bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular retention and degradation of human mutant variant of a α1‐antitrypsin in stably transfected Chinese hamster ovary cell lines
Authors: CICCARELLI Ernesto , ALONSO Maria Angeles , CRESTEIL Danielle , BOLLEN Alex , JACOBS Paul , ALVAREZ Fernando
Published: March 2005

Normal (PiM) and mutant (PiZ) variants of human α1‐antitrypsin (α1‐AT) cDNA, cloned into the pTnd eucaryotic expression vector, were used to derive recombinant Chinese hamster ovary cell lines permanently expressing the corresponding proteins.

Secretion, accumulation and glycosylation of PiM and PiZ α1‐AT proteins were studied in the presence of various transport‐impairing drugs. Pulse‐chase, followed by immunoprecipitation as well as immunofluorescence experiments showed that the PiZ α1‐AT undergoes continuous degradation that was prevented by Brefeldin A but not by incubation of cells at 16 °C. Moreover, monensin partially impaired the glycosylation of both PiM and PiZ α1‐AT but not their secretion nor the degradation of PiZ α1‐AT. Those results suggest that PiZ α1‐AT degradation occurs in the cis‐Golgi network, a compartment located between the endoplasmic reticulum and the Golgi stack. The process did not apparently involve lysosomes since it was insensitive to chloroquine. In addition, inhibition of PiM and PiZ α1‐AT glycosylation and secretion by tunicamycin did not result in the accumulation of the protein, but instead in its rapid lag‐free degradation. Treatment of cells with the A23187 ionophore, for a short (60 min) but not a long (24 h) period, improved the secretion of PiZ α1‐AT in a similar way as it affects retention of naturally endoplasmic‐reticulum‐resident proteins, suggesting that the small proportion of PiZ α1‐AT which is not degraded or secreted, but accumulates in the endoplasmic reticulum, is back transported as a partially glycosylated species from the post endoplasmic reticulum compartment in which degradation takes place.
1H‐NMR spectroscopy of βB2‐crystallin from bovine eye lens
Authors: CARVER John A. , COOPER Philip G. , TRUSCOTT Roger J. W.
Published: March 2005

1H‐NMR spectroscopic studies of a 46‐kDa homodimer, βB2‐crystallin, from bovine eye lens are presented. βB2‐crystallin has terminal extensions extending from globular N‐ and C‐terminal domains that are well resolved in the NMR spectra, whereas, in the main, resonances from the bulk of the protein are not observed. Using two‐dimensional NMR methods on βB2‐crystallin, its synthesised terminal extensions and a proteolysed sample of βB2‐crystallin with a portion of its C‐terminus removed, it was possible to assign resonances to most of the amino acids in the terminal extensions. One‐dimensional experiments at various pH values provided H‐2 chemical shifts for the three terminal extension histidines from which their pKa values were measured. It is concluded that the terminal extensions appear to be of little ordered conformation, are accessible to solvent and flex freely from the main body of the protein. The results of the NMR spectroscopic studies of βB2‐crystallin are in excellent agreement with those for the X‐ray crystal structure [Bax, B., Lapatto, R., Nalini, V., Driessen, H., Lindley, P. F., Mahadevan, D., Blundell, T. L. & Slingsby, C. (1990) Nature 347, 776–780]. No change in the spectrum of βB2‐crystallin was observed in the presence of calcium, suggesting that the termini are not involved in calcium binding.
4‐O‐(2‐Amino‐2‐deoxy‐α‐d‐glucopyranosyl)‐3‐deoxy‐d‐manno‐2‐octulosonic acid, a constituent of lipopolysaccharides of the genus Pectinatus
Authors: HELANDER Ilkka M. , MOLL Hermann , ZÄHRINGER Ulrich
Published: March 2005

A disaccharide containing GlcN and 3‐deoxy‐d‐manno‐2‐octulosonic acid (Kdo) was detected after acidic methanolysis and peracetylation of hydrofluoric‐acid‐treated smooth‐type and rough‐type lipopolysaccharide of Pectinatus cerevisiiphilus and Pectinatus frisingensis, which are strictly anaerobic bacteria capable of growing in packaged beer. The disaccharide was also found in alkalitreated lipopolysaccharide, but was not directly detectable from intact lipopolysaccharide. This suggested that the disaccharide carried a phosphate residue. The position of this phosphate was shown, by GLC/MS of appropriately degraded and derivatized samples, to be O6 of the GlcN. Methylation analysis of the purified disaccharide revealed that GlcN was linked to position 4 of Kdo. The acetylated derivative of the disaccharide was isolated in pure form, and, by 1H‐NMR and 13C‐NMR spectroscopy, it was confirmed to possess the structure α‐d‐GlcpN‐(1′→4)‐Kdo. In the lipopolysaccharide the amino group of GlcN is free.
The interactions of retinoids with retinol‐binding protein
Authors: MANOR Dan , CALLENDER Robert , NOY Noa
Published: March 2005

We have measured the pre‐resonance Raman spectrum of retinal, retinoic acid and retinol in dilute CCl4 solutions and when bound to the bovine‐serum retinol‐binding protein. The comparison reveals that the binding interactions does not involve any specific interactions of the head group and/or the polyene chain with a particular protein residue. The data indicate hydrogen bonding of bound retinal's head‐group oxygen to water, as well as some torsional angle change of its polyene chain upon binding.
Purification and characterization of extracellular, acidic chitinase isoenzymes from elicitor‐stimulated parsley cells
Authors: KIRSCH Christoph , HAHLBROCK Klaus , KOMBRINK Erich
Published: March 2005

