bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and characterization of a precursor form of lysosomal α‐glucosidase from human urine
Authors: OUDE ELFERINK Ronald P. J. , BROUWER‐KELDER Elisabeth M. , SURYA Inge , STRIJLAND Anneke , KROOS Marian , REUSER Arnold J. J. , TAGER Joseph M. 
Published: March 2005

1. A two‐step procedure is described for the isolation of lysosomal α‐glucosidase from human urine. In the second step, affinity chromatography on Sephadex G‐100, two fractions with acid α‐glucosidase activity were obtained.2. Fraction I contained α‐glucosidase of Mr 109000, whereas fraction II contained components of Mr 76000 and 70000.3. α‐Glucosidase in fraction I had an Mr similar to that of the precursor of α‐glucosidase detected in the medium of fibroblasts after labelling with [14C]leucine. The components in fraction II had Mr identical to those of the mature forms of α‐glucosidase found in placenta or cultured human skin fibroblasts.4. α‐Glucosidase in fraction I contained mannose 6‐phosphate (3.5 mol/mol polypeptide). No mannose 6‐phosphate was present in the components in fraction II.5. Fraction I, but not fraction II, was avidly endocytosed by α‐glucosidase‐deficient cultured human skin fibroblasts. Endocytosis of fraction I was inhibited by mannose 6‐phosphate.6. The pH optimum and Km values for p‐nitrophenyl α‐glucoside, maltose and glycogen of fractions I and II α‐glucosidase were almost identical. However, the activity with glycogen relative to that of either p‐nitrophenyl α‐glucoside or maltose was lower in fraction I than in fraction II.7. It is concluded that fraction I consists of the precursor form of α‐glucosidase and fraction II of the mature forms of the enzyme.8. The importance of urine as a source of precursors of lysosomal enzymes is discussed.
Pyruvate carboxylase from Aspergillus nidulans
Authors: OSMANI Stephen A. , MAYER Frank , MARSTON Fiona A. O. , SELMES Ian P. , SCRUTTON Michael C. 
Published: March 2005

1. A method is described for purification of pyruvate carboxylase from Aspergillus nidulans by affinity chromatography on monomeric avidin‐Sepharose. The purified enzyme is homogeneous as judged by electrophoretic and immunochemical analysis. The sub‐unit Mr determined by electrophoresis in the presence of sodium dodecyl sulphate is 133000 ± 5000.2. Electron microscopic analysis of purified A. nidulans pyruvate carboxylase after negative staining with uranyl acetate reveals the presence of molecules showing rhomboid and triangular projections as well as a projection showing two intensity maxima. A cleft running along the longitudinal axis of the sub‐unit is observed in the rhomboid and triangular projections. Interconversion between all three projections can be obtained in a tilt series.3. Significantly better preservation of molecular structure is obtained if A. nidulans pyruvate carboxylase is prepared for electron microscopy in the presence of acetyl‐CoA, 2‐oxoglutarate or as the enzyme‐avidin complex. l‐Aspartate has no significant effect when added alone but markedly decreases the enhanced preservation given by acetyl‐CoA. No marked alterations in molecular dimensions are caused by any of these additions.4. l‐Aspartate, but not 2‐oxoglutarate, enhances the rate of inactivation observed on incubation of A. nidulans pyruvate carboxylase at 4°C in the presence of 0.5 M KCl.5. Addition of l‐aspartate in low concentrations enhances the effectiveness of inhibition by 2‐oxoglutarate by causing a decrease in the value of [I]0.5 without affecting the Hill coefficient h or the extent of activity observed at saturating 2‐oxoglutarate concentrations. Conversely addition of low concentrations of 2‐oxoglutarate decreases the concentration of l‐aspartate required to give 50% inhibition but also causes a fall in h and an absolute increase in the extent of activity observed in the presence of saturating l‐aspartate concentrations.6. The data are consistent with the proposal that A. nidulans pyruvate carboxylase is a tetrameric molecule in which the four sub‐units are located at the corners of a tetrahedron. Metabolites which regulate the activity of the enzyme do not cause major alterations in this molecular structure but may alter its stability. The data presented also provide further evidence supporting the postulate that l‐aspartate and 2‐oxoglutarate occupy distinct regulatory sites on A. nidulans pyruvate carboxylase.
Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction
Authors: ANDERSSON Pia , KVASSMAN Jan , OLDÉN Bertil , PETTERSSON Gösta 
Published: March 2005

