bluuebunny/crossref_metadata_2025 huggingface dataset:
Inducible nitric‐oxide‐synthase mRNA is transiently expressed and destroyed by a cycloheximide‐sensitive process
Authors: EVANS Tom , CARPENTER Adam , COHEN Jonathan
Published: March 2005

Nitric oxide is a mediator of a wide range of physiological processes. It is produced by an enzyme family, the nitric‐oxide synthases, one form of which is induced in many cells following stimulation with cytokines and lipopolysaccharide. The aim of the experiments reported in this study was to investigate the regulation of mRNA expression for this inducible nitric‐oxide synthase in smooth muscle cells and macrophages. Stimulation of these cells with cytokines and lipopolysaccharide results in a marked elevation of nitric‐oxide‐synthase mRNA levels, which however do not remain elevated, but reach a maximum at 3–6 h after stimulation before returning to baseline levels over the next 20 h. Enzyme activity, however, remained virtually constant for 48 h following stimulation. Inspection of the 3′ untranslated segment of both murine and human inducible nitric‐oxide‐synthase mRNAs showed the presence of a conserved AU‐rich octanucleotide sequence, previously identified in cytokine and oncogene mRNAs and shown to mediate mRNA instability. A particular feature of the breakdown of mRNAs bearing this sequence is that degradation is prevented by protein‐synthesis inhibition. We show in this study that the half‐life of inducible nitric‐oxide‐synthase mRNA is 6 h and that in the presence of an inhibitor of protein synthesis this breakdown is prevented. Thus, the mRNA for inducible nitric‐oxide synthase shares some features in common with cytokines such as the transient expression and decay of its mRNA which can be prevented by protein‐synthesis inhibition.
Three‐dimensional structure of (1–71)bacterioopsin solubilized in methanol/chloroform and SDS micelles determined by 15N‐1H heteronuclear NMR spectroscopy
Authors: PERVUSHIN Konstantine V. , OREKHOV Vladislav Yu. , POPOV Alexander I. , MUSINA Larisa Yu. , ARSENIEV Alexander S.
Published: March 2005

Spatial structures of a chymotryptic fragment C2 (residues 1–71) of bacterioopsin from Halobacterium halobium, solubilized in a mixture of methanol/chloroform (1:1, by vol.) and 0.1 M 2HCO2NH4, or in perdeuterated sodium (2H)dodecyl sulfate (SDS) micelles in the presence of perdeuterated (2,2,2‐2H)trifluoroethanol, were determined by two‐dimensional and three‐dimensional heteronuclear 15N‐1H NMR techniques. The influence of (2,2,2‐2H)trifluoroethanol on the conformational dynamics of C2 in micelles and the effect of the salt (organic mixture) were studied. Under the best conditions, 1H and 15N resonances of 15N‐uniformly enriched protein were assigned in both milieus by homonuclear two‐dimensional NOE (NOESY) and two‐dimensional total‐correlated (TOCSY) spectra and heteronuclear three‐dimensional NOESY‐multiple‐quantum‐correlation (HMQC) and TOCSY‐HMQC spectra. 651 (organic mixture) and 520 (micelles) interproton‐distance constraints, derived from volumes of cross‐peaks in two‐dimensional NOESY and three‐dimensional NOESY‐HMQC spectra, along with deuterium exchange rates of amide groups measured in both milieus and 51 HN‐CαH coupling constants obtained in the case of the organic mixture, were used in the construction of C2 spatial structures. Obtained structures are similar in both milieus and have two right‐handed α‐helical regions stretching from Pro8 to Met32 and Phe42 to Tyr64 (organic mixture), and from Pro8 to Met32 and Ala39 to Leu62 (micelles). In micelles, the second α helix is terminated by C‐cap Gly63, adopting a conformation characteristic of a left‐handed helix. Residues Gly65 to Thr67 form the turn of a right‐handed helix. In the isotropic medium of the organic mixture, the C‐terminal region of residues 65–71 lacks an ordered structure. Torsion angles χ1 were unequivocally determined for 18 α‐helical residues in both milieus. In the isotropic organic mixture and anisotropic micellar system, C2 remains a compact structure with a characteristic size of 3.0–3.5 nm. C2 seems to be present in at least two conformational states, packed and unpacked. Using NMR data, along with the electron cryomicroscopy model of bacteriorhodopsin [Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckman, E. & Downing, K. H. (1990) J. Mol. Biol. 213, 899–929], we suggested a model for the conformation of C2 in this putative close‐packed state. However, no NOE contact between α helices was found in either milieu.
Characterisation and amino acid sequence of cytochrome c‐550 from Thiosphaera pantotropha
Authors: SAMYN Bart , BERKS Ben C. , PAGE M. Dudley , FERGUSON Stuart J. , VAN BEEUMEN Jozef J.
Published: March 2005

A cytochrome c‐550, with mid‐point potential +265 mV, has been purified from Thiosphaera pantotropha. The cytochrome was recognised by antibodies to Paracoccus denitrificans cytochrome c‐550, but the two proteins were not immunologically identical. Amino acid sequencing of the cytochrome c‐550 showed 85.9% and 95.5% identities, respectively, with the cytochromes c‐550 of P. denitrificans and Thiobacillus versutus; these are amongst the highest values reported for similarities between class I c‐type cytochromes of the c2 group. These similarities are consistent with the published values of 85% for the overall DNA similarity of P. denitrificans and T. pantotropha, but contrast with published 16S rRNA analyses which indicate identity between T. pantotropha and P. denitrificans and 97.5% similarity of T. versutus with these two organisms. Analysis by plasma‐desorption mass spectrometry of the peptide containing the haem‐binding motif isolated from the apocytochrome has shown that an Hg atom binds to one or both of the two thiol groups.
Accumulation of Reactive Oxygen Species and Oxidation of Cytokinin in Germinating Soybean Seeds
Authors: Gidrol Xavier , Lin Wen Shu , Dégousée Norbert , Yip Soo Fern , Kush Anil
Published: March 2005

