bluuebunny/crossref_metadata_2025_split huggingface dataset:
Temperature‐induced conformational transition in rabbit muscle aldolase studied by temperature dependence of sulfhydryl reactivity
Authors: HEYDUK Tomasz , KOCHMAN Marian 
Published: March 2005

A temperature‐induced non‐denaturing conformational transition in rabbit muscle aldolase has been as subject of discussion and controversy for some period of time. In this study the temperature dependence of the reactivity of aldolase SH groups is investigated in order to detect subtle changes in the enzyme conformation.For model thiol‐containing systems such as cysteine, glutathione and bovine serum albumin, linear Arrhenius plots have been obtained for the reaction with 5,5′‐dithiobis(2‐nitrobenzoic acid). On the other hand, for rabbit muscle pyruvate kinase, a protein which undergoes temperature‐induced conformational transition, the plot obtained is nonlinear with a break at the temperature (18 °C) close to that reported earlier. In the case of aldolase the Arrhenius plots for three slowly reacting SH groups (Cys‐72, 289, 338) and a fast reacting group (Cys‐239) are nonlinear with a break at about 26–27 °C. The fluorescence measurements show that a plot of the fluorescence intensity of tryptophan residues versus temperature exhibits a break at the same temperature. It is shown that the observed conformational change is fully reversible. In the presence of the competitive inhibitor hexitol 1,6‐bisphosphate, which is known to protect Cys‐72 and Cys‐338 from chemical modification, the Arrhenius plot exhibits a break for the fast reacting Cys‐239 residue and is linear for the slowly reacting Cys‐289. It is found that 0.6 M urea increases the transition temperature for all exposed SH groups of aldolase. The above results show that at several points in the aldolase molecule, including the active‐site region, an abrupt change of microenvironments takes place with temperature. The competitive inhibitor protects a portion of aldolase molecule against the thermal transition.
Lipopolysaccharides from Yersinia pestis
Authors: DALLA VENEZIA Nicole , MINKA Samuel , BRUNETEAU Maud , MAYER Hubert , MICHEL Georges 
Published: March 2005

The chemical structure of the lipid A of lipopolysaccharide I and II from Yersinia pestis, strain EV 40, was studied. It consists of a (1 → 6), β‐linked d‐glucosamine disaccharide which carries two phosphate groups; one phosphate is linked glycosidically with a glucosamine unit, the other one is linked to the non‐reducing glucosamine. Various degradation methods combined with 31P nuclear magnetic resonance spectroscopy showed that the esterbound phosphate group is linked to a 4‐aminoarabinosyl residue and the glycosidically linked phosphate group is linked to a d‐arabinofuranosyl residue in lipopolysaccharide II and to the phosphorylethanolamine in lipopolysaccharide I.The hydroxyl groups of the disaccharide are acylated by dodecanoic, hexadecenoic, 3‐hydroxytetradecanoic and 3‐dodecanoyloxytetradecanoic acids.The amino groups of the disaccharide carry 3‐hydroxytetradecanoic and 3‐dodecanoyloxytetradecanoic acids. In addition smaller amounts of 3‐tetradecanoyloxyltetradecanoic and 3‐hexadecanoyloxytetradecanoic acids are present in ester linkage.
Covalent cross‐linking of vasoactive intestinal peptide (VIP) to its receptor in intact colonic adenocarcinoma cells in culture (HT 29)
Authors: MULLER Jean‐Marc , LUIS José , FANTINI Jacques , ABADIE Brigitte , GIANNELLINI Fernand , MARVALDI Jacques , PICHON Jacques 
Published: March 2005

[1 2 5I]Monoiodinated vasoactive intestinal peptide (125I‐VIP) was cross‐linked with human colonic adenocarcinoma cells (HT 29 cells) grown as a monolayer using dithiobis(succinimidylpropionate) as cross‐linking reagent. The cross‐linked polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.A major polypeptide of Mr= 67000 was characterized and it behaved like a high‐affinity binding site for VIP according to the following data.