1. The interaction of liver alcohol dehydrogenase with NADH and aldehyde substrates has been characterized with respect to ternary‐complex formation by the apparently non‐preferred pathway which involves intermediate formation of binary enzyme · aldehyde complexes. Rate constant estimates are reported for dimethylamino‐cinnamaldehyde (DACA) binding to free enzyme and for NADH binding to the enzyme · DACA complex.2. The rate of NADH (or NAD+) association to liver alcohol dehydrogenase is not detectably affected by DACA binding to the enzyme, but the NADH dissociation rate decreases approximately by a factor of 6. The NADH‐induced increase in affinity of the enzyme for DACA is similarly attributable to a decreased dissociation rate rather than an increased association rate of the aldehyde. DACA dissociates much more rapidly than coenzyme from the enzyme · NADH · aldehyde complex and shows a higher association rate constant than NADH in its interaction with free enzyme.3. It is concluded from these results that the enzymic reduction of typical aldehyde substrates will conform to a rate equation which is experimentally indistinguishable from that of a compulsory‐order mechanism with coenzyme binding preceding substrate binding, and that this rate equation will obtain irrespective of which pathway for ternary‐complex formation is actually preferred. Rate equations provide no reliable information about the order of ligand binding in ternary‐complex systems.4. A flow analysis is presented which indicates that coenzyme and substrate are actually bound in random order to liver alcohol dehydrogenase during the enzymic reduction of aldehydes by NADH. The enzyme · aldehyde pathway for ternary‐complex formation is fully kinetically competent, and reaction flow via this pathway may predominate when aldehyde concentrations exceed those required for half‐saturation of free enzyme. Binary enzyme · aldehyde complexes are seemingly insignificant with respect to the rate behaviour of the enzyme, but may provide most significant and even predominant contributions to the catalytic reaction flow.
Resolution of bovine heart cytochrome c oxidase into smaller complexes by controlled subunit denaturation
Authors: CORBLEY Michael J. , AZZI Angelo 
Published: March 2005

Bovine heart cytochrome c oxidase has been partially denaturated under mild conditions with 0.1–0.25% lithium dodecyl sulfate and 0.05% Triton X‐100.From its reactivity towards CO and CN–, an unmasking of the heme a was inferred in this enzyme. The catalytic activity was lost during the denaturation and small spectral differences became visible. Spectra and ligand binding properties of the denatured enzyme were reversed by dilution in 2% Triton X‐100. This suggests that during the denaturation procedure the hemes were not displaced from their original sites.By gel filtration of the partially denatured enzyme the following complexes of subunits were obtained: I–III, I–II– III, II–IV–V–VI–VII and IV–V–VI–VII. The first three complexes retained almost all the heme, and their spectral characteristics were very similar to those of the partially denatured cytochrome c oxidase. The data, in combination with the information that subunit III does not contain heme [Saraste et al. (1980) FEBS Lett. 114, 35–38], suggest that the hemes are attached to subunit I and II.After denaturation of cytochrome c oxidase under more drastic conditions some of the heme was also found to be associated with the smaller subunits, but its spectral characteristics were radically altered, becoming almost identical to those of free heme.
Biosynthesis and compartmentalization of rat‐intestinal vitamin‐D‐dependent calcium‐binding protein
Authors: LEONARD Warren J. , STRAUSS Arnold W. , GO Mae F. , ALPERS David H. , GORDON Jeffrey I. 
Published: March 2005

We have purified the primary translation product of rat intestinal vitamin‐D‐dependent calcium‐binding protein mRNA from wheat germ and ascites cell‐free systems. We show that calcium‐binding protein is neither synthesized as a larger precursor nor likely to be exported from the intestinal epithelium. Our conclusions are based on the following observations. (1) The primary translation product, NH2‐terminally labeled with formyl[35S]methionine, comigrates with the mature cytoplasmic protein during electrophoresis through denaturing gels. (2) It does not possess a cleavable signal peptide sequence or internal signal equivalent as judged by co‐ and post‐translational cleavage assays in vitro. (3) The NH2 terminus of the cell‐free product is acetylated. (4) Comparison of the NH2‐terminal amino acid sequences of the primary translation product and cyanogen bromide peptides obtained from the blocked, purified cytoplasmic protein.The kinetics of calcium‐binding protein mRNA accumulation and decay in rachitic intestinal epithelium after primary and secondary stimulation with 1,25‐dihydroxycholecalciferol (calcitriol) were studied using the cell‐free translation system. The results are reminiscent of other steroid‐hormone‐inducible systems. Both the rate of mRNA accumulation and the peak response were greater after secondary stimulation.
The mechanism of inactivation of lipoxygenases by acetylenic fatty acids
Authors: KÜHN Hartmut , HOLZHÜTTER Hermann‐Georg , SCHEWE Tankred , HIEBSCH Christa , RAPOPORT Samuel M. 
Published: March 2005

