bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine‐biosynthetic protein CAD
Authors: CARREY Elizabeth A. , HARDIE D. Grahame 
Published: March 2005

We have examined the domain organization, and the locations of the sites phosphorylated by the cyclic‐AMP‐dependent protein kinase, in the multifunctional polypeptide of the pyrimidine‐biosynthetic protein, CAD. Fragments produced after limited proteolysis by elastase or trypsin were separated by SDS/polyacrylamide gel electrophoresis and transferred onto nitrocellulose. The blots were probed with antibodies raised against the core aspartate carbamoyltransferase (ACTase) and dihydroorotase (DHOase) fragments to locate fragments containing these domains, and we also examined the locations of the phosphorylation sites by complete tryptic digestion of blotted, 32P‐labelled fragments, followed by analytical isoelectric focussing.Our results are consistent with the domain order glutaminase(GLNase)‐carbamoyl‐phosphate synthetase‐(CPSase)‐DHOase‐ACTase, as suggested by recently reported homologies between the predicted amino acid sequence for the Drosophila rudimentary gene product, and monofunctional CPSases/ACTases/DHOases. In particular, the finding of a 95‐kDa elastase fragment which cross‐reacted with both anti‐DHOase and anti‐ACTase antibodies rules out the previously suggested domain order: DHOase‐GLNase‐CPSase‐ACTase.Phosphorylation by cyclic‐AMP‐dependent protein kinase accelerates cleavage of native CAD by both elastase and trypsin, and abolishes the protective effect of UTP. Site 1 is located close to the C‐terminal end of the 160‐kDa GLNase/CPSase region. Comparison with the predicted amino acid sequence of the Drosophila rudimentary gene revealed a strong homology between the tryptic peptide containing site 1 from hamster CAD, and a region at the extreme C‐terminal end of the CPSase II domain of the Drosophila enzyme. Alignment of the Drosophila sequence and that of rat liver CPSase I, which is not phosphorylated by cyclic‐AMP‐dependent protein kinase, revealed that this putative site 1 region is missing in CPSase I.Site 2 could not be located with certainty, either from the limited proteolysis data, or from comparison of the sequence around this site and the sequence of the rudimentary gene. There were also one or more previously undetected minor phosphorylation site(s) located in the protease‐sensitive hinge region between the DHOase and ACTase domains.
Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6‐bisphosphate in yeast
Authors: FRANÇOIS Jean , VAN SCHAFTINGEN Emile , HERS Henri‐Géry 
Published: March 2005

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500‐fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP‐agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [γ‐32P]ATP‐Mg and the catalytic subunit of cyclic‐AMP‐dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105‐kDa peptide and also, but only faintly, into a 162‐kDa subunit.A low‐Km, (0.1 μM) fructose‐2,6‐bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6‐[2‐32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300‐fold, had an apparent molecular mass of 110 kDa and was made of two 56‐kDa subunits. It was inhibited by fructose 6‐phosphate (Ki= 5 μM) and stimulated 2–3‐fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low‐Km fructose‐2,6‐bisphosphatase clearly separated from each other in all purification procedures used. A high‐Km (∼ 100 μM), apparently specific, fructose 2,6‐bisphosphatase was separated by anion‐exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6‐bisphosphate, which it converts to fructose 6‐phosphate and Pi, because it is not inhibited by fructose 6‐phosphate, glucose 6‐phosphate or Pi.Several other phosphatases able to hydrolyze fructose 2,6‐bisphosphate into a mixture of fructose 2‐phosphate, fructose 6‐phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6‐bisphosphate (Km, > 50 μM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.
Purification, properties and cation activation of galactosyltransferase from lactating‐rat mammary Golgi membranes
Authors: NAVARATNAM Naveenan , WARD Simon , FISHER Cynthia , KUHN Nicholas J. , KEEN Jeffery N. , FINDLAY John B. C. 
Published: March 2005

