bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reconstitution of Reduced Nicotinamide Adenine Dinucleotide Oxidase Activity with Menadione in Membrane Vesicles from the Menaquinone‐Deficient Bacillus subtilis AroD
Authors: BISSCHOP Arnold , KONINGS Wil N. 
Published: March 2005

Membrane vesicles from the menaquinone‐deficient Bacillus subtilis aroD contain a low content of menaquinone and consequently oxidize reduced nicotinamide adenine dinucleotide (NADH) at low rate. Supplementation of the membrane vesicles suspension with the menaquinone‐analogue menadione, results in an incorporation of menadione in the membranes. The incorporated menadione increases with the external menadione concentration up to a maximum of 7 nmol of menadione bound per mg membrane protein.The NADH oxidase activity of the membrane vesicles increases linearly with the menadione content and a 35‐fold stimulation is obtained in fully reconstituted membrane vesicles; this maximal NADH oxidase activity is about two‐fold higher than the NADH oxidase activity in membrane vesicles from wild‐type B.subtilis W23. Supplementation of membrane vesicles from B. subtilis W23 with menadione also results in a stimulation of the NADH oxidase activity but only a stimulation of 1.6‐fold is maximally obtained. The NADH oxidase activities in reconstituted B.subtilis aroD and B. subtilis W23 membrane vesicles are similarly affected by respiratory chain inhibitors, indicating that menadione occupies physiological sites of menaquinone.NADH and the non‐physiological electron donor ascorbate + phenazine methosulphate are the best energy sources for active amino acid transport in membrane vesicles from B.subtilis W23. Membrane vesicles from B subtilis aroD accumulate amino acids in the presence of ascorbate phenazine methosulphate, but not with NADH. However, membrane vesicles from this mutant, reconstituted with menadione, demonstrate NADH‐driven transport activity. This activity increases linearly with the NADH oxidase activity, but maximal transport activities are reached under conditions where the NADH oxidase activity is not yet maximal. These results indicate that the rate of energy supply is the limiting factor for transport at low NADH oxidase activities and that the transport system itself becomes the limiting factor under conditions of high NADH oxidase activities. Under energy‐limiting conditions 135–235 molecules of NADH have to be oxidized in order to transport one molecule of amino acid. At all levels of energy supply a competition by the different amino acid transport systems for the available energy could not be observed. These observations indicate that only a fraction of the energy, generated by the respiratory chain, is used for the transport of an amino acid and that the bulk of the energy dissipates via other channels in the membrane vesicles.
The Structure of Artemia salina Haemoglobins
Authors: MOENS Luc , KONDO Masatoshi 
Published: March 2005

Haemoglobins of Artemia salina were investigated during development and the results obtained are summarised as follows.

A new haemoglobin (haemoglobin N) was induced de novo in the hatching nauplius, but not in the prenauplius nor in the embryo.
Haemoglobin N seemed not to be synthesised in the adult.
The adult contained three distinct haemoglobins (haemoglobins I, II and III) in a quantitative ratio of 1: 6: 3, respectively, and these haemoglobins were not detected in nauplius.
Isoelectric points determined were 5.7, 5.6, 5.7. and 5.9, for haemoglobin N, I, II and III, respectively.
All four haemoglobins contained one large polypeptide (Mr= 1.1–1.3 × 105) and possibly two minor polypeptides (Mr= 8–9.5 × 104 and Mr= 5.4 × 104, respectively).
Phospholipid and Lipopolysaccharide in Proteus mirabilis and Its Stable Protoplast l‐Form
Authors: GMEINFR Jobst , MARTIN Hans Herbert 
Published: March 2005

