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Among the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers. Type III-A CRISPR-Cas system is a multi-component and multipronged immune effector, activated by viral RNA that previously has not been repurposed for disease detection owing in part to the complex enzyme reconstitution process and functionality. Here, we describe the construction and application of a virus detection method, based on an in vivo-reconstituted Type III-A CRISPR-Cas system. This system harnesses both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification that allow virus detection with high sensitivity and at multiple settings. We demonstrate the use of the Type III-A system-based method in detection of SARS-CoV-2 that reached 2000 copies/μl sensitivity in amplification-free and 60 copies/μl sensitivity via isothermal amplification within 30 min and diagnosed SARS-CoV-2-infected patients in both settings. The high sensitivity, flexible reaction conditions, and the small molecular-driven amplification make the Type III-A system a potentially unique nucleic acid detection method with broad applications., CRISPR-Cas based virus detection assays can be both rapid and sensitive. Here the authors use a Type III-A CRISPR-Cas system to detect SARS-CoV-2.

Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism and screen inhibition of viral spike protein-mediated fusion under the biosafety level 1conditions.

Ochratoxin A (OTA) is a common cereal mycotoxin that seriously threatens food safety and public health. Herein a horseradish peroxidase-nanobody fusion protein (HRP-Nb) retaining antibody and enzyme activity was obtained after inclusion body denaturation and renaturation and enzyme reconstitution, which served both as the primary antibody and reporter enzyme and was applied to develop a membrane-based dot immunoassay (HN-DIA) for OTA visual detection. Based on the optimal experimental conditions, the HN-DIA could be finished in 10 min with a cut-off limit of 50 μg kg−1 in rice and oat samples by eye. The HN-DIA showed high selectivity for OTA and had good accuracy and reproducibility in the recovery experiments. Spiked sample analysis results of the HN-DIA and high performance liquid chromatography (HPLC) correlated well with each other. Therefore, the proposed HN-DIA has the potential for rapid screening of OTA and other small molecule pollutants in food and the environment by naked eye.

The ability to switch on the activity of an enzyme through its spontaneous reconstitution has proven to be a valuable tool in fundamental studies of enzyme structure/reactivity relationships or in the design of artificial signal transduction systems in bioelectronics, synthetic biology, or bioanalytical applications. In particular, those based on the spontaneous reconstitution/activation of the apo-PQQ-dependent soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus were widely developed. However, the reconstitution mechanism of sGDH with its two cofactors, i.e. the pyrroloquinoline quinone (PQQ) and Ca2+, remains unknown. The objective here is to elucidate this mechanism by stopped-flow kinetics under single-turnover conditions. The reconstitution of sGDH exhibited biphasic kinetics, characteristic of a square reaction scheme associated to two activation pathways. From a complete kinetic analysis, we were able to fully predict the reconstitution dynamic, but also to demonstrate that when PQQ first binds to the apo-sGDH, it strongly impedes the access of Ca2+ to its enclosed position at the bottom of the enzyme binding site, thereby greatly slowing down the reconstitution rate of sGDH. This slow calcium insertion may purposely be accelerated by providing more flexibility to the Ca2+ binding loop through the specific mutation of the calcium coordinating P248 proline residue, reducing thus the kinetic barrier to calcium ion insertion. The dynamic nature of the reconstitution process is also supported by the observation of a clear loop shift and a reorganization of the hydrogen bonding network and van der Waals interactions observed in both active sites of the apo and holo forms, a structural change modulation that was revealed from the refined X-ray structure of apo-sGDH (PDB:5MIN)., 45 pages, 11 figures, 6 tables

A facile and efficient enzymatic reconstitution methodology has been proposed for high-catalysis peroxidase mimics by remolding the redox active centers of heme-containing proteins with the in-site biomineralized gold using hemoglobin (Hb) as a model. Catalytic hemin (Hem) was extracted from the active centers of Hb for the gold biomineralization and then reconstituted into apoHb to yield the Hem-Au@apoHb nanocomposites showing dramatically improved intrinsic catalysis and electrocatalysis over natural Hb and Hem. The biomineralized gold, on the one hand, would act as "nanowires" to promote the electron transferring of the nanocomposites. On the other hand, it would create a reactivity pathway to pre-organize and accumulate more substrates towards the active sites of the peroxidase mimics. Steady-state kinetics studies indicate that Hem-Au@apoHb could present much higher substrate affinity (lower Michaelis constants) and intrinsic catalysis even than some natural peroxidases. Moreover, the application feasibility of the prepared artificial enzymes was demonstrated by colorimetric assays and direct electrocatalysis for H2O2 sensing, showing a detection limitation low as 0.45μM. Importantly, such a catalysis active-center reconstitution protocol may circumvent the substantial improvement of the intrinsic catalysis and electrocatalysis of diverse heme-containing proteins or enyzmes toward the extensive applications in the chemical, enviromental, and biomedical catalysis fields.

Human phospholipid scramblase 1 (hPLSCR1) belongs to the ATP-independent class of phospholipid translocators which possess a single EF-hand-like Ca 2+-binding motif and also a C-terminal helix (CTH). The CTH domain of hPLSCR1 was believed to be a putative single transmembrane helix at the C-terminus. Recent homology modeling studies by Bateman et al. predicted that the hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this is not a true transmembrane helix [Bateman A, Finn RD, Sims PJ, Wiedmer T, Biegert A & Johannes S. Bioinformatics 2008, 25, 159]. To determine the exact function of the CTH of hPLSCR1, we deleted the CTH domain and determined: (a) whether CTH plays any role beyond membrane anchorage, (b) the functional consequences of CTH deletion, and (c) any conformational changes associated with CTH in a lipid environment. In vitro reconstitution studies confirm that the predicted CTH is required for membrane insertion and scrambling activity. CTH deletion caused a 50% decrease in binding affinity of Ca2+ for CTH-hPLSCR1 (Ka = 115 ?m) compared with hPLSCR1 (Ka = 249 ?m). Far UV-CD studies revealed that the CTH peptide adopts ?-helicity only in the presence of SDS micelles and negatively charged vesicles, indicating that electrostatic interactions are required for insertion of the peptide. CTH peptide-quenching studies confirm that the predicted CTH inserts into the membrane and its ability to interact with the membrane depends on the presence of charge interactions. TOXCAT assay revealed that CTH of hPLSCR1 does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion and Ca2+ coordination and also plays an important role in the functional conformation of hPLSCR1. Phospholipid scramblases constitute a family of homologous proteins named as hPLSCR1-hPLSCR4. The C-terminal helix (CTH) is presently close to the EF-hand motif in the scramblase homologues indicating that it may play an important role in Ca2+ binding. This study shows that CTH is required for membrane insertion, Ca2+ co-ordination and also plays an important role in the functional conformation of hPLSCR1. � 2013 FEBS.

