bluuebunny/crossref_metadata_2025 huggingface dataset:
Horse Liver Alcohol Dehydrogenase
Authors: Jörnvall Hans , Harris J. Ieuan
Published: March 2005

The ethanol‐active isoenzyme of alcohol dehydrogenase from horse liver has been carboxy‐methylated with iodo[2‐14C]acetate in 6 M guanidine hydrochloride. A study of the tryptic digest of the carboxymethylated enzyme has shown that it contains 14 unique sequences with [2‐14C]‐carboxymethylcysteine, two with tryptophan, one with C‐terminal phenylalanine and one with a blocked N‐terminal residue which represents the N‐terminal sequence in the protein chain. This number of unique residues found in peptides is in each case half the number previously found by direct amino acid analysis (based on a molecular weight of 80000) of the intact enzyme. These results support the view that the native isoenzyme is a dimer consisting of two similar and probably identical polypeptide chains each containing about 370 amino acid residues. Failure to demonstrate the presence of more than one type of chain by chromatography of the carboxymethylated enzyme on DEAE‐cellulose provides additional support for the dimer hypothesis. An alternative proposal [13] that the enzyme is composed of two pairs of dissimilar chains each with a molecular weight of 20000 appears to be incompatible with these results.
Subunits of the Small‐Intestinal Sucrase · Isomaltase Complex and Separation of Its Enzymatically Active Isomaltase Moiety
Authors: Cogoli Augusto , Eberle Alex , Sigrist Hans , Joss Christine , Robinson Etheria , Mosimann Hugo , Semenza Giorgio
Published: March 2005

The size and number of subunits of the sucrase · isomaltase complex (mol. wt approximately 220000) from rabbit small intestine were determined by dodecylsulfate‐polyacrylamide gel electrophoresis, gel filtration and fingerprint analysis of tryptic hydrolysates.

It was found that two subunits of similar size (estimated mol. wt 110000 to 120000 each) build up the sucrase · isomaltase complex, the one carrying sucrase, the other isomaltase activity.

The isolation of the active isomaltase moiety, along with small amounts of active sucrase as a byproduct, has been achieved.

Amino acid and carbohydrate analysis of the two subunits are reported.
On the Mode of Activation of the Catalytically Essential Sulfhydryl Group of Papain
Authors: Polgár László
Published: March 2005

The reaction of the thiol group of papain with chloroacetamide, iodoacetamide, d‐ and l‐2‐bromopropionamides was studied. In the acidic pH‐range the reaction rate is higher than expected for an ordinary SH‐group and shows a double‐sigmoid pH‐rate profile. In addition to the already described pKa of 8.5, we found a pKa of 4.0. This indicates that in the pH‐range where the enzyme is catalytically active the thiol group of Cys‐25 interacts with some amino acid side chain, presumably with the imidazole group of the neighboring His‐159. In the light of the X‐ray diffraction studies (Drenth et al., 1971), it is probable that the anomalous PKa of His‐159 is due to its interaction with Trp‐177.

The comparison of the pH‐rate profiles of d‐ and l‐2‐bromopropionamide reactions indicates that there is no sifgnificant alteration in the geometry of the active site around pH 4, whereas at slightly alkaline pH structural changes can be observed.

2H2O has no effect on the rate constants of the alkylation reaction. This rules out the possibility of general base catalysis by the imidazole group of His‐159.

The experimental data indicate that the dissociated thiol group forms an ion pair with the protonated imidazole group. On the basis of the formation of thiolate‐imidazolium ion pair, the mechanism of action of papain is reconsidered. It is concluded that the acylation and deacylation steps are not symmetrical processes, and they are one‐encounter type reactions. The interaction between His‐159 and Trp‐177 may be important in the catalysis.
A Simple Digital‐Computer Program for Estimating the Parameters of the Hill Equation
Authors: Atkins Gordon L.
Published: March 2005

Fitting the Hill equation to experimental data for the kinetics of allosteric enzymes by using the linear regression of the Hill plot can produce unreliable results because of the uncertainty of estimating the value for the maximum velocity of the reaction.

The theory of a method and the flow diagram for a simple digital computer program are given so that the Hill equation may be fitted directly to experimental data by minimising a sum of squares of residuals. All three parameters of the equation are precisely determined.

The program was evaluated using artificial data simulated using the Hill equation and sets of normally distributed pseudo‐random numbers. The simulated error corresponded to coefficients of variation of 1, 2, 5 and 10%. The data were for 20 equally spaced, 10 equally spaced, 10 geometrically spaced and 6 geometrically spaced values of substrate concentration.

The Hill equation was fitted to these data sets using two published computer programs, the present program and an improved version of the Hill plot. The performances of these four methods were compared with respect to the precision and the bias of the sets of estimated parameters.

The precision of the parameter estimates was investigated with respect to the quantity, the precision and the spacing of data.
Conformational Studies on Lipoamide Dehydrogenase from Pig Heart
Authors: van Muiswinkel‐Voetberg H. , Visser Jaap , Veeger Cornelis
Published: March 2005

Lipoamide dehydrogenase can be isolated as dimer in two forms: a dissociable and a non‐dissociable form. The non‐dissociable form has a higher activity. Both forms can be converted into each other.

The dissociable form can be converted into the non‐dissociable form, by heating to 70 °C, by treatment with NAD+, by alkaline pH or by 0.2 M phosphate buffer pH 7.2. The non‐dissociable form can be converted into the dissociable form by extensive removal of phosphate ions at low ionic strength, or freezing and thawing.

The monomer obtained upon dissociation is inactive. A reversible equilibrium exists between monomer and dimer; association constant 3–6 μM−1.

