bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid characterization of asparagine‐linked oligosaccharides isolated from glycoproteins using a carbohydrate analyzer
Authors: ANUMULA Kalyan Rao , TAYLOR Paul B. 
Published: March 2005

Chromatographic methods were developed for the separation and characterization of acidic (sialylated) and neutral (asialo‐complex and high‐mannose) oligosaccharides released from glycoproteins with peptide N‐glycosidase F, endo‐β‐N‐acetylglucosaminidase F and endo‐β‐N‐acetylglucosaminidase H using a carbohydrate analyzer (Dionex BioLC). All the carbohydrate separations were carried out on a polymeric pellicular anion‐exchange column HPIC‐AS6/CarboPac PA‐1 (Dionex) using only two eluants namely, 0.5 M NaOH and 3% acetic acid/NaOH pH 5.5, which were mixed with water to generate various gradients. Developed conditions for quantitative detection of carbohydrates with pulsed amperometry were necessary to obtain steady baselines at 0.1–0.3 μA output with suitable sensitivity (< 5 pmol) in separations employing a variety of acidic and alkaline sodium acetate gradients.Oligosaccharides released from heat‐denatured and trypsin‐treated glycoproteins were purified initially from large‐scale digestions (> 0.1 g) by extraction of peptide material into phenol/chloroform and finally by ionexchange chromatography of the aqueous phase. Oligosaccharides isolated from the peptide N‐glycosidase digests of bovine fetuin, human transferrin and α1‐acid glycoprotein gave multiple peaks in each charge group in separations based on the charge content at pH 5.5. Alkaline sodium acetate gradients were developed to obtain oligosaccharide maps of the glycoproteins within 60 min, in which separated oligosaccharides eluted in the order of neutral, mono‐, di‐, tri‐ and tetra‐sialylated species based on both charge, size and structure. Baseline separations were obtained with neutral oligosaccharide types but mixtures of high‐mannose and complex types were poorly resolved. The high‐mannose peaks were eliminated specifically from complex oligosaccharides by digesting with α‐mannosidase. Treatment with β‐galactosidase, βN‐acetylglucosaminidase and α‐mannosidase resulted in a decrease of the oligosaccharide elution times corresponding to the number of sugar residues lost, the profile of changes was highly reproducible. In contrast, treatment with α‐l‐fucosidase, endo‐β‐N‐acetylglucosaminidase F and endo‐β‐N‐acetylglucosaminidase H resulted in an increase in their corresponding oligosaccharide retention times similar to the presence of an additional sugar residue.Conditions developed for separation of the reduced oligosaccharides and also a mixture of monosaccharide to oligosaccharide containing about 15 sugar residues within 30 min were useful in determining the effect of endo‐and exo‐glycosidases on porcine thyroglobulin oligosaccharides. Changes in elution time of the oligosaccharides following specific glycosidase digestions combined with methylation analysis provided a rapid and sensitive tool for confirmation of the carbohydrate primary structures present in thyroglobulin.
Sensory transduction in eukaryotes
Authors: Van HAASTERT Peter J. M. , JANSSENS Pim M. W. , ERNEUX Christophe 
Published: March 2005

The organization of multicellular organisms depends on cell–cell communication. The signal molecules are often soluble components in the extracellular fluid, but also include odors and light. A large array of surface receptors is involved in the detection of these signals. Signals are then transduced across the plasma membrane so that enzymes at the inner face of the membrane are activated, producing second messengers, which by a complex network of interactions activate target proteins or genes [1].Vertebrate cells have been used to study hormone and neurotransmitter action, vision, the regulation of cell growth and differentiation. Sensory transduction in lower eukaryotes is predominantly used for other functions, notably cell attraction for mating and food seeking. By comparing sensory transduction in lower and higher eukaryotes general principles may be recognized that are found in all organisms and deviations that are present in specialised systems. This may also help to understand the differences between cell types within one organism and the importance of a particular pathway that may or may not be general. In a practical sense, microorganisms have the advantage of their easy genetic manipulation, which is especially advantageous for the identification of the function of large families of signal transducing components.
Inhibition of protein synthesis by antagonists of calmodulin in Ehrlich ascites tumor cells
Authors: KUMAR Rajeswari V. , PANNIERS Richard , WOLFMAN Alan , HENSHAW Edgar C. 
Published: March 2005

