bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear magnetic resonance studies of the old yellow enzyme
Authors: BEINERT Wolf‐Dieter , RÜTERJANS Heinz , MÜLLER Franz , BACHER Adelbert
Published: March 2005

The apoenzyme of NADPH oxidoreductase, ‘old yellow enzyme’, was reconstituted with selectively 13C‐enriched flavin mononucleotides and investigated by 13C NMR spectroscopy. The 13C NMR results confirm the results obtained by 15N NMR spectroscopy and yield additional information about the coenzyme‐apoenzyme interaction. A strong deshielding of the C(2) and C(4) atoms of enzyme‐bound FMN both in the oxidized and reduced state is observed, which is supposed to be induced by hydrogen‐bond formation between the protein and the two carbonyl groups at C(2) and C(4) of the isoalloxazine ring system. The chemical shifts of all 13C resonances of the flavin in the two‐electron‐reduced state indicate that the N(5) atom is sp3‐hybridized.

From 31P NMR measurements it is concluded that the FMN phosphate group is not accessible to bulk solvent. The unusual 31P chemical shift of FMN in old yellow enzyme seems to indicate a different binding mode of the FMN phosphate group in this enzyme as compared to the flavodoxins.

The 13C and 15N NMR data on the old‐yellow‐enzyme–phenolate complexes show that the atoms of the phenolate are more deshielded whereas the atoms of the enzyme‐bound isoalloxazine ring are more shielded upon complexation. A non‐linear correlation exists between the chemical shifts of the N(5) and the N(10) atoms and the pKa value of the phenolate derivative bound to the protein. Since the chemical shifts of N(5), N(10) and C(4a) are influenced most on complexation it is suggested that the phenolate is bound near the pyrazine ring of the isoalloxazine system. 15N NMR studies on the complex between FMN and 2‐aminobenzoic acid indicate that the structure of this complex differs from that of the old‐yellow‐enzyme–phenolate complexes.
Role of plasma membrane transport in hepatic glutamine metabolism
Authors: HÄUSSINGER Dieter , SOBOLL Sibylle , MEIJER Alfred J. , GEROK Wolfgang , TAGER Joseph M. , SIES Helmut
Published: March 2005

In livers of fed rats and in perfused livers supplied with a physiological portal glutamine concentration of 0.6 mM, the mitochondrial and cytosolic glutamine concentrations are 20 mM and 7 mM, respectively, thus, the mitochondrial/cytosolic glutamine concentration gradient is 2–3.

Uptake and release of glutamine by periportal and perivenous hepatocytes occurs predominantly by an Na+‐dependent transport system (so‐called system ‘N’).

Histidine in near‐physiological concentrations inhibits both glutamine uptake by periportal hepatocytes and its release by perivenous hepatocytes. This is not due to an inhibition of glutamine‐metabolizing enzymes by histidine or its metabolites. With physiological portal glutamine concentrations (0.6 mM), stimulation of glutaminase flux or of glutamine transaminase flux is followed by a decreased of hepatic glutamine levels to about 80% or 30%, respectively, glutamine levels are further decreased to 50% or 20% in the presence of histidine.

When glutamine is synthesized endogeneously (no glutamine added), the histidine‐induced inhibition of glutamine release is paralleled by a 210% increase of the hepatic tissue level of glutamine.

In experiments with and without methionine sulfoximine and in the absence of added glutamine, the glutamine content in the small perivenous hepatocyte population containing glutamine synthetase is estimated to be about 3.5 μmol/g wet weight and that in the periportal hepatocytes as low as 0.1 μmol/g wet weight.

In contrast to the prevailing view, it is concluded that glutamine transport across the plasma membrane of hepatocytes is a potential regulatory site in glutamine degradation and synthesis, especially under the influence of effectors like histidine.
Ribosomes are stalled during in vitro translation of alfalfa mosaic virus RNA 1
Authors: LINDHOUT Pim , NEELEMAN Lyda , VAN TOL Hans , VAN VLOTEN‐DOTING Lous
Published: March 2005

In the presence of plant tRNAs the full‐length translation product of alfalfa mosaic virus RNA 1 is produced in rabbit reticulocytes only at low mRNA concentration. At higher mRNA concentration translation is restricted to the 5′ half of RNA 1. At high mRNA concentration the full‐length product can be formed when additional plant tRNA and glutamine are supplied to the translation mixture. In contrast, in the presence of yeast or calf liver tRNA the translation pattern of alfalfa mosaic virus RNA 1 always results in the synthesis of the full‐length product. Pulse‐chase experiments in the presence of plant tRNAs show that the ribosomes pause at several positions in the 5′ half of RNA 1. The pausing time is different at the different ‘halting places’. Protein synthesis is resumed upon addition of glutamine, even when the addition is delayed for more than 3 h after the start of protein synthesis. Only one tRNA species, purified from wheat germ or tobacco, could promote full‐length translation of RNA 1. This tRNA can be charged with glutamine. Analysis of the position of glutamine codons on RNA 1 shows a correlation between the positions of the CAA codons and the halting places of the ribosomes. The CAA codon (for any other codon) on its own cannot be responsible for the pausing of the ribosomes, since a variety of RNAs, known to contain all sense codons, are translated efficiently in rabbit reticulocyte lysates in the presence of plant tRNAs. Apparently other elements can restrict decoding of normal codons during protein chain elongation.
Homologies and anomalies in primary structural patterns of nucleotide binding proteins
Authors: ARGOS Patrick , LEBERMAN Reuben
Published: March 2005