Dioxan and 2‐chloroethanol convert the enzyme into the dissociable form in a time‐dependent process. The rate of conversion is highly accelerated at high phosphate concentration. The presence of (NH4)2SO4, leads to denaturation.
Conformational Studies on Lipoamide Dehydrogenase from Pig Heart
Authors: van Muiswinkel‐Voetberg H. , Veeger Cornelis
Published: March 2005

No structural differences could be detected between the dissociable and non‐dissociable dimer of lipoamide dehydrogenase from pig heart.

Optical rotatory dispersion and circular dichroism studies show that upon dissociation the protein undergoes conformational changes, very probably accompanied by changes in α‐helix content. The monomeric apoenzyme probably has a higher α‐helix content than the holoenzyme.

Treatment of the non‐dissociable enzyme with dioxan leads to monomerisation, accompanied by similar changes in optical rotatory dispersion and circular dichroism parameters in the 260 to 300‐nm region as obtained by spontaneous dissociation. The perturbation spectrum shows changes around a tryptophyl residue.

Dissociation leads to significant changes in fluorescence characteristics. The protein fluorescence emission increases upon dissociation, the energy‐transfer from tryptophan to FAD increases. It could be shown that one tryptophyl residue becomes more exposed.

Both the dimer and the monomer undergo a series of temperature‐dependent conformational changes.

Upon reconstitution the dimer from apoenzyme and FAD, conformational changes occur during more than 48 h.
Conformational Studies on Lipoamide Dehydrogenase from Pig Heart
Authors: van Muiswinkel‐Voetberg H. , Veeger Cornelis
Published: March 2005

Lipoamide dehydrogenase binds sodium dodecylsulphate in its dimeric state at 25 °C in two phases. The first phase, 0–0.3 g/g protein, is bound purely to hydrophobic sites; the second binding phase, 0.3–1.4 g/g protein is less well characterized and is influenced by the ionic strength and the dissociation of a protonated group with pK 6.6. The effects can be reversed by cooling to 0 °C.

The spectral and kinetic parameters are only affected during the second binding phase, maximal effects are obtained, with the exception of fluorescence parameters, at 1.4 g/g protein. The changes in the absorption spectrum indicate that different parts of the flavin absorption spectrum are influenced by unequal dodecylsulphate concentrations. Fluorescence polarisation shows that FAD becomes less tightly bound. Upon prolonged incubation it dissociates.

Binding of dodecylsulphate has a large influence on the hydrodynamic parameters. Viscosity measurements indicate the existence of asymmetrical molecules; the sedimentation constant declines from 5.8 S to 2.6 S after an increase to 8.9 S. The results show that a dimeric dodecylsulphate · enzyme complex can be reactivated by removal of dodecylsulphate at 0 °C, the monomeric dodecylsulphate · protein complex remains inactive.
A Numerical Method for Acquisition and Processing of Steady‐State Kinetic Data Contained in an Entire Progression Curve
Authors: Bizzozero Spartaco A. , Kaiser August W. , Dutler Hans
Published: March 2005

A new method is described in which the information available from steady‐state kinetic experiments is thoroughly exploited. The procedure involves varying the concentration of substrate and product, measuring rates along the entire progression curve (general rates), and applying mathematical methods for data processing. Evidence is presented to show that the results are obtained from fewer experiments and that they are more accurate than those obtained by conventional methods. For α‐chymotrypsin‐catalyzed hydrolysis of N‐acetyl‐l‐phenylalanine methyl ester as a substrate the following constants were determined: the dissociation constant of the enzyme‐substrate complex, both rate constants of the acylation step and the forward rate constant of the deacylation step.
The Initiation of Protein Synthesis Directed by the RNA from Encephalomyocarditis Virus
Authors: Smith Alan E.
Published: March 2005

The initiation of protein synthesis directed by the RNA from encephalomyocarditis virus in a cell‐free system from Krebs II ascites cells has been investigated.

Ascites cell Met‐tRNAMet donates all the internal methionyl residues of the viral‐RNA‐specified polypeptide, whereas little incorporation from Met‐tRNAfMet is detected.

fMet‐tRNAfMet, which is not normally present in the cytoplasm of eukaryotic cells, donates formylmethionine into polypeptide in response to the viral RNA to give a product which contains a single N‐terminal sequence fMet‐Ala‐Thr‐, and is identical to the normal cell‐free polypeptide except for the N‐terminal formylmethionyl residue.

Short‐chain peptidyl‐tRNA synthesised in vitro in response to encephalomyocarditis virus RNA in the presence of low concentrations of sparsomycin, which inhibits chain elongation but not initiation, contains the N‐terminal sequence Met‐Ala‐Thr‐.

This data supports the notion that unblocked Met‐tRNAfMet is the universal initiator of protein synthesis in eukaryotes and donates a methionyl residue into the N‐terminal position of all proteins. The initiating methionyl residue is in most cases subsequently removed from the nascent polypeptide, but this can be inhibited either by an N‐terminal formyl group or by preventing the nascent polypeptide from growing long enough to be a substrate for the methionine aminopeptidase.

