bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunochemical Determination of Human Cholesterol 7α‐Hydroxylase
Authors: Maeda Yo‐rio , Eggertsen Gösta , Nyberg Björn , Setoguchi Toshiaki , Okuda Kyu‐Ichiro , Einarsson Kurt , Björkhem Ingemar 
Published: March 2005

To produce antibodies for determination of the protein mass of human cholesterol 7α‐hydroxylase, a fusion protein was prepared from an in‐frame fusion gene containing the cDNA for human cholesterol 7α‐hydroxylase near the 3′ terminus of the lacZ gene of Escherichia coli. The fusion protein was purified by (NH4)2SO4 fractionation and gel‐filtration chromatography on a Sephacryl column. Rabbits were immunized with this fusion protein and antisera were obtained.IgG was prepared by submitting antisera to chromatography on protein‐A‐Sepharose. Antibodies directed against bacterial proteins including β‐galactosidase were removed by affinity chromatography on a column to which bacterial proteins of E. coli containing β‐galactosidase had been immobilized. Evidence that the antibodies are indeed reactive against human liver cholesterol 7α‐hydroxylase was obtained by immunoblot analysis with human cholesterol 7α‐hydroxylase expressed in COS cells from the coding region of the human cholesterol 7α‐hydroxylase cDNA.The antiserum inhibited the activity of cholesterol 7α‐hydroxylase in human liver microsomes by approximately 70%. On immunoblotting of solubilized human liver microsomes, a positive band was obtained at a position corresponding to the protein mass for human cholesterol 7α‐hydroxylase. When calibration was performed using the fusion protein, a linear relationship was observed between the density and the amount of protein. Proportionality was also observed between the density and the amount of protein for microsomes of COS cells transfected with the coding region of the human cholesterol 7α‐hydroxylase cDNA.Liver microsomes from patients treated with cholestyramine (n= 3) were shown to contain levels of cholesterol 7α‐hydroxylase protein approximately twofold higher than those of liver microsomes from untreated patients (n= 6; P < 0.02), whereas cholesterol 7α‐hydroxylase activity was approximately sixfold higher in liver microsomes from the cholestyramine‐treated patients than in the corresponding preparations from the untreated patients (P <0.02). The higher activities observed in cholestyramine‐treated patients, therefore, cannot be explained only by an increased amount of protein, suggesting a posttransla‐tional mechanism to increase the activity of human cholesterol 7α‐hydroxylase in addition to the transcrip‐tional control.
Evidence for a Catalytic Role of Tyrosine 383 in the Peptidase Reaction of Leukotriene A4 Hydrolase
Authors: Blomster Martina , Wetterholm Anders , Mueller Martin J. , Haeggström Jesper Z. 
Published: March 2005

Leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme which catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B4 and which also possesses a peptidase activity. From sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA4 hydrolase has been suggested as a possible catalytic amino acid. To explore the potential role of this amino acid in catalysis, we replaced the tyrosine residue with phenylalanine, histidine or glutamine residues by site‐directed mutagenesis. The mutated cDNAs were expressed in Escherichia coli and the resulting recombinant proteins, named [Y383F]LTA4 hydrolase, [Y383H]LTA4 hydrolase and [Y383Q]LTA4 hydrolase, were purified to homogeneity to allow assays of both the epoxide hydrolase activity, i.e. the conversion of LTA4 into leukotriene B4, and the peptidase activity.None of the mutated proteins exhibited significant peptidase activities, all of them showing activities less than 0.3% that of the wild‐type enzyme. The epoxide hydrolase activity was not affected to the same degree and corresponded to 11, 16 and 17% that of the unmutated enzyme for [Y383F]LTA4 hydrolase, [Y383H]LTA4 hydrolase and [Y383Q]LTA4 hydrolase, respectively. Kinetic analysis was performed with the mutant [Y383Q]LTA4 hydrolase, which revealed an approximately 10‐fold increase in Km for leukotriene A4 compared to that for the unmutated enzyme. At high concentrations of substrate, the difference in enzyme velocity was only moderate, with Vmax values of 600 nmol · mg−1· min−1 and 1000 nmol · mg−1· min−1 for [Y383Q]LTA4 hydrolase and the wild‐type enzyme, respectively. No such effect of substrate concentration could be observed on the peptidase activity. As a positive control, we exchanged a glycine residue in position 386 for an alanine residue, and the recombinant protein, [G386A]LTA4 hydrolase retained 19% and 77% of the peptidase and epoxide hydrolase activities, respectively. The results from this study are consistent with a role for Tyr383 in the peptidase reaction of LTA4 hydrolase, where it may act as a proton donor in a general base mechanism. However, our data do not allow a similar interpretation for the mechanism involved in the hydrolysis of LTA4 into LTB4.
Phosphatidylinositol Hydrolysis and an Increase in Ca2+Concentration in the Signal‐Transduction Process Triggered by Murine FcγRIII are not Required for Protein Kinase C Translocation
Authors: Altrichter Jens , Guse Andreas H. , Resch Klaus , Brock Josef , Daëron Marc , Hückel Christian 
Published: March 2005

