bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational studies on the pancreatic polypeptide hormone family
Authors: GLOVER Ian D. , BARLOW David J. , PITTS James E. , WOOD Stephen P. , TICKLE Ian J. , BLUNDELL Thomas L. , TATEMOTO Kazuhiko , KIMMEL Joe R. , WOLLMER Axel , STRASSBURGER Wolfgang , ZHANG You‐Shang 
Published: March 2005

Pancreatic polypeptide has been extracted and sequenced from a wide range of species. The 36‐residue polypeptides have some hormonal characteristics, and show a high degree of sequence homology. Two recently isolated polypeptides, from porcine gut and brain, also show a high degree of sequence homology with the pancreatic polypeptides. It was proposed that these polypeptides were members of a related family.The X‐ray determined structure of one member of the family, turkey pancreatic polypeptide, is known to high resolution, but there is no structural information for the others. Studies designed to give an insight into the tertiary structure of these related molecules have been carried out, including model building using interactive computer graphics, circular dichroic spectroscopy and secondary structure prediction using a variety of algorithms.The results indicate that a compact globular conformation, similar to that observed in turkey pancreatic polypeptide may be adopted by all molecules and that this may be more highly conserved than the individual amino acid sequences.
An azide‐insensitive low‐affinity ATPase stimulated by Ca2+ or Mg2+ in basal‐lateral and brush border membranes of kidney cortex
Authors: ILSBROUX Ingrid , VANDUFFEL Luc , TEUCHY Henri , DE CUYPER Marcel 
Published: March 2005

Basal‐lateral and brush border membranes from pig kidney cortex were prepared by differential centrifugation followed by free‐flow electrophoresis. In each type of membrane, azide‐insensitive, low‐affinity Ca2+‐ATPase and Mg2+‐ATPase activities are demonstrated. A comparative study for both membranes further reveals the following analogies between these ATPase: (a) they show maximal activity between pH 8 and 8.5; (b) they exhibit Km values for Ca‐ATP or Mg‐ATP in the millimolar range and have a comparable low substrate specificity; (c) they are insensitive to 10 μM of vanadate, N,N′‐dicyclohexylcarbodiimide, diethylstillbestrol, quercetin, harmaline and amiloride. The partial inhibition by 1 mM of the various compounds is rather aspecific. In view of these similarities it is concluded that only one enzyme entity is responsible for the activity which is measured in both membrane types.The HCO3−‐stimulated Mg2+‐ATPase activity in pig kidney cortex was also studied. This enzyme, however, is clearly of mitochondrial origin since the HCO3−‐stimulation coincides with the distribution profile of succinate dehydrogenase, a mitochondrial marker; and since it is inhibited by azide.
Adenosine kinase from bovine adrenal medulla
Authors: ROTLLAN Pedro , MIRAS PORTUGAL Maria Teresa 
Published: March 2005

Adenosine kinase from bovine adrenal medulla was purified 1600‐fold by using ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration yielded a relative molecular mass around 42000 and Michaelis constants were 0.2 μM for adenosine and 20 μM for MgATP. The enzyme showed a broad specificity for purine nucleoside triphosphate as phosphate donors. Both free Mg2+ and ATP were inhibitors.AMP was a competitive inhibitor with regard to adenosine and a non‐competitive inhibitor versus MgATP, while ADP was a uncompetitive inhibitor with regard to adenosine and a non‐competitive inhibitor versus MgATP.Adenosine kinase was strongly inhibited by the bis(adenylyl) polyphosphates Ap4A and Ap5A. These compounds inhibited the enzyme competitively versus MgATP (Ki= 0.06 μM for Ap4A and 0.4 μM for Ap5A) and uncompetitively with regard to adenosine. The results of the kinetic analysis suggest an ordered bi‐bi mechanism, adenosine being the first substrate. The phosphorylation of adenosine was unaffected in the presence of vanadate ions.
Role of carbohydrates within variant surface glycoprotein of Trypanosoma congolense
Authors: REINWALD Erwin 
Published: March 2005

The effects of tunicamycin on different aspects of structure and biosynthesis of variant surface glycoprotein from Trypanosoma congolense have been studied.

