bluuebunny/crossref_metadata_2025_split huggingface dataset:
Electron allocation to H+ and N2 by nitrogenase in Rhizobium leguminosarum bacteroids
Authors: HAAKER Huub , WASSINK Hans 
Published: March 2005

Electron allocation to H+ and N2 by nitrogenase in intact Rhizobium leguminosarum bacteroids has been studied. Nitrogenase activity was measured in intact cells with succinate and oxygen substrates. When whole cell nitrogenase activity was inhibited by oxygen‐limitation or by the addition of the H+‐conducting ionophore carbonylcyanide m‐chlorophenylhydrazone, both inducing a low intracellular ATP/ADP ratio, the electron allocation to H+ was favoured over that to N2. When whole cell nitrogenase activity was inhibited by excess oxygen or by the addition of the K+‐conducting ionophore valinomycin, both inhibiting electron transport to nitrogenase without affecting the intracellular ATP/ADP ratio, no effect upon the electron allocation to H+ and N2 was observed.The whole cell experiments could be confirmed by experiments with bacteroids treated with hexadecyltri‐methylammonium bromide. Nitrogenase is highly active in these preparations with Na2S2O4 and MgATP as substrates. No effect was observed upon electron allocation to H− and N2 when nitrogenase was inhibited by limitation of reductant (Na2S2O4) or MgATP. Only when nitrogenase was inhibited by MgADP, electron allocation to H+ was favoured.The amount of nitrogenase component 1 and 2 in bacteroids was estimated with protein blotting, followed by an immunological detection. It was found that 17%± 3% of total bacteroid protein is component 1 and 12%±2% is component 2. The specific nitrogenase activity of bacteroids treated with hexadecyltrimethylammonium bromide is 178±62 nmol C2H4 formed · min−1· mg total protein−1. Despite the high protein concentrations, nitrogenase is not inhibited.With cell‐free extracts or with purified nitrogenase components isolated from R. leguminosarum bacteroids, electron allocation to H+ was favoured over that to N2, independently of the mechanism of inhibition. The discrepancies between the whole cell studies and those with isolated enzyme will be discussed with respect to the present mechanism of action of nitrogenase.
Studies on the transmembrane disposition of the neural cell adhesion molecule N‐CAM
Authors: GENNARINI Gianfranco , HIRN Michel , DEAGOSTINI‐BAZIN Hermine , GORIDIS Christo 
Published: March 2005

The transmembrane orientation of the polypeptide chains present in preparations of adult and neonatal mouse N‐CAM was studied using, as a model system, liposome‐inserted purified N‐CAM preparations. N‐CAM purified from adult or neonatal mouse brain was 125I‐labeled and reconstituted into artificial lipid vesicles. After trypsin digestion, the peptides that remained associated with the liposomes were isolated by floatation of the vesicles on sucrose gradients. In control experiments the liposomes were lysed before trypsin treatment. Large, overlapping peptides were obtained after this treatment, several of which were protected by the liposome membrane. Sialic‐acid‐bearing peptides were revealed by their sensitivity to neuraminidase. To distinguish between peptides corresponding to intracellular or extracellular domains use was made of the P61 and H28.123 monoclonal antibodies, which recognize determinants located on the cytoplasmic and the extracellular part of the molecules respectively.There was no indication that the N‐CAM chains were inserted in an inside‐out configuration. Peptides protected from trypsin attack by the liposomes and recognized only by P61 had Mr values of 92000, 42000 and 35000. The H28.123 determinant could be mapped to a 32000‐Mr peptide located close to the membrane at the vesicle's exterior. The bulk of the sialic acid seemed to be carried by a rather short sequence distal to the H28.123‐reactive peptide but at some distance from the N terminus. Fragments of very similar Mr were generated from young and adult material. However, a 45000‐Mr peptide from neonatal N‐CAM appeared to migrate in the high‐Mr region of sodium dodecyl sulfate/polyacrylamide gels in its fully sialylated form.It is concluded that (a) identical polypeptide chains are present in young and adult preparation, (b) the 180000‐Mr, 140000‐Mr and 120000‐Mr chains differ by the length of their cytoplasmic extensions and (c) the largest cytoplasmic sequences have a Mr close to 90000. A tentative linear model of the transmembrane topography of the N‐CAM polypeptides is presented.
Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine‐linked oligosaccharides
Authors: HETTKAMP Harald , LEGLER Günter , BAUSE Ernst 
Published: March 2005