Seed germination is an important developmental switch when quiescent seed cells initiate oxidative phosphorylation for further development and differentiation. During early imbibition of soybean seeds (Glycine max L. cv. Weber), a superoxide dismutase (SOD) activity peak was observed, in embryonic axes, after 6 h imbibition. Peroxidase activities, including catalase, were significantly increased after 12 h imbibition and during germination phase III. Catalase was the most efficient enzyme in catabolizing H2O2 in embryonic axes. When stored at 42°C and 100% relative humidity, seeds were stressed and lost their viability in a time‐dependent manner. A significant increase in the Cu, Zn‐superoxide‐dismutase activity, and to a lesser extent, Mn superoxide dismutase activity was observed during germination in low‐viability (stressed) seeds as compared to high‐viability (unstressed) seeds. Northern blot analysis confirmed that superoxide dismutase induction resulted from an accumulation of its transcripts in response to the production of O−2. The induction of catalase did not occur in low‐viability seeds, resulting in dramatic accumulation of H2O2. Using capillary electrophoresis, HPLC and NMR we found that the endogenous cytokinin, zeatin riboside, was present in large quantities in the high‐viability seeds, but it was oxidized into adenine in the low‐viability seeds. In vitro superoxide anion could also oxidize the cytokinin. Zeatin riboside, but not adenine, was found to act as a scavenger of superoxide anions and may help to maintain seed viability by detoxifying reactive oxygen species. Germination of stressed seeds was partially restored by the addition of exogenous cytokinin (zeatin riboside). Protection against oxidative stress by cytokinin seemed to be a general phenomenon, as Escherichia coli cells were also protected against superoxide stress in the presence of cytokinin.
Spectroscopic Observation of Renaturation Between Polynucleotides Interacting with RecA in the Presence of ATP Hydrolysis
Authors: Wittung Pernilla , Nordén Bengt , Takahashi Masayuki
Published: March 2005

To obtain mechanistic insights about RecA‐promoted base pairing between complementary polynucleotides, the complex formation of RecA with poly(dA) and poly(dT) in the presence of ATP (and ATP‐regenerating system) has been studied. The reaction was followed using a fluorescent probe, benzopyrenediolepoxide (BPDE), covalently attached to less than 1% of the adenine bases of poly(dA). BPDE is sensitive to its environment and has been found useful for detection of interactions between DNA strands, in the three binding positions of the RecA filament, in the presence of adenosine 5′‐O ‐3‐thiotriphosphate (ATP[S]) [Wittung, P., Nordén, B. & Takahashi, M. (1994) J. Biol. Chem. 269, 5799–58031. The emission spectrum of RecA:BPDE‐poly(dA) formed in the presence of ATP is similar to that observed with ATP[S] supporting similar structures of the complexes. However, the fluorescence anisotropy is considerably reduced, suggesting a higher degree of freedom of DNA in the presence of ATP hydrolysis. Upon addition of a complementary strand, poly(dT), to a preformed filament of RecA:BPDE‐poly(dA) in the presence of ATP, the fluorescence intensity slowly decreases and a change of emission profile consistent with Watson‐Crick base pairing is observed. This contrasts with the case of ATP[S] in which normal base pairing is never observed. Hence, ATP hydrolysis appears necessary for the RecA filament to be able to promote true renaturation. The renaturation reaction is found more effective when one of the complementary DNA strands is bound in the primary RecA DNA‐binding position and the other is added as the third strand, but the reaction can also occur between DNA strands in any combination of binding positions in the RecA filament. This observation suggests the importance of the third DNA‐binding position of the RecA filament. Renaturation between DNA strands in the other two combinations of binding positions is speculated to have a role in aborting the strand‐exchange reaction when the strands are insufficiently complementary.
Chemical Structure of the Lipid A Component of Lipopolysaccharides of the Genus Pectinatus
Authors: Helander Ilkka M. , Kilpeläinen Ilkka , Vaara Martti , Moran Anthony P. , Lindner Buko , Seydel Ulrich
Published: March 2005

The chemical structure of the lipid A components of smooth‐type lipopolysaccharides isolated from the type strains of strictly anaerobic beer‐spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were analyzed. The hydrophilic backbone of lipid A was shown, by controlled degradation of lipopolysaccharide combined with chemical assays and 31P‐NMR spectroscopy, to consist of the common β1‐6‐linked disaccharide of pyranosidic 2‐deoxy‐glucosamine (GlcN), phosphorylated at the glycosidic position and at position 4′. In de‐O ‐acylated lipopolysaccharide, the latter phosphate was shown to be quantitatively substituted with 4‐amino‐4‐deoxyarabinose, whereas the glycosidically linked phosphate was present as a monoester. Laser‐desorption mass spectrometry of free dephosphorylated lipid A revealed that the distal (non‐reducing) GlcN was substituted at positions 2′ and 3′ with (R)‐3‐(undecanoyloxy)tridecanoic acid, whereas the reducing GlcN carried two unsubstituted (R)‐3‐hydroxytetradecanoic acids at positions 2 and 3. The lipid A of both Pectinatus species were thus of the asymmetric hexaacyl type. The linkage of lipid A to polysaccharide in the lipopolysaccharide was relatively resistant to acid‐catalyzed hydrolysis, enabling the preparation of a dephosphorylated and deacylated saccharide backbone. Methylation analysis of the backbone revealed that position 6′ of the distal GlcN of lipid A was the attachment site of the polysaccharide. Despite the quantitative substitution of the lipid A 4′‐phosphate by 4‐amino‐4‐deoxyarabinose, which theoretically should render the bacteria resistant to polymyxin, P. cerevisiiphilus was shown to be susceptible to this antibiotic. P. cerevisiiphilus was, however, also susceptibile to vancomycin and bacitracin, indicating that the outer membrane of this bacterium does not act as an effective permeability barrier.
Isolation of the ALG5 Locus Encoding the UDP‐Glucose:Dolichyl‐Phosphate Glucosyltransferase from Saccharomyces cerevisiae
Authors: Heesen Stephan te , Lehle Ludwig , Weissmann Ariel , Aebi Markus
Published: March 2005

