bluuebunny/crossref_metadata_2025 huggingface dataset:
Fast Kinetic Studies on the Interaction of Cholinergic Agonists with the Membrane‐Bound Acetylcholine Receptor from Torpedo marmorata as Revealed by Quinacrine Fluorescence
Authors: GRÜNHAGEN Hans‐Heinrich , IWATSUBO Motohiro , CHANGEUX Jean‐Pierre
Published: March 2005

Rapid changes of fluorescence intensity take place after fast mixing of quinacrine‐labelled receptorrich membrane fragments from Torpedo marmorata with cholinergic effectors and are recorded in the time range of 1 – 1000 ms, accessible to the stopped‐flow technique. Agonists such as acetylcholine, phenyltrimethylammonium, carbamylcholine, suberyldicholine or choline cause the change, but not antagonists such as d‐tubocurarine, flaxedil or Naja nigricollisα‐toxin. In addition, preincubation of the membrane fragments with N. nigricollisα‐toxin blocks the response to agonists. Ceruleotoxin, a toxin from Bungarus ceruleus venom which blocks the postsynaptic response to acetylcholine at a site distinct from the α‐toxin site, causes a decrease of the apparent amplitude of the fast signal. In the presence of 0.05 mM tetram, an acetylcholinesterase inhibitor, the kinetics of the fast response to acetylcholine do not change; in the second or minute range and in the case of acetylcholine, however, a decrease of fluorescence intensity occurs in the absence of acetylcholinesterase inhibitor showing that the fast fluorescence signal is reversible.

Under the present experimental conditions the observed fast kinetics do not vary with the concentration of receptor sites or quinacrine but do vary with the concentration of agonist. The data can be analyzed in terms of the phenomenological reaction scheme , when k2»k3, if the fluorescence signal monitors the isomerization from A to A*. The following values of the constants are found: for acetylcholine: k4= (1.5 ± 0.5) s−1, k3= (60 ± 40) s−1, k2≥ 5 × 102 s−1 and k1≥ 107l·mol−1 s−1, K1=k2/k1= (7 ± 3) × 10−5 M, K=K1·k4/k3= (2 ± 1) × 10−6M; for carbamylcholine: k4= (0.2 ± 0.1) s−1, k3= (10 ± 5) s−1, k2× 102 s−1 and k1≥ 3 × 105 s−1, K1= (3 ± 1) × 10−4 M and K= (4 ± 3) × 10−6 M. The values of the apparent dissociation constants determined from the variation of the amplitudes of the fast effect for acetylcholine and carbamylcholine are respectively (1.0 ± 0.5) × 10−6 M and (5 ± 2) × 10−6 M. Some variability in the absolute values of the constants is noticed from one preparation to another and, for a given preparation, changes with age. Sets of kinetic constants have also been determined in the case of choline and suberyldicholine. At 20 °C, the Q10 is about 2.5 in the presence of 0.03 mM carbamylcholine and 1.3 in the presence of 0.01 M carbamylcholine; it is 1.3 with 0.05 M acetylcholine.

The data are interpreted in terms of a three‐state model and their physiological significance is discussed.
Adenosine Triphosphatase Activity Associated with Purified Cholinergic Synaptic Vesicles of Torpedo marmorata
Authors: BREER Heinz , MORRIS Stephen J. , WHITTAKER Victor P.
Published: March 2005

A rapid method for purifying Torpedo electric organ vesicles is described, which employs an iso‐osmotic continuous sucrose‐glycine gradient followed by chromatography on CPG‐10‐3000 porous glass beads.

The synaptic vesicles have a buoyant density of 1.057 g/ml. The purified vesicles are free of cholinesterase, lactate dehydrogenase and Na+,K+‐stimulated ATPase activity. They contain a ouabaininsensitive, Na+,K+‐inhibited, Mg2+,Ca2+‐stimulated ATPase activity. This is further stimulated by acetylcholine but not by choline.
Mechanism of the Prolyl Hydroxylase Reaction
Authors: MYLLYLÄ Raili , TUDERMAN Leena , KIVIRIKKO Kari I.
Published: March 2005

The kinetics of the prolyl hydroxylase reaction were studied with pure enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double‐reciprocal plots for all possible pairs of Fe2+, 2‐oxoglutarate, O2 and the polypeptide substrate, whereas parallel lines were obtained for pairs involving ascorbate with each substrate. In addition, parallel lines were obtained when the polypeptide substrate concentration was varied at different fixed 2‐oxoglutarate concentrations in the presence of saturating O2 concentration. Poly(L‐proline) was a competitive inhibitor with respect to the polypeptide substrate, but uncompetitive with respect to Fe2+ and 2‐oxoglutarate. High concentrations of the polypeptide substrate inhibited the reaction, this substrate inhibition being competitive with respect to Fe2‐ and 2‐oxoglutarate. Succinate, CO2 and collagen were product inhibitors, succinate inhibiting the reaction competitively with respect to 2‐oxoglutarate, but noncompetitively with respect to the other substrates, and collagen noncompetitively with respect to all substrates. The apparent Km and Ks values for the substrates and Ki values for the inhibitors are given.