Trimming glucosidase I and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p‐nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific α‐glucosidase(s) were inhibited by 1‐deoxynojirimycin with median inhibitory concentration of 3 μM, 20 μM, 12 μM, respectively. Discrimination between these enzymes was strongly enhanced by N‐alkylation of 1‐deoxynojirimycin and formed the basis for the design of the affinity ligand.Glucosidase I has been purified to homogeneity by affinity chromatography on AH‐Sepharose 4B with N‐carboxypentyl‐ 1–deoxynojirimycin as ligand. Sodium dodecyl sulfate gel eletrophoresis of the purified enzyme revealed a subunit molecular mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was ∼ 320–350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4°C in the presence of detergent (tl/2∼ 20 days) and showed high specificity for the hydrolysis of the terminal (α 1,2)‐linked glucose residue in the natural substrate Glc3‐Man9‐(GlcNAc)2.
Induction of phytoalexin synthesis in soybean. Stereospecific 3,9‐dihydroxypterocarpan 6a‐hydroxylase from elicitor‐induced soybean cell cultures
Authors: HAGMANN Marie‐Luise , HELLER Werner , GRISEBACH Hans 
Published: March 2005

A microsomal preparation from elicitor‐challenged soybean cell suspension cultures catalyzes an NADPH‐dependent and dioxygen‐dependent 6a‐hydroxylation of 3,9‐dihydroxypterocarpan to 3,6a,9‐trihydroxypterocarpan. The latter is a precursor for the soybean phytoalexin glyceollin. No reaction is observed with NADH. The 6a‐hydroxylase is inhibited by cytochrome c.Optical rotatory dispersion spectra of the enzymatic product formed from racemic dihydroxypterocarpan and of the remaining unreacted substrate proved that the product has the natural (6aS, 11aS)‐configuration and that hydroxylation proceeds with retention of configuration.The 6a‐hydroxylase was also found in elicitor‐challenged soybean seedlings. The results indicate that the 6a‐hydroxylase is specifically involved in the biosynthesis of glyceollin.
Comments on ‘Reaction mechanism of flavin‐dependent hydroxylation: evolution of a non‐imitable enzyme’ by C. M. Visser
Authors: Humphrey I. X. MAGER 
Published: March 2005

Dear Sir,Studies on bioorganic model systems have provided the evidence that dihydroflavin and tetrahydropteridine hydroperoxides can rearrange to other reactive peroxide forms including 4,4a‐dioxetanes. Such rearranged peroxides were already proposed as alternative hydroxylating agents in 1976. This original proposal is ignored in recent biochemical papers in which some incorrect claims and statements are made.
Lactoperoxidase, a dithionite ion dismutase
Authors: OHLSSON Per‐Ingvar 
Published: March 2005

The dithionite ion is catalytically disproportionated by lactoperoxidase with Km= 0.36 mM in 100 mM glycine HCl pH 3.0. The products formed are thiosulfate and hydrogensulfite ions. The rate of reaction is considerably increased at low pH with a pKa at 3–3.5 possibly indicating the involvement of a carboxyl group. The reaction is competitively inhibited by hydrogensulfite, Ki= 5.5 mM in 100 mM glycine HCl pH 3.50.Four different spectral forms of reduced lactoperoxidase appear during the reaction. The first two forms are found during the lag phase of the reaction. The third form, which is interpreted as a ternary complex, exists under the dismutation phase. After exhaustion of the substrate a visible spectrum similar to that of lactoperoxidase H2O2 compound III appears.A mechanistic model for the lactoperoxidase dismutation of the dithionite ion is proposed and discussed.
A synthetic hapten for induction of thymine‐dimer‐specific antibodies
Authors: KLOCKER Helmut , AUER Bernhard , BURTSCHER Helmut J. , HIRSCH‐KAUFFMANN Monica , SCHWEIGER Manfred 
Published: March 2005