Cells of the stable, protoplast L‐form of Proteus mirabilis contain 1.5 to 2 times more extractable lipid, mostly phospholipid, per dry weight than cells of the bacterial form.Under identical conditions of cultivation the qualitative and quantitative composition of the phospholipid is very similar in both cell forms. The range of mole percentages of individual phospholipid species is 76–80 for phosphatidylethanolamine, 10–13 for phosphatidylglycerol, 3.9–5.5 for diphosphatidylglycerol and 1.0–2.1 for lysophospholipid. However, all phospholipid species in the L‐form differ from those of the bacterial form by a lower content of long‐chain fatty acids and a higher content of short‐chain fatty acids.Growth of the L‐form in the presence of growth‐stimulating horse serum results in a change of phospholipid composition accompanied by the uptake of phosphotipid and fatty acids from the serum into L‐form phospholipid.L‐form protoplasts synthesize the same two types of Lipopolysaccharide, I and II, that were previously identified in the bacterial form of Proteus mirabilis. However, only small amounts of the more hydrophilic lipopolysaccharide II are present in the L‐form. Lipopolysaccharides from both cell forms have virtually identical polysaccharide compositions but differ strikingly in the relative content of fatty acids in their lipid‐A moieties. Molar ratios of tetradecanoic acid, hexadecanoic acid and 3‐hydroxytetradecanoic acid are 5:1:6 in the bacterial form and 5:0.1:6 in the L‐form grown in serum‐free medium.The observed differences between the bacterial form and the protoplast L‐form are interpreted as results of the adaptation of the L‐form to life in the state lacking an envelope by formation of a physically more stable but still sufficiently fluid protoplast membrane.A rapid method based on fatty acid analysis for the simultaneous quantitative determination of phospholipid and lipopolysaccharide content of whole cells is reported.
The Non‐Covalent Binding of Small Molecules by Ligandin
Authors: TIPPING Edward , KETTERER Brian , CHRISTODOULIDES Lucia , ENDERBY Graham 
Published: March 2005

Equilibrium dialysis studies have been made of the binding of a number of small molecules by rat ligandin. Direct measurements of binding together with competition experiments indicated that bromosulphophthalein, oestrone sulphate and dehydroepiandrosterone sulphate each bind at the same single primary binding site with association constants of 1.1 × 107. 6.6 × l05 and 2.6 × 105 l/mol respectively at pH 7.0, 10.16 M, 4 oC. As well as bromosulphophthalein and dehydroepiandrosterone sulphate, a number of strucurally similar organic anions including 2‐hydroxyoestradiol‐glutathione. oestrone glucuronide, N‐methyl‐4‐aininoazo benzene‐glutathione and several bile acids, were able to displace oestrone sulphate from ligandin in a manner consistent with competition at a single binding site. From these experiments association constants for the competing ligands were derived; these were in the range 1 × 104‐1 × 106 l/mol.
Ligandin was found to bind a number of compounds for which, because of their low aqueous solubilities relative to their binding affinities, complete binding isotherms could not be obtained. These included several steroids (but not cortisol), 20‐methylcholanthrene, diethylstilboestrol, oleate and palmitate. Oestrone sulphate was able to compete with these ligands for binding and the results of the competition experiments were interpretable in terms of 1 :1 competition at a single binding site.
In general the conjugation of non‐polar ligands with sulphate or glutathione resulted in increased affinities, but such increases were relatively small (∼ 15% in terms of free energy) implying that the main driving force for the binding of both the conjugated and unconjugated species was the hydrophobic effect. This conclusion is borne out by the oobservations that both oestrone and its sulphate showed slight increases in affinity with increase in ionic strength, as would be expected for hydrophobic interactions.
As well as non‐polar compounds and organic anions, ligandin was also found to bind sulphate and glucuronate to a measurable degree, and to interact quite strongly with glutathione. For the latter compound a single binding site was found with an associat.ion.constant of 1 × 105 l/mol. Glutathione was able to cause the dissociation of the ligandin‐oestrone sulphate complex, but this effect was not explicable in terms of simple 1 : 1 competition.
Both oestrone and oestrone sulphate were bound most strongly at pH 6‐ 7, the affinity of the protein for these ligands falling off quite sharply on either side of this maximum.
The affinities of ligandin for bromosulphophthalein, steroids and their conjugates, diethylstilboestrol and N,N‐dimethyl‐4‐aminoazohenzenc are similar in magnitude to those of serum albumin and aminoazodye‐binding protein A (B. Ketterer, E. Tipping, J. F. Hackney and D. Beale, 1976).
Crosslinking with Bifunctional Reagents as a Means for Studying the Symmetry of Oligomeric Proteins
Authors: HAJDU János , BARTHA Ferenc , FRIEDRICH Peter 
Published: March 2005

