bluuebunny/crossref_metadata_2025 huggingface dataset:
Zur Biosynthese des Chinazolinalkaloids Arborin
Authors: Johne Siegfried , Waiblinger Konrad , Gröger Detlef
Published: March 2005

The indian plant Glycosmis arborea (Roxb.) DC. (Rutaceae) contains alkaloids of different types. The main alkaloid of the Glycosmis leaves is the 4‐quinazolone derivative arborine (VI). Previously it has been shown that labelled anthranilic acid (X) and phenylalanine (XI) are specifically incorporated into this alkaloid. This study was performed in order to elucidate the possible pathway of arborine formation in vivo. During these investigations an improved procedure for the isolation of arborine was developed.

The following results were obtained: After feeding of [14CH3]methionine the N‐methyl group of VI was specifically labelled. The same result was obtained after the application of [14CH]3N‐methylanthranilic acid. These results seem to indicate that anthranilic acid gets methylated prior to the condensation with phenylalanine which leads to VI. But on the other hand after feeding of [2‐14C] glycosminine (V) labelled arborine was also isolated. However in this case the incorporation rate was much lower compared with those of the two other mentioned precursors. It should be mentioned that V is only sparingly soluble in solvents suitable for feeding experiments. The N‐1 atom of VI is provided by the nitrogen of anthranilic acid. The experiment with [3‐14C, 15N]phenylalanine shows clearly that with the exception of the carboxyl group the aliphatic side chain of XI is incorporated into arborine. Therefore phenylacetic acid can be excluded as an immediate precursor of VI. [U‐3H]anthranoylphenylalanine (XIV) was a very poor precursor of arborine. N‐Methyl‐N‐phenyl‐[14CO‐CH2]acetylanthranilamid (XV) was transformed with high efficiency in Glycosmis into arborine. However it is doubtful whether the N‐phenylacetyl derivative of N‐methylanthranilamide is a true precursor in arborine biosynthesis. Surprisingly it could be shown that the transformation of XV → VI takes also place in pea plants. Apparently the ring closure of XV is catalyzed by an enzym or enzymatic system which is unspecific and widely distributed.
Chemical Structure of Tubular and Glomerular Basement Membranes of Human Kidney
Authors: Mahieu Philippe , Winand Roger J.
Published: March 2005

Enzymatic digestion with pronase and collagenase of glomerular and tubular basement membranes isolated from human kidneys yields soluble glycopeptides which can be separated by gel filtration and ion exchange chromatography.

Two different glycopeptides were isolated from both types of membranes. One contains a disaccharide composed of an equimolecular quantity of glucose and galactose probably linked to hydroxylysine. The second contains a heterosaccharide composed of galactose, fucose, mannose, hexosamine and sialic acids. The heterosaccharide from glomerular basement membrane had a molecular weight of 3600 and twice as much sialic acid as the heterosaccharide from tubular basement membrane which had a molecular weight of 3400.
Separation of Contributions of Tryptophans and Tyrosines to the Ultraviolet Circular Dichroism Spectrum of Hen Egg‐White Lysozyme
Authors: Teichberg Vivian I. , Kay Cyril M. , Sharon Nathan
Published: March 2005

Ultraviolet circular dichroism measurements over the wavelength range of 190–300 nm have been carried out on hen egg‐white lysozyme and on an iodine‐oxidized derivative of lysozyme in which Tryptophan‐108 has been selectively modified. The contribution of Tryptophan‐108 to the aromatic circular dichroism profile has been ascertained from a difference spectrum generated between the native and the modified protein. This tryptophyl residue is exceptional in its properties, as it is the principal contributor to the circular dichroism pattern in this wavelength range. The chemically modified protein shows only minor differences from the native molecule in terms of the magnitude of the conformation‐dependent ellipticity bands at 209 and 192 nm. Separation of the partial contributions of tyrosines was obtained from circular dichroism difference spectra constructed from circular dichroism profiles of both the native and iodine oxidized proteins at pH 7 and 11.2. Oligomers of N‐acetyl‐d‐glucosamine, which are inhibitors of lysozyme, markedly affect the circular dichroism spectrum of the enzyme at the aromatic region. It is postulated that this effect on the spectrum is a result of a change in orientation of Tryptophan‐108, which occurs on binding of the inhibitors to the active site of the enzyme.

These results are discussed with respect to the proposed three‐dimensional model of lysozyme.
Inhibition and Modification of Human Carbonic Anhydrase B with Bromoacetate and Iodoacetamide
Authors: Whitney Philip L.
Published: March 2005

Bromoacetate and iodoacetamide reversibly inhibit human erythrocyte carbonic anhydrase B. The binding of these inhibitors is competitive with that of the other inhibitors, chloride and sulfanilamide. However, bromoacetate and iodoacetamide are only partially competitive with each other. That is, both can bind at the same time, but the binding of one inhibitor increases the Ki for the second by about 50%.

While bound as a reversible inhibitor, bromoacetate or iodoacetamide reacts with the basic form of a specific histidine residue in the enzyme. The half‐times of reaction are 0.44 h and 2.7 h, respectively. The pH‐dependence of reaction shows that the pK of this histidine is 5.6 when bromoacetate is bound as a reversible inhibitor, and the pK when iodoacetamide is bound is below 5.0.

Reaction with bromoacetate introduces a carboxymethyl group in the region of the active center. This group perturbs the active site of the enzyme. This specific carboxymethylation causes about a five‐fold decrease in esterase activity and a shift in the midpoint of the pH‐dependence curve of activity from about pH 7.3 to 9.2. The affinity for several inhibitors is decreased.

Results of titration of the carboxymethyl enzyme with acetazolamide are consistent with 1.0 binding sites per enzyme molecule. This is the same number of sites as for native enzyme, but the Ki is higher with modified enzyme.

