bluuebunny/crossref_metadata_2025 huggingface dataset:
Function and cellular localization of farnesoic acid O‐methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis
Authors: Gunawardene Y. I. N. Silva , Tobe S. S. , Bendena W. G. , Chow B. K. C. , Yagi K. J. , Chan S.‐M.
Published: March 2003

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O‐methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti‐FAMeT serum. FAMeT was localized in the neurosecretory cells of the X‐organ‐sinus gland complex of the eyestalk. As shown by RT‐PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X‐organ‐sinus gland.
Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia
Authors: Tengel Tobias , Sethson Ingmar , Francis Matthew S.
Published: March 2003

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid‐encoded type III secretion machine that permits the translocation of several anti‐host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre‐secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N‐terminus and in a predicted amphipathic domain located near the C‐terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C‐terminal amphipathic domain of YopD. A solution structure of YopD278−300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well‐defined amphipathic α helix ranging from Phe280 to Tyr291, followed by a type I β turn between residues Val292 and His295. The C‐terminal truncated peptides, YopD278−292 and YopD271−292, lacked helical structure, implicating the β turn in helix stability. An interaction between YopD278−300 and its cognate chaperone, LcrH, was observed by NMR through line‐broadening effects and chemical shift differences between the free peptide and the peptide–LcrH complex. These effects were not observed for the unstructured peptide, YopD278−292, which confirms that the α helical structure of the YopD amphipathic domain is a critical binding region of LcrH.
Detecting mitochondrial permeability transition by confocal imaging of intact cells pinocytically loaded with calcein
Authors: Jones Rachel A. , Smail Alison , Wilson Mark R.
Published: March 2003

When studied in vitro, mitochondrial permeability transition (MPT) is associated with an increase in mitochondrial permeability to solutes up to 1500 Da in mass and a loss of electrical potential difference across the inner mitochondrial membrane (Δψmit). The MPT has been implicated as being important in cellular calcium homeostasis, autophagy and cell death via necrosis and apoptosis. Thus, it is important to develop a valid technique for accurate measurement of this phenomenon in intact cells. We developed a procedure for the detection of MPT in intact cells that avoids the disadvantages associated with earlier approaches. In this new technique, unmodified (green‐fluorescent) calcein is simultaneously introduced into the cytosol of millions of cells by the process of pinocytic loading and, to identify the position of individual mitochondria and to measure Δψmit, the cells are counter‐stained with a red‐fluorescing potentiometric dye. Using this approach with a variety of cell types, we demonstrate that cytosolic calcein is excluded from normal polarized mitochondria but enters them during MPT. This technique may be valuable in studies investigating the cellular functions of MPT.
Unfolding and aggregation during the thermal denaturation of streptokinase
Authors: Azuaga Ana I. , Dobson Christopher M. , Mateo Pedro L. , Conejero‐Lara Francisco
Published: March 2003

The thermal denaturation of streptokinase from Streptococcus equisimilis (SK) together with that of a set of fragments encompassing each of its three domains has been investigated using differential scanning calorimetry (DSC). Analysis of the effects of pH, sample concentration and heating rates on the DSC thermograms has allowed us to find conditions where thermal unfolding occurs unequivocally under equilibrium. Under these conditions, pH 7.0 and a sample concentration of less than ≈ 1.5 mg·mL−1, or pH 8.0, the heat capacity curves of intact SK can be quantitatively described by three independent two‐state transitions, each of which compares well with the two‐state transition observed for the corresponding isolated SK domain. The results indicate that each structural domain of SK behaves as a single cooperative unfolding unit under equilibrium conditions. At pH 7.0 and high sample concentration, or at pH 6.0 at any concentration investigated, the thermal unfolding of domain A was accompanied by the time‐dependent formation of aggregates of SK. This produces a severe deformation of the DSC curves, which become concentration dependent and kinetically controlled, and thus precludes their proper analysis by standard deconvolution methods. A simple model involving time‐dependent, high‐order aggregation may account for the observed effects. Limited‐proteolysis experiments suggest that in the aggregates the N‐terminal segment 1–63 and the whole of SK domain C are at least partially structured, while domain B is highly unstructured. Unfolding of domain A, under conditions where the N‐terminal segment 1–63 has a high propensity for β sheet structure and a partially formed hydrophobic core, gives rise to rapid aggregation. It is likely that this region is able to act as a nucleus for the aggregation of the full‐length protein.
Supramolecular calsequestrin complex
Authors: Glover Louise , Quinn Sandra , Ryan Michelle , Pette Dirk , Ohlendieck Kay
Published: March 2003