Pseudomonas aeruginosa is a versatile bacterium that can grow using citronellol or leucine as sole carbon source. For both compounds the degradation pathways converge at the key enzyme 3-methylcrotonyl coenzyme-A carboxylase (MCCase). This enzyme is a complex formed by two subunits (α and β), encoded by the liuD and liuB genes, respectively; both are essential for enzyme function. Previously, both subunits had been separately expressed and then the complex re-constituted, however this methodology is laborious and produces low yield of active enzyme. In this work, the MCCase subunits were co-expressed in the same plasmid and purified in one step by affinity chromatography using the LiuD-His tag protein, interacting with the LiuB-S tag recombinant protein. The purified enzyme lost most of the activity within few hours of storage. The co-expressed subunits formed an (αβ)(4) complex that suffered a modification of its oligomerization state after storage, which probably contributed to the loss on activity observed. The recombinant MCCase enzyme presented optimum pH and temperature values of 9.0 and 30º C, respectively. Functionally, MCCase showed Michaelian kinetics behavior with a K(m) for its substrate and V(max) of 168 μM and 430 nmoles mg(-1)min(-1), respectively. The results suggest that the co-expression and co-purification of the subunits is a suitable procedure to obtain the active complex of the MCCase from Pseudomonas aeruginosa in a single step.

A new detection method for ligand–protein interactions was constructed using interaction-dependent native chemical ligation and enzyme reconstitution (IDNCL-ER) by employing ribonuclease S. Using a...

Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe–S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS.

Leukodystrophies are rare diseases caused by defects in the genes coding for lysosomal enzymes that degrade several glycosphingolipids. Gene therapy for leukodystrophies requires efficient distribution of the missing enzymes in CNS tissues to prevent demyelination and neurodegeneration. In this work, we targeted the external capsule (EC), a white matter region enriched in neuronal projections, with the aim of obtaining maximal protein distribution from a single injection site. We used bidirectional (bd) lentiviral vectors (LV) (bdLV) to ensure coordinate expression of a therapeutic gene (beta-galactocerebrosidase, GALC; arylsulfatase A, ARSA) and of a reporter gene, thus monitoring simultaneously transgene distribution and enzyme reconstitution. A single EC injection of bdLV.GALC in early symptomatic twitcher mice (a murine model of globoid cell leukodystrophy) resulted in rapid and robust expression of a functional GALC protein in the telencephalon, cerebellum, brainstem and spinal cord. This led to global rescue of enzymatic activity, significant reduction of tissue storage and decrease of activated astroglia and microglia. Widespread protein distribution and complete metabolic correction were also observed after EC injection of bdLV.ARSA in a mouse model of metachromatic leukodystrophy. Our data indicated axonal transport, distribution through cerebrospinal fluid flow and cross-correction as the mechanisms contributing to widespread bioavailability of GALC and ARSA proteins in CNS tissues. LV-mediated gene delivery of lysosomal enzymes by targeting highly interconnected CNS regions is a potentially effective strategy that, combined with a treatment able to target the PNS and peripheral organs, may provide significant therapeutic benefit to patients affected by leukodystrophies.

Over the past 15 years, the lysosomal storage diseases have become paradigms for the specific treatment of monogenic disorders, particularly those affecting children. This review summarizes the phenotypes and recent literature regarding enzyme reconstitution (replacement) therapy and outcomes for such treatable lysosomal storage diseases: Gaucher disease, Fabry disease, Pompe disease and the mucopolysaccharidoses.Recent clinical trials have shown that enzyme reconstitution therapy effectively treats many of the manifestations of the lysosomal storage diseases. When initiated early in the disease course, enzyme reconstitution therapy can reverse some disease manifestations, but may not completely alleviate the disease progression. Enzyme reconstitution therapy is generally well tolerated. Many adverse events are antibody-related, but can be managed without requiring cessation of enzyme reconstitution therapy. Documented IgE reactions, i.e. anaphylactoid, are quite rare (fewer than 1%).Enzyme reconstitution therapy is a safe and effective treatment modality available for several of the lysosomal storage diseases. Owing to the short history of enzyme reconstitution therapy, the long-term outcomes of enzyme reconstitution therapy-treated individuals are unknown and require further investigation. Medical professionals must learn to identify patients likely to benefit from these life-changing therapies so as to prevent many of the devastating, irreversible complications of the lysosomal storage diseases.

Filamentous fungi produce and secrete beta-N-acetylhexosaminidases, Hex, as important components of the binary chitinolytic systems involved in the formation of septa and hyphenation. Enzyme reconstitution experiments published previously indicate that Hex can occur in the form of two molecular species containing either one or two molecules of the propeptide noncovalently associated with the enzyme dimer. Here, we describe a novel mechanism for the regulation of the activity of Hex based on the association of their catalytic subunits with the large N-terminal propeptides in vivo. We show that the enzyme precursor is processed early in the biosynthesis, shortly after the addition of N-glycans through the action of a dibasic peptidase, cleaving both before and after the dibasic sequence. The processing site for this unique dibasic peptidase, different from that of kexins, is conserved among the beta-N-acetylhexosaminidases from filamentous fungi, and inhibition of the dibasic peptidase abrogates enzyme folding and activation. Binding of the released propeptide to the catalytic subunit of Hex is essential for its activation. An examination of the kinetics of Hex activation and dimerization in vitro allowed us to understand the unusually high efficiency of the assembly of this enzyme. We also report that the fungus is able to actively regulate the concentration of the processed propeptide in endoplasmic reticulum and thus the specific activity of the produced Hex. This novel regulatory mechanism enables the control of the catalytic activity and architecture of the secreted enzyme according to the needs of the producing cell at various stages of its growth cycle.

