bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissection of laminin by cathepsin G into its long‐arm and short‐arm structures and localization of regions involved in calcium dependent stabilization and self‐association
Authors: BRUCH Marcel , LANDWEHR Ruth , ENGEL Jürgen
Published: March 2005

Native laminin–nidogen complex isolated from mouse Engelbreth‐Holm‐Swarm tumor was treated with purified cathepsin G or leucocyte elastase, two neutral serine proteases which play a role in inflammatory processes accompanied by degradation of basement membranes. Both enzymes were found to be more active than porcine pancreatic elastase. In the absence of Ca2+, laminin fragments produced by leucocyte elastase resembled those formed by the pancreatic enzyme but at physiological concentrations of Ca2+ cleavage by cathepsin G was much more selective. Initially laminin (900 kDa) was cleaved at two major sites only with similar rates leading to three fragments. Fragment Cl–4 (about 550 kDa) comprises the intact three short arms of the molecule and fragment C8–9 (about 350 kDa) contains the entire triple‐coiled region by which its three chains are assembled and the major part of the terminal globular domain of the long arm. The remaining C‐terminal region of this domain was recovered as fragment C3 of about 50 kDa. Stabilization against proteolytic attack was restricted to the region of fragment Cl–4 and only this fragment exhibited strong Ca2+ dependent self‐association similar to that of intact laminin or of its complex with nidogen. The associative properties of fragment Cl–4 were dramatically diminished upon removal of the tip of one of the short arms comprising fragment 4. In addition, this provides a clear assignment of the important laminin function to a distinct domain in one of its short arms. The new fragment C8–9 may be employed for exploring the properties and possible functions of the upper long‐arm region which so far has not been available as a fragment.
Studies on the domain structure of the Salmonella/typhimurium AraC protein
Authors: LAUBLE Hanspeter , GEORGALIS Yannis , HEINEMANN Udo
Published: March 2005

The Salmonella typhimurium araC gene product is known to be susceptible to proteolytic degradation. Limited cleavage by trypsin, kallikrein, elastase and pronase E yields stable fragments comprising approximately the N‐terminal two thirds of the AraC protein. These fragments have in common the ability to dimerize in solution and to bind L‐arabinose and D‐fucose. Under appropriate conditions, hydrolysis of the AraC protein with Staphylococcus aureus V8 protease leads to a small C‐terminal fragment which is able to bind specifically to a synthetic ara consensus sequence. These results indicate that, as with several other prokaryotic gene regulatory proteins, the basic functions of effector binding, subunit interaction and specific DNA binding are segregated into distinct domains of the AraC protein.
The activity of GD3 synthase modulates the ganglioside pattern in rat liver
Authors: SCHÜZ‐HENNINGER Renate , ULLMER Elke , PRINZ Claudia , DECKER Karl
Published: March 2005

Variations of the ganglioside composition in the livers of Wistar rats correlated with the activity of GD3 synthase in the corresponding liver homogenates. With increasing enzyme activity, higher proportions of b‐series gangliosides (GD3, GD1b, GT1b) were detected. No significant changes in the activity of GM2 synthase or GM1 synthase were observed, indicating a regulatory function for GD3 synthase in this tissue. Young animals showed an average GD3 synthase activity of 0.5–0.6 nmol · h−1· mg protein−1 without sex‐dependent variations. Among the older animals, however, males expressed an activity five‐fold higher than females, suggesting that this enzyme might be affected by hormones.
Structural analogies between the 3′ tRNA‐like structure of brome mosaic virus RNA and yeast tRNATyr revealed by protection studies with yeast tyrosyl‐tRNA synthetase
Authors: PERRET Véronique , FLORENTZ Catherine , DREHER Theo , GIEGE Richard
Published: March 2005

Contacts between the tRNA‐like domain in brome mosaic virus RNA and yeast tyrosyl‐tRNA synthetase have been determined by footprinting with enzymatic probes. Regions in which the synthetase caused protections indicative of direct interaction coincide with loci identified by mutational studies as being important for efficient tyrosylation [Dreher, T. W. & Hall, T. C. (1988) J. Mol. Biol. 201, 41–55]. Additional extensive contacts were found upstream of the core of the tRNA‐like structure. In parallel, the contacts of yeast tRNATyr with its cognate synthetase were determined by the same methodology and comparison of protected nucleotides in the two RNAs has permitted the assignment of structural analogies between domains in the viral tRNA‐like structure and tRNATyr. Amino acid acceptor stems are similarly recognized by yeast tyrosyl‐tRNA synthetase in the two RNAs, indicating that the pseudoknotted fold in the viral RNA does not perturb the interaction with the synthetase. A further important analogy appears between the anticodon/D arm of the L‐conformation of tRNAs and a complex branched arm of the viral tRNA‐like structure. However, no apparent anticodon triplet exists in the viral RNA. These results suggest that the major determinants for tyrosylation of yeast tRNATyr lie outside the anticodon stem and loop, possibly in the amino acid acceptor stem.
Substitution of Val20 by Gly in elongation factor Tu
Authors: JACQUET Eric , PARMEGGIANI Andrea
Published: March 2005