UDP‐glucose:dolichyl‐phosphate glucosyltransferase is a transmembrane‐bound enzyme of the endoplasmic reticulum involved in protein N‐linked glycosylation. This enzyme catalyzes the transfer of glucose from UDP‐glucose to dolichyl phosphate. The structural gene encoding this transferase from Saccharomyces cerevisiae was isolated by complementation of an alg5‐1 mutation. DNA sequencing of ALG5 revealed an open‐reading frame of 1002 bases encoding a transmembrane protein of molecular mass 38.3 kDa. Overexpression of Alg5p in both yeast and Escherichia coli results in an increase of UDP‐glucose: dolichyl‐phosphate glucosyltransferase activity, whereas a deletion of the yeast gene leads to a loss of this activity and a concomitant underglycosylation of carboxypeptidase Y. The ALG5 protein has sequence similarity to the GDP‐mannose: dolichyl‐phosphate mannosyltransferase (Dpm1p) from S. cerevisiae. Topological studies indicate that UDP‐glucose:dolichyl‐phosphate glucosyltransferase is a transmembrane protein that spans the membrane several times.
Competitive Elution of Protein A Fusion Proteins Allows Specific Recovery Under Mild Conditions
Authors: Nilsson Joakim , Nilsson Peter , Williams Ylva , Pettersson Lena , Uhlén Mathias , Nygren Per‐Åke
Published: March 2005

A novel system is described for mild elution of fusion proteins by competitive elution. The approach is based on displacement of immobilized fusions containing a monovalent IgG‐binding staphylococcal protein A fragment (Z) from an IgG‐affinity matrix by a divalent fragment fused to a serum‐albumin‐binding region derived from streptococcal protein G. Using real‐time interaction analysis, the binding (Kaff) to polyclonal human IgG was found to be 3.3 (±0.4)×108M‐1 for divalent ZZ and 2.0 (±0.1)×107M‐1 for monovalent Z. This more than tenfold difference in binding strength ensures a high efficiency in the elution step. The competitor protein can specifically be removed and recovered from the elution mixture by subsequent passage through a human serum albumin(HSA)‐affinity column, leaving only the target fusion protein in the flow‐through fraction. Here, we show that a recombinant Klenow fragment of DNA polymerase I expressed in Escherichia coli can be recovered with high yield, and retained activity, from a crude bacterial lysate by IgG‐affinity chromatography using mild conditions during both binding and elution.
Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli
Authors: Sancho Elena , Tonge David W. , Hockney Robert C. , Booth Nuala A.
Published: March 2005

Plasminogen‐activator inhibitor type 1 (PAI‐l), the primary physiological inhibitor of tissue‐type plasminogen activator, is an unusual member of the serine protease inhibitor (serpin) superfamily in that it spontaneously converts to a latent form lacking activity. This latent form can be reactivated by denaturation and refolding, but the activation is usually incomplete and often leads to aggregation of the protein. In this study we have developed a high‐level expression system that leads to the accumulation of PAI‐1 at 30–50% total microbial protein. We have developed a single‐step purification protocol which can be completed in a few hours, yielding approximately 20 mg purified recombinant PAI‐Mitre culture. The purified PAI‐1 was 80–100% active and was stable upon incubation at 37°C with a half‐life of approximately 48 h. At 20°C, PAI‐1 activity was stable for a week and at 4°C it retained its activity completely for up to two months. Freezing caused significant loss of activity. The stability of PAI‐1 activity was found to be dependent on pH and ionic strength, being most stable at pH 5.6 and at an ionic strength of 1 M salt. We show that by a combination of high‐level expression and rapid purification under optimum conditions, it is possible to produce active and stable PAI‐1 in high yield.
The C‐Terminus of the Prostaglandin‐E‐Receptor EP3 Subtype is Essential for Activation of GTP‐Binding Protein
Authors: Irie Atsushi , Sugimoto Yukihiko , Namba Tsunehisa , Asano Tomiko , Ichikawa Atsushi , Negishi Manabu
Published: March 2005

Three isoforms of the mouse prostaglandin‐E‐receptor EP3 subtype (EP3), EP3α, EP3β, and EP3γ, with different C‐termini, which are produced through alternative splicing, showed different efficiencies with respect to heterotrimeric GTP‐binding protein activation and adenylate cyclase inhibition [Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, T., Honda, A., Watabe, A., Hirata, M., Narumiya, S. & Ichikawa, A. (1993) J. Biol. Chem. 268, 2712–2718; Irie, A., Sugimoto, Y., Namba, T., Harazono, A., Honda, A., Watabe, A., Negishi, M., Narumiya, S. & Ichikawa, A. (1993) Eur. J. Biochem. 217, 313–318]. To assess the role of the C‐terminus in GTP‐binding protein coupling, we truncated the C‐terminus of EP3 at an alternative splicing site and expressed the mutant receptor. The truncated receptor retained the ability to physically associate with Gi2, forming an agonist/receptor/Gi2 ternary complex, and to undergo the characteristic conversion of its agonist‐binding affinity, mediated by a guanine nucleotide from a low‐affinity state to a high‐affinity state. However, sulprostone, an EP3 agonist, failed not only to inhibit the forskolin‐induced CAMP accumulation in the mutant receptor‐expressing cells but also to stimulate the GTPase activity in the mutant receptor‐expressing cell membrane. These results indicated that the C‐terminus of EP3 is essential for the activation of GTP‐binding protein.
An Evaluation of Receptor‐Mediated Gene Transfer Using Synthetic DNA‐Ligand Complexes
Authors: Perales Jose C. , Ferkol Thomas , Molas Maria , Hanson Richard W.
Published: March 2005

