bluuebunny/crossref_metadata_2025 huggingface dataset:
Intestinal absorption of β‐lactam antibiotics and oligopeptides
Authors: KRAMER Werner , GIRBIG Frank , GUTJAHR Ulrike , KOWALEWSKI Simone , ADAM Friedhelm , SCHIEBLER Werner
Published: March 2005

The H+‐dependent uptake system responsible for the enteral absorption of oligopeptides and orally active β‐lactam antibiotics was functionally reconstituted into liposomes. Membrane proteins from rabbit small intestinal brush border membrane vesicles were solubilized with n‐octyl glucoside and incorporated into liposomes using a gel filtration method. At protein/lipid ratios of 1:10 and 1:40, the uptake of the orally active α‐amino‐cephalosporin, D‐cephalexin into proteoliposomes was stimulated by an inwardly directed H+ gradient and was protein‐dependent. In these proteoliposomes the binding protein for oligopeptides and β‐lactam antibiotics of Mr 127000 could be labeled by direct photoaffinity labeling with [3H]benzylpenicillin revealing an identical binding specificity as in the original brush border membrane vesicles.

The uptake system for β‐lactam antibiotics and oligopeptides showed a remarkable stereospecificity; only D‐cephalexin was taken up by intact brush border membrane vesicles, whereas the L‐enantiomer was not taken up to a significant extent. This stereospecificity for uptake was also seen after reconstitution of solubilized brush border membrane proteins into liposomes demonstrating a functional reconstitution of the peptide transporter. Both enantiomers however, bound to the 127‐kDa binding protein as was shown by a decrease in the extent of photoaffinity labeling of the 127‐kDa protein in the presence of both enantiomers.

After reconstitution of subfractions of brush border membrane proteins obtained by wheat germ lectin affinity chromatography into proteoliposomes, only liposomes containing the 127‐kDa binding protein showed a significant uptake of D‐cephalexin whereas the L‐enantiomer was not transported. The uptake rates for D‐cephalexin into proteoliposomes correlated with the content of 127‐kDa binding protein in these liposomes as was determined by specific photoaffinity labeling with [3H]benzylpenicillin. The purified 127‐kDa binding protein was also reconstituted into liposomes and its ability for specific binding of substrates as well as stereospecific uptake of cephalexin could be restored.

These results indicate that the binding protein for oligopeptides and β‐lactam antibiotics of Mr 127000 mediates the stereospecific and H+‐dependent transport of orally active β‐lactam antibiotics across the enterocyte brush border membrane. We therefore suggest that this 127‐kDa binding protein is the intestinal peptide transport system (or a component thereof).
The ICL1 gene from Saccharomyces cerevisiae
Authors: FERNÁNDEZ Ernestina , MORENO Fernando , RODICIO Rosaura
Published: March 2005

The glyoxylate cycle is essential for the utilization of C2 compounds by the yeast Saccharomyces cerevisiae. Within this cycle, isocitrate lyase catalyzes one of the key reactions. We obtained mutants lacking detectable isocitrate lyase activity, screening for their inability to grow on ethanol. Genetic and biochemical analysis suggested that they carried a defect in the structural gene, ICL1. The mutants were used for the isolation of this gene and it was located on a 3.1‐kb Bg/II‐SphI DNA fragment. We then constructed a deletion‐substitution mutant in the haploid yeast genome. It did not have any isocitrate lyase activity and lacked the ability to grow on ethanol as the sole carbon source. Both strands of a DNA fragment carrying the gene and its flanking regions were sequenced. An open reading frame of 1671 bp was detected, encoding a protein of 557 amino acids with a calculated molecular mass of 62515 Da. The deduced amino acid sequence shows extensive similarities to genes encoding isocitrate lyases from various organisms. Two putative cAMP‐dependent protein‐kinase phosphorylation sites may explain the susceptibility of the enzyme to carbon catabolite inactivation.
Molecular cloning and regulatory analysis of the cuticle‐degrading‐protease structural gene from the entomopathogenic fungus Metarhizium anisopliae
Authors: ST. LEGER Raymond J. , FRANK David C. , ROBERTS Donald W. , STAPLES Richard C.
Published: March 2005

The proteinaceous insect cuticle is an effective barrier against most microbes, but entomopathogenic fungi can breach it using extracellular proteases. We report here the isolation and characterization of a cDNA clone of the cuticle‐degrading protease (Pr1) of Metarhizium anisopliae. The cDNA sequence revealed that Pr1 is synthesized as a large precursor (40.3 kDa) containing a signal peptide, a propeptide and the mature protein predicted to have a molecular mass of 28.6 kDa. The primary structure of Pr1 has extensive similarity with enzymes of the subtilisin subclass of serine endopeptidases and the serine, histidine and aspartate components of the active site in subtilisins are preserved. Proteinase K demonstrated the closest sequence similarity to Pr1 (61%) but Pr1 was twofold more effective than proteinase K at degrading isolated cuticles of Manduca sexta and 33‐fold more effective at degrading structural proteins bound to the cuticle by covalent bonds. We postulate that the additional positively charged residues on the surface of the Pr1 molecule, as determined using proteinase K, may facilitate electrostatic binding to cuticle proteins which is a prerequisite for activity. Northern‐blot analysis of RNA and nuclear run‐on assays demonstrated transcriptional control of the expression of Pr1 during nutrient deprivation and during the formation of infection structures. Southern‐blot analysis demonstrated that genes with significant homologies to Metarhizium Pr1 were present in the entomopathogens Aspergillus flavus and Verticillium lecanii but not Zoophthora (=Erynia) radicans.
Selection and characterization of randomly produced mutants of gene V protein of bacteriophage M13
Authors: STASSEN Alphons P. M. , ZAMAN Guido J. R. , van DEURSEN Jan M. A. , SCHOENMAKERS John G. G. , KONINGS Ruud N. H.
Published: March 2005