Treatment of cultured parsley cells (Petroselinum crispum) with fungal elicitor caused large increases in the activities of chitinase and 1,3‐β‐glucanase. Chitinase activity accumulated predominantly in the culture medium, whereas 1,3‐β‐glucanase activity was located almost exclusively intracellularly. Extracellular chitinase activity was resolved into six different isoenzymes, all of which were purified and characterized. All six isoforms were acidic proteins (pI 3.8–5.3), with molecular mass 30–38 kDa. Four were exochitinases and two were endochitinases. The most abundant isoform also showed lysozyme activity. Three of the exochitinases were glycoproteins and two of these were reactive with an antiserum specific for xylose in complex glycosidic structures. The exochitinases constituted relatively small proportions of the total chitinase activity and may serve a different function in cellular metabolism compared to the more abundant endochitinases.
Cell‐specific expression of human β‐galactoside α2,6‐sialyltransferase transcripts differing in the 5′ untranslated region
Authors: AASHEIM Hans‐Christian , AAS‐ENG Dordi Anne , DEGGERDAL Arne , BLOMHOFF Heidi Kiil , FUNDERUD Steinar , SMELAND Erlend B.
Published: March 2005

In humans, two cDNAs have been isolated encoding β‐galactoside α2,6‐sialyltransferase, differing only in part of the 5′ untranslated region. Primer extension data show that the two cDNAs are near full‐length clones. RNase protection analysis of different cell types showed that the transcript corresponding to the α2,6‐sialyltransferase cDNA isolated from a B‐cell library resided only in mature B cells. In contrast, the transcript corresponding to the α2,6‐sialyltransferase cDNA isolated from a placenta library was found in all cells tested. Our results also indicate the existence of a third α2,6‐sialyltransferase transcript in the hepatoma cell line HepG2. Mature B cells were found to express high amounts of α2,6‐sialyltransferase mRNA, compared to other cell types tested, as shown by Northern blot analysis. Moreover there was an increased expression of β‐galactoside α2,6‐sialyltransferase mRNA in activated B cells compared to resting B cells. In vitro transcription and translation of the cDNAs resulted in a protein of 45 kDa, but the transcripts were translated with different efficiency, suggesting a role for the 5′ untranslated region in regulation of translation. We have also made an α2,6‐sialyltransferase construct lacking the specific 5′ regions of the two cDNAs. A transcript generated from this construct was translated more efficiently in vitro than the two α2,6‐sialyltransferase cDNAs.
Exchange reactions catalyzed by group‐transferring enzymes oppose the quantitation and the unravelling of the identity of the pentose pathway
Authors: FLANIGAN Ian , COLLINS J. Grant , ARORA Krishan K. , MacLEOD John K. , WILLIAMS John F.
Published: March 2005

The distributions and rates of transfer of carbon isotopes from a selection of specifically labelled ketosugar‐phosphate substrates by exchange reactions catalyzed by the pentose and photosynthetic carbon‐reduction‐pathway group‐transferring enzymes transketolase, transaldolase and aldolase have been measured using 13C‐NMR spectroscopy.

The rates of these exchange reactions were 5, 4 and 1.5 μmol min−1 mg−1 for transketolase exchange, transaldolase exchange and aldolase exchange, respectively.

A comparison of the exchange capacities contributed by the activities of these enzymes in three in vitro liver preparations with the maximum non‐oxidative pentose pathway flux rates of the preparations shows that transketolase and aldolase exchanges exceeded flux by 9–19 times in liver cytosol and acetone powder enzyme preparations and by 5 times in hepatocytes. Transaldolase was less effective in the comparison of exchange versus flux rates: transaldolase exchange exceeded flux by 1.6 and 5 in catalysis by liver cytosol and acetone powder preparations, respectively, but was only 0.6 times the flux in hepatocytes.

Values of group enzyme exchange and pathway flux rates in the above three preparations are important because of the feature role of liver and of these particular preparations in the establishment, elucidation and measurement of a proposed reaction scheme for the fat‐cell‐type pentose pathway in biochemistry.

It is the claim of this paper that the excess of exchange rate activity (particularly transketolase exchange) over pathway flux will overturn attempts to unravel, using isotopically labelled sugar substrates, the identity, reaction sequence and quantitative contribution of the pentose pathway to glucose metabolism.

The transketolase exchange reactions relative to the pentose pathway flux rates in normal, regenerating and foetal liver, Morris hepatomas, mammary carcinoma, melanoma, colonic epithelium, spinach chloroplasts and epididymal fat tissue show that transketolase exchange may exceed flux in these tissues by factors ranging over 5–600 times.

The confusion of pentose pathway theory by the effects of transketolase exchange action is illustrated by the 13C‐NMR spectrum of the hexose 6‐phosphate products of ribose 5‐phosphate dissimilation, formed after 30 min of liver enzyme action, and shows 13C‐labelling in carbons 1 and 3 of glucose 6‐phosphate with ratios which range over 2.1–6.4 rather than the mandatory value of 2 which is imposed by the theoretical mechanism of the pathway.
Low activity of the yeast cAMP‐dependent protein kinase catalytic subunit Tpk3 is due to the poor expression of the TPK3 gene
Authors: MAZÓN María J. , BEHRENS M. Margarita , MORGADO Eulalia , PORTILLO Francisco
Published: March 2005