The data also shows that encephalomyocarditis virus RNA, which codes for at least nine stable proteins in vivo, contains only one initiation site which is expressed in vitro. The polypeptide coded for immediately after the initiation site is not virus‐coat protein and is rapidly metabolised in vivo, suggesting that a lead‐in polypeptide precedes the first meaningful protein sequence.
Mechanism of Enzymatic Reduction of Steroid Double Bonds
Authors: Björkhem Ingemar , Holmberg Inger
Published: March 2005

The rate‐limiting step in the enzymatic reduction of double bonds in some steroids was studied with the aid of deuterated water and NADPH labeled in the A‐and B‐position with deuterium. The following reactions were studied: conversion of progesterone into 5α‐pregnane‐3, 20‐dione, catalyzed by rat liver microsomes; conversion of progesterone into 5β–pregnane‐3, 20‐dione, catalyzed by a partially purified Δ4‐3‐oxosteroid 5β‐reductase from rat liver, and conversion of 7‐dehydrocholesterol into cholesterol, catalyzed by rat liver microsomes fortified with a desalted 100000 ×gsupernatant fluid. It was concluded that the rate‐limiting step in the reductions catalyzed by the Δ4‐3‐oxosteroid 5β‐reductase and the Δ4‐3‐oxosteroid 5β‐reductase is the addition of a hydride ion from NADPH. Evidence was obtained to indicate that in the reaction catalyzed by 7‐dehydrocholesterol reductase the protonization step could be rate‐limiting.
Synthesis of Immunoglobulins by Membrane‐Bound Polysomes and Free Polysomes from Plasmacytoma Cells
Authors: Pryme Ian F. , Garatun‐Tjeldstö Oddvard , Birckbichler Paul J. , Weltman Joel K. , Dowben Robert M.
Published: March 2005

Free polysomes and membrane‐bound polysomes were prepared from tissue‐cultured mouse plasmacytoma cells and interacted with 125I‐labelled F(ab) fragments of rabbit antibody to the mouse myeloma proteins. Peaks of reactive polysomes were found in the 6–8 oligomer and 16–20 oligomer region of membrane‐bound polysomes but not of free polysomes. When used in an amino‐acid‐incorporating system in vitro, membrane‐bound polysomes gave rise primarily to light‐chain immunoglobulins which were identified after reduction and acetylation and separation on dodecylsulfate‐gel electrophoresis, but free polysomes did not. Polysomes associated with synthesis of immunoglobulin, a secreted protein, appear to be entirely in the membrane‐bound fraction.
Partition of Proteins in Two‐Phase Systems Containing Charged Poly(ethylene glycol)
Authors: Johansson Göte , Hartman Annika , Albertsson Per‐Åke
Published: March 2005

Proteins have been partitioned in dextran–poly(ethylene glycol)–water biphasic systems containing positively charged poly(ethylene glycol). The effect of pH, buffer concentration, polymer concentration, protein concentration and temperature on the partition has been investigated. Characteristic extraction profiles are obtained when the percentage of protein in the upper phase is plotted versus pH. By keeping the salt concentration low, very steep extraction profiles can be obtained. Thus, within an interval of one pH unit, a protein is transferred from one phase to the other. This pH‐interval is located close to the isoelectric point of the protein.

The possibility of resolving the components in a natural enzyme mixture, yeast lysate, by counter‐current distribution in this type of biphasic system has been investigated. According to activity measurements, several glycolytic enzymes are resolved by this procedure. Good separation is achieved. For some of the enzymes the activity is split into more than one peak in the counter‐current distribution.
The Rotational Diffusion of Albumin in Solutions of Connective‐Tissue Polysaccharides
Authors: Preston Barry N. , BjörnÖbrink  , Laurent Torvard C.
Published: March 2005

The rotational behaviour of dansyl‐labelled human serum albumin in solutions of connective tissue polysaccharides (a hyaluronic acid and a sulphated proteoglycan) has been studied by fluorescence depolarization measurements. Over a wide range of pH, including the physiological conditions, the presence of the polymers up to concentrations of 10 mg/ml has no effect on the relaxation time of the protein. At higher concentrations of the sulphated proteoglycan (20–30 mg/ml) a moderate suppression of the rotational diffusion is observed. At pH values below the isoionic point of the protein, a marked effect on the polarization occurs which is interpreted as an extensive associative interaction between the protein and the polysaccharide materials. A quantitative interpretation of the effect is attempted.
The Interaction of Glutamate Dehydrogenase and Malate Dehydrogenase with Phospholipid Membranes
Authors: Dodd George H.
Published: March 2005

The interaction between sonicated dispersions of purified phospholipids, corresponding to those found in the inner mitochondrial membrane, and the enzymes was studied by kinetic and spectroscopic methods.

With glutamate dehydrogenase the charge type of the lipid head group determines the extent of lipid‐protein complexing. Phosphatidylcholine did not significantly alter the activity of the enzyme at lipid concentrations up to 1 mM, and the nuclear magnetic resonance (NMR) spectrum of the lipid (1%, w/v) was not altered in the presence of enzyme (up to 20% w/v). Phosphatidylethanolamine did not affect the activity in the oxidation of glutamate, but interfered with the assay in the reverse direction, possibly owing to Schiff‐base formation with 2‐oxoglutarate. The negatively charged cardiolipin, which is found uniquely in the inner mitochondrial membrane, strongly and reversibly inhibited the enzyme (50% inhibition with 0.1–20 μM lipid, depending on conditions). Another anionic phospholipid, phosphatidylserine (which does not occur in mitochondria), also inhibited the enzyme, but less effectively (50% inhibition at about 8 μM). The lipid‐enzyme interaction decreases with increasing ionic strength, indicating an electrostatic contribution to the binding. NMR studies suggest that there is also an apolar interaction involving the lipid chains.

The studies with malate dehydrogenase concentrated on detecting possible stabilization of this enzyme through interaction with lipids. A clear‐cut stabilization was found with lysophosphatidylcholine. Under conditions such that the enzyme alone lost 50% on the initial activity in about 2 h, lysophosphatidylcholine gave a slight (20%) activation and this activity remained constant for several hours. NMR studies indicated that, as with the related enzyme 3‐hydroxybutyrate dehydrogenase, both the polar choline head group and the hydrocarbon chains are involved in the binding of the lipid to the protein.