In searches for homology among nucleotide binding proteins, recent reports have described primary structure alignments for stretches of 30 or so amino acid residues among a variety of proteins including the ras and src oncogene products. The significance of these sequence matches has been tested by searching in available data banks for certain conserved residue patterns resulting from the alignments. The tests suggest that alignments over these limited stretches are not necessarily justifiable and any implications for residues involved in nucleotide binding must be viewed with caution.
Secondary structure of the pore‐forming colicin A and its C‐terminal fragment
Authors: PATTUS Franc , HEITZ Frederic , MARTINEZ Carmen , PROVENCHER Steven W. , LAZDUNSKI Claude
Published: March 2005

Conformational investigations, using circular dichroism, on the pore‐forming protein, colicin A (Mr 60 000), and a C‐terminal bromelain fragment (Mr 20 000) were undertaken to estimate their secondary structure and to search for pH‐dependent conformational changes. Colicin A and the bromelain peptide are mainly α‐helical with an enrichment of the α‐helical content in the C‐terminal domain carrying the ionophoric activity. The non‐negligible β‐sheet structure in the C‐terminal domain is unstable and is easily transformed into α‐helix upon decreasing the polarity of the solvent. No evidence of pH‐dependent conformational modification, correlated with modification of colicin A activity, could be obtained. The secondary structure estimated on the basis of experimental data favoured a model in which the pore is built of a minimal number of six transmembrane α‐helical segements. Search for such segments in the amino acid sequence of the C‐terminal domain of colicin A was carried out by combining secondary structure prediction methods with hydrophobicity and hydrophobic moment calculations. Similar calculations on the C‐terminal domains of colicin E1 and IB indicate a common structure of the pores formed by colicin A, E1 and IB. Only two or three putative transmembrane segments could be selected in the sequences of colicin A, IB or E1. As a result, it is concluded that the channel is probably not built by a single colicin molecule but more likely by an oligomer.
Role of the Arg158 residue of the outer membrane PhoE pore protein of Escherichia coli K 12 in bacteriophage TC 45 recognition and in channel characteristics
Authors: KORTELAND Jaap , OVERBEEKE Nico , de GRAAFF Pieter , OVERDUIN Piet , LUGTENBERG Ben
Published: March 2005

In order to study the structure‐function relationship of the PhoE protein pore we have isolated five independent, TC 45‐resistant, phoE mutants all of which appeared to produce normal amounts of an electrophoretically altered PhoE protein, designated as PhoE* protein. Nucleotide sequence analysis of the DNA fragments carrying the mutations showed that the mutations all correspond to a G · C to A · T transition at the same place within the phoE gene resulting in a deduced change of amino acid residue arginine 158 into histidine. This result shows that the arginine 158 residue plays an important role in the interaction of the PhoE protein pore with phage TC 45. Moreover, studies on the channel properties of the PhoE* protein showed that the PhoE channel has lost part of its preference for negatively charged solutes, as a result of the arginine to histidine change. The results are discussed in terms of the structure‐function relationship of PhoE protein as well as in terms of the topological organization of the protein channel in the outer membrane.
The folding and mutual interaction of the domains of yeast 3‐phosphoglycerate kinase
Authors: ADAMS Benjamin , BURGESS Rowland J. , PAIN Roger H.
Published: March 2005

Analysis of the reversible unfolding of yeast phosphoglycerate kinase leads to the conclusion that the two lobes are capable of folding independently, consistent with the presence of intermediates on the folding pathway with a single domain folded. The domains have different free energies of stabilisation. Immunological cross‐reactivity, circular dichroism and thiol reactivity provide evidence for cyanogen bromide peptide 1–173, which comprises five‐sixths of the N‐terminal domain, containing sufficient information to refold into a native‐like structure which dimerises.
Phase equilibria in four lysophosphatidylcholine/water systems
Authors: ARVIDSON Gösta , BRENTEL Ingvar , KHAN Ali , LINDBLOM Göran , FONTELL Krister
Published: March 2005

The phase equilibria in four lysophosphatidylcholine/water systems were investigated at different temperatures. Each of the 1‐palmitoyl‐, 1‐stearoyl‐, 1‐oleoyl‐ and 1‐linoleoyl‐sn‐glycero‐3‐phosphocholines was dispersed in heavy water at different concentrations. The phase structures were determined by 2H‐, 14N‐ and 31P‐NMR, polarization microscopy and low‐angle X‐ray diffraction.

The phase diagrams of the oleoyl and linoleoyl systems were quite similar. At room temperature and with decreasing water content the isotropic micellar solution was followed by a hexagonal phase and then a cubic phase. Finally the lamellar phase appeared before the region of hydrated crystals. The same sequence of phases was observed in the stearoyl system at elevated temperatures. The palmitoyl system differed from the others: here a cubic phase followed after the micellar solution, then came a hexagonal phase and after this a lamellar phase. In general the lysophosphatidylcholines seem to behave similarly to the many soaps and detergents as they show the same sequence of isotropic micellar solution, hexagonal phase, lamellar phase with interspersed cubic phases.

The presently established phase diagrams demonstrate that the major lysophosphatidylcholines which may be generated by phospholipase A2 in mammalian cell membranes, viz. 1‐palmitoyl‐ and 1‐stearoyl‐glycerophosphocholines differ greatly in their packing properties. The extraordinary ability of 1‐palmitoyl‐glycerophosphocholine to form a cubic phase in equilibrium with a micellar solution is of particular interest with regard to the possible occurrence of cubic structures in biomembranes during the process of fusion.
Mitochondrial aldehyde dehydrogenase from human liver
Authors: HEMPEL John , KAISER Rudolf , JÖRNVALL Hans
Published: March 2005

The 500‐residue amino acid sequence of the subunit of mitochondrial human liver aldehyde dehydrogenase is reported. It is the first structure determined for this enzyme type from any species, and is based on peptides from treatments with trypsin, CNBr, staphylococcal Glu‐specific protease, and hydroxylamine. The chain is not blocked (in contrast to that of the acetylated cytosolic enzmye form), but shows N‐terminal processing heterogeneity over the first seven positions. Otherwise, no evidence for subunit microheterogeneities was obtained. The structure displays 68% positional identity with that of the corresponding cytosolic enzyme, and comparisons allow functional interpretations for several segments.