These and additional data would be consistent with a tentative reaction scheme involving an ordered binding of Fe2+, 2‐oxoglutarate, O2 and the polypeptide substrate to the enzyme in this order, the binding of Fe2+ being at thermodynamic equilibrium. The enzyme can also react directly with the polypeptide substrate or its analogue poly(L‐proline) under certain conditions, forming dead‐end complexes. The products are released only after the hydroxylation, possibly in the order: the hydroxylated polypeptide, CO2 and succinate. Ascorbate may react either with enzyme·Fe before the release of Fe2+ or with free enzyme before the binding of Fe2+, but a reaction with ascorbate at any stage after the release of the first product is not excluded. The mechanism proposed is not entirely identical with either of the main two previous suggestions for the mechanism of 2‐oxoglutarate dioxygenases.
Structure and Function of Ornithine Carbamoyltransferases
Authors: LEGRAIN Christiane , STALON Victor , NOULLEZ Jean‐Pierre , MERCENIER Annick , SIMON Jean‐Paul , BROMAN Kathleen , WIAME Jean‐Marie
Published: March 2005

The reaction catalyzed by ornithine carbamoyltransferase can participate in either the anabolism or the catabolism of arginine. The carbamoylation of ornithine, yielding citrulline, is involved in the biosynthetic sequences; the reverse reaction, the phosphorolysis of citrulline, is the second step of the arginine deiminase pathway.

The ornithine carbamoyltransferases of a number of microorganisms which can fulfil both of these functions have been studied in this work. This group of organisms was found to possess two distinct ornithine carbamoyltransferases. The functions of these enzymes were surmised by determining the type of genetic regulation to which they were subjected.

The kinetic properties of these various enzymes have been determined. All of them, regardless of the role they play in the cell, catalyze both the synthesis and arsenolysis of citrulline. The anabolic transferase of Pseudomonas is the only enzyme which displays functional irreversibility.

A comparison of the quaternary structure of these transferases was performed and reveals interesting features in relation to the metabolic function of these enzymes. All well‐characterized anabolic enzymes have low molecular weights (from 150000–105000) and are likely to be trimers. Catabolic enzymes, with the exception of those of Bacillus licheniformis and halobacterium salinarium, display much higher molecular weights and more elaborate quaternary structure.

The properties of these two groups of transferases are discussed in relation to their metabolic role in the cells.
A Cytoplasmic High Affinity Estrogen‐Binding Protein in the Embryonic Chicken Liver
Authors: GSCHWENDT Michael , KITTSTEIN Walter
Published: March 2005

1. Embryonic chicken liver contains cytoplasmic estrogen‐binding sites with high affinity for estradiol (Kd= 1 × 10−9 M) and with low capacity (200–1100 binding sites/cell, depending on the age of the embryo).

2. Only estrogenic hormones are bound with high affinity.

3. At least the estrogen‐binding moiety of these binding sites is a protein.

4. By agarose gel chromatography an apparent molecular weight of 50 000–60 000 daltons was determined, very similar to that of the nuclear estrogen‐binding protein.

5. In low salt the cytoplasmic binding sites exhibit a stong tendency to aggregate, which is abolished by mild trypsination.

6. A single injection of estradiol into the egg seems to cause a translocation of the cytoplasmic binding protein to the nucleus.

7. During the embryonic development the number of binding sites increases up to the age of 19 days. Thereafter it decreases again reaching a constant low level soon after hatching.

8. Data are presented to show that this low level of binding sites can not be due solely to a degradation or inhibition in vitro by a component eventually present in the liver homogenate of hatched animals.
The Isolation and Characterization of Differentially Phosphorylated Fractions of Phosphofructokinase from Rabbit Skeletal Muscle
Authors: HUSSEY Christopher R. , LIDDLE Peter F. , ARDRON David , KELLETT George L.
Published: March 2005

A preparation of phosphofructokinase from rabbit skeletal muscle is described which exploits the association‐dissociation properties of the enzyme. Phosphofructokinase so prepared is partially phosphorylated and may be fractionated into three distinct species with sedimentation coefficients of 30 S, 18 S and 13 S by chromatography on agarose gels, hydroxyaptite or DEAE‐cellulose. Measurements of alkali‐labile phosphate content (phosphoserine and/or phosphothreonine) show that fractions consisting almost exclusively of 30‐S species and fractions consisting predominantly of 18‐S and 13‐S species contain approximately 0.15 and 0.29 mol of phosphate per phosphofructokinase monomer (Mr= 80 000) respectively. The results are interpreted in terms of at least two 13‐S components which differ in their phosphate contents and also in their self‐association properties. The possible significance of phosphorylation is discussed.
Adenosylhomocysteinase from Yellow Lupin Seeds
Authors: GURANOWSKI Andrzej , PAWELKIEWICZ Jerzy
Published: March 2005

Adenosylhomocysteinase from yellow lupin seeds (Lupinus luteus) has been purified to homogeneity.

Active enzyme, Mr= 110 000, consists of two probably identical subunits with Mr= 55 000 as judged by gel filtration and dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of 2‐mercaptoethanol. The isoelectric point of the enzyme was shown to be 4.9 ± 0.1. It was demonstrated by disc and pore gradient electrophoresis that the most purified fraction formed multimers. The enzyme shows optimum activity at pH 8.5–9.0. Km values are 2.3 μM, 4.6 mM and 12 μM for adenosine, DL‐homocysteine and S‐adenosyl‐L‐homocysteine, respectively. The energy of activation for S‐adenosylhomocysteine synthesis was estimated as 14.4 kcal/mol (60.2 kJ/mol) and temperature coefficient as 2.4. The equilibrium constant for the hydrolysis of S‐adenosylhomocysteine amounts to 5 × 10−7 M. Anti‐sulfhydryl reagents such as p‐hydroxymercuribenzoate and N‐ethylmaleimide acted as irreversible inhibitors. The enzyme exhibits high specificity for homocysteine whereas some of the rare nucleosides tested could substitute for adenosine.
The Preparation of Ligandin with Glutathione‐S‐Transferase Activity from Porcine Liver Cytosol by Affinity Chromatography on Bromosulphophthalein‐Sepharose
Authors: GRAHNÉN Anders , SJÖHOLM Ingvar
Published: March 2005