Several recent publications indicate that Ca2+ is required for protein synthesis in mammalian cells, including the Ehrlich ascites tumor cell. The present communication examines whether the effects of Ca2+ might be mediated through calmodulin or a related protein. Four calmodulin antagonists belonging to different chemical categories were used to provide evidence of calmodulin involvement. Three of the antagonists inhibited protein synthesis in intact cells; 50% inhibitory concentrations were 10 μM calmidazolium, 12 μM N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W7) and 17.5 μM trifluoperazine (TFP). Initiation was preferentially inhibited as indicated by an increase in the 80S monomers accompanied by a significant disaggregation of polyribosomes. All the antagonists also inhibited protein synthesis initiation in the cell‐free protein‐synthesizing system; 50% inhibitory concentrations for compound 48/80, calmidazolium, TFP, and W7 were 10 μM, 125 μM, 300 μM and 500 μM, respectively. A weak analogue of W7 inhibited only 20% at 1000 μM. Inhibition in the cell‐free system was reversed by the addition of exogenous calmodulin in all four cases. The levels of 43S complexes were significantly elevated with all four antagonists, indicating a block in the utilization of 43S complexes.The similarity of the effects of four distinct classes of antagonists and their ready reversal by exogenous calmodulin leads us to suggest that there may be a role for calmodulin or a very similar calcium‐binding protein in protein synthesis.
Carbohydrate microheterogeneity of rat serotransferrin
Authors: SPIK Geneviève , CODDEVILLE Bernadette , STRECKER Gérard , MONTREUIL Jean , REGOECZI Erwin , CHINDEMI Paul A. , RUDOLPH John R. 
Published: March 2005

A previously established procedure [Regoeczi, E., Chindemi, P. A., Rudolph, J. R., Spik, G. & Montreuil, J. (1987) Biochem. Cell Biol. 65, 948 – 954] was used to isolate from three DEAE‐cellulose chromatographic fractions of diferric rat serotransferrin (rTf) subpopulations having discernible affinities for concanavalin A (ConA). These entities are designated rTf‐1 (not retarded by ConA column), rTf‐2 (retarded) and rTf‐3 (bound). Each rTf type was found to be endowed with carbohydrate sufficient to account for a single diantennary glycan/protein molecule. Glycan structures were determined on the glycopeptides by employing GLC/MS and 400‐MHz 1H‐NMR spectroscopy.All glycans possessed a common, trimannosyl‐N,N′‐diacetylchitobiose core with or without one L‐fucose α‐1,6‐linked to the Asn‐linked GlcNAc. However, there were differences in the antennae. Thus, in rTf‐3, both antennae were of the disialylated diantennary N‐acetyllactosamine type which is frequently encountered in other plasma glycoproteins. However, the α‐1,3‐Man‐linked antenna in rTf‐1 as well as rTf‐2 had the sequence: Neu5Ac(α2–3)Gal(β1–3)[Neu5Ac(α2–6)]GlcNAc(β1–2)Man.In addition, the α‐1,6‐Man‐linked antenna deviated in rTf‐2 from the standard structure by having the sequence: Neu5Ac(α2–3)Gal(β1–3)GlcNAc(β1–2)Man. The possible relevance of the above structures to the ConA binding of rTf is discussed. A further preparation, obtained from the most anionic DEAE‐cellulose fraction (peak V) of rTf contained several tetrasialylated diantennary glycans whose precise structures remain to be established in future studies.
Purification, biosynthesis and cellular localization of a major 125‐kDa glycophosphatidylinositol‐anchored membrane glycoprotein of Saccharomyces cerevisiae
Authors: FANKHAUSER Christoph , CONZELMANN Andreas 
Published: March 2005