Three genes TPK1, TPK2 and TPK3 encode in Saccharomyces cerevisiae distinct catalytic subunits of cAMP‐dependent protein kinase (cAPK). We have measured cAPK activity in vitro and, indirectly, in vivo in yeast strains carrying only one of the three TPK genes. The strain containing TPK3 as the only intact TPK gene showed nearly undetectable phosphorylating activity and no TPK3 mRNA could be detected, although the cells grow normally. Overexpression of TPK3 in a high copy vector or under the control of the inducible GAL1 promoter did not by itself result in a corresponding increase in activity but coexpression of BCY1, the gene coding for the regulatory subunit, was necessary in both cases to achieve high levels of phosphorylating activity. Moreover, BCY1 overexpression not only increased Tpk3 catalytic activity but also increased the amount of TPK3 mRNA detected in Northern blots.
Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor
Authors: ŠABOVÁ Ludmila , ZEMAN Igor , SUPEK Frantisek , KOLAROV Jordan
Published: March 2005

The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROX1 factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using lacZ as a reporter gene to determine the cis‐acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T‐rich segment adjacent to the consensus elements that are present in the 5′ flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROX1 factor.

The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADP/ATP translocation is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX5b, HEM13 and CYC7 that are negatively regulated by oxygen and heme.
Enzymes of anaerobic metabolism of phenolic compounds
Authors: BIEGERT Thomas , ALTENSCHMIDT Uwe , ECKERSKORN Christoph , FUCHS Georg
Published: March 2005

The initial step of anaerobic 4‐hydroxybenzoate and 3‐hydroxybenzoate degradation was studied in a denitrifying Pseudomonas sp. 4‐Hydroxybenzoate and 3‐hydroxybenzoate are converted into their coenzyme A (CoA) thioesters by two different specific coenzyme A ligases. 4‐Hydroxybenzoate‐CoA ligase (AMP‐forming) was purified 350‐fold. The ligase is active as a monomer of molecular mass 48 kDa, as determined by gel filtration and SDS/PAGE. At a pH optimum of 8.5, the apparent Km values for 4‐hydroxybenzoate, ATP, and coenzyme A are 37 μM, 77 μM, and 125 μM, respectively. The enzyme reacts specifically with 4‐hydroxybenzoate (100%) and 4‐aminobenzoate (30%). Other analogues of benzoate, notably 3‐ or 2‐hydroxybenzoate, are inactive, and 2,4‐dihydroxybenzoate and 2‐hydroxy‐4‐methylbenzoate act as competitive inhibitors (Ki= 1 μM). Polyclonal antibodies were raised and used in immunoblot assays to study the regulation of the expression of 4‐hydroxybenzoate‐CoA ligase. The ligase is synthesized when cells are grown anaerobically with 4‐hydroxybenzoate, phenol, or p‐cresol; phenol and p‐cresol are degraded via 4‐hydroxybenzoate. The enzyme is not present in cells grown aerobically with 4‐hydroyxbenzoate or anaerobically with benzoate or 4‐hydroxyphenylacetate.
Characterization of a clotting protein, isolated from plasma of the freshwater crayfish Pacifastacus leniusculus
Authors: KOPÁČEK Petr , HALL Martin , SÖDERHÄLL Kenneth
Published: March 2005

A protein responsible for clot formation was isolated from plasma of the crayfish Pacifastacus leniusculus, by repeated percipitation at low ionic strength, pH 6.0. The protein, here named clotting protein (CP), is a lipoglycoprotein, which consists of two 210‐kDa subunits, covalently associated by disulfide bonds. Preparations of the CP can form stable clots in the presence of crayfish haemocyte lysate supernatant, which contains endogenous, Ca2+‐dependent transglutaminase (TGase) activity. The covalent, TGase‐mediated polymerization of CP could clearly be visualized in SDS/PAGE under reducing conditions, where the 210‐kDa subunit is covalently cross‐linked into dimeric, trimeric and higher polymeric forms. The CP was shown to be a substrate for transglutaminases, since two different fluorescent TGase substrates, namely dansylcadaverine and a dansylated glutamine‐containing peptide, were incorporated into the CP subunit by active TGase. This indicates the presence of both glutamine and lysine residues in the CP, accessible for TGase cross‐linking. The amino acid composition and the N‐terminal amino acid sequence of the clotting protein was determined.
Responses of metabolic systems to large changes in enzyme activities and effectors
Authors: SMALL J. Rankin , KACSER Henrik
Published: March 2005

This first paper in a series investigates the problem of predicting and analysing the effects of large changes in enzyme activities or external nutrients/effectors on metabolic fluxes. We introduce the concept of a deviation index, D, which gives a measure of the relative change in a metabolic variable (e.g. flux) due to a large (non‐infinitesimal) relative change in a parameter (e.g. enzyme). Using simplifying kinetic assumptions we have found, for an unbranched metabolic chain, a direct relationship between deviation indices and flux control coefficients. This relationship provides a method to estimate flux control coefficients using a single large change in enzyme activity. We also provide a method of predicting the effects of, for example, DNA manipulation or other techniques for enzyme activity/concentration changes on metabolic fluxes. Up‐modulations of single enzymes rarely produce significant changes in fluxes. We show that combined changes of activity of a group of enzymes will produce a more than ‘additive’ response. We provide a method of predicting the effects of these combined changes, given either the flux control coefficients of the group of enzymes or the effects on the flux of changing the enzymes individually. A similar analysis is carried out for large changes in external nutrients or effectors. These amplification factors, f, give experimentally accessible estimates of the expected changes in metabolic variables. We provide three ‘case studies’ to illustrate our results.
Responses of metabolic systems to large changes in enzyme activities and effectors
Authors: SMALL J. Rankin , KACSER Henrik
Published: March 2005

We extend the analysis of unbranched chains (preceding paper) to large parameter changes in branched systems using linear kinetic assumptions. More complex relationships between flux control coefficients and deviation indices are established. In particular, the deviation index in such systems depends on more than one control coefficient as well as on the magnitude of the enzyme change. Non‐additivity of the indices is the general rule. Combined changes of groups of enzymes, whether co‐ordinate or not, have also been formulated. Control coefficients can be estimated from a small number of independent large‐change experiments. Alternatively, the amplification factors can be calculated given the knowledge of the control coefficients. A ‘case study’ using published data is presented.

