Side-chain Cleavage Reaction Research Articles

Biotransformation of hydrocortisone succinate with whole cell cultures of Monascus purpureus and Cunninghamella echinulata

Hydrocortisone succinate (1) is a synthetic anti-inflammatory drug and key intermediate in the synthesis of other steroidal drugs. This work is based on the fungal biotransformation of 1, using Monascus purpureus and Cunninghamella echinulata strains. Comopound 1 was transformed into four metabolites, identified as hydrocortisone (2), 11β-hydroxyandrost-4-en-3,17-dione (3), Δ1-cortienic acid (4), and hydrocortisone-17-succinate (5), obtained through side chain cleavage, hydrolysis, dehydrogenation, and oxidation reactions. These compounds have previously been synthesized either chemically or enzymatically from different precursors. Though this is not the first report on the biotransformation of 1, but it obviously is a first, where the biotransformed products of compound 1 have been characterized structurally with the help of modern spectroscopic techniques. It is noteworthy that these products have already shown biological potential, however a more thorough investigation of the anti-inflammatory properties of these metabolites would be of high value. These results not only emphasize upon the immense potential of biotransformation in catalysis of reactions, otherwise not-achievable chemically, but also holds promise for the development of novel anti-inflammatory compounds.

Efficient and stable Fe-Ce-Al catalyst for catalytic deoxygenation of lignin for phenol and hydrocarbon-rich fuel: Effect of the synthesis method

To develop a structure-tailoring catalyst for catalytic conversion of lignin for value-added chemicals, a series of novel Fe-Ce-Al metal oxide catalysts was synthesized via different methods to tailor activity and structure for catalytic pyrolysis of lignin to enhance hydrocarbon-rich bio-oil. The results revealed that FeCeAl-CO catalysts derived from coprecipitation method with smaller particle sizes exhibited excellent catalytic deoxygenation activity due to higher Lewis/Brønsted acid, reversible Ce3+/Ce4+ redox pairs, tailorable oxygen vacancies and promoted β-O-4, aromatic-OCH3 and side-chain cleavage. Additionally, coprecipitation method was facilitated to enhance hydrogen transfer, side-chain cleavage and aromatization reactions, while wet impregnation was beneficial to enhance demethoxylation and H-abstraction activity. During catalytic pyrolysis process, over 57.91% of hydrocarbon, including 20.21% and 25.71% for aromatics and olefins were achieved over FeCeAl-CO catalyst. Over 60.74% phenols and 52.48% alkylphenols were obtained over Fe-Ce/Al2O3-IM catalyst due to synergistic effect of FeOx and CeOx species. Fe-Ce-Al catalyst exhibited great activity and stability after fourth run, greater Brønsted acid-favored lignin cleavage and coke deposition, and metal active species leaching, oxidation and pore blockage were the key reasons for deactivation. Therefore, these findings could provide a cost-effective method for designing structure-tailoring catalysts for direct catalytic deoxygenation of lignin to generate hydrocarbon-rich upgrading bio-oil.

Catalytic valorization of Kraft lignin into feedstock chemicals with methyltrioxorhenium (MTO) catalyst in microbial electrochemical cell

In this study, the authors investigate a novel approach to valorize Kraft lignin using the catalyst Methyltrioxorhenium (MTO) in tandem with in-situ produced H2O2 in a Microbial Electrochemical Cell (MEC). This study demonstrates the in-situ oxidation of Kraft lignin using different concentrations of MTO catalyst (2 mM to 8 mM) and H2O2 (5.24 ± 0.40 mM to 8.91 ± 0.70 mM) in three MECs. The depolymerized Kraft lignin samples were characterized using FTIR, CHNS/O, and 1H NMR analysis. The MTO/H2O2 combination showed high selectivity towards the oxidation of Kraft lignin, resulting in both aromatic ring and side chain cleavage reactions and the production of valuable feedstock chemicals. The oxidation also led to a reduction of 68.42 % to 78.18 % in Chemical Oxygen Demand (COD) of lignin. The selective oxidation favored the recovery of Guaiacyl (G) unit-derived feedstock chemicals, with Guaiacol being the most abundant compound (45.04 mg/mL) among the quantified products by HPLC. Additionally, the system demonstrated high efficiency in anodic wastewater treatment, achieving BOD and COD removal rates ranging from 67.68 % to 72.55 %. This method showcases the use of a sustainable system in combination with a selective catalyst to produce valuable products from usually discarded Kraft lignin while simultaneously treating wastewater.

