Higher Detergent Concentrations Research Articles

Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast

The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

Aberrant Folding of a Mutant Stat5b Causes Growth Hormone Insensitivity and Proteasomal Dysfunction

A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were approximately 3-fold higher than Stat5bA630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5bA630P was <15% of Stat5bWT (3.5 h versus >24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5bA630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5bA630P could produce additional defects through inhibition of proteasome function.

A Highly Conserved Amino-terminal Region of Sonic Hedgehog Is Required for the Formation of Its Freely Diffusible Multimeric Form

Although members of the Hedgehog (Hh) family were initially described as morphogens, many of these early conclusions were based on experiments that used non-physiologically relevant forms of Hh. Native Hh is modified by cholesterol (HhNp) and palmitate. These hydrophobic modifications are responsible for the ability of Hh to associate with cellular membranes, a property that initially appeared inconsistent with its ability to act far from its site of synthesis. Although it is now clear that Hh family members are capable of acting directly in long-range signaling, the form of Hh capable of this activity remains controversial. We have previously provided evidence for a freely diffusible multimeric form of Sonic Hedgehog (Shh) termed s-ShhNp, which is capable of accumulating in a gradient fashion through a morphogenic field. Here, we provide further evidence that s-ShhNp is the physiologically relevant form of Shh. We show that the biological activity of freely diffusible ShhNp resides in its multimeric form and that this multimeric form is exceedingly stable, even to high concentrations of salt and detergent. Furthermore, we now validate the Shh-Shh interactions previously observed in the crystal structure of human Shh, showing that a highly conserved amino-terminal domain of Shh is important for the formation of s-ShhNp. We also conclusively show that palmitoylation is required for s-ShhNp formation. Thus, our results identify both protein-protein and protein-lipid interactions that are required for s-ShhNp formation, and provide the first structural analyses supporting the existence of Shh multimers.

Versatile Interactions of the Antimicrobial Peptide Novispirin with Detergents and Lipids

Novispirin G-10 is an 18-residue designed cationic peptide derived from the N-terminal part of an antimicrobial peptide from sheep. This derivative is more specific for bacteria than the parent peptide. We have analyzed Novispirin's interactions with various amphipathic molecules and find that a remarkably wide variety of conditions induce alpha-helical structure. Optimal structure induction by lipids occurs when the vesicles contain 40-80% anionic lipid, while pure anionic lipid vesicles induce aggregation. SDS also forms aggregates with Novispirin at submicellar concentrations but induces alpha-helical structures above the cmc. Both types of aggregates contain significant amounts of beta-sheet structure, highlighting the peptide's structural versatility. The cationic detergent LTAC has a relatively strong affinity for the cationic peptide despite the peptide's net positive charge of +7 at physiological pH and total lack of negatively charged side chains. Zwitterionic and nonionic detergents induce alpha-helical structures at several hundred millimolar detergent. We have solved the peptide structure in SDS and LTAB by NMR and find subtle differences compared to the structure in TFE, which we ascribe to the interaction with an amphiphilic environment. Novispirin is largely buried in the SDS-micelle, whereas it does not enter the LTAC-micelle but merely forms a dynamic equilibrium between surface-bound and nonbound Novispirin. Thus, electrostatic repulsion can be overruled by relatively high-detergent concentrations or by deprotonating a single critical side chain, despite the fact that Novispirin's ability to bind to amphiphiles and form alpha-helical structure is sensitive to the electrostatics of the amphiphilic environment. This emphasizes the versatility of cationic antimicrobial peptides' interactions with amphiphiles.

Analysis of proteins from a glioma cell line by using micro‐scale solution isoelectric focusing in combination with liquid chromatography/tandem mass spectrometry

In this study we have investigated whether micro-solution isoelectric focusing (microsol-IEF) can be used as a pre-fractionation step prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS) and if extensive sample purification of the different fractions is required. We found that, in spite of the high concentrations of buffer and detergents, no clean up of the digested microsol-IEF fractions was necessary before analysis by LC/MS/MS. We also concluded that it is possible to identify at least twice as many proteins in a glioma cell lysate with the combination of microsol-IEF and LC/MS/MS than with LC/MS/MS alone. Furthermore, most of the proteins that were identified from one microsol-IEF fraction by using analytical narrow-range two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were also identified by LC/MS/MS. Finally, we used the combination of microsol-IEF and LC/MS/MS to compare two sample preparation methods for glioma cells and found that several nuclear, mitochondria, and endoplasmic reticulum proteins were only present in the sample that had been subjected to lipid extraction by incubating the homogenized cells in chloroform/methanol/water.

Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry

Characterization of membrane proteins remains an analytical challenge because of difficulties associated with tedious isolation and purification. This study presents the utility of the polyvinylidene difluoride (PVDF) membrane for direct sub-proteome profiling and membrane protein characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The hydrophobic adsorption of protein, particularly membrane proteins, on the PVDF surface enables efficient on-PVDF washing to remove high concentrations of detergents and salts, such as up to 5% sodium dodecyl sulfate (SDS). The enhanced spectrum quality for MALDI detection is particularly notable for high molecular weight proteins. By using on-PVDF washing prior to MALDI detection, we obtained protein profiles of the detergent-containing and detergent-insoluble membrane fractions from Methylococcus capsulatus (Bath). Similar improvements of signal-to-noise ratios were shown on the MALDI spectra for proteins electroblotted from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) onto the PVDF membrane. We have applied this strategy to obtain intact molecular weights of the particulate methane monooxygenase (pMMO) composed of three intrinsic membrane-bound proteins, PmoA, PmoB, and PmoC. Together with peptide sequencing by tandem mass spectrometry, post-translational modifications including N-terminal acetylation of PmoA and PmoC and alternative C-terminal truncation of PmoB were identified. The above results show that PVDF-aided MALDI-MS can be an effective approach for profiling and characterization of membrane proteins.

Reconstitution of the isolated phosphate-transport system of pig-heart mitochondria.

The phosphate carrier of pig heart mitochondria has been isolated and reconstituted in liposomes. The highest specific activity for [32P]phosphate exchange was obtained with hydroxyapatite eluate from mitochondria, extracted with Triton X-114 in the presence of cardiolipin. This fraction, which is free from the ADP ATP-carrier, had a specific activity of 30 mumol 32Pi x min-1 x mg protein -1. The following conditions were found to inactivate the phosphate carrier irreversibly in the solubilized state: high ionic strength, high detergent concentrations and a high pH. The decrease of the activity by high detergent concentrations can be largely prevented by cardiolipin, present in the extraction buffer, suggesting a specific removal of this lipid by the detergent. After reconstitution in liposomes, the phosphate carrier is rather stable. The Arrhenius plot of the temperature-dependence of the reconstituted phosphate exchange showed different slopes above and below 27 degrees C. Between 0 degrees C and 27 degrees C the EA was 64 kJ . mol-1, between 27 degrees C and 42 degrees C 44 kJ . mol-1. The exchange of Pi followed a first-order kinetic.

Endonucleases in Yeast Mitochondria

An apurinic endonuclease activity has been characterized in yeast mitochondrial. It is dependent on Mg2+, stimulated by about 50% in the presence of 50 mM NaCl and inhibited at higher NaCl concentrations. It is located in the inner mitochondrial membrane and requires high concentrations of detergent (1.5-3% Triton X-100) to be extracted. The same treatment extracts several other endonuclease activities: the two Mg2+-dependent endonuclease activities cleaving double-stranded DNA at pH 7.5 and 5.4 respectively, the ethidium-bromide-stimulated endonuclease activity, the endonuclease activity cleaving single-stranded DNA at pH 7.l5 [Jacquemin-Sablon et al. (1979) Biochemistry, 18, 119-127], and a manganese-stimulated deoxyribonuclease activity cleaving double-stranded DNA at pH 7.5 which has been discovered during the present work. Another endonuclease activity cleaving double-stranded DNA at pH 7.5 in the presence of Mg2+, slightly stimulated by low NaCl concentrations and inhibited by ethidium bromide is extracted from the membrane pellet remaining after the treatment with 1.5% Triton X-100 by a second treatment with 1.5% Triton X-100 plus 1 M KCl. The presence in the mitochondrial membrane of this apurinic endonuclease activity indicates that, like nuclear and prokaryotic DNA, yeast mitochondrial DNA is also subject to specialized repair systems.

Bacteriophage P22 Tail Accessory Factor GP26 Is a Long Triple-stranded Coiled-coil

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.