A simple and rapid method for the purification of porcine ligandin with glutathione‐S‐transferase activity is presented. After ion‐exchange chromatography on DEAE‐Sephadex, ligandin is isolated from porcine liver cytosol by affinity chromatography on bromosulphophthalein‐Sepharose and gel filtration on Sephadex G‐100. Evidence is presented that the purified ligandin is homogeneous with respect to polyacrylamide‐gel electrophoresis (7.5%) and sodium dodecylsulphate‐gel electrophoresis.

Physico‐chemical investigations show that the purified ligandin has properties similar to those of ligandin isolated from other species with respect to molecular weight, amino‐acid composition, secondary structure and catalytic activity. As is the case for human and rat ligandin, porcine ligandin binds bilirubin. Evidence is also presented that porcine liver cytosol contains several bromosulphophthalein‐binding proteins with basic isoelectric points lacking catalytic activity.
Properties of the Complex between Histone H1 and Poly(ADP‐ribose) Synthesised in HeLa Cell Nuclei
Authors: STONE Peter R. , LORIMER Wishard S. , KIDWELL William R.
Published: March 2005

Preparations of H1 histone from HeLa cell nuclei incubated with [3H]NAD to permit poly(ADP‐ribose) synthesis were electrophoresed on polyacrylamide gels. The incorporated radioactivity migrated as a sharply defined peak in association with a protein band which moved more slowly than H1, the major protein component. The following observations indicate that this complex is composed of two molecules of H1 and a single chain of poly(ADP‐ribose) with one detectable covalent linkage of polymer to protein.

The [14C]arginine/[3H]lysine ratio is identical in H1 histone and in the protein moiety of the complex.

Protein is displaced from H1 histone to the complex during poly(ADP‐ribose) synthesis. At least 90% of the protein in the complex (stainable protein and labelled protein) is derived from H1.

Sedimentation rate studies indicate a molecular weight of the complex about twice that of H1 histone.

The average chain length of the polymer is 15 ADP‐ribose units and there are 7–8 ADP‐ribose units for each molecule of H1 histone in the ‘complex’.

Poly(ADP‐ribose) glycohydrolase, which hydrolyses the polymer exoglycosidically from the AMP terminus, degrades the complex producing ADP‐ribose and mono‐ADP‐ribosylated H1 histone which co‐electrophoreses with unmodified H1.

Although only one covalent linkage between protein and polymer has been detected, the ‘complex’ does not dissociate when electrophoresed on dodecylsulfate gels. Nor can the noncovalently linked H1 histone of the complex readily exchange with free H1. Complex formation does not occur when purified poly(ADP‐ribose) and H1 are mixed.
The Peptidoglycan Crosslinking Enzyme System in Streptomyces Strains R61, K15 and rimosus. Kinetic Coefficients Involved in the Interactions of the Membrane‐Bound Transpeptidase with Peptide Substrates and β‐Lactam Antibiotics
Authors: DUSART Jean , LEYH‐BOUILLE Mélina , GHUYSEN Jean‐Marie
Published: March 2005

The transpeptidation reaction performed by the membranes of Streptomyces strain R61 fits the general rate equation for an enzyme‐catalysed bimolecular reaction. The same membranes (E) interact with β‐lactams (I) to form inactive penicillin · enzyme · membrane complexes (EI*) of rather high stability, which subsequently break down (E + I → EI*→ E + degradation products). The enzyme is regenerated and the antibiotic is released in the form of an inactive metabolite. With benzylpenicillin, the degradation product is benzylpenicilloic acid. The reaction is heat‐labile. The first step of the reaction (E + I → EI*) is characterized by a second‐order rate constant (kformation in M−1 s−1) and the second step (EI*→ E + degradation products) by a first‐order rate constant (kbreakdown in s−1). The effects in vitro of various β‐lactams on the membrane‐bound transpeptidase, as expressed by the relevant kformation and kbreakdown values, parallel the effects in vivo of the same antibiotics as expressed by their ability to prevent the germination and growth of conidiospores. The kinetic parameters of the transpeptidase that was solubilized with N‐cetyl‐N,N,N‐trimethylammonium bromide with respect to its interaction with both peptide substrates and β‐lactam antibiotics are quantitatively different from those of the membrane‐bound enzyme. Moreover, the solubilized enzyme fragments benzylpenicillin with formation of phenylacetylglycine, a reaction which is similar to that catalysed by the exocellular R61 enzyme. The membranes of Streptomyces strains rimosus and K15 possess an active ‘classic’ penicillinase. They were not studied but the kinetic coefficients of the corresponding solubilized transpeptidases were determined and compared with those of the solubilized enzyme from strain R61.
Initial Stages of the Thermal Unfolding of Yeast Phenylalanine Transfer RNA as Studied by Chemical Modification: The Effect of Magnesium
Authors: RHODES Daniela
Published: March 2005

The early stages of thermal unfolding of the tertiary structure of yeast tRNAphe have been followed, in the presence and absence of Mg2+, by measuring changes in the chemical accessibility of the bases uracil and guanine. The reagent used in these studies is 1‐cyclohexyl 3‐[2‐morpholino(4)‐ethyl]carbodiimide methotosylate. 32P‐labelled tRNA was used so that the points of modification could be examined with ribonuclease digestion and established fingerprinting techniques.

