Biogenesis of endoplasmic reticulum membrane in rat liver cells. II. Discharge of the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 on the cytoplasmic side of the endoplasmic reticulum membrane.

The direction of discharge of the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 from bound polyribosomes of rough microsomes was investigated in order to elucidate the mechanism of separation of these membrane proteins from secretory proteins, which are also synthesized by the same class of ribosomes of rough endoplasmic reticulum. The nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 in intact rough microsomes were accessible to externally added 125I-Fab's against these proteins, and were susceptible to trypsin digestion, whereas the nascent peptides of serum albumin were not. The nascent peptides of these two microsomal proteins were released into the cytoplasm by puromycin treatment of intact rough microsomes, while the nascent peptides of serum albumin were retained in the microsomal lumen. These observations suggest that the nascent peptides of microsomal proteins, which are present on the cytoplasmic surface of the endoplasmic reticulum membrane, are exposed on the surface of microsomal vesicles, while those of secretory proteins are enclosed inside the vesicles. Therefore, the topographical separation of microsomal membrane proteins from secretory proteins is accomplished at the step of their synthesis by the bound polyribosomes of rough endoplasmic reticulum.     

Biogenesis of endoplasmic reticulum membrane in rat liver cells. III. Biosynthesis of NADPH-cytochrome c reductase and cytochrome b5 by loosely-bound ribosomes.

Membrane-bound ribosomes were separated into two distinct classes (loosely-bound and tightly-bound ribosomes) by treatment with 0.6 M KCl, 1 mM puromycin, 0.05% DOC, or 10 mM EDTA. It was also confirmed that any one of these reagents except for EDTA dissociated the same class of ribosomes from the membrane. A population of lighter microsomal vesicles was formed from rough microsomes upon the dissociation of loosely-bound ribosomes by treatment with these chemicals. Rough microsomes were subfractionated into lighter and heavier fractions, L-rMs and H-rMs, by centrifugation using a discontinuous gradient of sucrose consisting of 1.3 M, 1.5 M, and 2.1 M solutions. It was found that L-rMs was rich in loosely-bound ribosomes, whereas H-rMs contained a high proportion of tightly-bound ribosomes. It is likely that loosely-bound and tightly-bound ribosomes are heterogeneously distributed among rough microsomal vesicles. Loosely-bound ribosomes and tightly-bound ribosomes synthesize different kinds of proteins. Two microsomal membrane proteins, NADPH-cytochrome c reductase and cytochrome b5, were exclusively synthesized by loosely-bound ribosomes, whereas serum albumin, which is a major component of the secretory proteins of hepatocytes, was synthesized only by tightly-bound ribosomes. Since the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 are released from bound ribosomes to the cytoplasmic surface of endoplasmic reticulum, while those of secretory proteins are discharged into the lumen across the membrane, the strength of the association between ribosomes and microsomal membrane seems to be correlated with the direction of release of nascent peptides.     

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.     

Contributions of cytoplasmic free and membrane-bound ribosomes to the synthesis of mitochondrial cytochrome P-450(SCC) and P-450(11 beta) and microsomal cytochrome P-450(C-21) in bovine adrenal cortex

Rabbit antibodies against cytochrome P-450 (SCC), P-450 (11 beta), and P-450 (C-21) from bovine adrenal cortex were prepared, and it was confirmed that these three cytochrome P-450 species are immunologically distinct from one another. Cytoplasmic sites of synthesis of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) in bovine adrenal cortex were determined by examining the presence of their nascent peptides on isolated free and bound ribosomes. Nascent peptides were released in vitro from ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) were isolated by immunoprecipitation. The nascent peptides of these three cytochrome P-450 species were found in both free and bound ribosomal fractions, suggesting that they share common sites of synthesis in the cytoplasm. However, the nascent peptides of mitochondrial P-450 (SCC) and P-450 (11 beta) were more concentrated in the free ribosomal fraction, whereas those of microsomal P-450 (C-21) were more abundant in the bound ribosomal fraction. The nascent peptides of the three cytochrome P-450 species were released from the membrane-bound ribosomes of rough microsomes into the cytoplasmic surface of microsomal vesicles by puromycin treatment.     

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.     