Gene V protein of bacteriophage Ff (M13, f1, fd) is a master regulator of phage DNA replication and phage mRNA translation. It exerts these two functions by binding to single‐stranded viral DNA or to specific sequences in the 5′ ends of its target mRNAs, respectively. To study the structure/function relationship of gene V protein, M13 gene V was inserted in a phagemid expression vector and a library of missense and nonsense mutants was constructed by random chemical mutagenesis. Phagemids encoding gene V proteins with decreased biological activities were selected and the nucleotide sequences of their gene V fragments were determined. Furthermore, the mutant proteins were characterized both with respect to their ability to inhibit the production of phagemid DNA transducing particles and their ability to repress the translation of a chimeric lacZ reporter gene whose expression is controlled by the promoter and translational initiation signals of M13 gene II. From the data obtained, it can be deduced that the mechanism by which gene V protein binds to single‐stranded DNA differs from the mechanism by which it binds to its target sequence in the gene II mRNA.
Inactivation of human pancreatic lipase by 5‐dodecyldithio‐2‐nitrobenzoic acid
Authors: GARGOURI Youssef , CUDREY Claire , MEJDOUB Hafedh , VERGER Robert
Published: March 2005

Both thiol groups of native human pancreatic lipase can react with the new hydrophobic sulfhydryl reagent 5‐dodecyldithio‐2‐nitrobenzoic acid (Dod‐S‐NbS) in the absence of a denaturing agent. Here we describe for the first time the covalent and stoichiometric modification of the inaccessible SHII group of native pancreatic lipase, using a 16‐fold molar excess of this hydrophobic sulfhydryl reagent. A direct correlation was found to exist between the covalent modification of this SHII group and the loss of lipase activity. The question has not yet been answered, however, as to how Dod‐S‐NbS reaches the SHII‐containing residue, whereas classical hydrophilic sulfhydryl reagents are unable to do so. This difference in reactivity may be attributable to the hydrophobic character of Dod‐S‐NbS and its potential capacity to form aggregates inducing a conformational change in the lipase molecule.
Effect of KCl on the interactions between NADPH:cytochrome P‐450 reductase and either cytochrome c, cytochrome b5 or cytochrome P‐450 in octyl glucoside micelles
Authors: NISIMOTO Yukio , EDMONDSON Dale E.
Published: March 2005

Significant dissociation of FMN from NADPH:cytochrome P‐450 reductase resulted in loss of the activity for reduction of cytochrome b5 as well as cytochrome c and cytochrome P‐450. However, the ability to reduce these electron acceptors was greatly restored upon incubation of FMN‐depleted enzyme with added FMN. The reductions of cytochrome c and detergent‐solubilized cytochrome b5 by NADPH:cytochrome P‐450 reductase were greatly increased in the presence of high concentrations of KCl, although the stimulatory effect of the salt on cytochrome P‐450 reduction was less significant. No apparent effect of superoxide dismutase could be seen on the rate or extent of cytochrome reduction in solutions containing high‐salt concentrations. Complex formation of the flavoprotein with cytochrome c, which is known to be involved in the mechanism of non‐physiological electron transfer, caused a perturbation in the absorption spectrum in the Soret‐band region of cytochrome c, and its magnitude was enhanced by addition of KCl. Similarly, an appreciable increase in ellipticity in the Soret band of cytochrome c was observed upon binding with the flavoprotein. However, only small changes were found in absorption and circular dichroism spectra for the complex of NADPH:cytochrome P‐450 reductase with either cytochrome b5 or cytochrome P‐450. It is suggested that the high‐salt concentration allows closer contact between the heme and flavin prosthetic groups through hydrophobic‐hydrophobic interactions rather than electrostatic‐charge pairing between the flavoprotein and the cytochrome which causes a faster rate of electron transfer. Neither alterations in the chemical shift nor in the line width of the bound FMN and FAD phosphate resonances were observed upon complex formation of NADPH:cytochrome P‐450 reductase with the cytochrome.
Purification of bovine brain inositol‐1,4,5‐trisphosphate 5‐phosphatase
Authors: VERJANS Benoît , LECOCQ Raymond , MOREAU Colette , ERNEUX Christophe
Published: March 2005