The movement of intermediate metabolites as a consequence of large parameter changes can be dealt with in a similar manner.

Experimental methods for showing the admissibility of assuming the simplifying assumptions used are summarised. Some simulation studies show possible limits of the application of the approach and some aspects of the general, non‐linear, case are discussed. It is concluded that, although metabolic systems are in principle non‐linear, many behave, in practice, as quasi‐linear systems. The relationships established between deviation indices and control coefficients therefore provide a practical way of predicting the effects of large‐scale changes in parameters for many metabolic systems.
Synthesis of human parathyroid‐hormone‐related protein(1–141) in Saccharomyces cerevisiae
Authors: RIAN Edith , JEMTLAND Rune , OLSTAD Ole K. , GORDELADZE Jan O. , GAUTVIK Kaare M.
Published: March 2005

Gene fusions have been widely used in heterologous expression systems as a technique to stabilize the recombinant product against proteolysis, increase the translational initiation efficiency or to serve as an affinity handle for the purification of the protein. A further advantage is the potential to generate an authentic amino terminus of the foreign protein when this is vital for its biological activity, such as for the ability of human parathyroid‐hormone‐related protein (hPTHrP) to mediate activation of adenylate cyclase. We report here the construction and utility of a ubiquitin fusion protein system for production of the otherwise short‐lived hPTHrP(1–141) as a carboxyl extension to ubiquitin in yeast. A hybrid gene containing the hPTHrP(1–141) cDNA coding region fused in‐frame to the 3′ end of the yeast ubiquitin cDNA was constructed and expressed under the control of the regulatable yeast metallothionein promoter. The recombinant protein was purified to homogeneity and finally characterized by N‐terminal amino acid sequencing and amino acid composition analysis, demonstrating that the fusion protein was cleaved correctly and quantitatively in vivo by an ubiquitin‐specific yeast endoprotease to generate authentic hPTHrP(1–141). hPTHrP(1–141) stimulated adenylate cyclase in rat osteosarcoma cell membranes to the same extent as equimolar amounts of recombinant human parathyroid hormone(1–84) and [Tyr34]hPTHrP(1–34)amide. Thus, this expression cloning strategy permits the production of authentic, biologically active recombinant hPTHrP(1–141), and the procedure can easily be adapted to make PTHrP analogues for further studies of its domain‐specific activities and biological roles.
The primary structures and properties of non‐stomach lysozymes of sheep and cow, and implication for functional divergence of lysozyme
Authors: ITO Yuji , YAMADA Hidenori , NAKAMURA Miyuki , YOSHIKAWA Ayako , UEDA Tadashi , IMOTO Taiji
Published: March 2005

Lysozymes were purified from the homogenate of cow and sheep kidneys, and their amino‐acid sequences as well as some enzymic properties were determined. Like most mammalian lysozymes both sheep and cow kidney lysozymes are composed of 130 amino acids. The sequences of these two lysozymes are the most similar to each other (95% identity), the second most similar to the conventional mammalian lysozymes like human, rat and rabbit lysozymes (74–85% identity), and much less similar to their own stomach lysozymes (65–70% identity). Cow kidney lysozyme is also different from cow milk lysozyme (partial sequence), indicating that cow contains at least three kinds of chicken type lysozymes, that is kidney, milk and stomach lysozymes. The activities of cow and sheep kidney lysozymes were 3% and 29% against Micrococcus luteus at pH 7.0, ionic strength of 0.1 and 30 °C, and 57% and 84% against glycol chitin at pH 5.5 and 40 °C, which were expressed as percentages relative to hen lysozyme. The net charges of cow and sheep lysozymes at pH 7 were less positive (+1.5 and +2.5, respectively) than human and hen lysozymes (both +8.0) and rather close to the stomach ones (‐2 to 0). The decreased net positive charge observed in cow and sheep kidney lysozymes may suggest that the ruminant kidney lysozyme had functioned once as a digestive enzyme in the stomach of an ancestral ruminant.
Vigilin is a cytoplasmic protein
Authors: NEU‐YILIK Gabriele , ZORBAS Haralabos , GLOE Torsten R. , RAABE Hans‐Merten , HOPP‐CHRISTENSEN Torsen A. , MÜLLER Peter K.
Published: March 2005

A fusion protein composed of about two vigilin domains and β‐galactosidase was used to raise polyclonal antibodies which were affinity‐purified and employed for immunoblotting and immunohistochemistry. A protein of an apparent molecular mass of 155 kDa could be stained in extracts of a variety of cells from different species and organs. Immunohistological studies on single cells showed that vigilin is accumulated in the cytoplasm. During in vitro maintenance of primary cell cultures, as well as of a growth‐factor‐dependent cell line, vigilin expresion decreases and ceases in senescent cells. In contrast, vigilin is constitutively expressed in all other transformed cell lines of various origin studied so far. Vigilin expression can be induced in peripheral blood lymphocytes by mitogen stimulation. These observations suggest an involvement of vigilin in processes of cell activation. Immunoblot experiments demonstrating the presence of vigilin in a broad range of eukaryotes, indicate a high degree of evolutionary conservation.
Modification of the E1ATP binding site of Na+/K+‐ATPase by the chromium complex of adenosine 5′‐[β,γ‐methylene]triphosphate blocks the overall reaction but not the partial activities of the E2 conformation
Authors: HAMER Evelyn , SCHONER Wilhelm
Published: March 2005

