Properties of liposomal membranes containing lysolecithin

The diffusion of glucose from phospholipid membranes has been measured in the presence of serum albumin or methylated serum albumin. At neutral pH, serum albumin enhanced the rate at which glucose diffused from liposomes containing more than a certain amount of lysolecithin. Net charge of the membrane is not important for the reaction, since positively charged membranes containing stearylamine showed almost the same reactivity as negatively charged liposomes containing dicetyl phosphate. Carboxylmethylated albumin showed enhancement of the diffusion rate of glucose from negatively but not from positively charged liposomes. The amount of methylated albumin required to affect liposomes was much smaller than the amount of albumin required to damage liposomes containing lysolecithin. Cholesterol incorporation suppressed the sensitivity of liposomes to both proteins, albumin and methylated albumin. The effect of temperature and fatty acid composition of phospholipids on the sensitivity of liposomes to proteins suggests the importance of the fluidity of the membrane, especially in the case of methylated albumin.     

Purification and characterization of constituent testosterone 2 alpha-hydroxylase (cytochrome P450(2)alpha) from mouse liver

Hepatic microsomal testosterone/androstenedione 2 alpha-hydroxylase (i.e., cytochrome P450(2)alpha) was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-sephacel, and hydroxylapatite columns at heme absorbing 417 nm and by cytochrome P450 content, reactivity to a monoclonal antibody against female-specific rat cytochrome P450 2C12, and testosterone 2 alpha-hydroxylase activity. The catalytic activity of the purified cytochrome P450(2)alpha, exhibiting a high degree of regioselectivity and stereospecificity, was basically restricted to the 2 alpha-hydroxylation of testosterone and androstenedione; representing > 96% and > 92% of these respective metabolites. Polyclonal antibodies against cytochrome P450(2)alpha exhibited a dose-dependent and very selective inhibition of testosterone 2 alpha-hydroxylation. The specific cytochrome P450 content of the purified cytochrome P450(2)alpha fraction was 12.06 nmol/mg protein. The specific testosterone 2 alpha-hydroxylase activity of the purified protein was 14 nmol/min/nmol cytochrome P450, which was about 60-fold higher than the respective microsomes. The apparent subunit molecular weight of cytochrome P450(2)alpha was 51,000 and the protein appeared as a single band on sodium dodecyl sulfate polyacrylamide gels. The amino-terminal sequence analysis indicates that cytochrome P450(2)alpha is a member of the murine cytochrome P450 2d family.     

Purification and comparative characterization of cytochrome P-450scc (CYP XIA1) from sheep adrenocortical mitochondria

Cytochrome P-450scc (CYP XI A1) was purified from sheep adrenocortical mitochondria. The purified cytochrome was found to be homogeneous on SDS-polyacrylamide gel electrophoresis and to have a heme content of 20.8 nmol/mg of protein. Its amino acid composition and NH2-terminal amino acid sequence were determined, and compared with those of other known mammalian and fish cytochromes P-450scc. EPR spectra of the cytochrome P-450scc were measured for oxidized and NO-reduced forms in the presence or absence of cholesterol and/or adreno-ferredoxin. Spectral properties of these various forms were very similar to those of the bovine enzyme. Circular dichroism spectra of the purified sheep cytochrome P-450scc in the oxidized and dithionite-reduced forms, and of their complexed forms with cholesterol or adreno-ferredoxin were analyzed in the region from 200 to 700 nm. The difference CD spectrum of the oxidized cytochrome P-450scc complexed with adreno-ferredoxin minus the oxidized form suggests an increase in the high-spin form upon the addition of adreno-ferredoxin. This may suggest a direct influence of the adreno-ferredoxin binding to the heme moiety of the oxidized cytochrome P-450scc.     