Receptor‐mediated gene transfer is an attractive method for therapeutically correcting human genetic diseases since it permits the targeting of DNA to cellular receptors in specific tissues of adult animals. Genes introduced by this technique have been shown to be expressed in the target tissue for varying periods. However, to be useful for gene therapy, it is critical that both the chemical properties and physical interactions of the reagents involved in the design of the DNA delivery vehicle be rigorously characterized. In this review, we discuss the critical steps in the preparation of the DNA‐ligand complex and the factors involved in the delivery and regulated expression of a transgene in animal tissues. The feasibility of using this technique for the therapeutic delivery of genes to mammalian tissues will also be evaluated.
Multiple Proline Substitutions Cumulatively Thermostabilize Bacillus Cereus ATCC7064 Oligo‐1,6‐Glucosidase
Authors: Watanabe Kunihiko , Masuda Tomoko , Ohashi Hiroyuki , Mihara Hisaaki , Suzuki Yuzuru
Published: March 2005

Nine residues of Bacillus cereus ATCC7064 oligo‐1,6‐glucosidase were replaced stepwise with proline residues. Of the nine residues, Lys121, Glu208 and Glu290 were at second sites of β turns; Asn109, Glu175 and Thr261 were at N‐caps of α helices; Glu216, Glu270 and Glu378 were in coils within loops. The replacements were carried out in the order, Lys121→Pro, Glu175→Pro, Glu290–Pro, Glu208→Pro, Glu270→Pro, Glu378→Pro, Thr261→Pro, Glu216→Pro and Asn109 →Pro. The resultant nine active mutant enzymes contained 1–9 more proline residues than B. cereus oligo‐1,6‐glucosidase. The thermostability of these mutants was additively enhanced with the increase in the number of proline residues introduced. The increase in the thermostability was most remarkable when proline residues were introduced at second sites of β turns or at N‐caps of α helices. The above results afforded irrefragable proof for the proline rule as an effective principle for increasing protein thermostability [Suzuki, Y., Oishi, K., Nakano, H. & Nagayama, T. (1987) Appl. Microbiol Biotechnol. 26, 546–551].
Purification and Characterization of a Rhamnogalacturonase with Protopectinase Activity from Trametes sanguinea
Authors: Sakamoto Mitsuhiro , Shirane Yuichi , Naribayashi Ikuya , Kimura Katsumi , Morishita Naoichi , Sakamoto Tatsuji , Sakai Takuo
Published: March 2005

In a culture filtrate of Trametes sanguinea IFO 6490, we found a protopectin‐solubilizing enzyme, protopectinase‐T, that did not degrade polygalacturonic acid. The enzyme was purified to homogeneity with hydrophobia, cation‐exchange, anion‐exchange, and size‐exclusion chromatographies. It had an apparent molecular mass of 55 kDa by SDS/PAGE and 39 kDa by size‐exclusion chromatography on Superose 12. The isoelectric point was at pH 8.1. Protopectinase‐T was stable from pH 3.0 to 6.0 and at temperatures up to 50°C. The optimum pH for enzyme activity was 4.0 at 37°C, and the optimum temperature was 50°C at pH 5.0. Protopectinase‐T catalyzed the release of highly polymerized pectin from lemon peel protopectin.
Cathepsin B, a Cysteine Protease Implicated in Metastatic Progression, is also Expressed During Regression of the Rat Prostate and Mammary Glands
Authors: Guenette R. Sean , Mooibroek Marilyn , Wong Kim , Wong Paul , Tenniswood Martin
Published: March 2005

We have developed a novel library‐to‐library cross‐screening technology to clone unique mRNAs that are expressed during tissue regression. We have cloned a number of regression selected genes (RSG) that are expressed during the regression of the mammary gland and ventral prostate of the rat after the removal of the respective trophic hormone. In this investigation, we have characterized one of these genes, RSG‐2, that is homologous to cathepsin B, a thiol protease that has been previously identified as one of the extracellular proteases which is activated in metastatic cells. The steady‐state levels of RSG‐2 mRNA in the normal prostate are low but detectable. In the regressing prostate, RSG‐2 mRNA levels peak at 3–4 days after castration, at the time that tissue regression is maximal. The gene is induced in a similar fashion in the regressing mammary gland. Using in situ hybridization, we have established that RSG‐2 mRNA is expressed in the luminal epithelial cells of the prostate and mammary gland that are known to undergo active cell death, suggesting that it may be a general marker for secretory epithelial cell death. Analysis of the distribution of the cathepsin B protein by immunofluorescence microscopy demonstrates that there is diffuse, but punctate, expression of the protein in all of the luminal epithelial cells of the normal prostate and mammary gland. However, at early times after hormone ablation in both glands, the majority of the increase in cathepsin B protein appears to result from redistribution to the basal aspect of the cells. At later time points, there appears to be increased amounts of the protein which is localized to the apoptotic bodies. These results suggest that RSG‐2, or cathepsin B, is required for the local degradation of the basement membrane, which is one of the earliest morphologically recognizable events of active cell death.
Distinct Signaling Pathways Mediate Phorbol‐Ester‐Induced and Cytokine‐Induced Inhibition of Erythropoietin Gene Expression
Authors: Fandrey Joachim , Huwiler Andrea , Frede Stilla , Pfeilschifter Josef , Jelkmann Wolfgang
Published: March 2005

