bluuebunny/crossref_metadata_2025 huggingface dataset:
The origin of 1H NMR‐visible triacylglycerol in human neutrophils
Authors: Wright Lesley C. , Obbink Katrina L. Groot , Delikatny Edward J. , Santangelo Rosie T. , Sorrell Tania C.
Published: March 2003

Human neutrophils incubated for 1 h in vitro with 10% commercial pooled, human serum containing high levels of free fatty acids (1141 µm) displayed a distinct lipid signal, typical of triacylglycerol, in the 1H NMR spectrum. Concurrently their plasma membrane triacylglycerol mass increased 4.6‐fold with a selective rise in the content of palmitic and linoleic acids. Although qualitatively similar, these effects were much greater than those observed after incubating neutrophils with 50 µg·mL−1 of lipopolysaccharide in the presence of 10% AB serum with normal free fatty acid content (345 µm, LPS/S). Incubation of neutrophils with an artificial mixture of free fatty acids at concentrations found in commercial serum, or with the fatty acid fraction isolated from commercial serum increased the 1H NMR‐detectable triacylglycerol. The signal intensity of the 1H NMR‐detectable triacylglycerol depended on the triacylglycerol composition, and correlated with increased membrane triacylglycerol mass. Cellular uptake of 3H‐labelled palmitic or oleic acids increased in the presence of commercial serum but not with LPS/S, with little contribution in either case to the triacylglycerol pool that increased in mass. Pulse‐chase experiments demonstrated that with LPS/S and commercial serum, radiolabelled palmitic acid was preferentially incorporated into triacylglycerol located in the plasma membrane. This process could occur at the plasma membrane, as cytoplasts efficiently convert exogenous fatty acids into triacylglycerol. We propose that LPS/S and serum containing high levels of free fatty acid, important in conditions of sepsis and inflammation, may facilitate the sequestration of palmitic acid into triacylglycerol by different pathways. This triacylglycerol originates from exogenous and endogenous free fatty acids, is 1H NMR‐visible, and may have a role in regulating apoptosis.
Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase
Authors: Bouvier‐Navé Pierrette , Benveniste Pierre , Oelkers Peter , Sturley Stephen L. , Schaller Hubert
Published: March 2003

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full‐length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double‐mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild‐type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2–3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT‐transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2‐dihexanoyl 3‐oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light‐microscopy observations of leaf epidermis cells, stained with a lipid‐specific dye, showed the presence of lipid droplets in the cells of triacylglycerol‐overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase‐transformed plants.
Effects of noncatalytic residue mutations on substrate specificity and ligand binding of Thermobifida fusca endocellulase Cel6A
Authors: Zhang Sheng , Barr Brian K. , Wilson David B.
Published: March 2003

The availability of a high‐resolution structure of the Thermobifida fusca endocellulase Cel6A catalytic domain makes this enzyme ideal for structure‐based efforts to engineer cellulases with high activity on native cellulose. In order to determine the role of conserved, noncatalytic residues in cellulose hydrolysis, 14 mutations of six conserved residues in or near the Cel6A active‐site cleft were studied for their effects on catalytic activity, substrate specificity, processivity and ligand‐binding affinity. Eleven mutations were generated by site‐directed mutagenesis using PCR, while three were from previous studies. All the CD spectra of the mutant enzymes were indistinguishable from that of Cel6A indicating that the mutations did not dramatically change protein conformation. Seven mutations in four residues (H159, R237, K259 and E263) increased activity on carboxymethyl cellulose (CM‐cellulose), with K259H (in glucosyl subsite −2) creating the highest activity (370%). Interestingly, the other mutations in these residues reduced CM‐cellulose activity. Only the K259H enzyme retained more activity on acid‐swollen cellulose than on filter paper, suggesting that this mutation affected the rate‐limiting step in crystalline cellulose hydrolysis. All the mutations lowered activity on cellotriose and cellotetraose, but two mutations, both in subsite +1 (H159S and N190A), had higher kcat/Km values (6.6‐fold and 5.0‐fold, respectively) than Cel6A on 2,4‐dinitrophenyl‐β‐d‐cellobioside. Measurement of enzyme : ligand dissociation constants for three methylumbelliferyl oligosaccharides and cellotriose showed that all mutant enzymes bound these ligands either to the same extent as or more weakly than Cel6A. These results show that conserved noncatalytic residues can profoundly affect Cel6A activity and specificity.
Purification, characterization and structural analysis of an abundant β‐1,3‐glucanase from banana fruit
Authors: Peumans Willy J. , Barre Annick , Derycke Veerle , Rougé Pierre , Zhang Wenling , May Gregory D. , Delcour Jan A. , Van Leuven Fred , Van Damme Els J. M.
Published: March 2003

