation of 4 alpha-methyl groups are catalyzed by different terminal oxidases, but resolution affords enzymes of sufficient purity to carry out reconstitution experiments. A novel assay based on substrate-dependent increments of oxidation of alpha-NADH has been developed for measurement of 5-desaturase activity. Measurement of stoichiometry of 5-desaturase demonstrates that for each equivalent of cis-desaturation of delta 7-cholestenol, 1 eq of NADH is consumed. Along with strict dependence upon oxygen, this observation confirms, as suggested by previous workers, that the 5-desaturation is catalyzed by a mixed function oxidase rather than a dehydrogenase.     

Biosynthesis of gamma-linolenic acid in cotyledons and microsomal preparations of the developing seeds of common borage (Borago officinalis).

The developing seeds of Borago officinalis (common borage) accumulate a triacylglycerol oil that is relatively rich in the uncommon fatty acid gamma-linolenate (octadec-6,9,12-trienoic acid). Incubation of developing, whole, cotyledons with [14C]oleate and [14C]linoleate showed that the gamma-linolenate was synthesized by the sequential desaturation of oleate----linoleate----gamma-linolenate. Microsomal membrane preparations from the developing cotyledons contained an active delta 6-desaturase enzyme that catalysed the conversion of linoleate into gamma-linolenate. Experiments were designed to manipulate the [14C]linoleate content of the microsomal phosphatidylcholine. The [14C]linoleoyl phosphatidylcholine labelled in situ was converted into gamma-linolenoyl phosphatidylcholine in the presence of NADH. The substrate for the delta 6-desaturase in borage was, therefore, the linoleate in the complex microsomal lipid phosphatidylcholine, rather than, as in animals, the acyl-CoA. This was further confirmed in experiments that compared the specific radioactivity of the gamma-linolenate, in acyl-CoA and phosphatidylcholine, that was synthesized when [14C]linoleoyl-CoA was incubated with microsomal membranes, NADH and non-radioactive gamma-linolenoyl-CoA. The delta 6-desaturase was positionally specific and only utilized the linoleate in position 2 of sn-phosphatidylcholine. Analysis of the positional distribution of fatty acids in the endogenous microsomal sn-phosphatidylcholine showed that, whereas position 1 contained substantial linoleate, only small amounts of gamma-linolenate were present. The results shed further light on the synthesis of C18 polyunsaturated fatty acids in plants and in particular its relationship to the regulation of the acyl quality of the triacylglycerols in oilseeds.     

Increased response of liver microsomal delta 6-desaturase activity to dietary methionine in rats

The effects of dietary casein level (5-40%) on the liver microsomal phospholipid profile, delta 6-desaturase activity and related variables were investigated in rats to examine whether the dietary protein level affected the delta 6-desaturase activity through an alteration of the liver microsomal phospholipid profile. The effects of supplementing a 10% casein diet with certain amino acids were also investigated. The concentration of hepatic S-adenosylmethionine (SAM), the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) and the delta 6-desaturase activity in liver microsomes, and the ratio of arachidonate to linoleate of microsomal PC increased with increasing dietary casein level. There were significant correlations between the dietary methionine content and hepatic SAM concentration, hepatic SAM concentration and microsomal PE concentration, and microsomal PE concentration and delta 6-desaturase activity. Supplementation of the 10% casein diet with methionine significantly increased the hepatic SAM concentration, PC/PE ratio, delta 6-desaturase activity, and arachidonate/linoleate ratio, whereas cystine supplementation had no or little effect on these variables. These increases induced by methionine were significantly suppressed by additional glycine. The results obtained here, together with those in our previous report, suggest that quantity and type of dietary protein might affect the delta 6-desaturase activity through an alteration of the liver microsomal profile of phospholipids, especially PE, and that the alteration of phospholipid profile might be mediated by a hepatic SAM concentration that reflects the dietary methionine level.     