Hypoxia‐induced erythropoietin (Epo) production in vitro is suppressed by interleukin 1β (IL‐ 1β), tumor necrosis factor α (TNF) and phorbol esters. Herein, the Epo‐synthesizing human hepatoma cell line HepG2 was used to investigate whether protein kinase C (PKC) is involved in the inhibitory action of the cytokines. Within 1 h after the onset of hypoxia, Epo mRNA levels were markedly increased in untreated HepG2 cells as quantitated by competitive reverse transcription PCR. The cytokines IL‐1β and TNF prevented this hypoxia‐induced increase in Epo mRNA levels. In phorbol‐ester‐treated cells first inhibitory effects on Epo mRNA levels were observed only after 3 h. Western blot analyses revealed the presence of four isoenzymes of PKC in HepG2 cells. None of these isoenzymes was translocated in response to TNF or IL‐1β, suggesting that the cytokines do not activate PKC in HepG2 cells. In contrast, phorbol esters translocated and, upon prolonged exposure, down‐regulated PKC isoenzymes α and ɛ. Activation of protein kinase A by dibutyryl‐cAMP partially antagonized the cytokine‐dependent inhibition of Epo production but did not influence the inhibitory effect of phorbol esters. Endogenous cAMP levels in HepG2 cells were unchanged by cytokine treatment.

Obviously, at least two signaling pathways exist that can confer inhibition of Epo production in HepG2 cells. One of these may be mediated by down‐regulation of the PKC α or ɛ isoenzyme. The other pathway, however, which is triggered by IL‐1β and TNF, is independent of PKC.
C‐Terminal Proteolytic Degradation of Recombinant Desulfato‐Hirudin and Its Mutants in the Yeast Saccharomyces cerevisiae
Authors: Heim Jutta , Takabayashi Kenji , Meyhack Bernd , Märki Walter , Pohlig Gabriele
Published: March 2005

The potent thrombin inhibitor hirudin variant 1, originally isolated from the leech Hirudo medicinalis, was expressed in Saccharomyces cerevisiae under the control of a truncated glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter fragment. Fusion of the yeast acid phosphatase (PHO5) signal sequence to the hirudin gene led to quantitative secretion of recombinant desulfato‐hirudin variant 1 (r‐hirudin) into the extracellular medium in a growth‐dependent manner. In comparison to the genuine molecule, r‐hirudin lacks the sulfate group at the Tyr in position 63. Besides the full‐length protein of 65 amino acids (hir65), chemical analysis revealed the presence mainly of two derivatives lacking the last amino acid Gin (hir64) or the penultimate Leu (hir63) in addition. When expressing r‐hirudin in mutant strains defective in all but one of the three major known carboxypeptidases, it turned out that the vacuolar carboxypeptidase yscY as well as the α‐factor precursor‐processing carboxypeptidase, yscα, participate in the C‐terminal degradation of r‐hirudin. Direct involvement of yscY and yscα was confirmed by sequential disruption of their structural genes PRC1 and KEX1, respectively. Disruption of PRA1, coding for the yscY‐processing proteinase yscA, also abolished yscY‐mediated C‐terminal r‐hirudin degradation, but clearly reduced the overall expression yield. Since yscα is described to be highly specific for basic amino acids which are not present at the C‐terminus of r‐hirudin, a series of r‐hirudin mutants with changes in the C‐terminal amino acids were constructed and analysed for yscα‐mediated and yscY‐mediated degradation. Chromatographic analysis of the expression products confirmed the preference of yscα for basic amino acids, although Tyr, Leu and Gin were also hydrolysed. It could further be concluded that yscα might also be responsible for the C‐terminal degradation of recombinant atrial natriuretic factor and epidermal growth factor expressed in yeast.
In vitro Proteolytic Activity and Active‐Site Identification of the Human Cytomegalovirus Protease
Authors: Stevens John T. , Mapelli Claudio , Tsao Jonglin , Hail Mark , O'Boyle Donald , Weinheimer Steven P. , Diianni Carolyn L.
Published: March 2005

Human cytomegalovirus (HCMV) encodes a protease that cleaves itself and the HCMV assembly protein. Two proteolytic processing sites within the protease were identified at Ala 256–Ser 257 (release site) and Ala 643–Ser 644 (maturation site). Identification of rP5‐P4′ and mP4‐P6′ as the minimal peptide substrates spanning the release and maturation cleavage sites, respectively, demonstrated a requirement for residues flanking the conserved core in substrate recognition and hydrolysis, which are unique to HCMV. Kinetic parameters determined for release‐site‐derived and maturation‐site‐derived peptides revealed a 10‐fold increase in kcat/Km for a maturational peptide (mP4–P8′) over release‐site peptide (rP5‐P5′)‐Experimental results with a panel of class‐specific protease inhibitors were consistent with the protease being a member of the serine or cysteine family of proteases. Further investigation revealed that the HCMV protease activity decreased with incorporation of [14C]iodoacetic acid, but when approximately 4.5 mol 14C were incorporated/mol enzyme, the enzyme retained approximately 20% of its original activity, indicating that hydrolysis does not require a cysteine nucleophile. Analysis of diisopropyl‐fluorophosphate‐inactivated protease by mass spectrometry indicated a stoichiometry of 1 diisopropyl phosphate/protease molecule, suggesting that hydrolysis requires a single serine nucleophile. The residue modified by diisopropyl fluorophosphate was identified as Ser132 by modification with 3H‐labeled diisopropyl fluorophosphate, peptide mapping and Edman degradation. This residue and the region in which it is found is highly conserved among the herpes virus proteases. These data demonstrates that HCMV protease is a serine protease and that Ser132 is the active‐site nucleophile.
Correct Post‐Translational Modification and Stable Vacuolar Accumulation of Phytohemagglutinin Engineered to Contain Multiple Methionine Residues
Authors: Kjemtrup Susanne , Herman Eliot M. , Chrispeels Maarten J.
Published: March 2005

