bluuebunny/crossref_metadata_2025 huggingface dataset:
Immobilization of Yeast Microbodies by Inclusion with Photo‐crosslinkable Resins
Authors: TANAKA Atsuo , YASUHARA Shigeki , OSUMI Masako , FUKUI Saburo
Published: March 2005

Yeast microbodies containing FAD‐dependent alcohol oxidase, catalase and D‐amino acid oxidase were isolated from methanol‐grown cells of Kloeckera sp. 2201 and immobilized intact in matrices formed by a short‐time illumination of photo‐crosslinkable resin oligomers. The relative activities of catalase, alcohol oxidase and D‐amino acid oxidase of the gel‐entrapped microbodies were 36, 76 and 31% respectively as compared with those of free microbodies.

Immobilization enhanced the stability of catalase to a certain degree, but not that of alcohol oxidase. The pH/activity profiles of catalase and alcohol oxidase of the entrapped organelles showed more narrow pH optima than those of the free counterparts. D‐Amino acid oxidase in immobilized microbodies showed a somewhat higher Km value for D‐alanine than that in free ones. Immobilized microbodies oxidized two moles of methanol to form two moles of formaldehyde with consumption of one mole of molecular oxygen. Addition of 3‐amino‐1,2,4‐triazole, an inhibitor of catalase, reduced the formation of formaldehyde to half the amount without change in the amount of oxygen consumed, indicating the synergic action of alcohol oxidase and catalase in methanol oxidation in the microbodies of living yeast cells.
Properties of Human Erythrocyte Catalases after Crosslinking with Bifunctional Reagents Symmetry of the Quaternary Structure
Authors: HAJDU János , WYSS Sonja R. , AEBI Hugo
Published: March 2005

Normal erythrocyte catalase, the enzyme present in the blood of Swiss acatalasemic heterozygotes, and their hybrid produced in vitro, were studied after crosslinking with bifunctional reagents.

On theoretical grounds [cf. Hajdu, J., Bartha, F. & Friedrich, P. (1976) Eur. J. Biochem. 68, 373–383] it is inferred from the dodecylsulphate gel electrophoretic patterns obtained after treating catalase with diimidates of various chain lengths that the enzyme is an isologous tetramer (D2 symmetry). The minimal distances between crosslinkable primary amino groups across the three domains of bonding are different.

Reaction with diimidates causes a moderate loss of enzyme activity in all three enzyme types due to amidination rather than crosslink formation. On the other hand, crosslinking stabilizes the enzyme against urea and heat inactivation. This is most prominent with heterozygote acatalasemic catalase.

Crosslinking markedly prevents the development of peroxidase activity that can be elicited in catalases by urea treatment. The role of the quaternary structure of the protein in the relationship between catalase and peroxidase activities is discussed.
Development of Escherichia coli Virus T1: Repression of Host Gene Expression
Authors: WAGNER Erwin Friedrich , PONTA Helmut , SCHWEIGER Manfred
Published: March 2005

Host protein synthesis, measured either as amino acid incorporation into proteins or as enzyme synthesis, is inhibited rapidly after infection of Escherichia coli with T1. Analysis of this inhibition, using a technique which distinguishes between translation and transcription, revealed that translation of host mRNA is specifically blocked. Comparison of the time course of T1‐induced host repression with inhibition by the drugs rifampicin, nitrofurantoin and chloramphenicol showed that T1 affects the initiation step of host translation.

Intact membranes are apparently essential for host repression, suggesting a membrane‐mediated process. Concomitant viral protein synthesis is not required. The membrane‐altering principle is a constituent of the viral particle.
Amino Acid Sequences of Neurotoxins I and III of the Elapidae Snake Naja mossambica mossambica
Authors: GREGOIRE Jana , ROCHAT Hervé
Published: March 2005

The amino acid sequences of two neurotoxins of the African cobra Naja mossambica mossambica have been determined using almost uniquely phenylisothiocyanate degradation in a liquid protein sequencer programmed alternatively with ‘protein’ and ‘peptide’ programs. When compared to known sequences of so‐called ‘short’ neurotoxins belonging to other Elapidae snakes, neurotoxins I and III of Naja mossambica mossambica are very similar to the cobrotoxin, a neurotoxin isolated from the formosan cobra Naja atra atra.
Solution Conformational Analysis of 2′‐Amino‐2′‐Deoxyadenosine, 3′‐Amino‐3′‐Deoxyadenosine and Puromycin by Pulsed Nuclear‐Magnetic‐Resonance Methods
Authors: PLACH Harald , WESTHOF Eric , LÜDEMANN Hans‐Dietrich , MENGEL Rudolf
Published: March 2005

The solution conformation of 2′‐amino‐2′‐deoxyadenosine, 3′‐amino‐3′‐deoxyadenosine, and 3′‐amino‐3′‐deoxy‐6‐N,N‐dimethyladenosine have been determined by nuclear magnetic resonance in aqueous and ammonia solutions. The analysis of the ribose moiety is based on the two‐state S ⇄ N model of Altona and Sundaralingam. Longitudinal proton relaxation time and nuclear Overhauser enhancement measurements have been carried out in order to characterize the orientation of the base relative to the ribose. These studies indicate that 3′‐amino‐3′‐deoxyadenosine and 3′‐amino‐3′‐deoxy‐6‐N,N‐dimethyladenosine exist in solution preferentially in the N‐anti‐g+ conformations. On the other hand, 2′‐amino‐2′‐deoxyadenosine adopts the S‐syn‐g+/t conformation families. It appears that the base is restricted to the anti conformation in the first two compounds, while in 2′‐amino‐2′‐deoxyadenosine, one third of the molecules in the S state are in the anti range. These studies corroborate the previously proposed correlations between the N state of the ribose and the anti orientation of the base and between the S state of the ribose and the syn orientation of the base.
Purification and Properties of Citrate Lyase Ligase from Streptococcus diacetilactis
Authors: BOWIEN Susanne , GOTTSCHALK Gerhard
Published: March 2005

Citrate lyase ligase (acetate: SH‐[acyl‐carrier protein] enzyme ligase (AMP)) from Streptococcus diacetilactis was purified 920‐fold with a yield of 6.3%. The molecular weight of the enzyme was estimated to be 41000; the ligase consisted of one polypeptide chain.