Deglycosylated variant antigen becomes synthesized in vitro, is transported through the cell, and is deposited on the cell surface in equivalent amounts compared to the glycosylated species.
In contrast to the glycosylated molecule only marginal amounts of high‐molecular‐mass fragments can be removed from the parasitic cell by externally added proteases in the case of tunicamycin‐treated cells.
Most of the material removed by proteases from the cell surface of tunicamycin‐treated cells has a molecular mass lower than 2 kDa.
Many additional proteolytic cleavage sites become accessible after removal of the glycan chains. There is no indication that in the deglycosylated molecule the same prefential protease‐sensitive site exists as is found in the glycosylated species.

These results suggest that glycosylation of variant surface glycoprotein could be important for the survival of the parasite within the host organism.
Demonstration of an Mg2+‐induced conformational change by photoaffinity labelling of the high‐affinity ATP‐binding site of (Na++ K+)‐ATPase with 8‐azido‐ATP
Authors: SCHEINER‐BOBIS Georgios , SCHONER Wilhelm 
Published: March 2005

8‐Azido‐ATP (8‐N3ATP) is a substrate of (Na++ K+)‐ATPase from pork kidney and photoinactivates it by binding to the Mr= 100 000 α‐subunit. The photoinactivation requires the presence of Mg2+ even though 8‐azido‐ATP is recognized by the high‐affinity ATP binding site (Kd= 3.1 μM). K+ ions protect the enzyme against photoinactivation as does excess ATP.
To see whether the Mg2+‐requirement of the photoinactivation is due to the action of free Mg2+ or to the existence of an Mg · 8‐azido‐ATP complex, the action of the stable Mg · ATP complex analogue, chromium · 8‐N3ATP (Cr · 8‐N3ATP), was studied. Cr · 8‐N3ATP photoinactivates (Na++ K+)‐ATPase in the absence of Mg2+, but the photoinactivation is enhanced by Mg2+, indicating that the formation of a Mg · ATP complex is an absolute requirement for photoinactivation. However, the interaction of Mg2+ with a low‐affinity site also enhances the photoinactivation. It is therefore concluded that interactions with MgATP and free Mg induce conformational changes in the purine subsite of the high‐affinity ATP binding site.
Controlled trypsinolysis of the [α‐32P]8‐N3ATP‐phostolabelled enzyme in the presence of K− results in the formation of an Mr= 56000 radioactive peptide, whereas trypsinolysis of a [γ‐32P]Cr · ATP‐labelled enzyme under identical conditions forms an Mr= 41000 radioactive peptide. Extensive trypsinolysis of the [α‐32P] 8‐N3ATP‐photolabelled α‐subunit leads to the formation of a radioactive peptide of Mr= 1800.
Modification of subunit b of the Fo complex from Escherichia coli ATP synthase by a hydrophobic maleimide and its effects on Fo functions
Authors: SCHNEIDER Erwin , ALTENDORF Karlheinz 
Published: March 2005

Purified F0 from Escherichia coli ATP synthase was labelled with N‐(7‐dimethylamino‐4‐methyl‐coumarinyl)‐maleimide (DACM), a hydrophobic reagent which forms a stable, strongly fluorescent adduct with SH groups. Sodium dodecyl sulfate gel electrophoresis clearly demonstrated that subunit b was exclusively labelled, most likely at Cys‐21, the only cysteine residue in E. coli F0. The amount of two molecules of DACM bound per F0, which was calculated from the absorption spectrum at 380 nm, is in full agreement with the postulated stoichiometry of two copies of subunit b/F0 complex. Thus the label provides a useful tool for simply detecting subunit b in protein chemical studies.DACM‐labelled F0 was incorporated into liposomes and assayed for H+ translocating activity and its capacity to bind purified F1. Whereas the initial rate of H+ uptake was inhibited about 40% the reconstitution of a dicyclohexylcarbodiimide‐sensitive F1 F0 ATPase activity was completely unaffected. In a second set of experiments we reconstituted an F0 complex from DACM‐labelled purified subunit b and an ac complex. In contrast to the results obtained with intact, DACM‐labelled F0, both H+ translocating activity and F1 binding capacity were greatly reduced. Our data indicate that cysteine‐21, probably together with other amino acids, is involved in maintaining a proper interaction of the hydrophobic N‐terminal region of subunit b with the ac complex. This interplay seems to be a prerequisite for at least the in vitro assembly of a functional F0 complex.
Schistosoma mansoni surface glycoproteins
Authors: PAYARES Gilberto , SIMPSON Andrew J. G. 
Published: March 2005