As recently demonstrated by overlay assays using calsequestrin‐peroxidase conjugates, the major 63 kDa Ca2+‐binding protein of the sarcoplasmic reticulum forms complexes with itself, and with junctin (26 kDa), triadin (94 kDa) and the ryanodine receptor (560 kDa) [Glover, L., Culligan, K., Cala, S., Mulvey, C. & Ohlendieck, K. (2001) Biochim. Biophys. Acta1515, 120–132]. Here, we show that variations in the relative abundance of these four central elements of excitation–contraction coupling in different fiber types, and during chronic electrostimulation‐induced fiber type transitions, are reflected by distinct alterations in the calsequestrin overlay binding patterns. Comparative immunoblotting with antibodies to markers of the junctional sarcoplasmic reticulum, in combination with the calsequestrin overlay binding patterns, confirmed a lower ryanodine receptor expression in slow soleus muscle compared to fast fibers, and revealed a drastic reduction of the RyR1 isoform in chronic low‐frequency stimulated tibialis anterior muscle. The fast‐to‐slow transition process included a distinct reduction in fast calsequestrin and triadin and a concomitant reduction in calsequestrin binding to these sarcoplasmic reticulum elements. The calsequestrin‐binding protein junctin was not affected by the muscle transformation process. The increase in calsequestrin and decrease in junctin expression during postnatal development resulted in similar changes in the intensity of binding of the calsequestrin conjugate to these sarcoplasmic reticulum components. Aged skeletal muscle fibers tended towards reduced protein interactions within the calsequestrin complex. This agrees with the physiological concept that the key regulators of Ca2+ homeostasis exist in a supramolecular membrane assembly and that protein–protein interactions are affected by isoform shifting underlying the finely tuned adaptation of muscle fibers to changed functional demands.
Isolation and enzymatic characterization of lamjapin, the first ribosome‐inactivating protein from cryptogamic algal plant (Laminaria japonica A)
Authors: Liu Ren‐shui , Yang Jia‐hua , Liu Wang‐Yi
Published: March 2003

Lamjapin, a novel type Ι ribosome‐inactivating protein, has been isolated from kelp (Laminaria japonica A), a marine alga. This protein has been extensively purified through multiple chromatography columns. With a molecular mass of ≈ 36 kDa, lamjapin is slightly larger than the other known single‐chain ribosome‐inactivating proteins from the higher plants. Lamjapin can inhibit protein synthesis in rabbit reticulocyte lysate with an IC50 of 0.69 nm. It can depurinate at multiple sites of RNA in rat ribosome and produce the diagnostic R‐fragment and three additional larger fragments after the aniline reaction. Lamjapin can deadenylate specifically at the site A20 of the synthetic oligoribonucleotide (35‐mer) substrate that mimics the sarcin/ricin domain (SRD) of rat ribosomal 28S RNA. However, it cannot hydrolyze the N‐C glycosidic bond of guanosine, cytidine or uridine at the corresponding site of the A20 of three mutant SRD RNAs. Lamjapin exhibits the same base and position requirement as the ribosome‐inactivating proteins from higher plants. We conclude that lamjapin is an RNA N‐glycosidase that belongs to the ribosome‐inactivating protein family. This study reports for the first time that ribosome‐inactivating protein exists in the lower cryptogamic algal plant.
Studies into factors contributing to substrate specificity of membrane‐bound 3‐ketoacyl‐CoA synthases
Authors: Blacklock Brenda J. , Jaworski Jan G.
Published: March 2003

We are interested in constructing a model for the substrate‐binding site of fatty acid elongase‐1 3‐ketoacyl CoA synthase (FAE1 KCS), the enzyme responsible for production of very long chain fatty acids of plant seed oils. Arabidopsis thaliana and Brassica napus FAE1 KCS enzymes are highly homologous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length. We used in vivo and in vitro assays of Saccharomyces cerevisiae‐expressed FAE1 KCSs to demonstrate that the B. napus FAE1 KCS enzyme favors longer chain acyl substrates than the A. thaliana enzyme. Domains/residues responsible for substrate specificity were investigated by determining catalytic activity and substrate specificity of chimeric enzymes of A. thaliana and B. napus FAE1 KCS. The N‐terminal region, excluding the transmembrane domain, was shown to be involved in substrate specificity. One chimeric enzyme that included A. thaliana sequence from the N terminus to residue 114 and B. napus sequence from residue 115 to the C terminus had substrate specificity similar to that of A. thaliana FAE1 KCS. However, a K92R substitution in this chimeric enzyme changed the specificity to that of the B. napus enzyme without loss of catalytic activity. Thus, this study was successful in identifying a domain involved in determining substrate specificity in FAE1 KCS and in engineering an enzyme with novel activity.
Diffusion through channel derivatives of the Escherichia coli FhuA transport protein
Authors: Braun Michael , Killmann Helmut , Maier Elke , Benz Roland , Braun Volkmar
Published: March 2003

FhuA is a multifunctional protein in the outer membrane of Escherichia coli that actively transports [Fe3+]ferrichrome, the antibiotics albomycin and rifamycin CGP 4832, and mediates sensitivity of cells to the unrelated phages T5, T1, φ80 and UC‐1, and to colicin M and microcin J25. The energy source of active transport is the proton motive force of the cytoplasmic membrane that is required for all FhuA functions except for infection by phage T5. The FhuA crystal structure reveals 22 antiparallel transmembrane β‐strands that form a β‐barrel which is closed by a globular N‐terminal domain. FhuA still displays active transport and sensitivity to all ligands except microcin J25 when the globular domain (residues 5–160) is excised and supports weakly unspecific diffusion of substrates across the outer membrane. Here it is shown that isolated FhuAΔ5–160 supported diffusion of ions through artificial planar lipid bilayer membranes but did not form stable channels. The double mutant FhuAΔ5–160 Δ322–336 lacking in addition to the globular domain most of the large surface loop 4 which partially constricts the channel entrance, displayed an increased single‐channel conductance but formed no stable channels. It transported in vivo[Fe3+]ferrichrome with 45% of the rate of wild‐type FhuA and did not increase sensitivity of cells to antibiotics. In contrast, a second FhuA double mutant derivative which in addition to the globular domain contained a deletion of residues 335–355 comprising one‐third of surface loop 4 and half of the transmembrane β‐strand 8 formed stable channels in lipid bilayers with a large single‐channel conductance of 2.5 nS in 1 m KCl. Cells that synthesized FhuAΔ5–160 Δ335–355 showed an increased sensitivity to antibiotics and supported diffusion of maltodextrins, SDS and ferrichrome across the outer membrane. FhuAΔ5–160 Δ335–355 showed no FhuA specific functions such as active transport of [Fe3+]ferrichrome or sensitivity to the other FhuA ligands. It is concluded that FhuAΔ5–160 Δ335–355 assumes a conformation that is incompatible with any of the FhuA functions.
Prediction of temporal gene expression
Authors: Klipp Edda , Heinrich Reinhart , Holzhütter Hermann‐Georg
Published: March 2003