Two regions of protection by Mg2+ have been found. One is within the oligonucleotide U8‐A‐m2G10 and the other is in the vicinity of residue U‐59.

The tertiary interactions and the D stem are the most readily melted parts of the tertiary structure. In the absence of Mg2+ the region of U‐59 is the first part of the tertiary structure to become accessible to the reagent. This is closely followed by the opening up of the ‘wobble’ G · U base pair in the aminoacyl stem. Most of the triple interactions in the augmented D helix are also disrupted early in the melting. The region of intricate interactions between the invariant G‐G part of the D loop and the T‐Ψ‐C‐G loop contains the most stable set of tertiary structure interactions.
F1‐ATPase from Micrococcus sp. ATCC 398
Authors: RISI Sergio , HÖCKEL Michael , HULLA Franz W. , DOSE Klaus
Published: March 2005

The preparation of highly purified F1‐ATPase from Micrococcus sp. ATCC 398 by application of DEAE‐Sepharose CL‐6B chromatography as final step is described. This enzyme consists of five subunits of different molecular weight: α (65000), β (55000), γ (35000), δ (20000), and ∈ (17000). Disc electrophoresis on 5% polyacrylamide gels removes the ∈‐polypeptide yielding an active ATPase complex with four different subunits: α, β, γ, δ. Additionally, by variation of the ionic strength δ can be (partly) removed allowing the isolation by disc electrophoresis of an active ATPase complex which consists only of three different subunits α, β, and γ. If the DEAE‐Sepharose chromatography is carried out in the absence of diisopropyl phosphofluoridate (auto)proteolysis yields both an active ATPase with the subunits α+ (mol. wt 61000), β, γ, and δ and an inactive protein complex with the subunits α+, β, γ, δ, and two additional polypeptides a (mol. wt 38000) and b (mol. wt 23000). The latter two polypeptides are supposedly fragments of α+‐chains which have become partially cleaved by (auto)proteolysis.
Studies on the Mechanism of Action of Histone Kinase Dependent on Adenosine 3′:5′‐Monophosphate. Evidence for Involvement of Histidine and Lysine Residues in the Phosphotransferase Reaction
Authors: KOCHETKOV Sergei N. , BULARGCINA Tamara V. , SASHCHENKO Lidia P. , SEVERIN Evgenii S.
Published: March 2005

The reaction of the phosphate residue transfer catalysed by histone kinase dependent on adenosine 3′: 5′‐monophosphate (cyclic AMP) was studied. The phosphotransferase reaction was shown to obey the mechanism of ping‐pong bi‐bi type.

After incubation of the catalytic subunit of histone kinase with [γ‐32P]ATP the incorporation of one mole of [32P]phosphate per mole of protein was observed. The tryptic [32P]phosphohistidine‐containing peptide was isolated and its N‐terminus and amino acid composition were determined.

The 2′,3′‐dialdehyde derivative of ATP (oATP) was used as the affinity label for the catalytic subunit of cyclic‐AMP‐dependent histone kinase. The inhibitor formed an aldimine bond with ∈‐amino group of the lysine residue of the active site and was irreversibly bound to the enzyme after reduction by sodium borohydride with concurrent irreversible inactivation of the enzyme. After inactivation, about one mole of 14C‐labelled inhibitor was incorporated per mole of the enzyme. ATP effectively protected the catalytic subunit of histone kinase against inactivation by oATP.

Tryptic digestion of the enzyme‐inhibitor complex led to the isolation of the 14C‐labelled peptide of the active site of histone kinase.

Basing on these results, the role of histidine and lysine residues in the active site of the catalytic subunit of histone kinase was suggested.
Röntgenkleinwinkel‐Untersuchungen und Strukturmodell‐Betrachtungen an der Fettsäuresynthetase aus Schweineleber
Authors: FOLKHARD Waltraud , FELSER Birgit , PILZ Ingrid , KRATKY Otto , DUTLER Hans , VOGEL Hugo
Published: March 2005

Small‐Angle X‐Ray‐Scattering Investigation and Structural‐Model Study of the Fatty‐Acid Synthetase from Pig Liver

The structure of the fatty acid synthetase from pig liver was studied on models based upon structural and functional properties selected from pertinent results available from numerous investigations carried out with fatty acid synthetases from this and other sources. When comparing small‐angle X‐ray‐scattering curves calculated with these models and curves obtained from small‐angle X‐ray‐scattering experiments carried out with the pig‐liver enzyme, we tried to select a model which would lead to an acceptable correlation between the calculated and the experimental curves and at the same time fulfil the known structural and functional requirements. The comparison of the curves was started with a model of low complexity. The observed discrepancy, together with arguments from the structural and the functional properties, helped decide which is the next most reasonable model to be considered. This procedure was repeated for five models of increasing complexity. In the model which led to the best fit the multienzyme complex is composed of two halves in an assymetric conformation including hollow spaces. This highly anisotropic model would imply that the two halves change their conformation each time a synthetic cycle is completed and that the growing fatty acid is handed over from one half to the other.
Lipopolysaccharides de mutants thermosensibles de Escherichia coli K12
Authors: BRUNETEAU Maud , MICHEL Georges
Published: March 2005

Lipopolysaccharides from Thermosensitive Mutants of Escherichia coli K12

Thermosensitive mutants of Escherichia coli K12 were grown at 30°C and 40°C. The serologic properties and the composition of their lipopolysaccharides were investigated. The inhibition of hemagglutination by the lipopolysaccharides of various mutant strains was tested against the anti‐E. coli K12 CR34 system. An inhibition was observed with all the mutants but one, CR 34 T83 which had no inhibitory effect.