Galactosyltransferase was purified from Golgi membranes of lactating‐rat mammary gland and studied with respect to its physical and enzymic (lactose synthetase) properties.The enzyme occurred in both monomeric (43–46 kDa) and apparently dimeric (90 kDa) forms. It was very unstable except in the presence of phospholipid, detergent, or cations binding to site 2. The amino acid composition and the N‐terminal sequence closely resembled that of the human and bovine milk enzymes, particularly in respect to a Pro‐Pro‐Pro‐Pro sequence.Kinetic studies demonstrated a high‐affinity Mn2+‐binding site (1) essential for activity, and a low‐affinity Mn2+‐binding site (2) that could also bind spermidine or clupeine. Mn2+ binding at site 2 raised Vmax fivefold. Spermidine binding at site 2 enhanced Mn2+ binding at site 1, and influenced binding of glucose. At physiological glucose concentration, clupeine or spermidine activated nearly as well as 15 mM MnCl2 and are regarded as models of a natural cation activator that remains to be isolated. Evidence is given for an essential histidine residue in the galactosyltransferase.It is proposed that site 1 Mn2+ participates directly in the reaction mechanism, whereas site 2 is a regulator site allosterically activated by a basic protein.
Primary structure of neutral oligosaccharides derived from respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis, determined by combination of 500‐MHz 1H‐NMR spectroscopy and quantitative sugar analysis
Authors: KLEIN André , LAMBLIN Geneviève , LHERMITTE Michel , ROUSSEL Philippe , BREG Jan , VAN HALBEEK Herman , VLIEGENTHART Johannes F. G. 
Published: March 2005

Carbohydrate chains of respiratory‐mucus glycopeptides from a patient (blood group O) suffering from bronchiectasis with a Kartagener's syndrome have been released by alkaline borohydride treatment. Application of high‐performance liquid chromatography using subsequently two silica columns, one bonded with aminopropyl groups and the other with octadecyl groups, afforded 39 neutral fractions; 35 oligosaccharide‐alditol structures have been characterized by employing 500‐MHz 1H‐NMR spectroscopy in conjunction with sugar analysis.Here, 16 oligosaccharide structures, possessing a core consisting of Galβ(1→3)GalNAc‐ol with or without branching through a GlcNAc residue linked β(1→6) to the GalNAc residue (core type 2 or core type 1, respectively), are described. Ten oligosaccharide‐alditols with these types of cores (2, 3, 10a, 14, 7, 11a, 15a, 16a, 12 and 16c) have been identified previously in human bronchial mucins of patients suffering from cystic fibrosis [Lamblin, G., Boersma, A., Lhermitte, M., Roussel, P., Mutsaers, J. H. G. M., Van Halbeek, H. and Vliegenthart, J. F. G. (1984) Eur. J. Biochem. 143, 227–236].Of the remaining six compounds, one is a partial structure of oligosaccharides previously described:
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The structures 17a and 20 contain the Y determinant, i.e., Fucα(1→2)Galβ(1→4)[Fucα(1→3)]GlcNAcβ(1→). High‐resolution 1H‐NMR spectroscopy is able to distinguish whether the Y determinant is β(1→3) or β(1→6) linked in such oligosaccharide‐alditols.
Electrostatic interactions in the cellular dynamics of the interferon‐receptor complex
Authors: UZE Gilles , BANDU Marie‐Thérèse , EID Pierre , GRÜTTER Markus , MOGENSEN Knud E. 
Published: March 2005

Using membrane preparations of the interferon receptor, prepared from cells of the Burkitt line, Daudi, we have examined the binding of three human recombinant α‐interferons.

We discovered a binding titration of the interferons IFN‐αA and IFN‐αD in the pH range 6–9. Receptor binding, negligible at pH 6, rises to a maximum close to pH 9. We have shown that binding of IFN‐αA at basic pH is to the same receptors as at neutrality and that IFN‐receptor complexes extracted with digitonin are more stable at basic pH than they are at neutrality.
The recombinant interferon, IFN‐αB, shows little change of binding in the pH range 6–9. At its basic optimum the binding of IFN‐αA approaches that of IFN‐αB, while at neutral pH the binding of IFN‐αA is 3–4 times less. This difference at neutral pH is seen on intact cells as well as on membrane preparations. The specific activity of IFN‐αB is close to that of IFN‐αA, both of which are 10–20 times more active than IFN‐αD; and the binding titration is, therefore, independent of the initial binding affinities.
Using hybrid IFNs constructed from the DNA sequences of αD and αB, we have isolated the sequence responsible for the binding titration to the segment comprising amino acids 61–92. Examination of these sequences reveals that Lys‐84 is present in all the IFN‐α except IFN‐αB where it is replaced by Glu; and Tyr‐90, present in most of the common IFN‐α including αA and αD, is replaced by Asp in IFN‐αB. Lys and Tyr would normally titrate in the pH range 6–9. We conclude that the binding titration is due to an electrostatic interaction and we propose that the interaction is between IFN‐receptor complexes. The role of the interaction in the binding losses that accompany the antiproliferative effects of IFN is discussed.
Specific inhibition of simian virus 40 protein synthesis by heat and arsenite treatment
Authors: ANGELIDIS Charalampos E. , LAZARIDIS Ioannis , PAGOULATOS Gerassimos N. 
Published: March 2005