A computational approach is used to analyse temporal gene expression in the context of metabolic regulation. It is based on the assumption that cells developed optimal adaptation strategies to changing environmental conditions. Time‐dependent enzyme profiles are calculated which optimize the function of a metabolic pathway under the constraint of limited total enzyme amount. For linear model pathways it is shown that wave‐like enzyme profiles are optimal for a rapid substrate turnover. For the central metabolism of yeast cells enzyme profiles are calculated which ensure long‐term homeostasis of key metabolites under conditions of a diauxic shift. These enzyme profiles are in close correlation with observed gene expression data. Our results demonstrate that optimality principles help to rationalize observed gene expression profiles.
Bax‐induced cell death in yeast depends on mitochondrial lipid oxidation
Authors: Priault Muriel , Bessoule Jean‐Jacques , Grelaud‐Coq Angela , Camougrand Nadine , Manon Stéphen
Published: March 2003

The oxidant function of pro‐apoptotic protein Bax was investigated through heterologous expression in yeast. Direct measurements of fatty acid content show that Bax‐expression induces oxidation of mitochondrial lipids. This effect is prevented by the coexpression of Bcl‐xL. The oxidation actually could be followed on isolated mitochondria as respiration‐induced peroxidation of polyunsaturated cis‐parinaric acid and on whole cells as the increase in the amount of thiobarbituric acid‐reactive products. Treatments that increase the unsaturation ratio of lipids, making them more sensitive to oxidation, increase kinetics of Bax‐induced death. Conversely, inhibitors of lipid oxidation and treatments that decrease the unsaturation ratio of fatty acids decrease kinetics of Bax‐induced death. Taken together, these results show that Bax‐induced mitochondrial lipid oxidation is relevant to Bax‐induced cell death. Conversely, lipid oxidation is poorly related to the massive Bax‐induced superoxide and hydrogen peroxide accumulation, which occurs at the same time, as chemical or enzymatic scavenging of ROS does not prevent lipid oxidation nor has any effects on kinetics of Bax‐induced cell death. Whatever the origin of mitochondrial lipid oxidation, these data show that it represents a major step in the cascade of events leading to Bax‐induced cell death. These results are discussed in the light of the role of lipid oxidation both in mammalian apoptosis and in other forms of cell death in other organisms.
Brassica napus soluble epoxide hydrolase (BNSEH1)
Authors: Bellevik Stefan , Zhang Jiaming , Meijer Johan
Published: March 2003

Epoxide hydrolase (EC 3.3.2.3) in plants is involved in the metabolism of epoxy fatty acids and in mediating defence responses. We report the cloning of a full‐length epoxide hydrolase cDNA (BNSEH1) from oilseed rape (Brassica napus) obtained by screening of a cDNA library prepared from methyl jasmonate induced leaf tissue, and the 5′‐RACE technique. The cDNA encodes a soluble protein containing 318 amino acid residues. The identity on the protein level is 85% to an Arabidopsis soluble epoxide hydrolase (sEH) and 50–60% to sEHs cloned from other plants. A 5 × His tag was added to the N‐terminus of the BNSEH1 and the construct was over‐expressed in the yeast Pichia pastoris. The recombinant protein was recovered at high levels after Ni‐agarose chromatography of lysed cell extracts, had a molecular mass of 37 kDa on SDS/PAGE and cross‐reacted on Western blots with antibodies raised to a sEH from Arabidopsis thaliana. BNSEH1 was shown to be a monomer by gel filtration analysis. The activity was low towards cis‐stilbene oxide but much higher using trans‐stilbene oxide as substrate with Vmax of 0.47 µmol·min·mg−1, Km of 11 µm and kcat of 0.3 s−1. The optimum temperature of the recombinant enzyme was 55 °C and the optimum pH 6–7 for trans‐stilbene oxide hydrolysis. The isolation of BNSEH1 will facilitate metabolic engineering of epoxy fatty acid metabolism for functional studies of resistance and seed oil modification in this important oilcrop.
Role of three isoforms of phospholipase A2 in capacitative calcium influx in human T‐cells
Authors: Hichami Aziz , Joshi Beenu , Simonin Anne Marie , Khan Naim Akhtar
Published: March 2003