The acetylation of 1 mol of deacetyl‐citrate lyase to enzymatically active citrate lyase required 6 mol ATP. The formation of AMP and pyrophosphate in the acetylation reaction was demonstrated.

Citrate lyase ligase was specific for the lyase from S. diacetilactis and did not acetylate lyases from Rhodopseudomonas gelatinosa and Enterobacter aerogenes. The substrates acetate and ATP could be replaced by propionate and dATP, respectively.

The reaction rates for ATP, acetate and deacetyl‐citrate lyase followed Michaelis‐Menten kinetics (Km values: 26 μM for ATP, 25 mM for acetate and 38 nM for deacetyl‐citrate lyase).
Mechanism of the Prolyl Hydroxylase Reaction
Authors: TUDERMAN Leena , MYLLYLÄ Raili , KIVIRIKKO Kari I.
Published: March 2005

The co‐substrate requirements of prolyl hydroxylase were studied with pure enzyme from chick embryos. No hydroxylation occurred without added Fe2+, indicating that the enzyme does not retain iron sufficiently to catalyze any reaction. Zn2+ was an effective competitive inhibitor with respect to Fe2+, but was noncompetitive with respect to the polypeptide substrate and 2‐oxoglutarate, suggesting that it replaced iron in the active site of the enzyme.

The enzyme catalyzed the uncoupled decarboxylation of 2‐oxoglutarate at a rate of about 4 mol CO2 formed (mol enzyme)−1 min−1 in the presence of Fe2+, O2, and ascorbate but in the absence of the polypeptide substrate. This rate was about 1/80 of that observed in the presence of the substrate. Several compounds inhibited the enzyme competitively with respect to 2‐oxoglutarate but non‐competitively with respect to Fe2+. It seems that these two co‐substrates become bound at separate sites on the enzyme, and additional data suggested that these are distinct from the binding site of the polypeptide substrate.

The reaction was completely dependent on O2. Nitroblue tetrazolium was a competitive inhibitor with respect to O2, but noncompetitive with respect to the polypeptide substrate and all other co‐substrates. Epinehrine also inhibited the enzyme, but this inhibition was competitive with respect to Fe2+. The results suggest that nitroblue tetrazolium consumed an activated form of oxygen, whereas epinephrine acted primarily by binding Fe2+.

The reaction was completely dependent on ascorbate, and in contrast to previous data, this could not be significantly replaced by tetrahydrofolic acid or dithiothreitol. Dehydroascorbate replaced ascorbate in the presence of dithiothreitol but not in its absence. The results also indicate that ascorbate is not stoichiometrically consumed during the reaction.
The Use of Liver Transglutaminase for Protein Labeling
Authors: IWANIJ Victoria
Published: March 2005

Guinea‐pig transglutaminase catalyzes the formation of an amide bond between the γ‐carboxyl group of peptide‐bound glutamine and a variety of amines, including fluorescent or spin‐labeled derivatives. A systematic study was conducted with this enzyme in order to establish its usefulness as a protein‐labeling technique. Transglutaminase‐catalyzed amine incorporation was tested with isolated proteins, intact cells, membrane preparations, and virus particles. It was found that, due to its large molecular weight and its specificity, the transglutaminase reaction is limited to highly exposed glutamine residues. For example, proteins such as tubulin and α2‐macroglobulin were modified while immunoglobulins (IgG) from a variety of sources were not, despite their high glutamine content. The peptide hormone glucagon was modified by transglutaminase, which resulted in the loss of hormonal activity.

Most of the intact cells were not labeled with transglutaminase. Only in the case of human fibroblasts the labeling of a few heavy‐molecular‐weight proteins, including large external transformation‐sensitive (LETS) protein, occurred. However, isolated membrane preparations, such as erythrocyte ghosts, sarcoplamic reticulum and retinal disc membranes, were significantly labeled.

Intact influenza virions were susceptible to the transglutaminase‐catalyzed reaction; however, only hemaglutinin but not neuraminidase was labeled.

The transglutaminase reaction was found useful when specific and limited labeling is required. This technique should be particularly amenable for the introduction of fluorescent probes into membrane proteins and for elucidation of the structure‐function relationship of the proteins.
Development of a Microassay for Estradiol Receptors
Authors: BARBANEL Gérard , BORGNA Jean‐Louis , BONNAFOUS Jean‐Claude , MANI Jean‐Claude
Published: March 2005

The calculation of equilibrium binding constants for a specific receptor‐hormone interaction requires the exact determination of the unbound ligand concentration and the specifically bound concentration at equilibrium. These determinations usually require corrections for the contribution of non‐specific binding. We introduce several parameters allowing equilibrium concentrations to be calculated from experimental concentration values; these parameters can be measured for the particular receptor assay procedure used. These parameters are used in a microassay of estradiol‐receptor complex by selective retention on DE‐81 cellulose paper.
Characterization of the Cellular Binding of Exogenous Gangliosides
Authors: CALLIES Richard , SCHWARZMANN Günter , RADSAK Klaus , SIEGERT Rudolf , WIEGANDT Herbert
Published: March 2005

Experiments were performed to characterize the accumulation of different exogenous sialoglycolipids by cells.