The chromium complex of adenosine 5′‐[β,γ‐methylene]triphosphate, Cr(H2O)4AdoPP[CH2]P, inactivates Na+/K+‐ATPase from pig kidney at 37°C with an inactivation velocity constant of 7.1X10‐3min‐1 by binding to the high‐affinity ATP site (E1ATP site). The dissociation constant (Kd) of the analogue at this site is 26 μM, and of ATP 0.8 μM. Inactivation of the overall reaction of Na+/K+‐ATPase by Cr(H2O)4AdoPP[CH2]P did not alter the activities of the E2 conformational state such as K+‐activated p‐nitrophenylphosphatase, 86Rb+ occlusion and [3H]ouabain binding by the ‘backdoor’ phosphorylation. However, [3H]ouabain binding via the forwards reaction from E1ATP in the presence of Na++ Mg2+ is inhibited.

K+‐activated p‐nitrophenylphosphatase activity of the Cr(H2O)4AdoPP[CH2]P‐inactivated enzyme decreases when an MgATP analogue, the tetraammine cobalt complex of ATP, Co(NH3)4ATP, is used additionally to inactivate the E2ATP site. The enzyme activity of K+‐activated phosphatase is also lost if the β,γ‐bidentate chromium(III) complex of ATP, Cr(H2O)4ATP, which may form a stable E1‐chromo‐phosphointermediate, is used for the inactivation of Na+/K+‐ATPase.

We conclude that the phenomenon of a blockade of the overall reaction of Na+/K+‐ATPase by the formation of a stable E1· CrAdoPP[CH2]P complex, leading thereby to a loss of the partial activities of the E1 conformation, but not of the E2 conformation, is consistent with the postulate of an (αβ)2 diprotomeric nature of the sodium pump. The observation, moreover, that treatment of the sodium pump with Cr(H2O)4ATP but not with Cr(H2O)4AdoPP[CH2]P leads to an inactivation of K+‐activated phosphatase seems to indicate that the formation of a E1‐phosphointermediate affects the E2ATP site.
Regulation of the L‐arginine‐dependent and tetrahydrobiopterin‐dependent biosynthesis of nitric oxide in murine macrophages
Authors: SCHOEDON Gabriele , SCHNEEMANN Markus , HOFER Simone , GUERRERO Lourdes , BLAU Nenad , SCHAFFNER Andreas
Published: March 2005

Nitric oxide is a recently discovered biomolecule with a broad range of actions. The present study investigated the regulation of nitric oxide synthase activity by dexamethasone and the cofactor tetrahydrobiopterin in murine macrophages. The influence of the tetrahydrobiopterin biosynthesis inhibitors 2,4‐diamino‐6‐hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, and phenpro‐coumon, an inhibitor of sepiapterin reductase, on the synthesis of nitric oxide was investigated. Dexamethasone decreased the nitric oxide production due to direct inhibition of the induction of nitric oxide synthase and of GTP cyclohydrolase. Substitution of tetrahydrobiopterin via sepiapterin could not overcome the dexamethasone‐mediated inhibition. 2,4‐Diamino‐6‐hydroxypyrimidine abolished nitric oxide synthesis and synergized with dexamethasone, completely eliminating nitric oxide production. Phenprocoumon inhibited production of nitric oxide via interference with later steps of tetrahydrobiopterin biosynthesis. An exogenous supply of tetrahydrobiopterin through sepiapterin led to a further increase of nitric oxide production, even in fully activated macrophages. The amount of nitric oxide produced by murine macrophages is therefore limited by the amount of tetrahydrobiopterin present in the cells. Inhibitors of tetrahydrobiopterin biosynthesis could provide a novel approach for therapy of pathological conditions mediated by nitric oxide, such as septic shock.
Two different genes from Schwanniomyces occidentalis determine ribosomal resistance to cycloheximide
Authors: Del POZO Lourdes , ABARCA Dolores , HOENICKA Janet , JIMENEZ Antonio
Published: March 2005

Two genes (SCR1 and SCR2) encoding natural cycloheximide resistance in the budding yeast Schwanniomyces occidentalis have been cloned by expression in Saccharomyces cerevisiae. Both genes determine resistance to the inhibitory action of cycloheximide on the ribosome. SCR1 and SCR2 are present as single copies in Schwanniomyces occidentalis, where they map on chromosomes II and V, respectively. The nucleotide sequence of SCR2 contains an open reading frame of 321 nucleotides which is interrupted by an intron of 452 nucleotides. It encodes a polypeptide of 106 amino acids of molecular mass 12.25 kDa and pI 11.19. The deduced amino acid sequence shows a high degree of similarity to the L41 protein of the 60S ribosomal subunit from several eukaryotic organisms. The intron and the 5′non‐coding region of SCR2 possess conserved elements which are typical of yeast ribosomal protein genes. A single amino acid change determines the resistance or sensitive phenotype to cycloheximide of the 80S ribosome since replacement of Gln56 in L41 from Schwanniomyces with Pro, by site‐directed mutagenesis, confers cycloheximide sensitivity. SCR2 may serve as a practical yeast cloning marker if integrated in a multicopy plasmid.
Structural studies of the O‐antigenic polysaccharide of an enteroaggregative Escherichia coli strain
Authors: WEINTRAUB Andrej , LEONTEIN Karin , WIDMALM Göran , VIAL Pablo A. , LEVINE Myron M. , LINDBERG Alf A.
Published: March 2005