The yeast Saccharomyces cerevisiae has been shown to contain a major 125‐kDa membrane glycoprotein which is anchored in the lipid bilayer by a glycophosphatidylinositol anchor. This protein was purified to near homogeneity and was used to raise a rabbit antibody. Biosynthesis of the 125‐kDa protein was studied by immunoprecipitation of 35SO4‐labeled material from wild‐type cells or a secretion mutant (sec18) in which the vesicular traffic from the endoplasmic reticulum (ER) to the Golgi is blocked. The 125‐kDa protein is first made in the ER as a 105‐kDa precursor which already contains a glycophosphatidylinositol anchor and which is slowly transformed into the 125‐kDa form upon chase (t1/2∼ 10–15 min). The 105‐kDa precursor can be reduced to an 83‐kDa form by the enzymatic removal of N‐glycans. The removal of N‐glycans from the mature 125‐kDa protein yields a 95‐kDa species. Thus, removal of the N‐glycans does not reduce the ER and mature forms to the same molecular mass, indicating that not only elongation of N‐glycans but also another post‐translational modification takes place during maturation. Selective tagging of surface proteins by treatment of 35SO4‐labeled cells with trinitrobenzene sulfonic acid at 0°C followed by immunoprecipitation of the tagged proteins shows that the 125‐kDa protein, but not the 105‐kDa precursor, becomes transported to the cell surface. This tagging of cells after various lengths of chase also shows that the surface appearance of the protein is biphasic with about one half of the mature 125‐kDa protein remaining intracellular for over 2 h.Glycosylation and/or glycophosphatidylinositol anchor addition is important for the stability of the 125‐kDa protein since the protein remains undetectable in sec53, a temperature‐sensitive mutant which does not make GDP‐mannose at 37°C and does not add glycophosphatidylinositol anchors at 37°C.
Determination of the covalent structure of an N‐ and C‐terminally blocked glycoprotein from endocuticle of Locusta migratoria
Authors: TALBO Gert , HØJRUP Peter , RAHBEK‐NIELSEN Henrik , ANDERSEN Svend Olav , ROEPSTORFF Peter 
Published: March 2005

The complete structure of a protein isolated from endocuticle of sexually mature locusts, Locusta migratoria, has been determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The protein is extensively post‐translationally modified. The N‐terminal is 5‐oxoproline (pyroglutamic acid) and the C‐terminal proline residue is amidated. Furthermore, the protein is glycosylated by a single N‐acetyl‐galactosamine residue at one, two or three threonines. The N‐terminal sequence was obtained by analysing the N‐acetylated N,O‐permethylated derivative using plasma desorption mass spectrometry. The position and type of carbohydrate were determined by combining an HPLC‐based carbohydrate analysis with the peak pattern of the phenylthiohydantoin derivative in automatic sequencing and with mass information on peptides.The protein has pronounced similarity to cuticular proteins from larvae of diptera and lepidoptera, but only slight resemblance to the previously sequenced locust exocuticular proteins. This indicates a similarity between soft larval cuticles and locust endocuticle, a similarity which may extend to their mechanical properties.
Demonstration of a molybdenum‐ and vanadium‐independent nitrogenase in a nifHDK‐deletion mutant of Rhodobacter capsulatus
Authors: SCHNEIDER Klaus , MÜLLER Achim , SCHRAMM Uwe , KLIPP Werner 
Published: March 2005