The present study was conducted on human Jurkat T‐cell lines in order to elucidate the role of phospholipase A2 in capacitative calcium entry. We have employed thapsigargin (TG) that induces increases in [Ca2+]i by emptying the calcium pool of endoplasmic reticulum, followed by capacitative calcium entry. We designed a Ca2+ free/Ca2+ reintroduction (CFCR) protocol for the experiments, conducted in Ca2+‐free medium. By employing CFCR protocol, we observed that addition of exogenous arachidonic acid (AA) stimulated TG‐induced capacitative calcium influx. The liberation of endogenous AA and its autocrine action seems to be implicated during TG‐induced capacitative calcium influx: TG potentiates the induction of constitutively expressed mRNA of four PLA2 isoforms (type 1B, IV, V, VI), the inhibitors of the three PLA2 isotypes (type 1B, V, VI) inhibit TG‐induced release of [3H]AA into the extracellular medium, and finally, these PLA2 inhibitors do curtail TG‐stimulated capacitative calcium entry in these cells. These results suggest that stimulation of three isoforms of PLA2 by thapsigargin liberates free AA that, in turn, induces capacitative calcium influx in human T‐cells.
Molecular cloning of columbamine O‐methyltransferase from cultured Coptis japonica cells
Authors: Morishige Takashi , Dubouzet Emilyn , Choi Kum‐Boo , Yazaki Kazufumi , Sato Fumihiko
Published: March 2003

To identify all of the O‐methyltransferase genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica cells, we sequenced 1014 cDNA clones isolated from high‐alkaloid‐producing cultured cells of C. japonica. Among them, we found all three reported O‐methyltransferases and an O‐methyltransferase‐like cDNA clone (CJEST64). This cDNA was quite similar to S‐adenosyl‐l‐methionine:coclaurine 6‐O‐methyltransferase and S‐adenosyl‐l‐methionine:isoflavone 7‐O‐methyltransferase. As S‐adenosyl‐l‐methionine:columbamine O‐methyltransferase, which catalyzes the conversion of columbamine to palmatine, is one of the remaining unelucidated components in isoquinoline alkaloid biosynthesis in C. japonica, we heterologously expressed the protein in Escherichia coli and examined the activity of columbamine O‐methyltransferase. The recombinant protein clearly showed O‐methylation activity using columbamine, as well as (S)‐tetrahydrocolumbamine, (S)‐, (R,S)‐scoulerine and (R,S)‐2,3,9,10‐tetrahydroxyprotoberberine as substrates. This result clearly indicated that EST analysis was useful for isolating the candidate gene in a relatively well‐characterized biosynthetic pathway. The relationship between the structure and substrate recognition of the O‐methyltransferases involved in isoquinoline alkaloid biosynthesis, and a reconsideration of the biosynthetic pathway to palmatine are discussed.
Regulation of peptide‐chain elongation in mammalian cells
Authors: Browne Gareth J. , Proud Christopher G.
Published: March 2003

The elongation phase of mRNA translation is the stage at which the polypeptide is assembled and requires a substantial amount of metabolic energy. Translation elongation in mammals requires a set of nonribosomal proteins called eukaryotic elongation actors or eEFs. Several of these proteins are subject to phosphorylation in mammalian cells, including the factors eEF1A and eEF1B that are involved in recruitment of amino acyl‐tRNAs to the ribosome. eEF2, which mediates ribosomal translocation, is also phosphorylated and this inhibits its activity. The kinase acting on eEF2 is an unusual and specific one, whose activity is dependent on calcium ions and calmodulin. Recent work has shown that the activity of eEF2 kinase is regulated by MAP kinase signalling and by the nutrient‐sensitive mTOR signalling pathway, which serve to activate eEF2 in response to mitogenic or hormonal stimuli. Conversely, eEF2 is inactivated by phosphorylation in response to stimuli that increase energy demand or reduce its supply. This likely serves to slow down protein synthesis and thus conserve energy under such circumstances.
The C‐terminal domain of perfringolysin O is an essential cholesterol‐binding unit targeting to cholesterol‐rich microdomains
Authors: Shimada Yukiko , Maruya Mikako , Iwashita Shintaro , Ohno‐Iwashita Yoshiko
Published: March 2003

There is much evidence to indicate that cholesterol forms lateral membrane microdomains (rafts), and to suggest their important role in cellular signaling. However, no probe has been produced to analyze cholesterol behavior, especially cholesterol movement in rafts, in real time. To obtain a potent tool for analyzing cholesterol dynamics in rafts, we prepared and characterized several truncated fragments of θ‐toxin (perfringolysin O), a cholesterol‐binding cytolysin, whose chemically modified form has been recently shown to bind selectively to rafts. BIAcore and structural analyses demonstrate that the C‐terminal domain (domain 4) of the toxin is the smallest functional unit that has the same cholesterol‐binding activity as the full‐size toxin with structural stability. Cell membrane‐bound recombinant domain 4 was detected in the floating low‐density fractions and was found to be cofractionated with the raft‐associated protein Lck, indicating that recombinant domain 4 also binds selectively to cholesterol‐rich rafts. Furthermore, an enhanced green fluorescent protein‐domain 4 fusion protein stains membrane surfaces in a cholesterol‐dependent manner in living cells. Therefore, domain 4 of θ‐toxin is an essential cholesterol‐binding unit targeting to cholesterol in membrane rafts, providing a very useful tool for further studies on lipid rafts on cell surfaces and inside cells.
Thermolysin‐linearized microcin J25 retains the structured core of the native macrocyclic peptide and displays antimicrobial activity
Authors: Blond Alain , Cheminant Michel , Destoumieux‐Garzón Delphine , Ségalas‐Milazzo Isabelle , Peduzzi Jean , Goulard Christophe , Rebuffat Sylvie
Published: March 2003