The catalytic versatility of flavins encompasses the ability to catalyze one and two electron redox processes as well as dioxygen activation. In flavoenzymes, protein-cofactor interactions modulate flavin chemistry enabling these enzymes to perform a diverse set of biological roles. Flavin analogs, such as deoxyflavins, provide a convenient method for identifying which polar contacts to FMN are necessary for enzymatic activity. Iodotyrosine deiodinase (IYD) is responsible for the deiodination of byproducts from thyroxine biosynthesis (mono- and diiodotyrosine (MIT and DIT)) in the thyroid. A unique flavoenzyme, IYD, is one of few known aerobic enzymes able to catalysis reductive dehalogenation. Literature precedence for the deiodination mechanism of IYD has not been found. Detection of the flavin semiquinone (Flsq) intermediate during anaerobic reduction of IYD in the presence of substrate suggests deiodination occurs via two single electron transfer processes. Proposed one electron mechanisms for IYD involving formation of a substrate keto-tautomer are supported by the enzyme’s preference for the phenolate form of substrate during binding. Previous analysis of a co-crystal of IYD and MIT showed the involvement of substrate in extensive interactions with the FMN cofactor, including a hydrogen bond between the FMN ribityl 2ʹ-OH and the phenolic OH of MIT. This hydrogen bond has been hypothesized to activate substrate for deiodination. To probe the significance of the polar interaction between the ribityl 2ʹ-OH and the phenolic OH of MIT in IYD, 2-deoxyriboflavin was synthesized and enzymatically phosphorylated using riboflavin kinase. Reconstitution of human IYD (hIYD) with 2-deoxyFMN produced an enzyme with a significantly decreased affinity for MIT. Deiodinase activity was retained in 2-deoxyhIYD with a 10-fold decrease in catalytic efficiency. Detection of the Flsq was not observed during anaerobic reduction of 2-deoxyhIYD in the presence of mono-fluorotyrosine (MFT). The enzyme’s preference for the phenol versus phenolate form of substrate could not be determined due to the low solubility of DIT at concentrations necessary for pH dependent binding analysis. Removal of the ribityl 2ʹ-OH did not support turnover of O-methyl MIT, a substrate analog incapable of undergoing tautomerization.

Horseradish peroxidase (HRP) is a heme protein that acts specifically on H(2)O(2) as the electron acceptor. Hemin (Ferriprotoporhyrin-IX) is the prosthetic group of the enzyme. A direct molecular wire to the redox center of the enzyme is expected to enhance the electrochemical response of the enzyme. Native HRP was immobilized onto the surface of glassy carbon (GC) matrix using a 16-atom spacer arm. We have also immobilized the redox center of the enzyme (hemin) through one of the propionate groups onto the surface of glassy carbon matrix using an 11-atom spacer arm with amino terminus. Apoperoxidase was isolated according to the Teale's method and was allowed to reconstitute with the hemin-bound matrix for enzyme reconstitution. The HRP paste and reconstituted-HRP (rec-HRP) paste electrodes were used to study the electrochemical response to substrate H(2)O(2) using electrochemical techniques like cyclic voltammetry (CV) and flow injection (FI) studies. Flow injection studies using HRP paste electrode showed a linearity from 25 to 200 microM H(2)O(2). The rec-HRP paste showed approximately 100 times increase in the electron transfer rates compared to native HRP paste, and substrate linearity from 25 to 100 microM was observed.

In this study, we found that the full-length CYP2C8 (WT CYP2C8) and N-terminal truncated splice variant 3 (∼ 44-kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the x-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues variant 3 possessed nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, whereas variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854-Da substrate. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.

Reconstitution of enzymes onto surface tethered cofactors may be considered as a powerful tool both for design of biorecognition interfaces with a molecular-level control over spatial distribution and orientation of biosensing elements and for fundamental studies of their structure–function relationships. Here, reconstitution of horseradish peroxidase (HRP) on the heme cofactor covalently attached to the mixed self-assembled monolayers (SAM) on gold electrodes was studied. The efficiency of the holoenzyme formation was assessed by analysis of the hemin electrode reaction and bioelectrocatalytic activity of HRP. The formal potential, E 0 ′, of the electrode-tethered hemin approached −340 mV vs. Ag/AgCl, while the heterogeneous electron transfer (ET) rate constant, k s , for its redox transformation ranged between 245 and 340 s −1 , depending on the SAM composition. After enzyme reconstitution, both the E 0 ′ and k s values for the hemin ET reaction showed a very little variation, while the onset of electrocatalytic reduction of H 2 O 2 shifted by ca. 500 mV more positive by this approaching the potentials of compound I of native HRP. Both the onset potential and bioelectrocatalytic currents were lower than observed with native HRP adsorbed on gold (Ferapontova and Gorton, 2001), being consistent with a lower surface population of the holoenzyme and/or less efficient ET pathway.

The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 is an (αβ) 2 heterotetramer of two nonidentical subunits. These subunits are derived from nascent polypeptides that are cleaved proteolytically between Gly198 and Ser199 after the nascent polypeptides have been translocated into the periplasm. The activation mechanism of the GL-7-ACA acylase has been analyzed by both in vivo and in vitro expression studies, site-directed mutagenesis, in vitro renaturation of inactive enzyme precursors, and enzyme reconstitution. An active enzyme complex was found in the cytoplasm when its translocation into the periplasm was suppressed. In addition, the in vitro-expressed GL-7-ACA acylase was processed into α and β subunits, and the inactive enzyme aggregate of the precursor was also processed and became active during the renaturation step. Mutation of Ser199 to Cys199 and enzyme reconstitution allowed us to identify the secondary processing site that resides in the α subunit and to show that Ser199 of the β subunit is essential for these two sequential processing steps. Mass spectrometry clearly indicated that the secondary processing occurs at Gly189-Asp190. All of the data suggest that the enzyme is activated through a two-step autocatalytic process upon folding: the first step is an intramolecular cleavage of the precursor between Gly198 and Ser199 for generation of the α subunit, containing the spacer peptide, and the β subunit; the second is an intermolecular event, which is catalyzed by the N-terminal Ser (Ser199) of the β subunit and results in a further cleavage and the removal of the spacer peptide (Asp190 to Gly198).

A monomercury derivative of fluoresceine acetate (FMMA) was previously suggested as a specific reagent reacting with only one of four cysteine (Cys) residues in the α subunit of Escherichia coli RNA polymerase. Here, we analyzed the reactivity against FMMA of both isolated α subunit and α subunit assembled in the holoenzyme. In both cases, the highest reactivity was identified for Cys-269 positioned in the regulatory helix of C-terminal domain (CTD) which includes the contact sites for both class-I transcription factors and DNA UP elements. Substitution of Ala for both Cys-269 and Cys-176 completely eliminates the reactivity of α subunit against the fluorescent dye, supporting the prediction that another reactive amino acid under native conformation is Cys-176, which is positioned within or near the region important for α dimerization and its binding of β' subunit. In the isolated α subunit, the reactivity against FMMA is different between these two Cys residues and the order is from Cys-269 to Cys-176. Mutant α-subunits, bearing only one Cys residue at either 269 or 176, could be reconstituted into locally modified and active enzymes. This FMMA modification system may provide a tool suitable for studies of intra- and intermolecular interactions of this subunit. Proteins 30:183–192, 1998. © 1998 Wiley-Liss, Inc.