High specificity and sensitivity of thymine cyclobutane dimer (thy[]thy) detection were obtained by a radioimmunoassay. Attempts to raise thy[]thy‐monospecific antibodies with antigens produced according to conventional methods were unsuccessful. Thy[]thy‐specific antibodies could only be raised by using a new strategy to bind thy[]thy to protein: thymine was activated by trimethylsilylation and alkylated at N1 yielding N1‐thyminebutanoic acid which was dimerised by ultraviolet treatment. The resulting derivative of thymine cyclobutane dimer was coupled to bovine serum albumin by the active‐ester method. The new strategy appears to be generally applicable for binding haptens, such as DNA bases, photoproducts etc. to proteins via a derivative containing a carboxyl group. Immunisation of rabbits with the thy[]thy‐bovine‐serum‐albumin conjugate prepared by the new method resulted in a highly specific antiserum which allows detection of thy[]thy down to 0.06 p mol (15pg).The thy[]thy‐specific radioimmunoassay was applied to measure thy[]thy formed in human fibroblasts which were exposed to sunlight at altitudes of 600 m or 2300 m. The amounts of thy[]thy formed in an hour corresponded to doses of 14 J m−2 and 24 J m−2, respectively, of an ultraviolet light lamp emitting predominantly 245‐nm light.
Differences in the binding of sulfate, selenate and thiosulfate ions to bovine liver rhodanese, and a description of a binding site for ammonium and sodium ions
Authors: LIJK Leo J. , TORFS Chris A. , KALK Kor H. , DE MAEYER Marc C. H. , HOL Wim G. J. 
Published: March 2005

The binding of sulfate, selenate and thiosulfate by the sulfur‐transferase rhodanese (EC 2.8.1.1) in the crystalline state has been studied by X‐ray analysis at resolutions between 0.23 nm and 0.4 nm. The three ions appear to occupy a common site between the N" atoms of Arg‐29 and the main‐chain NH group of Glu‐148 at the surface of the enzyme molecule.A second binding site for the three ions is situated at the entrance to the active centre, between the side chains of Arg‐186 and Lys‐249. Selenate and thiosulfate are bound equally well at both anion‐binding sites. Sulfate, however, binds better at the first position, near Arg‐29, than at the second site near Arg‐186.In the complex of sulfur‐rhodanese with thiosulfate, the outer sulfur atom of the anion near the active centre points towards the extra sulfur atom which is bound as a persulfide to the Sγ of the essential Cys‐247. The distance between the outer sulfur atom of the thiosulfate ion and the persulfide sulfur atom appears to be about 0.3 nm.The thiosulfate difference Fourier also shows a distinct, localized conformational change involving residues 71, 72 and 249. This is the result of the replacement of an ammonium ion in the sulfate and selenate media by a sodium ion in the sodium thiosulfate solution. Rhodanese is apparently able to accomodate ions with different radii at this cation‐binding site by minor structural alterations.
Factors determining the relative contribution of the adenine‐nucleotide translocator and the ADP‐regenerating system to the control of oxidative phosphorylation in isolated rat‐liver mitochondria
Authors: WANDERS Ronald J. A. , GROEN Albert K. , VAN ROERMUND Carlo W. T. , TAGER Joseph M. 
Published: March 2005

The control exerted by the adenine nucleotide translocator and the ADP‐regenerating system on oxidative phosphorylation was studied in isolated rat‐liver mitochondria respiring with succinate.