Amount and type of dietary lipid modulate rat hepatic cytochrome P-450 activity

Cardiovascular responses to sustained and rhythmic (5 s on, 2 s off) forearm isometric exercise to fatigue at 40% maximal voluntary contraction (MVC) and to a period of arterial occlusion were investigated in elite rock climbers (CLIMB) as a trained population compared to non-climbing sedentary subjects (SED). Blood pressure (BP), monitored continuously by Finapres, and forearm blood flow, by venous occlusion plethysmography, were measured and used to calculate vascular conductance. During sustained exercise, times to fatigue were not different between CLIMB and SED. However, peak increases in systolic (S) BP were significantly lower in CLIMB [25 (13) mmHg; (3.3 (1.7) kPa] than in SED [48 (17) mmHg; (6.4 (2.3) kPa] (P < 0.05), with a similar trend for increases in diastolic (D) BP. Immediately after sustained exercise, forearm conductance was higher in CLIMB than SED (P < 0.05) for up to 2 min. During rhythmic exercise, times to fatigue were two fold longer in CLIMB than SED [853 (76) vs 420 (69) s, P < 0.05]. Increases in SBP were not different between groups except during the last quarter of exercise when they fell in CLIMB. Conductance both during and after rhythmic exercise was higher in CLIMB than in SED. Following a 10-min arterial occlusion, peak vascular conductance was significantly greater in CLIMB than SED [0.597 (0.084) vs 0.431 (0.035) ml x min(-1) x 100 ml(-1) x mmHg(-1); P < 0.05]. The attenuated BP response to sustained isometric exercise could be due in part to enhanced forearm vasodilatory capacity, which also supports greater endurance during rhythmic exercise by permitting greater functional hyperaemia in between contraction phases. Such adaptations would all facilitate the ability of rock climbers to perform their task of making repetitive sustained contractions.     

Angiotensin-II stimulates an increase in cAMP and expression of 17 alpha-hydroxylase cytochrome P450 in fetal bovine adrenocortical cells

While known to be a potent activator of phosphoinositidase C, angiotensin II (A-II) also causes a small but significant increase in cAMP production through the type 1 A-II (AT1) receptor in bovine adrenocortical cells (Mol Cell Endocrinol 81:33-41, 1991). We have carried out studies on primary cultures of fetal bovine adrenocortical cells to examine the effects of A-II on the expression of cytochrome P450 17 alpha-hydroxylase (P450c17), which is known to be regulated in a cAMP-dependent fashion. Prolonged treatment (48 h) of cells with A-II (10(-7) M) did not give rise to a detectable increase in P450c17 as measured by immunoblotting, although both A-II and the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA) attenuated the large increase in P450c17 induced by ACTH (10(-8) M). A-II alone (10(-7) M) however, caused a time-dependent increase in cAMP secretion, reaching 8-fold within 3 h. Prolonged treatment of cells with A-II also resulted in a 3-fold increase in P450c17 mRNA within 12 h (10(-7) M), and a dose-dependent increase in 17 alpha-hydroxylase activity within 48 h (16.4-fold max at 10(-7) M). The stimulatory actions of A-II alone (10(-7) M) on cAMP levels, P450c17 mRNA, and 17 alpha-hydroxylase activity were much smaller than in response to ACTH (10(-8) M), but were largely reproduced by TPA (10(-7) M), suggesting a role for protein kinase C in mediating these responses to A-II. These findings indirectly support the hypothesis that A-II alone can stimulate an increase in cAMP in adrenocortical cells. Such a stimulation of cAMP may then result in increased expression of steroidogenic enzymes, as we have shown is the case for P450c17 expression. However, A-II in the presence of ACTH appears to attenuate the ACTH-stimulated expression of P450c17.     