The effects of heat treatment of CV1 cells infected with simian virus 40 (SV40) on viral and cellular protein synthesis were investigated by one‐dimensional and two‐dimensional polyacrylamide gel electrophoresis.A 12‐h heat treatment during the late phase of the viral life‐cycle inhibits VP1 synthesis. No inhibition of normal cellular proteins is apparent, but heat‐shock proteins are strongly induced and accumulate in the cells. Inhibition of VP1 synthesis in infected cells is demonstrated to occur also after arsenite treatment, another agent known to induce heat‐shock proteins. Northern blot analysis of cytoplasmic RNA demonstrated a decrease in the abundance of late SV40 mRNAs thus showing that the inhibition occurs at the transcriptional or immediately post‐transcriptional level. Cumulative labeling with [3H]thymidine of viral DNA showed that the decrease in the abundance of late mRNAs is not due to a blocking of viral DNA synthesis. Immunofluorescence microscopy and immunoprecipitation analysis show that heat and arsenite treatments also affect the synthesis of T antigen.These results suggest that heat‐shock proteins may play a role in the inhibition of SV40 virus gene functions.
Rat liver alcohol dehydrogenase of class III
Authors: JULIÀ Pere , PARÉS Xavier , JÖRNVALL Hans 
Published: March 2005

The amino acid sequence of alcohol dehydrogenase of class III from rat liver (the enzyme ADH‐2) has been determined. This type of structure is quite different from those of both the class I and the class II alcohol dehydrogenases. The rat class III structure differs from the rat and human class I structures by 133–138 residues (exact value depending on species and isozyme type); and from that of human class II by 132 residues. In contrast, the rat/human species difference within the class III enzymes is only 21 residues. The protein was carboxymethylated with iodo[214C]acetate, and cleaved with CNBr and proteolytic enzymes. Peptides purified by exclusion chromatography and reverse‐phase high‐performance liquid chromatography were analyzed by degradation with a gas‐phase sequencer and with the manual 4‐N,N‐dimethylaminoazobenzene‐4′‐isothiocyanate double‐coupling method.The protein chain has 373 residues with a blocked N terminus. No evidence was obtained for heterogeneity. The rat ADH‐2 enzyme of class III contains an insertion of Cys at position 60 in relation to the class I enzymes, while the latter alcohol dehydrogenase in rat (ADH‐3) has another Cys insertion (at position 111) relative to ADH‐2. The structure deduced explains the characteristic differences of the class III alcohol dehydrogenase in relation to the other classes of alcohol dehydrogenase, including a high absorbance, an anodic electrophoretic mobility and special kinetic properties. The main amino acid substitutions are found in the catalytic domain and in the subunit interacting segments of the coenzyme‐binding domain, the latter explaining the lack of hybrid dimers between subunits of different classes. Several substitutions provide an enlarged and more hydrophilic substrate‐binding pocket, which appears compatible with a higher water content in the pocket and hence could possibly explain the higher Km for all substrates as compared with the corresponding values for the class I enzymes. Finally the class III structure supports evolutionary relationships suggesting that the three classes constitute clearly separate enzymes within the group of mammalian zinc‐containing alcohol dehydrogenases.
Purification of bovine liver cytosolic 5′‐nucleotidase
Authors: ZEKRI Mustapha , HARB Jean , BERNARD Serge , MEFLAH Khaled 
Published: March 2005