Murine class III receptors for IgG (mFcγRIII) are composed of an IgG‐binding α chain associated with a γ subunit dimer. These receptors have been shown to trigger the release of serotonin and tumor necrosis factor‐α [Daëron, M., Latour, S., Hückel, C., Bonnerot, C. & Fridman, W. H. (1992)Immunobiology 185, 159–174], and are involved in endocytosis and phagocytosis [Daëron, M., Malbec, O., Bonnerot, C., Latour, S., Segal, D. M. & Fridman, W. H. (1994)J. Immunol. 152, 783–792]. Using a transfection model where the cDNA encoding mFcγRIII was stably transfected into the rat basophilic leukemia cell line RBL‐2H3, we found that the functional efficiency of mFcγRIII is correlated with its ability to increase the intracellular Ca2+concentration and to stimulate inositol phosphate metabolism.The deletion of intracellular sequences of the α subunit did not alter the ability of mFcγRIII to trigger the Ca2+and phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] response. After substitution of the intracellular domain of mFcγRII for that of mFcγRIIIγ, but not that of mFcγRIIIα, the chimeric receptor was also able to trigger Ca2+and PtdIns(4,5)P2responses.In contrast, all transfected receptors induced protein kinase C translocation. Furthermore, dimerization of the receptor was sufficient for the initiation of this protein kinase C translocation while a further cross‐linking was necessary for the induction of the Ca2+and PtdIns(4,5)P2responses.Protein kinase C translocation therefore can be dissociated from Ca2+mobilization, PtdIns(4,5)P2turnover and mast cell secretory responses induced by murine FcγRIII.
Cloning, Expression and Characterization of Two Putative Calcium‐binding Sites in Human Non‐Erythroid α‐Spectrin
Authors: Lundberg Susanne , Björk Johanna , Löfvenberg Lars , Backman Lars 
Published: March 2005

The C‐terminus of α‐spectrins contains two putative calcium‐binding sites or EF‐hands. To characterize the binding, we have isolated clones from a human fetal liver cDNA library and expressed several fragments comprising either one or both of these sites. When the isolated clones were sequenced, we found that three consecutive nucleotides differed compared to the published sequence. The discrepancy affected two codons in the first of the two putative calcium sites. These codons translated into glutamate and phenylalanine, which are identical to the residues present at the same position in other α‐spectrins.In the presence of magnesium, only recombinant peptides comprising the second putative site bound calcium as determined by a calcium overlay assay. Although the first putative EF‐hand appeared to bind some calcium in the absence of magnesium, no binding could be detected under stringent conditions. Therefore, it is likely that the second EF‐hand constitutes the only functional calcium‐binding site in the C‐terminus of human non‐erythroid α‐spectrin. Since peptides comprising the second EF‐hand bound calcium nearly as well as intact spectrin, it is also apparent that the second EF‐hand constitutes the major binding site for calcium in spectrin. The relative change in negative ellipticity, induced by the binding of calcium, indicates a dissociation constant of approximately 120 μM.
Characterization of Monomeric 4‐Aminobutyrate Aminotransferase at Low pH
Authors: Pineda Teresa , Osei Yaa Difie , Churchich Jorge E. 
Published: March 2005