In bovine brain, two soluble inositol‐1,4,5‐trisphosphate (InsP3) 5‐phosphatases, which catalyse the dephosphorylation of InsP3 to inositol 1,4‐bisphosphate, have been separated by DEAE‐Sephacel. Type I, i.e. the first eluted enzyme, is the main soluble form and is reminiscent of the membrane‐bound enzyme by multiple criteria. Type I was purified to apparent homogeneity by a method involving chromatography on DEAE‐Sephacel, Blue‐Sepharose, Sephacryl S‐200, phosphocellulose, and C18 HPLC. A single protein band of 42–43 kDa was identified by SDS/PAGE, corresponding to the peak of maximal activity. InsP3 5‐phosphatase was purified to apparent homogeneity to a final yield of 45–50 μg protein. The minimal estimate value of the Vmax for InsP3 5‐phosphatase was in the range 20–35 μmol · min−1· mg protein−1.
Structural analysis of the N‐linked carbohydrate chains of the 55‐kDa glycoprotein family (PZP3) from porcine zona pellucida
Authors: NOGUCHI Satoru , HATANAKA Yumiko , TOBITA Tohru , NAKANO Minoru
Published: March 2005

The N‐linked oligosaccharides, released by hydrazinolysis from the major 55‐kDa family, PZP3, of porcine zona pellucida glycoproteins, were separated into neutral (28%) and acidic (72%) carbohydrate chains by anion‐exchange HPLC. By competition assay, it was shown that the mixture of neutral chains has the sperm‐receptor activity, while that of the acidic chains has no activity. Their carbohydrate structures were analyzed after the reducing ends were modified with 2‐aminopyridine. The neutral chains were fractionated into several components by reverse‐phase and normal‐phase HPLC. By sequential glycosidase digestion and 500‐MHz 1H‐NMR spectroscopy, the structures of three major components were determined. The structures of some of the minor components were analyzed only by sequential glycosidase digestion. By these analyses, it was found that a diantennary complex‐type structure with a fucose residue was predominant in the neutral chains. Furthermore, the analyses of the endo‐β‐galactosidase digests of the acidic chains revealed that the partially sulfated and sialylated N‐acetyllactosamines are linked to the non‐reducing ends of diantennary, triantennary, and tetra‐antennary complex‐type neutral chains, forming heterogeneous acidic chains.
Preferential synthesis of heptaacyl lipopolysaccharide by the ssc permeability mutant of Salmonella typhimurium
Authors: HELANDER Ilkka M. , HIRVAS Laura , TUOMINEN Jari , VAARA Martti
Published: March 2005

In Salmonella typhimurium, a chromosomal gene termed ssc has been shown to cause an antibiotic‐supersensitive phenotype. We studied the effect of the ssc gene on the chemical composition of the lipopolysaccharide component, using a thermosensitive ssc1 mutant (SH7622) that grows poorly at 42°C. Analysis of the lipopolysaccharide by various techniques including fast‐atom‐bombardment mass spectrometry of lipid A, and determination of the type of linkage of fatty acids, revealed a profound temperature‐dependent effect associated with the ssc1 mutation. At the non‐permissive temperature, SH7622 contained hexadecanoic acid in the majority of lipid A molecules, resulting in the exclusive presence of heptaacyl lipopolysaccharide. This effect was largely reversed by the introduction of the cloned wild‐type ssc gene to SH7622 and much reduced by growth of SH7622 at 37°C.
Recombinant human retinoic acid receptor α
Authors: KEIDEL Siegfried , RUPP Eva , SZARDENINGS Michael
Published: March 2005

The human retinoic acid receptor α was expressed in Escherichia coli. The recombinant protein was found to be very unstable in several E. coli strains, probably due to proteolysis. Conditions were established to obtain reasonable amounts of active protein for ligand and DNA binding studies. The recombinant receptor showed the expected DNA binding activities in gel‐retardation assays. Ligand binding properties were measured in a charcoal absorption assay. The dissociation constant for highly specific bound retinoic acid was found to be 0.67 nM. The affinity of several synthetic retinoids to the recombinant protein was determined and compared to their biological activity. Some of the values presented here differ significantly from those published earlier for the receptor or its isolated hormone‐binding domain.
A comparative study of the temperature dependence of the oxygen‐binding properties of mammalian hemoglobins
Authors: COLETTA Massimo , CLEMENTI Maria Elisabetta , ASCENZI Paolo , PETRUZZELLI Raffaele , CONDÒ Saverio Giovanni , GIARDINA Bruno
Published: March 2005

The effect of temperature on the oxygen‐binding properties of hemoglobin (Hb) from ruminants, such as ox, reindeer, musk ox, mouflon and egyptian water buffalo is compared to that of human adult Hb (HbA). A striking difference emerges where in the presence of chloride ions and in the absence of 2,3‐diphosphoglycerate [Gri(2,3)P2] a strongly reduced exothermic oxygenation process is observed for all ruminant Hb investigated with respect to HbA. Next, in the presence of physiological concentrations of Gri(2,3)P2, HbA displays a less exothermic oxygenation process, with values tending toward those observed in ruminant Hb [where Gri(2,3)P2 is not a physiological effector and for which the addition of Gri(2,3)P2 has essentially no effect on the oxygenation enthalpy].