Studies of the visible spectrum of cobaltous carbonic anhydrase B have revealed two pH‐dependent transitions. The first occurs below pH 7 and produces relatively small changes in the spectrum. Introduction of the carboxymethyl group has little effect on the first transition, but shifts the midpoint of the second transition from pH 7.1 to 9.2.

Several lines of evidence indicate that the carboxymethyl group in the modified enzyme occupies a position different from the bromoacetate ion in the reversible complex.
Structure primaire de la caséine αs1 bovine
Authors: Mercier Jean‐Claude , Grosclaude François , Ribadeau‐Dumas Bruno
Published: March 2005

Earlier work in this laboratory on the determination of the primary structure of bovine αs1 casein [1–5] established that this phosphoprotein is composed of only one single peptide chain of 198 residues. The sequence of 101 residues have already been determined: the substitution of Glu/Gly at position 191 in the COOH‐terminal region differentiates the B and C genetic variants [1–3]. The sequence of 53 amino acid residues in the NH2‐terminal region was also determined [5].

This publication deals with the sequence of the central part of the chain. Sequence determinations have been made on various tryptic [2], CNBr and chymotryptic [4] peptides obtained earlier, which have been further broken down with the help of endopeptidases or mild acid hydrolylysis into smaller fragments to which classical methods of degradation have been applied: exopeptidase hydrolysis and substractive Edman's degradation.

The results obtained and previously published results establish the sequence of 172 amino acid residues in the molecule. The residues of which the positions have not been established so far are situated in 2 regions of the chain, between position 46 to 52 and position 61 to 77. These 2 regions are characterized by the presence of phosphorus and their sequences will be dealt with in our final publication.
Enzymes from Human Gastric Mucosa Conferring Blood‐Group A and B Specificities upon Erythrocytes
Authors: Schenkel‐Brunner Helmut , Tuppy Hans
Published: March 2005

Enzyme preparations obtained from human gastric mucosa microsomes are able to provide human erythrocytes with blood‐group A and B specificities, the newly conferred serological properties being dependent on the blood‐group of the tissue donor. The time‐course and the extent of the enzyme‐catalyzed change in antigenic properties of the erythrocytes were assessed by the serum absorption method.

The enzyme preparation from gastric mucosa of group A1 individuals, when incubated with O or B erythrocytes in the presence of UDP‐acetylgalactosamine, rendered them agglutin‐able by anti‐A serum. The enzymatically converted erythrocytes behaved like A2 cells. After treatment with enzyme from B individuals and UDP‐galactose, O, A1 and A2 erythrocytes could be agglutinated by anti‐B serum. When the enzymes from A1 and B individuals were allowed to act together upon O erythrocytes in the presence of UDP‐acetylgalactosamine and UDP‐galactose, the red cells became susceptible to agglutination with both anti‐A and anti‐B serum. The blood‐group specificity of red cells was not changed upon exposure to microsomal extracts from O individuals.
The Binding of Americium and Plutonium to Bone Glycoproteins
Authors: Chipperfield Alan R. , Taylor David M.
Published: March 2005

By means of a gel filtration technique it is shown that plutonium and americium differ in their mode of binding to two glycoproteins isolated from bovine cortical bone. Plutonium binds more strongly than americium, plutonium binding is maximal at pH 6 and americium at pH 8, and sialic acid is involved in the binding of plutonium but not americium. The mechanisms by which these elements bind to glycoproteins in vitro are discussed.
Biosynthesis of Salmonella Lipopolysaccharide
Authors: Mühlradt Peter F.
Published: March 2005

Several transfer reactions involved in the biosynthesis of the core part of Salmonella lipopolysaccharide have been studied in a cell free system. This system contained three components: (1) enzymes which were solubilised by repeated washings of EDTA‐lysozyme spheroplasts; (2) acceptor which consisted of either a heated cell wall fraction sedimenting at 20 000 x g, or heated, partially lysed, EDTA‐lysozyme treated cells; and (3) radioactively labelled precursors ([32P]ATP, UDP‐[14C]Glc, UDP‐[14C]Gal).

The following results were obtained with this transfer system: (1) only the γ‐phosphate group of ATP is transferred to the heptose moiety of the core; (2) polyphosphate which is synthesised enzymatically in a a side‐reaction by the system is not involved in the heptose phosphorylation; (3) there is a definite sequence of addition of the core substituents glucose, phosphate, and galactose to the incomplete core stub. This sequence is as follows:

(Hep)2‐(KDO, ethanolamine, phosphate, lipid A) → Glc‐(Hep)2‐(KDO, ethanolamine, phosphate, lipid A) → Glc‐(Hep)2P‐(KDO, ethanolamine, phosphate, lipid A) → (Gal)2‐Glc‐(Hep)2P‐(KDO, ethanolamine, phosphate, lipid A).
Thermisches Schmelzen und optische Rotations‐Dispersion von DNA‐Hg2+‐Komplexen
Authors: Luck Gerhard , Zimmer Chritoph
Published: March 2005

The effect of Hg2+ on the secondary structure of various DNA's with different (G + C) content is investigated by optical rotatory dispersion and melting temperature measurements.

Marked deformations of the optical rotatory dispersion spectra can be observed already at 1 Hg2+ per 500 DNA‐P. The changes in specific rotations beyond 250 nm are more effective for AT‐rich than for GC‐rich DNA. Comparison to corresponding DNA‐Cu2+‐complexes indicates the greater tendency of Hg2+ to produce conformational changes in the DNA double‐helical structure due to its higher affinity to the bases. DNA from Proteus mirabilis (42 mole‐% G + C) shows an increase in its melting temperature around 0.02 and at 0.09 Hg2+/DNA‐P. At 6 nM Na+ the Tm increases up to 0.03 Hg2+/DNA‐P. Between 0.03 and 0.07 Hg2+/DNA‐P Tm decreases whereas awt higher Hg2+ concentrations melting tempeature increases agains. Similar behaviour was found with DNA's of varying GC content. The decrease of Tm is more effective for GC‐rich than for AT‐rich DNA's.