Recently we showed that the beta-naphthoflavone-inducible liver mitochondrial P450MT2 consists of two N-terminal truncated forms of the microsomal P4501A1, termed P450MT2a (+5/1A1) and MT2b (+33/1A1) [Addya et al. (1997) J. Cell Biol. 139, 589-599]. In the present study, we demonstrate that intact P4501A1 and the major mitochondrial form, P450MT2b (routinely referred to as P450MT2), show distinct substrate specificities and preference for different electron transport proteins. Enzyme reconstitution and spectral studies show that the wild-type adrenodoxin (Adx), but not the mutant Adx, binds to P450MT2 in a functionally productive manner (Kd = 0.6 microM) and induces a characteristic high-spin state. Adx binding to intact P4501A1 or +5/1A1 is less efficient as seen from spectral shift patterns (Kd = 1.8-2.0 microM) and reconstitution of enzyme activity. Use of Adx--Sepharose affinity matrix yielded < 90% pure P450MT2 (specific activity: 13.5 nmol/mg of protein) starting from a partially purified fraction of 10-15% purity, further demonstrating the specificity of P450MT2 and Adx interaction. Chemical cross-linking studies show that the bovine Adx forms heteroduplexes with both P450MT2 and intact P4501A1, though at different efficiencies. Our results show that Adx interacts with P450MT2 through its C-terminal acidic domain 2, while interaction with intact P4501A1 likely involves the N-terminal acidic domain 1. These results point to an interesting possibility that different electron transfer proteins may differently modulate the enzyme activity. Our results also demonstrate for the first time as to how a different mode of Adx interaction differently modulates the substrate specificities of the two P450 forms.

Glutamic acid 286 (E286; Escherichia coli cytochrome bo 3 numbering) in subunit I of the respiratory heme-copper oxidases is highly conserved and has been suggested to be involved in proton translocation. We report a technique of enzyme reconstitution that yields essentially unidirectionally oriented cytochrome bo 3 vesicles in which proton translocation can be measured. Such experiments are not feasible in the E286Q mutant due to strong inhibition of respiration, but this is not the case for the mutants E286D and E286C. The reconstituted E286D mutant enzyme readily translocates protons whereas E286C does not. Loss of proton translocation in the D135N mutant, but not in D135E or D407N, also is verified using proteoliposomes. Stopped-flow experiments show that the peroxy intermediate accumulates in the reaction of the E286Q and E286C mutant enzymes with O 2 . We conclude that an acidic function of the 286 locus is essential for the mechanism of proton translocation.

The steroidogenic activities in non-hyperfunctioning adrenal adenoma (NHFA) have not yet been fully examined. Steroid content both in adenoma and its non-tumorous adjacent adrenal tissue was measured by the HPLC system in 8 cases of NHFA and 2 cases of preclinical Cushing's syndrome (PCS). Activities of cytochrome P450c17alpha, P450c21, P450c11beta, P450c18 and aldosterone-synthesizing enzyme (P450aldo) were also determined by the enzyme reconstitution system. Steroid content in NHFA varied but no significant difference was seen between NHFA and normal control adrenals. Activities of all P450s except P450aldo were confirmed in NHFA, but they were almost equivalent to those in normal control adrenals. As no significant difference between NHFA and normal controls was observed in either steroid contents or P450s activities, the steroidogenic activity of NHFA is considered to be comparable to that of normal adrenals.

1. An αβ heterodimer of the F1-ATPase of Rhodospirillum rubrum (RF1) was isolated from extracts of chromatophores. This fragment of RF1 is highly active in restoring ATP hydrolysis and ATP synthesis to LiCl-treated chromatophores, maximal activity being reached at approximately 2 mol αβ per original RF1 molecule. Both α and β subunits of the heterodimer bind to chromatophore membranes on reconstitution. It was concluded that the αβ heterodimer was able to reconstitute RF1 in these membranes. 2. Reconstitutive activity in LiCl extracts purifies with the αβ heterodimer and away from the monomeric β subunit. Prolonged exposure to LiCl leads to irreversible dissociation of the heterodimer and loss of reconstitutive activity. It was concluded that the αβ heterodimer was required for reconstitution in LiCl-treated membranes, and that the monomeric β subunit was inactive. 3. The αβ heterodimer contains a non-catalytic nucleotide binding site. It can also catalyse CaATP and MgATP hydrolysis at similar rates, with K m values similar to RF1. However, hydrolysis is insensitive to activators/inhibitors of RF1 such as sulphite, octyl glucoside and azide. It was concluded that the isolated heterodimer represents a functional unit of RF1 but that, as it has a single catalytic site, catalytic cooperativity is absent.

Sphingolipids are intrinsic components of membrane lipid rafts. The abnormal accumulation of these molecules may introduce architectural and functional changes in these domains, leading to cellular dysfunction. Galactosylsphingosine (psychosine) is a pathogenic lipid raft-associated molecule whose accumulation leads to brain deterioration and irreversible neurological handicap in the incurable leukodystrophy Krabbe disease (KD). The relevance of clearing excessive levels of pathogenic psychosine from lipid rafts in therapy for KD has not been investigated. The work presented here demonstrates that psychosine inhibits raft-mediated endocytosis in neural cells. In addition, although in vitro enzyme reconstitution is sufficient for the reversal of related endocytic defects in affected neural cells, traditional in vivo enzyme therapies in the mouse model of KD appear to be insufficient for complete removal of pathogenic levels of raft-associated psychosine. This work describes a mechanism that may contribute to limiting the in vivo efficacy of traditional therapies for KD. V C 2010 Wiley-Liss, Inc.

Biofuel cells transform abundant raw materials into electrical power in the presence of biocatalysts, enzymes, or whole cell organisms. Biomaterials may participate in the biofuel cell activity by either producing fuel substrates or by catalyzing the electron transfer chain between the fuel substrates and oxidizers and the electrodes. Two types of biofuel cell elements are discussed in this review article: microbial-based biofuel cells and enzyme-based biofuel cells. Microorganisms may act as microreactors in fuel cells for the generation of the fuel products such as H2 or H2S. These fuel products may be generated apart from the biofuel cell and transported to its anodic compartment or, alternatively, may be directly generated in the anodic compartment of the biofuel cell. Different microbial-based biofuel cells are reviewed in the account. Enzymes are employed as catalysts for the activation of electron transfer chains between the fuel substrate and the anode in the anodic compartment, and between the oxidizer and the electrode in the cathodic compartment. In order to activate the electron transfer cascades between the enzymes and the electrodes, native electron carriers (co-factors) and artificial electron transfer mediators must be coupled to the biocatalytic transformations. By the nanoengineering of the electrode surfaces with co-factor/electron-relay/enzyme assemblies, integrated, electrically contacted, bioelectrocatalytic anodes and cathodes are tailored. Different enzyme-based biofuel cell configurations are described. The different parameters controlling biofuel cells efficiencies are discussed. The efficiencies of the different biofuel cell configurations are evaluated in terms of the limiting factors and kinetic features of the systems. Keywords: current–potential characteristic; cyclic voltammetry; electrocatalysis; electrode redox; electron transfer; electro–organic chemistry; fuel cells; current–voltage studies; biofuel cell; biocatalysis; biocatalyst; glucose oxidase; lactate dehydrogenase; cytochrome oxidase; apoenzyme; enzyme reconstitution; enzyme co-factor; electron transfer mediator; redox protein; cytochrome c

The Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1 have been implicated in the initial oxidation of high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs), forming cis-dihydrodiols. To clarify how these two RHOs are functionally different with respect to the degradation of HMW PAHs, we investigated their substrate specificities to 13 representative aromatic substrates (toluene, m-xylene, phthalate, biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, carbazole, and dibenzothiophene) by enzyme reconstitution studies of Escherichia coli. Both Nid systems were identified to be compatible with type V electron transport chain (ETC) components, consisting of a [3Fe-4S]-type ferredoxin and a glutathione reductase (GR)-type reductase. Metabolite profiles indicated that the Nid systems oxidize a wide range of aromatic hydrocarbon compounds, producing various isomeric dihydrodiol and phenolic compounds. NidAB and NidA3B3 showed the highest conversion rates for pyrene and fluoranthene, respectively, with high product regiospecificity, whereas other aromatic substrates were converted at relatively low regiospecificity. Structural characteristics of the active sites of the Nid systems were investigated and compared to those of other RHOs. The NidAB and NidA3B3 systems showed the largest substrate-binding pockets in the active sites, which satisfies spatial requirements for accepting HMW PAHs. Spatially conserved aromatic amino acids, Phe-Phe-Phe, in the substrate-binding pockets of the Nid systems appeared to play an important role in keeping aromatic substrates within the reactive distance from the iron atom, which allows each oxygen to attack the neighboring carbons., IMPORTANCE Since the discovery of microbial ring-hydroxylating oxygenases (RHOs) in 1970, the sequences, structures, and enzyme biochemistry, including enantiospecific products, of RHOs have been studied and discussed extensively from the perspective of biodegradation, biotransformation, and biocatalysis processes. However, with all that effort to elucidate the enzymatic mechanisms of RHOs, little is known about the biochemistry and enzymology underlying high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) degradation. We used Mycobacterium vanbaalenii PYR-1 Nid enzymes, the first type V RHO members to display an apparent substrate preference for HMW PAHs. Here, we examine the mechanism of the RHO reaction by integrating structural information of the NidAB and NidA3B3 enzymes with substrate and product data. This study gives us an understanding of how the model RHO systems of M. vanbaalenii PYR-1 metabolize HMW PAHs. The information obtained would also be helpful for successful application of RHO enzymes to the production of industrially and medically important chiral chemicals and the development of PAH bioremediation technologies.

An αβ heterodimer of the F 1 -ATPase of Rhodospirillum rubrum was isolated by extraction of chromatophores with LiCl. Each αβ heterodimer contains one tightly bound ADP, which is released upon removal of medium Mg 2+ . The dimer can be reversibly dissociated by removal of Mg 2+ -ions. The αβ heterodimer restores both ATP-synthetic and hydrolytic activities to LiCl-treated chromatophores, saturation being achieved at approximately 2 mmol αβ · mol BChl −1 . The heterodimer itself hydrolyses Mg-ATP with an activity distinct from RF 1 , being unaffected by azide or sulphite ions. The V max and K m (ATP) for this Mg 2+ -dependent activity were 110 ± 10 nmol · min −1 mg protein −1 and 100 ± 30 μM, respectively. The K m did not differ significantly from that of RF 1 .

During the transition of Escherichia coli cultures from exponential growth to stationary phase, the pre-existing RNA polymerase was found to be converted into at least three different holoenzyme forms, which could be separated by phosphocellulose column chromatography. The relative levels of these three holoenzyme forms changed depending on the phase of cell growth. The altered stationary phase forms of RNA polymerase showed promoter recognition properties that were different from those of the holoenzyme from exponentially growing cells. Enzyme reconstitution experiments showed that the altered promoter selectivity was due to modification of the core enzyme. We propose that modulation of RNA polymerase plays a role in the global switch of gene expression during the transition from exponential growth to stationary phase.

The proton-translocating ATP-synthase of chloroplasts CF0F1, was isolated and reconstituted into asolectin liposomes. CF0F1 can exist in at least four different states, oxidized or reduced, either inactive or active. These states are characterized by different kinetics of ADP binding: There is no binding of ADP to the inactive, oxidized state, the rate constant for ADP binding to the inactive, reduced state is 7 · 102M−1 · s−1. ADP binding to the active, reduced state occurs under deenergized conditions with 105 M−1 · s−1 and transforms the enzyme into the inactive, reduced state. Parallel to the ADP-dependent inactivation, the enzyme can also inactivate without ADP binding with a first-order rate constant of 7 · 10−3M−1 · s−1. With the active, reduced enzyme ATP-hydrolysis was measured under uni-site conditions as has been carried out with MF1 (Grubmeyer, C., Cross, R.C. and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092–12100). The rate constant for ATP binding is 106 M−1 · s−1, the ‘equilibrium constant’ on the enzyme EADPP i EATP is 0.4. The rate constants for Pi release and ADP release are 0.2 s−1 and 0.1 s−1, respectively. This indicates that the enzyme carries out a complete turnover under uni-site conditions with rates much higher than that reported for MF1.

Intramembrane proteolysis catalyzed by rhomboid proteases plays key roles in such diverse cell communication events as receptor tyrosine kinase signalling during animal development, and quorum sensing during bacterial growth. In these contexts, rhomboid proteins act in the signal-sending cell to activate signal precursor proteins and initiate the signalling event. Recent biochemical advances have culminated in the first high-resolution crystal structures of an intramembrane protease, and a pure enzyme reconstitution system for studying rhomboid activity. Functional studies have expanded the cellular role of rhomboid proteins to broad biological processes, including host-cell invasion by malaria parasites, which is the first implication of these enzymes as possible therapeutic targets in human disease