Substitution of V20 by G in the consensus element G18HVDHGK24 of EF‐Tu (referred to as EF‐TuG20) strongly influences the interaction with GDP as well as the GTPase activity [Jacquet, E. & Parmeggiani, A. (1988) EMBO J. 7, 2861–2867]. In an extension of this work we describe additional properties of the mutated factor, paying particular attention to the interaction with the macromolecular ligands. Our results show that the conformational transitions induced by the mutation strongly favor the regeneration of the active complex EF‐TuG20 · GTP, almost as effectively as with wild‐type EF‐Tu in the presence of elongation factor Ts. Addition of elongation factor Ts further enhances the rate of the GDP to GTP exchange of the mutated factor. Remarkably, EF‐TuG20 · GDP can support the enzymatic binding of aminoacyl‐tRNA to ribosome · mRNA at low MgCl2 concentration, an effect that with wild‐type EF‐Tu can only occur in the presence of kirromycin. Our results show that EF‐TuG20 · GDP shares common features with the GTP‐like conformation induced by kirromycin on wild‐type EF‐Tu. The ability of the ribosome to activate the EF‐TuG20 center for GTP hydrolysis is strongly decreased, while the stimulation by aminoacyl‐tRNA is conserved. The ribosomal activity is partially restored by addition of aminoacyl‐tRNA plus poly(U), showing that codon/anticodon interaction contribute to correct the anomalous interaction between ternary complex and ribosomes. The impaired activity of EF‐TuG20 in poly(Phe) synthesis is related to the degree of defective GTP hydrolysis and, most interestingly, it is characterized by a striking increase of the fidelity of translation at high MgCl2 concentration. This effect probably depends on a more selective recognition of the ternary complex by ribosome · mRNA, as a consequence of a longer pausing of EF‐TuG20 on the ribosome. In conclusion, position 20 in EF‐Tu is important for coordinating the allosteric mechanisms controlling the action of EF‐Tu and its ligands.
The 23S ribosomal RNA higher‐order structure of Pseudomonas cepacia and other prokaryotes
Authors: HÖPFL Peter , LUDWIG Wolfgang , SCHLEIFER Karl Heinz , LARSEN Niels
Published: March 2005

A 23S ribosomal RNA gene of Pseudomonas cepacia has been cloned and sequenced. A general higher‐order structure model based on earlier published models has been derived from comparative analysis of 23S‐like rRNAs of eubacteria, archaebacteria, organelles and eukaryotes. Differences between the previous models were carefully analyzed and controversial regions evaluated. Moderately large insertions and deletions have been found at new points in the secondary structure. The analysis of 50 published as well as unpublished 23S rRNA sequences provide additional proof for six of the seven previously suggested tertiary interactions within the 23S rRNA. P. cepacia is the first representative of the β subgroup of the Proteobacteria phylum whose 23S rRNA has been sequenced. A tree reflecting evolutionary relationships of prokaryotes was constructed. The topology of this tree is in good agreement with the 16S rRNA tree.
Role of the cell surface‐exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein
Authors: AGTERBERG Marja , ADRIAANSE Henriëtte , TIJHAAR Edwin , RESINK Annelies , TOMMASSEN Jan
Published: March 2005

To investigate the role of the cell surface‐exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface‐exposed regions of the protein. Intact cells expressing these mutant proteins were recognized by PhoE‐specific monoclonal antibodies, which recognize conformational epitopes on the cell surface‐exposed parts of the protein and/or were sensitive to a PhoE‐specific phage. This shows that the polypeptides were normally incorporated into the outer membrane. When the deletions extended four amino acid residues into the seventh presumed membrane‐spanning segment, the polypeptides accumulated in the periplasm. In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC. In contrast, the membrane‐spanning segments are essential for the assembly process.
Sulfation of two tyrosine‐residues in human complement S‐protein (vitronectin)
Authors: JENNE Dieter , HILLE Annette , STANLEY Keith K. , HUTTNER Wieland B.
Published: March 2005

Human S‐protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human heptoma‐derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine‐labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S‐protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S‐protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S‐protein. Examination of the S‐protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S‐protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S‐protein may stabilize the conformation of S‐protein in the absence of thrombin‐antithrombin III complexes and may play a role in its binding to thrombin‐antithrombin III complexes during coagulation.
Ligand‐induced conformational changes of citrate synthase studied with the fluorescent probe 8‐anilinonaphthalene 1‐sulfonate
Authors: KOLLMANN‐KOCH Angelika , EGGERER Hermann
Published: March 2005

Experiments are presented which show that oxaloacetate and analogs thereof with (R)‐malate substructure, on interaction with citrate synthase linked to synthase 8‐anilinonaphthalene 1‐sulfonate (ANS), induce identical conformational changes of a characteristic magnitude. A conformational change of lower magnitude is also produced on binding of CoASH or ATP to citrate synthase · ANS and is completed on addition of oxaloacetate. The significance of these ligand‐dependent conformational changes is discussed.
Inhibition of Desulfovibrio gigas hydrogenase with copper salts and other metal ions
Authors: FERNANDEZ Victor M. , RUA Maria L. , REYES Presentación , CAMMACK Richard , HATCHIKIAN E. Claude
Published: March 2005