A region with segments suggested to participate in coenzyme binding is the most highly conserved long segment of the entire structure (positions 194–274). Cys‐302, identified in the cytosolic enzyme in relation to the disulfiram reaction, is also present in the mitochondrial enzyme. A new model of the active site appears possible and involves a hydrophobic cleft. Near‐total lack of conservation of the N‐terminal segments may reflect a role of the N‐terminal region in signaling the transport of the mitochondrial protein chains. Non‐conservation of interior regions may reflect the differences between the two enzyme forms in subunit interactions, explaining the lack ofheterotetrameric molecules. The presence of some internal repeat structures is also noted as well as apparently general features of differences between cytosolic and mitochondrial enzymes.
Activation of dolichyl‐phospho‐mannose synthase by phospholipids
Authors: JENSEN John W. , SCHUTZBACH John S.
Published: March 2005

Dolichyl‐phospho‐mannose synthase, or GDPmannose:dolichyl‐phosphate mannosyltransferase (EC 2.4.1.83), was solubilized from rat liver microsomes with 1.0% Nonidet P‐40 and the enzyme was further purified by column chromatography on DEAE‐cellulose in the presence of 0.1 % Nonidet P‐40. The purified enzyme preparation (880‐fold over microsomes) was unstable in the presence of detergent and had no activity in the presence of Nonidet P‐40, Triton X‐100, octyl β‐glucoside, or deoxycholate. Detergent‐free enzyme was active in the presence of phosphatidylethanolamine (PtdEtn) and in the presence of phospholipid mixtures of PtdEtn and phosphatidylcholine (PtdCho) when the molar proportion of PtdCho was 70% or less. The enzyme was inactive in the presence of PtdCho alone. Unsaturated species of PtdEtn have a tendency to destabilize membrane bilayers [Cullis, P. R. & de Kruijff, B. (1978) Biochim. Biophys. Acta 507, 207–218] and we have shown that dolichol promotes the destabilizing effect of PtdEtn on membranes composed of PtdCho and PtdEtn [Jensen, J. W. & Schutzbach, J. S. (1984) Biochemistry 23, 1115–1119]. These results suggest that dolichyl‐P‐mannose synthase is optimally active in a phospholipid matrix that contains some component phospholipids that prefer non‐bilayer structural organization in isolation. Heat‐inactivation and sedimentation experiments demonstrated that the synthase associated with PtdEtn in the presence of dolichyl‐P. The PtdEtn‐reconstituted enzyme catalyzed the reversible transfer of mannose from GDP‐mannose to dolichyl‐P. The Km for GDP‐mannose was found to be 0.69 μM and the apparent Km for dolichyl‐P was 0.3 μM. GMP, GDP, and GTP inhibited mannosyltransfer 50% at concentrations of 16 μM, 1.3 μM and 3 μM respectively.
Solubilization of high‐affinity [3H]tryptamine‐binding sites from rat brain
Authors: BRÜNING Gerold , ROMMELSPACHER Hans
Published: March 2005

High‐affinity binding sites for [3H]tryptamine from rat brain were solubilized by treatment with detergents. Digitonin was found to be most efficient. Triton X‐100 and Chaps were less and Lubrol‐PX was even less effective. The properties of [3H]tryptamine binding were essentially unchanged by solubilization with digitonin. The equilibrium dissociation constant Kd was determined as 3.7 nM; the association and dissociation rates were ka= 0.019 nM−1 min−1 and kd= 0.032 min−1, respectively, resulting in a calculated Kd of 3.6 nM. The structure activity profile of membrane‐bound as well as solubilized binding sites was characterized by the high affinity of some β‐carbolines, especially harmaline, and the low affinity of 5‐hydroxytryptamine. On glycerol gradient centrifugation the digitonin‐[3H]tryptamine‐binding‐site complex sedimented at 12.8 S. Size‐exclusion chromatography on Sepharose 6B revealed a Stokes' radius of 5.9 nm.
Accumulation of UDP‐GlcNAc into intracellular vesicles and occurrence of a carrier‐mediated transport
Authors: CECCHELLI Roméo , CACAN René , VERBERT André
Published: March 2005

Metabolic labelling of mouse thymocytes with radioactive mannose or glucosamine leads to the formation of labelled GDP‐Man and UDP‐GlcNAc. Using isotonic ammonium chloride treatment which renders the plasma membrane of thymocytes permeable to sugar nucleotides, we demonstrate that, in contrast to GDP‐Man, a pool of UDP‐GlcNAc remains associated with the cells after plasma membrane permeabilization. These observations are confirmed in experiments in which permeabilized thymocytes are incubated with exogenous labelled GDP‐Man and UDP‐GlcNAc, and we show that only UDP‐GlcNAc is accumulated into sealed intracellular vesicles. This accumulation is a saturable process which can be inhibited by UDP, demonstrating the occurrence of a specific carrier. This transport mechanism can be blocked by covalent attachment of a non‐permeant inhibitor UDP‐dialdehyde without affecting the N‐acetylglucosaminyltransferase itself. The fact that this carrier‐mediated transport is not inhibited by tunicamycin indicates that this translocation process of UDP‐GlcNAc does not involve lipid intermediates.
About the specificity of photoinduced affinity labeling of Escherichia coli ribosomes by dihydrorosaramicin, a macrolide related to erythromycin
Authors: SIEGRIST Sylvie , MOREAU Nicole , LE GOFFIC François
Published: March 2005