A biphasic kinetics of accumulation appeared to be characteristic for intact primary and permanent monolayer cells and chicken erythrocytes whereas that of chicken erythrocyte ghosts was monophasic. Saturation levels could be reached with Galβ1→3 GalNAcβ1→4 Gal[3←2αNeuAc]‐β1→4 Glcβ1–1′Cer and Galβ1→3 GalNAcβ1→4 Gal[3←2αNeuAc8←2αNeuAc]β1→4 Glcβ1 – 1′Cer with concentrations of 100 and 200 nmol per ml culture medium, respectively. Under these conditions the amount of 1.3–1.4 nmol of sialoglycolipids per 100 μg of cellular protein were found to be cell‐bound after approximately two hours. In case of the ganglioside NeuAcα2→3Galβ1→4 Glcβ1 – 1′Cer no saturation could be reached.

The intermediate plateau of accumulation was abolished by the agents colchicine and sodium fluoride which on the other hand had no influence on the level of saturation. In contrast the presence of cytochalasin B, 2‐deoxy‐D‐glucose plus sodium azide, and glutardialdehyde as well as low temperature decreased the amount of incorporated gangliosides drastically. The effect of cytochalasin B was limited to monolayer cultures.

Mild trypsinisation liberated some 70–85% of cell‐associated gangliosides even after drug‐inhibited accumulation of gangliosides. Also some 87–95% of the cell‐bound ganglioside NeuAcα2→3 Galβ1–4 Glcβ1–1′Cer remained accessible to the action of exogenous sialidase, thus excluding gross internalisation.
Maintenance of the Ratio of α and β Globin Synthesis in Rabbit Reticulocytes
Authors: STEWART Andrew G. , LLOYD Malgorzata , ARNSTEIN Henry R. V.
Published: March 2005

The synthesis of rabbit α and β globins under various conditions was studied using intact reticulocytes and reticulocyte cell‐free systems. Raising the salt concentration of media in which reticulocytes were incubated with radioactive amino acids reduced the total protein synthesis but did not affect the ratio of α to β globins produced. Using a reticulocyte lysate which had been incubated with micrococcal nuclease to remove the endogenous globin messenger RNA activity, it was found that unlike the intact cell or the untreated lysate very little α globin was synthesised on adding purified globin mRNA. These results are discussed in terms of their compatibility with some proposed models of coordination of α and β globin production.
Characterization of the Isolated Transferase Subunit of Citrate Lyase as a CoA‐Transferase. Evidence against a Covalent Enzyme‐Substrate Intermediate
Authors: DIMROTH Peter , LOYAL Rosemarie , EGGERER Hermann
Published: March 2005

1. The largest subunit of citrate lyase from Klebsiella aerogenes represents an acetyl‐thio‐acyl carrier protein: citrate acyl carrier protein transferase but is shown to be also an acetyl‐CoA: citrate CoA‐transferase.

2. The enzyme catalyzes an exchange of acetyl residues between acetyl‐thio‐acyl carrier protein or acetyl‐CoA and acetate. The reaction occurs in the presence and, at a much lower rate, also in the absence of acetate. The citrate‐independent reaction was attributed to a substitution of citrate by acetate as a substrate analogue.

3. Additionally, the isolated enzyme, in the presence of acetyl‐CoA and citrate, catalyzed the formation of (3S)‐citryl‐CoA and was thus unequivocally shown to be a CoA‐transferase.

4. The catalytic efficiency of the enzyme in the CoA‐transfer reaction is similar to that of other CoA‐transferases. It was concluded that the transferase subunit of citrate lyase and the classical CoA‐transferases could belong to one family to enzymes.

5. Attempts to demonstrate this relationship were based on a comparison of the mechanism of action of the transferase subunit of citrate lyase with that established for CoA‐transferases. These form enzyme‐CoA and ‘anhydride’ intermediates.

6. Evidence is presented for the generation of ‘anhydride’ intermediates on the transferase subunit of citrate lyase. The ‘anhydrides’ are ‘citric acetic anhydride’ and ‘acetic anhydride’ in the acetyl exchange reactions with and without citrate, respectively.

7. In contrast to other CoA‐transferases, however, the transferase‐catalyzed CoA‐transfer from acetyl‐CoA to citrate, apparently involves no enzyme‐CoA intermediate. Neither the results of several different chemical approaches nor the kinetics were compatible with the formation of this intermediate.

8. An explanation is proposed and the consequences are discussed.
β‐D‐Galactoside Transport in Escherichia coli: Substrate Recognition
Authors: SANDERMANN Heinrich
Published: March 2005

1. A number of galactosides and other sugar compounds were examined as inhibitors of facilitated or active transport by the lactose permease system of Escherichia coli. Efficient inhibition required an α‐ or β‐anomeric galactopyranosyl ring of D‐configuration, a free 6‐hydroxyl group, and a certain aglycone size which was reached, for example, by monosaccharide or nitrophenyl substituents.

2. Aromatic α‐D‐galactopyranosides acted as high‐affinity inhibitors (Ki, below 50 μM). At lest two of them were not transported, in contrast of α‐galactoside disaccharides and to aromatic β‐D‐galactopyranosides.

3. β‐D‐galactoside transport was not significantly inhibited by specific inhibitors and transition‐state analogues of β‐galactosidase (D‐galactal, D galactonolactone).