The inactivation of soybean lipoxygenase by 5,8,11,14‐eicosatetraynoic acid was studied in detail. The inactivation was found to be time‐dependent and irreversible. A kinetic scheme, based on the assumption of a rapid inactivation of the enzyme‐product complex, yielded a Km value for 5,8,11,14‐eicosatetraynoic acid of 1.3 μM, which is about a tenth of that described for arachidonic acid, and a reaction constant k+2 of 0.006s−1, which is four orders of magnitude lower. The reasons for these differences are discussed. Several types of experimental evidence indicate that the first step of the enzyme inactivation is the conversion of 5,8,11,14‐eicosatetraynoic acid via a lipoxygenase reaction: (a) the conversion of radioactively labelled methyl ester of 5,8,11,14‐eicosatetraynoic acid to other products; (b) the oxygen requirement of the inactivation; (c) the competitive protective effect of linoleic acid; (d) the similarity of the activation energy for both the dioxygenation of linoleic acid and the enzyme inactivation by 5,8,11,14‐eicosatetraynoic acid; (e) the formation of one mole methionine sulfoxide/mole enzyme during the reaction with 5,8,11,14‐eicosatetraynoic acid, similar to the suicidal reaction of reticulocyte lipoxygenase with 13Ls‐hydroperoxy‐linoleic acid.These results, as well as the lack of covalent binding of 14C‐labelled 5,8,11,14‐eicosatetraynoic acid methyl ester, contradict the allene mechanism postulated by others [D. T. Downing, D. G. Ahern, and M. Bachta (1970) Biochem. Biophys. Res. Commun. 40, 218–223; K. H. Gibson (1977) Chem. Soc. Rev. 6, 489–510]. It is assumed that the susceptible methionine is located at the active centre of the enzyme.
Changes in membrane‐associated proteins during sporulation in Bacillus subtilis
Authors: RHAESE Hans J. , REICHENBACH Angelika , STAMMINGER Gudrun 
Published: March 2005

Membrane proteins from vegetative and sporulating cells of Bacillus subtilis were separated by the two‐dimensional gel electrophoresis system using isoelectric focusing and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (O'Farrell technique). Membrane proteins were isolated according to published procedures.The gels were stained with Coomassie blue. Three different concentrations of proteins were analyzed to detect even minor constituents. Over two hundred different membrane proteins were identified in vegetative cells by their isoelectric point (pI) and molecular weight (Mr). Analysis of membrane proteins from cells harvested during and at the end of logarithmic growth (A600∼ 0.8; T0) and every hour thereafter until T4 showed that in the wild‐type strain 55 proteins are degraded mostly at the beginning or sporulation. Many others (76 proteins) are newly synthesized during sporulation. About 16 proteins are synthesized at times during sporulation but again degraded within 1 h or less. Others (uncertain proteins, 65) are degraded and resynthesized again. This observation is in agreement with experiments previously published by Andreoli et al. [Andreoli, A. J., Kao, M., Chui, R., Cabrera, J., and Wong, S. K. S (1981) in Sporulation and Germination (Levinson, H. S., Sonenshein, A. L., and Tipper, D. J., eds) pp. 168–173, American Society for Microbiology, Washington] using Bacillus cereus.Experiments with the early blocked asporogenous mutant JH 649 (spoof) showed that few proteins (40%) are degraded and even fewer (30%) are newly synthesized between A600∼ 0.8 and T4.Protease inhibitors (phenylmethylsulfonyl fluoride, EDTA, o‐phenanthroline) have no effect on the protein patterns. The experiments presented here show that proteins involved in differentiation in B. subtilis can be identified by the two‐dimensional gel electrophoresis system and with the aid of asporogenous mutants.In order to assure that no cytoplasmic proteins are contaminating the membrane preparations, several cytoplasmic enzyme activities have been measured. Their concentration was found to be always below 0.005% of total protein, which is below the level of detection by Coomassie blue staining.
Differentiation of receptor sites for [3H]nitrendipine in chick hearts and physiological relation to the slow Ca2+ channel and to excitation‐contraction coupling
Authors: RENAUD Jean‐François , KAZAZOGLOU Théodossia , SCHMID Annie , ROMEY Georges , LAZDUNSKI Michel 
Published: March 2005