Chemical analysis of lipopolysaccharides and mass spectrometric analysis of their methylated derivatives indicated the presence of the same components in the various lipopolysaccharides: glucose, galactopyranose, galactofuranose, heptopyranose and heptofuranose. However the 2,3,4 tri‐O‐methyl glucose is missing in the lipopolysaccharide of the mutant T83. This result agrees with the absence of a substituent on the 6‐position of the non‐reducing core‐terminal glucose. The lipopolysaccharide of the T83 mutant has the complete core‐type of E. coli K12. The relations of mutations with modifications of the composition of inner and outer envelopes in various mutants are discussed.
Colivirus‐T3‐Coded S‐Adenosylmethionine Hydrolase
Authors: SPOEREL Nikolaus , HERRLICH Peter
Published: March 2005

Bacteriophage T3 induces an enzyme activity which hydrolyzes S‐adenosylmethionine. This S‐adenosylmethionine hydrolase is interesting, not only because of its unique activity, but also because the protein has to overcome host restriction [F. W. Studier and N. R. Movva (1976) J. Virol. 19, 136–145].

S‐Adenosylmethionine hydrolase was purified to homogeneity using affinity chromatography on S‐adenosylhomocysteine‐Sepharose. The enzyme occurs in two forms, A and B. Form A consists of the viral peptide chain only; its native and subunit molecular weight is 17000. Form B contains, in addition, a host subunit with a molecular weight of 49000. The host subunit does not modify S‐adenosylmethionine cleavage in vitro and no apparent relationship to the host‐restriction system could be detected.
Characterization of Pepsin Fragments of Basement Membrane Collagen Obtained from a Mouse Tumor
Authors: TIMPL Rupert , BRUCKNER Peter , FIETZEK Peter
Published: March 2005

Basement membrane collagen was purified from an acetic acid extract of a mouse tumor and subjected to pepsin treatment. At initial stages disulfide‐linked molecules, consisting of peptide chains with an apparent molecular weight of 140000, were formed which were some 15–20% smaller than the chains in the original protein. In a second stage further fragmentation occurred producing at least five peptides ranging in molecular weight from 27000 to 72000. Each of these peptides was isolated and characterised by amino acid composition, cyanogen bromide cleavage, circular dichroism and antigenic activity.

The data indicate that the major portion of the initial pepsin‐resistant fragment is degraded forming two triple‐helical segments, the disulfide‐linked fragment P3 and the cysteine‐free fragment P1 containing chains with molecular weights of 72000 and 55000 respectively. The other fragments (P2, P3B and P3C) were recovered at lower yields and may originate from a second, genetically distinct type of basement membrane collagen. This interpretation is supported by partial amino acid sequence analysis of the two non‐disulfide‐linked fragments (P1, P2). These sequences show that P1 and P2 are different and start with a non‐helical sequence followed by the helical triplet structure Gly‐X‐Y.

Studies on pepsin fragments of the insoluble tumor collagen demonstrated a pattern similar to that obtained with the soluble form except for the occurrence of disulfide‐linked α‐chain‐like material. These chains also have an amino acid composition characteristic for basement membrane collagen. Taken together, the findings suggest that the tumor produces several related collagens.
Suppression of Thermotropic Lipid Clustering in Tetrahymena Nuclear Membranes upon Ca2+/Mg2+ Induced Membrane Contraction
Authors: GIESE Günter , FROMME Inge , WUNDERLICH Frank
Published: March 2005

Nuclear membranes surrounding the macronuclei isolated from Tetrahymena cells grown at either 28 C or 18 C are induced to expand or contract laterally by Ca2+/Mg2+ (3/2) in final concentrations of 1 mM or 5 mM, respectively. Only expanded nuclear membranes are temperature‐responsive as revealed by freeze‐etch electron microscopy and electron spin resonance using 5‐doxylstearic acid as a spin label.

In expanded nuclear membranes from growth at 28°C, electron spin resonance detects, upon lowering the temperature, a biphasic decrease of the lipid fluidity in terms of 2TII with a discontinuity at ∼ 17°C. In this temperature range, freeze‐etch electron microscopy reveals an increase in the number of nuclear membrane fracture faces with smooth areas. We ascribe this response to a clustering of ordered lipid domains within a broad thermotropic fluid→ordered lipid phase separation [Wunderlich et al. (1978) Biochemistry, 17, 2005–2010].