Low-Temperature Oxidation of Heavy Oil Asphaltene with and without Catalyst.

In this study, the asphaltene extracted from Luntai heavy oil was oxidized by a mixture of propionic anhydride and hydrogen peroxide without and with a catalyst. Elemental analysis and infrared spectroscopy results indicated the occurrence of oxygen addition, condensation, and side chain cleavage reactions in the oxidation process. Oxidation products were divided into methanol solubles and methanol insolubles. The H/C and O/C atomic ratios of the MeOHS in the oxidation products without a catalyst were higher than those of the Luntai asphaltene. MeOHS had fewer aromatic rings than Luntai asphaltene. Compared with the oxidative reaction without a catalyst, the total mass of oxidation products and the proportion of MeOHS in oxidation products both increased after catalytic oxidation. This low-temperature oxidation technology can be used to upgrade asphaltenes, and thus can promote the exploitation and processing of heavy oil.

Aquatic photolysis of the pharmaceutical ambroxol: The role of 2,4-dibromoaniline chromophore and heavy atom effect of bromine

As one of the most effective expectorant class drugs, ambroxol (AMB) has been frequently used to treat acute and chronic bronchitis. Extensive use and human excretion result in the widespread occurrence of AMB in wastewater. Herein, we reported the photolysis of AMB in aqueous solution upon 254 nm ultraviolet radiation (UV254). Spectroscopic characterization showed that 2,4-dibromoaniline (DBA) moiety is the core chromophore of AMB. Quantum yield of DBA changed little at pH 4.0 - 9.0; however, AMB showed higher quantum yield at pH > 8.0. Both DBA and AMB have high photoreactivity, which can be attributed to the “heavy atom” effect of bromine substituents. The photolysis of AMB occurred through photoreduction, photoionization, photonucleophilic substitution, side-chain cleavage, and coupling reactions. Both AMB and DBA underwent debromination with yields reaching 80% under 3200 mJ cm−2 UV fluence. Photo-debromination occurred preferentially at the para-position. The presence of natural organic matter inhibited the photodegradation, mainly due to the light-screening effect. The photolysis of AMB was slightly enhanced in the presence of NO3− likely due to radical-induced oxidation. Bioluminescence inhibition assay revealed that photoproducts were not toxic. The results show that UV254 radiation with fluences relevant to advanced oxidation processes was effective for the abatement of AMB in wastewater. However, UV254 treatment of wastewater containing higher concentrations (˃ μg L−1) of AMB should be done with caution because the released Br− can be converted to toxic brominated disinfection byproducts and bromate in subsequent oxidation process.

Programed Thermoresponsive Polymers with Cleavage-Induced Phase Transition.

A new programed upper critical solution temperature-type thermoresponsive polymer was developed using water-soluble anionic polymer conjugates derived from polyallylamine and phthalic acid with cleavage-induced phase transition property. Intrinsic charge inversion from anion to cation of the polymer side chain is induced through a side chain cleavage reaction in acidic aqueous media. With the progress of side chain cleavage under fixed external conditions, the polymer conjugates express a thermoresponsive property, followed by shifting a phase boundary due to the change in polymer composition. When the phase transition boundary eventually reached the examined temperature, phase transition occurs under fixed external conditions. Such new insight obtained in this study opens up the new concept of time-programed stimuli-responsive polymer possessing a cleavage-induced phase transition.

Photo-induced fragmentation of tyrosine side chains in IgG4-Fc: Effect of protein sequence, conformation and glycan structure