Detection of a system of microsomal monooxygenases in the rodent malaria parasite Plasmodium berghei

A microsomal fraction from the cells of the malaria parasite of rodent Plasmodium berghei was obtained. The spectral properties of microsomal preparations suggest that P. berghei microsomes contain cytochromes b5 and P-420. Electrophoretic separation of microsomal proteins revealed the presence of proteins whose molecular mass corresponds to NADPH-cytochrome c reductase, cytochrome P-450 and epoxide hydratase. The activities of NADPH-cytochrome c reductase and benzpyrene hydroxylase were determined. The spectral parameters, electrophoretic data and enzymatic activities of microsomal proteins indicate that P. berghei cells contain a cytochrome P-450 monooxygenase system. The interrelationship between the activity of the microsomal monooxygenase system and the resistance of P. berghei cells to the antimalaria preparation chloroquine is discussed.     

Effect of monooxygenase reactions catalyzed by cytochrome P-450 on the microsomal membrane

Hydroxylation of dimethylaniline in rabbit liver microsomes is accompanied by inactivation of cytochrome P-450 and the formation of products inhibiting the catalytic activity of non-inactivated cytochrome P-450. Other enzymes and electron carriers of microsomal membrane (cytochrome b5, NADH-ferricyanide reductase, NADPH-cytochrome c and NADPH-cytochrome P-450 reductases) as well as glucose-6-phosphatase were not inactivated in the course of the monooxygenase reactions. Phospholipids and microsomal membrane proteins were also unaffected thereby. Consequently, the changes in the microsomal membrane during cytochrome P-450 dependent monooxygenase system functioning are confined to the inactivation of cytochrome P-450.     

Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase.

A flavoprotein catalyzing the reduction of cytochrome c by NADPH was solubilized and purified from microsomes of yeast grown anaerobically. The cytochrome c reductase had an apparent molecular weight of 70,000 daltons and contained one mole each of FAD and FMN per mole of enzyme. The reductase could reduce some redox dyes as well as cytochrome c, but could not catalyze the reduction of cytochrome b5. The reductase preparation also catalyzed the oxidation of NADPH with molecular oxygen in the presence of a catalytic amount of 2-methyl-1,4-naphthoquinone (menadione). The Michaelis constants of the reductase for NADPH and cytochrome c were determined to be 32.4 and 3.4 micron M, respectively, and the optimal pH for cytochrome c reduction was 7.8 to 8.0. It was concluded that yeast NADPH-cytochrome c reductase is in many respects similar to the liver microsomal reductase which acts as an NADPH-cytochrome P-450 reductase [EC 1.6.2.4].     

The effects of phosphatase on the components of the cytochrome P-450-dependent microsomal monooxygenase

Incubation of rabbit liver microsomes with alkaline phosphatase resulted in a marked decrease of NADPH-dependent monooxygenase activities. This decrease was found to be correlated with the decrease of NADPH-cytochrome c reductase activity catalyzed by NADPH-cytochrome P-450 reductase. Neither the content of cytochrome P-450, as determined from its CO difference spectrum, nor the peroxide-supported demethylase activity catalyzed by cytochrome P-450 alone was affected by the phosphatase treatment. NADH-cytochrome b5 reductase and cytochrome b5 were not affected by the phosphatase either. NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes lost its NADPH-dependent cytochrome c reductase activity upon incubation with phosphatase in a way similar to that of microsome-bound reductase. Flavin analysis showed that the phosphatase treatment caused a decrease of FMN with concomitant appearance of riboflavin. Alkaline phosphatase, therefore, inactivates the reductase by attacking its FMN, and the inactivation of the reductase, in turn, leads to a decrease of the microsomal monooxygenase activities.     