The observed changes in the optical ratotory dispersion and melting profiles of DNA‐Hg2+ complexes are explained in terms of a specific binding of Hg2+ to adjacent thymine bases and AT‐pairs in accordance with previous results obtained by other authors. At 6 nM Na+ Hg2+ may combine with phosphate sites up to 0.03 Hg2+/DNA‐P. Within the decreasing Tm range the destabilization is attributed to a preferred formation of complexes of the type thymine‐Hg2+‐thymine whereas the stabilization effect at higher metal ion concentrations is presumably due to cross‐link formation between AT paris of opposite stands.
Structure of Pig Skin Dermatan Suflate
Authors: Fransson Lars‐Åke , Malmström Anders
Published: March 2005

Thee copolymeric structure of pig skin dermatan sulfate was further studied with particular referene to residual glucuronic acid moieties present in the molecule after sequential treatment with testicular hyaluronidase and β‐glucuronidase. Subsequent treatment with chondroitinase‐AC lead to a further release of glucuronic acid in the form of disaccharides, tetrasaccharidies and higher oligosaccharides/glycopeptides. Since these products contained nonreducing teminal, unsaturated uronic acid residues, it was concluded that some of the residual glucuronic acid moieties were located in ultimate or penultimate reducing terminal chondroitin 4‐sulfate repeating periods of fragments obtained after cleavage with testicular hyalyronidase. Additional, single chondroitin 4‐sulfate repeating periods were present in the vicinity of the carbohydrate‐protein linkage region or elsewhere in the chain. The former periods were regularly obseved 2‐3 units apart from the linkage region whereas the latter periods occurred preferentially in sequences like…BBBBABABAAAA…(A and B are chondroitin 4‐sulfate and dermatan sulfate repeating periods, respectively).
The Effect of Temperature and Ionic Strength on the Electrophoretic Mobility of Yeast Mitochondrial RNA
Authors: Grivell Leslie A. , Reijnders Lucas , Borst Piet
Published: March 2005

The electrophoretic mobilities of mitochondrial and cell‐sap ribosomal RNAs from Saccharomyces carlsbergensis have been measured relative to the mobility of Escherichia coli rRNA in 2.4% polyacrylamide gels at various temperatures and ionic strengths. At 5° C and in 20 mM sodium acetate, 40 mM Tris‐acetate, 2 mM EDTA (pH 7.8), mitochondrial and cell‐sap rRNAs co‐migrated and were only partially resolved from E. coli rRNA. When the temperature of electrophoresis was increased the mobility of the mitochondrial rRNAs (and to a lesser extent of the cell‐sap rRNAs) decreased relative to the mobility of the E. coli rRNA species, reaching a minimum between 9 and 16° C. As a consequence the mitochondrial and cell‐sap rRNAs were completely resolved in this buffer at temperatures above 9° C. Similar effects of temperature on relative electrophoretic mobility were observed in 90 mM Tris‐borate buffer at pH 8.3 and the effect was even more pronounced at low ionic strength (10 mM NaCl, 5 mM Tris‐HCl, 2 mM EDTA, pH 7.5).

At low ionic strength the decrease in the relative electrophoretic mobility of mitochondrial rRNA with an increase in temperature was accompanied by a decrease in sedimentation coefficient from 21–22 S (large component) and 14 S (small component) at 5° C to 16 and 11.5 S at 20° C (calculated in relation to assumed S‐values of 23 and 16 S for the rRNA species of E. coli).

We conclude that the electrophoretic mobility of RNA in polyacrylamide gels is more dependent on secondary structure than previous work had suggested and that caution is needed to interpret the relative mobility of mitochondrial RNA on gels in terms of molecular weight. In addition, our results show that judicious variation of temperature and ionic strength may allow the separation on gels of RNA mixtures that cannot be resolved under standard conditions.
Thermodynamic and Kinetic Aspects of the Interconversion of Chemical and Osmotic Energies in Mitochondria
Authors: Azzone Giovanni Felice , Massari Stefano
Published: March 2005

The conversion of osmotic energy into ATP has the following properties:

a) K+, as effluent ion, can be replaced by Rb+, whereas when H+, as influent, is replaced by other cations, the synthesis is abolished.

b) Both the effluent cation and the influent H+ seem to provide energy for the synthesis. The minimal sum of ΔpH +ΔpK, compatible with the synthesis, is 2.5 units, corresponding to an osmotic potential of 3.45 kcalories per gram‐ion equivalent−1. The number of ion equivalents exchanged per ATP synthesized is dependent on the dimension of the osmotic potential.

c) The osmotic potential compatible with the ATP synthesis is partially affected by the phosphate potential.

The conversion of redox into osmotic energy has the following properties:

a) The number of K+ ion equivalents exchanged with protons per energy rich bond is dependent on the concentrations of H+ and K+ in the inner and outer aqueous phases, increasing up to a maximal value of 4.

b) The number of K+ ion equivalents exchanged with protons per energy rich bond increases parallel to the increase of the amount of valinomycin, up to the value of 4.

On the basis of available data the following thermodynamic parameters are evaluated:

a) The relationship between osmotic potential and ATP synthesis.

b) The relationship between redox and osmotic energy.

c) The osmotic potential barrier for ion translocation.