This account summarizes recent work on photo-induced side chain cleavage reactions of Tyr in IgG4-Fc. These processes are initiated via photo-ionization of Trp to a Trp radical cation, followed by one-electron oxidation of Tyr to a Tyr radical cation, where fragmentation of the Cα-Cβ bond leads to an intermediary glycyl radical. The latter converts into Gly via hydrogen atom transfer, or into various backbone cleavage products subsequent to the addition of oxygen. Site-directed mutation of Trp381 to Phe381 significantly reduces the yields of Tyr side chain cleavage products, underlining a critical role of Trp for these reactions. Likewise, conformational changes of IgG4-Fc induced by increasing contents of 1-propanol in the solution diminishes the yields of Tyr side chain cleavage products. Variations of the glycan structure at Asn297 had an effect on Tyr side chain cleavage, where larger glycan structures led to lower yields. This result is in interesting contrast to Trp side chain cleavage in IgG1-Fc, where larger glycan structures led to higher yields. The opposite trends for Trp and Tyr side chain cleavage as a function of glycan structure are consistent with a connection between the precursors for both reactions, radical cations of Trp and Tyr, related by electron transfer. The potential significance of these reactions for protein integrity in formulations of pharmaceutical proteins will be discussed.

Photoinduced Tyrosine Side Chain Fragmentation in IgG4-Fc: Mechanisms and Solvent Isotope Effects.

Immunoglobulin gamma (IgG) monoclonal antibodies (mAbs) are glycoproteins that have emerged as powerful and promising protein therapeutics. During the process of production, storage and transportation, exposure to ambient light is inevitable, which can cause protein physical and chemical degradation. For mechanistic studies of photodegradation, we have exposed IgG4-Fc to UV light. The photoirradiation of IgG4-Fc with monochromatic UVC light at λ = 254 nm and UVB light with λmax = 305 nm in air-saturated solutions revealed multiple photoproducts originating from tyrosine side chain fragmentation at Tyr300, Tyr373, and Tyr436. Tyr side chain fragmentation yielded either Gly or various backbone cleavage products, including glyoxal amide derivatives. A mechanism is proposed involving intermediate Tyr radical cation formation, either through direct light absorption of Tyr or through electron transfer to an initial Trp radical cation, followed by elimination of quinone methide. Product formation showed either no (cleavage of Tyr373) or significant (cleavage of Tyr436) inverse product solvent isotope effects (SIEs), indicating a role for proton transfer in the cleavage mechanism of Tyr436. The role of electron transfer in the cleavage of Tyr436 was further investigated through mutation of an adjacent Trp381. This is the first observation of a photoinduced Tyr side chain cleavage reactions in a protein.

Influence of ultrasonic assisted ionic liquid on heavy oil structure

The effect of ionic liquid concentration on the content of four components and structural composition of heavy oil was studied by using ultrasonic-assisted nickel based ionic liquid. The results reveals that compared with crude oil and individual ultrasonic crude oil, the combination of ultrasonic wave and ionic liquid can achieve synergistic effect. Saturate increased and aromatic decreased gradually as the increase of the concentration of ionic liquid. The structure of resin and asphaltene has been characterized by infrared and nuclear magnetic resonance spectroscopy. It has been indicated that the structure of polyaromatic compounds can be obviously changed and mainly occurrence in alkyl side chain cleavage and cycloalkanes ring-opening reaction.

Photonic Reactions Leading to Fluorescence in a Polymeric System Induced by the Photothermal Effect of Magnetite Nanoparticles Using a 780 nm Multiphoton Laser.

Recently, polymer-coated magnetite (Fe3 O4 ) nanoparticles (NPs) are extensively studied for applications in therapeutics or diagnostics using photothermal effect. Therefore, it is essential to understand the interactions between Fe3 O4 NPs and polymers when optical stimuli are applied. Herein, the photonic reactions of Fe3 O4 NPs and polymer composites upon application of a 780 nm multiphoton laser are analyzed. The photonic reactions produce unique results including fluorescence from conformationally changed polymer and low-temperature phase transformation of Fe3 O4 NPs. Typically, π-conjugated chains are formed, inducing fluorescence through a series of main and side-chain cleavage reactions of polymers with the aliphatic chain. In addition, fluorescence is detected in the cellular system by photonic reactions between Fe3 O4 NPs and biomolecules. After multiphoton laser irradiation, light emission is detected near the intracellular Fe3 O4 NPs, and a stronger intensity is observed in large-sized NPs.