In Rhodobacter capsulatus there exists, in addition to a conventional Mo‐containing nitrogenase, a second, Mo‐indendent nitrogenase which was demonstrated in wild‐type cells as well as in cells of a nifHDK− mutant. To construct this R. capsulatus mutant, a 4‐kb BglII–HindIII fragment encompassing nifK, nifD and most of the nifH coding region was substituted by an interposon coding for kanamycin resistance. The alternative nitrogenase is repressed by molybdenum. Mo concentration > 1 ppb in the growth medium prevented diazotrophic growth of nifHDK− cells and the expression of nitrogenase activity. The Mo‐independent nitrogenase was maximally derepressed in activated carbon‐treated media which contained < 0.05 ppb Mo, high concentrations of iron (1 mM ferric citrate) and serine as N source. Under N2‐fixing and optimal Mo‐deficient conditions, nifHDK− cells grew with a doubling time of 9 h. The highest activity achieved with whole cells was 1.2 nmol ethylene · min−1· mg protein−1. Vanadium neither stimulated nor inhibited growth and activity.The alternative nitrogenase reduced acetylene to both ethylene and ethane. With whole cells (nifHDK−) the proportion of ethane varied over 2–5% depending on the amount of residual traces of Mo in the medium. The addition of Mo to a growing, nitrogenase‐active culture resulted in a slow decrease of total activity but also in a simultaneous increase of ethane production up to 40%. In contrast, cell‐free extracts and the purified enzyme did not show any or only very little ethane formation (0–0.4%).Both enzyme components appeared to be very labile proteins. Component 2 lost almost all its activity during cell breakage. With component 1 in crude extracts, if complemented with the stable component 2 of the Mo‐nitrogenase from Xanthobacter autotrophicus, a recovery of 50% of the original whole cell activity could be achieved. During purification, component 1 (from the nifHDK− mutant) remained remarkably stable. The partially purified component 1 had a pH optimum (acetylene reduction) of 7.8–8.0, relatively high affinity to acetylene (Km= 0.055 mM) and was analyzed to contain 20 mol Fe atoms/mol protein, 0.2 mol Mo atoms and negligible amounts of V, W and Re. The dithionite‐reduced dinitrogenase appeared to be ESR‐silent. The results indicate that the alternative nitrogenase of R. capsulatus is not a vanadium enzyme but rather a heterometal‐free Fe‐nitrogenase or a nitrogenase with an as‐yet‐unidentified heterometal atom.
Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies
Authors: ORSINI Gaetano , BRANDAZZA Anna , SARMIENTOS Paolo , MOLINARI Antonio , LANSEN Jacqueline , CAUET Gilles 
Published: March 2005

Prourokinase is a plasminogen activator of 411 amino acids which displays a clot‐lysis activity through a fibrin‐dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction.The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain.In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (δ125‐prourokinase) in which 125 amino acids of the N‐terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first‐order rate constant of renaturation of 0.57 h−1 for δ125‐prourokinase and 0.25 h−1 for prourokinase).Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure, non‐glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and δ125‐prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria.After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160000 and 250000 IU/mg protein, respectively, for prourokinase and δ125‐prourokinase.As with prourokinase, the deletion mutant δ125‐prourokinase displays a zymogenic nature, being activated by plasmin to the active two‐chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.
The substrate specificity of protein kinases which phosphorylate the α subunit of eukaryotic initiation factor 2
Authors: PROUD Christopher G. , COLTHURST David R. , FERRARI Stefano , PINNA Lorenzo A. 
Published: March 2005

The α subunit of eukaryotic protein synthesis initiation factor (eIF‐2α) is phosphorylated at a single serine residue (Ser51) by two distinct and well‐characterised protein kinase, the haem‐controlled repressor (HCR) and the double‐stranded RNA‐activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51‐Arg‐Arg‐Arg‐Ile‐Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI.Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two‐dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP‐dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both ‘sites’ in clupeine Y1 and salmine A1, whereas HCR acted only on the N‐terminal cluster of serines in these protamines.The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C‐terminal clusters of arginines on the ‘target’ serine residue, provided that they were present at positions + 3 and/or + 4 relative to Ser51. However, peptides containing only N‐terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF‐2α and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.
Characterization of the cellular binding domain and the effects of monoclonal antibodies and thrombin inhibitors on the binding and internalization of the antithrombin‐III — thrombin complex by cultured cells
Authors: KNOLLER Sarah , SAVION Naphtali 
Published: March 2005