The delta 12-desaturase from the house cricket, Acheta domesticus (Orthoptera: Gryllidae): characterization and form of the substrate

A novel delta 12-desaturase from animals, which converts oleic acid (18:1n-9) to linoleic acid (18:2n-6), was characterized in the house cricket, Acheta domesticus. The delta 12-desaturase product, linoleic acid, was determined by silver nitrate thin-layer chromatography, radio-gas-liquid chromatography and radio-high-performance liquid chromatography with the latter being used for routine analyses. Enzyme activity was located in the microsomal fraction of whole insect homogenates. NADPH or NADH was required for activity, with NADPH being the more efficient electron donor. In short incubation times with oleoyl-CoA as substrate, the highest amount of product, linoleic acid, was found as linoleoyl-CoA. With longer incubation periods, most of the linoleic acid was recovered in the polar lipid fraction containing phospholipid. Preincubation of the microsomal preparation in the absence of NADPH, which allowed 90% of the oleoyl moiety to be transacylated into complex lipid, resulted in no detectable desaturation upon addition of NADPH. These data indicate that the oleic acid moiety used as substrate was in the form of a CoA derivative and not in the form of a phospholipid, as it is for the plant delta 12-desaturase. This is the first characterization of a delta 12-desaturase from an animal system and the first report of a delta 12-desaturase that uses oleoyl-CoA as substrate.     

Immunochemical evidence for the enzymatic difference of delta 6-desaturase from delta 9- and delta 5-desaturase in rat liver microsomes

The enzymatic properties of the three types of microsomal acyl-CoA desaturases, delta 6-, delta 9- and delta 5-desaturases, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (delta 6-desaturase). By the double immunodiffusion technique, the anti-delta 6-desaturase antibody showed a single precipitin line to the purified delta 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified delta 9-desaturase. The antibody even inhibited definitely delta 6-desaturase activity in microsomes, but neither stearoyl-CoA (delta 9-) nor eicosatrienoic acid (delta 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal delta 6-desaturase is different enzyme from desaturases delta 9- and delta 5.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Characterization, solubilization, and partial purification of NADPH-dependent delta 8,14-steroid 14-reductase

The membrane-bound enzyme of microsomes that catalyzes NADPH-dependent reduction of the 14-double bond of conjugated delta 8,14- and delta 7,14-sterols has been studied both as collected in microsomes from broken cell preparations of rat liver and after solubilization. Optimal incubation conditions for assay of the membrane-bound enzyme have been determined, and properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics, pH, addition of inhibitors, addition of glycerol phosphatides, and sterol substrate specificity. The 14-reductase is readily solubilized with a mixture of octylglucoside and taurodeoxycholic acid. The solubilized enzyme has been enriched by precipitation with polyethylene glycol and chromatography on DEAE-Sephacel and hydroxylapatite columns. The resulting partially purified enzyme has been obtained free of other microsomal enzymes of cholesterol biosynthesis: 4-methyl sterol oxidase, delta 5,7-sterol 7-reductase, delta 8,24-sterol 24-reductase, 3-ketosteroid reductase, and steroid 8----7-ene isomerase, plus microsomal cytochrome P-450, cytochrome P-450 reductase, cytochrome b5 reductase, and cytochrome b5. The partially purified enzyme is stimulated by addition of phospholipids. All of the properties exhibited by partially purified 14-reductase are consistent with the suggestion that the solubilized and enriched enzyme catalyzes the microsomal reduction of the 14-double bond of the sterol-conjugated dienes. However, presence of the enzyme does not prove that the sterol-conjugated dienes are obligatory precursors of cholesterol.     

Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.).

Cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata) was solubilized from microsomal membranes with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonic acid). Cytochrome P-450 was separated from NADPH: cytochrome c (P-450) reductase by ion-exchange chromatography on DEAE-Sephacel. NADPH:cytochrome c (P-450) reductase was further purified by affinity chromatography on 2',5'-ADP-Sepharose 4B. This procedure resulted in a 248-fold purification of the enzyme; on SDS/polyacrylamide-gel electrophoresis after silver staining, only one band, corresponding to a molecular mass of 80 kDa, was present. The digitoxin 12 beta-hydroxylase activity could be reconstituted by incubating partially purified cytochrome P-450 and NADPH:cytochrome c (P-450) reductase together with naturally occurring microsomal lipids and flavin nucleotides. This procedure yielded about 10% of the original amount of digitoxin 12 beta-hydroxylase.     