An abundant, catalytically active β‐1,3‐endoglucanase (EC 3.2.1.39) has been isolated from the pulp of ripe bananas. Biochemical analysis of the purified protein, molecular modelling, and molecular cloning of the corresponding gene indicate that this banana enzyme closely resembles previously characterized plant β‐glucanases with respect to its amino‐acid sequence, structure and biological activity. The results described in this paper demonstrate both the occurrence of an abundant active β‐1,3‐endoglucanases in fruits and also readdress the question of the possible involvement of these enzymes in the ripening and/or softening process.
Structure and dynamics of lipid‐associated states of apocytochrome c
Authors: Bryson Elzbieta A , Rankin Saffron E. , Goormaghtigh Erik , Ruysschaert Jean‐Marie , Watts Anthony , Pinheiro Teresa J. T.
Published: March 2003

Apocytochrome c (apocyt c), which in aqueous solution is largely unstructured, acquires an α‐helical conformation upon association with lipid membranes. The extent of α‐helix induced in apocyt c is lipid‐dependent and this folding process is driven by both electrostatic and hydrophobic lipid–protein interactions. The structural and dynamic properties of apocyt c in lipid membranes were investigated by attenuated total reflection Fourier transform infrared spectroscopy combined with amide H–D exchange kinetics. Apocyt c acquires a higher content of α‐helical structure with negatively charged membranes than with zwitterionic ones. For all membranes studied here, the helices of these partially folded states of apocyt c have a preferential orientation perpendicular to the plane of the lipid membrane. The H–D exchange revealed that a small fraction of amide protons of apocyt c, possibly associated with a stable folded domain protected by the lipid, remained protected from exchange over 20 min. However, a large fraction of amide protons exchanged in less than 20 min, indicating that the helical states of apocyt c in lipid membranes are very dynamic.
A protein kinase C‐independent pathway leading to c‐Jun‐dependent expression of 100‐kDa Ras GTPase‐activating protein in JEG‐3 human choriocarcinoma cells
Authors: Ye Fei , Bourgeade Marie‐Françoise , Cayre Yvon E. , Thang Ming Nguy
Published: March 2003

Although the 100‐kDa Ras GTPase‐activating protein (p100 RasGAP) has been reported to exist specifically in human placental trophoblasts, the molecular mechanisms responsible for regulating its expression remain unclear. In this study we used okadaic acid, an inhibitor of serine/threonine phosphatase 1 and 2 A, as a probe to explore the signaling pathway regulating the expression of p100 RasGAP in JEG‐3 human placental choriocarcinoma cells. Treatment of JEG‐3 cells with okadaic acid provoked dose‐ and time‐dependent stimulation of p100 RasGAP expression without marked modification of expression of p120 RasGAP, another isoform of RasGAP. Co‐treatment of cells with okadaic acid and the protein kinase C activator, phorbol 12‐myristate 13‐acetate, exerted an additive effect on p100 RasGAP induction. Moreover, the response of the p100 RasGAP de novo synthesis to okadaic acid was not affected by the selective inhibitor of protein kinase C, GF 109203X. Thus this study identified a novel signaling pathway regulating p100 RasGAP expression, which is independent of protein kinase C. In addition, okadaic acid treatment resulted in the activation of ERK2 (p42 MAP kinase) and the induction of both c‐Jun and c‐Fos proteins without activating JNK (c‐Jun NH2‐terminal kinase). Significantly, blockade of c‐Jun expression with antisense c‐jun oligonucleotides suppressed p100 RasGAP expression. Taken together, it is concluded that okadaic acid induces the expression of p100 RasGAP protein in JEG‐3 cells preceded by activation of ERK and AP‐1 cascade, and that this okadaic acid‐induced p100 RasGAP expression is independent of protein kinase C‐mediated pathway but requires c‐Jun/AP‐1 function.
His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis
Authors: Talwar Rashmi , Jagath Junutula R. , Rao N. Appaji , Savithri H. S.
Published: March 2003

The three‐dimensional structures of rabbit and human liver cytosolic serine hydroxymethyltransferase revealed that H231 interacts with the O3′ of pyridoxal‐5′‐phosphate and other residues at the active site such as S203, K257, H357 and R402 (numbering as per the human enzyme). This and the conserved nature of H231 in all serine hydroxymethyltransferases highlights its importance in catalysis and/or maintenance of oligomeric structure of the enzyme. In an attempt to decipher the role of H230 (H231 of the human enzyme) in the catalytic mechanism and/or maintenance of oligomeric structure of sheep liver serine hydroxymethyltransferase, the residue was mutated to arginine, phenylalanine, alanine, asparagine or tyrosine. Our results suggest that the nature of the amino acid substitution has a marked effect on the catalytic activity of the enzyme. H230R and H230F mutant proteins were completely inactive, dimeric and did not bind pyridoxal‐5′‐phosphate. On the other hand, mutation to alanine and asparagine retained the oligomeric structure and ability to bind pyridoxal‐5′‐phosphate. These mutants had only 2–3% catalytic activity. The side reactions like transamination and 5,6,7,8‐tetrahydrofolate independent aldol cleavage were much more severely affected. They were able to form the external aldimine with glycine and serine but the quinonoid intermediate was not observed upon the addition of 5,6,7,8‐tetrahydrofolate. Mutation to tyrosine did not affect the oligomeric structure and pyridoxal‐5′‐phosphate binding. The H230Y enzyme was 10% active and showed a correspondingly lower amount of quinonoid intermediate. The kcat / Km values for l‐serine and lallothreonine were 10‐fold and 174‐fold less for this mutant enzyme compared to the wild‐type protein. These results suggest that H230 is involved in the step prior to the formation of the quinonoid intermediate, possibly in orienting the pyridine ring of the cofactor, in order to facilitate effective proton abstraction.
Upregulation of cytosolic chaperonin CCT subunits during recovery from chemical stress that causes accumulation of unfolded proteins
Authors: Yokota Shin‐ichi , Yanagi Hideki , Yura Takashi , Kubota Hiroshi
Published: March 2003