Expanded nuclear membranes from growth at 18°C shift their lipid clustering temperature down to ∼ 12°C. Their lipid fluidity (2TII) is increased in comparison with the nuclear membranes from growth at 28°C. This is probably due to an increased fatty acid unsaturation of the polar lipids. Thin‐layer‐chromatography reveals the following major polar lipids in the nuclei from growth at 18°C (28°C): 33.9%(31.4%) phosphatidylethanolamine, 14.2%(13.4%) phosphatidylethylamine, 22.5%(18.0%) unidentified phospholipid, 21.4%(19.1%) phosphatidylcholine, and 6.1%(10.4%) ceramide‐phosphonoethylamine. The major fatty acids are mainly even‐numbered ranging between C12 and C18 as analyzed by gas chromatography. The 18°‐nuclei (28°‐nuclei) contain 67.1% (60.4%), 74.5%(68.4%), and 32.3%(43.5%) unsaturated fatty acids in their total lipids, polar lipids, and neutral lipids, respectively. The individual phospholipids, except phosphatidylcholine and ceramide‐phosphonoethylamine, are more unsaturated in the nuclei from growth at 18°C.

Contracted nuclear membranes from growth at 28°C and 18°C suppress thermotropic lipid clustering. They reveal an apparent higher lipid fluidity and percentage of fracture faces with smooth areas than the corresponding expanded nuclear membranes at 28°C and 18°C, respectively. The lipid clustering suppression is interpreted as being due to a change in lipid packing which in turn is induced by a Ca2+/Mg2+‐induced contraction of the Ca2+/Mg2+‐sensitive nucleoskeleton proteins associated with nuclear membranes.
The Reaction of Nitrogen Monoxide and of Nitrite with Deoxyhaemocyanin and Methaemocyanin of Helix pomatia
Authors: VERPLAETSE Jozef , VAN TORNOUT Philippe , DEFREYN Ghislain , WITTERS Raphaël , LONTIE René
Published: March 2005

Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and N2O in a fast reaction.

In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g= 4 and yielded a nitrosyl derivative with a characteristic g= 2 Cu(II) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra.

Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g= 4 with 7 hyperfine lines.
Reversible Inactivation of tRNA Nucleotidyltransferase from Baker's Yeast by tRNAPhe Containing Iodoacetamide‐Alkylated 2‐Thiocytidine in Normal and Additional Positions
Authors: KRÖGER Manfred , STERNBACH Hans , CRAMER Friedrich
Published: March 2005

2‐Thiocytidine 5′‐triphosphate, s2CTP, is able to replace CTP as a substrate for tRNA nucleotidyltransferase. s2CMP can be incorporated into both cytidine sites of the C‐C‐A terminus common to all tRNAs, and in the absence of ATP into at least two additional positions. This was shown by alkylation of the 2‐thiocytidine residues with iodo[14C]acetamide, total nucleoside analysis, microgel electrophoresis and analysis of RNase T1 fragments of these tRNAs. The incorporation of the 3′‐terminal AMP is not influenced by the additional s2CMP residues at pH 9.0. However, at pH 7.6 the additional s2CMP residues are hydrolysed and AMP can be incorporated into the normal position. Two different tRNAs with terminal 2‐thiocytidine alkylated by iodoacetamide inhibit tRNA nucleotidyltransferase. This inhibition is significantly slower if an elongated species is used compared to a tRNA with alkylated 2‐thiocytidine in the normal position 75. The addition of 2‐mercaptoethanol reactivates the enzyme and leads to a cytidine containing tRNA. This reaction identifies the attacking nucleophile of the enzyme as a cysteine residue, which is probably identical to a cysteine residue found in a similar experiment reported previously. The mechanism of the enzymatic and chemical reactions is discussed.
Ribosomal Phosphoproteins of Mouse Myeloma Cells
Authors: MARTINI Oskar H. W. , KRUPPA Joachim
Published: March 2005

Mouse myeloma cells (MPC 11) in culture, labelled with [32P]i for several hours, were subjected to hypertonic initiation block by raising the salt concentration of the culture medium from 110 to 210 mM NaCl.

The relative amounts of messenger‐free and bound ribosomes of the cells were estimated from ribosome profiles following sucrose density centrifugation. The initiation block caused the proportion of messenger‐bound ribosomes to decrease substantially. The extent of the decrease was larger under suboptimal (50%) than under satisfactory (25%) nutritional conditions.

Analysis of the ribosomal proteins by two‐dimensional gel electrophoresis, autoradiography and radiospectrometry showed, that in the small subunit only protein S3 had incorporated 32P and that S3 was the most strongly phosphorylated ribosomal protein in control cells. Upon salt treatment of the cells the radioactivity of S3 from both messenger‐free and bound ribosomes decreased to low levels. The lowest levels (15% of the controls) were reached when the nutritional conditions were suboptimal. The results show that there is a positive correlation between the number of ribosomes engaged in protein synthesis and the degree of phosphorylation of S3. However, phosphorylation of S3 does not seem to be a prerequisite for ribosomes to be attached to mRNA. Under all experimental conditions S3 from messenger‐bound ribosomes was about twice as strongly labelled as S3 from messenger‐free ribosomes.

The large‐subunit phosphoproteins were identified as L28, the protein thought to be homologous to the Escherichia coli proteins L7/L12, and as L5, which was only weakly labelled. In contrast to S3 the radioactivity of L28 increased slightly upon the tonicity shift.
270‐MHz 1H Nuclear‐Magnetic‐Resonance Study of Met‐enkephalin in Water
Authors: ZETTA Lucia , CABASSI Franco , TOMATIS Roberto , GUARNERI Mario
Published: March 2005

1H spectra at 270 MHz of the Met‐enkephalin pentapeptide (Tyr‐Gly‐Gly‐Phe‐Met) in aqueous solution, as a function of pH and of temperature, are reported.