The properties of interaction of the Ca2+ channel antagonist [3H]nitrendipine have been investigated in chick hearts at various stages of in ovo and post‐natal development and in cultured cells. The dissociation constant of the [3H]nitrendipine‐receptor complex is between 0.4 nM and 0.5 nM for intact ventricle and cultured cells. [3H]Nitrendipine binding is antagonized by nitrendipine analogs. The order of efficacy of the different dihydropyridine molecules is nitrendipine < nimodipine < nifedipine < nisoldipine with Kd values ranging from 0.5 to 4 nM. Inhibition of [3H]nitrendipine binding by other antiarrhythmic molecules like amiodarone, F13004 and bepridil was observed. Half‐maximum inhibitions (K0.5) were found for verapamil and D600 at concentrations between 0.23 and 0.26 μM.The potency of organic Ca2+ blockers to depress by 50% the maximum amplitude of spontaneous beating of heart cells is closely related to K0.5 values obtained from [3H]nitrendipine binding experiments.Electrophysiological results indicate that the slow channel is insensitive to nitrendipine at the younger stage of development (3‐day‐old) whereas, in adult like cells, nitrendipine (50 nM) abolished both slow action potential due to the slow Ca2+ channel and contraction. The maximum binding capacity for [3H]nitrendipine is found to increase during development of the embryonic heart from 40 fmol/mg protein at day 3 to 100 fmol/mg protein at day 14, to stay relatively stable until day 18. Then the number of sites increases rapidly to reach a second plateau at 210 fmol/mg protein on day 4 after hatching. Treatment with 6‐hydroxydopamine results in 35% increase in [3H]nitrendipine binding, whereas reserpine treatment is without effect. Developmental properties of nitrendipine‐sensitive Ca2+ channels have been compared with those of tetrodotoxin‐sensitive Na+ channels and muscarinic receptors. These results indicate that nitrendipine receptors exist at the early stage of development (3‐day‐old‐hearts) but that they do not correspond to functional slow Ca2+ channels, that in ovo development corresponds both to an increase of the number of [3H]nitrendipine receptors and to the transformation of silent Ca2+ channels into functional Ca2+ channels, and that there is a regulation of the level nitrendipine‐sensitive Ca2+ channels by innervation.
The interaction of neurotoxin derivatives with either acetylcholine receptor or a monoclonal antibody
Authors: ROUSSELET Annie , FAURE Grazyna , BOULAIN Jean‐Claude , MÉNEZ André 
Published: March 2005

Five derivatives of Naja nigricollis toxin α, spin‐labeled on a single amino group, were prepared. The toxin derivatives were purified to homogeneity by ion‐exchange and high‐pressure liquid chromatographies. The modified amino groups are localized at residue 1 and lysines 15, 27, 47 and 51. Competition data show that incorporation of spin label at residues 27 or 47 reduces the affinity of the toxin for the nicotinic acetylcholine receptor (AcChR), while incorporation at residues 1 or 15 diminishes toxin affinity for a monoclonal toxin‐specific immunoglobulin (M1). Classical and/or saturation transfer electron spin resonance (ESR) analysis was carried out on each derivative, either in the free state or bound to AcChR or M1. The data obtained give the following indications.

In the free state, the nitroxides incorporated at residues 1, 15, 47 and 51 have their own rapid motion, while that at residue 27 has no residual mobility and reflects the toxin rotation.
Binding of AcChR to the toxin reduces the motion of the nitroxide bound to Lys47. Binding of M1 to the toxin immobilizes the two nitroxides fixed on residues 1 and 15. ESR spectra show that Lys27‐bound nitroxide remains immobilized upon binding of either AcChR or M1.
The change in nitroxide immobilization observed upon AcChR or M1 binding correlates well with the variation of nitroxide accessibility to a water‐soluble paramagnetic N2+i ion.
Binding of the labeled Lys47 toxin derivative to AcChR yields a complex ESR signal, disclosing the existence of a physical difference between the two toxin binding sites on AcChR.

All the data indicate that AcChR and M1 bind at two topographically distinct sites on the toxin surface.
A temperature‐jump study of the electron transfer reactions in Hansenula anomala flavocytochrome b2
Authors: TEGONI Mariella , SILVESTRINI Maria Chiara , LABEYRIE Françoise , BRUNORI Maurizio 
Published: March 2005