These specific lipid · protein complexes are probably examples of allotopic interactions. This form of complexing may explain aspects of the behaviour of these enzymes in vivo.
Deoxyribonucleoside‐Triphosphate Pools and DNA Synthesis in Synchronized Hamster Cells
Authors: Skoog K. Lambert , Nordenskjöld Bo A. , Bjursell K. Gunnar
Published: March 2005

Cellular pools of all four deoxyribonucleoside triphosphates were determined in synchronized Chinese hamster cells obtained by mitotic selection or by the addition of complete medium to isoleucine‐starved cells. Using either technique the degree of synchrony is high and a maximum of more than 80% of the cells incorporated labeled thymidine during a short pulse period.

Cells not involved in DNA synthesis contained measurable pools of deoxyribonucleoside triphosphates, but the pools expanded when the cells entered S phase. At all time points the pool of dGTP was the largest while the dGTP pool was the smallest amounting to 1–5% of the size of the dCTP pool. The size of the dCTP pool was closely correlated to the rate of DNA synthesis while the other pools reached their highest values after DNA synthesis.
Uptake and Reduction of Radioactive Folate by Everted Sacs of Rat Small Intestine
Authors: Selhub Jacob , Brin Hanna , Grossowicz Nathan
Published: March 2005

Uptake of pteroylglutamie acid ([2‐14C]PteGlu) by everted sacs of rat small intestine was studied. Under conditions of zero initial concentration gradient, a net transport of PteGlu from the mucosal to the serosal side was found at initial concentrations not exceeding 7 μM.

Using a simplified chromatographic procedure for estimation of reduced folates it was found that part of the transported PteGlu was converted to reduced forms, tentatively identified as tetrahydropteroylglutamic acid (H4PteGlu) and its methyl and formyl derivatives. PteGlu was reduced throughout the small intestine. The reduction was time and concentration dependent with an apparent Km of 1.55 μM.

Low concentrations of methotrexate (23 μM) inhibited the reduction of PteGlu but did not affect its transport; at a much higher concentration (3 μM) methotrexate decreased the accumulation of folates in the tissue without significantly affecting the vitamin concentration in the serosal solution.
Evidence for Metabolic Compartmentation in Escherichia coli
Authors: Macnab Robert , Moses Vivian , Mowbray John
Published: March 2005

The incorporation into amino acids in protein of 14C from (a) glycerol and maltose, and (b) galactose and lactose, alone and in competition, has been measured in Escherichia coli during aerobic growth.

Glycerol and maltose contributed carbon equally to growth and to amino acids whose precursors are tricarboxylic acid cycle intermediates, but unequally to amino acids derived from glycolysis or the pentose phosphate pathway. Glycolytic compartmentalisation has been suggested and the possible nature of this is briefly discussed.

Lactose and galactose complemented one another as growth substrates. Free galactose, and galactose from lactose hydrolysis, contributed unequally to some amino acids, as did the glucose and galactose moieties when lactose was the sole substrate. A simple compartmented model of the cell has been adduced to explain these results. These data are in accord with an earlier suggestion of metabolic channelling of the oxidative carbohydrate pathways in E. coli.
Role of DNA · RNA Hybrids in Eukaryotes
Authors: DezÉLÉE Sybille , Sentenac André
Published: March 2005

Yeast RNA polymerase B was purified by glycerol density‐gradient centrifugation and phosphocellulose chromatography. The purified enzyme migrates as a main active band on polyacryl‐amide‐gel electrophoresis. No evidence was found in favor of the existence in yeast of two different RNA polymerases B as was the case for calf thymus or rat liver. RNA polymerase activity depends critically on the ionic conditions and on the molecular structure of the template. T4 or T7 phage DNA do not serve as template unless they have been previously denatured. The template activity of calf thymus DNA is drastically reduced after purification on nitrocellulose which eliminates DNA molecules containing single‐stranded regions. RNA chain initiation and elongation was studied with native and denatured calf‐thymus DNA as template. Using denatured DNA, RNA chains are initiated predominantly with GTP and, to a lesser extent, with ATP, whereas almost no chains were initiated with CTP or UTP. The low degree of RNA synthesis obtained with native DNA was due to a deficiency in chain initiation. The preferential transcription of denatured DNA as compared to native DNA is enhanced at low concentrations of nucleoside triphosphates, DNA and enzyme. The effect of various bacterial or mammalian RNA polymerase inhibitors on yeast polymerase was investigated.
Transport of Lactate and Succinate by Membrane Vesicles of Escherichia coli, Bacillus subtilis and a Pseudomonas Species
Authors: Matin Abdul , Konings Wilhelmus N.
Published: March 2005

Transport of d‐lactate, l‐lactate and succinate in isolated membrane vesicles of Escherichia coli, Bacillus subtilis and a fresh‐water Pseudomonas species occurs actively by specific transport systems which are linked to the electron transport chain. These substrates are not transported by the membrane vesicles unless an electron donor (reduced phenazine methosulfate, NADH, d‐lactate, l‐lactate, or succinate) is present in the reaction mixture; in the presence of an electron donor, the substrates or their oxidized forms are concentrated several‐fold inside the membrane vesicles; and inhibitors of the electron transport chain markedly inhibit this electron‐donor mediated transport. In addition, transport of pyruvate in membrane vesicles of E. coli, but not in those of B. subtilis and the Pseudomonas sp. is stimulated by reduced phenazine methosulfate.