The concentration of native VIP (0.5 nM) giving half‐maximum inhibition of 125I‐VIP covalent cross‐linking with this polypeptide was very similar to that giving half‐maximum displacement of 125I‐VIP on HT 29 cells (0.6 nM).
Glucagon or insulin was unable to inhibit the labelling of the Mr‐67000 component.
In our experimental conditions neither specific 125I‐VIP binding nor covalent labelling was observed with monolayers of Madin Darby canine kidney epithelial cells (MDCK cells) or African green monkey kidney fibroblasts (Vero cells) while the Mr‐67000 polypeptide was also characterized with human rectal adenocarcinoma cells (HRT 18 cells), known to possess the VIP receptor.
Preincubation of HT 29 cells with native VIP at 37°C, before 125I‐VIP binding and subsequent crosslinking reaction, decreased the labelling of the Mr‐67000 polypeptide up to 80%.

Assuming one molecule of 125I‐VIP cross‐linked per polypeptide, we have characterized, for the first time, a major polypeptide of Mr= 64000, which belongs to the high‐affinity VIP binding site of an intestinal human cell line.
Kinetic co‐operativity of monomeric mnemonical enzymes
Authors: RICARD Jacques , NOAT Georges 
Published: March 2005

The expression of the kinetic Hill coefficient for a two‐substrate, two‐product mnemonical enzyme has been derived. Its relation with the Λ coefficient, that is the slope of the reciprocal plots for 1[A] →0, has been established. The variation of this Hill coefficient, as a function of the second substrate and product concentrations, has been studied theoretically. Whereas the Λ coefficient does not vary as a function of the substrate and first product concentrations, the kinetic Hill coefficient does. If the enzyme is positively co‐operative, the Hill coefficient increases upon increasing the second substrate concentration and decreases if the first product concentration is increased. The converse is expected to occur if the enzyme displays a negative co‐operativity. The last product may either reverse a positive co‐operativity into a negative one or, alternatively, strengthen an already negative co‐operativity. The co‐operativity generated by the mnemonical model has been compared to the kinetic behaviour of a random model. These two models have been shown to be discriminated on the basis of the departure they show with respect to the Michaelis‐Menten behaviour.These theoretical considerations have been applied to previously published data, obtained with wheat germ hexokinase LI. This monomeric enzyme has a negative co‐operativity with respect to the preferred substrate, glucose. The Hill coefficient decreases with MgADP concentration and decreases with glucose‐6‐phosphate concentration. This is exactly what is to be exactly what is to be expected on the basis of the above theory of kinetic co‐operativity.
Structure of the capsular antigen of Neisseria meningitidis serogrup K
Authors: VAN DER KAADEN Arie , GERWIG Gerrit J. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. , TIESJEMA Rudy H. 
Published: March 2005

The capsular antigen isolated from the culture liquid of a Neisseria meningitidis serogroup K(1811) fermentation consists of the 2‐acetamido‐2‐deoxymannuronic acid disaccharide repeating unit as follows:
→3)‐β‐d‐ManpNAcA‐(1→4)‐β‐d‐ManpNAcA‐(1→The polysaccharide is O‐acetylated at the non‐glycosylated C‐4. Structural evidence has been obtained from sugar analysis, methylation analysis, as well as 1H and 13C NMR spectroscopy.
Regulation of flux through pyruvate dehydrogenase and pyruvate carboxylase in rat hepatocytes
Authors: AGIUS Loranne , ALBERTI K. George M. M. 
Published: March 2005