Downregulation of cytochrome P450scc as an initial adverse effect of adult exposure to diethylstilbestrol on testicular steroidogenesis

Reproductive toxicities and endocrine disruptions caused by chemicals in adult males are still poorly understood. It is our objectives to understand further details of the initial adverse effects leading severe testicular toxicities of a pharmaceutical endocrine disruptor, diethylstilbestrol (DES). Downregulations of both testicular regulatory proteins, such as the steroidogenic acute regulatory protein (StAR) and the peripheral benzodiazepine receptor (PBR), which play important roles in the transport of cholesterol into the mitochondria, and cytochrome P450 mediating the cholesterol side chain cleavage reaction (P450scc), were observed in the rat orally administered DES (340 μg/kg/2 days) for 2 weeks. We found that after only 1 week treatment with DES, the blood and testicular testosterone (TS) levels were drastically decreased without abnormalities of the StAR and PBR; however, the protein and mRNA levels of P450scc were diminished. Decrease in the conversion rate of cholesterol to pregnenolone was delayed in the in vitro assay using the testicular mitochondrial fraction from the rat treated with DES for 1 week. When the precursors in TS biosynthesis containing the testis were identified and determined by liquid chromatography-mass spectrometry analysis, decreased levels of all precursors except cholesterol were observed. In conclusion, suppressed cytochrome P450scc expression in adult male rat was identified as an initial target of DES in testicular steroidogenesis disorder leading reproductive toxicities.

Role of Positively Charged Residues Lys267, Lys270, and Arg411 of Cytochrome P450scc (Cyp11A1) in Interaction with Adrenodoxin

Cytochrome P450scc and adrenodoxin are redox proteins of the electron transfer chain of the inner mitochondrial membrane steroid hydroxylases. In the present work site-directed mutagenesis of the charged residues of cytochrome P450scc and adrenodoxin, which might be involved in interaction, was used to study the nature of electrostatic contacts between the hemeprotein and the ferredoxin. The target residues for mutagenesis were selected based on the theoretical model of cytochrome P450scc-adrenodoxin complex and previously reported chemical modification studies of cytochrome P450scc. In the present work, to clarify the molecular mechanism of hemeprotein interaction with ferredoxin, we constructed cytochrome P450scc Lys267, Lys270, and Arg411 mutants and Glu47 mutant of adrenodoxin and analyzed their possible role in electrostatic interaction and the role of these residues in the functional activity of the proteins. Charge neutralization at positions Lys267 or Lys270 of cytochrome P450scc causes no significant effect on the physicochemical and functional properties of cytochrome P450scc. However, cytochrome P450scc mutant Arg411Gln was found to exhibit decreased binding affinity to adrenodoxin and lower activity in the cholesterol side chain cleavage reaction. Studies of the functional properties of Glu47Gln and Glu47Arg adrenodoxin mutants indicate that a negatively charged residue in the loop covering the Fe2S2 cluster, being important for maintenance of the correct architecture of these structural elements of ferredoxin, is not directly involved in electrostatic interaction with cytochrome P450scc. Moreover, our results indicate the presence of at least two different binding (contact) sites on the proximal surface of cytochrome P450scc with different electrostatic input to interaction with adrenodoxin. In the binary complex, the positively charged sites of the proximal surface of cytochrome P450scc well correspond to the two negatively charged sites of adrenodoxin: the "interaction" domain site and the "core" domain site.

Diverse biohopanoid compositions of non-marine sediments

Biohopanoid compositions were determined for modern sediments from 27 lacustrine environments and 1 near coastal lagoon. GC–MS analysis of existing hopanols and those produced by a side-chain cleavage reaction (periodic acid and sodium borohydride) of the bacteriohopanepolyols provided quantitative data for bacteriohopanetetrol (BHT), bacteriohopanepentol, and composite tetra-, penta- and hexafunctionalised biohopanoids. Additional analysis of acetylated total extracts by HPLC–APCI–MS has allowed us to identify up to seven intact bacteriohopanepolyols in these environments, including the first direct observation of 35-aminobacteriohopanepentol in environmental samples. This method is complementary to the periodic acid approach in providing more specific compositional information. The lacustrine environments exhibit a wide range of compositions, many with high inputs of hexafunctionalised biohopanoids (up to 60% of total tetra-, penta- and hexafunctionalised biohopanoids), indicative of major contributions to the sediment from Type I methanotrophic bacteria. Several methylated biohopanoids were also detected in most of the samples with 2β as the dominant configuration of the methyl group; 2β-methylbacteriohopanetetrol was by far the most abundant and most commonly occurring compound of this type and comprised the most abundant bacteriohopanepolyol in one saline lake (Laguna Grande, Spain). Such compounds are believed to be derived from cyanobacteria, currently the only known quantitatively-significant source of 2β-methylated biohopanoids. Biohopanoids containing a methyl group at C-3 were also detected in a limited number of environments. These data demonstrate the wide variability in bacteriohopanepolyol compositions among depositional environments, and the potential of these compounds to preserve bacterial source information and, thereby, to record environmental change.