Cytosolic 5′‐nucleotidase from bovine liver has been purified to homogeneity. Two affinity chromatographies on concanavalin A and 5'AMP‐Sepharose columns result in a 12000‐fold purification. The sequential elution of glycoproteins from the concanavalin‐A – Sepharose column with methyl α‐D‐glucoside and methyl α‐D‐mannoside greatly increases the degree of purification of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows two subunits having apparent molecular masses of 65 kDa and 57 kDa respectively, while only one band at 70 kDa is observed in the case of the membrane‐bound 5′‐nucleotidase. Both the Stokes radii, measured by gel exclusion HPLC, and the sedimentation coefficient, determined by density gradient ultracentrifugation, indicate that the cytosolic enzyme is a heterodimer of about 130 kDa. This contrasts with the membrane‐bound 5′‐nucleotidase which is a homodimer of 140 kDa. Moreover, the antibodies raised against the membrane 5′‐nucleotidase inhibited the cytosolic form indicating that a common antigenic determinant(s) exists between the two isoenzymes. However, structural differences are revealed by immunoblotting. In the same way, the effect of lectins suggests that differences in the structure of the carbohydrate chains exist between the two isoenzymes.The purified cytosolic enzyme has lower affinity for the nucleotides than does the membrane enzyme. In addition, while ADP, [α,β‐CH2]ADP and ATP were strong competitive inhibitors of the membrane enzyme, ADP and ATP activate the cytosolic form and [α,β‐CH2]ADP has no effect. Moreover, two pH optima at 7.5 and 9.5 are observed in the cytosolic enzyme while only one at 7.5 occurred in the membrane form. Finally the exogenous cations, MgCl2 and MnCl2, are necessary for the maximal activity of the cytosolic but not of the membrane 5′‐nucleotidase. All these observations indicate that the two isoenzymes are different.
Characterization of cDNA clones for human glycophorin A
Authors: RAHUEL Cécile , LONDON Jacqueline , d'AURIOL Luc , MATTEI Marie‐Geneviève , TOURNAMILLE Christophe , SKRZYNIA Cécile , LEBOUC Yves , GALIBERT Francis , CARTRON Jean‐Pierre 
Published: March 2005

Glycophorin A is the major membrane sialoglycoprotein of human erythrocytes and represents a typical example of a transmembrane glycoprotein. The functional role of this cell‐surface component is not known but it represents a receptor for viruses, bacteria and parasites like Plasmodium falciparum.

Two cDNA clones encoding glycophorin A have been characterized from human fetal cDNA libraries. The longer cDNA extended from the coding region of glycophorin A (residues 4–131) to the 3′ untranslated region which included two polyadenylation signals and a poly(A) tail.
The structural gene for glycophorin A is located on chromosome 4, q28–q31 as shown by in situ hybridization, thus confirming the previous localization by genetic linkage analysis.
Three distinct mRNA species (1.0 kb, 1.7 kb and 2.2 kb) have been identified in erythroid spleen. Northern blot analyses with a probe directed against the 3′ untranslated region of the mRNAs indicated that all these species share a homologous 3′ non‐coding region and that the first polyadenylation signal downstream the stop codon is not used.
Preliminary studies by Southern blot analysis of the genomic DNA from normal En(a+) and rare En(a‐) donors suggest that the glycophorin A gene has a complex organization and is largely deleted in donors of the En(a‐) phenotype (Finnish type) who lack glycophorin A on their red cells.
Interaction of a spin‐labelled cholesterol derivative with the cytochrome P‐450SCC active site
Authors: LANGE Reinhard , MAURIN Luc , LARROQUE Christian , BIENVENÜE Alain 
Published: March 2005

The cholesterol analogue 25‐doxyl‐27‐nor‐cholesterol (CNO), was found to be a substrate for cytochrome P‐450scc. Upon incubation with the cytochrome P‐450scc electron transfer system, CNO is transformed to pregnenolone (Km= 33 μM, Vmax= 0.32 min−1). The pregnenolone formation from endogenous cholesterol is strongly inhibited by CNO (50% at 5 μM). It binds tightly to cytochrome P‐450scc as evidenced by a reversed type I spectral absorbance change (Kd= 5.9 μM) which is paralleled by a greater hyperfine splitting of the room‐temperature CNO ESR spectrum due to an enhanced probe immobilization (Kd= 1.9 μM). This finding is in accord with a rotational correlation time of about 10−7 s, which is close to the tumbling rate of the protein. At 110 K the CNO‐bound cytochrome P‐450ssc displays the ESR g‐values gx= 2.404/2.456, g,y= 2.245 and gz= 1.916; these are different from those of cholesterol‐liganded cytochrome P‐450scc and may thus serve as a marker for cytochrome P‐450ssc.Our data indicate that the stereospecificity of the cytochrome P‐450scc, side‐chain‐cleaving activity is not dependent on the nature of the cholesterol side‐chain termination (C25 to C27). The substrate binding site is however rather sensitive to a modification of the side chain. The doxyl ring confers a stronger affinity of the substrate to the enzyme. Upon binding it becomes embedded in the protein matrix, and we estimate that its final position is 0.6 – 1.0 nm from the heme moiety.
Purified subunit δ of chloroplast coupling factor CF1 reconstitutes photophosphorylation in partially CF1‐depleted membranes
Authors: ENGELBRECHT Siegfried , JUNGE Wolfgang 
Published: March 2005