4. The β‐D‐galactopyranoside, lactitol, and the α‐D‐galactopyranoside, galactinol, were not efficiently bound by the lactose permease system, although the maximal rate of uptake of lactitol was similar to that of lactose. By comparison with several structrally related D‐galactopyranosides, the decreased affinity was attributed to an effect of the membrane/water interface. A model for substrate recognition by the lactose permease system is presented.
Human α‐N‐Acetylglucosaminidase
Authors: von FIGURA Kurt
Published: March 2005

α‐N‐Acetylglucosaminidase was purified from human urine to a state of apparent homogeneity. α‐N‐Acetylglucosaminidase is a glycoprotein with an extensive charge heterogeneity. The molecular weight determined by gel filtation is 307 000. Polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate indicates molecular weight heterogeneity of isocharged forms of the purified enzyme. The enzyme has a pH optimum of 4.5 ± 0.3 and Km and V values of 0.14–0.74 mM, and 1.04–3.68 μmol mg−1 min−1 for three ary1 2‐acetamido‐2‐deoxy‐α‐D‐glucosides and UDP‐N‐acetylglucosamine. Heparan sulfate, heparin and dermatan sulfate are competitive inhibitors. The enzyme is inhibited by Hg2+ and Cu2+. ‐SH‐protective reagents and thiol reagents have no effect on the enzyme activity. Heating at 65°C and pH values below 5 inactivate the enzyme rapidly.
Thermodynamic Studies of Binary and Ternary Complexes of Pig‐Skeletal‐Muscle Lactate Dehydrogenase
Authors: HINZ Hans‐Jürgen , SCHMIDT Renate , SCHEURMANN Wolfgang , JAENICKE Rainer
Published: March 2005

The thermodynamics of formation of the various intermediary complexes in the reaction catalyzed by pig skeletal muscle lactate dehydrogenase (EC 1.1.1.27) has been investigated in 0.2 M potassium phosphate buffer, pH 7, over the temperature range 10–36°C. As with the analogous experiments on pig heart muscle lactate dehydrogenase [Schmid, F., Hinz, H.‐J. & Jaenicke, R. (1976) Biochemistry, 15, 3052–3059] the catalytic process was approximated by employing the substrate‐analogous inhibitors oxamate and oxalate instead of pyruvate and lactate, respectively, in the two reactions forming the ternary complexes lactate dehydrogenase · NADH · oxamate and lactate dehydrogenase · NAD · oxalate. The standard Gibbs free energy for association of oxamate with the binary lactate dehydrogenase · NADH complex has been derived from fluorescence titrations, while the corresponding value for the binding of oxalate to the lactate dehydrogenase · NAD complex resulted from ultraviolet difference spectroscopy studies. All reaction enthalpies stem from direct calorimetric measurements. The prominent feature of the calorimetric studies is the pronounced variation with temperature of binding enthalpies, showing a negative temperature coefficient of −1901 ± 46 (− 7954 ± 193) cal (J) · (mol of enzyme)−1· K−1 for the association of oxamate and the lactate dehydrogenase · NADH complex and of −1484 ± 80 (6209 ± 335) cal (J) · (mol of enzyme)−1· K−1 for the reaction between lactate dehydrogenase · NAD and oxalate. These negative Δcp values involved in the binding reactions are suggestive of structural alterations of the enzyme induced by ligand binding, which consist of a limited immobilization of the protein with respect to vibrational modes of freedom and/or partial reversal of hydrophobic hydration. These effects are somewhat larger for the pig skeletal muscle enzyme than for the pig heart muscle enzyme.
The Hyphal Wall of Mucor mucedo
Authors: DATEMA Roelf , WESSELS Joseph G. H. , VAN DEN ENDE Herman
Published: March 2005

Nitrous acid, which specifically depolymerises polymers containing hexosamines with a primary amino group, was used to analyse the hexosamine‐containing polymers in the hyphal wall of Mucor mucedo. N‐Acetylglucosamine was found to occur in three polymeric fractions. One fraction which was solubilished by HNO2 treatment contained N‐acetylglucosamine interspersed with glucosamine; no homopolymer of glucosamine (chitosan) was detected. Another fraction became HNO2‐soluble after treatment with pronase or alkali; this points to the occurrence of a heteropolymer containing N‐acetylglucosamine and glucosamine in which some of the glucosamine residues are linked to peptides via their amino groups. The residue remaining after pronase and HNO2 treatment appeared to consist of a homopolymer of N‐acetylglucosamine (chitin).
An Evaluation of Fitting Methods for the Sum of Two Hyperbolas: Application to Uptake Studies
Authors: BURNS Donald J. W. , TUCKER Stephen A.
Published: March 2005

Analysis of data in terms of the sum of two rectangular hyperbolas is frequently required in solute uptake studies. Four methods for such analysis have been compared. Three are based on least‐squares fitting whereas the fourth (partition method I) is an extension of a single hyperbola fitting procedure based on non‐parametric statistics. The four methods were tested using data sets which had been generated with two primary types of random, normal error in the dependent variable: one of constant error variance and the other of constant coefficient of variation. The methods were tested on further data sets which were obtained by incorporating single 10% bias errors at different positions in the original two sets. Partition method I consistently gave good estimates for the four parameters defining the double hyperbola and was highly insensitive to the bias errors. The least‐squares procedures performed well under conditions satisfying the least‐squares assumptions regarding error distribution, but frequently gave poor estimates when these assumptions did not hold. Our conclusion is that in view of the errors inherent in many solute uptake experiments it would usually be preferable to analyse data by a method such as partition method I rather than to rely on a least‐squares procedure.
Physico‐chemical Properties of a DNA Binding Protein: Escherichia coli Factor H1
Authors: SPASSKY Annick , BUC Henri C.
Published: March 2005

H1 factor is a heat‐stable protein found in large amounts in Escherichia coli. In vitro, this protein has been found to stimulate transcription of λ templates by E. coli DNA‐dependent RNA polymerase.

The subunit molecular weight of this factor has been re‐estimated and found to be 15500 ± 1000 in the presence of 2‐mercaptoethanol. Crosslinking experiments performed with dimethylsuberimidate in the presence or absence of the DNA template indicate the presence of multiples of the 15500‐Mr subunit up to the tetramer, the dimeric species being predominant. One cysteinyl residue per 15000‐Mr subunit is labeled by 4‐chloro‐7‐nitrobenzofurazan. This residue is not labeled if the factor is exposed to oxidizing conditions. In this case, three lysyl residues are titrated.