The regulation of flux through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) by fatty acids and glucagon was studied in situ, in intact hepatocyte suspensions.
The rate of pyruvate metabolized by carboxylation plus decarboxylation was determined from the incorporation of [1‐14C]pyruvate into 14CO2 plus [14C]glucose. The flux through PDH was determined from the rate of formation of 14CO2 from [1‐14C]pyruvate correted for other decarboxylation reactions (citrate cycle, phosphoenolpyruvate carboxykinase and malic enzyme), and the flux through PC was determined by substracting the flux through PDH from the total pyruvate metabolized.
With 0.5 mM pyruvate as substrate the ratio of flux through PDH/PC was 1.9 in hepatocytes from fed rats and 1.4 in hepatocytes from 24 h‐starved rats.
In hepatocytes from fed rats, octanoate (0.8 mM) and palmitate (0.5 mM) increased the flux through PDH (59–76%) and PC (80–83%) without altering the PDH/PC flux ratios. Glucagon did not affect the flux through PDH but it increased the flux through PC twofold, thereby decreasing the PDH/PC flux ratio to the value of hepatocytes from starved rats.
In hepatocytes from starved rats, fatty acids had similar effects on pyruvate metabolism as in hepatocytes from fed rats, however glucagon did not increase the flux through PC.
2[5(4‐Chlorophenyl)pentyl]oxirane‐2‐carboxylate (100 μM) an inhibitor of carnitine palmitoyl transferase I, reversed the palmitate‐stimulated but not the octanoate‐stimulated flux through PDH, in cells from fed rats, indicating that the effects of fatty acids on PDH are secondary to the β‐oxidation of fatty acids. This inhibitor also reversed the stimulatory effect of palmitate on PC and partially inhibited the flux through PC in the presence of octanoate suggesting an effect of POCA independent of fatty acid oxidation.
It is concluded that the effect of fatty acids on pyruvate metabolism are probably secondary to increased pyruvate uptake by mitochondria in exchange for acetoacetate. Glucagon favours the partitioning of pyruvate towards carboxylation, by increasing the flux through pyruvate carboxylase, without directly inhibiting the flux through PDH.
Action of serine carboxypeptidases on endopeptidase substrates, peptide‐4‐methyl‐coumaryl‐7‐amides
Authors: KUNUGI Shigeru , FUKUDA Mitsuhiro , HAYASHI Rikimaru 
Published: March 2005

Carboxypeptidase Y hydrolyzed N‐substituted peptide‐4‐methylcoumarin‐7‐amides (peptide‐NH‐Mec) at pH 7 by releasing 7‐amino‐4‐methylcoumarin (NH2‐Mec) which was then followed by carboxypeptidase action. In particular, a chymotrypsin‐directed substrate, Suc‐Leu‐Leu‐Val‐Tyr‐NH‐Mec, was hydrolyzed by the enzyme with a second‐order rate constant of 7200 M−1 S−1, which is compatible with the rate for an anilide substrate and some N‐substituted dipeptides. The activity was completely inhibited by phenylmethylsulfonyl fluoride and competitively depressed by the presence of an N‐substituted dipeptide. Dependences of kinetic parameters on pH were different from those of carboxypeptidase, esterase, amidase, and anilidase activities.
Carboxypeptidases P from Penicillium janthinellum and W from wheat also hydrolyzed some of these peptide‐NH‐Mec derivatives in a similar manner but at a rather low rate. Thus, the NH2‐Mec‐releasing activity may be considered to be intrinsic to serine carboxypeptidases in general.
Taking into consideration this endopeptidase‐like activity of serine carboxypeptidases, fluorogenic substrates should be used carefully to specify endopeptidases in crude extracts.
Metabolism of polyunsaturated fatty acids by rabbit reticulocytes
Authors: SALZMANN‐REINHARDT Ulrike , KÜHN Hartmut , WIESNER Rainer , RAPOPORT Samuel 
Published: March 2005

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1‐14C]linoleic acid and [1‐14C]arachidonic acid by three different pathways.

Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono‐ di‐ and triacylglycerols, but not cholesterol esters).
Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14‐eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15‐Hydroxy‐5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13‐hydroxy‐9,11(Z,E)‐octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via β‐oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids.
β‐Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via β‐oxidation to 14CO2. Addition of 5,8,11,14‐eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished β‐oxidation.

Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.
Purification and characterization of esterases D1 and D2 from human erythrocytes
Authors: MATSUO Kiyosato , KOBAYASHI Kunihiko , HAGIWARA Keiji , KAJII Tadashi 
Published: March 2005

Esterase D1 and esteras D2, two common esterase D (EC 3.1.1.1) isozymes, were isolated and purified from human erythrocytes. Their substrate specificity, pH profile and Km values were essentially identical. Their molecular mass was the same at 34 kDa on sodium dodecyl sulfate/polyacrylamide electrophoresis and at 27 kDa on Sephadex G‐100 gel filtration. Antisera to each of the esterase D1 and esterase D2 isozymes were successfully raised in chickens; each antiserum reacted identically with both isozymes. These findings indicate that the isozymes are close to each other in structure.The fact that the molecular mass of the esterase D1 and esterase D2 isozymes computed on sodium dodecyl sulfate/polyacrylamide electrophoresis was close to that obtained on Sephadex G‐100 gel filtration in nondissociating buffer indicates that the isozymes are not dimers bound by disulfide bonds or a noncovalent force. These facts together indicate that the esterase D isozymes are monomers, contrary to the prevailing view that they are dimers.
Autophagic‐lysosomal and mitochondrial sequestration of [14C]sucrose
Authors: TOLLESHAUG Helge , SEGLEN Per O. 
Published: March 2005