Alpha-hydroxylation of lignoceroyl-CoA in rat brain microsomes: involvement of NADPH-cytochrome c reductase and topical distribution

1. The enzymatic mechanism of the alpha-hydroxylation of lignoceroyl-CoA, an intermediate in the synthesis of hydroxyceramide, was studied. In the presence of NADPH, sphingosine and microsomes from 20-day-old rat brain, 14C from [1-14C]lignoceroyl-CoA was incorporated into hydroxyceramide. 2. The alpha-hydroxylation of lignoceroyl-CoA in rat brain microsomes was strongly inhibited by a rabbit anti-immunoglobulin G which was prepared against rat liver microsomal NADPH-cytochrome c reductase. However, anti-immunoglobulin G against cytochrome b5 did not inhibit the alpha-hydroxylase activity. 3. The alpha-hydroxylation activity was more sensitive to trypsin treatment than was NADPH-cytochrome c reductase in rat brain microsomes. This indicates that either alpha-hydroxylase itself or an unknown factor essential in alpha-hydroxylation is highly exposed to the surface of brain microsomes.     

PRODUCTION OF MEMBRANE WHORLS IN RAT LIVER BY SOME INHIBITORS OF PROTEIN SYNTHESIS

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [³H]leucine and of [³H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [³H]leucine incorporation into cytoplasmic membranes was inhibited, while [³H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.     

Palmitoyl-CoA Elongation in Brain Microsomes: Dependence on Cytochrome b5 and NADH-Cytochrome b5 Reductase

Experiments were performed to demonstrate the involvement of electron transport system in fatty acid elongation in rat brain microsomes. Mercuric chloride and p-chloromercuriphenylsulfonate, inhibitors on NADH-cytochrome b5 reductase, at 32 microM inhibited NADH-supported palmitoyl-CoA elongation to 30 and 60% of control activity, respectively, whereas NADPH-supported palmitoyl-CoA elongation was unaffected by these mercurials. An antibody to rat liver NADH-cytochrome b5 reductase inhibited brain microsomal NADH-cytochrome b5 reductase activity and NADH-dependent palmitoyl-CoA elongation. Treatment of brain microsomes with trypsin diminished the cytochrome b5 content; NADH- and NADPH-cytochrome c reductase activities were significantly decreased, but the decrease in NADH-cytochrome b5 reductase activity was relatively small. Whereas essentially no incorporation of malonyl-CoA into palmitoyl-CoA was observed with trypsin-treated microsomes, addition of detergent-solubilized cytochrome b5 resulted in a recovery of fatty acid elongation. These results indicate the presence of an electron transport system, NADH-NADH-cytochrome b5 reductase-cytochrome b5-fatty acid elongation, in brain microsomes.     

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.     

Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.     

NADPH-cytochrome P-450 reductase is not a component of the liver microsomal steroid 5-alpha reductase

Liver microsomal steroid 5-alpha-reduction is catalyzed by a NADPH-dependent enzyme system. The requirement of NADPH-cytochrome P-450 reductase to shuttle reduction equivalents from NADPH to steroid 5-alpha-reductase was investigated using an inhibitory antibody against NADPH-cytochrome P-450 reductase. This antibody preparation inhibited cytochrome c reduction in microsomes from female rat liver with an I50 of 0.75 mg antibody/mg of microsomal protein. Benzphetamine N-demethylation and testosterone 6-beta-hydroxylation, two cytochrome P-450-mediated oxidative reactions, were inhibited by the antibody. On the other hand, testosterone 5-alpha-reductase was not affected by the antibody. These results suggest that NADPH-cytochrome P-450 reductase is not an obligatory component of the liver microsomal steroid 5-alpha-reduction.     

Transfer of cytochrome b 5 and NADPH cytochrome c reductase between membranes

NADPH-cytochrome c reductase also reduces cytochrome b 5. The reduction is very slow when the proteins are in solution or bound to different membranes. Only when both proteins share a common membrane, is cytochrome b 5 reduced rapidly by NADPH. The difference in reaction rates indicates recombination on a common membrane of cytochrome b 5 and NADPH reductase originally bound to different vesicles. The recombination of the two proteins occurs with a variety of biological membranes (previously enriched with either reductase or cytochrome b 5) as well as with liposomes. We explain this process as protein transfer rather than vesicle fusion for several reasons: 1. The vesicles do not alter shape or size during incubation. 2. The rate of this process corresponds to the rate of incorporation of the single proteins into liposomes carrying the 'complementary' protein. 3. The exchange of proteins between biological membranes and liposomes occupied by protein does not change the density of either membrane. Protein transfer between membranes appears to be limited to those proteins which had spontaneously recombined with a preformed membrane. In contrast, proteins incorporated into liposomes by means of a detergent were not transferred, nor were endogenous cytochrome b 5 and NADPH-cytochrome c reductase transferred from microsomes to Golgi membranes or lipid vesicles. We conclude that the endogenous proteins and proteins incorporated in the presence of a detergent are linked to the membrane in another manner than the same proteins which had been inserted into a preformed membrane.     