H1 factor behaves as a DNA‐binding protein. We have detected binding to DNA by two independent methods: displacement of a fluorescently labeled factor by the native protein and retention of radioactive DNA on millipore filters in the presence of the factor. Under our experimental conditions (high ionic strength, absence of magnesium ions), the saturation function of λplac DNA as well as of wild‐type λ DNA has been found to be non‐cooperative. Saturation is reached when 300 ± 30 molecules of dimeric factor are bound per λ molecule, the average dissociation constant of the complex being 10 nM.

The dissociation time of the H1· DNA complex is less than 5 s at 37°C. The binding of this factor lowers the affinity of native DNA for ethidium bromide. In the presence of this intercalating dye, the solubility of the complex decreases drastically.
Change of Protein Reactivity in Mammalian Ribosomal Subunits as a Function of Temperature
Authors: REBOUD Anne‐Marie , BUISSON Monique , MARION Marie‐Jeanne , REBOUD Jean‐Paul
Published: March 2005

Reductive methylation of rat liver ribosomal subunits was carried out at different temperatures and the reaction was followed as a function of time. The percentage of protein lysine residues which were methylated reached a definite plateau for each temperature, and was considerably increased by heating (four times at 40°C). This increase was not observed when free ribosomal proteins were heated under the same conditions. Ribosomal subunits kept their biological activity (elongation steps of protein synthesis) even when methylated to a high extent. Half of the subunit activity was still found when 67% of the lysine residues were methylated.

Proteins were divided into different classes on the basis of their alkylation in response to temperature. Those which reacted poorly within subunits even at high temperature should interact directly with rRNA. The results suggest that the protein reactivity modifications induced by heating (up to 37°C), which were often totally or partially reversible, reflect a conformational change of the proteins themselves within the subunits. Besides protein unfolding, higher temperatures produced structural modification and inactivation of the subunits, which were restrained by the presence of aminoacyl‐tRNA.
Phosphorylation from Inorganic Phosphate and ATP Synthesis of Sarcoplasmic Membranes
Authors: BEIL Frank U. , von CHAK Dorothea , HASSELBACH Wilhelm
Published: March 2005

The incorporation of inorganic phosphate in the fragmented sarcoplasmic membranes induced by the removal of calcium ions bound to high affinity binding sites at the cytoplasmic surface of the membranes gives rise to the formation of two species of phosphoenzyme. The properties of the phosphoproteins formed depend on the absence or the presence of a gradient of calcium ions across the membranes. The phosphoenzymes differ by the affinity of the protein for phosphate, the enthalpy of formation, the kinetics of phosphate incorporation, and by the sensitivity to ionophores and ADP. In the absence of a calcium gradient less than 0.5 nmol phosphoenzyme per mg protein are formed in media containing < 5 mM phosphate at pH 7 and 10°C. Under the same conditions ∼ 2 nmol of phosphoenzyme per mg protein are formed with an initial rate of 0.5 nmol mg−1· s−1 when a calcium gradient exists. When the gradient is abolished by the addition of the ionophore X537A, the level of phosphoprotein drops to the same value as observed in the absence of a gradient. On addition of ADP at concentrations increasing from 0.3 to 10 μM continuous ATP formation is activated to its maximum rate, and simultaneously, the level of phosphoprotein declines. These concentrations of ADP scarcely affect phosphoprotein formed in the absence of a gradient, the phosphoryl residue of which is displaced when the concentration of ADP exceeds 10 μM without the formation of an equivalent amount of ATP. Minimum mechanisms for the formation of gradient‐independent and gradient‐dependent phosphoprotein are discussed.
Enzymes Synthesizing and Hydrolyzing Murein in Escherichia coli
Authors: GOODELL E. William , SCHWARZ Uli
Published: March 2005

Envelopes from regions of the cell which in vivo show very little, if any, murein synthesis were isolated using the minicell‐producing strain P678‐54. Envelopes from minicells, representing in fact cell ends, were able to synthesize murein and to carry out transpeptidation in vitro; also all four murein hydrolase activities tested, carboxypeptidase, endopeptidase, amidase and transglycosylase, were found to be present. The specific activities of the murein synthesizing and degrading enzymes in envelopes derived from cell poles and from actively growing cells were similar. The topological distribution of murein‐synthesizing enzymes and of murein hydrolases over the cell envelope is discussed.
The Influence of Localized Chemical Modifications of the Basic Pancreatic Trypsin Inhibitor on Static and Dynamic Aspects of the Molecular Conformation in Solution
Authors: WAGNER Gerhard , TSCHESCHE Harald , WÜTHRICH Kurt
Published: March 2005

High‐resolution 1H nuclear magnetic resonance was used to characterize the molecular conformations in solution of the basic pancreatic trypsin inhibitor and three chemical modifications of this protein. It was found that, except for local variations near the modification site, the average spatial structure was preserved after reduction of the disulfide bond 14–38, after cleavage of the reactive peptide bond Lys‐15–Ala‐16, and after removal of the residues Ala‐16 and Arg‐17 from the reactive‐site‐cleaved inhibitor. In all four species the average solution conformation corresponds closely to the crystal structure of the native inhibitor. The stability of the globular conformation in the modified proteins is, however, markedly reduced, i.e. the denaturation temperature is lowered and the interior amide protons exchange more rapidly with the solvent. In contrast, the internal mobility of the aromatic rings is essentially unaffected by these modifications of the covalent structure. These observations and inspection of the molecular structure of the native inhibitor provide direct evidence for the occurrence of hydrophobic stability domains within these relatively small proteins. Functional roles of hydrophobic clusters in the architecture of globular proteins are discussed.
Similarities between Immunoglobulins and Transplantation Antigens in Amino‐Acid Sequence and Disulfide‐Bond Distribution
Authors: WIMAN Klas , TRÄGÅRDH Lena , RASK Lars , PETERSON Per A.
Published: March 2005

