Yeast Microsomes Research Articles

Expression of Rat Liver Cytochrome P-450MC cDNA inSaccharomyces cerevisiae

Rat liver cytochrome P-450MC cDNA was inserted between the ADH1 promoter and terminator regions of the yeast expression vector pAAH5. On introduction of the resulting recombinant plasmid pAMC1, Saccharomyces cerevisiae cells synthesized up to 8 X 10(5) molecules per cell of the cytochrome P-450MC protein, most of which was localized in yeast microsomes. Approximately half of the synthesized cytochrome contained heme in the enzyme molecule. These formed a functional electron-transport chain in the microsomes which exhibited aryl hydrocarbon hydroxylase activity toward benzo[a]pyrene.

Purification of intact and nicked forms of a zinc-containing, Mg2+-dependent, low Km cyclic AMP phosphodiesterase from bakers' yeast.

A low Km cyclic AMP phosphodiesterase was purified to homogeneity from microsomes of bakers' yeast. "Intact" enzyme, purified from microsomes prepared in the presence of the protease inhibitor phenylmethylsulfonyl fluoride, had a specific activity of 0.6 mumol/min/mg of protein (30 degrees C, pH 8.0, 1 microM cyclic AMP), a pI of 6.65 +/- 0.15, and a molecular weight of 61,000 determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Gel filtration of native enzyme suggested it is a monomer. When phenylmethylsulfonyl fluoride was omitted, a product ("nicked" enzyme) was obtained with a specific activity of 1.2 mumol/min/mg of protein, the same pI, and a similar amino acid composition; but gel electrophoresis now showed two bands, with molecular weights of 45,000 and about 17,000, together with a small amount of the 61,000 band. Apart from the higher specific activity of the nicked enzyme, no difference was found between the catalytic properties of the two enzyme forms. Between 40 nM and 1 microM cyclic AMP, an apparent Km of 170 nM was observed at pH 8.0, but at higher cyclic AMP concentrations (2-30 microM), Hofstee plots curved upwards. Cyclic deoxy-AMP was a substrate, but cyclic GMP was not and did not affect the activity towards cyclic AMP. Both enzyme forms contained tightly bound zinc. The metal chelators, 8-hydroxyquinoline and orthophenanthroline , caused progressive partial inactivation of the enzyme and a decrease in its affinity for cyclic AMP. Dialysis against Zn2+, Cu2+, Co2+, or Mn2+ (but not Mg2+ or Ni2+) reversed these changes.

Binding of azole fungicides related to diclobutrazol to cytochrome P‐450

AbstractFungicides containing the imidazole and triazole groups are known to block the 14α‐demethylation reaction in ergosterol biosynthesis, which is a cytochrome P‐450 enzyme system. Fungicides related to diclobutrazol [(2RS, 3RS)‐1‐(2,4‐dichlorophenyl)‐4,4‐dimethyl‐2‐(1,2,4‐triazol‐1‐yl)pentan‐3‐ol] bind to cytochrome P‐450 in rat liver microsomes, whole yeast cells, yeast microsomes and to a partially purified cytochrome P‐450 from yeast, with Type II spectral changes. The most fungicidally active isomer (2R, 3R) shows greater binding than the less active (2S, 3S)‐enantiomer to yeast microsomes; when the cytochrome P‐450 was purified, a preparation was obtained to which binding more closely matched the fungicidal activity. Binding to rat liver microsomes does not reflect the fungicidal activity of the compounds.

Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. II. Lanosterol metabolism by purified P-450(14)DM and by intact microsomes.