A molecular mechanism for the interconversion of osmotic and chemical energies is discussed.
Carbethoxylation of Polynucleotides
Authors: ÖBerg Bo
Published: March 2005

Diethyl pyrocarbonate has been shown to react with poly(A), poly(C) and poly(G) but poly(U) does not react. Diethyl pyrocarbonate appears to carbethoxylate amino groups on the bases. Poly(A) can bind one carbethoxy group per base but the binding capacity of poly(G) and poly(C) under non‐denaturing conditions is 0.32 and 0.23, respectively. The difference in binding between poly(A) on one hand and poly(G) and poly(C) on the other may depend on conformation of the polynucleotides. The double helix poly(A) · poly(U) is essentially not carbethoxylated and native adenovirus DNA does neither bind carbethoxy groups. Carbethoxylation of poliovirus RNA leading to a 3‐fold decrease in infectivity resulted in an average of 11 carbethoxy groups per RNA molecule. The specificity of the carbethoxylation for single strands was used to estimate the helix content of transfer RNA. Denatured yeast tRNAAla was carbethoxylated on 55 out of 56 bases containing amino groups. 18 carbethoxy groups could be introduced under native conditions in the absence of magnesium, compared with 16 carbethoxy groups in the presence of magnesium. This is in agreement with the number of A, G and C in single‐stranded regions of the clover‐leaf model. Unfractionated yeast tRNA was carbethoxylated on 29 and 20 bases respectively in the absence and presence of magnesium. The results suggest that carbethoxylation could be used to determine secondary structure of RNA.
Isoleucyl‐tRNA Synthetase
Authors: Penzer Geoffrey R. , Bennett Edward L. , Calvin Melvin
Published: March 2005

Fluorescence properties of purified isoleucyl‐tRNA synthetase isolated from Escherichia coli B have been studied. No changes in the quantum yield, energy or polarisation of the emission were detected in the presence (either individually or in combinations) of the substrates and cofactors required for activation of L‐isoleucine.

In 2.5 M urea enzyme activity and intrinsic fluorescence intensity (at 340 nm) each decrease with time, showing similar kinetics and rate constants. The rate of this decay is reduced in the presence of ligands which can bind to the enzyme and the effect has been used to measure dissociation constants for enzyme‐ligand complexes. Values have been obtained for the complexes between enzyme and l‐isoleucine (Kdiss= 25 μM), l‐valine (Kdiss=300μM), ATP(Kdiss= 150 μM) and PPi (Kdiss= 200 μM) at 25°. The effects of ionic strength, and the temperature dependence and urea concentration dependence of l‐isoleucine binding have also been studied. Magnesium ions, which are required for catalysis, do not greatly affect the binding of single substrates, but changes are seen in the presence of ATP and l‐isoleucine together. The magnesium ion concentration dependence of this effect (half‐point about 200 μM) and the equilibrium constant for l‐isoleucine activation (2 μM) have both been measured.

The reliability of the methods has been discussed. Results have been interpreted in terms of current theories of amino acid activation. The binding parameters are sufficient to explain the stability of enzyme bound l‐isoleucyladenylate without invoking conformation changes. This is consistent with the absence of substrate induced fluorescence changes. Magnesium effects are explained in terms of reduced electrostatic repulsion between reactants bearing like charges.
The Respiratory System of Azotobacter vinelandii
Authors: Ackrell Brian A. C. , Jones Colin W.
Published: March 2005

The growth and respiration of Azotobacter vinelandii, together with the composition and activity of its membrance‐bound respiratory system are compared during nitrogen‐fixing batch growth at high and low aeration.

The aeration rate determines the length of the lag period, the extent of logarithmic growth and the respiratory activity of the culture; logarithmic growth ceases when the dissolved oxygen concentration drops to approximately 32 μM. Aconsiderable increase in whole cell respiratory activity is observed on changing from low to high aeration conditions.

During logarithmic, non‐oxygen‐limited growth, oxidase activities and respiratory carrier concentrations, with the exception of cytochrome o, remain low; the concentration of cytochrome o starts to rise when the ambient oxygen concentration falls to approximately 120 μM. Phosphorylation efficiencies, particularly at site I, are very low at high ambient oxygen concentrations but increase as the oxygen concentration decreases. Site I P/2e ratio is maximal (approx. 0.5) only when the oxygen concentration becomes growth limiting.

At the turnover point into non‐logarithmic, oxygen‐limited growth, respiratory carrier concentrations increase considerably; primary dehydrogenase activities increase by 2‐fold whereas the activity of the minor phosphorylating terminal pathway increases by 9–13 fold.

A working hypothesis is proposed to account for the ability of the ambient oxygen concentration to control both the activity and the energy conservation efficiency of A. vinelandii respiration.
Molecular Complexes between Cytochrome b2 [Yeast L(+)Lactate: Cytochrome c Oxidoreductase] and Cytochrome c in the Crystalline State and in Solution
Authors: Baudras Alain , Krupa Mireille , Labeyrie Francoise
Published: March 2005

In the crystalline state and in solution, cytochrome b2 (yeast l‐lactate: cytochrome c oxidoreductase) is shown to form a very stable complex with cytochrome c (from horse heart or yeast). This complex is characterized by a cytochrome c/cytochrome b2 heme ratio of about 0.25 which corresponds to the binding of one cytochrome c molecule to one tetraheme enzyme unit. Its formation is pH sensitive and prevented by salts but not by antimycin A. The prosthetic flavin group of cytochrome b2 is not required for the binding of cytochrome c.
Optical Rotatory Dispersion of Alkali‐Denatured Cytochrome Oxidase and Heme a‐Polylysine Complexes
Authors: Yong Fook Choy , King Tsoo E.
Published: March 2005