Activation, Inhibition, and Destabilization of Thermomyces lanuginosus Lipase by Detergents

Lipases catalyze the hydrolysis of triglycerides and are activated at the water-lipid interface. Thus, their interaction with amphiphiles such as detergents is relevant for an understanding of their enzymatic mechanism. In this study, we have characterized the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and thermal stability of Thermomyces lanuginosus lipase (TlL). For all detergents, low concentrations enhance the activity of TlL toward p-nitrophenyl butyrate by more than an order of magnitude; at higher detergent concentrations, the activity declines, leveling off close to the value measured in the absence of detergent. Surprisingly, these phenomena mainly involve monomeric detergent, as activation and inhibition occur well below the cmc for the nonionic and zwitterionic detergents. For anionic and cationic detergents, activation straddles the monomer-micelle transition. The data can be fitted to a three state interaction model, comprising free TlL in the absence of detergent, an activated complex with TlL at low detergent concentrations, and an enzyme-inhibiting complex at higher concentrations. For detergents with the same headgroup, there is an excellent correspondence between carbon chain length and ability to activate and inhibit TlL. However, the headgroup and number of chains also modulate these effects, dividing the detergents overall into three broad groups with rising activation and inhibition ability, namely, anionic and cationic detergents, nonionic and single-chain zwitterionic detergents, and double-chain zwitterionic detergents. As expected, only anionic and cationic detergents lead to a significant decrease in lipase thermal stability. Since nonionic detergents activate TlL without destabilizing the protein, activation/inhibition and destabilization must be independent processes. We conclude that lipase-detergent interactions occur at many independent levels and are governed by a combination of general and structurally specific interactions. Furthermore, activation of TlL by detergents apparently does not involve the classical interfacial activation phenomenon as monomeric detergent molecules are in most cases responsible for the observed increase in activity.

The caudal complex of Giardia lamblia and its relation to motility

This paper presents a detailed study of the caudal complex of Giardia lamblia and its relation to movements observed in this region. The caudal complex of Giardia, composed of axonemes from the caudal flagella plus associated microtubular sheets, was investigated by light, electron microscopy, and 3D reconstruction tools. By the use of video-microscopy and digital image processing techniques, we were able to visualize in detail the caudal movements. A non-ionic detergent, Triton X-100, was used to isolate the complex that was afterwards analyzed by video-microscopy and transmission electron microscopy (TEM). We showed for the first time, using video-microscopy, that the intracellular portion of the caudal flagella axonemes presented motility, even after the disrupture of the cell membrane, contrasting with the caudal flagella themselves, that do not show active beating pattern. To check if actin filaments play a role in the above described movements, as previously supposed, we incubated the cells with jasplakinolide, a drug that induces the disruption of actin filaments in living cells. The experiments demonstrated that the drug did not affect the caudal motility. The analysis of the caudal complex by transmission electron microscopy (TEM) revealed that, even after the exposure to higher detergent concentrations, the connections between their components remained intact. The information obtained by TEM and 3D reconstruction tools showed that the region between both nuclei marks the intracellular end of the caudal complex, which proceeds toward the caudal portion of the cell following its longitudinal axis, where the axonemes emerge as the caudal flagella. The results obtained from video-microscopy assays of the isolated beating complex together with the 3D reconstruction data indicated that the internal portion of the caudal flagella is the force-generator of the movements in this region.

Removal of detergent from protein fractions.

The solubilization of biological membranes by detergents plays an important role in the identification, characterization, and extraction of integral membrane proteins. The solubilization process involves a number of intermediate states starting with the desta-bilization of membrane lipids and followed by the creation of membrane fragments, which finally results in the formation of protomers (). The most commonly used detergents in the biological sciences are described in Chapter 29 and several review articles exist on solubilization procedures (, , ). Because the initial extraction of intrinisic membrane proteins usually involves high detergent concentrations, excess detergent has to be removed or exchanged for another type of detergent at later stages of preparative or analytical procedures involving the solubilized protein fraction. The efficiency of certain chromatographic procedures is often significantly improved by lowering the overall detergent concentration or by exchanging one type of detergent for another. Lectin chromatography, a powerful technique to affinity-purify subsets of glycoproteins (see Chapter 18), appears to be especially sensitive to high concentrations of a variety of detergents (). Furthermore, because high concentrations and/or certain types of detergent interfere with many physical and chemical analyses and detergents also exhibit undesirable side effects on highly sensitive cell biological assays, detergent removal is, in many cases, of central importance for retaining the biological activity of isolated membrane proteins. Another important area of detergent removal is reconstitution studies with hydrophobic membrane proteins that exhibit vectorial transport ().

Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods.

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.

New insights into the respiratory chain of plant mitochondria. Supercomplexes and a unique composition of complex II.