The polysaccharide part of the lipopolysaccharide obtained from an enteroaggregative Escherichia coli strain isolated from a young child with diarrhoea in Santiago, Chile (strain 73–1) was investigated. Sugar and methylation analyses of native and partially degraded polysaccharide together with 1H‐NMR and 13C‐NMR spectroscopy revealed that the polysaccharide is built of pentasaccharide repeating units. The structure of the repeating unit of E. coli strain 73–1 O‐polysaccharide is
Determination of the nuclear‐magnetic‐resonance solution structure of cardiotoxin CTX IIb from Naja mossambica mossambica
Authors: O'CONNELL John F. , BOUGIS Pierre E. , WÜTHRICH Kurt
Published: March 2005

The NMR structure of cardiotoxin CTX IIb from Naja mossambica mossambica in aqueous solution was determined from a total of 593 nuclear Overhauser enhancement distance constraints and 135 dihedral angle constraints, which were collected using two‐dimensional homonuclear 1H‐NMR experiments. Structure calculations were performed with the program DIANA, using the redundant dihedral angle constraints strategy for improved convergence, followed by restrained energy minimization with the program FANTOM and a modified version of the program AMBER. The CTX IIb structure is represented by a group of 20 conformers with an average root‐mean‐square deviation relative to the mean solution structure of 0.072 nm for the backbone atoms, and 0.116 nm for all heavy atoms. The molecular structure of CTX IIb is characterized by a three‐stranded β‐sheet made up of residues 20–26, 32–39 and 48–54, and a two‐stranded β‐sheet composed of residues 1–5 and 10–14. A cluster of four disulfide bonds, 3–21, 14–38, 42–53 and 54–59, form the core of the molecule and crosslink the individual polypeptide strands. The NMR structure is similar to the previously reported X‐ray crystal structure of the cardiotoxin CTX VII4 from the same species. Differences between the two structures were noted in the tips of the two loops formed by residues 6–9 and 27–31, which connect the β‐strand 1–5 with 10–14, and 20–26 with 32–39, respectively. For these loops the NMR data also indicate significantly increased dynamic disorder in the solution structure. These observations are discussed with respect to earlier suggestions by others that these two loops are essential structural elements for function and specificity of a wide variety of homologous toxins.
The mechanism of substrate and coenzyme binding to clostridial glutamate dehydrogenase during oxidative deamination
Authors: BASSO Luiz A. , ENGEL Paul C. , WALMSLEY Adrian R.
Published: March 2005

The binding of NAD+ and L‐Glutamate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been investigated by stopped‐flow fluorescence spectroscopy. The formation of the binary complexes produces little change in the protein fluorescence but formation of the ternary complex results in quenching of its fluorescence with a maximum value of 40%. This finding, coupled with the finding that a step prior to hydride transfer but subsequent to ternary complex formation is rate limiting, has enabled us to monitor the kinetics of ternary complex formation in detail. The ternary complex can be formed via the GDH–NAD+ or the GDH‐L‐Glu binary complexes, but the route via the GDH‐NAD+ binary complex is the preferred pathway. The equilibrium and rate constants for the formation of the two binary complexes and the ternary complex formed via the two possible pathways have been determined. These studies have revealed an interaction between the coenzyme‐binding site and the substrate‐binding site, which lead to a decrease in the binding constant for the second substrate binding to the enzyme. The free energy coupling between the binary and ternary complexes is about 2.4–2.8 kJ · mol−1. We propose that there is a further isomerisation of the ternary complex, which is rate limiting for the steady‐state turnover of the enzyme. Formation of this complex is characterised by an increased negative interaction, with a free energy coupling between these complexes of 6.3–11.6 kJ · mol−1.
On the activation mechanism of the H+‐ATP synthase and unusual thermodynamic properties in the alkalophilic cyanobacterium Spirulina platensis
Authors: BAKELS Ronald H. A. , van WALRAVEN Hendrika S. , KRAB Klass , SCHOLTS Marijke J. C. , KRAAYENHOF Ruud
Published: March 2005

The activation requirements and thermodynamic characteristics of ATP synthase from the alkalophilic cyanobacterium Spirulina platensis were studied in coupled membrane vesicles. Activation by methanol increased the Vmax, while the Km for MgATP was unaffected (0.7 mM). We propose that in Sp. platensis, as in chloroplasts, the activating effect of methanol is based on perturbation of the γ‐ɛ subunit interaction. Light‐driven ATP synthesis by membrane vesicles of Sp. platensis was stimulated by dithiotreitol. The characteristics of the activation of the ATP synthase by the proton electrochemical potential difference (H+) were analyzed on the basis of the uncoupled rates of ATP hydrolysis as a function of a previously applied proton gradient. Two values of H+, at which 50% of the enzyme is active, were found; 13–14 kJ·mol−1 for untreated membrane vesicles, and 4–8 kJ·mol−1 for light‐treated and dithiotreitol‐treated membrane vesicles. These values are lower than the corresponding values for the oxidized and reduced forms, respectively, of the chloroplast enzyme. Although no bulk proton gradient could be observed, membrane vesicles of Sp. platensis were able to maintain an equilibrium phosphate potential (Gp) of 40–43.5 kJ·mol−1, comparable to values found for Synechococcus 6716 and Anabaena 7120 membrane vesicles. Acid/base‐transition experiments showed that the thermodynamic threshold, H+, for ATP synthesis, catalyzed by light‐treated and dithiotreitol‐treated Spirulina membrane vesicles, was less than 5 kJ·mol−1. The activation characteristics and the low thermodynamic threshold allow ATP synthesis to occur at low H+ values. The findings are discussed, both with repect to differences and similarities with the enzymes from chloroplasts and other cyanobacteria, and with respect to the alkalophilic properties of Sp. platensis.
Regulation of low‐density‐lipoprotein receptors in the human hepatoma cell line Hep G2
Authors: KAMPS Jan A. A. M. , VAN BERKEL Theo J. C.
Published: March 2005