Microcin J25 (MccJ25) is the single macrocyclic antimicrobial peptide belonging to the ribosomally synthesized class of microcins that are secreted by Enterobacteriaceae. It showed potent antibacterial activity against several Salmonella and Escherichia strains and exhibited a compact three‐dimensional structure [Blond et al. (2001) Eur. J. Biochem., 268, 2124–2133]. The molecular mechanisms involved in the biosynthesis, folding and mode of action of MccJ25 are still unknown. We have investigated the structure and the antimicrobial activity of thermolysin‐linearized MccJ25 (MccJ25‐L1−21: VGIGTPISFY10GGGAGHVPEY20F), as well as two synthetic analogs, sMccJ25‐L1−21 (sequence of the thermolysin‐cleaved MccJ25) and sMccJ25‐L12−11 (C‐terminal sequence of the MccJ25 precursor: G12GAGHVPEYF21V1GIGTPISFYG11). The three‐dimensional solution structure of MccJ25‐L1−21, determined by two‐dimensional NMR, consists of a boot‐shaped hairpin‐like well‐defined 8–19 region flanked by disordered N and C termini. This structure is remarkably similar to that of cyclic MccJ25, and includes a short double‐stranded antiparallel β‐sheet (8–10/17–19) perpendicular to a loop (Gly11–His16). The thermolysin‐linearized MccJ25‐L1−21 had antibacterial activity against E. coli and S. enteritidis strains, while both synthetic analogues lacked activity and organized structure. We show that the 8–10/17–19 β‐sheet, as well as the Gly11–His16 loop are required for moderate antibacterial activity and that the Phe21–Pro6 loop and the MccJ25 macrocyclic backbone are necessary for complete antibacterial activity. We also reveal a highly stable 8–19 structured core present in both the native MccJ25 and the thermolysin‐linearized peptide, which is maintained under thermolysin treatment and resists highly denaturing conditions.
High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine
Authors: Masson Patrick , Nachon Florian , Bartels Cynthia F. , Froment Marie‐Therese , Ribes Fabien , Matthews Cedric , Lockridge Oksana
Published: March 2003

Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as α‐chymotrypsin. Enzymes of this type are generally considered to be inactive at pH values below 5, because the histidine member of the catalytic triad becomes protonated. We have found that butyrylcholinesterase retains activity at pH ≤ 5, under conditions of excess substrate activation. This low‐pH activity appears with wild‐type butyrylcholinesterase as well as with all mutants we examined: A328G, A328I, A328F, A328Y, A328W, E197Q, L286W, V288W and Y332A (residue A328 is at the bottom of the active‐site gorge, near the π‐cation‐binding site; E197 is next to the active‐site serine S198; L286 and V288 form the acyl‐binding pocket; and Y332 is a component of the peripheral anionic site). For example, the kcat value at pH 5.0 for activity in the presence of excess substrate was 32 900 ± 4400 min−1 for wild‐type, 55 200 ± 1600 min−1 for A328F, and 28 700 ± 700 min−1 for A328W. This activity is titratable, with pKa values of 6.0–6.6, suggesting that the catalytic histidine is protonated at pH 5. The existence of activity when the catalytic histidine is protonated indicates that the catalytic‐triad mechanism of butyrylcholinesterase does not operate for catalysis at low pH. The mechanism explaining the catalytic behaviour of butyrylcholinesterase at low pH in the presence of excess substrate remains to be elucidated.
On the molecular basis of the recognition of angiotensin II (AII)
Authors: Tzakos Andreas G. , Bonvin Alexandre M. J. J. , Troganis Anasstasios , Cordopatis Paul , Amzel Mario L. , Gerothanassis Ioannis P. , van Nuland Nico A. J.
Published: March 2003

The high‐resolution 3D structure of the octapeptide hormone angiotensin II (AII) in aqueous solution has been obtained by simulated annealing calculations, using high‐resolution NMR‐derived restraints. After final refinement in explicit water, a family of 13 structures was obtained with a backbone RMSD of 0.73 ± 0.23 Å. AII adopts a fairly compact folded structure, with its C‐terminus and N‐terminus approaching to within ≈ 7.2 Å of each other. The side chains of Arg2, Tyr4, Ile5 and His6 are oriented on one side of a plane defined by the peptide backbone, and the Val3 and Pro7 are pointing in opposite directions. The stabilization of the folded conformation can be explained by the stacking of the Val3 side chain with the Pro7 ring and by a hydrophobic cluster formed by the Tyr4, Ile5 and His6 side chains. Comparison between the NMR‐derived structure of AII in aqueous solution and the refined crystal structure of the complex of AII with a high‐affinity mAb (Fab131) [Garcia, K.C., Ronco, P.M., Verroust, P.J., Brunger, A.T., Amzel, L.M. (1992) Science257, 502–507] provides important quantitative information on two common structural features: (a) a U‐shaped structure of the Tyr4‐Ile5‐His6‐Pro7 sequence, which is the most immunogenic epitope of the peptide, with the Asp1 side chain oriented towards the interior of the turn approaching the C‐terminus; (b) an Asx‐turn‐like motif with the side chain aspartate carboxyl group hydrogen‐bonded to the main chain NH group of Arg2. It can be concluded that small rearrangements of the epitope 4–7 in the solution structure of AII are required by a mean value of 0.76 ± 0.03 Å for structure alignment and ≈ 1.27 ± 0.02 Å for sequence alignment with the X‐ray structure of AII bound to the mAb Fab131. These data are interpreted in terms of a biological ‘nucleus’ conformation of the hormone in solution, which requires a limited number of structural rearrangements for receptor–antigen recognition and binding.
Overexpression in Escherichia coli of the rnf genes from Rhodobacter capsulatus
Authors: Jouanneau Yves , Jeong Ho‐Sang , Hugo Nicolas , Meyer Christine , Willison John C.
Published: March 2003