Papain‐solubilized HLA‐A, HLA‐B and HLA‐C antigens (referred to as HLA antigens) were cleaved by limited proteolysis to yield two large fragments. The NH2‐terminal 20000‐Mr fragment contains one immunoglobulin‐like disulfide bridge which encompasses some 55–60 amino acid residues. The other fragment, with a molecular weight of about 12000, is devoid of carbohydrate but contains an immunoglobulin‐like disulfide bond. Fragments of similar sizes were also obtained when the isolated HLA antigen heavy chain was subjected to acid cleavage in 70% formic acid. The combined amino acid content of the isolated 22000‐Mr and 11000‐Mr acid cleavage fragments was very similar, if not identical, to that of the intact 33000‐Mr HLA antigen heavy chain. These data suggested that the acid preferentially cleaved a single aspartyl‐prolyl bond in the heavy chain, as supported by the fact that the 11000‐Mr fragment had proline as its NH2‐terminal residue. Each one of the two acid cleavage fragments contained an immunoglobulin‐like disulfide bridge. While the 41 NH2‐terminal residues of the intact HLA antigen heavy chain did not display any homology with immunoglobulin sequences, the 31 NH2‐terminal residues of the 11000‐Mr acid cleavage fragment displayed as much homology with β2‐microglobulin and the constant immunoglobulin homology regions as they did among themselves. This information strongly suggests that immunoglobulins, β2‐microglobulin and the HLA antigen heavy chains have had a related evolution. Moreover, the HLA antigen heavy chain has most probably arisen by a gene duplication event involving the same ancestral gene which gave rise to the immunoglobulin chains.
Enzymic Synthesis, Characterisation and Nuclear‐Magnetic‐Resonance Spectra of pppA2′p5′A2′p5′A and Related Oligonucleotides: Comparison with Chemically Synthesised Material
Authors: MARTIN Edward M. , BIRDSALL Nigel J. M. , BROWN Ronald E. , KERR Ian M.
Published: March 2005

We have previously shown that a low‐molecular‐weight inhibitor (pppA2′p5′A2′p5′A) of cell‐free protein synthesis, effective at subnanomolar concentrations, is formed on incubation of extracts from interferon‐treated cells or rabbit reticulocytes with double‐stranded RNA and ATP. The inhibitors synthesised with the reticulocyte or interferon‐mediated enzymes, i.e. (2′‐5′)An synthetases, behave identically on thin‐layer chromatography and electrophoresis and have the same biological activity in the inhibition of cell‐free protein synthesis. Here we show that they also behave identically on chromatography on DEAE‐Sephadex in the presence of urea and yield identical products on enzymic and sequential degradation by periodate oxidation and β‐elimination. As previously shown with the interferon inhibitor, preparations of the reticulocyte pppA2′p5′A2′p5′A also include the corresponding biologically inactive dimer, pppA2′p5′A, and biologically active tetramer, ppp(A2′p)35′A, and occasionally higher oligomers in decreasing amounts. Further purification of the pppA2′p5′A2′p5′A yielded small amounts of the corresponding 5′‐diphosphates and 5′‐monophosphates. The 5′‐diphosphate has the same specific biological activity in the inhibition of cell‐free protein synthesis as the triphosphate. The 5′‐monophosphate is essentially inactive.

Milligram quantities of the reticulocyte inhibitor can be conveniently synthesised by incubating the (2′‐5′)An synthetase from these cells bound either to poly(I) · poly(C)‐Sepharose or poly(I) · poly(C)‐paper with ATP. Simple methods for the fractionation of the resultant (2′‐5′)An, into its individual components are described. Proton and phosphorus nuclear magnetic resonance spectra were obtained for the putative pppA2′p5′A2′p5′A and the corresponding 5′‐monophosphate and 5′‐diphosphate from this material and for the A2′p5′A2′p5′A ′core′ derived from these by treatment with bacterial alkaline phosphatase. The spectra were compared with those for chemically synthesised A2′p5′A2′p5′A and commercially available A3′p5′A3′p5′A, AMP, ADP and ATP. The results are in accord with the proposed structure.

Phosphorylation of chemically synthesised A2′p5′A2′p5′A yielded material with chromatographic properties and a specific biological activity in the inhibition of cell‐free protein synthesis apparently identical to those of biologically synthesised pppA2′p5′A2′p5′A.
Formate‐Nitrite Reduction in Escherichia coli K12
Authors: ABOU‐JAOUDÉ Antoine , PASCAL Marie‐Claire , CHIPPAUX Marc
Published: March 2005

Components of the electron transfer pathway from formate to nitrite were identified and compared with those of the formate‐nitrate system. Information was derived from studies on various mutants, different electron donors, and effects of inhibitors. An explanation is proposed for the loss activity in spheroplasts or cell free extract.

Formate and d‐lactate with E′o (pH 7) values of –420 mV and –180 mV respectively reduce both nitrite and nitrate. On the contrary ascorbate/phenazine methosulfate (+ 80 mV) does not reduce nitrite.

Formate dehydrogenase and hemin‐deficient mutants lack both formate nitrite and nitrate reductase activities. Quinone‐lacking mutants lost 40% of the formate‐nitrite reduction and all of the formate‐nitrate reductase activity. Strains affected on nitrate‐reductase cytochrome b or nitrate reductase enzyme have retained a normal capacity to reduce nitrite with formate. Mutant nir197 has simultaneously lost the formate‐nitrite reductase activity and the biosynthesis of the cytochrome c552.

With HpHOQnO one inhibitory site was found. Ultraviolet irradiation revealed the presence of quinones at more than one site. Carbon monoxide inhibited formate‐nitrite reductase but not d‐lactate‐nitrite reductase activity. This gas had no effect on the formate dehydrogenase. Action of inhibitors on the redox steady state showed the presence in the electron pathway of cytochrome c552 as well as a b‐type cytochrome.