Photoactivation of the [3H]dihydrorosaramicin chromophore at a wavelength above 300 nm allows the covalent attachment of the macrolide antibiotic to the bacterial ribosome. Bidimensional electrophoresis shows that the radioactivity is mainly associated with proteins L1, L5, L6, L15, L18, L19, S1, S3, S4, S5 and S9. When photoincorporation of the drug is conducted in the presence of puromycin as effector of [3H]dihydrorosaramicin‐binding sites, a decrease in the labeling of most proteins is observed, except for L18 and L19, which are radiolabeled to a larger extent. These results allow us to speculate that L18 and L19 belong to the high‐affinity binding site of rosaramicin antibiotic.
Cross‐linking of bovine mitochondrial H+‐ATPase by copper–o‐phenanthroline
Authors: TOROK Katalin , JOSHI Saroj
Published: March 2005

The nearest neighbor relationships between the F0 subunits of bovine mitochondrial H+‐ATPase were studied by using copper–o‐phenanthroline, an SH‐oxidizing cross‐linking reagent. The cross‐linked samples of purified H+‐ATPase, F1‐ATPase or F0 were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‐PAGE) and the disulfide cross‐linked polypeptides were identified by enzyme‐linked immunosorbent assay and immunoblot transfer using subunit specific antisera. SDS‐PAGE of H+‐ATPase showed several cross‐links, although none involved subunits of F0 sector linked to those of F1. Both H+‐ATPase and F0 showed formation of a 45‐kDa product. Upon reduction, the 45‐kDa component gave rise to a 21‐kDa band, identified as oligomycin‐sensitivity‐conferring protein (OSCP), and a 24‐kDa band. These two proteins thus appear to be near neighbors with their cysteine residues in close proximity with each other. Under the conditions of cross‐linking, there was a concentration‐dependent decrease in the Pi‐ATP exchange activity of the intact H+ ‐ATPase as well as of H+‐ATPase reconstituted with copper–o‐phenanthroline‐treated F0 and untreated F1. The site of inhibition appeared to residue in the F0 sector. Loss of Pi‐ATP exchange occurred at the same time as formation of the 45‐kDa product. Our present data showing copper–o‐phenanthroline‐induced interactions of the 24‐kDa protein with the OSCP and simultaneous inactivation of Pi‐ATP exchange activity of the complex strengthen earlier suggestions [Hadikusumo, R. G., Hertzog, P. J. & Marzuki, S. (1984) Biochim. Biophys. Acta 765, 258–267] that the 24‐kDa protein may be a bona fide subunit of F0.
The role of the proton motive force and electron flow in solute transport in Escherichia coli
Authors: ELFERINK Marieke G. L. , HELLINGWERF Klaas J. , KONINGS Wil N.
Published: March 2005

Transport of lactose and methyl β‐d‐thiogalactopyranoside, a melibiose analogue, was studied in intact cells of Escherichia coli. A proton motive force could drive the translocation of these solutes via these two transport systems, but the initial rates and steady‐state levels of solute accumulation increased upon initiation of electron transfer. When the absolute value of the proton motive force was decreased by ionophores the steady‐state levels of lactose accumulation did not decrease as expected if thermodynamic equilibrium with the proton motive force had existed. Accumulation of lactose was also observed in the absence of any measurable proton motive force as long as electron transfer took place. Since both proton/lactose and sodium/methyl β‐d‐thiogalactopyranoside symport showed the same characteristics, an explanation based on local proton diffusion pathways is unlikely.
Comparison of ornithine transcarbamylase from rat liver and intestine
Authors: WRAIGHT Christopher , LINGELBACH Klaus , HOOGENRAAD Nicholas
Published: March 2005

Ornithine transcarbamylase (OTCase) was purified from the small intestine of rat and the properties of the gut enzyme were compared with those of the enzyme from liver. The enzymes from both sources bound to the transition‐state analog inhibitor, δ‐N‐(phosphonoacetyl)‐L‐ornithine, immobilized on Sepharose and eluted with carbamyl phosphate as a homogeneous preparation. The specific activities of the pure enzymes were 966 μmol min−1 mg−1 and 928 μmol min−1 mg−1 from liver and gut respectively, and the molecular mass, based on electrophoretic mobility, was 38000 Da. The isoelectric point of the enzymes from both sources was 7.3. The enzymes from both sources cross‐react to the same extent with antibodies against the liver enzyme on Western transfers and the size of the mRNA was identical on Northern transfers probed with a cDNA for the liver enzyme. Although OTCase is apparently the same gene product in both liver and gut, the enzyme levels respond differently to alterations in the protein content of the diet. OTCase in liver increased from 0.76 μmol min−1μg−1 DNA on 15% casein to 1.3 μmol min−1μg−1 DNA on 60% casein (P < 0.01) whereas in small intestine the level decreased from 8.8 nmol min−1μg DNA on 15% casein to 5.7 nmol min−1 DNA on 60% casein (P < 0.05). When expressed on a fresh‐weight basis, the enzyme activity in liver shows the characteristic increase with increasing protein, whereas the activity in gut does not. The connection between these differences in gene expression and the different physiological roles of OTCase in liver and gut is discussed.
Metabolism of 2‐oxoaldehydes in yeasts
Authors: INOUE Yoshiharu , WATANABE Kunihiko , SHIMOSAKA Makoto , SAIKUSA Toshihiko , FUKUDA Yasuki , MURATA Kousaku , KIMURA Akira
Published: March 2005