Spirulina-acyl-lipid desaturases are membrane-bound enzymes found in thylakoid and plasma membranes. These enzymes carry out the fatty acid desaturation process of Spirulina to yield gamma-linolenic acid (GLA) as the final desaturation product. In this study, Spirulina-Delta(6) desaturase encoded by the desD gene was heterologously expressed and characterized in Saccharomyces cerevisiae. We then conducted site-directed mutagenesis of the histidine residues in the three histidine boxes to determine the role of these amino acid residues in the enzyme function. Our results showed that while four mutants showed complete loss of Delta(6)-desaturase activity and two mutants showed only trace of the activity, the enzyme activity could be partially restored by chemical rescue using exogenously provided imidazole. This study reveals that the histidine residues (which have imidazole as their functional group) in the conserved clusters play a critical role in Delta(6)-desaturase activity, possibly by providing a di-iron catalytic center. In our previous study, this enzyme was expressed in Escherichia coli. The results reveal that the enzyme can function only in the presence of an exogenous cofactor, ferredoxin, provided in vitro. This evidence suggests that baker's yeast has a cofactor that can complement ferredoxin, thought to act as an electron donor for the Delta(6) desaturation in cyanobacteria, including Spirulina. The electron donor of the Spirulina-Delta(6) desaturation in yeast is more likely to be cytochrome b(5), which is absent in E. coli. This means that the enzyme expressed in S. cerevisiae can catalyze the biosynthesis of the product, GLA, in vivo.

In this paper, the reconstitution of Na,K-ATPase in liposomes (formed by single or mixed phospholipids and cholesterol) was investigated and the enzyme orientation was determined on kinetic basis using only specific inhibitors of ATP hydrolysis. A condition of foremost importance for enzyme reconstitution is the achievement of complete solubilization of the lipid in the initial stage of the cosolubilization process for the subsequent formation of the liposomes and/or proteoliposomes. PC-liposomes showed that increasing the fatty acid chain length increases the percentage of Na,K-ATPase incorporated. The average diameter of the proteoliposomes also increases in proportion, reaching a maximum with phospholipids with 16 carbon chains, resulting in 75.1% protein reconstitution and 319.4 nm diameter size, respectively. Binary lipid systems with PC and PE were efficient for incorporation of Na,K-ATPase, depending on the lipid:protein ratio used, varying from 15 to 80% recovery of total ATPase activity. The best results for Na,K-ATPase reconstitution using PC and PE mixture were obtained using a lipid:lipid ratio 1:1 (w/w) and lipid:protein 1:3 (w/w). Integrity studies using calcein release mediated by detergent or alamethicin, in association with inhibition of ATPase activity (ouabain and vanadate) showed that the enzyme is oriented inside-out in DPPC:DPPE proteoliposomes. In these vesicular systems, the enzyme is reconstituted with about 78.9% ATPase activity recovery and 89% protein incorporation, with an average diameter of 140 nm. Systems constituted by DPPC:DPPE, DPPC:DLOPE or DLOPC:DLOPE showed approximately 80, 71 and 70% of recovery of total ATPase activity, but no homogeneity in the distribution of Na,K-ATPase orientation. Reconstitution of Na,K-ATPase in DPPC:DPPE:cholesterol or DPPC:DLOPE:cholesterol systems (55% of cholesterol) showed recovery of about 86 and 82%, respectively, of its total ATPase activity. The results point to an important effect of the lipid acyl chain length and lipid-protein ratio in relation to the composition of the lipid matrix to finely tune the structural asymmetry and the amount of enzyme that can be incorporated a lipid bilayer vesicle while preserving membrane permeability.

CYP1A is known to play important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics in animals including fish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7- methoxyresorufin in a reconstituted system containing purified fish P450 reductase and lipid. In addition, effects of each individual components of the reconstituted system, i.e., CYP1A1 and P450 reductase on 7- methoxyresorufin O-demethylase (MROD) activity were studied. 7- ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by ?- naphthoflavone (ANF). At 0.5 and 2.5 µM, ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline, ethylmorphine, N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cross-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD activities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 antibodies showed very weak cross-reactivity with two proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver microsomal EROD activity was weakly inhibited by the anti-L. saliens liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues purified from other teleost species but functionally and immunochemically distinct from mammalian CYP1A1. (C) 2000 Elsevier Science Inc.

Development of an effective therapy to prevent prostate cancer (PCa) progression to castrate-resistant PCa (CRPCa) remains an unmet medical need, mainly due to a poor understanding of the mechanism of PCa progression. Reactive oxygen species (ROS) are produced in high amounts in PCa cells. ROS play a major role in the development and progression of PCa. We have published that the activated androgen receptor (AR)-JunD complex induces spermidine/spermine N1-acetyl transferase (SSAT). SSAT is the first, and a regulatory, enzyme in a major polyamine catabolism pathway that leads to an over-production of ROS, specifically in the polyamine-rich PCa cells. A focus of our current research is to identify compounds that specifically target and block steps in this newly discovered ROS production pathway downstream to AR activation. Such compounds have the potential to become new targeted therapeutic agents offering minimal side effects for preventing progression to CRPCa in patients with early stage progressing PCa. Here we present data on drug-like small molecule inhibitors of the AR-JunD interaction that initiates this ROS-generating pathway in PCa. A novel high throughput assay based on Gaussia Luciferase enzyme reconstitution via protein-protein interaction was used to screen NCI Diversity Set and Life Chemicals libraries of drug-like small molecules to identify inhibitors of the AR-JunD interaction. The 27 selected hits were categorized as antiandrogens or non-antiandrogens through the use of a published fluorescence polarization assay. Sixteen non-antiandrogens, acting downstream of AR activation, were chosen for further study. In vitro studies entailed the measurement of the ability of the compounds to inhibit androgen-independent and dependent growth as well as androgen-induced ROS in human PCa cells. The top four compounds exhibited significant inhibition of growth and androgen-induced ROS at concentrations of 5 uM or less. Immunocytochemistry (ICC) studies were performed to determine the ability of the selected compounds to specifically inhibit the AR-JunD co-translocation to the nuclei. Based on the cell culture and ICC data, lead compounds were selected for pharmacokinetics studies to determine oral bioavailability. Detailed pharmacokinetic data of selected top compounds showing their bioavailability after intra venous and oral administration will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2844. doi:1538-7445.AM2012-2844