The regulation of low‐density‐lipoprotein(LDL)‐receptor activity by 4β‐phorbol 12‐myristate 13‐acetate (PMA) and LDL was investigated in the human hepatoma cell line Hep G2. Treatment of Hep G2 cells for 22 h with PMA results in an 18.6‐fold increase in the amount of LDL‐binding sites on the cell surface. The rate of turnover of LDL receptors was not significantly altered upon PMA treatment. Treatment of cultured rat parenchymal cells and human parenchymal cells with PMA did not lead to increased binding of LDL to these cells, suggesting that protein‐kinase‐C‐mediated regulation of LDL‐receptor activity is specific for Hep G2 cells and that in this aspect of regulation of LDL receptors, Hep G2 cells do not reflect human hepatocytes. The down‐regulation of LDL receptors by a 22‐h prior incubation with LDL in PMA‐treated Hep G2 cells, in which LDL receptors are upregulated, is more effective than in non‐treated cells. Prior incubation of control Hep G2 cells with an excess of LDL caused a partial down‐regulation to 33% of the initial level of receptor binding. In PMA‐treated Hep G2 cells an excess of non‐labeled LDL, led to a down‐regulation to 13% of the PMA‐induced level. Prior incubation of Hep G2 cells with LDL in the presence of PMA led to a 2.3‐fold increase of intracellular cholesteryl esters and a 9.1‐fold increase in acyl‐CoA:cholesterol‐acyltransferase (ACAT) activity. In control cells, LDL prior incubation led to a 1.6‐fold increase in intracellular cholesteryl esters and a 1.8‐fold increase of ACAT activity. It is concluded that in Hep G2 cells LDL itself can be an effective suppressor of the expression of LDL receptors, provided that the initial amount of receptor allows an adequate intracellular delivery of cholesterol to its sterol‐regulatory site.
FAD and substrate analogs as probes for lysine N6‐hydroxylase from Escherichia coli EN 222
Authors: MACHEROUX Peter , PLATTNER Hans Jürgen , ROMAGUERA Antonio , DIEKMANN Hans
Published: March 2005

Lysine N6‐hydroxylase catalyzes the hydroxylation of the N‐terminal amino function of l‐lysine at the expense of NADPH and molecular oxygen. The enzyme also requires FAD for its catalytic activity. Unlike other flavoprotein monooxygenases, binding of FAD is rather weak with a Kd of 30 μM at 4°C. The spectral properties of FAD bound to lysine N6‐hydroxylase are very similar to free oxidized FAD. In the absence of substrate, the enzyme has an NADPH oxidase activity which results in the generation of hydrogen peroxide. With increasing concentration of l‐lysine, the NADPH oxidase activity is enhanced up to 10‐fold and the generation of hydrogen peroxide decreases. At the same time, the substrate is hydroxylated. Km values for l‐lysine and FAD were determined as 105 μM and 0.7 μM, respectively. Utilizing FAD analogs, we could demonstrate that l‐lysine exerts its effector role mostly on the reductive half reaction of the overall catalytic cycle. Prolonged incubation of the enzyme with either 8‐chloro‐ or 8‐fluoro‐FAD gave rise to a covalently attached flavin which is formed as a result of the nucleophilic attack of a thiolate on the 8‐position of the flavin. Several lines of evidence indicate that the reaction takes place in the FAD binding site of the protein. The substrate specificity was investigated using amino acids with various lengths of side chain. L‐Lysine and derivatives with similar side chain length are hydroxylated by lysine N6‐hydroxylase. Ornithine, the lower homolog of lysine, was not hydroxylated and did not affect the NADPH oxidase activity of the enzyme. On the other hand, homolysine accelerated the rate of NADPH oxidation but was not hydroxylated. Additional requirements for effecient hydroxylation were also investigated using a variety of substrate analogs. From these studies a schematic structure of the active site of the enzyme was deduced. Sequence comparison of the FAD binding site of various flavoproteins revealed possible factors for weak binding of the cofactor in the case of lysine N6‐hydroxylase.
Evidence for a ternary interaction between α‐actinin, (meta)vinculin and acidic‐phospholipid bilayers
Authors: NIGGLI Verena , GIMONA Mario
Published: March 2005