Disruption of rat hepatic microsomal electron transport chains by the selenium-containing anti-inflammatory agent Ebselen

The influence of Ebselen, an organoselenium anti-inflammatory agent, on the two electron transport chains present in rat liver microsomes has been studied. At low micromolar concentrations, Ebselen markedly inhibited the flow of reducing equivalents from NADPH-cytochrome P450 reductase to both its natural electron acceptor, cytochrome P450, and its artificial electron acceptor, cytochrome c. Similarly, the microsomal NADH-cytochrome c reductase system consisting of cytochrome b5 and its flavoprotein, NADH-cytochrome b5 reductase, was also significantly inhibited by Ebselen. The inhibition appears to be due to the inability of the reduced pyridine nucleotide to transfer electrons to the flavin (FAD and/or FMN) in the flavoprotein reductase. This was shown with the purified NADPH-cytochrome P450 reductase, which in the presence of Ebselen was not converted to the semiquinone form following the addition of NADPH. The addition of Ebselen to a suspension of hepatic microsomes from either untreated or phenobarbital-treated rats did not result in any spectral change characteristic of type I, type II, or reverse type I.     

Mixed function oxidases in kidney and duodenum of camel,guinea pig and rat

The activities of the drug-metabolizing enzymes, aniline 4-hydroxylase, benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase have been measured in vitro in kidneys and duodenum of camels (Camelus dromedarius), guinea pigs (Cavia porcellus) and rats (Rattus norvegicus). In these species, levels of hepatic microsomal parameters namely microsomal protein, cytochrome P(450), cytochrome b(5) and NADPH-cytochrome c reductase have also been determined. In general, camels seemed to have the lowest enzyme activity when compared to rats and guinea pigs. Rats showed the highest activity in NADPH-cytochrome c reductase, aniline 4-hydroxylase and ethoxycoumarin O-deethylase among these species. However, guinea pigs showed the highest enzyme activity in cytochrome P(450), cytochrome b(5) and benzphetamine N-demethylase.     

Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes.

The dehydrogenation reaction of cholest-7-en-3beta-ol (I) to cholesta-5,7-dien-3beta-ol (II) in the presence of NADH was studied in rat liver microsomes and in microsomal acetone powder preparations, using [3alpha-3H]cholest-7-en-3beta-ol. It was found that the reaction was inhibited by menadione, adenosine diphosphate, potassium ferricyanide, and cytochrome c while p-cresol had no effect. These results indicated the participation of a microsomal electron transport system in the dehydrogenation of cholest-7-en-3beta-ol. The conversion of cholest-7-en-3beta-ol to cholesta-5,7-dien-3beta-ol was also observed in the absence of NADH when ascorbic acid was included in the incubation mixture. However, the ascorbic acid-catalyzed dehydrogenation was not inhibited by potassium ferricyanide. Immunological evidence that microsomal cytochrome b5 is involved in the dehydrogenation of (I) to (II) was obtained. Antibodies specific for rat liver microsomal cytochrome b5 were elicited in rabbits. The anticytochrome b5 immunoglobulin fraction inhibited rat liver microsomal NADH-cytochrome c reductase but not NADPH-cytochrome c reductase. Also, the extent of reduction of cytochrome b5 was not affected by the antibodies. The conversion of (I) to (II) by rat liver microsomes was inhibited (73%) by anticytochrome b5 immunoglobulin at a ratio of microsomal protein:immunoglobulin of 1:5.6. These results are consistent with the participation of microsomal cytochrome b5 in the introduction of the C-5 double bond in cholesterol biosynthesis. A close analogy of the microsomal dehydrogenation of fatty acids and of cholest-7-en-3beta-ol is apparent and this suggests a possible similarity in the mechanisms of the two reactions.     

Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants.

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.