The effect of several transition metals on the activity of Desulfovibrio gigas hydrogenase has been studied. Co(II) and Ni(II) at a concentration of 1 mM did not modify the activity of the enzyme; nor did they affect the pattern of activation/deactivation. Cu(II) inhibited the active hydrogenase, prepared by treatment with hydrogen, but had little effect on the ‘unready’ enzyme unless a reductant such as ascorbate was present, in which case inactivation took place either in air or under argon. Hg(II) also inactivated the enzyme irreversible in the ‘unready’ state without the requirement for reductants. The reaction of H2 uptake with methyl viologen was much more sensitive to inhibition than the H2/tritium exchange activity. EPR spectra of this preparation showed that the rates of decline were [3Fe‐4S] signal > H2‐uptake activity > Ni‐A signal. Similar results were obtained when the protein was treated with Hg(II). The results demonstrate that the [3Fe‐4S] cluster is not essential for H2‐uptake activity with methyl viologen, but the integrity of [4Fe‐4S] clusters is probably necessary to catalyze the reduction of methyl viologen with hydrogen. D. gigas hydrogenase was found to be highly resistant to digestion by proteases.
Phosphorylation of low molecular mass cytosolic proteins by protein kinase C and protein kinase A in the rabbit exocrine pancreas
Authors: EDERVEEN Antoine G. H. , VAN DER LEEST Jos V. M. , VAN EMST‐DE VRIES Sjenet E. , DE PONT Jan Joep H. H. M.
Published: March 2005

Subcellular fractionation of rabbit pancreatic acini was performed to study the distribution of endogenous substrates for protein kinase C. Substrates for protein kinase C were found to be predominantly low molecular mass proteins of cytosolic origin. At least three of these soluble substrates, with molecular masses of 17‐19 kDa, were relatively heavily phosphorylated by endogenous as well as purified pancreatic protein kinase C. In the same molecular mass range, 16‐18 kDa, soluble proteins were also phosphorylated by protein kinase A. Moreover, addition of cyclic AMP under conditions that activated protein kinase C gave a more than additive labelling of these low molecular mass proteins. The latter observation may be of interest in view of the potentiating effect cyclic‐AMP‐activated protein kinase A has on amylase secretion stimulated by secretagogues which increase free cytosolic Ca2+ and activate protein kinase C.
Degradation of ornithine decarboxylase in mammalian cells is ATP dependent but ubiquitin independent
Authors: ROSENBERG‐HASSON Yael , BERCOVICH Zippi , CIECHANOVER Aaron , KAHANA Chaim
Published: March 2005

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines in mammalian cells is characterized by an extremely short half‐life. In the present study, ODC degradation was investigated in 653‐1 mouse myeloma cells that overproduce ODC and in ts85 cells that are thermosensitive for conjugation of ubiquitin to target proteins. Addition of 2‐deoxyglucose and dinitrophenol (agents that efficiently deplete cellular ATP) to the growth medium of these cells inhibited ODC degradation. In contrast, chloroquine and leupeptin, inhibitors of intralysosomal proteolysis, did not affect ODC degradation. Shifting ts85 cells to 42°C (a non‐permissive temperature that inhibited conjugation of ubiquitin to target proteins) did not prevent ODC degradation. The ATP‐dependent degradation of ODC in 653‐1 cells was inhibited substantially by Nα‐tosyl‐L‐lysine chloromethane (TosPheMeCl), iodoacetamide and o‐phenanthroline. These results suggest that ODC degradation occurs via a non‐lysosomal, ATP‐requiring and ubiquitin‐independent cellular proteolytic mechanism, and that serine proteases and enzymes containing sulphydryl groups and metalloenzyme(s) may be involved in this process.
The reaction of hemin with H2O2
Authors: KREMER Mordechai L.
Published: March 2005

The kinetics of formation of the dominant intermediate (CII) formed between hemin and H2O2 has been studied by the stopped‐flow method. CII is preceded by a precursor (CI) for which a steady state is established at an early stage of the reaction. The formation of CI from hemin and H2O2 causes only a marginal change in the optical absorbance (A). The transition CI°CII is accompanied by a substantial decrease of A in the Soret region. Relevant rate constants (or combinations of them) and the molar absorption coefficients of the intermediates at 400 nm have been determined. The absorption spectrum of CII in the Soret region has been evaluated. Aspects of the catalysis of decomposition of H2O2 by hemin in relation to the Fe3+ ion and catalase are discussed.
Analysis of magnesium, europium and lead binding sites in methionine initiator and elongator tRNAs by specific metal‐ion‐induced cleavages
Authors: CIESIOŁKA Jerzy , WRZESINSKI Jan , GÓRNICKI Piotr , PODKOWIŃSKI Jan , KRZYŻOSIAK Włodzimierz J.
Published: March 2005