Different from HbA, the intrinsically less exothermic oxygen binding seems to be independent of the experimental conditions for ruminant Hb, underlying specific structural characteristics which might be responsible for this feature.
Pigment‐binding properties of mutant light‐harvesting chlorophyll‐a/b‐binding protein
Authors: PAULSEN Harald , HOBE Stephan
Published: March 2005

Light‐harvesting chlorophyll‐a/b‐binding protein (LHCP), overexpressed in Escherichia coli, can be reconstituted with pigments to yield complexes that are structurally very similar to light‐harvesting complex II (LHCII) isolated from thylakoids [Paulsen, H., Rümler, U. & Rüdiger, W. (1990) Planta 181, 204–211]. In order to analyze which domains of the protein are involved in pigment binding, we reconstituted deletion mutants of LHCP with pigments and characterized the resulting complexes regarding their pigment composition and spectroscopic properties. Series of progressive deletions from either end of the protein revealed that most of the N‐terminal and part of the C‐terminal hydrophilic regions of LHCP are dispensible for pigment binding. In either deletion series, the deletions that completely abolished reconstitution could be narrowed down to segments of five amino acids that do not contain histidine, asparagine, or glutamine. All mutants either formed complexes with both pigment composition and spectroscopic properties very similar to those of light‐harvesting complex II isolated from thylakoids, or they did not form any stable complexes at all. There is no indication of a segment of LHCP binding a subset of LHCII pigments. We conclude that the stabilization of LHCP‐pigment complexes is highly synergetic rather than based on individual pigment‐binding sites provided by the protein.
Kinetic modeling of Sendai virus fusion with PC‐12 cells
Authors: PEDROSO DE LIMA Maria da Conceição , RAMALHO‐SANTOS João , MARTINS Maria de Fátima , PATO DE CARVALHO Arsélio , BAIROS Vasco , NIR Shlomo
Published: March 2005

We have studied the fusion activity of Sendai virus, a lipid‐enveloped paramyxovirus, towards a line of adherent cells designated PC‐12. Fusion was monitored by the dequenching of octadecylrhodamine, a fluorescent non‐exchangeable probe. The results were analysed with a mass action kinetic model which could explain and predict the kinetics of virus–cell fusion. When the temperature was lowered from 37°C to 25°C, a sharp inhibition of the fusion process was observed, probably reflecting a constraint in the movement of viral glycoproteins at low temperatures. The rate constants of adhesion and fusion were reduced 3.5‐fold and 7‐fold, respectively, as the temperature was lowered from 37°C to 25°C. The fusion process seemed essentially pH‐independent, unlike the case of liposomes and erythrocyte ghosts. Preincubation of the virus in the absence of target cell membranes at neutral and alkaline pH (37°C, 30 min) did not affect the fusion process. However, a similar preincubation of the virus at pH = 5.0 resulted in marked, though slow, inhibition in fusion with the fusion rate constant being reduced 8‐fold. Viral preincubation for 5 min in the same acidic conditions yielded a mild inhibition of fusogenic activity, while preincubation in the cold (4°C, 30 min) did not alter viral fusion activity. These acid‐induced inhibitory effects could not be fully reversed by further viral preincubation at pH = 7.4 (37°C, 30 min). Changes in internal pH as well as endocytic activity of PC‐12 cells had small effect on the fusion process, thus indicating that Sendai virus fuses primarily with the plasma membranes.
Control of plasminogen‐activator inhibitor type 2 gene expression in the differentiation of monocytic cells
Authors: ANTALIS T. M. , DICKINSON J. L.
Published: March 2005

Plasminogen‐activator inhibitor type 2 (PAI‐2) is a potent and primary inhibitor of urokinase‐type plasminogen activator. Its production in monocytic cells is thought to play an important role in the control of localized proteolysis at sites of invasion as occurs in the control of inflammatory processes, tumor invasion and cellular differentiation. Therefore, we have investigated the mechanisms responsible for the regulation of PAI‐2 gene expression in differentiating monocytic cells using the human promyelocytic cell line, HL‐60, as a model. These cells are induced to differentiate to a macrophage‐like phenotype in response to phorbol ester [4‐phorbol‐12‐myristate 13‐acetate (PMA)]. The levels of PAI‐2 mRNA are barely detectable in undifferentiated cells, however, activation with PMA is associated with a rapid induction of PAI‐2 transcripts, reaching a maximum of 25‐fold in 4 h. Nuclear run on assays demonstrate that this induction is related primarily to an enhanced rate of gene transcription. Inhibition of de novo protein synthesis by cycloheximide increases PAI‐2 mRNA levels in both resting (sevenfold) and PMA‐treated cells (fivefold) after 4 h, but has no detectable effect on the rate of PAI‐2 gene transcription. The initial apparent half‐life of the induced PAI‐2 mRNA, determined by actinomycin‐d‐decay experiments, is very short, 32 min, suggesting rapid turnover. Furthermore, the PAI‐2 mRNA transcript is stabilized in the presence of cycloheximide, with a fourfold increase in the observed half‐life.