The chaperonin containing TCP‐1 (CCT) is a molecular chaperone consisting of eight subunit species and assists in the folding of actin, tubulin and some other cytosolic proteins. We examined the stress response of CCT subunit proteins in mammalian cultured cells using chemical stressors that cause accumulation of unfolded proteins. Levels of CCT subunit proteins in HeLa cells were coordinately and transiently upregulated under continuous chemical stress with sodium arsenite. CCT subunit levels in several mammalian cell lines were also upregulated during recovery from chemical stress caused by sodium arsenite or a proline analogue, l‐azetidine‐2‐carboxylic acid. Several unidentified proteins that were newly synthesized and associated with CCT were found to increase concomitantly with CCT subunits themselves and known substrates during recovery from the stress. These results suggest that CCT plays important roles in the recovery of cells from protein damage by assisting in the folding of proteins that are actively synthesized and/or renatured during this period.
Aspartic protease inhibitors
Authors: Tossi Alessandro , Bonin Irena , Antcheva Nikolinka , Norbedo Stefano , Benedetti Fabio , Miertus Stanislav , Nair Anil C. , Maliar Tibor , Dal Bello Federico , Palù Giorgio , Romeo Domenico
Published: March 2003

Aspartic proteases play key roles in a variety of pathologies, including acquired immunodeficiency syndrome. Peptidomimetic inhibitors can act as drugs to combat these pathologies. We have developed an integrated methodology for preparing human immunodeficiency virus (HIV)‐1 aspartic protease diaminodiol inhibitors, based on a computational method that predicts the potential inhibitory activity of the designed structures in terms of calculated enzyme–inhibitor complexation energies. This is combined with a versatile synthetic strategy that couples a high degree of stereochemical control in the central diaminodiol module with complete flexibility in the choice of side chains in the core and in flanking residues. A series of 23 tetrameric, pentameric and hexameric inhibitors, with a wide range of calculated relative complexation energies (−47.2 to +117 kJ·mol−1) and predicted hydrophobicities (logPo/w = 1.8–8.4) was thus assembled from readily available amino acids and carboxylic acids. The IC50 values for these compounds ranged from 3.2 nm to 90 µm, allowing study of correlations between structure and activity, and individuation of factors other than calculated complexation energies that determine the inhibition potency. Multivariable regression analysis revealed the importance of side‐chain bulkiness and rigidity at the P2, P2′ positions, suggesting possible improvements for the prediction process used to select candidate structures.
The proteome of Mycoplasma genitalium
Authors: Wasinger Valerie C. , Pollack J. Dennis , Humphery‐Smith Ian
Published: March 2003

Mycoplasma genitalium is the smallest member of the class Mollicutes, with a genome size of 580 kb. It has the potential to express 480 gene products, and is therefore considered to be an excellent model to assess: (a) the minimum metabolism required by a free living cell; and (b) proteomic technologies and the information obtained by proteome analysis. Here, we report on the most complete proteome observed at 73% (expected proteome), and analysed at 33% (reported proteome). The use of four overlapping pH windows in conjunction with SDS/PAGE has allowed 427 distinct proteins to be resolved in association with the exponential growth of M. genitalium. Proof of expression for 201 proteins of sufficient abundance on silver stained two‐dimensional gels was obtained using peptide mass fingerprinting (PMF) of which 158 were identified. The potential for gene product modification in even the simplest known self‐replicating organism was quantified at a ratio of 1.22 : 1, more proteins than genes. A reduction in protein expression of 42% was observed for post‐exponentially‐grown cells. DnaK, GroEL, DNA gyrase, and a cytadherence accessory protein were significantly elevated, while some ribosomal proteins were reduced in relative abundance. The strengths and weaknesses of techniques employed were assessed with respect to the observed and predicted proteome derived from DNA sequence information. Proteomics was shown to provide a perspective into the biochemical and metabolic activities of this organism, beyond that obtainable by sequencing alone.
Structural characterization of the oligosaccharide chains of human α1‐microglobulin from urine and amniotic fluid
Authors: Amoresano Angela , Minchiotti Lorenzo , Cosulich Maria E. , Campagnoli Monica , Pucci Piero , Andolfo Annapaola , Gianazza Elisabetta , Galliano Monica
Published: March 2003