Purification and characterization of constituent androstenedione 15 alpha-hydroxylase (cytochrome P450(15 alpha AD)) from mouse liver. Sex- and tissue-dependent expression

Hepatic microsomal androstenedione 15 alpha-hydroxylase (i.e.cytochrome P450(15)alpha AD was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-Sephacel, and hydroxylapatite columns at heme absorbing 417 nm, by cytochrome P450 content, reactivity to monoclonal antibody against female-specific rat cytochrome P450 2C12, and androstenedione 15 alpha-hydroxylase activity. The catalytic activity for androgens of the purified cytochrome P450(15)alpha AD, exhibiting a high degree of regioselectivity and stereospecificity, was restricted to the 7 alpha- and 15 alpha-hydroxylation of androstenedione, representing, respectively, > 5% and > 93% of the total metabolites. Polyclonal antibodies against cytochrome P450(15)alpha AD exhibited a concentration-dependent and very selective inhibition of hepatic microsomal androstenedione 7 alpha- and 15 alpha-hydroxylation and a 60% inhibition of benzphetamine demethylation, the latter drug appearing to be a much more effective substrate than androgens. Cytochrome P450(15)alpha AD accounted for about 3% of the total P450 in female mouse liver microsomes. The apparent subunit molecular weight of P450(15)alpha AD was 53,000, and the protein appeared as a single band or sodium dodecyl sulfate-polyacrylamide gels. The isoform was intensely expressed in both liver and lung of CD-1 female mice and was female-predominant in the livers of five or eight strains examined; it was sex-independent in the remaining three strains. Amino-terminal sequence analysis indicates that cytochrome P450(15)alpha AD is a member of the murine cytochrome P450 2c subfamily.     

Modulation of ovarian cytochrome P450 17 alpha-hydroxylase and cytochrome aromatase messenger ribonucleic acid by prolactin in the domestic turkey

The effect of exogenous ovine prolactin (oPRL) on preovulatory follicle P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA abundance was investigated in turkeys. Ovine PRL (124 IU/hen per day) was injected i.m. into four sets (n = 8) of laying turkeys for 2, 4, 8, or 14 days. Vehicle was injected into control hens for 8 days (n = 8). Blood samples were collected and serum was assayed for LH, progesterone (P), testosterone (T), and estradiol (E). Theca layers from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest preovulatory follicles and from small white follicles (SWF) were examined for C17 and ARO mRNA contents. The number of atretic follicles increased from 0 (vehicle-injected controls) to 9 (14-day-oPRL-injected hens). Serum E, T, and LH levels decreased, while P levels remained unchanged. There was a transient increase in theca C17 mRNA abundance of 2- and 4-day-oPRL-treated hen follicles. Cytochrome P450 ARO mRNA levels were reduced in SWF and F7 in response to oPRL. Thecal C17 and ARO mRNA content was reduced during follicular maturation in laying hens. ARO mRNA was not detectable in granulosa cells. The progressive decline in C17 and ARO mRNA content associated with follicular maturation as well as the absence of ARO mRNA in granulosa cells is consistent with the secretory activity of P, T, and E in preovulatory follicles. These findings suggest that reduced circulating E may be a consequence of suppressed ARO gene expression whereas the oPRL suppression of T secretion may not be coupled to C17 gene expression.     

Preparation and properties of partially purified pulmonary cytochrome P-450 from rabbits

Cytochrome P-450 from rabbit pulmonary microsomes was purified approximately 32-fold. The purification method involved solubilization of microsomes using sodium cholate, and recovery of cytochrome P-450 in the precipitate formed between 25 to 42% saturation of the digested microsomes with ammonium sulfate in the absence of glycerol. Further purification was achieved by chromatography on DEAE-cellulose and hydroxylapatite using Emulgen 913 as an eluent. Partially purified preparations containing up to 7.4 nmol of cytochrome P-450 per mg of protein were essentially free of NADPH-cytochrome c reductase activity and cytochromes b5 and P-420. However, epoxide hydrase was found to co-purify with cytochrome P-450. The CO-difference spectrum of dithionite-reduced purified cytochrome showed the expected peak at 450 nm. However, the magnitude of the peak was dependent on added microsomal lipid fraction in the assay medium. Purified pulmonary cytochrome P-450 formed typical types I and II substrate difference spectra with benzphetamine and pyridine, respectively. Sodium dodecyl sulfate-gel electrophoresis of partially purified cytochrome P-450 gave two major bands when stained with Coomassie blue. The faster moving band which contained peroxidase activity had an estimated molecular weight of 49,000 +/- 1,200. The cytochrome P-450 fraction, when combined with solubilized pulmonary microsomal NADPH-cytochrome c reductase and lipid fractions, was active in the O-deethylation of 7-ethoxycoumarin and the N-demethylation of benzphetamine.     