The specificity of cleavages in yeast and lupin initiator and elongator methionine tRNAs induced by magnesium, europium and lead has been analysed and compared with known patterns of yeast tRNAPhe hydrolysis. The strong D‐loop cleavages occur in methionine elongator tRNAs at similar positions and with comparable efficiency to those found in tRNAPhe, while the sites of weak anticodon loop cuts, identical in methionine elongator tRNAs, differ from those found in tRNAPhe. Methionine initiator tRNAs differ from their elongator counterparts: (a) they are cleaved in the D‐loop with much lower efficiency; (b) they are cleaved in the variable loop which is completely resistant to hydrolysis in elongator tRNAs; (c) cleavages in the anticodon loop are stronger in initiator tRNAs and they are located mostly at the 5′ side of the loop whereas in elongator tRNAs they occur mostly at the opposite, 3′ side of the loop. The distinct pattern of the anticodon loop cleavages is considered to be related to different conformations of the anticodon loop in the two types of methionine tRNAs.
Type I procollagen C‐proteinase from mouse fibroblasts
Authors: KESSLER Efrat , ADAR Rivka
Published: March 2005

The enzyme procollagen C‐proteinase removes the carboxy‐terminal propeptide from procollagen. In the present study we describe an improved procedure for the purification of this enzyme. From the medium of cultured mouse fibroblasts, consisting of ammonium sulfate precipitation, gel filtration and affinity chromatography on a lysyl‐Sepharose column, followed by chromatography on a column of Sepharose coupled to the carboxy‐terminal propeptide of type I procollagen (PP‐Sepharose). This procedure yielded a practically homogeneous, 18 500‐fold‐purified enzyme preparation and the molecular mass of the purified C‐proteinase as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis was 80 kDa. The lysyl‐Sepharose step separated the enzyme from the majority of the contaminating proteins, including a 55‐kDa protein which was further purified by PP‐Sepharose chromatography and identified as an additional form of the 36‐kDa and 34‐kDa procollagen C‐proteinase enhancer proteins described before [Adar et al. (1986) Collagen Relat. Res. 6, 267–277]. It enhanced the C‐proteinase activity, bound to the carboxyl propeptide of type I procollagen, cross‐reacted immunologically with the 36‐kDa as well as the 34‐kDa enhancer proteins, and in common with the latter proteins, it was glycosylated. In the course of PP‐Sepharose chromatography, a large proportion of the 55‐kDa protein disappeared with the concomitant appearance of the smaller enhancer proteins. All these findings suggest that the 55‐kDa protein is a precursor of the low molecular mass enhancer proteins. Also suggested from this study is that lysyl‐Sepharose chromatography is a highly beneficial purification step which may find use in the purification of the C‐proteinase from other sources as well.
Expression of recombinant diaminopimelate epimerase in Escherichia coli
Authors: HIGGINS William , TARDIF Chantal , RICHAUD Catherine , KRIVANEK Michèle A. , CARDIN Alan
Published: March 2005

Recombinant diaminopimelate epimerase is overproduced to give 1% of soluble protein when grown under the appropriate conditions in Escherichia coli. This compares with 0.02% of the constitutive level of wild‐type enzyme. A new purification procedure now yields milligram quantities of homogeneous enzyme of high specific activity (192 U/mg). This has enabled sufficient amounts of enzyme both to compare with wild‐type enzyme and to enable active site modification studies to be performed. Incubation of the enzyme with 2‐(4‐amino‐4‐carboxybutyl)‐2‐aziridine‐carboxylic acid (AZIDAP), results in time‐dependent irreversible inhibition. Tryptic digestion of the inactivated enzyme and peptide‐mapping show that AZIDAP is specifically and covalently bound to the enzyme at a unique peptide. Determination of the amino acid sequence of this peptide and comparison with the sequence deduced from the DNA sequence of the dapF gene shows that Cys73 is labelled. Finally based on limited sequence similarities around this cysteine and active‐site cysteines of proline racemase and 1‐hydroxyproline 2‐epimerase, together with mechanistic considerations, we propose that all three non‐pyridoxalphosphate‐containing racemases/epimerases derive from a common evolutionary origin.
Gangliosides of sea urchin embryos
Authors: ZVEZDINA Natalia D. , SADYKOVA Kamila A. , MARTYNOVA Larisa E. , PROKAZOVA Nina V. , MIKHAILOV Alexander T. , BUZNIKOV Gennady A. , BERGELSON Lev D.
Published: March 2005

The influence of antibodies to gangliosides of sea urchin Strongylocentrotus intermedius eggs on early embryos of this species was studied. γ‐Globulins were isolated from rabbit anti‐ganglioside serum by micropreparative electrophoresis. These γ‐globulins produced anomalies in the development of embryos permeabilized in Triton X‐100. The anomalies were not observed when anti‐ganglioside γ‐globulins were added to the incubation medium together with gangliosides or when the permeabilized embryos were incubated with γ‐globulins of normal rabbit serum.

Pretreatment of S. intermedius embryos with serotonin, tryptamine or some other indole derivatives led to the disappearance of ganglioside determinants from the cell surface and sharply increased immunofluorescence within the cell. Such pretreatment of embryos increased the amount of cell‐associated gangliosides more than threefold as compared to untreated embryos. Serotonin was shown to bind specifically to sea urchin gangliosides immobilized on octyl‐Sepharose.