The analysis of the chemical shifts, line widths, coupling constants and rotamer populations around χ1 and χ2, suggests the existence of three conformational regions in the pH range 1–10, due to some rearrangements of the first two amino acids, Tyr‐1 and Gly‐2. A first conformational transition occurs corresponding to the N‐terminus titration, with an exchange rate between conformational states which is not too fast. The second transition occurs concomitantly with the phenolic group titration.

The conformational states of the cationic and zwitterionic forms are probably very similar and are characterized by the absence of intramolecular hydrogen bonds and of head‐to‐tail interactions. A bent structure, with the Tyr‐1 ring Facing the Gly‐2 could contribute to the equilibrium of the conformational states of Met‐enkephalin in the pH range 8–10. This structure could be destabilized as a consequence of the phenolic group deprotonation.

The Met‐5 and Phe‐4 residues appear to have little sensitivity to the conformational transitions of the backbone and both show a settling of the side chains corresponding to the carboxyl group titration, thus showing a reciprocal steric hindrance.

The temperature coefficients of the chemical shifts of amide protons at acidic pH are in the range of solvated peptides. In the temperature range 0–75°C, the 3JαNH coupling constants were independent of temperature.
Isolation and Characterization of a Native Placental Basement‐Membrane Collagen and Its Component α Chains
Authors: GLANVILLE Robert W. , RAUTER Anton , FIETZEK Peter P.
Published: March 2005

Native type IV collagen was isolated from human placenta using pepsin solubilisation followed by fractional salt precipitation and chromatographic purification. The native preparation was characterised using amino acid analyses, disc gel electrophoresis, segment‐long‐spacing crystallites and immunological methods. Two component α chains were isolated with molecular weights of approximately 95000 and 70000. Cyanogen bromide digests of these chains indicated that they are not related to any of the known α chains of interstitial collagens or to the recently described collagen containing αA and αB chains. They are also not related to one another and are therefore probably fragments of two genetically distinct type IV collagen α chains.
Characterization of the Isoenzymes of Pig‐Liver Esterase 1. Chemical Studies
Authors: HEYMANN Eberhard , JUNGE Wolfgang
Published: March 2005

Three different subunits of highly purified pig liver esterase (EC 3.1.1.1) can be separated by analytical dodecyl sulfate electrophoresis, though their relative mobilities are very similar. The same subunit bands are obtained with microsomes, in which the esterases have been labeled with the specific active‐site‐directed inhibitor bis(4‐nitro‐[14C]phenyl)phosphate. The heterogeneity of the native trimeric enzyme is much more complex, as is demonstrated by isoelectric focussing and polyacrylamide gel electrophoresis. Fractions of esterase which were partially separated by preparative isoelectric focussing show differences in their subunit composition, their amino acid analyses, their tryptic peptide maps, and their C‐terminal amino acids. From these experiments various features of the differing esterase subunits can be deduced. Based on the chemical results and on various experiments which did not indicate any secondary modification of the protein side‐chains, the molecular basis of the esterase heterogeneity is discussed. We conclude that the native trimeric esterase is a mixture of numerous hybrids of at least three protein subunits with differing but closely related primary sequences. A comparison of the relative specificity of various preparations of pig liver microsomes indicates that genetic differences concerning the composition of liver esterase exist between individuals.
Characterization of the Isoenzymes of Pig‐Liver Esterase 2. Kinetic Studies
Authors: JUNGE Wolfgang , HEYMANN Eberhard
Published: March 2005

The kinetic properties of two of the partially separated isoenzymes I and V of pig liver esterase were studied. The cholinesterase‐like isoenzyme I hydrolyses butyrylcholine as well as various other esters and aromatic amides. This isoenzyme is sensitive to 0.01 mM physostigmine and to fluoride. The second type (isoenzyme V) has the features of the so‐called aliesterase: it acts preferentially on short‐chain aliphatic esters, does not hydrolyse butyrylcholine and has only low activity towards amides. Further differences exist with regard to sensitivity towards organophosphorous inhibitors, to the influence of organic solvents and to the pH optimum. Transacylation reactions with methanol as an acceptor are mainly catalyzed by the isoenzyme of the aliesterase type.

The esterase forms III and IV, which are located between isoenzymes I and V on the isoelectric focussing column, show kinetic features similar to those of a mixture of I and V. A stepwise increase or decrease, respectively, of certain kinetic properties, e.g. specific activities, is observed for the sequence I, III, IV, V. A quantitative comparison of the kinetic properties supports our proposed subunit model [1], according to which the main components of these four esterase fractions are the trimers γγγ, αγγ, ααγ and ααα.

These results offer explanations for many of the very complex and often controversial results formerly obtained with the heterogeneous pig liver esterase.
The Molecular Size and Shape of the Folch‐Pi Apoprotein in Aqueous and Organic Solvents
Authors: LAVIALLE Françoise , de FORESTA Béatrice , VACHER Monique , NICOT Claude , ALFSEN Annette
Published: March 2005

In 1% acetic acid, sedimentation velocity measurements and equilibrium ultracentrifuge experiments demonstrate that the Folch‐Pi apoprotein is not monodisperse. The weight‐average molecular weight calculated from ultracentrifuge experiments and combining sedimentation coefficient and viscosity measurements, ranged from 64000 to 80000. The intrinsic viscosity value suggests an asymetric shape for the apoprotein if a low value of hydration is considered.

In dioxan/1% acetic acid (2:3, v/v) a smaller sedimentation coefficient was found, the intrinsic viscosity value remaining identical to that in 1% acetic acid.