The formate‐nitrite electron pathway proposed shares with that of nitrate a membrane‐bound part constituted by formate‐dehydrogenase, cytochrome b, and quinones. It also involves a soluble specific part containing at least the cytochrome c552. It is suggested that the loss of a soluble periplasmic element or its dilution lead to the loss of the activity, as is the case in spheroplasts and osmotic‐shock‐treated cells or in cell‐free extracts and sucrose‐treated cells.
Protein S1 from Escherichia coli Ribosomes: An Improved Isolation Procedure and Shape Determination by Small‐Angle X‐Ray Scattering
Authors: LABISCHINSKI Harald , SUBRAMANIAN Alap‐Raman
Published: March 2005

Ribosomal protein S1 from Escherichia coli was studied in solution by small‐angle X‐ray scattering and the following parameters were obtained. The radius of gyration R= 8.0 ± 0.2 nm; largest diameter D= 28 nm; molecular weight = (8–9) × 104. The data also yielded (with the assumption of a rigid particle with almost constant electron density) two radii of gyration of cross‐section Rq1= 2.5 ± 0.1 nm and Rq2= 1.05 ± 0.05 nm and molecular volume = 140 nm3. The experimental scattering curve of S1 was compared with the theoretical scattering curves for several rigid triaxial homogeneous bodies and the closest fit was given by that of a flat elliptical cylinder with the dimensions of 4.5 nm and 0.88 nm for the two semiaxes and 26.5 nm for height. The results from the present X‐ray scattering studies and those from limited proteolytic digestion of protein S1 [J. Mol. Biol. 127, 41–54, (1979)] support the notion that the structure of protein S1 is organized into two distinct subdomains within its elongated overall shape.

Protein S1 was purified for this study by an efficient procedure which yielded 12 mg S1/g ribosomes. The isolated protein was fully active in functional tests both before and after X‐ray irradiation.
Effects of Cytosol on Transcription in Isolated Simian‐Virus‐40‐Infected Nuclei
Authors: ICEKSON Isaac , YANIV Moshe
Published: March 2005

The transcription in vitro of nuclei isolated from monkey kidney cells infected with simian virus 40 was stimulated by a cytosol fraction from the same uninfected cells. Transcription in nuclei was inhibited 60–80% by 0.1 μg/ml of α‐amanitin, in the presence or in the absence of the cytosol preparation.

Treatment of nuclei after transcription in vitro with 0.02 M EDTA preferentially solubilized viral‐specific RNA partially bound to the viral chromatin.

The increase in incorporation into total RNA seen in the presence of cytosol was not paralleled by any detectable increase in transcription of viral sequences as shown by RNA · DNA hybridizations.
Dissociation and Reassembly of Limulus polyphemus Hemocyanin
Authors: BIJLHOLT Martha M. C. , VAN BRUGGEN Ernst F. J. , BONAVENTURA Joseph
Published: March 2005

Limulus polyphemus hemocyanin is a 3.3 × 106‐Mr protein containing 48 subunits in an assemblage of eight hexamers. The molecule can be dissociated into monomers and dimers at pH 8.9 in the presence of 0.01 M EDTA. These subunits are heterogeneous and can be separated into five zones (I–V) by DEAE‐Sephadex chromatography.

Reassembly experiments were carried out with varied subunit mixtures, based on different combinations of the five chromatographic zones, in order to study the structural role of the diverse subunits in the eight‐hexamer molecule. The reassembly products were analysed by electron microscopy and ultracentrifugation. No structural role for zone I could be found. Zone V and possibly zone II are needed to form structures larger than hexamers. Absence of zone III causes irregular aggregation of hexamers. Zone IV and perhaps zone II are needed to make eight‐hexamer molecules from four‐hexamer molecules. From these results we conclude that there is a high degree of subunit specificity in the inter‐subunit contacts in the native Limulus hemocyanin molecule.
Characterization of Porins from the Outer Membrane of Salmonella typhimurium 1. Chemical Analysis
Authors: TOKUNAGA Hiroko , TOKUNAGA Masao , NAKAE Taiji
Published: March 2005

The three species of channel‐forming outer membrane proteins, porins, have been purified to homogeneity from mutant strains of Salmonella typhimurium which produce single species of porin. Purification was by stepwise solubilization with dodecylsulfate or guanidine thiocyanate, gel filtration, and preparative gel electrophoresis. Amino acid compositions and tryptic peptide maps of the three species of porins showed close resemblance, but at the same time clear differences among them. The number of amino acid residues in the porins purified from the strains SH5551, SH6377 and SH6017 were 361, 354 and 345, and their calculated molecular weights 39800, 39300 and 38000, respectively. Amino‐terminal and carboxyl‐terminal amino acids in all three species of porins appeared to be alanine and phenylalanine, respectively. Neither half‐cystine nor hexosamine was found in these preparation of porins. The isoelectric points of porins from the strains SH5551, SH6377 and SH6017, determined by isoelectric focusing, showed slight differences from each other. These results, and the genetic experiments from another laboratory, suggest that the three species of porins in Salmonella typhimurium are distinct polypeptides, probably coded for by distinct structural genes, which might have been derived from the same ancestral gene.
Characterization of Porins from the Outer Membrane of Salmonella typhimurium 2. Physical Properties of the Functional Oligomeric Aggregates
Authors: TOKUNAGA Masao , TOKUNAGA Hiroko , OKAJIMA Yoshiaki , NAKAE Taiji
Published: March 2005