NAD‐dependent lactaldehyde dehydrogenase, catalyzing an oxidation of lactaldehyde to lactate, was purified approximately 70‐fold from cell extracts of Saccharomyces cerevisiae with a 28% yield of activity. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The relative molecular mass of the enzyme was estimated to be 40000 on Sephadex G‐150 column chromatography and on sodium sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.5, 60°C and specifically oxidized L‐lactaldehyde to L‐lactate in the presence of NAD. The Km values for L‐lactaldehyde and NAD were 10 mM and 2.9 mM, respectively. The purest enzyme was extremely unstable and almost completely inactivated during storage at ‐20°C, pH 7.5. For the reactivation of the enzyme, halide ions such as Cl−, I− and Br− were required.
Identification of 2‐keto‐3‐deoxy‐1,7‐dicarboxyheptonic acid as a constituent of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305
Authors: BRADE Helmut , RIETSCHEL Ernst Th.
Published: March 2005

A 3‐deoxyaldulosonic acid, not previously described, was identified as a component of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305: 2‐keto‐3‐deoxy‐1,7‐dicarboxyheptonic acid. This compound, whose steric configuration has not been determined, was released from lipopolysaccharide after acid hydrolysis as monosaccharide and in the form of a glucose‐substituted disaccharide. It was identified by combined gasliquid chromatography/mass spectrometry using various derivatization procedures. The presence of a 2‐keto‐3‐deoxyaldulosonic acid was established from the typical fragmentation pattern of the C1–C6 moiety which was identical to that of similarly similarly derivatized authentic 2‐keto‐3‐deoxy‐D‐Mannooctonic acid (dOclA). Location of the keto and deoxy function at C2 and C3, respectively, was deduced from the fragmentation pattern of the deuterolabelled derivatives after reduction of the keto and carboxy groups. The presence of two carboxy groups was ascertained after carboxy reduction with sodium boro‐(2H)hydride of the carbonyl‐reduced and permethylated derivative whereby the molecular masses were found to differ by 4 Da. Further proof for the presence of two carboxy groups was obtained by alkaline transesterification of the carbonyl‐reduced and permethylated derivative with ethanolic sodium ethylate: it was shown that two methyl ester groups had been transesterified since the molecular mass was shifted higher by 28 Da. Substitution by a D‐glucopyranosyl residue in position 4 was established by methylation analysis performed on the carbonyl‐ and carboxyl‐reduced and permethylated dissaccharide. The D‐configuration of the glucosyl residue was determined by its reactivity with D‐glucose oxidase.

Thus, the structure of the disaccharide is 4‐O‐glucopyranosyl‐2‐keto‐3‐deoxy‐1,7‐dicarboxyheptonic acid.
Bovine serum brevin
Authors: SOUA Zohra , PORTE Francoise , HARRICANE Marie‐Cecile , FEINBERG Jeanne , CAPONY Jean‐Paul
Published: March 2005

Brevin, an actin‐severing protein present in serum from numerous mammals, has been purified to homogeneity from bovine serum, using hydrophobic chromatography as the last purification step. The physicochemical parameters of brevin have been established and some of them studied in the absence and presence of Ca2+. Brevin exhibits an apparent Stokes radius, RS, of 3.4 nm, an intrinsic sedimentation coefficient S°20, w, of 4.8 S and 4.4 S in the absence and presence of Ca2+ respectively, indicative of calcium‐induced conformational change. The native molecular mass of brevin was found to be 68 kDa and the hydrodynamic data suggest that the protein is an asymmetric molecule. Sedimentation equilibrium studies demonstrated that Ca2+ affects the shape (asymmetry) of brevin without altering its molecular mass. Limited tryptic and chymotryptic digestion of brevin distinguishes the Ca2+‐induced conformation from the EGTA one. No change in the electrophoretic migration of brevin was seen upon Ca2+ addition. Several isoforms were detected by two‐dimensional gel electrophoresis. Brevin increases the rate of nucleation of actin but decreases the rate of elongation of the filaments and the steady‐state viscosity of F‐actin in substoïchiometric amounts, as measured by viscometric assays under high shear conditions. Electron microscopic examination documents these effects. Brevin produces shorter actin filaments and binds to the ‘barbed’ end of filaments to which monomers add preferentially during elongation, as demonstrated by indirect immunogold staining of antibodies against brevin. Filament elongation occurs only at the slowly growing end. An enzyme‐linked immunosorbent assay was developed and used to detect and quantify brevin and related proteins in extracts of different bovine cells and tissue. Liver and smooth muscles were found to contain the highest amounts of the severing protein.
Pig thyroid spectrin
Authors: REGNOUF Francoise , NGUYEN Eric , CASSOLY Robert , PRADEL Louise‐Anne
Published: March 2005

Thyroid spectrin has been obtained pure from pig thyroid glands. This protien, composed of two non‐identical polypeptide chains of 240 kDa and 235 kDa, appears to possess the same structural and immunological properties as well as the same calmodulin and actin‐binding properties as brain spectrin. Through cross‐linking of actin filaments it is a potent gelation factor for F‐actin solutions. It represents one of the major protein of the cytoskeleton underlying the thyroid plasma membrane together with myosin, α‐actinin and actin.
Purification, properties and kinetic mechanism of flavonol 8‐O‐methyltransferase from Lotus corniculatus L.
Authors: JAY Maurice , DE LUCA Vincenzo , IBRAHIM Ragai K.
Published: March 2005