These observations suggest that cell‐surface gangliosides, after binding drugs, are internalized and that serotonin and its antagonists inhibit the transport of newly synthesized gangliosides to the cell‐surface membrane.
Phosphagen kinase evolution
Authors: RATTO Anne , SHAPIRO Bennett M. , CHRISTEN Richard
Published: March 2005

Arginine kinase and creatine kinase that catalyze the transfer of a phosphate group between ATP and arginine and creatine, respectively, play an important role in cellular energetics. In contrast to most animals which exhibit a single phosphagen kinase activity (creatine kinase in chordates and arginine kinase in protostomians), echinoderms exhibit both arginine kinase and creatine kinase activities, sometimes in the same tissue. In contrast to chordates in which creatine kinases are dimers (consisting of two subunits of 40 kDa) and protostomians in which arginine kinases are usually monomers (40 kDa), echinoids contain specific phosphagen kinases: a dimeric arginine kinase (consisting of two subunits of 42 kDa) in eggs and a monomeric creatine kinase (145 kDa) in sperm. We have examined echinoderms from the five existing classes (echinoids, asteroids, ophiuroids, holothurians and crinoids) for the expression of these specific phosphagen kinases in different tissues. Gel filtration was used to determine the molecular masses of the native enzymes. Antibodies specific for arginine kinase or for creatine kinase were used to characterize the subunit composition of arginine kinase and creatine kinase after SDS/PAGE and transfer. In all echinoderms analyzed, arginine kinase always occurred as an enzyme of about 81 kDa consisting of two subunits of 42 kDa and creatine kinase as a monomeric enzyme of 140–155 kDa. The occurrence in echinoderms of both phosphagen kinases with distinct specificities and specific molecular structures is discussed from both a developmental and evolutionary point of view.
Pregnenolone synthesis from cholesterol and hydroxycholesterols by mitochondria from ovaries following the stimulation of immature rats with pregnant mare's serum gonadotropin and human choriogonadotropin
Authors: TUCKEY Robert C. , ATKINSON Helen C.
Published: March 2005

The rate of pregnenolone synthesis by cytochrome P‐450scc was measured in mitochondria isolated from ovaries of immature rats treated with pregnant mare's serum gonadotropin and human choriogonadotropin. Using cholesterol, 25‐hydroxycholesterol, 20α‐hydroxycholesterol, (22R)‐22‐hydroxycholesterol and (22R)‐20α,22‐dihydroxycholesterol as substrates, we have determined that the first hydroxylation of cholesterol, in the 22R position, is rate limiting in pregnenolone synthesis. It proceeds at only 22% of the rate of either of the subsequent two hydroxylations.

25‐Hydroxycholesterol proved to be a suitable substrate for determining the maximum rate of pregnenolone synthesis by cytochrome P‐450scc in isolated mitochondria. The maximum rate was 13 mol steroid · min−1· mol cytochrome P‐450−1scc and did not change after the follicles in the immature ovary had been stimulated to mature and luteinize with gonadotropin.

Using endogenous cholesterol in isolated mitochondria as substrate, the time course of pregnenolone synthesis was the same during the follicular phase as in the luteal stage of gonadotropin‐induced development. We conclude that during the artificially induced development of follicles in the immature ovary, the major cause of the increase in the rate of pregnenolone synthesis is the increase in the cytochrome P‐450scc content of the mitochondria, rather than changes in the catalytic activity of cytochrome P‐450scc or the cholesterol availability to the cytochrome.
Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells
Authors: PFEIFFER Günter , SCHMIDT Martin , STRUBE Karl‐Hermann , GEYER Rudolf
Published: March 2005

Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6‐3H]‐glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo‐β‐N‐acetylglucosaminidase H or peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F and fractionated by high‐performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H‐NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N‐terminal amino acid sequencing.

The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5–8 mannose residues as well as small amounts of hybrid‐type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N‐acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(α1–3) substituents and/or sulfate groups linked to position six of β‐galactosyl residues forming NeuAc(α2–3)[HO3S–6]Gal(β1–4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (α2–3)‐ or (α2–6)‐linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated.
13C and 15N NMR and time‐resolved fluorescence depolarization study of bovine carbonic anhydrase—4‐methylbenzenesulfonamide complex
Authors: JARVET Jüri , OLIVSON Abira , METS Ülo , POOGA Margus , AGURAIUJA Reet , LIPPMAA Endel
Published: March 2005

The influence of the binding of the high‐affinity inhibitor, 4‐methylbenzenesulfonamide, to the active site of bovine carbonic anhydrase B was studied by 15N‐ and 13C‐NMR spectroscopy. The rotational correlation time dependence on temperature and concentration of the complex was determined by time‐resolved fluorescence depolarization measurements.

Our experiment provides evidence that the stoichiometry of the interaction of 4‐methylbenzenesulfonamide with carbonic anhydrase B is 1:1 and the inhibitor is bound in anionic form. The 15N‐NMR relaxation parameters confirm our previous conclusions about the presence of librational motions in the active site of carbonic anhydrase and indicate that the internal motion in the enzyme‐inhibitor complex is more restricted than the backbone motion in the uncomplexed native enzyme.
Amount and turnover rate of the F0F1‐ATPase and the stoichiometry of its inhibition by oligomycin in Rhodospirillum rubrum chromatophores
Authors: NORLING Birgitta , STRID Åke , TOURIKAS Christos , NYRÉN Pål
Published: March 2005