Activity of the membrane‐bound lactate and succinate dehydrogenases is not essential for the transport of lactate and succinate, since inhibitors of these activities have little effect on reduced phenazine methosulfate‐ or NADH‐energized transport of these substrates; and the Km values for oxidation of these substrates by the membrane vesicles are 30‐ to 80‐fold higher than the Km values for their transport.

d‐Lactate and l‐lactate cause strong inhibition of the transport of each other in the three organisms, suggesting that the two substrates share a common transport system. In general, transport of these substrates is not inhibited by dicarboxylic acids. In contrast, transport of succinate is strongly inhibited by other dicarboxylic acids in the three organisms, suggesting that these substrates may share a common transport system. The transport systems for lactate and succinate are repressed by glucose.
Affinity Elution as a Purification Method for Aminoacyl‐tRNA Synthetases
Authors: von der Haar Friedrich
Published: March 2005

Aminoacyl‐tRNA synthetases adsorbed on phosphocellulose can be specifically eluted with purified tRNA. This method we call affinity elution in analogy to affinity chromatography. The advantage of the method is the use of enzyme‐ligand affinity without fixation of the ligand to a polymer support. As an example, purification of phenylalanyl‐tRNA synthetase, valyl‐tRNA synthetase, seryl‐tRNA synthetase and isoleucyl‐tRNA synthetase from baker's yeast is described. The first two aminoacyl‐tRNA synthetases were obtained as apparently homogeneous proteins with purification factors 537 and 246, respectively.
Yeast l‐Lactate Dehydrogenase (Cytochrome b2)
Authors: Guiard Bernard , Groudinsky Olga , Lederer Florence
Published: March 2005

As has been known for some time, proteolysis of cytochrome b2 yields a small protein fragment (cytochrome b2 core) which still carries the heme. This paper describes the separation of the various bands which can be seen upon electrophoresis of the small hemoprotein. The isoelectric points of the two main bands are 6.40 and 5.55. Amino acid analyses show that they have about 80 and 100 residues, respectively. End group studies using in particular carboxypeptidase C demonstrated that both bands have lysine as N‐ and C‐terminal residues. Altogether, the data indicated that the various bands come from splits of the main chain at different positions. The significance of these results is discussed in relation with what is currently known about the structure of cytochrome b2.
The Kinetics of Renaturation of 5‐S RNA from Escherichia coli in the Presence of Mg2+ Ions
Authors: Richards Edward G. , Lecanidou Rena , Geroch Mary E.
Published: March 2005

The kinetics of renaturation of denatured 5‐S RNA from Escherichia coli have been studied by estimating the composition of reaction mixtures from absorbance profiles of stained zones containing the two forms separated by polyacrylamide‐gel electrophoresis. The kinetics are first order and proceed to completion irrespective of temperature or Mg2+ ion concentration, but they depend strongly on temperature. Linear Arrhenius plots yield an activation energy of 65 kcal/mol RNA. It is suggested that about nine base pairs of the denatured form dissociate and about the same number reform in a different configuration in the renatured conformer.
A Study of Several Bonds Hypersensitive to Proteases in a Complex Flavohemoenzyme, Yeast Cytochrome b2
Authors: Naslin Lillian , Spyridakis Athanase , Labeyrie Françoise
Published: March 2005

A detailed study of the polypeptide fragments formed, and of their evolution during the first stages of the trypsinolysis of two homologous active cytochrome b2 molecules, was carried out. These cytochromes are complex flavoheme‐l‐lactate dehydrogenases extracted from different yeasts and having different molar activities.

Our purpose was to gather information concerning the bonds, which, in the native proteins, are the most sensitive to trypsin (termed here protease‐hypersensitive bonds). The products resulting from the fist hydrolytic events were thus analysed using disc‐electrophoresis in sodium dodecylsulfate according to the method of Shapiro et al. (1967). Electrophoretic patterns were related to the molecular weight and to the amount of each intermediate species as a function of the extent of reaction. Schemes of the series of events, (including several rate constants) were derived from this set of data. A tentative map of the homologous loci of protease‐hypersensitive bonds along the chains of the two cytochrome b2 molecules is proposed. Considering that such bonds which are very reactive towards trypsin are certainly located on flexible and thus loosely structured regions at the surface of the molecule, this map gives a first insight into the overall secondary and tertiary structure of the chain which includes globules of high internal stability.

An important finding was that the relative reactivities of the various protease‐hypersensitive bonds identified, depend on conditions such as ionic strength and the binding of a ligand which probably changes the conformation. The protein transitions are therefore possibly described in terms of modulation of the loci of these bonds and of their reactivity.
Covalent Binding of Lipid to Protein
Authors: Hantke Klaus , Braun Volkmar
Published: March 2005

A new structure of a lipid and its covalent linkage to a protein (murein‐lipoprotein of the Escherichia coli outer membrane) is described. Glycerylcysteine (S‐(propane‐2′,3′‐diol)‐3‐thio‐2‐aminopropanic acid) at the N‐terminal end of the polypeptide chain is the attachment site of two ester‐bound fatty acids. An additional fatty acid is bound as amide to the N‐terminal group. The diglyceride residue on the cysteine could be derived from the phospholipid pathway since the fatty acid composition is very similar to that of the phospholipids from the same cells. In contrast, 65% of the amide‐linked fatty acid is palmitate. The main fatty acids are palmitic acid (53%), cis‐vaccenic acid (20.7%), 9,10‐methylene‐hexadecanoic acid (10.6%) and palmitoleic acid (9.4%).