Human α1‐microglobulin (α1‐m; also called protein HC), a glycoprotein belonging to the lipocalin superfamily, was isolated by sequential anion‐exchange chromatography and gel filtration from the urine of hemodialized patients and from amniotic fluid collected in the week 16–18 of pregnancy. The carbohydrate chains of the protein purified from the two sources, which are organized in two Asn‐linked and one Thr‐linked oligosaccharides, were structurally characterized using matrix‐assisted laser desorption ionization and electrospray mass spectrometry. The glycans attached to Thr5 are differently truncated NeuHexHexNAc sequences, and O‐glycosylation in the amniotic fluid protein is only partial. Asn96 has both diantennary and triantennary structures attached in the case of urinary α1‐m and only diantennary glycans in the amniotic fluid protein. The main carbohydrate units attached to Asn17 are in both proteins monosialylated and disialylated diantennary glycans. The position of the oligosaccharide chains in a three‐dimensional model of the protein, produced using the automated Swiss‐Model service, is also discussed.
Determinants of substrate specificity of a second non‐neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis
Authors: Hussein Ayman S. , Smith Angela M. , Chacón Matilde R. , Selkirk Murray E.
Published: March 2003

We recently reported on a non‐neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describe the primary structure and enzymatic properties of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite. As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met).

A recombinant form of AChE C was highly expressed by Pichia pastoris. The enzyme was monomeric and hydrophilic, and displayed a marked preference for acetylthiocholine as substrate. A double mutation (W302F/W345F, corresponding to positions 290 and 331 in Torpedo) rendered the enzyme 10‐fold less sensitive to excess substrate inhibition and two times less susceptible to the bis quaternary inhibitor BW284C51, but did not radically affect substrate specificity or sensitivity to the ‘peripheral site’ inhibitor propidium iodide. In contrast, a triple mutant (M300G/W302F/W345F) efficiently hydrolysed propionylthiocholine and butyrylthiocholine in addition to acetylthiocholine, while remaining insensitive to the butyrylcholinesterase‐specific inhibitor iso‐OMPA and displaying a similar profile of excess substrate inhibition as the double mutant. These data highlight a conserved pattern of active site architecture for nematode secreted AChEs characterized to date, and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other invertebrate AChEs.
Purification and characterization of active recombinant human napsin A
Authors: Schauer‐Vukasinovic Vesna , Bur Daniel , Kitas Eric , Schlatter Daniel , Rossé Gerard , Lahm Hans‐Werner , Giller Thomas
Published: March 2003

Recombinant human napsin A expressed in human embryonic kidney 293 cells was purified to homogeneity by a single‐step procedure using part of napsin A propeptide as affinity ligand. N‐Terminal amino‐acid sequencing of the purified enzyme identified the mature form of napsin A. Treatment of purified napsin A with endoglycosidases F and H resulted in a decrease in its molecular mass from 39 kDa to ≈ 37 kDa, confirming that napsin A is glycosylated. The kinetic properties were analyzed by using two fluorogenic synthetic substrates K(Dabsyl)‐TSLLMAAPQ–Lucifer yellow (DS1) and K(Dabsyl)‐TSVLMAAPQ–Lucifer yellow (DS3). The Km values obtained were 1.7 µm and 6.2 µm, respectively. A substrate‐specificity study using a napsin A‐targeted peptide library confirmed the preference of napsin A for hydrophobic residues at positions P1 and P1′. Adjacent positions, P2–P4 and P2′–P4′, appeared less restricted in distribution of amino acids. A pH optimum between 4.0 and 5.5 at room temperature was determined. The purified enzyme was fully active for more than 10 h at pH 5.0 and 6.0, while a half‐life of 4 h was determined at pH 7.0 and 37 °C.
Propeptide dependent activation of the Antarctic krill euphauserase precursor produced in yeast
Authors: Kristjánsdóttir Sigrídur , Gudmundsdóttir Ágústa
Published: March 2003

Euphauserase is a brachyurin type digestive enzyme isolated from Antarctic krill. The brachyurins belong to clan SA of the S1 family of serine endopeptidases. In this study, we demonstrate that the precursor form of recombinant euphauserase, termed pro‐r‐euphauserase, can be successfully expressed in Pichia pastoris. The presence of most of the 51‐residue euphauserase propeptide is essential during expression, under the growth conditions of Pichia. The propeptide may be required either for correct folding or processing of the enzyme. Cod trypsin generates a fully active r‐euphauserase from its precursor, which appears to be identical to the native enzyme. The mature r‐euphauserase sequence contains 250 amino‐acid residues including a 13‐residue activation peptide, which seems to be attached to the molecule by a disulfide bond. Euphauserase shares an average sequence identity of 62% with its type I brachyurin analogue, crab collagenase I. However, the identity between these two sequences is much higher in the regions shown to be important for the broad substrate specificity and collagen binding of crab collagenase I. The type I brachyurins share only 30–40% identities with the type II brachyurins and trypsins. The low isoelectric point of euphauserase, with a calculated pI value of 3.9, is typical for the type I brachyurins.
Oxidation of aromatic sulfides by lignin peroxidase from Phanerochaete chrysosporium
Authors: Baciocchi Enrico , Gerini M. Francesca , Harvey Patricia J. , Lanzalunga Osvaldo , Mancinelli Simona
Published: March 2003