The rnf genes of Rhodobacter capsulatus, essential for nitrogen fixation, are thought to encode a system for electron transport to nitrogenase. In the present study, we have attempted to overexpress the rnf genes in Escherichia coli to investigate the molecular properties of the corresponding proteins. Corrections were made to the published DNA sequence of the rnf operon, resulting in the identification of two genes, rnfG and rnfH. The rnfABCDGEH operon thus comprises seven genes and shows similarities in gene arrangement and deduced protein sequences to homologous regions in the genomes of Haemophilus influenzae and E. coli. Four of the rnf gene products were found to be similar in sequence to components of an Na+‐dependent NADH :ubiquinone oxidoreductase from Vibrio alginolyticus. Three of the rnf genes were successfully overexpressed in E. coli as His‐tagged polypeptides, whereas the products of rnfA, rnfD and rnfE, predicted to be transmembrane proteins, could not be stably maintained in E. coli. The rnfB and rnfC gene products were isolated as two brown proteins with apparent molecular‐mass values of 25 kDa and 55 kDa, respectively. RnfB was shown to contain one [2Fe‐2S] cluster, based on absorption spectrophotometry, EPR spectroscopy and iron content. Recombinant RnfC contained at least one iron‐sulfur cluster, most likely of the [4Fe‐4S] type. Unambiguous identification of the prosthetic groups was, however, precluded by the extreme instability of this protein. In R. capsulatus, RnfB and RnfC were found by immunoblot analysis to be tightly bound to the membrane, despite their hydrophilic character. The RnfB and RnfC proteins were absent in mutant strains bearing insertions at various positions within the rnfABCDGEH operon, suggesting that their stability depends on the cosynthesis of the other rnf gene products. We observed that iron limitation during growth resulted in a decrease both in the cellular content of RnfB and in the level of transcription of the rnfABCDGEH operon, indicating that the expression of this operon is regulated as a function of iron availability
Purification, characterisation and reaction mechanism of monofunctional 2‐hydroxypentadienoic acid hydratase from Escherichia coli
Authors: Pollard John R. , Bugg Timothy D. H.
Published: March 2003

2‐Hydroxypentadienoic acid hydratase is found on many bacterial catabolic pathways responsible for the degradation of aromatic compounds. Monofunctional 2‐hydroxypentadienoic acid hydratase from Escherichia coli has been purified 3800‐fold to homogeneity, using enzymatically generated 2‐hydroxypentadienoic acid as substrate. The purified 28‐kDa protein requires a divalent metal ion for activity, optimum activity being obtained with Mn2+. Steady‐state kinetic parameters were measured (Km =41 ± 4 μM, kcat = 450 s−1), the enzyme exhibiting substrate inhibition at high substrate concentrations. The pH/rate profile and inhibition by group‐specific reagents were examined, and evidence was obtained for essential cysteine and tryptophan residues. An amino acid sequence alignment of the inferred amino acid sequence with nine other sequences was carried out and revealed several conserved sequence motifs. The substrate for the enzymatic reaction was found to be the dienol tautomer of 2‐hydroxypentadienoic acid. Analysis of the reaction products by HPLC confirmed the identity of the 4‐hydroxy‐2‐ketopentanoic acid product. Analogues of possible reaction intermediates were tested as inhibitors, and sodium oxalate was found to act as a potent enzyme inhibitor (Ki = 4.9 ± 0.7 μM). The potent inhibition by oxalate is consistent with a mechanism in which tautomerisation to 2‐ketopent‐3‐enoic acid takes place at the active site, followed by conjugate addition of water.
Insulin stimulation of phosphorylation of elongation factor 1 (eEF‐1) enhances elongation activity
Authors: Chang Yu‐Wen Edith , Traugh Jolinda A.
Published: March 2003

To examine the role of phosphorylation of the elongation factor eEF‐1 in regulation of translation, 32P‐labeled 3T3‐L1 cells were deprived of serum, then incubated in the presence or absence of 10 nM insulin for 15 min. eEF‐1 was purified by affinity chromatography on tRNA‐Sepharose and shown to be phosphorylated on the α, β and δ subunits. Phosphorylation of eEF‐1α was stimulated sixfold in response to insulin, β was stimulated fourfold and δ was threefold. The rate of elongation assayed with eEF‐1 from insulin‐stimulated cells was over twofold greater than with eEF‐1 from serum‐deprived cells. When eEF‐1 from insulin‐treated cells was subjected to two‐dimensional tryptic phosphopeptide mapping, nine phosphopeptides were obtained with the α subunit, one with the β subunit and three with the δ subunit. When compared with phosphopeptide maps of α, β and δ subunits of eEF‐1 phosphorylated in vitro by the insulin‐stimulated multipotential protein kinase, the maps of the β and δ subunits were identical. Five phosphopeptides obtained with the α subunit in vivo were identical to those obtained with S6 kinase in vitro; the remainder were unique. To examine whether protein kinase C had a role in phosphorylation of eEF‐1 in response to insulin, protein kinase C was down‐regulated by prolonged exposure of 3T3‐L1 cells to 4β‐phorbol 12‐myristate 13‐acetate (PMA). Phosphorylation of the α, β and δ subunits was stimulated 2.5‐fold in response to insulin, with elongation activity stimulated to a similar extent, suggesting that protein kinase C had no effect on stimulation of elongation in response to insulin. Thus, stimulation of eEF‐1 activity in response to insulin appears to be mediated primarily by multipotential S6 kinase. This data is consistent with previous studies on stimulation of initiation via phosphorylation of initiation factors by multipotential S6 kinase [Morley, S. J. & Traugh, J. A. (1993) Biochemie(Paris) 95, 985−989].
Role of an acidic compartment in tumor‐necrosis‐factor‐α−induced production of ceramide, activation of caspase‐3 and apoptosis
Authors: Monney Laurent , Olivier Reynald , Otter Isabelle , Jansen Burkhard , Poirier Guy G. , Borner Christoph
Published: March 2003