Steroidogenic factor-1 controls the aldose reductase akr1b7 gene promoter in transgenic mice through an atypical binding site.

Aldo-keto-reductase 1B7/mouse vas deferens protein (AKR1B7/MVDP) is expressed in rodent steroidogenic glands and in the mouse vas deferens. In steroidogenic organs, AKR1B7/MVDP scavenges isocaproaldehyde produced from the cholesterol side-chain cleavage reaction. Akr1b7/mvdp is responsive to ACTH in adrenals and to androgens in vas deferens. Using transgenic mice, we previously delimited the regulatory DNA sequences necessary for expression in both organs and identified by cell transfections, a cryptic steroidogenic factor-1 (SF-1) response element (SFRE) at -102 that overlaps a proximal androgen-responsive element. To address its in vivo functions in adrenals, we devised a transgenic mouse study using wild-type and mutant akr1b7 promoters driving the chloramphenol acetyltransferase reporter gene. Adrenal expression in adults was impaired in all lines mutant for -102 SFRE. This effect is linked to impaired SF-1 binding and not to impaired androgen receptor binding, because akr1b7 expression is not affected in adrenals of androgen receptor-defective Tfm mice. Triphasic developmental patterns of both AKR1B7 and wild-type transgene expression paralleled changes in SF-1 levels/binding activity; expression was maximal in late embryos, minimal in 6- to 15-d-old neonates, and thereafter progressively restored. Differences in developmental expression between wild-type and mutant transgenes revealed that requirement for the -102 SFRE appears stage specific, as its integrity is an absolute prerequisite for reinduction of gene expression after postnatal d 15. Further, mutation of this site did not affect transgene responsiveness to ACTH. These findings demonstrate a new function for SFRE in vivo, via influencing promoter sensibility to postnatal changes of SF-1 contents, in controlling promoter strength in adults without affecting adrenal targeting, hormonal control, or early gene expression.

Formation and functioning of fused cholesterol side-chain cleavage enzymes.

We studied the properties of various fused combinations of the components of the mitochondrial cholesterol side-chain cleavage system including cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). When recombinant DNAs encoding these constructs were expressed in Escherichia coli, both cholesterol side-chain cleavage activity and sensitivity to intracellular proteolysis of the three-component fusions depended on the species of origin and the arrangement of the constituents. To understand the assembly of the catalytic domains in the fused molecules, we analyzed the catalytic properties of three two-component fusions: P450scc-Adx, Adx-P450scc, and AdR-Adx. We examined the ability of each fusion to carry out the side-chain cleavage reaction in the presence of the corresponding missing component of the whole system and examined the dependence of this reaction on the presence of exogenously added individual components of the double fusions. This analysis indicated that the active centers in the double fusions are either unable to interact or are misfolded; in some cases they were inaccessible to exogenous partners. Our data suggest that when fusion proteins containing P450scc, Adx, and AdR undergo protein folding, the corresponding catalytic domains are not formed independently of each other. Thus, the correct folding and catalytic activity of each domain is determined interactively and not independently.

High-flux mitochondrial cholesterol trafficking, a specialized function of the adrenal cortex.