Most legume seed storage proteins are deficient in sulfur amino acids. In this study, we demonstrate that replacing specific amino acid residues of a seed protein with methionine residues at positions known to be occupied by methionine residues in homologous proteins, is an effective strategy to create methionine‐enriched seed proteins. Mutant phytohemagglutinin polypeptides with three or four methionine residues were found to undergo correct post‐translational modifications in transformed cultured tobacco cells and to accumulate stably in the protein storage vacuoles of transgenic tobacco seeds.
N5‐Methyltetrahydromethanopterin:Coenzyme M Methyltransferase from Methanobacterium thermoautotrophicum
Authors: Gärtner Peter , Weiss David S. , Harms Ulrike , Thauer Rudolf K.
Published: March 2005

N
5‐Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane associated, corrinoidcontaming enzyme complex which uses a methyl‐transfer reaction to drive an energy‐conserving sodium ion pump. The purified methyltransferase from Methanobacterium thermoautotrophicum (strain Marburg) exhibited a rhombic EPR signal indicative of a base–on cob(II)amide. In this form, the enzyme was almost completely inactive. Upon addition of Ti(III)citrate, which is a one–electron reductant known to reduce corrinoids to the cob(I)amide form, the EPR signal was completely quenched. In the reduced form, the enzyme was active. When the purified complex was incubated in the presence of both Ti(III) and N5‐methyltetrahydromethanopterin (CH3‐H4MPT), enzyme‐bound Co‐methyl‐5′‐hydroxybenzimidazolyl cob(III)amide was formed. Upon incubation of the methylated enzyme with either tetrahydromethanopterin or coenzyme M, the enzyme was demethylated with the concomitant formation of CH3–H4MPT and methyl‐coenzyme M, respectively. Enzyme demethylation, in contrast to enzyme methylation, was not dependent on the presence of Ti(III). Methyl transfer from the methylated enzyme to coenzyme M was essentially irreversible. These results are interpreted to that the purified enzyme complex is active only when the enzyme–bound corrinoid is in the reduced cob(I)amide form, and that methyl transfer from CH3‐H4MPT to coenzyme M proceeds via nucleophilic attack of the cobalt(I) on the N5‐methyl substituent of CH3‐H4MPT, forming an enzyme‐bound CH3‐corrinoid as intermediate.

Methyl–coenzyme M formation from CH3‐H4MPT and coenzyme M, as catalyzed by the purified methyltransferase, was stimulated by sodium ions, half‐maximal activity being obtained at approximately 50 μM Na+. We therefore infer that the methyltransferase, as isolated, is capable of vectorial sodium ion translocation.
Peripheral Nerve Sphingomyelin and Cerebroside are Both Formed Via two Metabolically and Kinetically Distinct Pathways In vivo
Authors: Heape Anthony M. , Boiron Françoise , Bessoule Jean‐Jacques , Cassagne Claude
Published: March 2005

We have studied the labeling kinetics of peripheral nerve sphingolipids in vivo. The kinetic analysis of the labeling profiles observed for the various sphingolipids demonstrated that 90% of cerebrosides, but only 30% of sphingomyelin, were synthesized via a de novo synthesized ceramide intermediate following the injection of 1–4 pmol [3H]palmitate into mouse sciatic nerves. The remaining sphingolipid labeling (30% of the total) was due to direct acylation events, using free fatty acids originating from a pool different from those implicated in the de novo ceramide pathway. Direct acylation events ceased within 1 h following substrate administration, while labeling via the ceramide pathway continued through 5 h. The results provide the first in vivo demonstration that the formation of cerebrosides and sphingomyelin in peripheral nerves in situ can be simultaneously assured via two metabolically and kinetically distinct pathways that employ different fatty acid pools.
Expression and Characterization of a Very‐Late Antigen‐4 (α4β1) Integrin‐Binding Fragment of Vascular Cell‐Adhesion Molecule‐1
Authors: Dudgeon Timothy J. , Bottomley Matthew J. , Driscoll Paul C. , Humphries Martin J. , Mould A. Paul , Wingfield George I. , Clements John M.
Published: March 2005

We have used an Escherichia coli expression system to produce forms of vascular cell‐adhesion molecule‐1 (VCAM‐1) containing the first two and three supposed immunoglobulin‐like domains. A form consisting of the first two domains of VCAM‐1 is shown to promote very‐late antigen‐4‐dependent spreading of a melanoma cell line comparable to that found for the equivalent region in the full seven‐domain form. Preliminary structural analysis by CD and NMR is consistent with an immunoglobulin fold which is predicted from sequence comparison studies.
Further Evidence for Multiple Pathways in Soluble Methane‐Monooxygenase‐Catalysed Oxidations from the Measurement of Deuterium Kinetic Isotope Effects
Authors: Wilkins Patricia C. , Dalton Howard , Samuel Christopher J. , Green Jeffrey
Published: March 2005

The data from the deuterium isotope experiments in this study show that the primary kinetic isotope effect for methane oxidation catalysed by soluble methane monooxygenase from Methylococcus capsulatus (Bath) is very small, <2. In contrast, the primary kinetic isotope effect for –CH3 group oxidation in toluene is large, >7. A mechanistic pathway in which a substrate radical is formed from hydrogen atom abstraction by a ferryl species is believed to operate for CH4, the toluene –CH3 group and similar alkanes. Direct oxygen atom addition, rather than H atom abstraction, is indicated for aromatic ring oxidations in benzene and toluene and for styrene oxide formation from styrene. Thus, more than one mechanistic pathway appears to operate in soluble methane‐monooxygenase‐catalysed reactions and, in some cases, the pathway chosen may be dictated by the substrate.