Surface glycoproteins from newly transformed schistosomula of Schistosoma mansoni have been identified by surface radioiodination and lectin‐affinity chromatography. From the glycoconjugates bound by the three lectins used, concanavalin A, peanut agglutinin and fucose‐binding protein, only in the concanavalin‐A‐bound fractions were glycoproteins identified. Changes in concanavalin‐A‐binding glycoproteins were detected after transformation and early maturation of the schistosomula.Some glycoproteins disappeared (Mr 38000, 29000 and 25000), some appeared independently of host molecules (Mr 19000), others only appeared after culture in human serum (Mr 45000).Two major glycoproteins of Mr 32000 and 16000 were detected on all stages examined. Within the total set of surface glycoproteins identified on 3‐h schistosomula only the strong Mr‐38000–32000 complex was found to be antigenic. Thus many major low‐molecular‐mass surface glycoproteins of the parasite are not recognised as antigens by immune animals. The separation of only the Mr‐38000–32000 antigens by concanavalin A affinity chromatography indicates the feasibility of using this method in conjunction with immunoaffinity columns to purity these molecules.
Heterogeneity in the metabolism of the arachidonoyl molecular species of glycerophospholipids of rabbit alveolar macrophages
Authors: NAKAGAWA Yasuhito , KURIHARA Koichi , SUGIURA Takayuki , WAKU Keizo 
Published: March 2005

The relative incorporation of [3H]arachidonic acid (20:4) into individual molecular species containing 20:4 at the 2 position (18:1 ‐ 20:4, 16:0 ‐ 20:4 and 18:0 ‐ 20:4 species) of diacyl and ether‐linked glycerophosphocholine, glycerophosphoethanolamine and glycerophosphoinositol of rabbit alveolar macrophages has been measured by reversed‐phase high‐performance liquid chromatography (HPLC).The rate of incorporation of [3H]20:4 into the molecular species of glycerophospholipids was greatly influenced by their structures. The reversed‐phase HPLC analysis allowed elucidation of the influence of structural differences, such as the nature of the polar head group, the fatty chain at the 1 position and the chemical form of the bond of the fatty chain attached at the 1 position on the uptake of [3H]20:4 by comparison of the specific radioactivities of arachidonoyl molecular species having the same structures, except that one of the three kinds of moiety was different. The specific radioactivities of the molecular species containing choline head groups were significantly higher than those containing ethanolamine and inositol moieties. The specific radioactivities of diacyl molecular species were considerably higher than those of ether‐linked molecular species. The nature of the fatty chain attached at the 1 position also influenced the uptake of [3H]20:4 into glycerophospholipids. The arachidonoyl molecular species containing 18:1 at the 1 position were preferentially labelled with [3H]20:4 as compared to the corresponding 16:0 ‐ 20:4 and 18:0 ‐ 20:4 species either of diacyl or ether‐linked glycerophospholipids.The present results suggest that the acyltransferase involved in the incorporation of 20:4 into glycerophospholipids has selectivity for the structures of glycerophospholipids and the order of selectivity of this enzyme for the arachidonoyl molecular species, deduced in the present experiments, was as follows: choline head group > ethanolamine and inositol groups, acyl bond > ether and vinyl ether bonds, 18:1 fatty chain > 16:0 and 18:0 fatty chains at the 1 position. Comparison of the metabolic activities of all major arachidonoyl molecular species of glycerophospholipids having a single structure is reported here for the first time.
Synthesis, intracellular processing and secretion of thrombospondin in human endothelial cells
Authors: VISCHER Peter , BEECK Hannelore , VOSS Bruno 
Published: March 2005