A project to systematically investigate respiratory supercomplexes in plant mitochondria was initiated. Mitochondrial fractions from Arabidopsis, potato (Solanum tuberosum), bean (Phaseolus vulgaris), and barley (Hordeum vulgare) were carefully treated with various concentrations of the nonionic detergents dodecylmaltoside, Triton X-100, or digitonin, and proteins were subsequently separated by (a) Blue-native polyacrylamide gel electrophoresis (PAGE), (b) two-dimensional Blue-native/sodium dodecyl sulfate-PAGE, and (c) two-dimensional Blue-native/Blue-native PAGE. Three high molecular mass complexes of 1,100, 1,500, and 3,000 kD are visible on one-dimensional Blue native gels, which were identified by separations on second gel dimensions and protein analyses by mass spectrometry. The 1,100-kD complex represents dimeric ATP synthase and is only stable under very low concentrations of detergents. In contrast, the 1,500-kD complex is stable at medium and even high concentrations of detergents and includes the complexes I and III(2). Depending on the investigated organism, 50% to 90% of complex I forms part of this supercomplex if solubilized with digitonin. The 3,000-kD complex, which also includes the complexes I and III, is of low abundance and most likely has a III(4)I(2) structure. The complexes IV, II, and the alternative oxidase were not part of supercomplexes under all conditions applied. Digitonin proved to be the ideal detergent for supercomplex stabilization and also allows optimal visualization of the complexes II and IV on Blue-native gels. Complex II unexpectedly was found to be composed of seven subunits, and complex IV is present in two different forms on the Blue-native gels, the larger of which comprises additional subunits including a 32-kD protein resembling COX VIb from other organisms. We speculate that supercomplex formation between the complexes I and III limits access of alternative oxidase to its substrate ubiquinol and possibly regulates alternative respiration. The data of this investigation are available at http://www.gartenbau.uni-hannover.de/genetik/braun/AMPP.

Human CYP1A1 allelic variants: baculovirus expression and purification, hydrodynamic, spectral, and catalytical properties and their potency in the formation of all- trans-retinoic acid

Three human cytochrome P450 1A1 (CYP1A1) allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V), and CYP1A1.4 (T461N), were expressed as C-terminal His-tagged fusions including a thrombin cleavage site in Spodoptera frugiperda insect cells by baculovirus infection. The variants were expressed with 30–90 nmol (1.8–5.4 mg) spectrally active cytochrome P450 per one liter of culture and purified to electrophoretic homogeneity by Ni–agarose chromatography. The recombinant variants were structurally characterized by UV/Vis, ultracentrifugation, and EPR. Optical and EPR spectra showed all three variants predominantly in high spin state; moreover, EPR indicated changes in the electronic structure of the heme iron of the two mutant variants. Sedimentation equilibrium experiments demonstrated the purified variants in dimeric state in the presence of 0.2% emulgen + 0.05% cholate. Higher detergent concentration, the presence of imidazole, and cleavage of the His-tag led to monomerization. Catalytic activity of all purified variants was reconstituted with purified human NADPH-P450 reductase and dilaurylphosphatidylcholine. Enzyme kinetics of ethoxyresorufin O-deethylation revealed similar K m ( ≈0.4 μM ) for all variants but slightly different V max values (CYP1A1.1: 4.2, CYP1A1.2: 7.0, and CYP1A1.4: 3.0 nmol/min/nmol CYP1A1). The extended C-terminus influenced the enzymatic activity only slightly. All three variants are able to produce significant amounts of all- trans-retinoic acid from all- trans-retinal with V max of 4.0, 3.3, and 5.6 nmol/min/nmol CYP1A1 and K m values of 111, 83, and 250 μM for CYP1A1.1, CYP1A1.2, and CYP1A1.4, respectively. Availability of the three purified human CYP1A1 variants should facilitate further characterization of their role in metabolism of endogenous and exogenous compounds as well as structural studies.

Membrane perturbations induced by the apoptotic Bax protein.