A reconstituted monooxygenase system containing a form of cytochrome P-450, termed P-450(14)DM, and NADPH-cytochrome P-450 reductase, both purified from yeast microsomes, catalyzed the conversion of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-01) to a sterol metabolite in the presence of NADPH and molecular oxygen. This conversion did not occur anaerobically or when either P-450(14)DM, the reductase, or NADPH was omitted from the system. In both free and trimethylsilylated forms, this metabolite showed a relative retention time (relative to lanosterol) of 1.10 in gas chromatography on OV-17 columns. Comparison of its mass spectrum and retention time with those of lanosterol and 4,4-dimethylzymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) indicated that the metabolite was 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol. Upon aerobic incubation of microsomes from semianaerobically grown yeast cells in the presence of NADPH and cyanide, endogenous lanosterol was converted to 4,4-dimethylzymosterol. This metabolism was inhibited by CO, metyrapone, SKF-525A, and antibodies to P-450(14)DM. It is concluded that in yeast microsomes lanosterol is 14 alpha-demethylated by a P-450(14)DM-containing monooxygenase system to give rise to 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol, which is then reduced to 4,4-dimethylzymosterol by an NADPH-linked reductase.

Import of proteins into mitochondria. In vitro studies on the biogenesis of the outer membrane.

The yeast mitochondrial outer membrane contains a major 29-kilodalton protein which is encoded in the nucleus. When this polypeptide is synthesized in vitro and then incubated with isolated yeast mitochondria, it binds to the organelles and becomes resistant to externally added trypsin. Post-translational insertion of the 29-kilodalton protein in vitro is not accompanied by NH2-terminal processing, appears to require neither ATP nor a transmembrane electrochemical potential, and is also observed with purified outer membranes, but not with yeast microsomes. In vitro insertion of the 29-kilodalton polypeptide is thus in several respects different from that of polypeptides destined for the internal compartments of the mitochondrion.

Purification and characterization of intact cytochrome b5 from yeast microsomes

A method for purification of detergent-solubilized cytochrome b 5 to gel electrophoretic homogeneity from yeast ( Saccharomyces cerevisiae) microsomes is described. The purified preparation shows the same absorption spectra as the trypsin-solubilized cytochrome (Y. Yoshida, H. Kumaoka, and R. Sato J. Biochem. 75, 1211–1219 (1974)) in the visible and Soret regions. The detergent-solubilized cytochrome is an amphipathic protein having a monomeric molecular weight of about 18,000 and exists as a hexa- or heptameric aggregate ( M r 122,000) in aqueous media. In the presence of low concentrations of Triton X-100, it interacts effectively with the intact form of NADH-cytochrome b 5 reductase purified either from yeast microsomes or from rabbit liver microsomes. Upon trypsin digestion, it is converted to a heme-containing, hydrophilic fragment ( M r 13,000) which retains the spectral characteristics of the original cytochrome, does not form aggregates, and interacts with the reductase only poorly. It is concluded that the preparation purified in this study represents the intact form of yeast cytochrome b 5 consisting of a hydrophilic, heme-containing moiety ( M r 13,000) and a hydrophobic, membrane-binding tail ( M r 5000).

Involvement of cytochrome b5 and a cyanide-sensitive monooxygenase in the 4-demethylation of 4,4-dimethyl-zymosterol by yeast microsomes

According to Ohba et al. (Ohba M., Sato R., Yoshida Y., Nishino T. and Katsuki H. (1978) Biochem. Biophys. Res. Commun. 85, 21–27), yeast microsomes catalyze the removal of three methyl groups attached to the C-4 and C-14 positions of [1,7,15,22,26,30- 14C]lanosterol (4,4,14α-trimethyl-5α-cholesta-8, 24-dien-3β-ol) in the presence of NADPH, NAD + and molecular oxygen, concomitant with the liberation of 14CO 2 derived from C-30 (one of the two methyl groups at the C-4 position). In this process the methyl group at the C-14 position is first removed in a cyanide-insensitive reaction and then the two methyl groups at the C-4 position are removed by a cyanide-sensitive enzyme system. In this study it was found that the 14CO 2 formation from the 14C-labeled lanosterol was inhibited by antibodies to yeast cytochrome b 5 and by palmitoyl-CoA, a substrate of the cytochrome b 5-containing fatty acyl-CoA desaturase system of yeast microsomes. However, neither the antibodies nor palmitoyl-CoA inhibited the conversion of lanosterol to 4,4-dimethyl zymosterol (4,4-dimethyl-5α-cholesta-8,24-dien-3β-ol). It is concluded that cytochrome b 5 and a cyanide-sensitive enzyme are involved in the 4-demethylation of 4,4-dimethylzymosterol, but not the 14α-demethylation of lanosterol, by yeast microsomes. It is suggested that a cyanide-sensitive enzyme acts as the terminal 4-demethylase and cytochrome b 5 transfers reducing equivalents from NADPH to the terminal enzyme, as in the case of fatty acyl-CoA desaturation. The cyanide sensitivity of the 4-demethylation was, however, much greater than that of the desaturation.