The structure of glycerylcysteine was established by chemical degradation of the compound containing the fatty acids, by incorporation studies of [35S]sulfate, [35S]cysteine, [14C]cystine, [methyl‐3H]methionine and [2‐3H]glycerol and by chemical synthesis. Glycerylcysteine was isolated as a constituent of the peptide (Ser)‐Ser‐Am‐Ala‐Lys. The C‐terminal part of the lipopeptide sequence overlaps with the N‐terminal end of the known sequence of the polypeptide chain. Its N‐terminal position was confirmed by isolation of the N‐terminal polypeptide fragment (position 1–31) after cleavage with cyanogen bromide. This contained the lipid.
Carbon‐13 Nuclear‐Magnetic‐Resonance and Infrared‐Absorption Spectroscopy of Valinomycin and Its Alkali‐Ion Complexes
Authors: Grell Ernst , Funck Theodor , Sautee Hanspeter
Published: March 2005

The 13C nuclear magnetic resonance spectra of the membrane‐active antibiotic valinomycin and its alkali‐ion complexes are interpreted. From the 13C chemical shifts and infrared absorptions of the highly stable K+, Rb+ and Cs+ complexes in methanol it is concluded that the ligand conformations of these complexes must be similar. The essential features are the coordination of all ester carbonyl groups with the cation in addition to the formation of intramolecular hydrogen bonds between the amide groups. In contrast to these results the weaker Na+ complex of the antibiotic exhibits different spectral properties which are more similar to those of the uncomplexed antibiotic in polar solvents thus indicating the existence of a considerably different ligand conformation in this complex. The structure of the Na+ complex can be characterized by a reduced number of coordinated ester carbonyl groups and intramolecular hydrogen bonds between the amide groups as compared to the complexes with larger cations.

The conformational rearrangements occurring during the complexation of cations are compared to those observed for the uncomplexed valinomycin in solvent systems of different polarities. Evidence is presented that the interaction of the uncomplexed antibiotic with polar solvent molecules leads to conformational rearrangements which can be characterized by a step‐wise opening of a closed structure stabilized by intramolecular hydrogen bond formation.
Some Evidence on the Site of Action of Plastocyanin in the Photosynthetic Electron‐Transport Chain between Cytochromes ƒ and P700
Authors: PlesniČAR Marijana , Bendall Derek S.
Published: March 2005

The effect of plastocyanin on the light‐induced steady‐state changes of cytochrome ƒ in chloroplast particles that were depleted of plastocyanin has been studied. It was observed that the extent of cytochrome ƒ photooxidation in sonic particles in the steady‐state during a short exposure to long‐wave red light was approximately 30% of that in chloroplasts. The addition of a catalytic amount of plastocyanin to the sonic particles stimulated cytochrome ƒ photooxidation to 80% of its steady‐state value in untreated chloroplasts. The reduction of cytochrome ƒ by short‐wave red light was not inhibited by sonic treatment of chloroplasts.

The rate of cytochrome ƒ photooxidation in the presence of a catalytic amount of detergent‐treated chloroplasts was proportional to the amount of plastocyanin added.

The enhancement of cytochrome ƒ photooxidation either in sonic particles or in digitonin‐treated particles after an addition of plastocyanin was decreased in the presence of the copper‐chelating agent sodium diethyldithiocarbamate. Plastocyanin preincubated with sodium diethyldithiocarbamate showed less ability to stimulate cytochrome c photooxidation by detergent‐treated chloroplasts.
Nomogram for Calculation of Centrifugal Data
Authors: Rasmussen Hans N.
Published: March 2005

The nomogram presented makes possible the calculation of time integral of squared angular velocity, sedimentation coefficient, centrifugal acceleration, time integral of centrifugal acceleration, and fraction of material remaining in the supernatant. The theoretical and practical aspects of reproduction of a particular centrifugation are discussed, and it is suggested that critical differential centrifugations are specified by minimal and maximal radii of rotation, speed of rotation, and time of centrifugation.
Observation par microscopie électronique des formes allongées et globulaires de l'acétylcholinestérase de gymnote (Electrophorus electricus)
Authors: Rieger François , Bon Et Suzanne , MassouliÉ Jean , Cartaud Jean
Published: March 2005

Electron micrographs of the various molecular forms of acetylcholinesterase (as previously characterized) are presented. The “globular” forms G and G' appear as tetramers, and dimers, respectively, whereas the “native” forms, A, C, and D show a structure “en grappe” where a cluster of globules (respectively 4, 6 to 8, and at least 10) is associated with a semi‐rigid “tail”. The globules seem identical in all forms and probably represent the sub‐units of the enzyme (Mw 60000—80000). The structures are 50 nm long in their greatest dimension; the tail is 2—3 nm thick and its visible part is longest in A and shortest in D; presumably part of it is masked by the head. These results explain our previous physico‐chemical observations which had led us to propose that the native forms were asymmetric in structure. We discuss a possible model of the sub‐unit structure for the “native” forms: A, C and D are proposed to consist of identical tails associated with respectively one, two, and three tetramers. This hypothesis provides a rationale for the occurrence of three well‐defined “native” acetylcholinesterases, as well as for the derived “globular” forms, the dimer and the tetramer. We further propose that the interactions inside the structures are not equivalent for the two kinds of subunits: for example, one of them might be linked directly to the tail, the other only through the tetramer interactions.
The Inhibitory Effect of AMP on the Activation Reactions of the Amino Acids Involved in Gramicidin S Biosynthesis
Authors: Kristensen Tom , Gilhuus‐Moe Carl Christian , Zimmer Trine‐Lise , Laland Søren G.
Published: March 2005

