Collagenolytic activity of rabbit V2-carcinoma growing at multiple sites.

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.     

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 b₅, NADH-cytochrome b₅ 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 cytochrome P-450 (P-45017α,lyase)from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome b5

We have reported (Kominami S., Shinzawa K. and Takemori S. (1982) Biochem. Biophys. Res. Commun. 109, 916–921) that a cytochrome P-450 purified from guinea pig adrenal microsomes shows 17α-hydroxylase and C-17,20-lyase activities in a reconstituted system with NADPH-cytochrome P-450 reductase. The homogeneity of the purified cytochrome P-450 was examined with the following methods: isoelectric focusing, immunoelectrophoresis and affinity chromatography on cytochrome b₅-immobilized Sepharose. It was found that progesterone competitively inhibited C-17,20-lyase reaction and that progesterone was converted into androstenedione by 17α-hydroxylation followed by the lyase reaction. These results indicate that the dual activities are carried out by a single enzyme (P-450_17α,lyase). P-450_17α,lyase had the maximum activity at pH 6.1 both for 17α-hydroxylation (6.0 nmol/min per nmol of P-450) and the lyase reaction (11.0 nmol/min per nmol of P-450). Upon addition of cytochrome b₅ to the reconstituted system, the optimal pH for 17α-hydroxylation was shifted to 7.0 and that of the lyase reaction to 6.6. The maximum activities at these optimal pH values were almost the same in the presence or absence of cytochrome b₅. With the addition of cytochrome b₅, both the activities were stimulated above pH 6.3–6.5 and were suppressed below pH 6.3–6.5. These results indicate that cytochrome b₅ plays some important role in controlling the dual activities of P-450_17α,lyase.     

Woodward's reagent K reacts with histidine and cysteine residues inEscherichia coli andSaccharomyces cerevisiae phosphoenolpyruvate carboxykinases

The reaction of Woordward's reagent K (WRK) with model amino acids and proteins has been analyzed. Our results indicate that WRK forms 340-nm-absorbing adducts with sulfhydryl- and imidazol-containing compounds, but not with carboxylic acid derivatives, in agreement with Llamaset al. [(1986),J. Am. Chem. Soc. 108, 5543–5548], but not with Sinha and Brewer [(1985),Anal. Biochem. 151, 327–333]. The chemical modification ofEscherichia coli andSaccharomyces cerevisiae phosphoenolpyruvate carboxykinases with WRK leads to an increase in the absorption at 340 nm, and we have demonstrated its reaction with His and Cys residues in these proteins. These results caution against claims of glutamic or aspartic acid modification by WRK based on the absorption at 340 nm of protein-WRK adducts.     

Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes

Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b₅, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens.     

Effect of phase transitions in hydrated 1,2-dipalmitoylphosphatidylethanolamine bilayers on the spin probe order parameter

The ‘main’ phase transition L_β→L_α of hydrated 1,2-dipalmitoylphosphatidylethanolamine (DPPE) bilayers in excess water affects the ESR order parameter S₃₃ of N-cetyl-N,N-dimethyl-N-tempoylammonium bromide (CAT-16), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) spin probes. The ‘pretransition’ and ‘subtransition’ suggested to occur in hydrated DPPE by Chowdhry et al. [(1984) Biophys. J. 45, 901–904] and Silvius et al. [(1986) Biochemistry 25, 4249–4258], respectively, affect exclusively the S₃₃ of CAT-16, but not that of 5-DSA and 16-DSA spin probes. The subtransition occurs about 15 ± 1°C below the main transition.     