Effect of glucosylation of lipid intermediates on oligosaccharide transfer in solubilized microsomes from Saccharomyces cerevisiae.

Glc3Man9GlcNAc2-P-P-dolichol and Man9GlcNAc2-P-P-dolichol isolated from Saccharomyces cerevisiae are substrates for the N-glycosylation of endogenous proteins in Triton X-100-solubilized yeast microsomes. The solubilized oligosaccharide transferase requires Mn2+ for activity; neither Mg2+ nor Ca2+ is an effective substitute. The pH optimum of the transfer reaction is between 6.5 and 7.5. Unlike animal systems, which utilize glucosylated oligosaccharide-lipid to a much greater extent than the unglucosylated species as a donor in the transferase rection, yeast extracts transfer more than 70% of Glc3Man9GlcNAc2 and Man9GlcNAc2 from their respective oligosaccharide-lipids to proteins. However, the rate of N-glycosylation in vitro is approximately 25-fold faster with Glc3Man9GlcNAc2-P-P-dolichol than with Man9GlcNAc2-P-P-dolichol. The apparent Km value for the glucosylated species is 75 nM, while that for the unglucosylated glycolipid is 55 nM.

Mutant and immunochemical studies on the involvement of cytochrome b5 in fatty acid desaturation by yeast microsomes

The involvement of cytochrome b 5 in palmitoyl-CoA desaturation by yeast microsomes was studied by using yeast mutants requiring unsaturated fatty acids and an antibody to yeast cytochrome b 5. The mutants used were an unsaturated fatty acid auxotroph (strain E5) and a pleiotropic mutant (strain Ole 3) which requires either Tween 80 and ergosterol or δ-aminolevulinic acid for growth. Microsomes from the wild-type strain possessed both the desaturase activity and cytochrome b 5, whereas those from mutant E5 contained the cytochrome but lacked the desaturase activity. Microsomes from mutant Ole 3 grown with Tween 80 plus ergosterol were devoid of both the desaturase activity and cytochrome b 5, but those from δ-aminolevulinic acid-grown mutant Ole 3 contained cytochrome b 5 and catalyzed the desaturation. The cytochrome b 5 content in microsomes from mutant Ole 3 could be varied by changing the δ-aminolevulinic acid concentration in the growth medium, and the desaturase activity of the microsomes increased as their cytochrome b 5 content was increased. The antibody to yeast cytochrome b 5, but not the control γ-globulin fraction, inhibited the NADH-cytochrome c reductase and NADH-dependent desaturase activities of the wild-type microsomes. It is concluded that cytochrome b 5 is actually involved in the desaturase system of yeast microsomes.

The 14α—demethylation of lanosterol by a reconstituted cytochrome P-450 system from yeast microsomes

Incubation of lanosterol (4,4,14α-trimethyl-5α-cholesta-8,24-dien-3β-ol) with a reconstituted system consisting of cytochrome P-450 and NADPH-cytochrome P-450 reductase, both purified from yeast microsomes, in the presence of NADPH and molecular oxygen resulted in the formation of a sterol product. This product showed a relative retention time to lanosterol of 1.10 on gas-liquid chromatography with a 1% OV-17 column, and was identified as 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol by comparing its mass spectrum with that of lanosterol. It is concluded that the cytochrome P-450-containing monooxygenase system of yeast microsomes catalyzes the 14α-demethylation of lanosterol.