Temperature‐jump experiments on flavocytochrome b2 were carried out at different levels of heme reduction at pH 7.0 and 6.0, and as a function of pyruvate concentration.The relaxation, corresponding to an increase in the concentration of reduced heme, is in no case a simple process. At pH 7.0 the mean reciprocal relaxation time is 1/τ*= 190 s−1, independent of enzyme concentration, wavelength of observation and percentage of heme reduction. Flavin semiquinone has been identified as the major electron donor to the heme in this process. At the same pH the presence of pyruvate in the millimolar concentration range increases the relaxation rate and affects its amplitude. The latter effect could be accounted for by a change in redox equilibria between heme and flavin upon pyruvate binding.At pH 6.0 the relaxation pattern depends more clearly on the level of heme reduction. A rapid process (τ−1= 2500 s−1), predominant at high percentages of reduced heme, has been assigned to the reduction of heme by flavin hydroquinone, while the slower process (τ−1= 350 s−1), essentially the only one present at or below 50% of heme reduction, has been ascribed to the reduction of heme by flavin semiquinone. These results are discussed in relation to the catalytic mechanism of the enzyme.
Calmodulin content and activity of Ca2+‐ATPase and phospholipase A2 in rat Kupffer cells
Authors: BIRMELIN Manfred , MARME Dieter , FERBER Ernst , DECKER Karl 
Published: March 2005

A protein resembling calmodulin was isolated from non‐parenchymal and parenchymal cells of rat liver by affinity chromatography. The biological activity of the purified protein was assessed by the bovine brain cAMP phosphodiesterase assay. A highly sensitive radioimmunoassay as well as the cAMP phosphodiesterase method were employed to determine the calmodulin content of crude extracts from monolayer cultures of rat Kupffer cells and hepatocytes.An ATP‐dependent, calmodulin‐enhanced calcium transport was demonstrated in a membrane fraction of the non‐parenchymal cells. Phospholipase A2 activity specific for 2‐arachidonoyl phosphatide and with a pH optimum of 8.1 was measured in homogenized Kupffer cells; it was stimulated by agents previously shown to enhance prostaglandin synthesis in Kupffer cells, e.g. zymosan particles and lipopolysaccharide isolated from Salmonella minnesota. The increase in activity was completely prevented by pretreatment with or simultaneous addition of R 24571, a known calmodulin antagonist. However, if this inhibitor or calmodulin was added to the cell‐free extract phospholipase A2 activity was not influenced. Phospholipase A1 activity could be detected at pH 5 only, showing a slight decrease in the homogenate of stimulated macrophages. Acyltransferase activity was high but independent of treatment of the Kupffer cells.
Blockade of the galactose‐binding sites of ricin by its linkage to antibody
Authors: THORPE Philip E. , ROSS Walter C. J. , BROWN Alex N. F. , MYERS Christopher D. , CUMBER Alan J. , FOXWELL Brian M. J. , FORRESTER J. Tony 
Published: March 2005

A method is described for preparing specific cytotoxic agents by linking intact ricin to antibodies in a manner that produces obstruction of the galactose‐binding sites on the B chain of the toxin and so diminishes the capacity of the conjugate to bind non‐specifically to cells. The conjugates were synthesised by reacting iodoacetylated ricin with thiolated immunoglobulin and the components of conjugate with reduced galactose‐binding capacity were separated by affinity chromatography on Sepharose (a β‐galactosyl matrix) and asialofetuin‐Sepharose.Fluorescence‐activated cell sorter (FACS) analyses revealed that the fraction of a monoclonal anti‐Thy1.1‐ricin conjugate that passed through a Sepharose column had markedly diminished capacity to bind non‐specifically to Thy1.2‐expressing CBA thymocytes and EL4 lymphoma cells. The fraction of conjugate that passed through an asialofetuin‐Sepharose column displayed no detectable non‐specific binding. Both fractions of conjugate were potent cytotoxic agents for Thy1.1‐expressing AKR‐A lymphoma cells in tissue culture. They reduced the [3H]leucine incorporation of the cells by 50% at a concentration of 2–5 pM. Comparable inhibition of EL4 cells was only achieved with 3000–7500‐fold greater concentrations of conjugate. By contrast, the fraction of anti‐Thy1.1‐ricin that retained Sepharose‐binding capacity showed marked non‐specific binding and toxicity to EL4 cells.A conjugate with diminished galactose‐binding capacity was also prepared from the W3/25 monoclonal antibody which recognises an antigen upon helper T‐lymphocytes in the rat. It elicited powerful and specific toxic effects upon W3/25 antigen‐expressing rat T‐leukaemia cells. This finding is of particular importance because isolated ricin A‐chain disulphide‐linked to W3/25 antibody is not cytotoxic. The property of the B‐chain in intact ricin conjugates that facilitates delivery of the A‐chain to the cytosol thus appears to be independent of galactose recognition.It is concluded that the ‘blocked’ ricin conjugates combine the advantages of high potency, which is often lacking in antibody‐A‐chain conjugates, with high specificity, which previously was lacking in intact ricin conjugates.