Rhomboid proteins are membrane-immersed enzymes with the remarkable ability to hydrolyze protein segments within the membrane. This form of intramembrane proteolysis plays key roles in cell communication including EGF signalling during animal development, and quorum sensing during bacterial growth. We have been investigating rhomboid protease mechanism using a pure enzyme reconstitution system that we developed, coupled with high-resolution crystal structures of a model bacterial rhomboid protease (1, 2). Intramembrane proteolysis is catalyzed within a hydrated cavity that opens to the outside of the cell and allows water to enter, but is protected laterally from membrane lipids by a ring of protein segments. Using a structure-function approach, we subsequently identified one transmembrane helix and overlying loop that provide a rate-limiting movement to gate substrate entry laterally from the membrane (3). By integrating reconstitution into defined bilayer environments with spectroscopic interrogation, we recently discovered that rhomboid proteases rely on the natural biophysical constraints of the membrane to achieve site-specific proteolysis. Since rhomboid proteins play critical roles in the infective cycles of several protozoan parasites (4,5,6), understanding the protease mechanism may provide therapeutic opportunities (7).1. Urban, S. and M.S. Wolfe. (2005). Proc. Natl. Acad. Sci. USA, 102(6):1883-1888.2. Wu Z., Yan N., Feng L., Oberstein A., Yan H., Baker R. P., Gu L., Jeffrey P.D., Urban S., and Y. Shi. (2006). Nature Structural and Molecular Biology 13: 1084-1091.3. Baker, R.P., Young K., Feng L., Shi Y. and S. Urban. (2007). Proc. Natl. Acad. Sci. USA, 104: 8257-62.4. Baker, R.P., Wijetilaka R, and S. Urban. (2006). PLoS Pathogens 10(2): e113; 922-32.5. Baxt, L., Baker, R.P., Singh, U. and S. Urban. (2008). Genes & Development 22(12): 1636-46.6. Urban S. (2009). Nature Reviews Microbiology 7: 411-23.7. Urban S. (2010). The Biochemical Journal 425(3): 501-512.

Development of an effective therapy to prevent prostate cancer (PCa) progression to castrate-resistant PCa (CRPCa) remains an unmet medical need, mainly due to a poor understanding of the mechanism of PCa progression. Reactive oxygen species (ROS) are produced in high amounts in PCa cells and play a major role in PCa development and progression. We have published that activated androgen receptor (AR)-JunD complex induces spermidine/spermine N1-acetyl transferase (SSAT), the first and regulatory enzyme in a major polyamine catabolism pathway that yields over-production of ROS specifically in the polyamine-rich PCa cells. Our recent data further suggest an intriguing mechanism of PCa progression, where AR-JunD induced SSAT expression and consequent upregulation of the transcription factor NF-κB may set up an autocrine feed forward loop of SSAT-ROS-NFκB-SSAT that can sustain ROS production and PCa cell proliferation in the absence of androgen. A focus of our current research is to identify compounds that specifically target and block steps in this novel pathway downstream to AR activation and thereby have potential to be new targeted therapeutic agents to prevent PCa progression to CRPCa in early stage progressing PCa patients with minimal side effects. In the research presented here we utilized a novel high throughput screen (HTS) assay to find compounds that prevent the initiating AR-JunD interaction step in this ROS generating pathway and tested their efficacy in PCa cells. A high throughput assay based on Gaussia Luciferase enzyme reconstitution via protein-protein interaction was used to screen the NCI diversity set library of drug like molecules to identify inhibitors of the AR-JunD interaction. Selected hits were further screened to determine their ability to bind to the AR using a published fluorescence polarization assay in order to categorize the molecules as non-antiandrogens or antiandrogens. As we intend to target the pathway downstream of AR activation, we focused on non-antiandrogens, further testing them for efficacy against androgen induced ROS generation and effect on cell growth in PCa cells in vitro by our published DCFH dye oxidation and DNA fluorescence assays. Of the 14 hits from the HTS assay of the NCI diversity set library, nine small molecules were chosen based on their drug-like chemical characteristics for further studies along with two synthesized analogs of one of the selected small molecules. We have categorized six agents as non-antiandrogens and five as having some antiandrogenic activity. Our ROS assay identified a lead non-antiandrogen compound that blocks androgen induced ROS production in PCA cells at less than 1uM concentration. This agent also significantly inhibited androgen-independent growth of PCa cells at 4uM. Data for all compounds will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2583. doi:10.1158/1538-7445.AM2011-2583

Soluble (Na++K+)-ATPase consisting predominantly of αβ-units with Mr below 170 000 was prepared by incubating pure membrane-bound (Na++K+)-ATPase (35–48 μmol Pi/min per mg protein) from the outer renal medulla with the non-ionic detergent dodecyloctaethyleneglycol monoether (C12E8). (Na++K+)-ATPase and potassium phosphatase remained fully active in the detergent solution at C12E8/protein ratios of 2.5–3, at which 50–70% of the membrane protein was solubilized. The soluble protomeric (Na++K+)-ATPase was reconstituted to Na+, K+ pumps in phospholipid vesicles by the freeze-thaw sonication procedure. Protein solubilization was complete at C12E8/protein ratios of 5–6, at the expense of partial inactivation, but (Na++K+)-ATPase and potassium phosphatase could be reactivated after binding of C12E8 to Bio-Beads SM2. At C12E8/protein ratios higher than 6 the activities were irreversibly lost. Inactivation could be explained by delipidation. It was not due to subunit dissociation since only small changes in sedimentation velocities were seen when the C12E8/protein ratio was increased from 2.9 to 46. As determined immediately after solubilization, S20,w was 7.4 S for the fully active (Na++K+)-ATPase, 7.3 S for the partially active particle, and 6.5 S for the inactive particle at high C12E8/protein ratios. The maximum molecular masses determined by analytical ultracentrifugation were 141 000–170 000 dalton for these protein particles. Secondary aggregation occurred during column chromatography, with formation of enzymatically active (αβ)2-dimers or (αβ)3-trimers with S20,w=10–12 S and apparent molecular masses in the range 273 000–386 000 daltons. This may reflect non-specific time-dependent aggregation of the detergent micelles.

Ethanolaminephosphotransferase (CDPethanolamine; 1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) has been purified in active form from rat brain microsomes by a two-step chromatographic procedure. Enzyme preparations characterized by high specific activity and stability were obtained supplementing the solubilization and elution buffers, containing 1% Triton X-100, with 0.01% 2,6-di-tert-butyl-4-methylphenol. The specific activity of the purified enzyme was about 1200-times higher than that of the crude solubilized enzyme. The lipid dependence of ethanolaminephosphotransferase was studied both in the presence of Triton X-100 and in detergent-free enzyme preparations. The activity of the detergent-solubilized ethanolaminesphosphotransferase was strongly modulated by phospholipids. The kinetic behaviour of the enzyme was also dependent on the lipids contained in the aggregates obtained by removal of the detergent from detergent/lipid/protein suspensions. A regulatory role of phospholipids on the activity of the membrane-bound ethanolaminephosphotransferase is discussed.