The induction of cytochrome P-448 dependent benzo(a)pyrene hydroxylase in Saccharomycescerevisiae

When grown in high concentrations of glucose, the yeast $\text{Saccharomyces}\text{cerevisiae}$ produces a microsomal cytochrome P-450 monooxygenase system which is capable of hydroxylating benzo(a)pyrene. The addition of benzo(a)pyrene to the yeast during growth causes only a small increase in cytochrome P-448 levels but results in a dramatic improvement in the apparent kinetics of benzo(a)pyrene hydroxylation as measured by a decrease in the Michaelis constant and an increase in maximal velocity. Dimethylnitrosamine, phenobarbital and 3-methylcholanthrene also induce this enzyme to various degrees. Yeast pretreatment with β-naphthoflavone did not affect this enzyme, yet pretreatment with lanosterol resulted in a decreased affinity for benzo(a)pyrene. The addition of benzo(a)pyrene to yeast growing at low glucose concentration does not induce cytochrome P-448. The implications of these findings with regard to the presence of multiple forms of cytochromes $\text{P-448}\text{P-450}$ in yeast are briefly discussed.     

Specificity of antisera directed against rat liver cytochrome P-448

A partially purified preparation of hepatic cytochrome P-448 from 3-methylcholanthrene treated rats was used to produce antisera in rabbits. Using both Ouchterlony double diffusion and quantitative immunoprecipitation analysis, this antisera was found to be more specific for cytochrome P-448 than for cytochrome P-450 from phenobarbital induced rats. The antisera did not form precipitin bands with the following rat liver microsomal proteins: cytochrome b₅, NADH-cytochrome b₅ reductase, NADPH-cytochrome c reductase or epoxide hydrase.     

Benzpyrene hydroxylase activity in hepatic microsomal and solubilized systems containing rabbit or rat cytochrome P-448 or P-450

Treatment of adult, male rabbits and rats with 3-methylcholanthrene results in the formation of hepatic microsomal cytochrome P-448. In the rat, this occurs coincidently with an increase in hepatic microsomal benzpyrene hydroxylase activity. In the rabbit, benzpyrene hydroxylase activity is decreased following treatment with 3-methylcholanthrene. Benzpyrene hydroxylase activity in solubilized, reconstituted mixed-function oxidase systems containing rat cytochrome P-448 is about seven times higher than in systems containing rabbit cytochrome P-448. Evidence obtained by spectral analysis suggests that rabbit P-448 is combined with a type I compound. Residual ¹⁴C-3-methylcholanthrene does not appear to be responsible for the differences observed between rat and rabbit cytochrome P-448.     

A -glucuronidase deficiency mucopolysaccharidosis: studies in cultured fibroblasts

Cytochrome P-450 cannot be detected spectrophotometrically in testis mitochondria of untreated rats because of the high cytochrome a₃ to Cytochrome P-450 ratio. Injection of human chorionic gonadotrophin (HCG) causes a large increase in mitochondrial cytochrome P-450. After 14 days injection, mitochondrial cytochrome P-450 levels are increased 15- to 30-fold (from 0.007 to 0.134 nmoles/mg protein) over control levels. Levels of cytochrome a + a₃ are not altered by this treatment. Mitochondrial cytochrome P-450 can also be demonstrated by injection of HCG into rats which were hypophysectomized 24 days previously. During hypophysectorny the mitochondrial cytochromes c + c_i, a + a₃ and mitochondrial protein decay with halflives of 14, 16, and 15.5 days, respectively. HCG treatment for 8 days increases mitochondrial cytochrome P-450 (from < 0.003 to 0.24 nmoles/mg protein) without altering the levels of the other mitochondrial cytochromes. The control of cytochrome P-450 levels in the mitochondria by HCG suggests that the level of this key component of cholesterol side-chain cleavage enzyme may be of importance in the regulation of steroidogenesis in the testis.     

Site-Directed Mutagenesis in Basic Amino Acid Residues of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V _max/K _mADP. These results indicate that Arg76 is not involved in CO₂ binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413–418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207–210], does not perform an essential function for the enzyme activity.     

Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.     