4‐Aminobutyrate aminotransferase undergoes a reversible process of association/dissociation at low pH. At pH 5.0, monomeric species exist predominantly in solution as revealed by FPLC and time‐dependent emission anisotropy measurements. The observed rotational correlation time at pH 5.0, фobs= 25 ns, corresponds to a compact spherical unit of 52 kDa. An increase in the net charge of the macromolecule at pH 5.0 is responsible for destabilization of the dimeric structure, (WEL≈ 41.84 kJ/mol), but the dissociation of the protein does not perturb the secondary structure as revealed by CD measurements.The fluorescent probe 1‐anilinonaphthalene‐8‐sulfonate (ANS), bound to hydrophobic sites of the enzyme, was used to monitor the kinetics of protein dissociation by stopped‐flow spectroscopy. The dissociation of the dimeric structure at pH 5.0 was characterized by a relaxation time of 18 ms. The rate of association of monomeric subunits at pH 7.0 was too fast to be detected in the stopped‐flow instrument. These observations have some bearing on the mechanism of reconstitution of dimeric structures of 4‐aminobutyrate aminotransferase in the cell.
cDNA Cloning and Tissue‐specific Regulation of Expression of Rat Calcium‐binding Protein 65/67
Authors: Fan Hua , Josić Djuro , Lim Yow‐Pin , Reutter Werner 
Published: March 2005

We isolated a cDNA encoding the rat membrane‐associated 65/67‐kDa calcium‐binding protein, CBP 65/67, from a λ ZAP II cDNA‐expression library of rat liver by immunoscreening using monospecific polyclonal anti‐(CBP 65/67) antibodies and monoclonal anti‐(CBP 65/67) IgG. The product of this cDNA expressed in Escherichia coli was confirmed as CBP 65/67 both by immunostaining and by comparison of the molecular mass with the CBP 65/67 isolated from rat liver by SDS/PAGE. The cDNA sequence and the deduced amino acid sequence of CBP 65/67 both show a high degree of identity to human p68 and human calelectrin, which belong to a family of calcium‐dependent, membrane‐associated, phospholipid‐binding proteins, called annexins. This means that CBP 65/67 is a homolog of the two human proteins just mentioned above. We are not aware that a rat annexin VI has previously been isolated and sequenced.The mRNA expression of CBP 65/67 in different rat organs during development was investigated by Northern blot analysis. In adult tissues, high mRNA levels of CBP 65/67 were found in lung, heart, muscle, spleen and especially in thymus and pancreas, whereas in liver, kidney, intestine, stomach and brain only low levels of CBP 65/67 mRNA could be detected. The amount of mRNA during tissue development in kidney, stomach and muscle showed only slight changes. In contrast, a significant increase of CBP 65/67 expression was observed in liver, lung, heart and brain. In most of the organs investigated, the level of mRNA correlated closely with the level of protein expression, indicating that the expression of CBP 65/67 in most organs is controlled primarily at the transcriptional level.
Characterization of Blood‐Group‐ABO(H)‐Active Glycosphingolipids in Type‐AB Human Erythrocytes
Authors: Kushi Yasunori , Tsunoda Atsunobu , Komatsuzaki Atsushi , Watanabe Kiyohiro , Kasama Takeshi , Handa Shizuo 
Published: March 2005

Neutral glycolipids in Folch's upper phase were isolated from human erythrocyte membranes of 22 individuals with blood type AB. On immunostaining by TLC with anti‐A IgG, all reactive glycolipids in type A corresponded to reactive glycolipids in type‐AB erythrocytes. With anti‐B IgM, all reactive glycolipids in type‐B erythrocytes also corresponded to reactive glycolipids in type‐AB erythrocytes. By comparison of the reactivity to that of the anti‐A and anti‐B antibodies, it was found that, in type‐AB erythrocytes, all glycolipids reactive with either one of the anti‐A or anti‐B antibodies were detected in both type‐A and type‐B erythrocytes, and that A‐active glycolipids had higher Rf values than B‐active glycolipids on TLC plates. A series of glycolipids reactive with both antibodies were purified from the Folch's upper neutral glycolipid fraction of erythrocyte membranes by column chromatography, and was characterized by TLC‐immunostaining and negative secondary‐ion mass spectrometry. The results strongly suggested that A‐active and B‐active carbohydrate chain epitopes existed separately as glycolipid molecules in blood‐type‐AB erythrocytes. It was also confirmed that these phenotypes observed in erythrocyte membranes were exhibited by blood‐group‐active glycosphingolipids in the small intestine of blood‐type‐AB individuals.Furthermore, upon treatment of fractions obtained from silicic acid column chromatography with α‐N ‐acetylhexosaminidase or α‐galactosidase, a branched hybrid‐type molecule with both A and B determinants was not detected.
Thermodynamic Analysis of the Binding of 5‐fluoro‐2′‐deoxyuridine 5′‐monophosphate to Thymidylate Synthase Over a Range of Temperatures
Authors: García‐Fuentes Luis , Reche Pedro , López‐Mayorga Obdulio , Santi Daniel V. , González‐Pacanowska Dolores , Barón Carmen 
Published: March 2005