The amount of F1‐ATPase in chromatophores from Rhodospirillum rubrum was determined by Western blotting using anti‐RrF1 rabbit antibodies. 9.1 mmol F1 (mol bacteriochlorophyll)−1 was obtained or 14% of the total protein content of the chromatophores. The turnover rate of the F0F1‐ATPase was 17 molecules ATP s−1 during synthesis, 2 molecules ATP s−1 during hydrolysis under coupled conditions with Mg2+ as the divalent cation, and 7 molecules ATP s−1 during hydrolysis in the presence of carbonyl cyanide p‐trifluoromethoxy‐phenylhydrazone. Binding of 1 mol oligomycin/mol F0F1‐ATPase was found to inhibit the activities of the enzyme completely. A single binding site was found with a Kd of approximately 2 μM.
Protein–protein interactions in microsomal cytochrome P‐450 isozyme LM2 and their effect on substrate binding
Authors: HILDEBRANDT Peter , GARDA Horacio , STIER Anton , BACHMANOVA Galina I. , KANAEVA Irina P. , ARCHAKOV Alexander I.
Published: March 2005

The effects of protein–protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P‐450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2‐dependent catalytic activities of the two states have been compared.

The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P‐450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P‐450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin‐state equilibrium. This indicates that in the oligomer the substrate‐binding pocket is converted by protein–protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five‐coordinated high‐spin configuration. In the monomer the substrate‐binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P‐450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein–protein interactions.

H2O2‐dependent N‐demethylation of benzphetamine and aniline p‐hydroxylation by cytochrome P‐450 LM2 did not depend on its state of aggregation.
Developmental expression of Cu,Zn superoxide dismutase in Xenopus
Authors: MONTESANO Luisa , CARRÌ Maria Teresa , MARIOTTINI Paolo , AMALDI Francesco , ROTILIO Giuseppe
Published: March 2005

cDNA clones for Xenopus laevis Cu,Zn‐superoxide dismutase were isolated, sequenced and used as probes to study the expression of the corresponding gene during oogenesis and embryogenesis; Cu,Zn‐superoxide dismutase activity was also monitored throughout development. It has been observed that its mRNA is actively synthesized during early oogenesis, reaching a maximum level at stage II, and is utilized through oogenesis. This results in an accumulation of enzyme activity during oocyte growth, paralleling the accumulation of the several other cellular components which are stored in the oocyte to be utilized later on by the developing embryo. In fact, Cu,Zn‐superoxide dismutase activity is present at an approximately constant level until late embryonic development, while its mRNA disappears soon after fertilization to be accumulated again only during the last part of embryogenesis. This developmental expression behaviour can be viewed as typical of an housekeeping function and suggests that Cu,Zn‐superoxide dismutase activity is a constant need of the cell rather than being subject to regulation by oxygen metabolism.
Crosslinking of cytochrome c and cytochrome b5 with a water‐soluble carbodiimide
Authors: MAUK Marcia R. , MAUK A. Grant
Published: March 2005

A water soluble carbodiimide, 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC), has been used to crosslink horse heart cytochrome c and trypsin‐solubilized bovine liver microsomal cytochrome b5. The reaction was conducted under a variety of solution conditions, and the products were purified by a combination of gel filtration and ion‐exchange chromatography. Under all conditions of pH, ionic strength, EDC/protein ratio and reaction time that were studied, multiple 1:1 crosslinked complexes were observed with no evidence of a single, dominant species. Acetate, which is often used as a quencher of such reactions, was found to increase the complexity of the reaction products, presumably through EDC‐promoted coupling to cytochrome c. Hydroxylamine treatment of the crosslinked complexes, a procedure frequently used to reverse EDC modification of tyrosyl residues, did not reduce the number of crosslinked components observed. The cytochrome b5 heme group was readily extracted from each of the 1:1 crosslinked complexes by standard techniques, so the crosslinking of heme propionate 7 with Lys79 of cytochrome c that might have been anticipated on the basis of molecular graphics modeling [Salemme, F. R. (1976) J. Mol. Biol. 102, 563–568] was not evident from this analysis.

Analysis of HPLC tryptic peptide maps produced from crosslinked complexes revealed reduced specificity of trypsin in hydrolysis of EDC‐crosslinked protein‐protein complexes and unsatisfactory resolution of crosslinked or branched peptides. Nevertheless, it was possible to demonstrate that residues 52–72 of cytochrome b5, a region predicted to be critical to interaction with cytochrome b5 [Salemme, F. R. (1976) J. Mol. Biol. 102, 563–568] was absent from all peptide maps of 1:1 cytochrome c· cytochrome b5 complexes. Based on these results and a review of the literature involving EDC crosslinking of electron transfer proteins, we conclude that the techniques available for specific protein hydrolysis and separation of crosslinked peptides are not adequate to permit routine unambiguous identification of crosslinking sites in carbodiimide‐crosslinked complexes.
Variability within mammalian sorbitol dehydrogenases
Authors: KARLSSON Christina , MARET Wolfgang , AULD Douglas S. , HÖÖG Jan‐Olov , JÖRNVALL Hans
Published: March 2005