The cytoskeletal component vinculin has been demonstrated by hydrophobic photoradiolabelling, to insert into bilayers containing acidic phospholipids and trace amounts of a photoactivatable analogue of lecithin. It is shown in this study that the higher‐molecular‐mass variant metavinculin and α‐actinin, also share this property. α‐Actinin and vinculin were also shown to associate with phosphatidylserine liposomes by chromatography of protein/lipid mixtures on a Bio‐Gel A‐5m column. Furthermore, interesting differences in the behaviour of binary mixtures of these proteins, in the presence of phosphatidylserine liposomes, are shown. Thus, incubation of α‐actinin with vinculin or metavinculin, prior to the addition of liposomes, strongly inhibited the photoradiolabelling of α‐actinin under conditions in which the liposome surface was non‐limiting, but enhanced the labelling of vinculin. In contrast, vinculin and metavinculin did not mutually influence their labelling. Using gel‐filtration chromatography, it was shown that α‐actinin still bound to the vinculin‐liposome complex, under conditions similar to those used for hydrophobic photolabelling with a non‐limiting lipid surface. In the presence of limiting amounts of liposomes, the α‐actinin/vinculin ratio was markedly decreased in the liposome fractions. Our results suggest the formation of a ternary complex consisting of vinculin, α‐actinin and phospholipids. In this complex, both proteins interact at the bilayer, resulting in an altered conformation of the two proteins and, as a consequence, in modified bilayer interactions.
Chemical structures of the core regions of Campylobacter jejuni serotypes O:1, O:4, O:23, and O:36 lipopolysaccharides
Authors: ASPINALL Gerald O. , McDONALD Armando G. , RAJU T. Shantha , PANG Henrianna , MORAN Anthony P. , PENNER John L.
Published: March 2005

Complete structures, including the location of N‐acetylneuraminic acid (Neu5Ac) residues, were assigned for the core regions of Campylobacter jejuni serotypes O:1, O:4, and O:23 and O:36 lipopolysaccharides (LPS). In continuation of earlier studies, structure determinations of liberated oligosaccharides and, where necessary, of intact LPS, were by 1H‐NMR spectroscopy, Smith degradation, chromium trioxide and enzymic degradations, in conjunction with methylation studies supported by fast‐atom‐bombardment mass spectrometry and linkage analyses by gas chromatography/mass spectrometry. It was concluded on the basis of the following structures, in which each was linked 1→5 to a terminal 3‐deoxy‐D‐manno‐octulosonic acid residue, that the core regions with qualititatively similar sugar compositions showed serotypic differences in one or more of their sequences, linkage types, and anomeric configurations:

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The outer regions of each structure carry Neu5Ac residues linked 2→3 to available β‐D‐Galp residues and show striking similarities with various glycosphingolipids of the ganglioside family. However, Neu5Ac epitopes are not apparently involved in determining serospecificity.
Chemical structure of the core region of Campylobacter jejuni serotype O:2 lipopolysaccharide
Authors: ASPINALL Gerald O. , McDONALD Armando G. , RAJU T. Shantha , PANG Henrianna , KURJANCZYK Linda A. , PENNER John L. , MORAN Anthony P.
Published: March 2005

The complete structure for the core region of Campylobacter jejuni serotype O:2 lipopolysaccharide (LPS) was assigned through studies on derivatives of the liberated oligosaccharide (OS 2) and the intact LPS. Structure determinations were performed using 1H‐NMR spectroscopy, methylation studies supported by fast‐atom‐bombardment mass spectrometry and linkage analysis by gas chromatography/mass spectrometry, Smith degradation, and oxidation with chromium trioxide. It was concluded that complete oligosaccharide chains had the following structure:

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Role of tyrosine 129 in the active site of spinach glycolate oxidase
Authors: MACHEROUX Peter , KIEWEG Volker , MASSEY Vincent , SÖDERLIND Eskil , STENBERG Kaj , LINDQVIST Ylva
Published: March 2005

The enzymatic properties and the three‐dimensional structure of spinach glycolate oxidase which has the active‐site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the mutant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (ɛmax= 11400 M−1 cm−1). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F mutant does not lower the pKa of the N(3) of oxidized flavin as does the wild‐type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612–4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild‐type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the mutant enzyme. Typical features of wild‐type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)‐C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)‐sulfite adducts, are all conserved in the Y129F mutant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild‐type activity. The lower turnover number for the mutant of 0.74 s−1 versus 20 s−1 for the wild‐type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady‐state analysis gave Km values of 1.5 mM and 7 μM for glycolate and oxygen, respectively. The Km for glycolate is slightly higher than that found for wild‐type glycolate oxidase (1 mM) whereas the Km for oxygen is much lower. As was the case for wild‐type glycolate oxidase, reduction was found to be the rate‐limiting step in catalysis, with a rate of 0.63 s−1. The kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilization of the transition state.
The primary structure of branched‐chain α‐oxo acid dehydrogenase from Bacillus subtilis and its similarity to other α‐oxo acid dehydrogenases
Authors: WANG Ge‐Fu , KURIKI Takashi , ROY Kenneth L. , KANEDA Toshi
Published: March 2005

The bfmB muant of Bacillus subtilis requires branched short‐chain carboxylic acids for growth because the organism is known to be defective in branced‐chain α‐oxo acid dehydrogenase. The DNA in the region of bfmB has now been cloned and sequenced, and the gene has been analyzed. The results show that there are three open reading frames in the area, each of which is preceded by a putative ribosome binding site, and the last of which is followed by a putative transcription termination site with inverted repeats. The amino acid sequences deduced by analysis of the reading frames are highly similar (with 32–49% identity) to the E1α, E1β and E2 components of pyruvate, 2‐oxoglutarate and branched‐chain α‐oxo acid dehydrogenases from different sources. The thiamin diphosphate binding, putative subunit interaction and phosphorylation sites of the E1α of four reported branched‐chain α‐oxo acid dehydrogenases from different sources are very similar to those of the first open reading frame (E1α) of bfmB. A similar result is also obtained with the lipoylbinding site (lysine) and its domain of the E2 component of α‐oxo acid dehydrogenases from different sources. The present data, along with the reported biochemical data, lead to the conclusion that bfmB encodes a branched‐chain α‐oxo acid dehydrogenase, which is composed of E1α, E1β and E2 genes. This organization is identical to that of the 2‐oxoglutarate dehydrogenase in B. subtilis.