The reaction of H2O2 with 4‐substituted aryl alkyl sulfides (4‐XC6H4SR), catalysed by lignin peroxidase (LiP) from Phanerochaete chrysosporium, leads to the formation of sulfoxides, accompanied by diaryl disulfides. The yields of sulfoxide are greater than 95% when X = OMe, but decrease significantly as the electron donating power of the substituent decreases. No reaction is observed for X = CN. The bulkiness of the R group has very little influence on the efficiency of the reaction, except for R = tBu. The reaction exhibits enantioselectivity (up to 62% enantiomeric excess with X = Br, with preferential formation of the sulfoxide with S configuration). Enantioselectivity decreases with increasing electron density of the sulfide. Experiments in O show partial or no incorporation of the labelled oxygen into the sulfoxide, with the extent of incorporation decreasing as the ring substituents become more electron‐withdrawing. On the basis of these results, it is suggested that LiP compound I (formed by reaction between the native enzyme and H2O2), reacts with the sulfide to form a sulfide radical cation and LiP compound II. The radical cation is then converted to sulfoxide either by reaction with the medium or by a reaction with compound II, the competition between these two pathways depending on the stability of the radical cation.
Role of ligand substitution on long‐range electron transfer in azurins
Authors: Farver Ole , Jeuken Lars J. C. , Canters Gerard W. , Pecht Israel
Published: March 2003

Azurin contains two potential redox sites, a copper centre and, at the opposite end of the molecule, a cystine disulfide (RSSR). Intramolecular electron transfer between a pulse radiolytically produced RSSR− radical anion and the blue Cu(II) ion was studied in a series of azurins in which single‐site mutations were introduced into the copper ligand sphere. In the Met121His mutant, the rate constant for intramolecular electron transfer is half that of the corresponding wild‐type azurin. In the His46Gly and His117Gly mutants, a water molecule is co‐ordinated to the copper ion when no external ligands are added. Both these mutants also exhibit slower intramolecular electron transfer than the corresponding wild‐type azurin. However, for the His117Gly mutant in the presence of excess imidazole, an azurin–imidazole complex is formed and the intramolecular electron‐transfer rate increases considerably, becoming threefold faster than that observed in the native protein. Activation parameters for all these electron‐transfer processes were determined and combined with data from earlier studies on intramolecular electron transfer in wild‐type and single‐site‐mutated azurins. A linear relationship between activation enthalpy and activation entropy was observed. These results are discussed in terms of reorganization energies, driving force and possible electron‐transfer pathways.
Mast cell tryptase from pig lungs triggers infection by pneumotropic Sendai and influenza A viruses
Authors: Chen Ye , Shiota Mayumi , Ohuchi Masanobu , Towatari Takae , Tashiro Junko , Murakami Meiko , Yano Mihiro , Yang Bing , Kido Hiroshi
Published: March 2003

A novel trypsin‐type serine proteinase, which processes the precursors of the envelope fusion glycoproteins of pneumotropic Sendai and human influenza A viruses, was purified to homogeneity from pig lungs. On SDS/PAGE, the purified enzyme gave a protein band corresponding to about 32 kDa, and has an apparent molecular mass of 120 kDa, as determined by gel permeation chromatography. Immunohistochemical staining with antibodies against this enzyme revealed that the enzyme is located in pig lung mast cells. The N‐terminal 44‐amino‐acid sequence of the enzyme exhibits about 80% identity with those of mast cell tryptases from other species. Of the inhibitors tested, di‐isopropyl fluorophosphate, antipain, leupeptin, benzamidine and a few proteinaceous inhibitors, such as mucus protease inhibitor and aprotinin, inhibited this enzyme activity. Heparin stabilized the enzyme, but high‐ionic‐strength conditions did not, unlike for human mast cell tryptase. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and slowly processed hemagglutinin of human influenza A virus, and triggered the infectivity of Sendai virus in a dose‐dependent manner, although human mast cell tryptase β and rat mast cell tryptase (rat MCP‐7) from lungs did not process these fusion glycoproteins at all. These results suggest that mast cell tryptase in pig lungs is the possible trigger of the pneumotropic virus infections.
Conversion of pregnenolone to DHEA by human 17α‐hydroxylase/17,20‐lyase (P450c17)
Authors: Soucy Penny ,   Van Luu‐The
Published: March 2003