The binding of 5‐fluoro‐2′‐deoxyuridine 5′‐monophosphate (FdUMP) to Lactobacillus casei recombinant thymidylate synthase has been studied by isothermal titration microcalorimetry at pH 7.1 over the temperature range 16–35°C. Calorimetric measurements in various buffer systems with different heats of ionization suggest that a proton uptake is involved in the binding process of the nucleotide. In the temperature range investigated, the mol protons bound/mol nucleotide increases as the temperature decreases.A model of two equal and independent sites fits well with the binding isotherms for thymidylate synthase. The binding constants, the changes in Gibbs energy, enthalpy, and entropy/site for FdUMP binding were calculated at each temperature. The results show that the binding is driven by both enthalpy and entropy contributions in the range 16–35°C. The enthalpy changes become more negative as the temperature increases, with CP=–170±20 J K1 (mol FdUMP bound)−1. The behavior of the system supports the observation that FdUMP binds to thymidylate synthase without producing profound conformational changes in the protein dimer.
Identification of Factor‐XIIIa‐Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand Factor
Authors: Takagi Junichi , Aoyama Toshihiro , Ueki Shoko , Ohba Hirotsugu , Saito Yuji , Lorand Laszlo 
Published: March 2005

von Willebrand factor is a large multimeric plasma protein which plays important roles in platelet aggregation, blood coagulation and probably also in the adhesion of endothelial cells. A 100‐kDa propeptide, called the propolypeptide of von Willebrand factor (pp‐vWF), is generated during biosynthesis. We found that pp‐vWF served as a substrate for transglutaminases including human factor XIIIa and guinea pig liver transglutaminase [Usui, T., Takagi, J. & Saito, Y. (1993) J. Biol. Chem. 268, 12311–123161. As such, it could form cross‐linked copolymers with the extracellular matrix protein, laminin, making it all the more likely that pp‐vWF plays a role in cell adhesion phenomena [Takagi, J., Sudo, Y., Saito, T. & Saito, Y. (1994) Eur. J. Biochem. 222, 861–867].In this work, we identified the Gln residues in pp‐vWF specifically reacting with blood coagulation factor XIIIa as amine acceptors. The fluorescent amine, dansylcadaverine, was employed for labeling the enzyme‐reactive sites of the protein. Following partial proteolysis, fragments containing the labeled Gln residues were isolated by passage through an anti‐dansyl affinity chromatographic column. Amino acid sequence analyses of the fragments revealed that, out of about 40 Gln residues in pp‐vWF, only four could be modified in the factor‐XIIIa‐catalyzed reaction.
The Contribution of Glucose Cycling to the Maintenance of Steady‐State Levels of Lactate by Hepatocytes During Glycolysis and Gluconeogenesis
Authors: Phillips John W. , Clark Dallas G. , Henly Debra C. , Berry Michael N. 
Published: March 2005