Purification and characterization of the cytochrome P-448 component of a benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae

Cytochrome P-448, a type of cytochrome P-450, from brewer's yeast (Saccharomyces cerevisiae) grown under conditions of glucose repression was isolated and purified. Triton X-100 in very low concentration proved to be very effective in stabilizing P-448 in the microsomal fraction and later prevented its conversion to cytochrome P-420 through solubilization with various ionic and nonionic detergents. Highest yields were obtained with 1% sodium cholate, in the presence of 0.1% Triton X-100 and reduced glutathione. A novel combination of hydrophobic adsorption and other chromatographic techniques was used for the purification of cytochrome P-448. These involve the use of amino octyl-Sepharose 4B, instead of the low-yielding aminohexyl derivative, followed by the fast-running hydroxyapatite-cellulose column. Finally, the use of DEAE-Sephacel was found to increase greatly the purity of the cytochrome P-448 obtained. The molecular weight of this preparation was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (M_r, 55,500). Using the known molar extinction coefficient of the carbon monoxide-difference spectrum the estimate of degree of purity of cytochrome P-448 obtained by this purification procedure was between 88 and 97%. Electrophoresis also showed that this preparation was completely homogeneous and assays showed that it was also completely free of cytochrome b_s, cytochrome c reductase and cytochrome P-420. Purified cytochrome P-448 reconstituted with cytochrome P-450 (cytochrome c) reductase, isolated from yeast, showed 10-fold higher aryl hydrocarbon hydroxylase activity with benzo[a]pyrene as a substrate than the corresponding microsomal fraction enzyme. Kinetics of benzo[a]pyrene hydroxylation were determined: K_m (33 μm) was comparable with that reported for purified hepatic cytochrome P-448. The number of binding sites of microsomal and purified cytochromes P-450 (from liver of phenobarbital-induced rats) and yeast cytochrome P-448 with benzo[a]pyrene has been determined using and equilibrium gel filtration method. There is one binding site in each case (contrast with six sites for microsomal enzymes). The Scatchard plot gives number of binding sites, apparent association constants (K), and the equivalent dissociation constants (K_s). Comparison is made with spectral dissociation constants for these enzymes and benzo[a]pyrene. Thus the proportion bound, dissociation constant (K_s), and stoichiometry of rat liver (phenobarbital induced) and yeast cytochrome P-448 with benzo[a]pyrene were compared with corresponding values for microsomal fractions of both systems. Purified enzymes had higher K_s values in both cases, and the proportion of enzyme that bound benzo[a]pyrene was high (53%) for liver and this value is 100% for purified enzyme from yeast, which is the same as the value obtained for the microsomal enzyme from yeast.     

Reconstitution of the reduced nicotinamide adenine dinucleotide phosphate- and reduced nicotinamide adenine dinucleotide-peroxidase activities from solubilized components of rat liver microsomes.

The liver microsomal enzyme system that catalyzes the oxidation of NADPH by organic hydroperoxides has been solubilized and resolved by the use of detergents into fractions containing NADPH-cytochrome c reductase, cytochrome P-450 (or P-448), and microsomal lipid. Partially purified cytochromes P-450 and P-448, free of the reductase and of cytochrome b₅, were prepared from liver microsomes of rats pretreated with phenobarbital (PB) and 3-methylcholanthrene (3-MC), respectively, and reconstituted separately with the reductase and lipid fractions prepared from PB-treated animals to yield enzymically active preparations functional in cumene hydroperoxide-dependent NADPH oxidation. The reductase, cytochrome P-450 (or P-448), and lipid fractions were all required for maximal catalytic activity. Detergent-purified cytochrome b₅ when added to the complete system did not enhance the reaction rate. However, the partially purified cytochrome P-450 (or P-448) preparation was by itself capable of supporting the NADPH-peroxidase reaction but at a lower rate (25% of the maximal velocity) than the complete system. Other heme compounds such as hematin, methemoglobin, metmyoglobin, and ferricytochrome c could also act as comparable catalysts for the peroxidation of NADPH by cumene hydroperoxide and in these reactions, NADH was able to substitute for NADPH. The microsomal NADH-dependent peroxidase activity was also reconstituted from solubilized components of liver microsomes and was found to require NADH-cytochrome b₅ reductase, cytochrome P-450 (or P-448), lipid, and cytochrome b₅ for maximal catalytic activity. These results lend support to our earlier hypothesis that two distinct electron transport pathways operate in NADPH- and NADH-dependent hydroperoxide decomposition in liver microsomes.     