Some 1-[(benzofuran-2-yl)methyl]imidazoles as inhibitors of 17 alpha-hydroxylase: 17, 20-lyase (P450 17) and their specificity patterns

Four 1-[(benzofuran-2-yl)methyl]imidazoles (1-4) have been evaluated as in-vitro inhibitors of human testicular and bovine adrenal microsomal 17 alpha-hydroxylase: 17,20-lyase (P450 17) as potential anti-prostatic agents. Their specificity towards other steroidogenic and liver enzymes has been compared with that of ketoconazole. All four compounds were inhibitors of the testicular enzyme (2, IC50 (concentration resulting in 50% inhibition) 0.185 microM; 4, IC50 0.18 microM) but less potent than ketoconazole (IC50 0.03 microM). Towards bovine adrenal enzyme 2 and 4 were 35- and 31-fold more potent than ketoconazole (IC50 = 39.8 microM). Compound 2 is a useful lead compound but although less potent than ketoconazole towards P450SCC and P450 11 beta, but not P450C21, at the enhanced dose required for equivalent effects in-vivo on P450 17 it is likely that cortisol and aldosterone production will be affected to a greater extent than with ketoconazole.     

Purification and properties of a plasminogen activator from pig heart

An improved procedure is described for the purification of plasminogen activator from pig heart. The initial purification steps were similar to these described previously (Bachmann, F., Fletcher, A. P., Alkjaersig, N., and Sherry, S. (1964) Biochemistry 3, 1578--1585). Use of a novel extraction medium containing EDTA, cysteine, and 2,3-dimercaptopropanol-1 facilitated the removal of large amounts of inert proteins prior to gel filtration on Bio-Gel P-150. The final product had a specific activity of 120,000 to 160,000 CTA units/mg of protein (CTA, Committee on Thrombolytic Agents of the National Heart Institute). Total purification over pig heart was 25,000 to 30,000-fold, average recovery compared to the initial extract was 6 to 8%. Polyacrylamide gel electrophoresis revealed a major and two minor components. The molecular weight of the activator determined by gel filtration was 51,500 +/- 3,400 for the major activity component and 48,000 for a minor component which was partially separated from the major peak in eight of nine chromatography runs. A gamma-globulin fraction of antiserum against purified activator neutralized the biological activity of the activator on fibrin plates. Immunoelectrophoresis of gel-filtered activator revealed only one anodic component.     

Antigenic mapping of bacterial and animal cytochromes P-450

A peptide scanning (PEPSCAN) approach was used for antigenic mapping of two hepatic microsomal cytochromes P450 (rab1A2 and rab2B4) and the microbial cytochrome from Pseudomonas putida (P450 101 or P450cam). This approach includes simultaneous synthesis of pin-linked overlapping hexapeptides covering the whole sequences of three P450s and testing them by ELISA with corresponding polyclonal antisera. Microsomal cytochrome P450 maps were shown to vary depending on an antiserum used for testing the peptides, however, the most active linear B-epitopes were revealed with antisera from two animal species used. P450 linear B-epitopes were classified into individual and group-specific epitopes. While almost all P450 101 linear antigenic determinants are unique for this protein, rab1A2 and rab2B4 contain epitopes both individual for each protein, and subfamily- or even family-specific epitopes. These results point out the possibility of producing both monospecific and group-specific antipeptide antibodies against different P450s. The antigenic map of P450 101 was superimposed on the structural-functional map of this protein. Its linear B-epitopes were shown to coincide with boundaries of secondary structure elements, with surface-located, water accessible regions and with sites responsible for intermolecular interactions in the Pseudomonas putida monooxygenase system. Several known or predicted functionally active sites in microsomal cytochrome P450 rab1A2 and rab2B4 were also shown to coincide with linear B-epitopes. The peculiarities of epitope locations in the protein tertiary structure will allow to predict antigenic regions starting from protein structural information and vice versa, to structural protein models in accordance with antigenic mapping results. Antigenic regions which coincide with sites responsible for intermolecular interactions in monooxygenase systems may be synthesized as separate peptides and used as blockers of such interactions.     