Kinetic studies show that AMP inhibits the activation of all the amino acids participating in the formation of gramicidin S. The activation was measured by the amino‐acid‐dependent ATP‐[32P] pyrophosphate exchange reaction. AMP acts as a competitive inhibitor to ATP and a non‐competitive inhibitor to the amino acids. The large difference in the effect of AMP on the activation of l‐phenylalanine and d‐phenylalanine makes it probable that the activation of the two isomers takes place on different sites on the enzyme.
The Acetylcholinesterase of Bungarus fasciatus Venom
Authors: Kumar Vijay , Elliott Willard Buford
Published: March 2005

Acetylcholinesterase from Bungarus fasciatus venom has been prepared free of electrophoretically or immunologically detectable contaminating proteins. A specific activity of 270 mmol acetylcholine hydrolysed per h per mg was observed for the homogenous enzyme. A molecular weight of 126000 as determined by sedimentation and diffusion, has been found. The enzyme is split into two apparently equal‐sized subunits by 8M urea—1 M NaCl. Active‐site measurements revealed two active sites per enzyme molecule. The enzyme exhibited substrate inhibition and had a Km of 141.7 μM for acetylcholine. The enzyme was not inhibited by phenylmethylsulfonyl fluoride.
Structures of Fucose‐Containing Glycolipids with H and B Blood‐Group Activity and of Sialic Acid and Glucosamine‐Containing Glycolipid of Human‐Erythrocyte Membrane
Authors: Kościelak Jerzy , Piasek Andrzej , Górniak Halina , Gardas Andrzej , Gregor Anita
Published: March 2005

Fucose‐containing glycolipids with H and B blood group activity as well as sialic acid and glucosamine‐containing glycolipid were isolated from human erythrocytes. The sequences and linkages of carbohydrate residues in these glycolipids were determined by partial acid hydrolysis, periodate oxidation and methylation studies. Anomeric linkage were ascribed to those materials on the basis of their optical rotations, infrared spectra, susceptibility to enzymes and immunological activities.

The structures of glycolipid H‐I and H‐II were respectively fucopyranosyl‐α‐(1 → 2)‐galacto‐pyranosyl‐β‐(1 → 4)‐2‐acetamido‐2‐deoxy‐glucopyranosyl‐β‐(1 → 3)‐galactopyranosyl‐β‐(1 → 4)‐glucopyranosyl‐β‐(1 → 1)‐ceramide; fucopyranosyl‐α‐(1 → 2)‐galactopyranosyl‐β‐(1 → 4)‐2‐acetamido‐2‐deoxy‐glucopyranosyl‐β‐(1 → 3)‐galactopyranosyl‐β‐(1 → 4)‐2‐acetamido‐2‐deoxy‐glucopyranosyl‐β‐(1 → 3)‐galactopyranosyl‐β‐(1 → 4)‐glucopyranosyl‐β‐(1 → 1)‐ceramide. The structure of glycolipid B‐I was galactopyranosyl‐α‐(1 → 3)‐[fucopyranosyl‐α‐(1 → 2)]‐galactopyranosyl‐β‐(1 → 4)‐2‐acetamido‐2‐deoxy‐glucopyranosyl‐β‐(1 → 3)‐galactopyranosyl‐β‐(1 → 4)‐glucopyranosyl‐β‐(1 → 1)‐ceramide. The structure of sialic acid and glucosamine‐containing glycolipid was N‐acetyl‐neuraminosyl‐α‐(2 → 3)‐galactopyranosyl‐β‐(1 → 4)‐2‐acetamido‐2‐deoxy‐glucopyranosyl‐β‐(1 → 3)‐galactopyranosyl‐β‐(1 → 4)‐glucopyranosyl‐β‐(1 → 1)‐ceramide. The exclusive presence of 4′‐galactosyl‐N‐acetylglucosamine in four erythrocyte glycolipids makes it likely that the (1 → 3) linkage between galactose and N‐acetyl‐glucosamine residues is missing from all membrane glycolipids which comprise these two sugars. Therefore the formation of Lea and Leb active structures is not possible in membrane glycolipids and all Lewis activity of erythrocytes must be derived from glycolipids absorbed from blood plasma.
Light Scattering in the Study of Associating Macromolecules
Authors: ÖBrink Björn , Sundelöf Lars‐Olof
Published: March 2005

Equations dealing with light scattering of a reacting three‐component system have been derived. The theoretical development is essentially a transcription of already existing theory in a form which simplifies the practical data treatment in certain cases. The equations have been used in the analysis of interactions between collagen and glycosaminoglycans. It was found that the composition of the complexes varied with different types of glycosaminoglycans and also with their molecular weights.
Sites Along the Electron‐Transport Chain Controlled by the Energy‐Conversion System in Chloroplasts
Authors: Eisenbach Michael , Carmeli Chanoch
Published: March 2005

The light‐induced absorbance changes of electron transport carriers were measured in intact (class I) chloroplasts which catalyzed oxygen evolution with phosphoglyceric acid or CO2 as electron acceptors. In these chloroplasts the reduction of cytochrome b563, induced by light at 720 nm, increased in the presence of ADP or in the presence of uncouplers such as gramicidin and NH4Cl.

In the presence of ADP there was a decrease in the light‐induced reduction of C550 and in the oxidation of cytochrome f. The effect of ADP was augmented when phosphoglyceric acid was used as an electron acceptor in class I chloroplasts. A decrease in the photoreduction of C550 was also observed in the presence of the uncoupler gramicidin in class I and class II, with benzylviologen as an electron acceptor, and in class II chloroplasts with ferricyanide.