Most previous studies using reconstituted systems and fast kinetics suggest that the conversion of pregnenolone to dehydroepiandrosterone (DHEA; the precursor of androgen and estrogen biosynthesis) by P450c17 does not require the release of the intermediate 17α‐OHPreg (a precursor of cortisol biosynthesis). With such a mechanism, it is difficult to conceive how high amounts of DHEA may be produced in some cells or tissues, such as the testis and cells from the adrenal reticularis, while in other tissues such as the fasciculata zone, high levels of 17α‐OHPreg are synthesized. In this report, we address this matter using intact transfected cells, which better reflect the actual cellular conditions. Furthermore, by using transfected cells, we can conveniently analyze human enzymes, as we are not restricted by the availability of human tissues as in the case of methods using purified or partially purified enzymes. Using intact HEK‐293 cells transfected with human P450c17 in culture, we showed, in a time course study of the transformation of pregnenolone, that there is an accumulation of 17α‐OHPreg, and that, subsequently, the accumulated 17α‐OHPreg decreases with a concomitant increase in DHEA production. The DHEA/17α‐OHPreg ratio changes from 0.1 :1 after 1 h incubation to 50 : 1 after 20 h. This result strongly suggests that the transformation of Preg to DHEA proceeds through two steps in which DHEA is produced from the released intermediate 17α‐OHPreg. We also show that high levels of substrate vs. enzyme concentration will lead to high hydroxylase activity whereas the reverse will increase the lyase activity. The result is in good agreement with recent observations suggesting that surrounding enzymes and steroids could modulate the lyase activity. Cotransfection of vectors expressing cytochrome b5 and NADPH cytochrome P450 reductase indicates that both are required for an optimum production of DHEA.
Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli
Authors: Bizouarn Tania , Fjellström Ola , Axelsson Magnus , Korneenko Tatyana V. , Pestov Nikolay B. , Ivanova Maria V. , Egorov Maxim V. , Shakhparonov Michail , Rydström Jan
Published: March 2003

Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the α and the β subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease‐treated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His‐tagged intact and trypsin‐treated E. coli transhydrogenase.

Despite harsh treatments with, e.g. detergents and denaturing agents, the α and β subunits remained tightly associated. A monoclonal antibody directed towards the α subunit was strongly inhibitory, an effect that was relieved by added rrI. In addition, rrI also reactivated the trypsin‐digested E. coli enzyme in which domain I had been partly removed. This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II.

Replacement of domain I of intact, as well as trypsin‐digested, E. coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3‐acetyl‐pyridine‐NAD+ by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions.
Effects of volatile anesthetic on the Ca2+‐ATPase activation by dimerization
Authors: Lopez Maria M. , Zelent Bogumil , Kosk‐Kosicka Danuta
Published: March 2003

The phenomenological distance‐dependent quenching (DDQ) model was employed to investigate the character of the interaction between volatile anesthetics (VAs) and the plasma membrane Ca2+‐ATPase (PMCA). The simultaneous analysis of the frequency‐domain and steady‐state data of tryptophan (Trp) fluorescence quenching by a VA points to a specific character of the apparent quenching effect of the VA, possibly arising from a significant contribution of static quenching. The apparent contributions of both static and dynamic quenching may be due to VA binding in the PMCA, which results in the modification of the conformational substates of the enzyme. To characterize further the molecular consequences of VA binding, we investigated its effects on the process of PMCA activation by self‐association. VA shifted the equilibrium from enzyme dimers to monomers, as monitored by the loss of fluorescence energy transfer. The shift was apparently due to the VA‐induced decrease in the affinity of PMCA molecules for self‐association. Addition of a large molecular mass dextran to increase the proximity between enzyme monomers induced re‐association of the VA‐impaired PMCA, while the Ca2+‐ATPase activity was not recovered. The results are congruent with a dual VA effect on PMCA, a shift in the monomer/dimer equilibrium, and an inactivation of both monomers and dimers.
Transient N‐acetylgalactosaminylation of mannosyl phosphate side chain in Paramecium primaurelia glycosylphosphatidylinositols
Authors: Azzouz Nahid , Gerold Peter , Schmidt Jörg , Capdeville Yvonne , Schwarz Ralph T.
Published: March 2003

The surface antigens of the free‐living protozoan Paramecium primaurelia belong to the family of glycosylphosphatidylinositol (GPtdIns)‐anchored proteins. Using a cell‐free system prepared from P. primaurelia, we have described the structure and biosynthetic pathway for GPtdIns glycolipids. The core glycans of the polar glycolipids are modified by a mannosyl phosphate side chain. The data suggest that the mannosyl phosphate side chain is added onto the core glycan in two steps. The first step involves the phosphorylation of the GPtdIns trimannosyl conserved core glycan via an ATP‐dependent kinase, prior to the addition of the mannose linked to the phosphate group. We show that dolichol phosphate mannose is the donor of all mannose residues including the mannose linked to phosphate. Furthermore, we were able to identify in vitro a hydrophilic intermediate containing an additional N‐acetylgalactosamine linked to the mannosyl phosphate side chain. The addition of this purified hydrophilic radiolabelled intermediate into the cell‐free system leads to a loss of the GalNAc residue and its conversion to the penultimate intermediate having only mannosyl phosphate as a side chain. Together the data indicate that the GalNAc‐containing intermediate is a transitional intermediate. We suggest that the GalNAc‐containing intermediate is essential for biosynthesis and maturation of GPtdIns precursors. It is hypothesized that this oligosaccharide processing in the course of GPtdIns biosynthesis is required for the translocation of GPtdIns from the cytoplasmic side of the endoplasmic reticulum to the luminal side.
Assembly of heterodimeric luciferase after de novo synthesis of subunits in rabbit reticulocyte lysate involves Hsc70 and Hsp40 at a post‐translational stage
Authors: Tyedmers Jens , Kruse Monika , Lerner Monika , Demand Jens , Höhfeld Jörg , Solsbacher Jens , Volkmer Jörg , Zimmermann Richard
Published: March 2003