A method based upon the principle that unlike domains of bonding are reflected in different re‐activities and distribution of residues that can be crosslinked, has been elaborated for the determination of symmetry of oligomeric proteins.The derivation of theoretical curves for the prediction of crosslinking patterns of tetramers produced by reaction with a bifunctional reagent and subsequent sodium‐dodecylsulphate‐gel electrophoretic analysis is presented. Based upon the theory the symmetry properties of a tetramer, to the extent whether it is an isologous or heterologous associationn, can be deduced by a simple calculation.Crosslinking patterns obtained with rabbit muscle aldolase and pig muscle lactate dehydrogenase after treatment with a series of diimidoesters of increasing chain length are evaluated and shown to be consistent with the expectations for isologous tetramers. From the patterns obtained with the various reagents the distances between lysyl residues located nearest to each other in different subunits in the two proteins could also be determined.
An Approach to the Antigenic Structure of Arginine Kinase and Creatine Kinase
Authors: BENYAMIN Yves , GUILLOU Yvonne , DESORMEAU‐BEDOT Jean‐Pierre , ROBIN Yvonne 
Published: March 2005

The antigenic structure of arginine kinase and creatine kinase has been approached using chemical modifications and enzymatic cleavage.Mild performic oxidation that, with a restricted number of oxidized amino acid residues, results for both enzymes in a severe decrease of the helical structure and in a large increase of the protein‐solvent interactions, affects differently their antigenic reactivity: when compared with antisera to the homologous native enzymes, arginine kinase and its oxidized derivative cross‐react fully, while creatine kinase and its oxidized derivative cross‐react only about 30%. The persistence of the antigenic reactivity of arginine kinase through drastic structural alterations is confirmed by the high inhibitory capacity (about 80%) of crude tryptic hydrolyzates towards the combination of arginine kinase with its specific antibodies. Tryptic peptides of creatine kinase, obtained in the same conditions, inhibit weakly (about 12 %) the homologous antigen‐antibody interaction.The participation of the lysines in the antigenicity of arginine kinase and creatine kinase is suggested by the enhanced inhibitory capacity of the tryptic hydrolyzates when the cleavage is restricted to the arginyl peptide bounds, and was verified for arginine kinase through assays with lysine‐modified derivatives
Molecular Forms of Electrophorus Acetylcholinesterase
Authors: BON Suzanne , HUET Marianne , LEMONNIER Marguerite , RIEGER. François , MASSOULIÉ Jean 
Published: March 2005

Molecular weights for the series of six Electrophorus acetylcholinesterase forms have been determined either by the sedimentation‐diffusion equilibrium method or, particularly in the case of the very scarce G′ and G″ forms, from their Stokes radius and sedimentation coefficient values.Both methods are in excellent agreement. The results provide good evidence for the model previously proposed, G″, G′ and G containing one, two and four subunits, whereas A, C and D possess, in addition to respectively one, two and three tetrameric sets of such subunits, a structural element, the tail. Although the amino acid composition of ‘tailed’ and globular forms did not reveal any significant feature of this element, its mass, about 100000 daltons, could be deduced from a comparison of molecular weights for the two classes of acetylcholinesterase forms. This value is in close agreement with electron microscopic data. The tail is thought to consist of three 30000‐dalton strands.
Kinetic Investigation of the Staphylococcal Protease‐Catalyzed Hydrolysis of Synthetic Substrates
Authors: HOUMARD Jean 
Published: March 2005

In investigating the staphylococcal protease‐catalyzed hydrolysis of N‐tert‐butoxycarbonyl‐l‐glutamate α‐phenyl ester, N‐benzyloxycarbonyl‐l‐glutamate α‐phenyl ester and N‐benzyloxycarbonyl‐l‐glutamate α‐p‐nitroanilide, we obtained kinetic evidence consistent with the formation of an acyl‐enzyme intermediate. We found that addition of a nucleophile, such as methanol, led to the partition of the common acyl‐enzyme intermediate between water and the alcohol. With N‐benzyloxycarbonyl‐l‐glutamate α‐phenyl ester, a specific ester substrate, deacylation was shown to be the rate‐limiting step.By studying the kcat/Km ratio of these hydrolyses as a function of pH, we have shown that two ionizable groups on the enzyme are essential to the catalytic process. One of these groups has a pK of 6.58 and the other, a pK of 8.25. The assignment of these pK values is discussed in connection with the known features of the serine proteinase reaction mechanism.In addition, monovalent anions were shown to inhibit staphylococcal protease hydrolyses. They seem to compete with the negative charge of the substrate, thus inhibiting its binding on the enzyme molecule. Finally we compared the kinetic parameters obtained with five proteases isolated from different strains of Staphylococcus aureus.
p‐Nitroanilides of 3‐Carboxypropionyl‐peptides
Authors: KASAFÍREK Evžen , FRIČ Pǐemysl , SLABÝ Jan , MALIŠ František 
Published: March 2005