The role of cytochrome b5 in delta 12 desaturation of oleic acid by microsomes of safflower (Carthamus tinctorius L.)

The electron donors for the membrane-bound fatty acid desaturases of higher plants have not previously been identified. In order to assess the participation of cytochrome b5 in microsomal fatty acid desaturation, the cytoplasmic domain of microsomal cytochrome b5 was purified from Brassica oleracea, and murine polyclonal antibodies were prepared. The IgG fraction from ascites fluid inhibited 62% of NADH-dependent cytochrome c reduction in safflower (Carthamus tinctorius L.) microsomes. These antibodies also blocked desaturation of oleic acid to linoleic acid in lipids of C. tinctorius microsomes by 93%, suggesting that cytochrome b5 is the electron donor for the delta 12 desaturase.     

Requirements of delta 9 and delta 12 fatty acid desaturation in Neurospora

Microsomes prepared from the wild-type strain and lipid auxotrophs of Neurospora were analyzed for delta 9 - (stearoyl-CoA) and delta 12 - (oleoyl-CoA) desaturase activities. The wild-type delta 9-desaturase was found to have a 20-fold higher specific activity and 2-fold lower activation energy than the delta 12-desaturase. In addition, delta 12-desaturase had higher Km app values for oleoyl-CoA and for NADH than the equivalent values for delta 9-desaturase. These properties were correlated with a rate-limiting role of delta 12-desaturase in the production of 18:2, the major fatty acid of Neurospora. The delta 12-desaturase also exhibited a higher tolerance to pH changes and to cyanide than did the delta 9-desaturase. Both activities could be measured in the same reaction mixture using stearoyl-CoA as the substrate, indicating a coupling of the two enzymes. Enrichment of cellular membranes of the wild-type Neurospora with 18:0 and 18:1, 18:2, 18:3 fatty acids led to the conclusion that the presence of excess substrate in the membrane induces activation of the appropriate desaturase. These experiments also suggested that the membrane fluidity, as determined by the degree of unsaturation of membrane fatty acids, may influence the activities of the desaturating enzymes. Perturbation of the polar head groups of the membrane phospholipids indicated that the correct composition of anionic phospholipids is an absolute requirement for the function of both desaturases. These studies show that the activities of the delta 9-desaturase and the delta 12-desaturase are regulated by a variety of factors and that the delta 12-desaturase is subjected to less stringent controls than the delta 9-desaturase.     

Effects of dietary eritadenine on delta6-desaturase activity and fatty acid profiles of several lipids in rats fed different fats

Effects of dietary eritadenine on liver microsomal delta6-desaturase activity and the fatty acid profile of phosphatidylcholine, cholesteryl esters, and triglycerides of liver microsomes or plasma were investigated in rats fed different fats (palm oil, olive oil, and safflower oil). The activity of delta6-desaturase was influenced by both dietary fat types and eritadenine. In rats fed control diets, delta6-desaturase activity was higher in the order of the palm oil, olive oil, and safflower oil groups. In rats fed eritadenine-supplemented diets, the enzyme activity was markedly decreased to a constant level irrespective of dietary fat type. The 20:4n-6/18:2n-6 ratio of phosphatidylcholine and cholesteryl esters, as compared with triglycerides, was highly sensitive to eritadenine. The results suggest that the activity of delta6-desaturase is regulated by dietary fats and eritadenine independently, and that the effect of eritadenine is stronger than that of dietary fats.     

