Characterizing the Stability of Angiotensin II in 0.9% Sodium Chloride Using High Performance Liquid Chromatography and Liquid Chromatography Tandem Mass Spectrometry.

The purpose of this study was to evaluate the stability of angiotensin II in 0.9% sodium chloride for up to 5 days. We prepared angiotensin II dilutions, by aseptically diluting 2.

Non-covalent inhibitors of thioredoxin glutathione reductase with schistosomicidal activity in vivo.

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target.

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry.

Aiming to elucidate the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) on drug metabolism, we explored the array of its protein-protein interactions (interactome) in human liver microsomes (HLM) with chemical crosslinking mass spectrometry (CXMS). Our strategy employs membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide and 4-(-succinimidylcarboxy)benzophenone. Exposure of bait-incorporated HLM samples to light was followed by isolating the His-tagged bait protein and its crosslinked aggregates on Ni-NTA agarose.

Probing functional interactions between cytochromes P450 with principal component analysis of substrate saturation profiles and targeted proteomics.

We investigated the correspondence between drug metabolism routes and the composition of the P450 ensemble in human liver microsomes (HLM). As a probe, we used Coumarin 152 (C152), a fluorogenic substrate metabolized by multiple P450 species. Studying the substrate-saturation profiles (SSP) in seven pooled HLM preparations, we sought to correlate them with the P450 pool's composition characterized by targeted proteomics.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4.

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples.

Nonadditivity in human microsomal drug metabolism revealed in a study with coumarin 152, a polyspecific cytochrome P450 substrate.

We closely characterized 7-Dimethylamino-4-trifluromethylcoumarin (Coumarin 152, C152), a substrate metabolized by multiple P450 species, to establish a new fluorogenic probe for the studies of functional integration in the cytochrome P450 ensemble. Scanning fluorescence spectroscopy and LC/MS-MS were used to characterize the products of N-demethylation of C152 and optimize their fluorometric detection. The metabolism of C152 by the individual P450 species was characterized using the microsomes containing cDNA-expressed enzymes.

Toward a systems approach to cytochrome P450 ensemble: interactions of CYP2E1 with other P450 species and their impact on CYP1A2.

In this study, we investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system, we used CYP2E1-enriched human liver microsomes (HLM) obtained by the incorporation of purified CYP2E1. Using a technique based on homo-FRET in oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with HLM result in the formation of its mixed oligomers with other P450 species present in the microsomal membrane.

Characterization of two steroid hydroxylases from different Streptomyces spp. and their ligand-bound and -unbound crystal structures.

Bacterial cytochrome P450 (CYP) enzymes are involved in the hydroxylation of various endogenous substrates while using a heme molecule as a cofactor. CYPs have gained biotechnological interest as useful biocatalysts capable of altering chemical structures by adding a hydroxyl group in a regiospecific manner. Here, we identified, purified, and characterized two CYP154C4 proteins from Streptomyces sp.

Bacterial CYP154C8 catalyzes carbon-carbon bond cleavage in steroids.

Here, we report the first bacterial cytochrome P450, CYP154C8, that catalyzes the C-C bond cleavage reaction of steroids. A major change in product distribution is observed with CYP154C8, when the reactions are supported by NADPH and spinach redox partners ferredoxin and ferredoxin reductase, compared with previously reported reactions supported by NADH and redox partners containing putidaredoxin and putidaredoxin reductase. The NMR-based structural elucidation of reaction products reveals 21-hydroxyprednisone as the major product for prednisone, while the other product is identified as 1-dehydroadrenosterone obtained due to C-C bond cleavage.

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution.

CYP154C8 catalyzes the hydroxylation of diverse steroids, as has previously been demonstrated, by using an NADH-dependent system including putidaredoxin and putidaredoxin reductase as redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8 reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase displayed catalytic activity different from that of the NADH-dependent system. The NADPH-dependent system showed multistep oxidation of progesterone and other substrates including androstenedione, testosterone, and nandrolone.

Tracking Down a New Steroid-Hydroxylating Promiscuous Cytochrome P450: CYP154C8 from Streptomyces sp. W2233-SM.

CYP154C8 from Streptomyces sp. has been identified as a new cytochrome P450 with substrate flexibility towards different sets of steroids. In vitro treatment of these steroids with CYP154C8 revealed interesting product formation patterns with the same group of steroids.

Crystal Structure and Functional Characterization of a Cytochrome P450 (CYP106A2) from sp. PAMC 23377.

Bacterial cytochrome P450 (CYP) steroid hydroxylases are effectively useful in the pharmaceutical industry for introducing hydroxyl groups to a wide range of steroids. We found a putative CYP steroid hydroxylase (CYP106A2) from the bacterium sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, showing 94% sequence similarity with CYP106A2 ( ATCC 13368).