A novel O‐methyltransferase catalyzing the transfer of the methyl group of S‐adenosyl‐L‐methionine to the 8‐hydroxyl group of flavonols was purified about 1200‐fold from Lotus flower buds, by precipitation with ammonium sulfate and successive chromatography on columns of Sephadex G‐100, S‐adenosyl‐L‐homocysteine‐Agarose, hydroxyapatite and Polybuffer ion exchanger. The enzyme exhibited strict specificity for position 8 of 8‐hydroxyquercetin and 8‐hydroxykaempferol, a pH optimum at 7.9, a pI value of 5.5, an Mr of 55X103 and required Mg2+ and SH groups for activity. The Km values for 8‐hydroxykaempferol and S‐adenosyl‐L‐methionine were 1.3 μM and 53 μM, respectively. The data obtained from substrate interaction and product inhibition studies are expected for a steady‐state ordered bi‐bi mechanism, with 8‐hydroxyflavonol binding before S‐adenosyl‐L‐methionine followed by the release of S‐adenosyl‐L‐homocysteine and 8‐methoxyflavonol. An alternative mechanism that may also fit the data is the mono‐iso Theorell‐Chance with the inverse binding sequence and an isomerization step of the free enzyme.
Activation of glycolysis by insulin with a sequential increase of the 6‐phosphofructo‐2‐kinase activity, fructose‐2,6‐bisphosphate level and pyruvate kinase activity in cultured rat hepatocytes
Authors: PROBST Irmelin , UNTHAN‐FECHNER Kirsten
Published: March 2005

The involvement of 6‐phosphofructo‐2‐kinase, fructose 2,6‐bisphosphate [Fru(2,6)P2] and pyruvate kinase in the insulin‐dependent short‐term activation of glycolysis was studied in primary cultures of rat hepatocytes. The short‐term influence of insulin on these parameters was dependent on the insulin concentration used for the longterm culture. Cells were cultured either with 10 nM or 0.1 nM insulin for 48 h, and are referred to as ‘insulin cells’ and ‘control cells’, respectively.

1. Insulin cells exhibited a high level of Fru(2,6)P2. Addition of insulin to insulin cells led to an immediate stimulation of glycolysis (two‐fold) and activation of pyruvate kinase. The concentration of Fru(2,6)P2 and activity of 6‐phosphofructo‐2‐kinase remained constant.

2. Control cells exhibited a very low level of Fru(2,6)P2 and low activity of 6‐phosphofructo‐2‐kinase directly after the medium change. However, both parameters increased during a 1–2‐h−incubation in the absence of insulin. Although the level of Fru(2,6)P2 thus changed up to tenfold the glycolytic rate remained at a constant value.

3. Addition of insulin to control cells led to a 5–8‐fold stimulation of glycolysis but only after a 30–90‐min lag phase. During this lag period insulin strongly increased sequentially the 6‐phosphofructo‐2‐kinase, the level of Fru(2,6)P2 and the pyruvate kinase activity. The activation of the latter enzyme slightly preceded the onset of the insulin‐stimulated glycolysis.

4. Addition of insulin to control cells, which were preincubated for 3 h in the absence of insulin and in which the Fru(2,6)P2 level had risen insulin‐independently, led to an immediate increase in glycolysis without a lag phase.

It is concluded that in this insulin‐sensitive cell system: (a) the changes of glycolytic flux did not correlate with changes in the level of total Fru(2,6)P2 either in insulin or in control cells; (b) an increase in the Fru(2,6)P2 concentration was not obligatory for the insulin‐dependent stimulation of glycolysis in insulin cells; (c) activation of pyruvate kinase and thus glycolysis by insulin did not proceed unless the Fru(2,6)P2 level had been elevated above a threshold level.

The lack of correlation between total Fru(2,6)P2 levels and the glycolytic flux and the apparent existence of a threshold concentration for Fru(2,6)P2 suggest a permissive action for this effector in enzyme interconversion.
Purification and characterization of GDP‐D‐mannose 4,6‐dehydratase from porcine thyroid
Authors: BROSCHAT Kay O. , CHANG Sulie , SERIF George
Published: March 2005

The enzyme GDP‐D‐mannose 4, 6‐dehydratase has been purified 1500‐fold from porcine thyroid tisue. The enzyme exhibits a molecular mass of 251000 Da as determined by sedimentation techniques. Its subunit size was determined as 41500 Da by dodecyl sulfate gel electrophoresis. The enzyme has a Km of 3.3 μM with respect to GDP‐D‐mannose and appears specific with respect to this substrate. The enzyme appears to be inhibited by guanine nucleotides and by guanine nucleotide sugars. It is particularly susceptible to inhibition by GDP‐L‐fucose. It is suggested that this compound may have a physiological function as an end‐product feedback inhibitor.
Protein‐protein interactions and antigenic relationships between the components of 4‐methoxybenzoate monooxygenase and of benzene 1,2‐dioxygenase from Pseudomonas putida
Authors: EICH Franz , GEARY Philip John , BERNHARDT Frithjof‐Hans
Published: March 2005

The investigations presented in this paper were performed on two enzyme systems from Pseudomonas putida: (a) 4‐methoxybenzoate monooxygenase, consisting of a NADH:putidamonooxin oxidoreductase and putidamonooxin, the oxygen‐activating component, and (b) benzene 1,2‐dioxygenase, a three‐component enzyme system with an NADH: ferredoxin oxidoreductase, functioning together with a plant‐type ferredoxin as electron‐transport chain, and an oxygen‐activating component similar to putidamonooxin in its active sites. The influence of temperature, ionic strength, and pH on the activities of 4‐methoxybenzoate monooxygenase and of NADH:putidamonooxin oxidoreductase were investigated. The studies revealed that the activity of 4‐methoxy‐benzoate monooxygenase is determined by the behaviour of the reductase. Spectroscopic measurements showed that the interaction between the two components of 4‐methoxybenzoate monooxygenase influences the opticalabsorption behaviour of one or both components. As a criterion for the affinity between the two components of 4‐methoxybenzoate monooxygenase, the Km value of the reductase for putidamonooxin was determined and found to be 31 ± 11 μM. Antibodies against both components of 4‐methoxybenzoate monooxygenase were obtained from rabbits. The antibodies against putidamonooxin inhibited the O‐demethylation reaction (up to 80%) and also the reduction of putidamonooxin by the reductase (up to 40%). The antibodies against putidamonooxin did not interact with the oxygen‐activating component of benzene 1,2‐dioxygenase. The electrontransport chains of 4‐methoxybenzoate monooxygenase and benzene 1,2‐dioxygenase could not be replaced by one another without a complete loss of enzyme activity.
Calcium release from intact calmodulin and calmodulin fragment 78–148 measured by stopped‐flow fluorescence with 2‐p‐toluidinylnaphthalene sulfonate
Authors: SUKO Josef , PIDLICH Johann , BERTEL Oswald
Published: March 2005