The biosynthesis of thrombospondin, a glycoprotein first described in platelets, has been studied in human endothelial cells. This glycoprotein has a molecular mass of 450 kDa. It is secreted and incorporated into the extracellular matrix of several cell types in culture. Pulse‐chase experiments with [3H]leucine were performed and the synthesis and secretion of the glycoprotein was studied by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results of these experiments show that the three subunits of thrombospondin are identical in molecular mass. During synthesis there is a small but significant increase in molecular mass within 20 min after pulse labeling. The early form of thrombospondin is sensitive to endoglucosaminidase H treatment, indicating that a transformation of the oligosaccharide structures from ‘high‐mannose’ to ‘complex’ structures takes place. Within 60 min after synthesis only the mature form of the glycoprotein is secreted into the medium. In the presence of tunicamycin, an inhibitor of N‐glycosylation, there is a reduction in molecular mass of the subunit from 165 kDa to 155 kDa.Pulse‐chase experiments in the presence of tunicamycin supported the conclusion that the carbohydrate part is processed during biosynthesis. Inhibition of glycosylation had a pronounced effect on the secretion of thrombospondin. The decreased occurrence of thrombospondin in the culture medium seemed to be due to a high intracellular degradation rate of unglycosylated thrombospondin. Characterization of the glycopeptide structures of thrombospondin metabolically labeled with [3H]mannose by Bio‐Gel P‐4 and concanavalin‐A–Sepharose column chromatography revealed that the oligosaccharide structures of the cellular and secreted forms of thrombospondin differ in their composition.
Structural and functional studies of hemoglobin Poissy a2β256 (D7) Gly → Arg and 86 (F2) Ala → Pro
Authors: LACOMBE Claire , CRAESCU Constantin T. , BLOUQUIT Yves , KISTER Jean , POYART Claude , DELANOE‐GARIN José , AROUS Nicole , BARDAKDJIAN Josiane , RIOU Jean , ROSA Jean , SCHAEFFER Corinne , GALACTEROS Frédéric 
Published: March 2005

Hemoglobin Poissy α2β256 (D7) Gly → Arg and 86 (F2) Ala Pro, is a new variant of the β chain with two substitutions within the second exon of the corresponding gene. The electrophoretic mobilities are identical to those of Hb Hamadan α2β256 (D7) Gly → Arg as is the fingerprint of the tryptic hydrolysate of the two abnormal β chains. The second substitution β86 Ala → Pro was detected by high‐pressure liquid chromatography.Hb Poissy has a threefold increase in oxygen affinity with low Hill coefficient and diminished Bohr effect, which are restored to normal upon addition of 2,3‐bisphosphoglycerate. Since the functional properties of Hb Hamadan (β56 Gly → Arg) have been described as normal, the abnormal function of Hb Poissy may be attributed to the β86 (F2) Ala → Pro substitution.Hb Poissy exhibits a mild instability and a greater reactivity of the thiol groups of the β93 (F9) Cys residues in the deoxy form than does Hb A. The oxidation rate of Hb Poissy is biphasic indicating a large inequivalence between the α and β hemes. Thereafter NMR studies demonstrated that the β86 Ala Pro substitution produces a displacement of the F helix closer to the heme plane and a large increase in the dynamic fluctuations of the tertiary structure on the proximal side of the β hemes. These results lead to the conclusion that the β86 Ala Pro susbstitution produces a destabilization of the F helix extending downwards to the FG corner and altering both the β hemes and the α1β2 contacts.
Enzymatic basis for the calcium‐induced decrease of membrane protein methyl esterification in intact erythrocytes
Authors: GALLETTI Patrizia , INGROSSO Diego , IARDINO Patrizia , MANNA Caterina , PONTONI Gabriele , ZAPPIA Vincenzo 
Published: March 2005

The effect of Ca2+ loading, induced by the ionophore A23187, on methyl esterification of membrane proteins (i.e. bands 2.1, 3, 4.1 and 4.5) has been investigated in intact human erythrocytes.When the cells were incubated with l‐[methyl‐3H]methionine, 40 μM CaCl2 and 10 μM A23187 induce a 50% inhibition of membrane protein methyl esterification. This effect is selectively due to the increased intracellular Ca2+ concentration, as it is antagonized by 10 μM EGTA, and other divalent cations such as Mn2+ do not exert any inhibition.In order to clarify the mechanism(s) of the reported inhibition, the various events involved in the methyl esterification process in vivo were analyzed.

l‐Methionine uptake as well as protein methylase II activity are not directly affected by altered intracellular Ca2+ concentrations.Conversely in the Ca2+‐loaded erythrocytes the conversion of [3H]methionine into [3H]AdoMet, catalyzed by AdoMet synthetase, decreases up to 25%.
When the undialyzed erythrocyte cytosolic fraction is assayed in vitro for AdoMet synthetase the activity of the enzyme from the CaCl2/A23187‐treated erythrocytes is significantly lower than the control, up to 5 μM ATP. This result suggests that in the Ca2+‐loaded erythrocytes the ATP intracellular concentration is significantly lowered.
The direct evaluation of ATP intracellular concentration, by HPLC, confirms a significant drop of ATP level, as a consequence of the Ca2+ loading.
The removal of Ca2+ from the cells quantitatively restores both the AdoMet synthesis and the methyl esterification levels.