The optical rotatory dispersion of alkali denatured cytochrome oxidase, the heme a‐poly‐l‐lysine complex in the presence and absence of Emasol‐1130 (polyoxyethylene‐sorbitan monolaurate), the heme a‐poly‐d‐lysine complex and the protoheme‐poly‐l‐lysine complex were studied in the Soret region at pH 11.6–12.0. No anomalous rotations were observed in the oxidized form of either the denatured oxidase or any of the synthetic heme a complexes. The reduced form of the denatured oxidase exhibited a coupled‐type dispersion curve having a prominent trough at 437.5 nm flanked by two small peaks. Carbon monoxide shifted the trough to 435 nm. These optical rotatory dispersion profiles which differed from those given by the native enzyme and heme a‐poly‐l‐lysine complexes were similar at least qualitatively to those exhibited by heme a‐poly‐d‐lysine complex. The amplitude of rotations for both the denatured enzyme and the heme a‐poly‐d‐lysine complex was several fold smaller than those of heme a‐poly‐l‐lysine complexes. The optical rotatory dispersion spectra of the heme a‐poly‐d‐lysine complex in the reduced and carbon monoxide forms were found not to be mirror images of those of the corresponding l complex.

These results are interpreted to mean that alkali causes drastic conformational changes in the enzyme molecule and bring about effective heme‐heme interactions, that the orientation of the heme a molecule in the denatured enzyme differs from that observed in the synthetic complex, and that at least one of the ligands coordinated to the heme iron in the denatured enzyme is a lysyl residue.

Optical rotatory dispersion titrations of the heme a‐poly‐l‐lysine complex and its carbon monoxide derivative at constant heme a concentration showed a linear increase in rotation with increase of polylysine concentration but the rotation then decreased in excess polylysine. Similar anomalous titration behavior was observed for heme a‐poly‐l‐lysine in Emasol. In contrast, protoheme‐poly‐l‐lysine and the carbon monoxide treated heme a‐poly‐l‐lysine in Emasol gave the conventional rectangular hyperbolic titration curve. These results suggest that the observed optical rotation of heme a‐polylysine complexes are complicated and are affected to a great extent by the aggregative properties of heme a molecules. Protoheme which lacks the large hydrophobic side chain and the formyl group, exhibits no such anomaly when complexed with polylysine.
Latex Particle Agglutination in the Immunochemical System Human Serum Albumin–Anti‐Human Serum Albumin Rabbit Serum
Authors: Deželić Gjuro , Deželić Nada , Muić Nikola , Pende Berislav
Published: March 2005

The stability of monodisperse polystyrene latex particles in the immunochemical system human serum albumin–rabbit anti‐human albumin serum was studied by using a photometric method for the detection of latex particle agglutination. The latex particles were coated with human serum albumin and mixed with dilutions of both a hyperimmune and a normal rabbit serum. The experiments were carried out with latex particles of different size at varying pH, ionic strength and temperature. From the agglutination curves it was possible to derive conclusions on the specific and nonspecific immunochemical interactions with latex particles as well as on the effects of electrolyte coagulation. Maximum sensitivity of the latex reagent was found at pH values between 8 and 9. At pH 6 the latex reagent becomes unstable, at pH 4 the immunochemical interactions disappear, and at pH > 11 the immunochemical reaction becomes indistinct. Thermal inactivation could eliminate some of the nonspecific interactions, but the most efficient method was in experimenting at high ionic strengths (I= 0.5).
Incorporation in vitro of [14C]Leucine into the Mitochondrial Protein of Lucilia cuprina
Authors: Williams Keith L. , Birt L. Michael
Published: March 2005

The energy requirements for incorporation in vitro of [14C]leucine into sterile mitochondria isolated from thoracic flight muscles of newly‐emerged Lucilia cuprina have been studied.

There is an absolute requirement for addition of either ADP or ATP. Oxidisable substrates (pyruvate, DL‐α‐glycerophosphate, proline), or an ATP‐generating system increased incorporation above that observed with added ADP or ATP alone. Both the rate and duration of incorporation are strongly dependent on the concentration of adenine nucleotides used. Experiments with oligomycin and 2,4‐dinitrophenol suggest that energy for incorporation is provided as ATP.

With non‐sterile preparations, incorporation was, independent of added adenine nucleotides, and almost constant for up to four hours with no added substrate or adenine nucleotide. It was inhibited by added substrate and also by added amino acids, especially isoleucine and valine.
Optical Rotatory Studies on Nucleic Acid Derivatives n–π* Transitions and the Relation of the B2u Cotton Effect to Conformation in Pyrimidine and Azapyrimidine Nucleosides
Authors: Rogers Gordon T. , Ulbricht's Tilo L. V.
Published: March 2005

It is shown by solvent studies that O2:2′‐ and O2:5′‐cyclouridine derivatives give a small long wavelength Cotton effect which is due to an n–π* transition. The sign of the B2u Cotton effect (corresponding to the main ultraviolet absorption band) is as predicted by the previously formulated rule for pyrimidine nucleosides. Cotton effects due to n–π* transitions have also been observed in a number of 6‐azauridine derivatives.

Optical rotatory dispersion and circular dichroic data for a variety of azapyrimidine nucleosides are presented, and discussed in relation to corresponding data for pyrimidine nucleosides. The sign of the B2u Cotton effect in azapyrimidine nucleosides (which is negative in 6‐β‐d‐azauridine) can be explained on the basis of conformational considerations, or by assuming that the direction of the transition moment is different from that in pyrimidine nucleosides, owing to the presence of an additional nitrogen in the ring. Both of these possible explanations are illustrated by appropriate projection diagrams relating the base‐sugar torsion angle ΨCN to the B2u Cotton effect.
Ribosomal Proteins
Authors: Hindennach Ingrid , Kaltschmidt Eberhard , Wittmann Heinz‐Günter
Published: March 2005

50 S ribosomal subunits of Escherichia coli obtained by zonal centrifugation in B XV rotors were treated with various concentrations of LiCl in the absence and presence of urea. This treatment resulted in three protein fractions, each of which was subjected to CM‐cellulose chromatography. Gel filtration in Sephadex G‐100 (and preparative polyacrylamide electrophoresis) were used for further separation. The purity of the isolated proteins was better than 95% as shown by four methods. Proteins were isolated in yields of 6–90 mg depending mainly on the number and kind of purification steps.