The ATP synthase of chloroplasts consists of the proton channel, CF0, and the catalytic part, CF1, which carries nucleotide‐binding sites on subunits α and β. The still poorly understood interaction between CF0 and the catalytic sites on CF1 is mediated by the smaller subunits γ, δ and ɛ of CF1. We investigated the ability of purified δ to block proton leakage through CF0 channels after their exposure by removal of the CF1 counterpart.Thylakoids were partially depleted of CF1 by EDTA treatment. This increased their proton permeability and thereby reduced the rate of photophosphorylation. Subunit δ was isolated and purified by FPLC [Engelbrecht, S. and Junge, W. (1987) FEBS Lett. 219, 321–325]. Addition of δ to EDTA‐treated thylakoids reconstituted high rates of phenazine‐methosulfate‐mediated photophosphorylation. Since δ does not interact with nucleotides by itself, the reconstitution was due to a reduction of the proton leakage through open CF0, channels. The molar ratio of purified δ over exposed CF0, which started to elicit this effect, was 3:1. However, if δ was added together with purified CF1 lacking δ, in a 1:1 molar ratio, the relative amount over exposed CF0 was as low as 0. 06. This corroborated our previous conclusion [Lill, H., Engelbrecht, S., Schönknecht, G. and Junge, W. (1986) Eur. J. Biochem. 160, 627–634] that only a very small fraction of exposed CF0 was actually proton‐conducting but with a very high unit conductance. CF1 including δ was apparently rebound preferentially to open CF0 channels.Although the ability of δ to control proton conduction through CF0 was evident, it remains to be established whether δ acts as a gated proton valve or as a conformational transducer in the integral CF0CF1 ATPase.
Sulfite oxidase from chicken liver
Authors: RITZMANN Martin , BOSSHARD Hans Rudolf 
Published: March 2005

The mitochondrial enzyme sulfite oxidase catalyzes the oxidation of cytochrome c by sulfite. The reaction is inhibited when the enzyme is treated with N‐cyclohexyl‐N′‐[2‐(N‐methylmorpholino)‐ethyl]carbodiimide p‐toluenesulfonate (CMC). Inhibition follows the conversion of two carboxyl groups to N‐acylurea derivatives. The two groups are about equally reactive toward this inhibitor and blocking of either group abolishes electron transfer to cytochrome c.The rate of inactivation is almost the same in the presence of cytochrome c and under conditions where, on average, 89% of the enzyme is bound to cytochrome c. Therefore, the functional groups are not likely to be at the cytochrome c binding site. There are two equal and non‐interacting cytochrome c binding sites per sulfite oxidase monomer. The Kd is 7.5 μM at pH 6.0 and low ionic strength.The data are difficult to reconcile with binding of cytochrome c to a cluster of acidic residues in the area of the heme b prosthetic group, as was envisaged for the cytochrome‐bs–cytochrome c complex [Salemme, F. R. (1976) J. Mol. Biol. 102, 563–568].An improved method for the purification of sulfite oxidase from chicken liver, using affinity chromatography on cytochrome c–Sepharose, is described.
Characterization of the protein which binds insulin‐like growth factor in human serum
Authors: RAPP Reinhard , DEGER Arno , BLUM Werner , KOCH Rüdiger , WEBER Ulrich 
Published: March 2005

The binding of the 125I‐labelled insulin‐like growth factors I and II (l25I‐IGF I and 125I‐IGF II) to the high‐molecular‐mass binding protein of human serum was characterized. With diluted human serum both growth factors showed optimal specific binding at 4°C and pH 5–6. When 0.1% Triton X‐100 was present in the incubation buffer an increase in the affinity of the IGF‐binding protein was induced, which produced an enhanced binding of IGF I and IGF II. Competition experiments with various peptide hormones revealed that the native IGF‐binding protein complex binds both the IGF I and IGF II with high specificity. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF I and IGF II respectively, indicating that in human serum only a single class of non‐interacting binding sites is present. At optimal binding conditions the dissociation constants were determined to be 0.28 × 10−9 M for IGF I binding and 0.66 × 10−9 M for IGF II.Human serum was gel‐filtered on Sepharose CL‐6B at neutral pH and the eluate was assayed for binding activity with both IGF I and IGF II. One peak with an apparent molecular mass of 175 kDa and a Stokes radius of 4.8 nm was determined for both growth factors. Thus, our data suggest that human serum contains one class of high‐molecular‐mass binding protein with comparable binding characteristics for IGF I and IGF II.