Involvement of cytochrome P-450 and a cyanide-sensitive enzyme in different steps of lanosterol demethylation by yeast microsomes

In the presence of NADPH, NAD+, and molecular oxygen, microsomes prepared from Saccharomycescerevisiae converted [1,7,15,22,26,30-14C]lanosterol to 4,4-dimethylzymosterol, 4-methylzymosterol, and zymosterol, and this conversion was accompanied by the liberation of 14CO2 derived from the methyl group (C-30) at the 4-position. The 14CO2 formation was inhibited by antibodies to yeast cytochrome P-450 and by cyanide. Gas chromatographic evidence indicated that the antibodies inhibited the conversion of lanosterol to 4,4-dimethylzymosterol, whereas the demethylation of the latter to 4-methylzymosterol was sensitive to cyanide. It is concluded that cytochrome P-450 and a cyanide-sensitive enzyme are involved in the 14α- and 4-demethylations of lanosterol, respectively, by yeast microsomes.

Squalene synthetase. Stoichiometry and kinetics of presqualene pyrophosphate and squalene synthesis by yeast microsomes

Squalene synthetase. Stoichiometry and kinetics of presqualene pyrophosphate and squalene synthesis by yeast microsomes

Squalene synthetase. Solubilization from yeast microsomes of a phospholipid-requiring enzyme

Squalene synthetase was solubilized from yeast microsomal membranes with deoxycholate. Solubilized enzyme was associated with one or more proteins with s20, w = 3.3 S, Stokes' radius = 40 A, and computed molecular weight = 54,500. In the presence of detergent the enzyme was catalytically inactive and unstable to heat. When detergent was removed with cholestyramine resin, both phases of squalene synthesis (farnesyl pyrophosphate leads to presqualene pyrophosphate leads to squalene) were recovered, and the enzyme was reaggregated to form sedimentable particles with a density of approximately 1.16 g/ml. Both activities were lost to variable extent upon chromatography over Sephadex G-200 in the presence of 0.2% deoxycholate, but could be recovered if phosphatidylcholine or phosphatidylethanolamine (but not phosphatidylserine or phosphatidylinositol) were added to fractions before removal of detergent. There was an apparently absolute requirement for phospholipid by the enzyme. The proteins catalyzing the two phases of squalene synthesis could not be resolved from one another and behaved in an identical fashion throughout a variety of manipulations.

Interaction of lanosterol to cytochrome P-450 purified from yeast microsomes: Evidence for contribution of cytochrome P-450 to lanosterol metabolism

Interaction between lanosterol and cytochrome P-450 purified from microsomes of anaerobically-grown Saccharomyces cerevisiae was studied. Lanosterol (4,4,14α-trimethyl-5α-cholesta-8,24-dien-3β-ol) stimulated the oxidation of NADPH by molecular oxygen in the presence of cytochrome P-450 and NADPH-cytochrome P-450 reductase both purified from S. cerevisiae microsomes. Lanosterol stimulated the reduction of cytochrome P-450 by NADPH with the cytochrome P-450 reductase, and induced Type I spectral change of cytochrome P-450. These observations suggest that lanosterol interacts to the substrate region of cytochrome P-450 of S. cerevisiae. Based on these facts, possible role of cytochrome P-450 in lanosterol metabolism in yeast cell is discussed.

Lipid Intermediates in the Synthesis of the Inner Core of Yeast Mannan

The synthesis by yeast microsomes of compounds that are probably dolichol-pyrophosphate derivatives containing N,N'-diacetylchitobiose and several mannose residues is described. However, the presence of monosaccharide residues other than N-acetylglucosamine and mannose has not been ruled out. The amount of the lipid derivatives synthesized was enhanced by the addition to the incubation mixture of an organic-solvent-soluble extract from rat liver known to contain dolicholpyrophosphate oligosaccharides. Incubation of these derivatives with yeast microsomes led to the transfer of about fifteen percent of their saccharide moieties to endogenous proteins. The oligosaccharides released from the dolichol derivatives by mild acid hydrolysis could serve as primers for the synthesis of a polysaccharide having the characteristics of mannan outer-chain. It is suggested that the dolichol-pyrophosphate derivatives described in this paper are intermediates in the synthesis of mannan inner core.