Instead of fractionation with LiCl prior to column chromatography, precipitation of extracted proteins at various concentrations of ammonium sulfate was tested. This method is useful if a given protein, e.g. from mutants is wanted in large quantity.
Studies on the Mechanism and Stereospecificity of the Urocanase Reaction
Authors: Kaeppeli Franz , Rétey Jänos
Published: March 2005

Nuclear‐magnetic‐resonance spectroscopy was used to follow the course of the urocanase reaction. It was shown that 3‐(4′‐hydroxyimidazol‐5′‐yl)‐propionate is the true enzymic product and that it spontaneously tautomerises to recemic 3‐(imidazol‐4′‐one‐5′‐yl)‐propionate.

Conversion of urocanate in deuterium oxide by a crude‐enzyme preparation from Pseudomonas putida led to a product containing two adjacent CH2H‐groups. Oxidative degradation of this product afforded optically pur (RR)‐[2H2]succinic acid. This finding defines the sterochmistry of proton addition to the double bond of urocate.
Substrate Complexes of Phenylalanyl‐tRNA Synthetase from Escherichia coli
Authors: Kosakowski Heinz M. , Böck August
Published: March 2005

The stoichiometry of substrate binding to phenylalanyl‐tRNA synthetase from Escherichia coli K10 was investigated. Isolation of the phenylalanyl · adenylate · enzyme complex by means of gel filtration revealed that phenylalanine and the nucleotide are bound in about equimolar amounts and in a stoichiometric ratio. Stable complexes between tRNAPhe and phenylalanyl‐tRNA synthetase could be separated from free tRNAPhevia sucrose‐gradient zone‐centrifugation or gel filtration. Determination of the amount of tRNAPhe bound to phenylalanyl‐tRNA synthetase (molecular weight 181000) by absorption at 260 nm or by the amino‐acid‐acceptance activity yielded ratios close to one. tRNAPhe· enzyme complexes were most stable at pH values below 6.5; complex formation between phenylalanyl‐tRNA synthetase and non‐cognate tRNA species was not observed. For determination of the affinity and ratio of binding of phenylalanine the non‐equilibrium dialysis method was employed: a Kd of 30 μM was obtained; by the criteria of this method one binding site for the amino acid could be demonstrated.

Phenylalanyl‐tRNA synthetase of E. coli is protected against heat inactivation by Mg, ATP, ATP and phenylalanine or tRNAPhe. By means of the protection constant, the apparent affinity constant for tRNAPhe was determined to be 3 × 107 M−1 at pH 6.0 and 37 °C. The dependence of the π‐values obtained on pH indicates that the affinity of phenylalanyl‐tRNA synthetase for tRNA drastically increases at more acid pH. The same tendency was obtained upon determination of the pH‐dependence of the Km values for tRNAPhe.

Out of the 24 SH‐groups of phenylalanyl‐tRNA synthetase which are reacting with 5,5′‐dithio‐bis(2‐nitrobenzoic acid) in the presence of 8 M urea only less than one half of them are oxidized on the native enzyme. The kinetics of titration of the sulfhydryl is grossly changed in the presence of ATP, ATP and phenylalanine or tRNAPhe. In addition, in the presence of ATP and phenylalanine or of tRNAPhe 2‐3 SH‐groups are completely protected from oxidation. Under this condition loss of activity which accompanies the SH‐group oxidation is greatly diminished.
Stereochemistry of the Citrate‐Lyase Reaction
Authors: Buckel Wolfgang , Lenz Helmut , Wunderwald Peter , Buschmeier Verena , Eggerer Hermann , Gottschalk Gerhard
Published: March 2005

Using a medium of tritiated water for the conversion of fumarate to malate and using a medium of deuterium oxide for the cleavage of citrate, chiral acetate was generated enzymically in the sequence fumarate → malate → oxaloacetate → citrate → acetate.

Acetate of opposite chirality was produced in the same sequence by replacing fumarate and tritiated water by [2,3‐3H2]fumarate and ordinary water, respectively.

The configuration of acetate generated in (1) was shown to be R, and the configuration of acetate generated in (2) was shown in to be S.

It was concluded that citrate lyase cleaves citrate with inversion of configuration at the methylene group which becomes methyl.
The Synthesis and Properties of the Sepharose‐Bound tRNA Nucleotidyltransferase
Authors: Litvak Simon , Tarrago‐Litvak Laura , Carre Danièle S. , Chapeville François
Published: March 2005

The synthesis of an insoluble tRNA nucleotidyltransferase by covalent coupling of the enzyme to cyanogen bromide‐activated sepharose is described. The sepharose‐bound enzyme is active in the repair of the pCpCpA sequence of tRNA. Most of its properties are identical to the soluble enzyme although its stability, regarding thermal inactivation and trypsin digestion, is increased. However, tRNA, which protects the soluble enzyme against thermal denaturation and protease digestion, surprisingly does not protect the sepharose‐bound enzyme.
Response of Phosphopyruvate Carboxylase to Tryptophan Metabolites and Metal Ions
Authors: Snoke Roy E. , Johnston James B. , Lardy Henry A.
Published: March 2005

Tryptophan, administered to rats, inhibits gluconeogenesis, but increases the activity of hepatic phosphopyruvate carboxylase when assayed in vitro. Quinolinate, a metabolite of tryptophan, inhibits gluconeogenesis in perfused livers, and inhibits phosphopyruvate carboxylase in liver cytosol from tryptophan‐treated rats but not from normal rats.