The primary structure of sorbitol dehydrogenase from human liver has been determined by peptide analysis in order to relate the variability of this enzyme to that of the others within the alcohol dehydrogenase family. The structure obtained reveals 355 residues with an acyl‐blocked N‐terminus and an unexpected microheterogeneity at position 237 (Gln/Leu). The residue identity between sheep and human liver sorbitol dehydrogenase is 89%. This variability is similar to that of class I alcohol dehydrogenases, but distinctly different from that of class III alcohol dehydrogenases, the structures of which are much more conserved. Consequently, class III alcohol dehydrogenase is thus far unique within this family of dehydrogenases, suggesting a particularly strict requirement for that structure. The variability within sorbitol dehydrogenase involves all segments of the molecule but is largely at surface positions and clusters in one such region, covering positions 214–240, corresponding to a segment of the coenzyme‐binding domain. Ligands to the active‐site zinc and most residues lining the coenzyme‐binding and substrate‐binding pockets are conserved. However, provided conformational models are reliable, a charge difference may affect the interactions at the inner part of the substrate pocket, another charge difference may affect the interdomain region, and a size difference the adenine pocket. The primary structure of human liver sorbitol dehydrogenase further shows that the absence of three of the four ligands to a second zinc atom present in alcohol dehydrogenases is a general property of sorbitol dehydrogenase.
O‐specific polysaccharides of Hafnia alvei lipopolysaccharides isolated from two serologically related strains: ATCC 13337 and 1187
Authors: GAMIAN Andrzej , ROMANOWSKA Elzbieta , OPFERKUCH Hans J. , HAUCK Michael , DABROWSKI Janusz
Published: March 2005

The O‐specific polysaccharides of Hafnia alvei ATCC 13337 standard strain and 1187 strain have been isolated and characterized. By means of 1H‐NMR spectroscopy, methylation analysis and periodate oxidation, the repeating unit of the polysaccharides could be allocated the respective structures:

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where Acyl = D‐3‐hydroxybutyryl, and 3‐O‐acetylation was to about 66%.

The structural similarity of the polysaccharides was confirmed in the serological study: their epitopes were determined and the importance of various structural elements for the serological specificity was discussed.
Effect of glycosylation inhibitors on the structure and function of the murine transferrin receptor
Authors: RALTON Julie E. , JACKSON Heather J. , ZANONI Maureen , GLEESON Paul A.
Published: March 2005

The murine transferrin receptor is a disulphide‐linked dimer with three N‐glycosylation sites. We have investigated the structural and functional properties of the transferrin receptor from murine plasmacytoma cells (NS‐1 cells) treated with the glycosylation inhibitor, tunicamycin and the glycosylation‐processing inhibitors, swainsonine and castanospermine.

Tunicamycin (1 μg/ml) inhibited mannose incorporation in NS‐1 cells by > 90%, but also inhibited me‐thionine incorporation by up to 50%. Both swainsonine (1 μg/ml) and castanospermine (50 μg/ml) resulted in mannose incorporation > 100% of untreated cells and neither drug affected methionine incorporation.

Incubation of NS‐1 cells with tunicamycin resulted in a shift in the apparent molecular mass of the transferrin receptor from 96 kDa and 94 kDa to approximately 82 kDa.

Peptide N‐glycosidase F digestion of the receptor from untreated cells resulted in the fully deglycosylated 82 kDa component as well as an 87 kDa component which represents partially deglycosylated receptor resistant to peptide N‐glycosidase F digestion.

The receptor from swainsonine‐treated cells was equally sensitive to peptide N‐glycosidase F and endo‐β‐N‐acetylglucosaminidase H (endo H; resulting in both 87‐kDa and 82‐kDa components), whereas the receptor from castanospermine‐treated cells was only partially sensitive to endo H.

Analysis of mannose‐ and fucose‐labelled cellular glycopeptides by concanavalin‐A ‐ Sepharose chromatography showed that swainsonine (1 μg/ml) treatment resulted in approximately 90% inhibition of the synthesis of complex N‐glycans and an accumulation of fucosylated hybrid structures. In contrast, castanospermine (100 μg/ml) treatment resulted in only partial inhibition (60%) of the synthesis of complex N‐glycans.

Analysis of the receptor from tunicamycin, swainsonine and castanospermine treated cells under non‐reducing conditions showed a single component corresponding to the dimer, indicating that dimerisation of newly synthesisted murine receptor is independent of carbohydrate.

The non‐glycosylated receptor from tunicamycin‐treated cells appears to bind transferrin as demonstrated by interaction with transferrin‐Sepharose.

Surface expression of the receptor was not significantly altered in the presence of either swainsonine or castanospermine as judged by flow cytometry.
Distribution analyses of chain substituents of lipoteichoic acids by chemical degradation
Authors: SCHUREK Josef , FISCHER Werner
Published: March 2005

The lipoteichoic acid fromLactococcus lactisKiel 48337 was analyzed. It had 61% of its glycerophosphate residues substituted with α‐d‐galactopyranosyl residues. Non‐substituted glycerophosphate residues were split off by two alkaline hydrolyses and an intermediate enzymatic phosphomonoester cleavage. The resulting (GalGroP)nGroGal and (GalGroP)nGlc2Gro oligomers were separated by chromatography on DEAE‐Sephadex into 10 pairs of molecular species withnfrom 1 to 10. The relative frequencies of GalGro and these oligomers were close to the values calculated by computer simulation for a random distribution of chain substituents. A similar series of oligomers was obtained in one step by hydrolysis of the lipoteichoic acid with 98% (by vol.) acetic acid. Due to side reactions, the picture was less precise but nevertheless indicative of the same distribution pattern.