Change in activity of microsomal cytochrome P-450-dependent monooxygenases from rat liver exposed to chronic low doses of radiation

The activity changes of cytochrome P-450-dependent monooxygenases of hepatic microsomal fraction were studied in, experimental animals after prolonged application (1 month) of low radiation doses (0.258 mkl/kg). The obtained results show the increase in catalytic activities of main forms of cytochrome P-450, participating in steroid hydroxylation, as well as the decrease in total content of cytochrome P-450 in hepatic microsomal fraction and the lowering of its demethylase activity.     

Differential redox and electron-transfer properties of purified yeast, plant and human NADPH-cytochrome P-450 reductases highly modulate cytochrome P-450 activities

Saccharomyces, human and two Arabidopsis (ATR1 and ATR2) NADPH-P-450 reductases were expressed in yeast, purified to homogeneity and used to raise antibodies. Among the P-450-reductases, ATR2 contrasted by its very low FMN affinity and required a thiol-reducing agent for efficient cofactor binding to the FMN-depleted enzyme. Analysis of reductase kinetic properties using artificial acceptors and different salt conditions suggested marked differences between reductases in their FAD and FMN environments and confirmed the unusual properties of the ATR2 FMN-binding domain. Courses of flavin reductions by NADPH were analysed by rapid kinetic studies. The human enzyme was characterized by a FAD reduction rate sixfold to tenfold slower than values for the three other reductases. Following the fast phase of reduction, expected accumulation of flavin semiquinone was observed for the human and ATR1 but not for ATR2 and the yeast reductases. Consistently, redox potential for the FMN semiquinone/reduced couple in the yeast enzyme was found to be more positive than the value for the FMN oxidized/semiquinone couple. This situation was reminiscent of similar inversion observed in bacterial P-450 BM3 reductase. Affinities of reductases for rabbit P-450 2B4 and supported monooxygenase activities in reconstituted systems highly depended on the reductase source. The human enzyme exhibited the highest affinity but supported the lowest kcat whereas the yeast reductase gave the best kcat but with the lowest affinity. ATR1 exhibited both high affinity and efficiency. No simple relation was found between reductase activities with artificial and natural (P-450) acceptors. Thus marked differences in kinetic and redox parameters between reductases dramatically affect their respective abilities to to support P-450 functions.     

An infrared study of the carbon monoxide complexes of cytochrome P-450 and cytochrome P-420

The infrared stretch vibrations (upsilonCO) of the CO-complexes of cytochrome P-450 and cytochrome P-420 have been determined from infrared difference spectra. The CO-complexes exhibit IR-bands at 1949 cm-1 and 1966 cm-1 with half widths of approximately 17 cm-1 and approximately 20 cm-1 respectively. These results are compared with the CO-stretch frequencies of other haemoproteins and discussed with respect to specific interactions of the CO-ligand with the protein moiety and to the ligand trans to CO of the cytochromes.     