The results demonstrate that PAI‐2 gene expression is regulated through post‐transcriptional mechanisms in undifferentiated cells, while both transcriptional and post‐transcriptional events govern the level of PAI‐2 transcripts in cells differentiated along the monocytic pathway. Destabilization of the PAI‐2 transcript may be associated with (A + U)‐rich sequences found in the 3′‐untranslated region of PAI‐2 mRNA. The short half life and rapid, strong induction of PAI‐2 point to an important, perhaps crucial, role in the differentiation of monocyte cells.
Isolation and characterization of two biologically active O‐glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae
Authors: OLSTAD Ole Kristoffer , REPPE Sjur , GABRIELSEN Odd S. , HARTMANIS Maris , BLINGSMO Ola R. , GAUTVIK Vigdis T. , HAFLAN Anne Karine , CHRISTENSEN Terje B. , ØYEN Tordis B. , GAUTVIK Kaare M.
Published: March 2005

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast‐culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the O‐glycosylated forms that were separated by isocratic reverse‐phase HPLC, two mannose‐linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N‐terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O‐glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone‐sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C‐terminal O‐glycosylation of hPTH with upto seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.
Inclusion bodies of the thermophilic endoglucanase D from Clostridium thermocellum are made of native enzyme that resists 8 M urea
Authors: CHAFFOTTE Alain F. , GUILLOU Yvonne , GOLDBERG Michel E.
Published: March 2005

Endoglucanase D from Clostridium thermocellum was purified from inclusion bodies formed upon its overproduction in Escherichia coli, using 5 M urea as a solubilizing solution. We examined the effects of denaturing agents upon the stability of the pure soluble enzyme as a function of the temperature. At room temperature, guanidinium chloride induces an irreversible denaturation. By comparison, we observed no structural or functional effects at room temperature using high concentrations of urea as denaturing agent. The irreversible denaturation process observed with guanidinium chloride also occurs with urea but only at elevated temperature (≤60°C); in 6 M urea, the activation energy of the denaturation reaction is decreased by a factor of only 1.8. We interpret the high resistance of this protein to urea as reflecting a reduced flexibility of its structure at normal temperatures which should be correlated to the thermophilic origin of this protein.
Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium
Authors: PLAGA Wulf , LOTTSPEICH Friedrich , OESTERHELT Dieter
Published: March 2005

An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant.

The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin‐diphosphate‐binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.
Identification and characterization of the murine cell surface receptor for the urokinase‐type plasminogen activator
Authors: SOLBERG Helene , LØBER Dorte , ERIKSEN Jens , PLOUG Michael , RØNNE Ebbe , BEHRENDT Niels , DANØ Keld , HØYER‐HANSEN Gunilla
Published: March 2005

Cell‐binding experiments have indicated that murine cells on their surface have specific binding sites for mouse urokinase‐type plasminogen activator (u‐PA). In contrast to the human system, chemical cross‐linking studies with an iodinated ligand did not yield any covalent adducts in the murine system, but in ligand‐blotting analysis, two mouse u‐PA‐binding proteins could be visualized. To confirm that these proteins are the murine counterpart of the human u‐PA receptor (u‐PAR), a peptide was derived from the murine cDNA clone assigned to represent the murine u‐PAR due to cross‐hybridization and pronounced sequence similarity with human u‐PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Danø, K. (1991) J. Cell Biol. 115, 1763–1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand‐blotting analysis. Binding of mouse u‐PA to its receptor showed species specificity in ligand‐blotting analysis, since mouse u‐PA did not bind to human u‐PAR and human u‐PA did not bind to mouse u‐PAR. The apparent Mr of mouse u‐PAR varied between different mouse cell lines and ranged over Mr 45000–60000. In four of the cell lines, mouse u‐PA bound to two mouse u‐PAR variant proteins, whereas in the other two cell lines studied, there was only one mouse u‐PA‐binding protein. In the monocyte macrophage cell line P388D.1, trypsin‐treatment of intact cells could remove only the large mouse u‐PAR variant (Mr 60000) indicating that only this type was a cell‐surface‐exposed molecule. The smaller mouse u‐PAR variant (Mr 45000), was deglycosylated by the enzyme endo‐β‐N‐acetylglucosaminidase H and is probably an intracellular precursor form carrying only high‐mannose carbohydrate. Deglycosylation of this variant yielded a polypeptide with an apparent Mr of about 30000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u‐PAR. Receptor‐bound mouse u‐PA could be released by phosphatidylinositol‐specific phospholipase C treatment, indicating that mouse u‐PAR is attached to the cell surface by glycosylphosphatidylinositol. Purification of the two mouse u‐PAR variant proteins by diisopropylfluorophosphate‐inactivated mouse u‐PA ‐ Sepharose affinity chromatography yielded two silver‐stained bands when analysed by SDS/PAGE, corresponding in electrophoretic mobility to those seen by ligand‐blotting analysis.