The uncoupler gramicidin caused an increase in the light‐induced reduction of cytochrome b569. Based on these effects of ADP or uncoupler on the steady state of the light‐induced redox level of the carriers, we proposed sites along the electron transport chain in which the rate of electron flow is controlled by the energy conversion system.
An Unusual Type of Bacterial Cell‐Wall in a Mutant of Bacillus subtilis 168
Authors: Leduc Mireille , van Heijenoort Jean , Kaminski Marie , Szulmajster Jekisiel
Published: March 2005

It was previously shown that in the cell walls of the Cbl‐1 mutant of Bacillus subtilis 168 wt, teichoic acid was replaced by a protein of high molecular weight which accounted for about 50% of the cell wall material. The structure of this unusual bacterial cell wall was further investigated. The autolysin associated with the cell walls of the mutant was characterized as a muramidase and thus differed from that of the parent strain. Furthermore, a proteolytic activity was detected in these cell walls. Several procedures were examined for the isolation and purification of the Cbl‐1 cell wall protein and peptidoglycan. The protein was readily isolated by treating the cell walls with 6 M lithium chloride and it was shown to consist of two non‐covalently associated chains which could be separeted by either dodecylsulfate or urea. Treatment of the cell walls with 2% sodium dodecylsulfate yielded an insoluble protein‐free fraction presenting the characteristic composition of a peptidoglycan and the well‐known morphology of rod‐shaped ghosts. As in the case of the cell walls of B. subtilis 168 wt, this fraction contained galactosamine and an excess of glucosamine.
Occurrence of 2‐Amino‐2‐deoxy‐hexuronic Acids as Constituents of Vibrio parahaemolyticus K15 Antigen
Authors: Torii Mitsuo , Sakakibara Keiko , Kuroda Koichi
Published: March 2005

Acidic capsular polysaccharide of Vibrio parahaemolyticus A55 (05: K15) was esterified with ethylene oxide and then reduced with sodium borohydride. Hydrolysis of the reduced material gave two hexosamines identical with mannosamine (2‐amino‐2‐deoxy‐mannose) and gulosamine (2‐amino‐2‐deoxy‐gulose) on paper and ion‐exchange chromatograms. This fact suggested that the K15 antigen contained mannosaminuronic acid (2‐amino‐2‐deoxy‐mannuronic acid) and gulosaminuronic acid (2‐amino‐2‐deoxy‐guluronic acid).

Direct acid hydrolysis of the antigen gave two 2‐amino‐2‐deoxy‐hexuronic acids. These two were identified as mannosaminuronic and gulosaminouronic acids by paper chromatography and paper electrophoresis, being compared with synthesized standard samples.

Titration of acid form of the polysaccharide indicated that about 91.5% of the polysaccharide was composed of the N‐acetyl‐hexosaminuronic acids.
Biosynthesis of the Crystal Protein of Bacillus thuringiensis var. tolworth
Authors: Gould Hannah J. , Loviny Thérèse F. L. , Vasu Sabin‐Sorin , Herbert Barry N.
Published: March 2005

In the accompanying paper, we present evidence to show that the biosynthesis of the two characteristic polypeptide constituents “A” and “B” of the crystal protein toxin of Bacillus thuringiensis var. tolworth, is restricted to a short interval, from 16–18 h, in the mid‐stationary phase of the growth cycle of 24 h in culture. Here we examine the question of the kinetics of biosynthesis of the corresponding crystal protein messenger RNA. We have taken the direct approach of assaying a series of RNAs isolated from B. thuringiensis cultures at varying times of culture for messenger activity in a heterologous Escherichia coli, subcellular system. The product of RNA‐stimulated protein synthesis in vitro was characterized by immunoprecipitation with antiserum against purified crystal protein and by polyacrylamide‐gel electrophoresis of the antigen‐antibody precipitates dissolved in the detergent sodium dodecylsulfate. RNA from B. thuringiensis, but not that of Bacillus cerens, stimulates the synthesis of protein which specifically precipitates with crystal protein antiserum. The time of extraction of RNA from B. thuringiensis does not appear to be critical in determining its messenger activity: all samples taken in the interval of 11–20 h contained messenger RNA for crystal protein by the criterion of specific immunoprecipitation. In each case a significant proportion of the synthesized protein migrated in the region of the smaller‐molecular‐weight component A (55000) of the crystal protein in the electrophoretic separation. However, hardly any synthesized protein co‐migrated with the larger‐molecular‐weight component B (120000) of this protein. Since all the labelled protein precipitated by the crystal protein antiserum was larger than about 15000 molecular weight we conclude that mainly large fragments of the crystal protein were synthesized in the E. coli system. We failed in our attempts to isolate polysomes as a source of messenger RNA from stationary‐phase cells. Thus it was not possible to determine by experiments similar to those described above whether the crystal protein messenge is stored in ribosomes or in some other fraction of the cell when it is not being translated.
Induction of Polyoma DNA Synthesis by Injection into Frog‐Egg Cytoplasm
Authors: Laskey Ronald A. , Gurdon John B.
Published: March 2005

Covalently closed circular DNA from polyoma virus (component I) was injected into enucleated unfertilized eggs of Xenopus laevis together with [3H]thymidine. Polyoma DNA stimulated [3H]thymidine incorporation and part of the labelled product had the sedimentation characteristics of component I polyoma DNA. Polyoma component I DNA was prepared with a density label (5‐bromo deoxyuridine) in one strand and tritium in the other. After injection into egg cytoplasm, part of the labelled material changed buoyant density to that of normal (light) double‐stranded polyoma DNA, indicating the synthesis of new DNA strands rather than terminal addition or partial strand repair. 14C‐labelled component I polyoma DNA of hybrid buoyant density was prepared and injected together with [3H]thymidine. Part of the newly synthesized material was complementary to, and hydrogen bonded to, the heavy parental strands. Degradation of the injected DNA could not be detected suggesting that the strand synthesis observed was due to replication of the injected DNA rather than nick translation.