Major pathway of imipramine metabolism is catalyzed by cytochromes P-450 1A2 and P-450 3A4 in human liver

The metabolism of imipramine by human liver microsomes was examined using a combination of five strategies. Human hepatic microsomes produced N-desmethylimipramine (84%), 2-hydroxyimipramine (10%), and 10-hydroxyimipramine (6%). Preincubation of human hepatocytes in culture with beta-naphthoflavone and macrolides exclusively induced the formation of desmethylimpramine (552%, p < 0.05, and 234%, p < 0.003, respectively). Correlations were obtained between rates of imipramine demethylation and cytochrome P-450 (P-450) 1A2 (r = 0.88, p < 0.001) and P-450 3A (r = 0.80, p < 0.02) concentrations in human liver microsomal preparations from 13 different subjects. Anti-P-450 1A2 and anti-P-450 3A antibodies selectively inhibited N-demethylation (80% and 60%, respectively). N-Demethylation was completely inhibited when anti-1A2 and anti-3A were added simultaneously. Kinetic studies with human microsomes confirm the contribution of two different enzymes in the N-demethylation. The Km of 1A2 was similar to the high affinity Km in human liver microsomes, whereas the Km of 3A was similar to the low affinity Km in human liver microsomes. P-450 1A2 was apparently more efficient than 3A4 (lower Km and higher Vmax) but was expressed in much lower concentration. Human P-450s 1A2 and 3A4 expressed in yeast efficiently produced desmethylimipramine. These results suggest that P-450 1A2 and P-450 3A4 are the major enzymes involved in imipramine N-demethylation in human hepatic microsomes. Similar experiments were conducted using P-450 2D6, and they confirmed that P-450 2D6 catalyzes imipramine 2-hydroxylation. Interindividual variations in 3A4 hepatic content may explain the large variations in imipramine blood levels observed after uniform dosages and thus may explain the variations in clinical efficacy. Caution might be advised in the clinical use of tricyclic antidepressants when drugs are also administered that induce or inhibit P-450s 3A4 and 1A2.     

Androgen synthesis in a songbird: a study of cyp17 (17alpha-hydroxylase/C17,20-lyase) activity in the zebra finch

Extracellular nucleotides, e.g., ATP, ADP, and UTP, are important signaling molecules which elicit various physiological responses in different tissues. Their degradation is catalyzed by ectonucleotidases which are located on cell surfaces. Most tissues have a mixed population of ectonucleotidases. In this report, the ATP and ADP hydrolyzing ectonucleotidases of chicken gizzard smooth muscle and liver plasma membranes were studied. The two membranes exhibited marked differences in the ratio of ATPase/ADPase activities, activation by divalent cations, thermal stability, responses to detergents and cross-linking agents, and sensitivity to several enzyme inhibitors. The ATPase activity of chicken gizzard membranes is (i) labile to heat and detergents; (ii) activated by concanavalin A and disuccinimidyl suberate, both cross-linking agents; (iii) inhibited by mercurials; and (iv) insensitive to high concentrations of azide, a known inhibitor of ecto-ATP diphosphohydrolases (ecto-ATP/Dase). In contrast, the liver membrane ATPase and ADPase activities are more stable to treatment by heat and detergents and insensitive to cross-linking agents and mercurials, but are inhibited by azide. A low ADP hydrolase activity in the gizzard membranes could be distinguished from both the gizzard ATPase and the liver ATPase/ADPase. This ADP hydrolase, which is markedly stimulated by NBD-Cl, accounts for most of the ADP hydrolysis activity in gizzard membranes. It is concluded that the major ectonucleotidase in the gizzard membranes is an ecto-ATPase whereas that in the liver membranes is an ecto-ATP/Dase. That both membranes contain a mixed population of the ecto-ATPase and ecto-ATP/Dase, but in different proportions, is further demonstrated by immunochemical characterization. The different composition of ectonucleotidases in the two membranes is expected to have an important effect on the regulation of hydrolysis of extracellular ATP as well as the concentration of extracellular adenine nucleotides in the gizzard and liver tissues.