Studies of the delta 12 desaturase of Carthamus tinctorius L

The delta 12 desaturase of developing safflower seeds responsible for the conversion of an oleoyl moiety to the linoleoyl moiety of phospholipids was further characterized. The protein concentration of the microsomal preparation, the oleoyl-CoA concentration (the primary substrate), short incubation periods, and the addition of lysophospholipids must be controlled to obtain optimal desaturation. No evidence could be obtained to implicate cytochrome b5 as the intermediate electron carrier. Attempts to solubilize the desaturase with a variety of detergents and chaotropic reagents were not successful. Brief exposure of the microsomal preparation to trypsin resulted in rapid loss of activity. The overall evidence would suggest that the delta 12 desaturase requires a reductant (NADPH), a NADPH:electron carrier reductase, an electron carrier, a specific desaturase, and an acyltransferase with oleoyl-CoA as the substrate to acylate lysophospholipid to the active oleoyl phospholipids (presumably phosphatidylcholine or phosphatidylethanolamine). The complexity of this system suggests that purification of the components and a reassembling of the purified components will be difficult.     

Evidence for a second microsomal trans-2-enoyl coenzyme A reductase in rat liver. NADPH-specific short chain reductase

Evidence for the existence of a previously unknown rat hepatic microsomal reductase, short chain trans-2-enoyl-CoA reductase (SC reductase) is presented. This reductase has a specific requirement for NADPH, is unable to utilize NADH, and catalyzes the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA to butyric acid and hexenoic acid at a rate of 5 and 65 nmol per min per mg of microsomal protein, respectively. Highly purified NADPH cytochrome P-450 reductase incorporated into liposomes prepared from dilauroyl phosphatidylcholine in the presence or absence of cytochrome P-450 possesses no SC reductase activity. These liposomal preparations did, however, catalyze mixed function oxidations of benzphetamine and testosterone. Rabbit antibody to rat liver NADPH cytochrome P-450 reductase had little to no effect on the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA, suggesting that the SC reductase accepts reducing equivalents directly from NADPH. When acetoacetyl-CoA was incubated with hepatic microsomes and either NADH or NADPH, no formation of butyrate was detected; however, when both cofactors were present, a rate of formation of 3 nmol of butyrate was determined per min per mg of microsomal protein. These results suggest the presence of a previously unknown short chain beta-ketoreductase which catalyzes the reduction of short chain beta-keto acids, only in the presence of NADH. Our results also indicate that the electrons from NADH to the beta-ketoreductase bypass cytochrome b5. The physiological significance is discussed in terms of lipogenesis and ketone body utilization by the liver.     

B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes

Fractions of plasma membranes, Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope were isolated from rat liver and were characterized by electron microsocpe and biochemical methods. The purity of the fractions was controlled by morphometry and by marker enzyme activities. Amounts of cytochromes b5, P-450, and P-420 were measured, as well as the NADPH- and NADPH-cytochrome c reductase activities. The pigments of the microsomal electron transport system were found in all membrane fractions in relatively high amounts, thus excluding an origin by microsomal contamination. Purified preparations of plasma membrane and Golgi apparatus contained approximately 30% of the cytochrome b5 and cytochrome P-450 + P-420 found in ER membranes. Plasma membranes were also characterized by a high ratio of P-420/450. Degradation of cytochromes P-450 and P-420 was relatively rapid in all fractions, except in the ER. Cytochrome b5 extracted from plasma membranes was spectrophotometrically and enzymatically indistinguishable from ER cytochrome b5. However, immunnlogical characterization with rabbit antibodies against the trypsin-resistant core of microsomal cytochrome b5 showed the presence of at least two types of cytochrome b5 in ER membranes, in contrast to the plasma membranes in which only one of these components was detected. This immunological differentiation also demonstrates that the plasma membrane-bound cytochrome b5 is endogenous to this membrane and does not reflect contamination by ER elements. We conclude that cytochromes b5, P-450, and P-420 are not confined only to ER and nuclear membranes but also occur in signficant amounts in Golgi apparatus and plasma membranes. The findings are discussed in relation to observations of similar redox components in Golgi apparatus, secretory vesicles, and plasma membranes of other cells.     