Fourteen 3‐carboxypropionyl‐tripeptide p‐nitroanilides of the general formula 3‐carboxypropionyl‐alanyl‐alanyl‐Y p‐nitroanilide (Y = glycine, norvaline, S‐methylcysteine, valine, norleucine. S‐ethylcysteine, methionine, leucine, isoleucine, phenylalanine, tyrosine, S‐benzylcysteine, Cx‐phenylglycine, and proline) were synthesized and their cleavage by elastase, trypsin, and chymotrypsin (Km, kcat, and kcat/Km) was determined.The significance of amino acid residues in the position of Y was evaluated firstly with respect to their structure (topographically), and secondly with respect to their free energy (thermodynamically). The alanine residue substrate was cleaved best by elastase, the phenylalanine substrate by chymotrypsin. Trypsin cleaved two substrates only, that is those containing a phenylalanine and a tyrosine residue.The optimum length of the elastolytic substrates was studied in a series of N‐3‐carboxypropionyl‐(Ala)n‐p‐nitroanilides (n= 1, 2, 3, 4, 5), N‐3‐carboxypropionyl‐(Gly)n‐p‐nitroanilides (n= 1, 2, 3), and in p‐nitroanilides of fatty acids with two to seven carbon atoms. Elastase cleaved tri, tetra, and pentapeptides of alanine. p‐Nitroanilides of the glycine series, as well as p‐nitroanilides of fatty acids were riot cleaved. 3‐Carboxypropionyl‐tetra‐alanine‐p‐nitroanilide was the most suitable substrate so far found for elastase cleavage; it is not cleaved by trypsin nor chymotrypsin. The optimum distance between Y and the terminal anionic carboxyl residue was found to be 1.8 nm in clastolytic substrates.
Some Properties of Phospholipid Exchange Proteins from Rat Liver
Authors: LUMB Roger H. , KLOOSTERMAN Aat D. , WIRTZ Karel W. A. , VAN DEENEN Laurens L. M. 
Published: March 2005

The phospholipid exchange proteins of rat liver that catalyze the transfer of phosphatidylcholine and phosphatidylinositol from rat liver microsomes to liposomes, have been purified and characterized.Two proteins were detected with dual specificities catalyzing the transfer of both phosphatidylinositol and phosphatidylcholine. Both proteins showed a strong preference for phosphatidylinositol transferring 8 to 9 times as much of the microsomal phosphatidylinositol pool as the microsomal phosphatidylcholine pool. The two proteins had iso‐electric points of 5.1 and 5.3 and were purified 300‐fold and 500‐fold, respectively.A protein that catalyzed specifically the transfer of phosphatidylcholine, was purified 7000‐fold. This protein had an iso‐electric point of 8.4 and a molecular weight of approximately 16000 calculated from Sephadex G‐50 chromatography and sodium dodecylsulfate‐polyacrylamide gel electrophoresis; the amino acid composition was determined. An antiserum against this protein was raised in rabbits. Treatment of a rat liver supernatant fraction with the antiserum immunoglobulin fraction demonstrated that 60 % of the phosphatidylcholine transfer activity is due to this protein.
Daunorubicin Inhibition of DNA‐Dependent RNA Polymerases from Ehrlich Ascites Tumor Cells
Authors: BARTHELEMY‐CLAVEY Véronique , MOLINIER Catherine , AUBEL‐SADRON Geneviève , MARAL René 
Published: March 2005

Two different forms of DNA‐dependent RNA polymerase have been solubilized and purified from nuclei of Ehrlich ascites tumor cells. The purification procedure involves ammonium sulfate precipitation and gel filtration on Sephadex G‐25. The separation of A and B activities is achieved by chromatography on DEAE‐cellulose. Nuclei arc prepared from cells, sensitive or resistant to daunorubicin. RNA polymerases A and B have an absolute requirement of divalent cations for activity. Native DNAs are better templates than heat‐denatured DNAs for RNA polymerase A. On the contrary heat‐denatured DNA is more transcribed than the native one by RNA polymerase B. The low level of transcription of total and nucleolar ascites DNAs is due to the DNA, the same results being obtained with ascites and calf thymus RNA polymerases A and B.The inhibitory action of daunorubicin on RNA polyrnerases A and B from Ehrlich ascites tumor cells has been studied in vitro. The same results are obtained with enzymes extracted from sensitive or resistant cells. Daunorubicin does not inhibit the binding of the RNA polymerases to the DNA template, but prevents the transformation of the DNA daunorubicin RNA‐polymerase unstable complex into the highly stable one. This inactive ternary complex has a dissociation rate faster than the stable complex formed without daunorubicin. The size of the RNA synthesized in the presence or absence of daunorubicin is the same.
An Improved Procedure for Automated Edman Degradation Used for Determination of the N‐Terminal Amino Acid Sequence of Human Transcobalamin I and Human Intrinsic Factor
Authors: THOMSEN Johannes , BUCHER Ditlef , BRUNFELDT Kay , NEXØ Ebba , OLESEN Henrik 
Published: March 2005