In the soluble methane‐monooxygenase‐catalysed oxidation of toluene the rates of: (a) substrate dissociation from the enzyme‐substrate complex, (b) product formation and (c) product release (benzyl alcohol and p ‐cresol) from the enzyme‐product complex are comparable in magnitude. Therefore all three of these steps are partially rate–determining in the soluble methane monooxygenase catalytic cycle for toluene oxidation.
Phosphorylation of Inositol 1,4,5–Trisphosphate Analogues by 3‐Kinase and Dephosphorylation of Inositol 1,3,4,5‐Tetrakisphosphate Analogues by 5‐Phosphatase
Authors: Van Duken Peter , Lammers Aleida A. , Ozaki Shoichiro , Potter Barry V. L. , Erneux Christophe , Van Haastert Peter J. M.
Published: March 2005

A series of 32P‐labeled d‐myo ‐inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4] analogues was enzymically prepared from the corresponding d‐myo–inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] analogues using recombinant rat brain Ins(1,4,5)P3 3‐kinase and [γ‐32P]ATP. Ins(1,4,5)P3 analogues with bulky groups at the 2‐OH position, substitutions of phosphates by thiophosphates and d‐6‐deoxy‐myo ‐Ins(1,4,5)P3 were tested. Using [3H]Ins(1,4,5)P3 and ATPγS, a [3H]Ins(1,3,4,5)P4 analogue with a thiophosphate at the D‐3 position was prepared. The D‐4 and/or D‐5 phosphate group seemed to be important for 3‐kinase activity, while the OH group at position 6 was not crucial. The addition of bulky groups at the 2‐OH position did not prevent phosphorylation.

The labeled Ins(1,3,4,5)P4 analogues were purified and their degradation by type‐I Ins(1,4,5)P3/Ins(1,3,4,5)P4 5‐phosphatase was compared with the degradation of Ins(1,3,4,5)P4. Substitution of the phosphate group at positions 1 or 3 by a thiophosphate, or the addition of bulky groups at the 2‐OH position did not prevent degradation. d‐6–Deoxy‐myo ‐inositol 1,3,4,5‐tetrakisphosphate could not be degraded by the 5‐phosphatase, indicating the importance of the 6‐OH group for 5‐phosphatase action. d‐6‐Deoxy‐myo–inositol 1,3,4,5‐tetrakisphosphate could be an important tool in elucidating the cellular functions of Ins(1,3,4,5)P4
Activation of the O–2‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System
Authors: Fuchs Alexandra , Dagher Marie‐Claire , Jouan Alain , Vignais Pierre V.
Published: March 2005

The neutrophil NADPH oxidase activation factors, p47, p67 and the small guanosine‐nucleotide‐binding regulatory (G) protein Rac1, were expressed in a baculovirus/insect cell system and purified. In coinfection experiments in which Sf9 cells overexpressed concomitantly p47, p67 and Rac1, the latter was not detected in the p47–p67 complex. The propensity of p47 and p67 to associate together was used to purify recombinant p67 from baculovirus–infected Sf9 cells. 20% of the overexpressed Rac1 in infected Sf9 cells was prenylated and was extracted with low doses of detergent from membranes. Elicitation of full oxidase activity on crude neutrophil membranes using a cell–free system required addition of recombinant p47 and p67, but not that of Rac. In contrast, in the case of KCl‐washed membranes, addition of Rac, prenylated or unprocessed, together with p47 and p67 was found to enhance oxidase activation up to fivefold. In all experiments, the amount of added arachidonic acid was optimized. In contrast to prenylated Rac, non‐prenylated Rac had to be loaded with guanosine 5′‐(3‐thiotriphosphate) (GTP[SJ]) to exhibit full activation efficiency. In the cell‐free system used, Rac was shown to be the mediator of the GTP[S] effect. The results suggest that the plasma membrane of resting neutrophils contains a sufficient amount of prenylated Rac for efficient oxidase activation. We therefore propose that Rac has a membrane‐associated role and helps to dock and position p47 and p67 on the flavocytochrome b component of the oxidase complex.
Pancreatic Carboxylester Lipase from Atlantic Salmon (Salmo salar)
Authors: Gjellesvik Dag R. , Lorens James B. , Male Rune
Published: March 2005

We report the isolation and characterization of a 1795‐bp cDNA fragment encoding Atlantic salmon pancreatic carboxylester lipase from salmon pancreas mRNA. The nearly full‐length cDNA contained a 540‐amino‐acid open‐reading frame, encompassing the mature protein (by similarity to mammalian carboxylester lipase enzymes). The salmon carboxylester lipase primary structure shared 58% identity with mammalian carboxylester lipases, lacking the praline rich C‐terminal repeats found in human and rat carboxylester lipases. Congruent with other esterase B type enzymes, the salmon carboxylester lipase contained a canonical serine‐esterase catalytic triad motif consisting of serine, histidine and aspartic acid. Computer‐assisted modelling of the tertiary structure for salmon carboxylester lipase was conducted using acetylcholine esterase (Torpedo californica) as a template structure. The model, in conjunction with sequence comparisons and available enzymological data, has been used to locate putative bile‐salt‐binding and lipid‐binding sites. The carboxylester lipase enzymes contain a unique, highly conserved insert region that may be associated with bile‐salt binding. In the model structure, this region is located close to the active site, and contains a tyrosine residue with an adjacent carboxylester‐lipase‐conserved arginine. These traits have previously been predicted for the non‐specific (regarding bile‐salt hydroxylation) bile‐salt‐binding site in carboxylester lipase enzymes. At this site, a dihydroxy or trihydroxy bile‐salt molecule may bind the tyrosine via hydrophobic interactions, the anionic bile‐salt head group may bind the arginine, while hydrogen bonding between the bile‐salt 12α hydroxy group and an adjacent aspargine residue is possible. The model does not contain an active site ‘lid’ structure as found in other lipases. The carboxylester lipase structural homolog to the ‘flap’ of the lipases from Geotrichum candidum and Candida rugosa contains a carboxylester‐lipase‐conserved deletion that renders this region unable to cover the active site. Instead, the shortening of this loop leads to solvent exposure of the carboxylester lipase insert region, an additional indication of the functional importance of this region.
A Blue Non‐Heme Iron Protein from Desulfovibrio gigas
Authors: Chen Liang , Sharma Pardeep , Le Gall Jean , Mariano Aida M. , Teixeira Miguel , Xavier António V.
Published: March 2005