[14C]Sucrose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered by sedimentable subcellular particles during incubation of the cells at 37°C. The sedimentation characteristics of particle‐associated [14C]sucrose were different from the lysosomal marker enzyme acid phosphatase, suggesting an involvement of organelles of greater size than the average lysosome.Isopycnic banding in isotonic metrizamide/sucrose density gradients resolved two major peaks of radioactivity: a light peak (1.08‐1.10 g/ml) coinciding with lysosomal marker enzymes, and a dense peak (1.15 g/ml), coinciding with a mitochondrial marker enzyme. The dense peak was preferentially associated with large‐size particles having the sedimentation properties of mitochondria, and it was resistant to the detergent digitonin at a concentration which extracted all of the radioactivity in the light peak. Similarly the autophagy inhibitor 3‐methyladenine prevented accumulation of [14C]sucrose in the light peak, while the radioactivity in the dense peak was unaffected. We therefore tentatively conclude that the light peak represents autophagic sequestration of [14C]sucrose into lysosomes (and probably autophagosomes) while the dense peak represents a mitochondrial uptake unrelated to autophagy.
High‐uptake and low‐uptake forms of proteoglycans secreted by arterial smooth muscle cells
Authors: SCHMIDT Annette , BUDDECKE Eckhart 
Published: March 2005

Cultured arterial smooth muscle cells synthesize and secrete two types of sulfated proteoglycans, designated as proteoglycan A and B, into the culture medium. They are isolated as immunologically distinct monomers with relative molecular masses of 280 × 103 and 180 × 103 and are characterized as chondroitin‐sulfate‐rich (A) and dermatan‐sulfate‐rich (B) proteoglycans. Both proteoglycan A and B were labelled with [35S]sulfate and used for studies of endocytosis.
Uptake of proteoglycan B by arterial smooth muscle cells shows saturable kinetics. At saturation (500 μ) one cell may endocytose up to 1.5 × 106 proteoglycan B molecules/h. Proteoglycan A is internalized at a 10‐fold lower rate. No saturation kinetics were observed at high proteoglycan A concentrations (500 μ).
Endocytosis of proteoglycan B in the presence of an excess of proteoglycan A and vice versa suggest that proteoglycan A and B do not compete for the same receptor site. Free hyaluronate or chondroitin sulfate do not inhibit the uptake of proteoglycan B or A.
The results suggest that proteoglycan B is internalized by arterial smooth muscle cells via a high‐affinity receptor‐mediated process, whereas proteoglycan A is taken up by fluid endocytosis and/or by low‐affinity endocytotic processes.
Steady‐state kinetics of skeletal muscle myosin light chain kinase indicate a strong down regulation by products
Authors: GEUSS Ursula , MAYR Georg W. , HEILMEYER Ludwig M. G. 
Published: March 2005

The kinetic behaviour of myosin light chain kinase isolated from skeletal muscle was studied under steadystate conditions using highly purified phosphorylatable light chains 2 (LC2).Forward reaction, product inhibition, and reverse reaction data indicate a sequential mechanism which can be interpreted best by a rapid‐equilibrium random bi‐bi reaction model. The forward reaction parameters are KATP= 150 μM, KLC2= 5.3 μM, and Ki, LC2= 7.6 μM. The enzyme forms a dead‐end complex with ADP and light chain 2; Kd, ADP of this complex is 50 μM. The forward reaction is also strongly inhibited by the phosphorylated light chain 2, Ki, LC2P is 1.5 μM. An equilibrium constant Keq of about 70 can be calculated from the kinetic parameters which agrees with the directly measured value of about 60.The role of the two inhibitory mechanisms in the regulation of the enzyme and of the high energy of the light chain phosphate bond as deducible from Keq are discussed.
Solubilization and reconstitution of the membrane‐bound carotenogenic enzymes from daffodil chromoplasts
Authors: BEYER Peter , WEISS Günter , KLEINIG Hans 
Published: March 2005