A P-450, designated P-450a, with high testosterone 7 alpha-hydroxylase activity was purified from rat liver microsomes. Specific polyclonal antibody against this P-450 was used to screen a lambda gt11 expression cDNA library and a 1687-base pair cDNA was isolated and sequenced. The deduced protein had 492 amino acids, a calculated Mr of 56,016, and it shared 51 and 45% amino acid similarities to P-450e and P-450f, respectively. Regions of similarity were distributed in distinct areas of high and low similarity along the P-450a primary sequence. P-450a cDNA was introduced into yeast cells using the expression vector pAAH5, and the resultant yeast microsomes contained both a protein of identical electrophoretic mobility to that of rat P-450a and testosterone 7 alpha-hydroxylase activity. These results confirm enzyme reconstitution data and antibody inhibition data that P-450a possesses testosterone 7 alpha-hydroxylase activity. The antibody and cDNA probes were used to examine the mechanism of regulation of P-450a by inducers and during development. P-450a and its mRNA were present at low level in newborn rats and increased to maximal level at 1 week of age in both males and females. At age 12 weeks, however, the P-450a level decreased in males but remained elevated in females. Concomitant with the decrease in P-450a in adult males was an increase in level of another immunologically related P-450. In adult male rats, P-450a was induced almost 5-fold by administration of 3-methylcholanthrene and this induction was the result of an increase in its mRNA. These results establish testosterone 7 alpha-hydroxylase as a member of the P-450e gene family that is developmentally regulated, sex-dependent, and markedly inducible by 3-methylcholanthrene.

This review will attempt to cover the present information on the multiple forms of eukaryotic DNA-dependent RNA polymerases, both at the structural and functional level. Nuclear RNA polymerases constitute a group of three large multimeric enzymes, each with a different and complex subunit structure and distinct specificity. The review will include a detailed description of their molecular structure. The current approaches to elucidate subunit function via chemical modification, phosphorylation, enzyme reconstitution, immunological studies, and mutant analysis will be described. In vitro reconstituted systems are available for the accurate transcription of cloned genes coding for rRNA, tRNA, 5 SRNA, and mRNA. These systems will be described with special attention to the cellular factors required for specific transcription. A section on future prospects will address questions concerning the significance of the complex subunit structure of the nuclear enzymes; the organization and regulation of the gene coding for RNA polymerase subunits; the obtention of mutants affected at the level of factors, or RNA polymerases; the mechanism of template recognition by factors and RNA polymerase.

Temperature-sensitive mutants of Escherichia coli that are unable to grow at high temperature can be obtained among those selected for resistance to streptovaricin or rifampicin at low temperature (Yura et al., 1973). One of these mutants (KY5323) that was supposed to carry a single mutation affecting both rifampicin resistance and temperature sensitivity was further investigated. Using purified RNA polymerase preparations obtained from the mutant and the wild type, it was found that the activity for RNA chain elongation is more sensitive to heat treatment than that for RNA chain initiation or DNA binding, and that the mutant enzyme is significantly more labile than the wild-type enzyme with respect to RNA chain elongation, when heat treatment is carried out at high salt concentration. These results, taken together with those of the enzyme reconstitution experiments, strongly suggest that the beta subunit of the polymerase is directly involved in both RNA chain initiation and elongation reactions. Enzyme reconstitution experiments using isolated subunits derived from the mutant and the wild-type polymerases demonstrate that the alteration of beta subunit is primarily responsible for both rifampicin resistance and thermolability of the mutant enzyme. In addition, the results suggested the apparent alteration of both beta and alpha subunits in this mutant. Extensive transduction experiments provided genetic evidence that are consistent with the view that the strain KY5323 carries a second mutation affecting the beta subunit, beside the primary mutation affecting the beta subunit. The hypothetical beta subunit mutation seems to modify quantitatively the rifampicin resistance caused by the beta subunit mutation.

The membrane-bound coupling factor (BF 1 ) of chromatophores from the photosynthetic bacerium Rhodopseudomonas sphaeroides was covalently labeled with the triplet probe eosin-isothiocyanate. The labeled enzyme was isolated and functionally reconstituted into depleted chromatophores from the same bacterium. ATPase and ATP synthase activities of the reconstituted vesicles were strongly dependent on the labeling conditions, decreasing at increasing load of eosin molecules per BF 1 . When labeling was carried out in the dark and in the presence of ATP, one molecule of eosin isothiocyanate was bound per BF 1 and the activities catalyzed by the reconstituted and labeled enzyme were as high as in untreated chromatophores. Upon light energization of the chromatophore membrane, a large conformational change of BF 1 could be detected by using the triplet probe as a spectroscopic tool. The domain flexibility and rotational mobility of the reconstituted coupling enzyme were directly related to the enhancement of the ATPase activity induced by light. Both the light-stimulated ATPase activity and conformational changes could be prevented by addition of ADP or oligomycin and affected to the same extent by uncouplers and inhibitors of electron transport. Moreover, the detected conformational changes were reversible in time, appearing with a half-time of 10 ms upon illumination of the chromatophores, and disappearing with a half-time of 70 ms in the dark. The results obtained prove the feasibility of the spectroscopic technique in detecting conformational changes of the membrane-bound BF 1 , similarly to what already has been observed for chloroplast coupling factor (Wagner, R. and Junge, W. (1980) FEBS Lett. 114, 327–333), and add to the possibility of characterizing, by this method, energy transduction at a molecular level.

Several known forms of bilayer vesicles of dimyristoylphosphatidylcholine exhibit the gel to liquid-crystalline phase transition in the temperature range convenient for membrane enzyme reconstitution studies. This warrants a systematic investigation of their physical characteristics and their phase transition behaviors. We have employed electron microscopy, gel chromatography, 31P nuclear magnetic resonance, differential scanning microcalorimetry, and fluorescence spectroscopy to determine several physical parameters of the limiting size microvesicle (260 +/- 40 A), the larger vesicle form (900 +/- 100A) of Enoch and Strittmatter [Enoch, H. G., & Strittmatter, P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 145], the multilamellar vesicle, and, in particular, an ATPase-trigger-fused macrovesicle (950 +/- 200 A). This latter vesicle form was produced by a spontaneous fusion of the complex of the plasma membrane ATPase of Schizosaccharomyces pombe and the lipid microvesicles at a low ratio of enzyme to vesicle concentrations, and at a low temperature (around 10 degrees C). The ATPase-trigger-fused vesicles are unilamellar and have an intact ionic permeation barrier at 30 degrees C and a gel to liquid-crystalline transition temperature at 24.4 degrees C with a transition heat of 5.64 kcal/mol. Thus, this vesicle form should be a valuable tool for studying possible proton-pumping activity of this ATPase. In contrast to data found in the literature, which show lack of the pretransition for unilamellar microvesicles, we have observed the pretransition around 15 degrees C for all the vesicle forms examined. Moreover, the transition widths of unilamellar vesicles are much broader than those of the multilamellar vesicles, suggesting that in the latter system interlayer interactions may contribute to the cooperativity of the transition.