Tumor necrosis factor‐α (TNF‐α) apoptosis by recruiting a complex of cytosolic proteins at its plasma membrane receptor. Among them is caspase‐8, an interleukin‐1β−converting enzyme (ICE)‐like protease that initiates an amplified protease cascade to activate the cell‐death machinery. The latter comprises at least caspase‐3 and caspase‐7, which execute cell death by cleaving numerous protein substrates, including poly(ADP‐ribose) polymerase. In addition, TNF‐α stimulates the production of ceramide, which also activates the death machinery. Whether the signaling pathways elicited by caspase‐8 and ceramide proceed independently or intersect at a specific subcellular site is unknown. Using the lysosomotropic agent NH4Cl and the vesicularization inhibitor brefeldin A, we show here the convergence of TNF‐α‐induced death signaling on an acidic, subcellular compartment reminiscent of lysosomes. This compartment generates at least two signaling pathways that account for the caspase‐3 activation and apoptosis induced by TNFα, one involving ceramide and caspase‐unrelated adapter molecules and another involving yet unknown lysosomal mediators. The apoptosis inhibitor Bcl‐2 specifically acts on the ceramide‐activated pathway to block caspase‐3 activation and apoptosis. The latter result explains why Bcl‐2 only partially blocks TNF‐α‐induced apoptosis.
Human aldehyde dehydrogenase gene family
Authors: Yoshida Akira , Rzhetsky Andrey , Hsu Lily C. , Chang Cheng
Published: March 2003

Twelve aldehyde dehydrogenase (ALDH) genes have been identified in humans. These genes, located on different chromosomes, encode a group of enzymes which oxidizes varieties of aliphatic and aromatic aldehydes. Metabolic disorders and clinical problems associated with mutations of ALDH1, ALDH2, ALDH4, ALDH10 and succinic semialdehyde (SSDH) genes have been emerged. Comparison of the human ALDHs indicates a wide range of divergency (> 80−< 15 % identity at the protein sequence level) among them. However, several protein regions, some of which are implicated in functional activities, are conserved in the family members.

The phylogenic tree constructed of 56 ALDH sequences of humans, animals, fungi, protozoa and eubacteria, suggests that the present‐day human ALDH genes were derived from four ancestral genes that existed prior to the divergence of Eubacteria and Eukaryotes. The neighbor‐joining tree derived from 12 human ALDHs and antiquitin indicates that diversification within the ALDH1/2/5/6 gene cluster occurred during the Neoproterozoic period (about 800 million years ago). Duplication in the ALDH 3/10/7/8 gene cluster occurred in Phanerozoic period (about 300 million years ago). Separations of ALDH3/ALDH10 and that of ALDH7/ALDH8 had occurred during the period of appearance and radiation of mammalian species.
Glycyl‐tRNA synthetase from Thermus thermophilus
Authors: Mazauric Marie‐Hélène , Keith Gérard , Logan Derek , Kreutzer Roland , Giegé Richard , Kern Daniel
Published: March 2003

The tRNA glycylation system is amongst the most complex aminoacylation systems since neither the oligomeric structure of the enzymes nor the discriminator base in tRNAs are conserved in the phylae. To understand better this structural diversity and its functional consequences, the prokaryotic glycylation system from Thermus thermophilus, an extreme thermophile, was investigated and its structural and functional inter‐relations with those of other origins analyzed. Alignments of the protein sequence of the dimeric thermophilic glycyl‐tRNA synthetase (Gly‐tRNA synthetase) derived from its gene with sequences of other dimeric Gly‐tRNA synthetases revealed an atypical character of motif 1 in all these class 2 synthetases. Interestingly, the sequence of the prokaryotic thermophilic enzyme resembles eukaryotic and archaebacterial Gly‐tRNA synthetases, which are all dimeric, and diverges drastically from the tetrameric enzymes from other prokaryotes.

Cross aminoacylations with tRNAs and synthetases of different origins provided information about functional interrelations between the glycylation systems. Efficient glycylations involving partners from T. thermophilus and Escherichia coli showed conservation of the recognition process in prokaryotes despite strong structural variations of the synthetases. However, Gly‐tRNA synthetase from T. thermophilus acylates eukaryotic tRNAGly while the charging ability of the E. coli enzyme is restricted to prokaryotic tRNAGly. A similar behaviour is found in eukaryotic systems where the restricted species specificity for tRNA glycylation of mammalian Gly‐tRNA synthetase contrasts with the relaxed specificity of the yeast enzyme. The consensus sequence of the tRNAs charged by the various Gly‐tRNA synthetases reveals conservation of only G1−C72 in the acceptor arm, C35 and C36 in the anticodon, and the (G10−Y25)‐G45 triplet involved in tRNA folding. Conservation of these nucleotides indicates their key role in glycylation and suggests that they were part of the ancestral glycine identity set.