Heterodimeric luciferase from Vibrio harveyi had been established as a unique model enzyme for direct measurements of the effects of molecular chaperones and folding catalysts on protein folding and subunit assembly after de novo synthesis of subunits in rabbit reticulocyte lysate. It was observed that luciferase assembly can be separated in time from synthesis of the two subunits and that under these post‐translational conditions assembly was inhibited by either ATP depletion or inhibition of peptidylprolyl cis/trans isomerases, that is, by addition of cyclosporin A or FK506. Furthermore, it was observed that the inhibitory effect of FK506 on luciferase assembly can be suppressed by addition of purified cyclophilin, thereby providing the first direct evidence for the involvement of peptidylprolyl cis/trans isomerases in protein biogenesis in the eukaryotic cytosol. Here the ATP requirement in luciferase assembly has been characterized. Depletion of either Hsp90 or CCT from reticulocyte lysate did not interfere with luciferase assembly. However, addition of purified Hsc70 stimulated luciferase assembly. While addition of purified Hsp40 did not have any effect on luciferase assembly, the stimulatory effect of Hsc70 was further increased by Hsp40. Thus, after synthesis of the two subunits in reticulocyte lysate assembly of heterodimeric luciferase involves Hsc70 and its co‐chaperone Hsp40. Therefore, Hsc70 aids protein biogenesis in the eukaryotic cytosol not only at the levels of nascent polypeptide chains and precursor proteins that have to be kept competent for transport into cell organelles, but also at the level of subunits that have to be kept competent for assembly.
The assembly pathway of outer membrane protein PhoE of Escherichia coli
Authors: Jansen Carmen , Heutink Marja , Tommassen Jan , de Cock Hans
Published: March 2003

The assembly of the wild‐type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse–chase experiments, approximately half of the wild‐type PhoE molecules assembled within the 30‐s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin‐sensitive monomer, a trypsin‐sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N‐dimethyldodecylamine‐N‐oxide (LDAO). A mutant PhoE protein, ΔF330, which lacks the C‐terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the ΔF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild‐type protein. Therefore, we propose that the conserved C‐terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane‐spanning amphipathic β strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.
Calcium‐dependent protein kinase from maize seedlings activated by phospholipids
Authors: Szczegielniak Jadwiga , Liwosz Aneta , Jurkowski Ireneusz , Loog Mart , Dobrowolska Grażyna , Ek Pia , Harmon Alice C. , Muszyńska Grażyna
Published: March 2003

A calcium‐ and phospholipid‐dependent protein kinase of apparent molecular mass 54 kDa (designated ZmCPKp54) was partially purified from etiolated maize seedlings. Activity of ZmCPKp54 is stimulated by phosphatidylserine and phosphatidylinositol, but is not essentially affected by diolein and phorbol esters. The enzyme cross‐reacts with polyclonal antibodies against the calmodulin like‐domain of the calcium‐dependent protein kinase, but not with antibodies against catalytic or regulatory domains of protein kinase C. ZmCPKp54 is not able to phosphorylate the specific substrates of protein kinase C (MARCKS peptide and protein kinase C substrate peptide derived from pseudosubstrate sequence) and its activity is not inhibited by specific PKC inhibitors (bisindolylmaleimide, protein kinase C pseudosubstrate inhibitory peptide). The substrate specificity and sensitivity to the inhibitors of the maize enzyme resembles calcium‐dependent protein kinase. The biochemical and immunological properties indicate that ZmCPKp54 belongs to the calcium‐dependent protein kinase family.
Zymogen activation in the streptokinase–plasminogen complex
Authors: Wang Shunguang , Reed Guy L. , Hedstrom Lizbeth
Published: March 2003

Plasminogen (Plgn) is usually activated by proteolysis of the Arg561–Val562 bond. The amino group of Val562 forms a salt‐bridge with Asp740, which triggers a conformational change producing the active protease plasmin (Pm). In contrast, streptokinase (SK) binds to Plgn to produce an initial inactive complex (SK·Plgn) which subsequently rearranges to an active complex (SK·Plgn*) although the Arg561–Val562 bond remains intact. Therefore another residue must substitute for the amino group of Val562 and provide a counterion for Asp740 in this active complex. Two candidates for this counterion have been suggested: Ile1 of streptokinase and Lys698 of Plgn. We have investigated the reaction of SK mutants and variants of the protease domain of microplasminogen (µPlgn) in order to determine if either of these residues is the counterion. The mutation of Ile1 of SK decreases the activity of SK·Plgn* by 100‐fold (Ile1Val) to ≥ 104‐fold (Ile1→Ala, Gly, Trp or Lys). None of these mutations perturb the binding affinity of SK, which suggests that Ile1 is not required for formation of SK·Plgn but is necessary for SK·Plgn*. The substitution of Lys698 of µPlgn decreases the activity of SK·Plgn* by only 10–60‐fold. In contrast with the Ile1 substitutions, the Lys698 mutations also decreased the dissociation constant of the SK complex by 15–50‐fold. These observations suggest that Lys698 is involved in formation of the initial SK·Plgn complex. These results support the hypothesis that Ile1 provides the counterion for Asp740.
Evidence that the lizard helospectin peptides are O‐glycosylated
Authors: Vandermeers‐Piret Marie‐Claire , Vandermeers André , Gourlet Philippe , Ali Mohamed H. , Waelbroeck Magali , Robberecht Patrick
Published: March 2003