The present identification and characterization of mouse u‐PAR will facilitate experimental studies in vivo on the role of u‐PAR and u‐PA/u‐PAR interaction in normal and pathological conditions, including cancer invasion.
Characterization of 4‐hydroxyphenylpyruvate dioxygenase
Authors: RÜETSCHI Ulla , ODELHÖG Birgit , LINDSTEDT Sven , BARROS‐SÖDERLING Jane , PERSSON Bengt , JÖRNVALL Hans
Published: March 2005

The primary structure of Pseudomonas 4‐hydroxyphenylpyruvate dioxygenase was determined. Sequence degradation of the intact protein and of peptides from three different digests of the carboxymethylated protein established a 357‐residue polypeptide chain with a free α‐amino group. Hydroxylamine cleavage at a single Asn‐Gly sequence was useful. Comparisons with known structures in data banks revealed no close relationship with other characterized proteins. The human enzyme has a related composition, suggesting that also the eukaryotic form belongs to this protein type, but with a blocked N‐terminus like in many other eukaryotic intracellular proteins. Secondary structure predictions suggest an α/β mixed structure, fairly typical of globular proteins, without long segments of hydrophobicity or charge, although a region in the middle of the C‐terminal third of the subunit appears to have the most extreme properties. A ferric centre, correlating with enzyme activity and absorbance at 595 nm, has previously been assigned to tyrosinate coordination. The Tyr and His distributions, and the position of a single Cys residue, all suggest a few likely sites, outside the C‐terminal segment, for this centre.
Concentration and regulation of cyclic nucleotides, cyclic‐nucleotide‐dependent protein kinases and one of their major substrates in human platelets
Authors: EIGENTHALER Martin , NOLTE Christine , HALBRÜGGE Maria , WALTER Ulrich
Published: March 2005

Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46‐kDa protein (vasodilator‐stimulated phosphoprotein, VASP) mediated by cAMP‐dependent protein kinase (PKA) and cGMP‐dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA‐ and PKG‐mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4 μM cAMP and 3.1 μM catalytic subunit of PKA, which is equivalent to 6.2 μM cAMP‐binding sites due to PKA. Unstimulated washed human platelets also contain 0.4 μM cGMP and 7.3 μM PKG monomer, equivalent to 14.6 μM cGMP‐binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25 μM.

Treatment of washed human platelets with 10 μM (or 10 nM) prostaglandin E1 (PGE1) elevated the intracellular cAMP concentration to 27 μM (10 μM with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46‐kDa protein) to the phosphorylated form (50‐kDa protein).

Treatment of washed human platelets with 100 μM (or 1 μM) sodium nitroprusside elevated the platelet cGMP level to 4 μM (0.9 μM with 1 μM sodium nitroprusside) within 2 min, accompanied by a less‐rapid VASP phosphorylation of 45% (27% with 1 μM sodium nitroprusside). PGE1 and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increases in cAMP levels are required for activation of most of PKA, whereas even several‐fold increases in platelet cGMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA‐ and cGMP/PKG‐regulated VASP phosphorylation are components of an efficient and sensitive signal‐transduction pathway, most likely involved in the inhibition of platelet activation. In particular, the high concentration of the converter enzymes PKA and PKG suggest that rapidity in reaching steady‐state levels of phosphorylation in response to cAMP‐ or cGMP‐elevating agents is of considerable importance for the regulation of human platelets.
A single‐residue exchange gives human recombinant ββ alcohol dehydrogenase γγ isozyme properties
Authors: HÖÖG Jan‐Olov , EKLUND Hans , JÖRNVALL Hans
Published: March 2005

Amino acid residue 48 in human alcohol dehydrogenase constitutes one of 21 residue differences between the common, adult‐type isozyme subunits β and γ. It is at the inner part of the substrate pocket and has been ascribed a role in hydrogen‐bond formation with both the substrate and coenzyme. In order to allow direct evaluation of its importance, Thr48 of a recombinant non‐acetylated β subunit was mutated to Ser (as in the γ subunit) or Ala (as in no native form, and not allowing side‐chain hydrogen bonds), and the proteins were expressed in Escherichia coli. The two non‐acetylated recombinant proteins, the β48T form and the mutant β48S, gave enzymatically active enzymes with indistinguishable specific activities towards ethanol, whereas the mutant β48A showed no enzymatic activity.

The most striking differences between dimers with the β subunit and the β48S subunit (both non‐acetylated) were observed with cyclohexanol, hydroxysteroids, methanol and ethanol. With cyclohexanol, the Km was lowered from 11 mM to 280 μM, and the kcat/Km ratio, although still less than that for the γγ isozyme, was increased 80‐fold. Similarly, β48S could use 3β‐hydroxy‐5β‐androstan‐17‐one as substrate, like γγ, although again with a catalytic efficiency much less than that for the γγ isozyme. Furthermore, testosterone inhibited β48S to 50% at a concentration of 100 μM, whereas the ββ form was not inhibited. All these results show that residue 48 is responsible for a large part of the differences between the two isozymes ββ and γγ of human class‐I alcohol dehydrogenase. The form with the inactive β48A subunit was possible to purify by AMP‐Sepharose chromatography, suggesting the presence of a functional NAD‐binding site. The enzymatic measurements, demonstrating a transition from one isozyme activity to that characteristic of another, confirmed that a side‐chain hydroxyl in residue 48 is required for activity, and interpretation by computer modelling showed marked differences at the active site.
A simple procedure for tenascin purification
Authors: SAGINATI Marco , SIRI Annalisa , BALZA Enrica , PONASSI Marco , ZARDI Luciano
Published: March 2005

Here we describe a two‐step procedure for purification of human tenascin from conditioned medium of the SK‐MEL‐28 human melanoma cell line.