Transport of electrons from mitochondria to microsomes in the reconstituted system of cell organelles

The reduction of cytochrome P-450--CO complex in the presence of various agents in the reconstituted system of liver cell organelles was studied. The reconstituted system was obtained by the preincubation of isolated liver microsomes and mitochondria of the rats kept on a prolonged phenobarbital diet. The addition of glutamate (but not succinate), NAD+ and amytal (or rotenone) to the reconstituted system caused a 40-50% reduction of NADPH-reducible cytochrome P-450. The inhibitor of mitochondrial NADH-cytochrome b5 reductase dicumarol prevented the cytochrome P-450 reduction in the presence of glutamate, NAD+ and amytal but did not affect the reduction of cytochrome P-450 by the added NADH. It was concluded that the electron transfer from the NAD-dependent substrates of the inner mitochondrial respiratory chain to the microsomal cytochrome P-450 occurs with the participation of non-bound NAD and cytochrome b5 of the outer mitochondrial membrane on the condition that the membranes of the two main oxidative systems are in tight contact.     

Evidence for an oleoyl phosphatidylcholine desaturase in microsomal preparations from cotyledons of safflower (Carthamus tinctorius) seed.

1. [14C]Oleoyl-CoA was metabolized rapidly and essentially completely by microsomal preparations from developing safflower (Carthamus tinctorius) cotyledons, and most of the [14C]oleate was incorporated into 3-sn-phosphatidylcholine. 2. In aerobic reaction mixtures containing NADH2 the [14C]oleate in 3-sn-phosphatidylcholine was converted into [14C]linoleate without any change in the specific radioactivity of the lipid. Over a 60 min incubation period the extent of conversion of [14C]oleoyl phosphatidylcholine into [14C]linoleoyl phosphatidylcholine was generally greater than 60%. The rate of desaturation of endogenous [14C]oleoyl phosphatidylcholine labelled from [14C]oleoyl-CoA was much greater that of exogenous [14C]dioleoyl phosphatidylcholine the specific radioactivity of the oleoyl moiety of the lipid remained constant, indicating that labelled and unlabelled oleate were desaturated at the same rate. On this assumption an initial rate of desaturation of about 15 nmol of oleate desaturated/min per mumol of 3-sn-phosphatidylcholine was estimated. 4. [14C]Oleate esterified at positions 1 and 2 of both endogenous and exogenous 3-sn-phosphatidylcholine was desaturated. 5. Attempts to demonstrate the presence of an oleoyl-CoA desaturase in safflower microsomal fractions by the appearance of linoleoyl-CoA in reaction mixtures were inconclusive.     

An enzyme-linked immunoadsorbent assay for measuring cytochrome b5 and NADPH-cytochrome P-450 reductase in rat liver microsomal fractions. Evidence for functionally inactive protein.

Immunoreactive cytochrome b5 and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from rat liver microsomal fractions were measured by using an enzyme-linked immunoadsorbent assay (e.l.i.s.a.) as a function of age, sex and type of inducer (phenobarbital or 3-methylcholanthrene), and the values were compared with those obtained by spectral measurement (for cytochrome b5) or enzymic assay (for reductase). In untreated animals, there was more cytochrome b5 and NADPH-cytochrome P-450 reductase when measured by an e.l.i.s.a. than was seen spectrally or enzymically. However, for microsomal preparations from phenobarbital-pretreated animals, spectrally obtained values for cytochrome b5 and immunoreactive-cytochrome b5 values were similar. Values from control animals suggest that there is about 20-30% more immunoreactive cytochrome b5 than that which is spectrally detectable.     