The adrenal cortex is a tissue of excess in terms of both cholesterol metabolism and cholesterol exchange with the circulation. Exceptionally high levels of lipoprotein receptors in this highly vascularized tissue provide ready access to dietary cholesterol, allowing the adrenocortical cells to maintain impressive stores of cytoplasmic cholesterol ester (CE) droplets. Tightly packed among the CE droplets are specialized mitochondria, carrying in their inner membranes high levels of the cytochrome P450scc (CYP11A1). This enzyme carries out the so-called side chain cleavage reaction, consuming cholesterol to produce pregnenolone, the precursor of cortisol and all other steroids. Glucocorticoid synthesis is tightly regulated at the level of cholesterol metabolism, which responds to ACTH stimulation over a period of minutes and ceases equally quickly when this hormone is removed. Remarkably, this dynamic process is modulated under most circumstances not by control of the intrinsic enzymatic activity of P450scc, but rather by substrate availability. For this reason, cholesterol transport within the mitochondrion has emerged as the key control point for steroidogenesis. The adrenal cortex is not alone in requiring efficient and controlled delivery of cholesterol into mitochondria. Other steroidogenic cells, including several cell types in the ovary, the Leydig cells of the testis, and a subset of hippocampal neurons (1), also employ P450scc to produce pregnenolone and a variety of downstream steroid hormones or neurosteroids. In vertebrates ranging from birds and fish (2) to mammals, these various cell types all express a short-lived mitochondrial import factor now called the steroidogenic acute regulatory protein (StAR), which mediates this process. Here, I examine the often confusing literature on StAR’s mechanism of action, particularly in light of recent work establishing the importance of other players, and I present a model for StAR’s interaction with cholesterol and with some of these other proteins. I also discuss the insights into mitochondrial function that have come from the analysis of patients with congenital adrenal hyperplasia (CAH), who lack this factor. Finally, I consider the multi-tiered regulation of StAR and related proteins in adrenocortical cells and other steroidogenic cell types.

Site-directed mutagenesis of cytochrome b5 for studies of its interaction with cytochrome P450.

We have shown earlier that microsomal cytochrome b5 can form a specific complex with mitochondrial cytochrome P450 (cytochrome P450scc). The formation of the complex between these two heme proteins was proved spectrophotometrically, by affinity chromatography on immobilized cytochrome b5, and by measuring the cholesterol side-chain cleavage activity of cytochrome P450scc in a reconstituted system in the presence of cytochrome b5. To further study the mechanism of interaction of these heme proteins and evaluate the role of negatively charged amino acid residues Glu42, Glu48, and Asp65 of cytochrome b5, which are located at the site responsible for interaction with electron transfer partners, we used site-directed mutagenesis to replace residues Glu42 and Glu48 with lysine and residue Asp65 with alanine. The resulting mutant forms of cytochrome b5 were expressed in E. coli, and full-length and truncated forms (shortened from the C-terminal sequence due to cleavage of 40 amino acid residues) of these cytochrome b5 mutants were purified. Addition of the truncated forms of cytochrome b5 (which do not contain the hydrophobic C-terminal sequence responsible for interaction with the membrane) to the reconstituted system containing cytochrome P450scc caused practically no stimulation of catalytic activity, indicating an important role of the hydrophobic fragment of cytochrome b5 in its interaction with cytochrome P450scc. However, full-length cytochrome b5 and the full-length Glu48Lys and Asp65Ala mutant forms of cytochrome b5 stimulated the cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc by 100%, suggesting that residues Glu48 and Asp65 of cytochrome b5 are not directly involved in its interaction with cytochrome P450scc. The replacement of Glu42 for lysine, however, made the Glu42Lys mutant form of cytochrome b5 about 40% less effective in stimulation of the cholesterol side-chain cleavage activity of cytochrome P450scc, indicating that residue Glu42 of cytochrome b5 is involved in electrostatic interactions with cytochrome P450scc. Residues Glu42 and Glu48 of cytochrome b5 appear to participate in electrostatic interaction with microsomal type cytochrome P450.

Real-time fluorescence analysis on molecular mechanisms for regulation of cytochrome P450scc activity upon steroidogenic stimulation in adrenocortical cells