Characterization of the heme of cytochrome P-450 using gas chromatography-mass spectrometry

The heme moieties of cytochromes P-450 and P₁-450 (P-448) have been characterized. CO-binding particles, devoid of cytochrome b₅, were prepared from normal or 3-methylcholanthrene-treated animals. Heme was removed by acid-acetone treatment of the CO-binding particles and crystallized. Heme isolated from hemoglobin of the corresponding animals served as a control. Reductive degradation (hydriodic acid) followed by gas chromatography/mass spectrometry analysis indicated the presence of opso-, crypto-, hemo-, and phyllopyrrole. Visible spectra of the iron-free tetrapyrroles isolated from microsomal heme and hemoglobin were identical and showed typical aetioporphyrin spectra. Finally, the mass spectra of the tetrapyrrole dimethyl esters isolated from microsomal heme and hemoglobin were identical to authentic protoporphyrin IX dimethyl ester. These data strongly suggest that the heme of cytochrome P-450 and P₁-450 are identical and are the same the same as that of hemoglobin, namely protoporphyrin IX.     

Stereoselective interaction of an azole antifungal agent with its target, lanosterol 14 alpha-demethylase (cytochrome P-45014DM): a model study with stereoisomers of triadimenol and purified cytochrome P-45014DM from yeast

The effect of the four triadimenol stereoisomers on the purified yeast lanosterol 14 alpha-demethylase (cytochrome P-45014DM), the primary target of azole antifungal agents, was studied. (1S,2R)-Triadimenol was the most potent demethylase inhibitor and bound quantitatively to the enzyme below 0.05 microM. This isomer also interfered with the chemical reduction of cytochrome P-45014DM and the binding of CO to the cytochrome. The other isomers showed a lower inhibitory effect on the enzyme, and the order of activity was (1R,2R) greater than (1R,2S) greater than or equal to (1S,2S). Based on these findings and the reported preferred conformations for the triadimenol stereoisomers (Anderson, N.H. et al., Pestic. Sci. 15:310-316, 1984), it is predicted that orientation of the hydrophobic tert-butyl and p-chlorophenyl groups relative to the azole nitrogen is important to fit the antifungal agent in the active site of the demethylase.     

Cytoplasm calcium-binding proteins of germ cells and embryos of the sea urchin

Synchronous, demonstrative, easily reproducible fertilization with the following embryonic development makes the process in the sea urchin extremely attractive for studying many biological enigmas. In particular, germ and embryonic cells of the sea urchin present a wide opportunity for investigating different associated phenomena launched by an increase in concentration of Ca²⁺ in cells ([Ca²⁺]_i).Ca²⁺ ions participate in the activation of diverse processes of respiration and sperm motility (Shapiro et al., 1990; Brokaw, 1991), chemotaxis of spermatozoa to components of the egg jelly (Ward et al., 1985), acrosomal reaction (Trimmer et al., 1986; Shapiro et al., 1990), cortical reaction, formation of the fertilization membrane (Sasaki, 1984; Sardet and Chang, 1987), cellular division in the embryo (Poenie et al., 1985; Silver, 1986; Whitaker and Patel, 1990), their adhesion (McClay and Matranga, 1986), differentiation and formation of spicules (Mitsunaga et al., 1988) and metamorphosis (Carpenter et al., 1984).The present review combines information on the function of calcium-binding proteins and their targets, calmodulin regulation of NAD-kinase, exocytosis of cortical granules, Ca²⁺- and calmodulin-dependent protein phosphatase, Ca²⁺-dependent protein phosphorylation, regulation of ion-exchanger in the germ and embryonic cells as well as Ca²⁺- and calmodulin control of sperm motility in sea urchins.