Six forms of helospectin (a vasoactive intestinal peptide analogue) were purified from the venom of the Heloderma horridum lizard. Their identification was performed by combining sequencing by automated Edman degradation and electrospray mass spectrometry analysis on the complete peptides and their tryptic fragments. The products resulting from the action of an O‐glycosidase were also analysed. Two forms were identified as the previously named Hs1 and Hs2 of 38 and 37 amino‐acid residues, respectively. Two forms corresponded to Hs1 and Hs2 O‐glycosylated by a N‐acetylhexosamine‐hexose motif attached to the Ser32 residue. Two other forms were not completely characterized but might correspond to the O‐glycosylated forms bearing a phosphate or a sulfate group. The glycosylation did not affect the capacity of the helospectins to recognize and to activate the human and the rat VPAC1 and VPAC2 receptors.
Suitable experimental conditions are required to characterize interferon‐α2b synthetic peptides
Authors: Blank Viviana C. , Peña Clara , Roguin Leonor P.
Published: March 2003

The binding and antiproliferative activities of synthetic peptides 29–35 and 122–139 of interferon‐α2b, both of which contain a cysteine residue in their sequences, were studied in the presence or absence of a dissociation medium containing mainly urea, dithiothreitol and 2‐mercaptoethanol. Although interferon‐α2b peptides either did not modify or slightly increased 125I‐labelled interferon‐α2b specific binding to WISH cell‐membrane receptors in the absence of dissociation medium, significant binding inhibition was obtained when both peptides were assayed in dissociation medium. Furthermore, also in the presence of dissociating agents, the two fragments inhibited cell growth in a concentration‐dependent manner, the 122–139 sequence being more effective than the 29–35 sequence. No additive effect on interferon binding and cell proliferation was observed when both peptides were added simultaneously. Results obtained after submitting peptide 122–139 to gel filtration or PAGE under different experimental conditions showed the presence of dimers and/or noncovalent aggregates arising from intermolecular disulfide bridges or hydrophobic interactions. Thus, our results indicated that peptide effects on 125I‐labelled interferon‐α2b binding and WISH cell proliferation were clearly manifested when the amount of monomeric species increased, showing that suitable experimental conditions should be used to study peptide behavior. The ability of both peptides to effectively trigger an interferon‐specific biological action, such as cell growth inhibition, strongly suggested that 29–35 and 122–139 interferon‐α2b fragments constitute the conformational epitope or mimotope that interacts with the cytokine‐specific receptor.
Plant mercaptopyruvate sulfurtransferases
Authors: Nakamura Tatsuo , Yamaguchi Yube , Sano Hiroshi
Published: March 2003

Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) and thiosulfate sulfurtransferase (TST, rhodanese, EC 2.8.1.1) are evolutionarily related enzymes that catalyze the transfer of sulfur ions from mercaptopyruvate and thiosulfate, respectively, to cyanide ions. We have isolated and characterized two cDNAs, AtMST1 and AtMST2, that are Arabidopsis homologs of TST and MST from other organisms. Deduced amino‐acid sequences showed similarity to each other, although MST1 has a N‐terminal extension of 57 amino acids containing a targeting sequence. MST1 and MST2 are located in mitochondria and cytoplasm, respectively, as shown by immunoblot analysis of subcellular fractions and by green fluorescent protein (GFP) analysis. However, some regions of MST1 fused to GFP were found to target not only mitochondria, but also chloroplasts, suggesting that the regions on the targeting sequence recognized by protein import systems of mitochondria and chloroplasts are not identical. Recombinant proteins, expressed in Escherichia coli, exhibited MST/TST activity ratios determined from kcat/Km values of 11 and 26 for MST1 and MST2, respectively. This indicates that the proteins encoded by both AtMST1 and AtMST2 are MST rather than TST type. One of the hypotheses proposed so far for the physiological function of MST and TST concerns iron–sulfur cluster assembly. In order to address this possibility, a T‐DNA insertion Arabidopsis mutant, in which the AtMST1 was disrupted, was isolated by PCR screening of T‐DNA mutant libraries. However, the mutation had no effect on levels of iron–sulfur enzyme activities, suggesting that MST1 is not directly involved in iron–sulfur cluster assembly.
A tribute to sulfur
Authors: Beinert Helmut
Published: March 2003

Recent progress in a number of areas of biochemistry and biology has drawn attention to the critical importance of sulfur in the biosynthesis of vital cofactors and active sites in proteins, and in the complex reaction mechanisms often involved. This brief review is intended as a broad overview of this currently rapidly moving field of sulfur biochemistry, for those who are interested or are involved in one or the other aspect of it, a synopsis by one who has stumbled into this field from several directions in the course of time. Only for iron are metal–sulfur relationships discussed in detail, as the iron–sulfur subfield is one of the most active areas.