The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin–Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5–300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin‐sulfate‐containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3–4 mg tenascin which corresponds to a yield of about 40–50%.

This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.
Accelerated secretion of human lysozyme with a disulfide bond mutation
Authors: OMURA Fumihiko , OTSU Mieko , KIKUCHI Masakazu
Published: March 2005

The mutant human lysozyme, [Ala77, Ala95]lysozyme, in which the disulfide bond Cys77 – Cys95 is eliminated, is known to exhibit increased secretion in yeast, compared to wild‐type human lysozyme [Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M. & Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962–967]. To investigate this phenomenon, mammalian cells were used to analyze the secretion kinetics of [Ala77, Ala95]lysozyme and wild‐type human lysozyme. The secretion rate of [Ala77, Ala95]lysozyme during the 150‐min chase period was significantly accelerated [half‐life (t1/2) = 29 min] compared to that of wild‐type human lysozyme (t1/2= 83 min), when expressed at the same levels within the cells. In contrast, after the 150‐min chase, the rates of disappearance of both wild‐type and mutant human lysozymes within the cells were similar, and considerably slower (t1/2= 220 min), respectively. The remaining intracellular wild‐type human lysozyme was localized mainly in the endoplasmic reticulum, whereas accelerated transport of the [Ala77, Ala95]lysozyme mutant protein from the endoplasmic reticulum to the Golgi apparatus was observed. Also in yeast cells, similar secretion kinetics and the differences in t1/2 for wild‐type and mutant human lysozymes during the early chase period were observed. The two‐phase kinetics of disappearance of intracellular human lysozymes suggest that only a proportion of the proteins becomes secretion competent soon after synthesis and is completely secreted during the early chase period, whereas others enter the distinct, slow pathways of intracellular transport and/or degradation. Increased secretion of [Ala77, Ala95]lysozyme is possibly due to enhanced competence for secretion acquired in the endoplasmic reticulum at the early stage of transport events, which is closely connected with the removal of a disulfide bond.
Collapsed intermediates in the reconstitution of dimeric aspartate aminotransferase from Escherichia coli
Authors: LEISTLER Bernd , HEROLD Marzell , KIRSCHNER Kasper
Published: March 2005

Aspartate aminotransferase from Escherichia coli, which had been denatured by guanidinium chloride, refolded and reassembled to active dimers in two distinct phases. The unfolded monomer U collapsed within 20 s to an intermediate I* that was inactive, fluoresced more strongly than, but had the same peptide CD signal as the native dimer. The formation of crosslinkable dimers, as well as the recovery of enzyme activity, occurred with a biphasic progress curve which was independent of protein concentration. The half‐lives of the two phases were 100 s and 2000 s. The data are consistent with a three‐step mechanism, in which the overall rate of reassembly is determined by an isomerization of I* to the assembly‐competent monomer M. The latter does not accumulate because it dimerizes rapidly to the active enzyme (D). Reassembly of the enzyme from the compact intermediate M*, which is stable at 1.0 M guanidinium chloride, also proceeded in a rapid and a slow phase. Moreover, the formation of M* from the unfolded state was rapid, whereas its refolding to the native dimer was slow. Both the transient intermediate I* and the equilibrium intermediate M* qualify as ‘collapsed intermediate’ or ‘molten globule’ states.
Studies on protein‐DNA interactions using the resonant recognition model
Authors: COSIC Irena , HEARN Milton T. W.
Published: March 2005

The structural features of protein‐DNA interactions have been evaluated using a new information theory algorithm for the analysis of protein structure/function dependence: the so‐called resonant recognition model. The physicochemical basis of this analysis was firstly validated with the trp‐repressor – operator interaction as a well‐defined example. The amino acid and structural features predicted by these procedures to be crucial for repressor‐operator interaction were found to be clustered around the known three‐dimensional structure of the active site of the trp repressor. Similar methods of analysis have been extended to the less‐well‐defined example of the Ha‐ras p21 protein family. The results of this analysis have indicated two distinct interactive regions in p21, one associated with the guanine‐nucleotide‐binding site, whilst the second is proposed to be associated with a binding site for an activator protein. These studies indicate that the p21 protein, besides the ability to function as a plasma‐membrane‐associated guanine‐nucleotide‐binding regulatory protein and bind free guanine nucleotides in the cytoplasm, has the structural ability to bind guanine incorporated in DNA. Thus, p21‐related proteins may have the potential to function as an DNA‐binding and regulating protein with the mode of upstream DNA binding closely related to their oncogenic function.
Inhomogeneous broadening and kinetic carbon monoxide isotope effects in low‐temperature carbon monoxide recombination with myoglobin and hemoglobin
Authors: LEGARTH Jens Brøbech , ULSTRUP Jens , ZAKARAYA Merab G.
Published: March 2005