The membrane‐bound carotenogenic enzymes of daffodil (Narcissus pseudonarcissus) chromoplast membranes, i.e. dehydrogenation, cis‐trans isomerization and cyclization activities, were solubilized in an enzymatically inactive form using the zwitterionic detergent 3‐[(3‐cholamidopropyl)‐dimethylamino]‐1‐propanesulfonate (Chaps).
Full enzymatic activities were regained upon reconstitution of the solubilized proteins into liposomes. These preparations converted radiolabelled cis‐phytoene into β‐carotene in a high yield. The reconstituted enzymatic sequence behaved as a tight ‘assembly line’.
In the enzymatic sequence of the reconstituted system the cis‐trans isomerization reaction occurred on the stage of cis‐phytofluene after a dehydrogenation of cis‐phytoene.
Modification of nuclear matrix proteins by ADP‐ribosylation
Authors: WĘSIERSKA‐GĄDEK Józefa , SAUERMANN Georg 
Published: March 2005

Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP‐ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography.In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP‐ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography.By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa.Approximately 2% of the total nuclear ADP‐ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix‐associated enzyme catalyzes the incorporation of [32P]ADP‐ribose into acid‐insoluble products of molecular mass 116 kDa and above, in a 3‐aminobenzamide‐inhibited, time‐dependent reaction.The possible function of ADP‐ribosylation of nuclear matrix proteins and of the attachment of ADP‐ribosyltransferase to the nuclear matrix in the regulation of matrix‐associated biochemical processes is discussed.
Nitrous oxide reductase from denitrifying
Authors: COYLE Catherine L. , ZUMFT Walter G. , KRONECK Peter M. H. , KÖRNER Heinz , JAKOB Wolfgang 
Published: March 2005

Nitrous oxide reductase from the denitrifying bacterium Pseudomonas perfectomarina has been isolated and purified to homogeneity. The enzyme contained about eight copper atoms/120 KDa and was composed of two presumably identical subunits. The isoelectric point was 5.1.Several spectroscopically distinct forms of the enzyme were identified. A ‘pink’ form of the enzyme was obtained when the purification was done aerobically. The specific activity of this species was around 30 nkat/mg protein as measured by the nitrous‐dependent oxidation of photochemically reduced benzyl viologen. A ‘purple’ form of the enzyme, whose catalytic acticity was 2–5‐fold higher, was obtained when the purification was done anaerobically. The activity of both forms of the enzyme was substantially increased by dialyzing the protein against 2‐(N‐cyclohexylamino)ethanesulfonate buffer at pH ∼ 10. A maximal activity of 1000 nkat/mg protein has been obtained for the purple form using this procedure. A ‘blue’, enzymatically inactive form of the enzyme resulted when either the pink or the purple species was exposed to excess dithionite or ascorbate.Anaerobic, potentiometric titrations of both the purple and the pink form of the enzyme gave a Nernst factor, n540, of 0.95 and a midpoint potential, E′0.540 of +260 mV (vs SHE, 25°C, Tris/HCl buffer, pH 7.5).Electron paramagnetic resonance (EPR) and optical spectra of N2O reductase suggested the presence of an unusual type 1 copper center. Type 2 copper was absent. The hyperfine splitting in the g‖ region consisted of a seven‐line pattern. In the presence of excess of reductant, a broad EPR signal with g values at 2.18 and 2.06 was observed. The EPR spectra of the pink and purple forms of the enzyme were similar; however, the spectrum of the purple form was better resolved with g‖= 2.18 (A‖= 3.83 mT) and g⊥= 2.03 (A⊥= 2.8 mT).Most of the copper in N2O reductase was removed by anaerobic dialysis against KCN. Reaction of the apoprotein with Cu(en)2SO4 partially regenerated the optical and EPR spectra of the holoprotein; the resulting protein was enzymatically inactive.Monospecific antibodies against the copper protein strongly inhibited the N2O reductase activity of purified samples and cell‐free extracts.