The data provide indirect evidence that the alanine ester substituents of the native lipoteichoic acid (Ala/P= 0.38) occupy the free positions between the galactosylated oligomers and are therefore themselves distributed randomly.
Changes in myosin heavy chain isoforms during chronic low‐frequency stimulation of rat fast hindlimb muscles
Authors: TERMIN Angelika , STARON Robert S. , PETTE Dirk
Published: March 2005

Fast‐twitch rat muscles contain three fast myosin heavy chains (HC) which can be separated by density gradient gel electrophoresis. Their mobility increases in the order of HCIIa < HCIId < HCIIb. In contrast to the rabbit, where chronic low‐frequency nerve stimulation induces a fast‐to‐slow conversion, stimulation for up to 56 days does not lead to appreciable increases in the relative concentration of the slow myosin heavy chain HCI in rat fast‐twitch muscles. However, chronic stimulation of rat fast‐twitch muscle does evoke a rearrangement of the fast myosin heavy chain isoform pattern with a progressive decrease in HCIIb and progressive increases in HCIIa and HCIId. As judged from the time course and extent of these transitions, it appears that HCIId is an intermediate form between HCIIb and HCIIa. Single‐fiber analyses of normal muscles make it possible to assign these heavy chain isoforms to histochemically defined fiber types IIB, IID, and IIA. The stimulation‐induced fiber transformations produce numerous hybrid fibers displaying more than one myosin heavy chain isoform. Some transforming fibers contain up to four different myosin heavy chain isoforms.
Specific programs of myosin expression in the postnatal development of rat muscles
Authors: d'ALBIS Anne , COUTEAUX René , JANMOT Chantal , ROULET Agnès
Published: March 2005

The expression of myosin during postnatal development was studied in a dozen muscles of the rat. All muscles displayed the usual sequential transitions from embryonic to neonatal and to adult isomyosins. However, we observed that these transitions did not take place uniformly. Thus, half‐transition times for the appearance of the adult intermediate and fast myosin extended from seven days for diaphragm, the most precocious muscle of all those examined, to 23 days for male rat masseter. Besides the large differences between their half‐transition times, we noticed that the transition curves displayed different slopes, covering different periods. Differences between muscles mainly affected the neonatal‐to‐adult transition rather than the embryonic‐to‐neonatal transition, since the embryonic‐type myosin disappeared from all muscles examined except for one, at about the same time, by the end of the first week after birth. In addition, the appearance of slow myosin varied for each muscle and did not follow curves parallel to those for intermediate and fast myosins. These results indicate that each muscle of the rat is subjected to a specific program of myosin isoform transitions during postnatal development.
Cloning and chromosomal localization of Drosophila cDNA encoding the catalytic subunit of protein phosphatase 1α
Authors: DOMBRADI Viktor , AXTON J. Myles , GLOVER David M. , COHEN Patricia T. W.
Published: March 2005

A 1.2‐kb clone containing the full coding sequence of a protein phosphatase 1 catalytic subunit has been isolated from a Drosophila head cDNA library. It encodes a polypeptide of 302 amino acids with a molecular mass of 34.5 kDa. The predicted protein sequence is 92% identical (94% similar) to rabbit protein phosphatase 1α (PP‐1α) demonstrating strict conservation of the phosphatase catalytic subunit over a considerable evolutionary distance. Abundant 1.6‐kb and 2.5‐kb mRNA transcripts were detected troughout Drosophila development. The clone hybridised to four sites on Drosophila salivary gland polytene chromosomes. The major site is at 87B6‐12 on the right arm of chromosome 3. In addition, there are three secondary sites, one on the same chromosome at 96A2‐5 and two on the X chromosome at 9C1‐2 and 13C1‐2. Isolation of a further cDNA clone, hybridising to 9C1‐2 and encoding part of the catalytic subunit 88% similar to Drosophila PP‐1α, proves the existence of at least two transcriptionally active genes for protein phosphatase 1.
Structural homology between glycophorins C and D of human erythrocytes
Authors: EL‐MALIKI Bouchra , BLANCHARD Dominique , DAHR Wolfgang , BEYREUTHER Konrad , CARTRON Jean‐Pierre
Published: March 2005

Glycophorin C (GPC) and D (GPD) are minor glycoproteins which are believed to be important for the structural integrity of the red cell membrane.

We have investigated the structural relationship between these glycoproteins by both immunological and structural investigations:

A rabbit anti‐serum produced against GPD reacts strongly with GPC and the abnormal glycoproteins of Gerbich: –2, –3 and Gerbich: –2,3 red cells, and recognizes most probably the homologous C‐terminal portions of GPC and GPD. The two molecules however differ at their N‐terminus.

One‐dimensional mapping of the peptides obtained after tryptic, chymotryptic, V8 protease or acid cleavage of 125 I‐labelled GPC and GPD, indicated that GPC and GPD are structurally related but some differences were found indicating that additional peptides were generated from GPC.

The partial primary structure of GPD was determined. The sequencing data are consistent with the assumption that GPD represents an abridged version of GPC that comprises residues approximately 21/29–128 and exhibits a N‐terminal residue that is blocked by an as yet undefined group.