These features are discussed in the context of the phylogenic connections between prokaryotes, eukaryotes, and archaebacteria, and of the particular place of T. thermophilus in this phylogeny.
Mitochondria and apoptosis
Authors: Mignotte Bernard , Vayssiere Jean‐Luc
Published: March 2003

Programmed cell death serves as a major mechanism for the precise regulation of cell numbers and as a defense mechanism to remove unwanted and potentially dangerous cells. Despite the striking heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes, and this form of programmed cell death has been termed apoptosis.

Genetic studies in Caenorhabditis elegans had led to the identification of cell death genes (ced). The genes ced‐3 and ced‐4 are essential for cell death; ced‐9 antagonizes the activities of ced‐3 and ced‐4, and thereby protects cells that should survive from any accidental activation of the death program. Caspases (cysteine aspartases) are the mammalian homologues of CED‐3. CED‐9 protein is homologous to a family of many members termed the Bcl‐2 family (Bcl‐2s) in reference to the first discovered mammalian cell death regulator. In both worm and mammalian cells, the antiapoptotic members of the Bcl‐2 family act upstream of the execution caspases somehow preventing their proteolytic processing into active killers.

Two main mechanisms of action have been proposed to connect Bcl‐2s to caspases. In the first one, antiapoptotic Bcl‐2s would maintain cell survival by dragging caspases to intracellular membranes (probably the mitochondrial membrane) and by preventing their activation. The recently described mammalian protein Apaf‐1 (apoptosis protease‐activating factor 1) could be the mammalian equivalent of CED‐4 and could be the physical link between Bcl‐2s and caspases. In the second one, Bcl‐2 would act by regulating the release from mitochondria of some caspases activators : cytochrome c and/or AIF (apoptosis‐inducing factor). This crucial position of mitochondria in programmed cell death control is reinforced by the observation that mitochondria contribute to apoptosis signaling via the production of reactive oxygen species. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of programmed cell death. In this review, we examine the data concerning the mitochondrial features of apoptosis. Furthermore, we discuss the possibility that the mechanism originally involved in the maintenance of the symbiosis between the bacterial ancestor of the mitochondria and the host cell precursor of eukaryotes, provided the basis for the actual mechanism controlling cell survival.
Purification and characterization of N‐acetylglucosamine kinase from rat liver
Authors: Hinderlich Stephan , Nöhring Sabine , Weise Christoph , Franke Peter , Stäsche Roger , Reutter Werner
Published: March 2003

N‐Acetylglucosamine, a major sugar in complex carbohydrates, enters the pathways of aminosugar metabolism by the action of N‐acetylglucosamine kinase (
EC2.7.1.59). In this study we report the purification to homogeneity of GlcNAc kinase from rat liver cytosol using salmine sulfate precipitation, chromatography on phenyl‐Sepharose, ATP‐agarose and MonoQ, and finally gel filtration on Superdex 200. It was characterized as a dimer of 39‐kDa subunits. About 25 % of the amino acid sequence of GlcNAc kinase was established by peptide mapping. Part of the ATP‐binding site of GlcNAc kinase was identified by sequence comparison with related hexokinases, including the bifunctional enzyme UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (
EC5.1.3.14/2.7.1.60), the key enzyme of N‐acetylneuraminic acid biosynthesis in rat liver.

The Cys residues in the active sites of GlcNAc kinase and ManNAc kinase were characterized by chemical modification with N‐ethylmaleimide, iodoacetamide and 5,5′‐dithiobis(2‐nitrobenzoic acid). The finding that the substrates GlcNAc and ManNAc protected their respective enzymes from inhibition by the above sulfhydryl reagents indicates that Cys residues are located in or near the active sites of both enzymes. Use of the specific dithiol‐modifying chemical reagents, sodium meta‐periodate, sodium meta‐arsenite/2,3‐dimercaptopropanol and diazenedicarboxylic acid bis‐N,N′‐dimethylamide revealed that the active sites of GlcNAc kinase and ManNAc kinase possess at least one pair of vicinal thiols. Chemical treatment of UDP−GlcNAc 2‐epimerase provided no evidence for the presence of cysteine in the active site of this enzyme. From the incorporation of N‐[3H]ethylmaleimide into GlcNAc kinase in the absence and presence of ligands we estimated that the active site of GlcNAc kinase contains two Cys residues.
Purification and properties of an extremely thermostable membrane‐bound sulfur‐reducing complex from the hyperthermophilic Pyrodictium abyssi
Authors: Dirmeier Reinhard , Keller Martin , Frey Gerhard , Huber Harald , Stetter Karl O.
Published: March 2003

The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 gains energy by reducing sulfur with H2 to H2S. From this hyperthermophile, a sulfur‐reducing complex catalyzing this reaction was purified 13.5‐fold. The native complex exhibited a brownish‐yellow colour and showed an apparent molecular mass of 520 kDa. SDS/PAGE revealed the presence of nine different major polypeptides with apparent molecular masses of 82, 72, 65, 50, 47, 42, 40, 30 and 24 kDa. The native complex contained 50−55 mol acid‐labile sulfur, 50−55 mol iron, 1.6 mol nickel, 1.2 mol copper, 2.8 mol cytochrome b and 0.3 mol cytochrome c (all per mol native complex). The temperature optimum of the H2 : sulfur oxidoreductase complex was 100 +C, which is consistent with the physiological growth optimum of the native organism. The complex is extremely heat stable. During 5 h incubation at 100 +C, no decrease in H2S‐forming activity could be observed.