Metal‐ion activation and quinolinate inhibition of hepatic cytosol phosphopyruvate carboxylase has been investigated in vitro. Of the cations tested, only Fe2+, Co2+, Cd2+ and Mn2+ appreciably increased this enzymic activity. Quinolinate inhibited the activity of divalent cation‐treated enzyme in the order Co2+ Fe2+ Cd2+ and did not inhibit the Mn2+‐treated phosphopyruvate carboxylase. Other tryptophan metabolites, metal chelators, and structural analogs of quinolinate have been tested, but only quinolinate lowered the activity of the Fe2+‐treated enzyme substantially.

The elevated phosphopyruvate carboxylase activity in cytosol of tryptophan‐treated rats was lowered in vitro by quinolinate to the same extent as was the activity of the Fe2+‐treated enzyme from normal rats. Ferrous iron is proposed as the physiologically important cation and quinolinate the important metabolite responsible for altering the activity of phoshopyruvate carboxylase in vivo following tryptophan administration.
Studies of Glutamate Dehydrogenase
Authors: Markau Klaus , Schneider Jens , Sund Horst
Published: March 2005

The investigation of the association‐dissociation equilibrium of beef liver glutamate dehydrogenase by light‐scattering measurements over a wide range of protein concentration (0.02 to 50 mg/ml) has shown that this equilibrium can be described as an open equilibrium (Mi+n Mh⇋ Mi+h, i and h≥ 1, M = oligomer) with identical equilibrium constants for all steps and without any limit. The dependence of the apparent weight‐average molecular weight on the protein concentration reaches a maximum at about 9 mg/ml. The decrease of the molecular weight at higher protein concentrations must be attributed to effects of nonideality (as expressed in relation to a first approximation by the second virial coefficient). At 20°C and in M/15 sodium phosphate buffer the equilibrium constant was found to be 9.0 × 105 M−1 (corresponding to a free energy change of 7.8 kcal/mol for each consecutive step in the association reaction) and the second virial coefficient to be 9 nmol × 1 × g−2. The calculation of the dependence of the reduced viscosity on the protein concentration based on these results conforms with the experimental data. However, it was found that the length of the oligomer obtained from light‐scattering measurements was dependent on the wavelength. The reason for this phenomenon is not quite obvious.

Low pH and low ionic strength promote the association of the oligomers. It was found that the sedimentation coefficient at pH 6.2 and an ionic strength of 0.04 is 31 S (34 S in the presence of 4.4 mM NAD+) compared to 27 S at an ionic strength of 0.115. The pH dependence of the sedimentation coefficient at low ionic strength indicates the participation of a group in the association with a pK near 7, possibly an imidazole group. At high ionic strength the sedimentation coefficient is almost pH independent.

From the results it may be concluded that, in the interaction between the oligomers at low ionic strength, ionic effects are involved that are shielded at higher ionic strength.

GTP and NADH cause the dissociation into the oligomer characterized by an s020,w value of 13.0 ± 0.3. The dissociation is complete in the presence of 20 mM GTP and NADH. The result that the s020,w values of the oligomer are the same in the absence and in the presence of effectors, GTP/NADH, conforms to our X‐ray small‐angle measurements.
Inactivation, Stabilization and Some Properties of ATP: Glutamine Synthetase Adenylyltransferase from Escherichia coli B
Authors: Wolf Dieter , Ebner Eberhard , Hinze Helga
Published: March 2005

ATP: glutamine synthetase adenylyltransferase loses activity after purification, forming aggregates with high molecular weight. This inactivation can be prevented by bovine serum albumin. Freshly prepared adenylyltransferase of low specific activity is reactivated by bovine serum albumin. It is shown that the effect of bovine serum albumin on adenylyltransferase is due to a protein—protein interaction. A mechanism for the inactivation, reactivation and stabilization is proposed.

The molecular weight of adenylyltransferase was estimated by ultracentrifugation to be 115000. The isoelectric point is 4.2 to 4.3. Adenylyltransferase was found to be specific for native glutamine synthetase.
Subunit Structure of L‐Lactate Dehydrogenase (Cytochrome b2) of Saccharomyces cerevisiae
Authors: Mevel‐Ninio Maryvonne
Published: March 2005

The molecular weights of yeast L‐lactate dehydrogenase (cytochrome b2) and of its polypeptide chains have been measured by ultracentrifugation. The intact protein was found to have a molecular weight of 240000 ± 10000 by sedimentation equilibrium measurements according to the Yphantis high‐speed method. The solution obtained by dissolving the protein in 6 M guanidine (with added 2‐mercaptoethanol) behaved as if it were monodisperse, giving Mr= 31300 ± 2000, by approach‐to‐equilibrium measurements. This result indicates that the “58 600 unit” associated to one set of prosthetic groups, heme and flavin, is made up of two chains, and thus the whole molecule contains a total of eight chains, identical or not. However, as the viscosity of the medium was reduced (using 2 M guanidine) the paucidispersity of the cytochrome b2 solution was revealed. The mixture was shown to consist of two kinds of chains distinct in molecular weight. From these experiments the molecular weight of the light chain was found to be 22600 ± 1500. After partial separation of the two chains by gel filtration through Sephadex G‐100, the molecular weight of the heavy chain was estimated as 39700 ± 3000.