Real-time fluorescence analysis revealed that the activity of cytochrome P450scc was related to Ca 2+ signals arising from extracellular NADPH, ACTH and ATP stimulation in adrenocortical fasciculata cells. The side-chain cleavage reaction by cytochrome P450scc was measured with 3β-hydroxy-22,23-bisnor-5-cholenyl ether (cholesterol–resorufin) by observing the distinct increase in fluorescence upon conversion of cholesterol–resorufin to resorufin and pregnenolone. Adrenocorticotropic hormone (ACTH) induced a relatively small stimulation of the P450scc activity. A significant production of resorufin was revealed after stimulation of cell cultures with 100 pM, 1 nM of ACTH for 3 h. On the other hand, extracellular NADPH was found to rapidly and greatly stimulate the resorufin production in intact cells immediately after the addition of 50–500 μM NADPH. The extracellular NADPH stimulation was prevented by the addition of thapsigargin and EGTA which abolished Ca 2+ oscillations induced by NADPH. Suramin, a specific antagonist of the P 2y type ATP receptor, also completely abolished the NADPH-induced cholesterol–resorufin conversion. These results imply that extracellular NADPH (membrane impermeable) produced Ca 2+ oscillations through its binding to ATP receptor thereby stimulating the activity of P450scc. The application of 45–500 μM extracellular ATP to cells did not, however, significantly increase the resorufin production. These three stimulators produced very different types of Ca 2+ signals. ACTH induced mainly a series of Ca 2+ spikes superimposed on a long-lasting basal Ca 2+ elevation. The Ca 2+ signals induced by NADPH showed predominantly a series of Ca 2+ spikes without elevation of the basal Ca 2+ concentration. Only long-lasting Ca 2+ elevation was induced by extracellular ATP. The stimulation of cytochrome P450scc may thus be correlated with the different patterns of Ca 2+ signals.

Environmental influence on the biohopanoid composition of recent sediments

Bacteriohopanepolyols are the biological precursors of extended hopane biomarkers which are ubiquitous in the geosphere. The abundance and composition of these biohopanoids were determined for modern sediments from eight depositional environments by GC-MS analysis of hopanols derived from a side-chain cleavage reaction (periodic acid and sodium borohydride) of the bacteriohopanepolyols. We report quantitative data for bacteriohopanetetrol (BHT), composite tetrafunctionalised biohopanoids, pentafunctionalised biohopanoids and hexafunctionalised biohopanoids. BHT was found in concentrations higher than previously reported (up to ca. 1500 μg/g TOC), but comprised only a relatively small proportion (0–26%) of the total biohopanoids in all except one sample. Composite tetrafunctionalised biohopanoids were the dominant group of bacteriohopanepolyols in all other sediments, and are likely to be a major source of the geologically occurring extended hopanes. Hexafunctionalised biohopanoids were most abundant in lake sediments, particularly two small highly productive stratified lakes (where they comprised 20–40% of total biohopanoids). This feature is interpreted to be due to greater contribution to the organic matter of these sediments from Type I methanotrophic bacteria (which produce dominantly hexafunctionalised biohopanoids), consistent with the environmental settings. In the marine environments where sulphate reduction is dominant, hexafunctionalised biohopanoids are relatively minor constituents (2–6% of total biohopanoids). These data comprise the first clear demonstration that bacteriohopanepolyols vary in composition between depositional environments, and have the potential to preserve specific bacterial source information in sedimentary organic matter.

Enzymatic properties of vesicle‐reconstituted human cytochrome P450SCC (CYP11A1)

The recently reported heterologous expression and purification of both human cytochrome P450SCC and adrenodoxin [Woods, S.T., Sadleir, J., Downs, T., Triantopoulos, T., Haedlam, M.J. & Tuckey, R.C. (1998) Arch. Biochem. Biophys. 353, 109-115] has enabled us to perform studies with the membrane-reconstituted human enzymes to better understand the side-chain cleavage reaction in humans. Human P450SCC was successfully reconstituted into dioleoylphosphatidylcholine vesicles with and without cardiolipin and its enzymatic properties characterized in the membrane-bound state. Enhancement of the P450SCC activity and significant activation by cardiolipin were observed when human adrenodoxin instead of bovine adrenodoxin was used as electron donor. In the absence of cardiolipin, Km for cholesterol was decreased twice in the case of human adrenodoxin indicating enhanced cholesterol binding. On the other hand, in the presence of cardiolipin in the membrane both Km and V for cholesterol were decreased with human adrenodoxin as electron donor. Kinetic analysis of the interaction between human P450SCC and its redox partners provided evidence for enhanced binding of the human electron donor to human P450SCC indicated by both an increased V and decreased Kd for human adrenodoxin compared with the values with bovine adrenodoxin. Because no similar effects were observed in Tween 20 micelles, these results suggest that the phospholipid membrane may play an important role in the interaction of human adrenodoxin with human P450SCC.