The apoptotic protein Bax, in oligomeric form, is effective in promoting both leakage and lipid mixing in liposomes composed of cardiolipin and phosphatidylethanolamine and/or phosphatidylcholine, upon the addition of calcium. In contrast, monomeric Bax is not active. At low concentrations at which caspase-8-cut Bid (tBid) alone has little effect on leakage, tBid augments the leakage caused by monomeric Bax. When solutions of oligomeric Bax are diluted to lower detergent concentrations than those required for Bax oligomerization, the protein is initially active in inducing liposomal leakage, indicating that the potency of the oligomeric form is not a consequence of being initially added to the liposomes in a high detergent concentration. However, in solutions of low detergent concentration, in the absence of liposomes, the oligomer gradually loses its lytic potency. This is accompanied by a loss of binding of bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulphonic acid), indicating the loss of exposed hydrophobic sites, as well as a loss of the ability of the protein to translocate to membranes. Membrane translocation was measured by an energy-transfer assay. It was demonstrated that membrane binding was greatly enhanced by oligomerization and by the presence of calcium. Thus the membrane-active form of Bax is unstable in the absence of detergent or lipid. In addition, we find that translocation to the membrane is enhanced by oligomerization as well as by the presence of high concentrations of calcium.

Effects of Detergents on in vitro Growth of Diatom Alga Thalassiosira pseudonana

The growth of the diatom alga Thalassiosira pseudonana was studied when exposed to an environment polluted by a detergent. We determined concentrations that inhibit cell division (10 mg/l) instead of algae growth (0.1 and 1 mg/l. It was shown that T. pseudonana can adapt to high detergent concentrations. The stimulation of the growth of Thalassiosira within a range of 0.03–0.08 mg/l concentration has been registered.

Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer

Two-dimensional crystals of a membrane protein, the proton ATPase from plant plasma membranes, have been obtained by a new strategy based on the use of functionalized, fluorinated lipids spread at the air-water interface. Monolayers of the fluorinated lipids are stable even in the presence of high concentrations of various detergents as was established by ellipsometry measurements. A nickel functionalized fluorinated lipid was spread into a monolayer at the air-water interface. The overexpressed His-tagged ATPase solubilized by detergents was added to the subphase. 2D crystals of the membrane protein, embedded in a lipid bilayer, formed as the detergent was removed by adsorption. Electron microscopy indicated that the 2D crystals were single layers with dimensions of 10 μm or more. Image processing yielded a projection map at 9 Å resolution, showing three well-separated domains of the membrane-embedded proton ATPase.

Influence of ligand binding to human cytochrome P-450 1A2: conformational activation and stabilization by α-naphthoflavone

Human cytochrome P-450 ( P-450) 1A2 expressed in Escherichia coli is readily converted into non-native cytochrome P-420 ( P-420) in the presence of detergents. α-Naphthoflavone (ANF) has been used to prevent P-450 1A2 inactivation to P-420 during purification. However, the mechanism by which ANF modulates P-450 1A2 is not clearly understood. We observed that recombinant human P-450 1A2 prepared in the absence of ANF has an approx. 5 times higher maximum catalytic activity in the O-deethylation of 7-ethoxycoumarin than that in the presence of ANF, with the same K m values. The results revealed that the enzyme purified with ANF is not catalytically fully active, indicating that ANF tightly binds to the enzyme, only to be dissociated by heat denaturation. Furthermore, the inactive P-420 form of the enzyme could be reconverted to P-450 by ANF in high concentrations of detergents. The reconversion was concentration-dependent, confirming ANF-induced regeneration of active P-450 1A2. The reconversion coincided with the conformational change of the enzyme including increased α-helix content. The conformation of P-450 1A2 was also stabilized by ANF, resulting in an approx. 5°C increase in thermal stability.

Characterization of the reversible nature of the reaction catalyzed by sphingolipid ceramide N‐deacylase

Sphingolipid ceramide N-deacylase catalyzes a reversible reaction in which the amide linkages of the ceramides of various sphingolipids are cleaved or synthesized. Hydrolysis of sphingolipids by the enzyme proceeded efficiently at acidic pH in the presence of high concentrations of detergents, whereas the reverse reaction tended to be favored at neutral pH with a decrease in the detergent concentration. Although the catalytic efficiency (V(max)/K(m)) of the hydrolysis and reverse reactions was changed mainly by the concentration of detergents in the reaction mixture, V(max) and K(m) for the reverse reaction were relatively higher than those for the forward reaction, irrespective of the detergent concentration. The reverse reaction proceeded most efficiently when the molar ratio of lyso-sphingolipids and fatty acids was fixed at 1 : 1-2, the yield of the reaction exceeding 70-80%. The reverse and exchange (transacylation) reactions did not require ATP, CoA, metal ions or addition of organic solvents. Studies using inhibitors and chemical modifiers of the enzyme protein suggested that both the hydrolysis and condensation reactions are catalyzed at the same catalytic domain. These results indicate that the reverse hydrolysis reaction of the enzyme is unique, being completely different from those of lipases, proteases and glycosidases reported to date.