The possible role of altered ATP intracellular concentrations as a regulatory factor in the AdoMet‐dependent reactions as well as in post‐translational protein methylation related to the ageing process is also discussed.
Laurate binding to human serum albumin
Authors: PEDERSEN Anders Overgaard , HUST Birthe , ANDERSEN Signe , NIELSEN Frede , BRODERSEN Rolf 
Published: March 2005

Multiple binding of laurate (n‐dodecanoate) to human serum albumin was studied by a kinetic dialysis method. With this method the concentration of unbound ligand can be determined by measuring the rate of exchange of radioactive label across a dialysis membrane under conditions of equilibirium.Determination of the rate constant of exchange, in the absence of albumin, revealed that laurate does not change its state of aggregation over a concentration range from 0.1 μM to 500 μM, indicating the prevalence of a monomer.Binding of laurate to albumin, at pH 7.5, 37°C, was investigated through 220 determinations of binding equilibria in the range of 0–10 mol laurate/mol albumin, corresponding to a concentration range of unbound laurate from 1 nM to 0.1 mM.Binding data were analyzed in terms of stepwise binding. Considering the stochastic errors of the experimental data, we generated a variety of possible (equal goodness of fit) solutions to the stoichiometric binding equation. This analysis resulted in reasonably well‐defined values for the first two step constants, with an indication of negative interaction. The variation of the higher step constants excluded any mechanistic conclusions, although the binding isotherms, defined by these varying sets of binding constants, fitted the experimental data well within a chosen probability limit.
Leukotriene C4 metabolism by hepatoma cells deficient in the uptake of cysteinyl leukotrienes
Authors: WECKBECKER Gisbert , KEPPLER Dietrich O. R. 
Published: March 2005

Uptake and metabolism of the cysteinyl leukotrienes C4 and E4 (LTC4 and LTE4) were studied in AS‐30D hepatoma cell suspensions and compared with rat hepatocytes. The hepatoma cells were deficient in the uptake of [3H]LTC4 and [3H]LTE4 but took up, in control experiments, l‐[14C]glutamine and [14C]adenosine in a time‐dependent manner. By contrast, isolated hepatocyte suspensions incubated under the same conditions took up [3H]LTC4 and [3H]LTE4 as well as l‐[14C]glutamine and [14C]adenosine.The hepatoma cells deficient in the uptake of cysteinyl leukotrienes metabolized extracellular [3H]LTC4 to [3H]LTD4 and to [3H]LTE4. Addition of acivicin, an inhibitor of γ‐glutamyltransferase, largely prevented metabolism of [3H]LTC4 by the hepatoma cells. Sonication of the cells did not enhance the formation of [3H]LTD4 and [3H]LTE4 from [3H]LTC4.We conclude that ectoenzymes of AS‐30D hepatoma cells catalyze the conversion of LTC4 to LTE4 via LTD4. As compared to hepatocytes, these neoplastic cells have lost the uptake system for cysteinyl leukotrienes and may serve in studies on leukotriene metabolism by cell‐surface enzymes.
Proteins from the prokaryotic nucleoid
Authors: LOSSO M. Adele , PAWLIK Roman T. , CANONACO Maria A. , GUALERZI Claudio O. 
Published: March 2005

Escherichia coli DNA‐binding proteins NS1, NS2 and NS (NS1 + NS2) react with the protein‐protein bifunctinol cross‐linking reagents dimethylsuberimidate and dimethyladipimidate to yield oligomers up to hexamers. The former reagent, with the longer arm, is more efficient than the other shorter one. Both one‐ and two‐dimensional gel electrophorses show that the cross‐linked trimers are homogeneous, while the dimers appear heterogeneous, suggesting that at least two types of dimers but geometrically equivalent trimers are formed. In the presence of DNA, the cross‐linking reaction with either reagent yields fewer dimers and more of the larger products.The yield of cross‐linked products of various sizes was determined for NS1, NS2 and NS as a function of the protein concentration (0.03–3000 μM), From the results obtained in these experiments, we derived a model of quaternary structure in which dimers and tetramers are predominant in very dilute solutions of the proteins. Above a critical concentration (10–50 μM), interactions among tetramers become increasingly important, yielding octamers and perhaps larger products. Our data do not support a recently proposed model in which the DNA is packaged around a protein disc consisting of 8–10 NS dimers.