An improved procedure for automated Edman degradation is presented. Three programs are described, one with double cleavage and two with single cleavage. The programs presented are characterized by a reversed delivery scheme for buffer, and phenyl isothiocyanate, and by reduced cleavage times. The modified procedures applied on automated Edman degradation of the vitamin B12‐binding proteins human transcobalamin I and human intrinsic factor, containing approximately 390 and 350 amino residues respectively, gave the following N‐terminal amino acid sequences:Human transcobalamin IGlu‐Ile‐Cys‐Glu‐Val‐Ser‐Glu‐Glu‐Asn‐Tyr‐Ile‐Arg‐Leu‐Lys‐Pro‐Leu‐Leu‐Asn‐Thr‐Met‐Ile‐Gln‐Ser‐Asn‐Tyr‐Asn‐?‐Gly‐Human intrinsic factorSer‐Thr‐Gln‐Thr‐Gln‐Ser‐Ser‐Cys‐Ser‐Val‐Pro‐Ser‐Ala‐Gln‐Glu‐Pro‐Leu‐Val‐Asn‐Gly‐Ile‐Gln‐?‐Leu‐Met‐Glu‐Thr‐The background accumulation seems to be related not only to the length of the polypeptide chain being degraded, but also to the content of serine (and possibly threonine). A possible N O acyl shift during the cleavage is a tentative explanation. The programs here represented lead to a significant reduction in background compared to conventional programs and allowed considerable prolongation of the degradations.
Epoxides Derived from Various Polycyclic Hydrocarbons as Substrates of Homogeneous and Microsome‐Bound Epoxide Hydratase
Authors: BENTLEY Philip , SCHMASSMANN Hansueli , SIMS Peter , OESCH Franz 
Published: March 2005

A general assay for epoxide hydratase using epoxides derived from polycyclic aromatic hydrocarbons as substrates is described. Addition of dimethylsulphoxide to the incubation mixture after incubation allows unreacted epoxide and its phenolic by‐product to be extracted into light petroleum whilst the product dihydrodiol remained in the aqueous phase. The product was then extracted into ethyl acetate and estimated radiochemically. This assay gave low extraction blanks (0.8–3.8%) when six K‐region epoxides of polycyclic hydrocarbons were used, with high recoveries of the corresponding dihydrodiol in the ethyl acetate phase (65–89%). Radiochromatograms demonstrated that all the radioactivity in the ethyl acetate extracts of active incubations above that of boiled enzyme blanks was confined to a single band that always cochromatographed with the authentic trans‐dihydrodiol.Using this assay, the kinetic parameters of six K‐region epoxides were estimated. In all cases the apparent Km was low (2–5.9 μM). This is about 100‐fold lower than the known apparent Km of epoxide hydratase for styrene oxide, an alkene oxide that is widely used as a substrate for epoxide hydratase. The rate of hydration varied with the substrate. Thus the maximum velocity for hydration of phenanthrene 9,10‐oxide > 7‐methylbenz[a]anthracene 5,6‐oxide:∼ benz[a]anthracene 5,6‐oxide ∼ benzo[a]pyrene 4,5‐oxide > 3‐methylcholanthrene 11,12‐oxide > dibenz[a,h] anthracene 5,6‐oxide. This relationship between the individual epoxides was found in microsomal fractions from both rat skin and rat liver, although the activity was always much lower in skin microsomes. All six arene oxides derived from polycyclic hydrocarbons were substrates for the homogeneous epoxide hydratase that was isolated from rat liver microsomal fractions using styrene oxide, an alkene oxide, as substrate to follow the purification.