Calcium release from high and low‐affinity calcium‐binding sites of intact bovine brain calmodulin (CaM) and from the tryptic fragment 78–148, purified by high‐pressure liquid chromatography, containing only the high‐affinity calcium‐binding sites, was determined by fluorescence stopped‐flow with 2‐p‐toluidinylnaphthalene sulfonate (TNS). The tryptic fragments 1–77 and 78–148 each contain a calcium‐dependent TNS‐binding site, as shown by the calcium‐dependent increase in TNS fluorescence. The rate of the monophasic fluorescence decrease in endogenous tyrosine on calcium dissociation from intact calcium‐saturated calmodulin (Kobs 10.8 s−1 and 3.2 s−1 at 25°C and 10°C respectively) as well as the rate of equivalent slow phase of the biphasic decrease in TNS fluorescence (Ksiowobs 10.6 s−1 and 3.0 s−1 at 25°C and 10°C respectively) and the rate of the solely monophasic decrease in TNS fluorescence, obtained with fragment 78–148 (Kobs 10.7 −1 and 3.5 s−1 at 25°C and 10°C respectively), were identical, indicating that the rate of the conformational change associated with calcium release from the high‐affinity calcium‐binding sites on the C‐terminal half of calmodulin is not influenced by the N‐terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed with intact calmodulin only (Kfastobs 280 s−1 at 10°C) but not with fragment 78–148, is most probably due to the conformational change associated with calcium release from low‐affinity sites on the N‐terminal half.

The calmodulin fragments 1–77 and 78–148 neither activated calcium/calmodulin‐dependent protein kinase of cardiac sarcoplasmic reticulum nor inhibited calmodulin‐dependent activation at a concentration approximately 1000‐fold greater (5 μM) than that of the calmodulin required for half‐maximum activation (5.9 nM at 0.8 mM Ca+2 and 5 mM Mg2+) of calmodulin‐dependent phosphoester formation.
Isolation and partial characterization of the messenger RNA encoding the B880 holochrome protein of Rhodospirillum rubrum
Authors: BÉLANGER Gilles , BÉRARD Jacques , GINGRAS Gabriel
Published: March 2005

The B880 holochrome messenger RNA was extracted from cultures of the photosynthetic bacterium Rhodospirillum rubrum. It was purified by chromatography on Sepharose 4B followed by sucrose density gradient centrifugation. The purified fractions were shown to program an Escherichia coli cell‐free system into synthesizing both the α and the ß polypeptides of the holochrome. The translation products were identified by immunoprecipitation with specific antibodies raised against these polypeptides. The latter are effective competitors with the translation products for antigen‐antibody complex formation. The purest mRNA preparations contained approximately 33% holochrome messenger RNA activity. Its most probable size, as determined by agarose gel electrophoresis in the presence of 6 M urea or methylmercuric hydroxide, is approximately 620 nucleotides. Since the combined sizes of the α and ß polypeptides add up to only 106 amino acid residues, we conclude that the holochrome mRNA is most probably polycistronic.
Comparative structural and lectin‐binding studies on γ‐glutamyltransferase from human adult liver, fetal liver and primary hepatoma
Authors: SELVARAJ Periasamy , BALASUBRAMANIAN Kunissery A.
Published: March 2005

γ‐Glutamyltransferase was purified to apparent homogeniety from human adult liver, fetal liver and hepatoma by deoxycholate extraction, immunoaffinity chromatography, papain digestion, phenyl‐Sepharose chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme from all three sources had an apparent Mr of 82000 by Sephadex G‐150 gel filtration and on dodecyl sulphate/polyacrylamide gel electrophoresis two nonidentical subunits of Mr 57000 and 23000 were obtained. The pI of all three forms was 3.85 and after neuraminidase treatment they each gave at least five bands with pI values ranging over 5.9–6.6. Sialic acid content was 188 (adult liver), 182 (fetal liver) and 188 (hepatoma) nmol/mg protein. Total neutral sugar content was 702 (adult and fetal liver) and 700 (hepatoma) nmol/mg protein. The hexosamine content of the enzyme from all the three sources was the same (354 nmol/mg protein) and galactosamine was absent. Partially purified hydrophobic and hydrophilic forms of γ‐glutamyltransferase from all the three sources were precipitated by Concanavalin A, Ricinus communis agglutinin and wheat germ agglutinin. These results show that γ‐glutamyltransferase from human adult liver, fetal liver and hepatoma are structurally similar and that the elevated levels found in fetuses and hepatoma are only a quantitative increase and are not due to a new isoenzyme.
Erabutoxin b
Authors: BOURNE Philip E. , SATO Atsushi , CORFIELD Peter W. R. , ROSEN Lawrence S. , BIRKEN Steven , LOW Barbara W.
Published: March 2005