We have analyzed the non‐exponential kinetics, the temperature variation, and the CO isotope effects of the CO recombination reactions with myoglobin and single‐chain hemoglobin. The analysis rests on multiphonon quantum‐mechanical chemical‐rate theory combined with static inhomogeneous broadening of either the reorganization free energy or the reaction Gibbs free energy. The simplest specific model which can account for all the data contains an inhomogeneous distribution function of width 0.2 – 0.3 eV, independent of temperature down to the tunnel transition at about 20 K, two discrete nuclear coordinates of low vibrational frequency (60 – 150 cm−1) representing iron‐heme and CO bending motion, the CO stretching motion of frequency about 2000 cm−1, and additional inhomogeneous broadening of the protein and CO bending configuration below the tunnel transition temperature. The model appears somewhat involved but in return provides corresponding insight in the dynamics of this important class of processes.
The carboxy‐terminal lysine of αB‐crystallin is an amine‐donor substrate for tissue transglutaminase
Authors: GROENEN Patricia J. T. A. , BLOEMENDAL Hans , de JONG Wilfried W.
Published: March 2005

A hexapeptide, corresponding to the sequence around the glutamine in βA3‐crystallin that functions as amine‐acceptor for transglutaminase, was synthesized. This peptide was biotinylated and used as a probe to identify amine‐donor substrates for transglutaminase among lens proteins. It was found that Ca2+‐activated transglutaminase linked this peptide not only to several β‐crystallins but, unexpectedly, also to αB‐crystallin. The C‐terminal lysine residue of αB‐crystalline could be identified as the site of linkage. This strengthens the notion that, at least in crystallins, all transglutaminase substrate residues are located in terminal extensions of the polypeptides. It was shown that in lens homogenate, αB‐crystallin can be covalently crosslinked to β‐crystallins by transglutaminase. The transglutaminase‐mediated crosslinking of αB‐crystallin may have implications for its involvement in normal and pathological processes in lens and other tissues.
Isolation and chemical characterization of a novel insulin‐related neuropeptide from the freshwater snail, Lymnaea stagnalis
Authors: Wan LI Ka , GERAERTS Wijnand P. M.
Published: March 2005

A novel molluscan insulin‐related peptide (MIP) III, has been isolated from alcohol extracts of the neurohaemal area of the cerebral neuroendocrine light‐green neurones of Lymnaea stagnalis. MIP III was purified by sequential high‐performance gel‐permeation chromatography followed by reverse‐phase HPLC. MIP III is a heterodimer connected by disulphide bonds. Edman degradation analysis and subsequent alignment with the A and B chains of the previously identified MIP I and II showed that the 24‐amino‐acid peptide with the sequence pQSRPSIVC(E)CCFNQCTVQ(E)LLAYC represents the MIP III A chain, and the 37‐amino‐acid peptide sequence TTQHTCSILSRPHPRGLCGSTLANMVQWLCSTYTTSS the B chain. The overall amino acid sequence of MIP III shows about 50% similarity with those of MIP I and II, and only 20–40% similarity with other peptides of the insulin superfamily. Important structural features, e.g. disulphide bridges and the hydrophobic core, are conserved in MIP III.
Regulation of plant cell‐wall pectin methyl esterase by polyamines — Interactions with the effects of metal ions
Authors: CHARNAY Dominique , NARI Joannès , NOAT Georges
Published: March 2005

The kinetic study of the de‐esterification of natural pectin by soya bean or orange pectin methyl esterase shows that the rate of the reaction is highly controlled by the presence of polyamines. The reaction rate versus the polyamine concentration is a bell‐shaped curve similar to that which is obtained when the concentration of salts is varied in the reaction mixture. However polyamines, in particular the largest ones, are more efficient than salts. The results may be interpreted by assuming that polyamines mainly interact with the negative charges of the pectic substrate which condition the binding of the pectin methyl esterase. Activating effects were observed at polyamine concentrations that have been shown to exist in the plant cell wall in vivo. Thus, polyamines may act as efficient regulators of the cell‐wall pH via the control of the electrostatic cell‐wall potential. If such is the case, they might have a role in all regulatory mechanisms in which cell‐wall enzymes are involved.
Apparent lack of N‐glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum
Authors: DIECKMANN‐SCHUPPERT Angela , BENDER Stephanie , ODENTHAL‐SCHNITTLER Maria , BAUSE Ernst , SCHWARZ Ralph T.
Published: March 2005

This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum, and the presence in the infected erythrocyte of the respective precursors.

In in vitro cultures, P. falciparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein‐glycosylation reactions. Gel‐filtration analysis of sugar‐labeled malarial proteins before and after specific cleavage of N‐glycans or O‐glycans, respectively, revealed the majority of the protein‐bound sugar label to be incorporated into O‐glycans, but only little (7–12% of the glucosamine label) or no N‐glycans were found. Analysis of the nucleotide sugar and sugar‐phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP‐mannose was below the detection limit. The analysis of organic‐solvent extracts of sugar‐labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N‐glycosylation. Consistently, the amount of UDP‐N‐acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl‐transferase activity was not detectable in a lysate of P. falciparum, using exogenous glycosyl donors and acceptors.

Our studies show that O‐glycosylation is the major form of protein glycosylation in intraerythrocytic P. falciparum, whereas there is little or no protein N‐glycosylation. A part of these studies has been published in abstract form [Dieckmann‐Schuppert, A., Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe‐Seyler 372, 645].