Electron-transport cytochrome P-450 system is involved in the microsomal metabolism of the carcinogen chromate

The kinetics of chromate reduction by liver microsomes isolated from rats pretreated with phenobarbital or 3-methylcholanthrene with NADPH or NADH cofactor have been followed. Induction of cytochrome P-450 and NADPH-cytochrome P-450 reductase activity in microsomes by phenobarbital pretreatment caused a decrease in the apparent chromate-enzyme dissociation constant, Km, and an increase in the apparent second-order rate constant, kcat/Km, but did not affect the kcat of NADPH-mediated microsomal metabolism of chromate. Induction of cytochrome P-448 in microsomes by 3-methylcholanthrene pretreatment did not affect the kinetics of NADPH-mediated reduction of chromate by microsomes. The kinetics of NADH-mediated microsomal chromate reduction were unaffected by the drug treatments. The effects of specific enzyme inhibitors on the kinetics of microsomal chromate reduction have been determined. 2'-AMP and 3-pyridinealdehyde-NAD, inhibitors of NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase, inhibited the rate of microsomal reduction of chromate with NADPH and NADH. Metyrapone and carbon monoxide, specific inhibitors of cytochrome P-450, inhibited the rate of NADPH-mediated microsomal reduction of chromate, whereas high concentrations of dimethyl-sulfoxide (0.5 M) enhanced the rate. These results suggest that the electron-transport cytochrome P-450 system is involved in the reduction of chromate by microsomal systems. The NADPH and NADH cofactors supply reducing equivalents ultimately to cytochrome P-450 which functions as a reductase in chromate metabolism. The lower oxidation state(s) produced upon chromate reduction may represent the ultimate carcinogenic form(s) of chromium. These studies provide evidence for the role of cytochrome P-450 in the activation of inorganic carcinogens.     

Subfractionation of rat liver microsomes by immunoprecipitation and immunoadsorption methods.

Rabbit antisera were prepared against cytochrome b5 and NADPH-cytochrome c reductase [EC 1.6.2.4] purified from rat liver microsomes, and utilized in examining the distribution of these and other membrane-bound enzymes among the vesicles of rat liver microsomal preparations by immunoprecipitation and immunoadsorption methods. Smooth microsomes with an average vesicular size of 200 nm (diameter) and sonicated smooth microsomes with an average diameter of 40-60 nm were used in subfractionation experiments. Immunoprecipitation of microsomal vesicles with anti-cytochrome b5 immunoglobulin failed to show any separation of the microsomes into fractions having different enzyme compositions. Cytochrome b5 was apparently distributed among all vesicles even when sonicated microsomes were used. When the antibody against NADPH-cytochrome c reductase was used, however, immunoadsorption of microsomes on Sepharose-bound antibody produced some separation of NADPH-cytochrome c reductase and cytochrome P-450 from NADH-cytochrome b5 reductase and cytochrome b5. The separation was more pronounced when sonicated microsomes were used. These results indicate microheterogeneity of the microsomal membrane, and suggest the clustering of NADPH-cytochrome c reductase and cytochrome P-450 molecules in the membrane.     

Neutrophil microsomes biosynthesize linoleate epoxide (9,10-epoxy-12-octadecenoate), a biological active substance

We demonstrated that linoleate epoxide (9,10-epoxy-12-octadecenoate) exists in human burned skin and in lung lavages in patients with adult respiratory distress syndrome. This epoxide shows a highly toxic effect on cellular function. Thus, it was given the name leukotoxin. In this communication, we reveal that neutrophils from various sources such as guinea-pig peritonea and canine or human blood biosynthesize linoleate epoxide from linoleate as a substrate. From the reaction mixture of neutrophils with linoleate, a leukotoxin isomer, 12,13-epoxy-9-octadecenoate, and a 'non-toxic' hydroxy derivative of linoleate, 9-hydroxy-12-octadecenoate, were detected. Biosynthesis of leukotoxin by neutrophils was substantially enhanced by osmotic activation or by a calcium-ionophore, A23187. Microsomes prepared from neutrophils could oxygenate linoleate to leukotoxin in the presence of NADPH. In liver or kidney microsomal reaction mixture, leukotoxin could be detected only in the presence of an epoxide hydrolase inhibitor, epoxytrichloropropane. As biosynthesis of leukotoxin was sensitive to carbon monooxide, it was concluded that cytochrome P-450 dependent monooxygenase is responsible for the biosynthesis. Elucidation of the biosynthesis pathway of leukotoxin might contribute to the treatment of diseases associated with neutrophil recruitment.     

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.