NADPH-cytochrome P-450 reductase of yeast microsomes

NADPH-cytochrome c reductase of yeast microsomes was purified to apparent homogeneity by solubilization with sodium cholate, ammonium sulfate fractionation, and chromatography with hydroxylapatite and diethylaminoethyl cellulose. The purified preparation exhibited an apparent molecular weight of 83,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reductase contained one molecule each of flavin-adenine dinucleotide and riboflavin 5′-phosphate, though these were dissociative from the apoenzyme. The purified reductase showed a specific activity of 120 to 140 μmol/min/mg of protein for cytochrome c as the electron acceptor. The reductase could reduce yeast cytochrome P-450, though with a relatively slow rate. The reductase also reacted with rabbit liver cytochrome P-450 and supported the cytochrome P-450-dependent benzphetamine N-demethylation. It can, therefore, be concluded that the NADPH-cytochrome c reductase is assigned for the cytochrome P-450 reductase of yeast. The enzyme could also reduce the detergent-solubilized cytochrome b 5 of yeast. So, this reductase must contribute to the electron transfer from NADPH to cytochrome b 5 that observed in the yeast microsomes.

A highly purified preparation of cytochrome P-450 from microsomes of anaerobically grown yeast

Cytochrome P-450 was purified from microsomes of anaerobically grown yeast to a specific content of 12–15 nmoles per mg of protein with a yield of 10–30%. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis, the purified preparation yielded a major protein band having a molecular weight of about 51,000 together with a few faint bands. It was free from cytochrome b 5, NADH-cytochrome b 5 reductase, and NADPH-cytochrome c (P-450) reductase. In the oxidized state it exhibited a low-spin type absorption spectrum, and its reduced CO complex showed a Soret peak at 447–448 nm. It was reducible by NADPH in the presence of an NADPH-cytochrome c reductase preparation purified from yeast microsomes. Its conversion to the cytochrome P-420 form was much slower than that of hepatic 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].

Fatty acid desaturase system of yeast microsomes: Involvement of cytochrome b5-containing electron-transport chain

The oxidative desaturation of palmitoyl CoA by microsomes from anaerobically grown Saccharomyces cerevisiae has been studied by using NADH as electron donor. The desaturation product was identified as palmitoleic acid by periodate oxidation. The desaturase activity was sensitive to relatively high concentrations of cyanide; the concentration of cyanide causing half-maximal inhibition was determined to be 7.1 m m. The rate of reoxidation of cytochrome b 5 in NADH-reduced microsomes was stimulated by the addition of palmitoyl CoA, and the amount of cytochrome b 5 reoxidized by the palmitoyl CoA added could be closely correlated to the amount of palmitoleate formed. No stimulation of the reoxidation of cytochrome b 5 was induced by palmitoyl CoA in microsomes prepared from the desaturase-repressed cells and from a desaturase-deficient mutant, strain KD-20. It is concluded that the fatty acyl CoA desaturase system of yeast microsomes involves cytochrome b 5 as an electron carrier and that the terminal desaturase is sensitive to relatively high concentrations of cyanide.

Studies on the microsomal electron-transport system of anaerobically grown yeast. III. Spectral characterization of cytochrome P-450.

A carbon monoxide-binding pigment which shows an absorption peak at about 450 nm in the reduced carbon monoxide difference spectrum was purified from the microsomal fraction of yeast grown anaerobically. The spectral characteristics of the pigment were practically identical with those of cytochrome P-450 of hepatic microsomes, especially from polycyclic hydrocarbon-induced animals. The pigment was denatured to P-420, and bound with ethyl isocyanide in the reduced state. Although Type I spectral change was not evident, the pigment showed Type II and modified Type II spectral changes upon binding with some organic compounds, as in the case of hepatic cytochrome P-450. These observations clearly indicate that the carbon monoxide-binding pigment of yeast microsomes may be designated as cytochrome P-450 of yeast.