When hepatocytes from fasted rats were incubated with 10 mM glucose, there was a linear accumulation of lactate and pyruvate for about 80 min after which steady‐state concentrations of these metabolites became established. The rate of glycolysis, determined with [6‐3H]glucose, was constant over the entire incubation period and was 50% greater than that calculated from carbon balance studies. This suggests that one‐third of the glycolytic products formed were recycled to glucose. To enable study of the factors associated with the generation and maintenance of the lactate steady state and to measure accurately the carbon balance, incubations were performed using supraphysiological concentrations of glucose (20–80 mM). Under these conditions the initial rate of lactate accumulation and its concentration at steady state were shown to be dependent on the concentration of extracellular glucose.Rates of glycolysis were also measured using 40 mM [6‐3H]glucose and [U‐14C]glucose added alone, or in combination with a steady‐state lactate concentration (3 mM). There was no effect on the rate of glycolysis determined with [6‐3H]glucose, even when lactate was present in the medium. The difference in rates between measurements with the two isotopes reflects the apparent degree of glucose recycling which in the absence and presence of added lactate increased from 0.26 to 0.54 μmol C3 equivalents · min−1· g−1 respectively. Identical studies employing [U‐14C]lactate showed that glucose and CO2 were the major products of lactate metabolism under steady‐state conditions and that the formation of lactate from [U‐14C]glucose exactly balanced the rate of lactate removal as a result of oxidation and gluconeogenesis. These studies provide evidence for the concomitant operation of glycolysis and gluconeogenesis, even in the presence of high glucose concentrations. They also demonstrate that lactate steady states are achieved not by the cessation of glycolysis but rather by the removal of lactate and pyruvate at a rate equal to that of their production.
Kinetics of β‐1,3 Glucan Interaction at the Donor and Acceptor Sites of the Fungal Glucosyltransferase Encoded by the BGL2 Gene
Authors: Goldman Robert C. , Sullivan Patrick A. , Zakula Dorothy , Capobianco John O. 
Published: March 2005

Formation of branched glucan, glucan‐glucan cross links, and glucan‐chitin cross links most likely involves the action of fungal wall glucanases and transglycosylases. We developed an HPLC assay using radiolabeled substrates in order to study the kinetics of interaction of donor and acceptor molecules with a glucosyltransferase present in the cell walls of both Saccharomyces cerevisiae and Candida albicans. Purified transferase first forms an activated intermediate from a donor β‐1,3 glucan, releasing free disaccharide. The activated intermediate is transferred, in the presence of an appropriate acceptor β‐1,3 glucan, yielding a linear glucan containing a β‐1,6 linkage at the transfer site [Yu, L., Goldman, R., Sullivan, P., Walker, G. & Fesik, S. W. (1993) J. Biomol. NMR 3, 429–441], An apparent Km of 0.41 mM for the acceptor site was determined using laminaritetraose as the acceptor. An apparent Km of 31 mM for the donor site was determined using increasing concentrations of laminaripentaose, and monitoring formation of laminaribiose. The enzyme functioned as a glucanase at low concentrations of acceptor molecules, with excess H2O competing for reaction at the activated donor site, thus resulting in hydrolysis.However, as the concentration of acceptor increased, the reaction shifted from hydrolysis to glucosyltransfer. The reaction appeared specific for β‐1,3 glucan as acceptor, in as much as no transfer was detected when either hexa‐N‐acetyl‐chitohexaose or maltooligosaccharides were used as acceptors. The roles of such an enzymic activity in cell wall metabolism is discussed in terms of repair, cross linking and incorporation of newly synthesized chains of β‐1,3 glucan into the previously existing cell wall structure.
Solution Structure of the Oxidized 2[4Fe‐4S] Ferredoxin from Clostridium Pasteurianum
Authors: Bertini Ivano , Donaire Antonio , Feinberg Benjamin A. , Luchinat Claudio , Picciolp Mario , Yuan Huaiping 
Published: March 2005

Following the recently developed approach to the solution structure of paramagnetic high‐potential iron‐sulfur proteins, the three‐dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 1H‐NMR. The X‐ray structure is not available. The protein contains 55 amino acids and two [4Fe‐4S] clusters. In the oxidized state, the clusters have S= 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non‐selective T1 smaller than 25 ms.The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology both in achieving an extended assignment and in obtaining a suitable number of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar connectivities, partially detected through tailored experiments, 3JHN‐Hα, H‐bond constraints and dihedral angle constraints on the Cys X2 angles have been generated by using a recently derived Karplus‐type relationship for the hyperfine shifts of cysteine βCH2protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root‐mean‐square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi urici ferredoxin [Duée, E. D., Fanchon, E., Vicat, J., Sieker, L. C., Meyer, J. & Moulis, J.‐M. (1994) J. Mol Biol. 243, 683–695]. The two proteins are very similar in the overall folding, secondary structure elements and side‐chain orientations. The Cα root‐mean‐square deviation values between the X‐ray‐determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3–53). Discrepancies in residues 26–28 may arise from the disorder observed in the X‐ray structure in that region.