At intermediate rates of respiration the flux control coefficient (control strength) of the adenine nucleotide translocator on respiration was much higher with creatine/creatine kinase than with glucose/hexokinase as the ADP‐regenerating system. In contrast, at the same rate of respiration the flux control coefficient of creatine kinase was much lower than that of hexokinase.
On addition of a small amount of carboxyatractyloside to mitochondria respiring in the presence of glucose/hexokinase or creatine/creatine kinase, the rate of respiration decreased abruptly and then increased again to a new steady state which was lower with creatine/creatine kinase than with glucose/hexokinase. In the new steady state, the extramitochondrial ATP/ADP ratio was lower with glucose/hexokinase than with creatine/creatine kinase.
At the same rate of respiration, the elasticity coefficient of creatine kinase towards the extramitochondrial ATP/ADP ratio was much higher than that of hexokinase.
The connectivity theorem [Kacser, H. and Burns, J. A. (1973) in Rate Control of Biological Processes (Davies, D. D., ed.) pp. 65–104, Cambridge University Press, London] which relates the flux control coefficients of two adjacent enzymes to their elasticity coefficients towards the common intermediate, provides an explanation for the difference in flux control coefficient of the adenine nucleotide translocator with the two ADP‐regenerating systems.
Using principles developed by R. Heinrich and T. A. Rapoport [BioSystems 7, 130–136 (1975)], the flux control coefficients of the adenine nucleotide translocator and hexokinase on phosphorylation were also calculated from the elasticity coefficients of these enzymes towards the extramitochondrial ATP/ADP ratio and the controls exerted by these enzymes on the extramitochondrial ATP/ADP ratio. The calculated values were approximately 30% lower than the values measured directly.
The elasticity coefficient of the adenine nucleotide translocator towards the extramitochondrial ATP/ADP ratio was determined, using either the connectivity theorem of Kacser and Burns (loe. cit.) or a new connectivity theorem developed by Westerhoff (following paper in this journal). Good agreement was obtained using the different methods of calculation.
It is concluded that flux through the adenine nucleotide translocator, although in principle dependent on ([ATP]/[ADP])out, ([ATP]/[ADP])in and ΔΨ, is regulated primarily by ([ATP]/[ADP])out under the conditions used in this study.
Cyclic nucleotide specificity of the activator and catalytic sites of a cGMP‐stimulated cGMP phosphodiesterase from Dictyostelium discoideum
Authors: KESBEKE Fanja , BARANIAK Janina , BULGAKOV Roman , JASTORFF Bernd , MORR Michael , PETRIDIS Georg , STEC Wojciech J. , SEELA Frank , VAN HAASTERT Peter J. M. 
Published: March 2005

The cellular slime mold Dictyostelium discoideum has an intracellular phosphodiesterase which specifically hydrolyzes cGMP. The enzyme is activated by low cGMP concentrations, and is involved in the reduction of chemoattractant‐mediated elevations of cGMP levels. The interaction of 20 cGMP derivatives with the activator site and with the catalytic site of the enzyme has been investigated. Binding of cGMP to the activator site is strongly reduced (more than 80‐fold) if cGMP is no longer able to form a hydrogen bond at N2H2 or O2H. Modifications at N7, C8, O3′ and O5′ induce only a small reduction of binding affinity. A cyclic phosphate structure, as well as a negatively charged oxygen atom at phosphorus, are essential to obtain activation of the enzyme. Substitution of the axial exocyclic oxygen atom by sulphur is tolerated; modification of the equatorial oxygen atom reduces the binding activity of cGMP to the activator site by 90‐fold.Binding of cGMP to the catalytic site is strongly reduced if cGMP is modified at N1H, C6O, C8 and O3′, while modifications at N2H2, N3, N7, O2′H, and O5′ have minor effects. Both exocyclic oxygen atoms are important to obtain binding of cGMP to the catalytic site. The results indicate that activation of the enzyme by cGMP and hydrolysis of cGMP occur at different sites of the enzyme. cGMP is recognized at these sites by different types of molecular interaction between cGMP and the protein.cGMP derivatives at concentrations which saturate the activator site do not induce the same degree of activation of the enzyme (activation 2.3–6.6‐fold). The binding affinities of the analogues for the activator site and their maximal activation are not correlated. Our results suggest that the enzyme is activated because cGMP bound to the activator site stabilizes a state of the enzyme which has a higher affinity for cGMP at the catalytic site.
Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls
Authors: CRASNIER Martine , MOUSTACAS Anne‐Marie , RICARD Jacques 
Published: March 2005