Antithrombin III (AT) binds to cultured cells mainly as a complex with thrombin or other serine proteases rather than in its free form. This implies that, upon complex formation, a new determinant appears on the AT molecule which is recognized by the cells. Fragmentation of AT by cyanogen bromide exposes this determinant and an 8‐kDa fragment is recognized by cultured cells. The binding of this fragment to cultured cells is inhibited by antithrombin‐III — thrombin (AT‐T) complex, but not by free AT. The putative cellular binding domain of AT‐T is located over amino acid residues 253–314 of AT, in the large loop close to the carboxy‐terminus of the molecule.The cell‐associated AT‐T is internalized and degraded, forming the thrombin‐modified AT (ATM). This process is inhibited by lysosomal degradation inhibitors, such as chloroquine or benzamidine. Thus, the appearance of ATM in cells is not a result of its binding to the cell surface, but probably a result of lysosomal degradation of cell‐associated AT‐T. Another degradation product, with a molecular mass of 43 kDa, appears in cells along with the appearance of ATM. Hirudin, a specific inhibitor of thrombin, inhibits the cellular internalization of AT‐T complexes, indicating a possible role for thrombin activity in the internalization process of complexes.Monoclonal antibodies (mAb) against AT, whose epitopes on the AT molecule have been located, affect the binding of AT‐T to cultured cells. Two mAb, A10 whose epitope is found outside of the cellular binding site, and B108 whose epitope may overlap this sequence, totally inhibit the binding of the complex to cells. Inhibition of binding results either from steric hindrance or induced changes in the binding site.
Immunology, biosynthesis and in vivo assembly of the branched‐chain 2‐oxoacid dehydrogenase complex from bovine kidney
Authors: CLARKSON George H. D. , LINDSAY J. Gordon 
Published: March 2005

Specific, polyclonal antisera have been raised to the native branched‐chain 2‐oxoacid dehydrogenase complex (BCOADC) from bovine kidney and each of its three constituent enzymes: E1, the substrate‐specific 2‐oxoacid dehydrogenase; E2, the multimeric dihydrolipoamide acyltransferase ‘core’ enzyme and E3, dihydrolipoamide dehydrogenase. Purified BCOADC, isolated by selective poly(ethyleneglycol) precipitation and hydroxyapatite chromatography, contains only traces of endogenous E3 as detected by a requirement for this enzyme in assaying overall complex activity and by immunoblotting criteria. A weak antibody response was elicited by the E1β subunit relative to the E2 and E1α polypeptides employing either purified E1 or BCOADC as antigens.Anti‐BCOADC serum showed no cross‐reaction with high levels of pig heart E3 indicating the absence of antibody directed against this component. However, immunoprecipitates of mature BCOADC from detergent extracts of NBL‐1 (bovine kidney) or PK‐15 (porcine kidney) cell lines incubated for 3–4 h in the presence of [35S]methionine contained an additional 55000‐Mr species which was identified as E3 on the basis of immunocompetition studies.Accumulation of newly synthesised [35S]methionine‐labelled precursors for E2, E1α and E3 was achieved by incubation of PK‐15 cells for 4 h in the presence of uncouplers of oxidative phosphorylation. Pre‐E2 exhibited an apparent Mr value of 56 500, pre‐E1α, 49 000 and pre‐E3, 57 000 compared to subunit Mr values of 50 000, 46 000 and 55 000, respectively, for the mature polypeptides. Thus, like the equivalent lipoate acyltransferases of the mammalian pyruvate dehydrogenase (PDC) and 2‐oxoglutarate dehydrogenase (OGDC) complexes, pre‐E2 of BCOADC characteristically contains an extended presequence.In NBL‐1 cells, pre‐E2 was found to be unstable since no cytoplasmic pool of this precursor could be detected; moreover, processed E1α was not assembled into intact BCOADC as evidenced by the absence of E2 or E3 in immunoprecipitates with anti‐(BCOADC) serum after a 45‐min ‘chase’ period in the absence of uncoupler. Dihydrolipoamide dehydrogenase (E3), in its precursor state, was not present in immune complexes with anti‐(BCOADC) serum, indicating that its co‐precipitation with mature complex is by virtue of its high affinity for assembled complex in vivo whereas no equivalent interaction of pre‐E3 with its companion precursors occurs prior to mitochondrial import.