A novel iron‐containing blue protein, named neelaredoxin, was isolated from the sulfate‐reducing bacterium Desulfovibrio gigas. It is a monomeric protein with a molecular mass of 15 kDa containing two iron atoms/molecule. The N‐terminal sequence of neelaredoxin has similarity to the second domain of desulfoferrodoxin, a protein purified from Desulfovibrio vulgaris Hildenborough. This finding supports the hypothesis that the gene coding for desulfoferrodoxin (rbo) might have arisen from a gene fusion [Brumlik, M. J., Leroy, G., Bruschi, M. & Voordouw, G. (1990) J. Bacteriol. 172, 7289–7292]. The visible spectrum exhibits a single band at 666 nm, responsible for the blue color of the protein, which is completely bleached upon reduction with sodium ascorbate. In the oxidized state the EPR spectrum is complex, exhibiting well‐resolved features at g = 7.6, 7.0, 5.9, and 5.8 which are assigned to two high‐spin (S= 5/2) mononuclear‐iron (III) centers with different rhombic distortions (E/D≈ 0.05 and ≈ 0.08). The two iron atoms contribute identically to the visible spectrum as judged from visible redox titrations, from which a reduction potential of + 190 mV was determined for both iron sites at pH 7.5. At high pH the visible and the EPR spectra become pH‐dependent with a pKa above 9: the 666–nm band shifts to 590 nm and the EPR signals are converted into a signal with gmax∼ 4.7. Neelaredoxin is readily reduced both by H2/hydrogenase/cytochrome c3 and by NADH/NADH–rubredoxin oxidoreductase.
Pathogen‐Defence Gene prp1–1 from Potato Encodes an Auxin‐Responsive Glutathione S‐Transferase
Authors: Hahn Karoline , Strittmatter Günter
Published: March 2005

Genetic studies have previously implicated the prp1 gene family in the defence of potato against infection with the late blight fungus Phytophthora infestans. Here, we show that the concentrations of PRP1 mRNA as well as protein rapidly increase in potato leaves after fungal infection and stay at high levels during an extended period of the infection cycle. After separation of subcellular components by differential centrifugation, PRP1 protein was identified in the cytosolic fraction. Expression studies with chimeric promoter/β‐glucuronidase gene constructs in transgenic potato plants provided evidence that transcription of the prp1‐1 gene, representing one member of the prp1 gene family, is at least partly responsible for the accumulation of PRP1 mRNA and protein upon fungal infection. After expression of the prp1‐1 coding sequence in Escherichia coli, the corresponding 26–kDa protein exhibited glutathione S‐transferase activity with Km values of 9.8 mM and 0.11 mM for the artificial standard substrate 1‐chloro‐2,4–dinitrobenzene and glutathione, respectively. Photoaffinity labeling of the protein with tritiated 5–azido‐indole‐3–acetic acid suggested that the phytohormone indole‐3–acetic acid or a structurally related compound serve as a regulator or substrate of the prp1‐1 encoded glutathione S–transferase. This assumption was further supported by the inhibitory effect of the phytohormone on the enzyme activity in vitro. The implications of these findings for a potential involvement of indole‐3–acetic acid in the control of defence reactions are discussed.
Purification and Properties of Alternanase, a Novel Endo‐α‐1,3‐α‐1,6‐d‐Glucanase
Authors: Biely Peter , Côté Gregory L. , Burgess‐Cassler Anthony
Published: March 2005

A newly isolated soil bacterium strain NRRL B‐21195, tentatively identified as a Bacillus species, was found to be a constitutive producer of a novel type of glycanase that hydrolyses in an endo‐fashion the polysaccharide alternan, an α‐1,3‐α‐1,6‐d‐glucan, referred to in the literature as B‐1355 dextran (fraction S), synthesized from sucrose by alternansucrase of Leuconostoc mesenteroides. The glycanase, named alternanase, has been purified to homogeneity from a cell‐free culture fluid of the bacillus grown in a liquid medium containing d‐glucose, and has been characterized. The enzyme has a molecular mass of 110000 Da (SDS/PAGE) and an isoelectric point of approximately 4.0. Optimum activity occurs at pH 7 and at a temperature of 40°C. The enzyme is stable up to 50°C but loses activity rapidly at 60°C. Its action is inhibited by EDTA and stimulated by Ca2+. The enzyme requires, for its action, d‐glucan chains in which α‐1,3‐linkages alternate with α‐1,6‐linkages; i.e., it is specific for alternan. Monitoring of alternan hydrolysis by determination of liberated reducing sugars pointed to an unusually low extent of hydrolysis and a low specific activity of the enzyme. As shown in the accompanying paper [Côté, G. L. & Biely, P. (1994) Eur. J. Biochem. 226, 641–648] the reason for this finding is that the main hydrolytic products are non‐reducing, novel types of cyclic oligosaccharides.
A Method for Increasing the Concentration of a Specific Internal Metabolite in Steady‐State Systems
Authors: Small J. Rankin , Kacser Henrik
Published: March 2005

An approach is described by which it is possible to increase the concentration of any internal metabolite without affecting the concentrations of other metabolites and fluxes in the organism. This approach requires the manipulation of only a limited number of enzyme activities. The method shows which enzymes to manipulate and the extent of the manipulation required to achieve a given increase in a chosen metabolite. A case study involving tryptophan overproduction in Saccharomyces cerevisae is given as a practical example of how this method could be used.