We have purified to homogeneity, from mutant strains of Salmonella typhimurium, the small oligomers of porin that confer permeability channels to artificial vesicle membranes reconstituted from phospholipids and lipopolysaccharide. The molecular weights of the porin oligomers from the strains SH5551 and SH6017 appeared to be 130000 and 125000, respectively, and those of the monomers were 41000 and 37500, respectively, when determined by sedimentation equilibrium in the presence of dodecylsulfate. It was thus concluded that the functional porin oligomers consisted of three identical subunits. The Stokes' radius of the trimer · dodecylsulfate complex was around 5 nm. The trimer bound less dodecylsulfate than the monomer. The trimer · dodecylsulfate complex retained at room temperature the native conformation of porin, which is rich in β‐structure. When the trimers were dissociated further by various treatments, only the porin monomers were recovered in significant amounts, and the permeability‐conferring activity was lost simultaneously. We propose, therefore, that the trimer is the minimal functional unit of porin that is capable of forming permeability channels in the outer membrane of Salmonella typhimurium.
Fatty‐Acid Synthesis in Lactating‐Goat Mammary Gland 2. Medium‐Chain Fatty Acid Synthesis
Authors: GRUNNET Inger , KNUDSEN Jens
Published: March 2005

Addition of goat, rat, rabbit and cow mammary microsomal fraction to fatty acid synthetase of goat mammary gland induced C10 fatty acid synthesis.

When the microsomal fractions from rat, rabbit and cow mammary gland were incubated with their respective fatty acid synthetases only the cow enzyme synthesized significant amounts of C10 compared to the purified fatty acid synthetases alone.

The goat mammary microsomal fraction was not able to induce C10 synthesis by rat and rabbit mammary‐gland fatty acid synthetase, but only by the goat and cow enzyme.

Both goat and cow mammary‐gland fatty acid synthetase have in contrast to the rabbit and rat enzymes the ability to hydrolyse medium‐chain acyl‐thioesters. We therefore suggest a novel mechanism for medium‐chain fatty acid synthesis in lactating‐goat mammary gland. This mechanism involves an activation of the medium‐chain acyl‐thioester hydrolase within the fatty acid synthetase of goat mammary gland by an unknown microsomal factor.
Actin in Mammalian Lens
Authors: KIBBELAAR Mac A. , SELTEN‐VERSTEEGEN Anne‐Marie E. , DUNIA Irène , BENEDETTI E. Lucio , BLOEMENDAL Hans
Published: March 2005

In this paper evidence is provided that one of the protein components of the water‐soluble fraction of the calf lens binds specifically to deoxyribonuclease I (DNAse I). On the basis of this property, the polypeptide could be purified by applying DNAse I affinity chromatography. Concomitantly a protein of Mr 55000 and a rather large amount of α‐crystallin copurify with this polypeptide, which has a molecular weight of 42000.

Highly purified 42000‐Mr protein was also obtained by extraction of the water‐insoluble fraction of the calf lens with 2‐{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid followed by gel filtration. Amino acid analyses, peptide mapping and electron microscopy show that the protein obtained from both lens fractions is identical to non‐muscle actin. Furthermore the amino acid composition of the 55000‐Mr protein is identical to hog stomach skeletin and very similar to calf brain desmin.
On the Nature of Biochemically Generated Hydroxyl Radicals Studies using the Bleaching of p‐Nitrosodimethylaniline as a Direct Assay Method
Authors: BORS Wolf , MICHEL Christa , SARAN Manfred
Published: March 2005

An efficient scavenger for radiolytically generated hydroxyl (OH) radicals, p‐nitrosodimethyl‐aniline, was used to try to substantiate the presence of this oxygen radical species in several biochemical systems. Most of these systems which were investigated had previously been assumed to generate OH radicals, e.g. the autoxidation of 6‐hydroxydopamine, the hydroxylating system NADH/phenazine methosulfate, and the oxidation of xanthine or acetaldehyde by xanthine oxidase.

We did not observe inhibition of the bleaching of p‐nitrosodimethylaniline in oxygenated solutions by other scavengers of OH radicals nor, in the case of xanthine/xanthine oxidase, by catalase and superoxide dismutase. We therefore conclude that, under biochemical conditions as opposed to radiolysis or photolysis, no freely diffusable OH radicals are formed. Rather, a strongly oxidizing OH‐analogous complex is considered to represent the p‐nitrosodimethylaniline‐detectable species formed under these conditions.
Structure of the Disaccharide Chain of Galactosyl‐N‐acetylgalactosaminyl‐protein Synthesized in vitro
Authors: VAN DEN EIJNDEN Dirk H. , BARNEVELD Rudolf A. , SCHIPHORST Wietske E. C. M.
Published: March 2005

In order to obtain a [14C]galactosyl‐N‐acetylgalactosaminyl‐protein which would be useful as an acceptor in studies on the specificity of glycosyltransferases, a porcine submaxillary gland microsomal galactosyltransferase preparation was used for the galactosylation in vitro of N‐acetylgalactos‐aminyl‐protein (desialylated ovine submaxillary mucin). The newly formed oligosaccharide unit was obtained as a reduced disaccharide after alkaline borohydride treatment of the [14C]galactosyl‐N‐acetylgalactosaminyl‐protein product and as glycopeptides by proteolytic digestion of the glyco‐protein. The reduced disaccharide consisted of equimolar amounts of galactose and N‐acetylgalactosaminitol and was characterized by thin‐layer chromatography, high‐voltage electrophoresis and gas‐liquid chromatography. Periodate oxidation studies on the reduced disaccharide revealed that [14C]galactose was linked to position C‐3 on the N‐acetylgalactosaminyl residue. Digestion of the reduced disaccharide and the glycopeptides with galactosidases gave equivocal results as to the anomeric configuration of the [14C]galactose residue. Nuclear magnetic resonance of the reduced disaccharide, however, definitely indicated that the configuration was β. The specificity of the porcine submaxillary gland galactosyltransferase thus can be defined as a uridine diphosphogalactose: α‐d‐N‐acetylgalactosaminyl‐protein β→ 3 transferase activity.