The crystal structure of the protein postsynaptic neurotoxin, erabutoxin b, has been refined at 0.140‐nm resolution (R= 0.22) by restrained least‐squares and interactive computer graphics. The study has established complete structural identity of the two sea‐snake venom toxins, erabutoxin b and neurotoxin b, isolated from Laticauda semifasciata snakes taken in different Pacific Ocean waters. Two chemical‐sequence inversion errors in erabutoxin b have been discovered during refinement, corrected and subsequently confirmed in both erabutoxin b and erabutoxin a by chemical analysis. The correct sequences are His6‐Gln7, hitherto unsuspected, and Ser18‐Pro19. The Sequence correction His6‐Gln7 explains the anomalous results of 1H NMR solution studies and those of early chemical modification experiments, which were in conflict with the previously published three‐dimensional structure of erabutoxin b. On refinement, the five stranded beta sheet described earlier is now shown to be discontinuous, split into a two‐stranded beta loop and a three‐stranded beta sheet. Unique features of the Pro44‐Gly49 peripheral segment have now been identified. 51 water molecule positions have been located.
Fluorescent anticytokinins as a probe for binding
Authors: HAMAGUCHI Nobuko , IWAMURA Hajime , FUJITA Toshio
Published: March 2005

4‐Substituted 2‐methylthiopyrido[2,3‐d]pyrimidines, a series of recently developed anticytokinins, have been found in fluoresce strongly in water and to be useful as probes for binding studies. The binding activity of the soluble proteins and particulate fraction of tobacco callus cells to the biologically most active member of the family, 4‐n‐butylamino‐2‐methylthiopyrido[2,3‐d]pyrimidine (BAMPP), was studied fluorimetrically. We found that the binding activity is better monitored in terms of saturable binding rather than in terms of the amount of bound ligand, a conventional method used in isolation studies of hormone receptor proteins. Using this technique we isolated two kinds of high‐affinity cytokinin‐binding proteins from the soluble fraction and identified a high‐affinity binding site on ribosomes.
Interaction of actinogelin with actin
Authors: OHTAKI Tetsuya , TSUKITA Sachiko , MIMURA Naotoshi , TSUKITA Shoichiro , ASANO Akira
Published: March 2005

Nucleation activity of actin polymerization of actinogelin, a calcium‐ sensitive F‐actin cross‐linking protein from rat liver, was measured by a fluorescence enhancement method using pyrenyl‐actin and by high shear viscometry. No stimulation of nucleation by the addition of actinogelin was observed under several ionic conditions using the fluorescent method. Similar results were also obtained by viscometry. Therefore, it can be concluded that actinogelin has no nucleation activity for actin polymerization.

By electron microscopy, it was found that actinogelin molecule has a dumbbell shape, binds to side of F‐actin through its end(s), and cross‐links actin filaments by binding with its two ends. It was also found that meshwork formation occurred in low Ca2+ conditions from F‐actin and actinogelin. Under non‐gelling high Ca2+ conditions, binding of actinogelin along the side of F‐actin with its one end was still detected in accordance with the binding assay using ultracentrifugation and protein determination.

Under low Ca2+ conditions, the critical gelling concentration of actinogelin measured by low shear viscometry at 20°C was 6 μg/ml for 250 μg/ml of actin. Comparing this value with those of the other actin cross‐linking proteins, it was found that actinogelin was one of proteins with the highest gelation activity. On the other hand, gelation activity of actinogelin in high Ca2+ conditions was one order of magnitude lower; than 50 μg/ml of the protein was required for gelation. At 37°C, gelation activity of actinogelin at low Ca2+ concentration was decreased to about a quarter of that at 20°C, but this was still higher than that of gizzard α‐actinin at 20°C. Thus, role of actinogelin as an efficient and Ca2+ ‐regulated cross‐linker of microfilaments was substantiated.
An assessment of the importance of intralysosomal and of α‐amylolytic glycogenolysis in the liver of normal rats and of rats with a glycogen‐storage disease
Authors: VANDEBROECK Alfons , BOLLEN Mathieu , DE WULF Henri , STALMANS Willy
Published: March 2005

Mechanisms of glycogenolysis have been investigated in a comparative study with Wistar rats and gsd rats, which maintain a high glycogen concentration in the liver as a result of a genetic deficiency of phosphorylase kinase.

In Wistar hepatocytes the rate of glycogenolysis, as modulated by glucagon and by glucose, was proportional to the concentration of phosphorylase a. In suspensions of gsd hepatocytes the rate of glycogenolysis was far too high as compared with the low level of phosphorylase a; in addition, only a minor fraction of the glycogen lost was recovered as glucose and lactate, owing to the accumulation of oligosaccharides. When the gsd hepatocytes were incubated in the presence of an inhibitor of α‐amylase (BAY e 4609) glycogenolysis and the formation of oligosaccharides virtually ceased; the production of glucose plus lactate; already modest in the absence of BAY e 4609, was further decreased by 40%, owing to the suppression of a pathway for glucose production by the successive actions of α‐amylase and α‐glucosidase.

Evidence was obtained that gsd hepatocytes are more fragile, and that amylolytic of glycogen occurred in damaged cells and/or in the extracellular medium. This may even occur in vivo, since quick‐frozen liver samples from anesthetized gsd rats contained severalfold higher concentrations of oligosaccharides than did similar samples from Wistar rats. However, administration of a hepatotoxic agent (CCl4) caused hepatic glycogen depletion in Wistar rats, but not in gsd rats.

The administration of phloridzin and of vinblastine, which have been proposed to induce glycogenolysis in the lysosomal system, did not decrease the hepatic glycogen level in gsd rats.

Taken together, the data indicate that only the phosphorolytic degradaion of glycogen is metabolically important, and that α‐amylolysis is an indication of an increased fragility of gsd hepatocytes, which becomes prominent when these cells are incubated in vitro.