Preliminary experiments indicate that the heavy component can bind quantitatively the heme prosthetic group. The FMN appears to be bound to the light component.
Precipitation of Lipopolysaccharides from Rough Mutants of Salmonella typhimurium by an A‐Hemagglutinin from Helix pomatia
Authors: Hammarström Sten , Lindberg Alf A. , Robertsson Eva Stina
Published: March 2005

Purified Helix pomatia A‐hemagglutinin precipitated lipopolysaccharides of Salmonella typhimurium rough mutants of chemotype Ra and Rb. No precipitation was obtained with lipopoly‐saccharide of the parent smooth strain or of Rc, Rd and Re mutants or with isolated lipid A. Reactivity of lipopolysaccharide from some of the Ra and Rb mutants was increased when precipitated in a mixture of tetrahydrofuran—phosphate‐buffered saline (1:3, by vol.). However lipopolysaccharide from nonreactive mutants was still negative in the latter medium. Precipitating ability of lipopolysaccharide from Ra and Rb mutants was retained after treatment of the lipopolysaccharide with sodium borohydride (or NaOH).

Precipitation was inhibited by N‐acetyl‐D‐galactosamine but not as easily by D‐galactose or D‐mannose. The lipopolysaccharide from the Ra mutant TV160 displaced a labelled blood‐group‐A‐active pentasaccharide from the combining site of the hemagglutinin. Inhibition studies indicated that the interaction was weak.

Core polysaccharides prepared by acid hydrolysis and gel filtration from the lipopolysaccharide of the Ra mutant SH180 and the Rb mutant TV160 inhibited precipitation. On a molar basis the SH180 core polysaccharide was approximately as active as α‐D‐Gal‐(1→6)‐D‐Glc while the TV160 fragment was five times more active.

The precipitating ability of TV160 lipopolysaccharide was abolished after treatment with the enzyme D‐galactose oxidase.

The structural element in the core region in common for all reactive lipopolysaccharide is nonreducing D‐Gal linked α(1→6) to D‐Glc. It is suggested that this residue is the “immunodominant” sugar responsible for precipitation. In the TV160 lipopolysaccharide there is an additional nonreducing D‐Gal residue linked α(1→3) to the same D‐Glc. It is possible that the presence of this residue increases the binding.

The experiments suggest that precipitation occurs when both reagents are polyvalent although each individual interaction may be very weak.
Recherches biochimiques sur l'oogenése
Authors: Denis Herman , Mairy Marianne
Published: March 2005

As found previously, 70 to 75% of the RNA present in previtellogenic oocytes of Xenopus laevis (average diameter: 50 to 150 μm) is 5‐S RNA and transfer RNA. In this paper, we show that most of the RNA of the small oocytes is stored in nucleoprotein particles sedimenting at 58 S and 42 S. These particles are no longer detectable in vitellogenic oocytes (average diameter: 375 to 1100 μm) which accumulate ribosomes very rapidly.

The 58‐S particles contain mainly high‐molecular‐weight RNA. This RNA has a base composition close to that of DNA. Its main component sediments at about 19 S. The 42‐S particles contain more than half of the RNA and a quarter of the protein present in homogenates of small oocytes. The only RNA species found in the 42‐S particles are 5‐S RNA and tRNA. The molar ratio of tRNA to 5‐S RNA in the 42‐S particles is 3:1.

5‐S RNA is distributed as follows in the small oocytes: less than 1% of the total cell content is bound to the ribosomes; about 50% is in the 42‐S particles and the remainder is apparently free in the cell sap. By its base composition and its hybridization properties 5‐S RNA from the 42‐S particles cannot be distinguished from 5‐S RNA of the cell sap and from 5‐S RNA of somatic cells.

About 90% of the total tRNA content of the oocyte is included in the 42‐S particles. The remaining 10% is either free or bound to the ribosomes. No difference was noticed in the hybridization properties of tRNA which is inside the 42‐S particles and tRNA which is outside these particles.

The 42‐S particles present in homogenates of previtellogenic oocytes are not artifacts. These particles are probably storage particles in which 5‐S RNA and tRNA produced in excess during the initial period of oogenesis are kept for many months until the ribosomes begin to accumulate. The role of the 58‐S particles remains unknown.
Recherches biochimiques sur l'oogenèse
Authors: Mairy Marianne , Denis Herman
Published: March 2005

Almost half of the RNA present in previtellogenic oocytes of Xenopus laevis is 5‐S RNA. Less than 1% of this RNA is immediately included in the ribosomes. 5‐S RNA made in excess in the small oocytes is not degraded but stored and progressively taken up by the ribosomes which accumulate very rapidly after the beginning of vitellogenesis.

The amount of 5‐S RNA stored in the small oocyte is not sufficient to meet the needs of the cell until the end of its growth since 5‐S RNA continues to be synthesized during the second part of oogenesis. The ribosomes assembled during this period incorporate both newly‐made 5‐S RNA and 5‐S RNA which has been synthesized months before. Newly‐made 5‐S RNA seems to be taken up by the ribosomes somewhat more rapidly than “old” 5‐S RNA.
The Denaturation of Muscle Phosphorylase b by Urea
Authors: Chignell Derek A. , Azhir Arasteh , Gratzer Walter B.
Published: March 2005

The denaturation of reduced rabbit muscle phosphorylase b by urea has been studied. Two intermediates of unfolding can be discerned in the denaturation profile, the first of which is stable near 4 M urea, the second at about 7 M urea. The first transition is accompanied by a large increase in fluorescence from the pyridoxamine chromophore. This form of the protein is prone to aggregation, and becomes rapidly insoluble at higher protein concentrations than are used for fluorescence. Both intermediates can be observed by circular dichroism. The second and third transitions are associated with different types of denaturation difference spectra, reflecting exposure respectively of tryptophan in generating the intermediate, and predominantly of tyrosine in converting it to what is presumed to be the fully random state. It is shown by the cross‐linking technique of Davis and Stark that the protein is essentially monomeric at urea concentration of 1 M and upwards, except for the formation of an aggregated fraction at all higher concentrations, which is also apparent in the analytical ultracentrifuge.