At ‘low’ ionic strength, acid phosphatase bound to plant cell walls exhibits an apparent negative co‐operativity, whereas it displays classic Michaelis‐Menten kinetics in free solution. Conversely, at ‘high’ ionic strength, the bound enzyme and the soluble enzyme behave identically. This apparent negative co‐operativity is explained by the existence of an electrostatic partition of the charged substrate by the fixed negative charges of the cell wall. Raising the ionic strength suppresses these electrostatic repulsion effects.Calcium may be removed from the cell walls by acid treatment and the acid phosphatase is apparently strongly inhibited. This inhibition occurs together with an increased apparent negative co‐operativity of the enzyme. Incubating cell wall fragments previously depleted of calcium with CaCl2 restores the initial behaviour of the enzyme. Calcium, which tightly binds to cell wall pectic compounds, has by itself no effect on the enzyme in free solution. It affects the net charge of the cell wall and therefore the amplitude of electrostatic repulsion effects. Non‐linear least‐square fitting methods make it possible to estimate the density of fixed negative charges as well as the electrostatic partition coefficient, for both the ‘native’ and ‘calcium‐deprived’ cell wall fragments.It may be shown directly that calcium loading and unloading in the cell wall controls the electrostatic effects, by monitoring proton extrusion from cell wall fragments upon raising the ionic strength. Proton outflux in the bulk phase is considerably enhanced upon removal of calcium from the cell walls.The main conclusion is that loading and unloading of calcium during cell elongation and division may regulate the activity of cell wall enzymes.
Effects of retinol on chromatin structure
Authors: FERRARI Nicoletta , VIDALI Giorgio 
Published: March 2005

The effect of retinol is studied in 3T3 cultured cells. The vitamin induces a decreased rate of cell proliferation and an augmented sensitivity of chromatin to DNase I digestion.Biochemical analyses of chromosomal components establish that the rates of radioactive acetate uptake and turnover on histones are increased leaving unaltered the steady‐state level of histone acetylation. The presence of retinol in the culture medium also causes the disappearance of a protein of Mr 20000, which is co‐extracted with the high‐mobility‐group proteins.The observed changes in chromatin structure and composition are reversible when retinol is removed from the culture medium.
Antiviral activity in L1210 cells of liposome‐encapsulated (2′‐5′)oligo(adenylate) analogues
Authors: BAYARD Bernard , LESERMAN Lee D. , BISBAL Catherine , LEBLEU Bernard 
Published: March 2005

Evidence is available for a role of a (2′‐5′)(A)n‐activated endoribonuclease (RNase L) in the antiviral activity of interferon for several RNA viruses. (2′‐5′)(A)n and their analogues might thus provide an interesting alternative to exogenous interferons or their inducers in antiviral chemotherapy.In addition, the evaluation of the activity of (2′‐5′)(A)n as mediators of interferon's biological activities or as cell growth regulators requires biochemical studies using agonists or antagonists of the system. Non‐disruptive techniques for the introduction of (2′‐5′)(A)n and their analogues into cell lines or tissues are required for these studies since these highly charged compounds are cell impermeable.(2′‐5′)(A)n oligomers and analogues of increased stability towards phosphodiesterases were derived by chemical modification of their 2′ end and encapsulated in protein‐A‐bearing liposomes. The specific delivery of liposome contents into L1210 mouse leukemic cells was achieved with the help of monoclonal antibodies directed against the appropriate class I major histocompatibility complex‐encoded proteins expressed by these cells. This intracellular delivery led to transient inhibition of protein synthesis and an antiviral activity, both compatible with activation of RNase L. This activity was enhanced for the analogues designed to resist degradation, with respect to the natural product.
Human serum α1‐antichymotrypsin is an inhibitor of pancreatic elastases
Authors: LAINE Anne , DAVRIL Monique , RABAUD Michel , VERCAIGNE‐MARKO Dominique , HAYEM Annette 
Published: March 2005

Incubation of human serum α1‐antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. α1‐Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor:enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when α1‐antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1).Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equimolar complex (Mr near 77000) and the release of a fragment migrating as a peptide of Mr near 5000. Moreover a free proteolytically modified form of α1‐antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when α1‐antichymotrypsin reacts with porcine elastase than with human elastase. As a consequence of our findings, the specificity of α1‐antichymotrypsin, so far limited to the inhibition of chymotrypsin‐like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work.A contribution of α1‐antichymotrypsin to the regulatory balance between plasma inhibitors and human pancreatic elastase 2 in pancreatic diseases is suggested.