In pure 2‐chloroethanol, light‐scattering experiments led to a molecular weight of 165000 indicating that even in this solvent the protein is not monomeric. Intrinsic viscosity and light scattering measurements on the one hand, primary sequence on the other hand (six proline residues per monomer of Mr 23500) suggest that the molecule in 2‐chloroethanol may consist of rod‐like segments with flexible junctions.
Inactivation and Disassembly of the Pyruvate Dehydrogenase Multienzyme Complex from Bovine Kidney by Limited Proteolysis with an Enzyme from Rat Liver
Authors: KRESZE Georg‐B. , STEBER Liesel
Published: March 2005

Mammalian pyruvate dehydrogenase multienzyme complex is inactivated when treated with a leupeptin‐sensitive enzyme (termed ‘inactivase’) obtained from rat liver lysosomes. However, the inactivation of the overall reaction does not affect any of the component activities of the enzyme complex.

By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation. The dissociation occurs as a consequence of limited proteolysis of the lipoate acetyltransferase core of the multienzyme complex. Isolated nicked acetyltransferase retains its complete enzymatic activity and behaves as a high‐molecular‐weight aggregate. The lipoamide dehydrogenase and pyruvate dehydrogenase components, however, are not cleaved by the inactivase.
Optical Probe Responses on Sarcoplasmic Reticulum Oxacarbocyanines as Probes of Membrane Potential
Authors: BEELER Troy , RUSSELL James T. , MARTONOSI Anthony
Published: March 2005

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on saracoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential.

3.3′‐Diethyloxodicarbocyanine responds to ATP‐induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump‐mediated Ca2 fluxes.

A large change in the absorbance of 3,3′‐diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient.

The maximum of the potential‐dependent absorbance change is at 575–580 nm. The potentials generated by valinomycin or ionophore A23187 are short‐lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.
Rearrangements in the Course of Ribonuclease Hydrolysis of Pre‐messenger Ribonucleoproteins A Warning
Authors: STÉVENIN James , GATTONI Renata , DIVILLIERS Ginette , JACOB Monique
Published: March 2005

A study of the action of ribonuclease on 30–50‐S monoparticles prepared from pre‐messenger ribonucleoprotein (pre‐mRNA · protein) was started in order to elucidate the structure of mono‐particles. A ribonucleoprotein complex containing mostly 30000–38000–Mr proteins of pI 7–9 (α class) persisted under conditions where other proteins (23000–110000 Mr, pI 5–8.5, β class) were released. An unexpected increase of sedimentation coefficient accompanied the formation of the ribonucleoprotein complex. The extent of increase varied with the initial size of the mono‐particles, reaching 45% for 30‐S monoparticles. The ribonucleoprotein complexes designated here as 40–45‐S α‐ribonucleoproteins were more homogeneous in size than the original monoparticles. Electron microscopic examination showed that the sedimentation shift corresponded to an increase of the actual size of the particles, not to flattening or change of shape. Therefore, the 40–45s α‐ribonucleoprotein is not a pre‐existing unit of pre‐mRNA · protein but arises from specific rearrangements probably between small α ribonucleoproteins formed by fragmentation of monoparticles. In addition to the 40–45S α‐ribonucleoproteins, large protein aggregates corresponding to 15% of the monoparticle proteins were formed upon ribonuclease hydrolysis. Their major proteins were neutral, suggesting that the aggregates might be precipitates of proteins at pH close to the pI. Ribonuclease being a widespread cellular enzyme, partial rearrangements may occur during preparation and handling of pre‐mRNA · protein. It is particularly crucial to remark that the 40–45S α‐ribonucleoprotein which does not pre‐exist might be mistaken for a pre‐mRNA · protein unit.
Methionyl‐tRNAfMet Deacylase
Authors: ANDERSEN Klaus B.
Published: March 2005

A methionyl‐tRNAfMet deacylase was found in ribosomal salt wash from cultured human cells of the HeLa line. This enzyme was purified by the use of DEAE‐cellulose, ammonium sulfate precipitation, gel filtration and isoelectric focusing, and appears to be a protein with a native molecular weight of 80000, which consists of two 40000‐Mr subunits.

The mechanism of the Met‐tRNAfMet deacylase is shown to involve end‐product inhibition by the deacylated form of MettRNAfMet. The methionyl‐tRNAfMet deacylase is rather specific for Met‐tRNAfMet as opposed to Met‐tRNAmMet, has a KCl optimum of 85 mM, is inhibited by MgCl2 and is inhibited by GTP and NAD+ at physiological concentration. 40‐S and 60‐S subunits inhibit the enzyme, possibly by binding to it.

The stability of translational initiation complexes, containing methionyl‐tRNAfMet, was investigated in the presence of the enzyme. Purified ternary complex was slowly broken down by the enzyme, while the 40‐S‐subunit · Met‐tRNAfMet complex was stable in the presence of enzyme. The 80‐S complex formed with A‐U‐G trinucleotide as the message molecule was broken down, whereas the 80‐S complex formed with globin mRNA was stable in the presence of the enzyme. The physiological role of this enzyme is unclear, but it might act to regulate initiation by deacylating Met‐tRNAfMet.
Mössbauer Spectroscopic Investigations of Photodissociated Myoglobin‐CO at Low Temperatures
Authors: MARCOLIN Hans‐Ernst , RESCHKE Reiner , TRAUTWEIN Alfred
Published: March 2005

Myoglobin‐CO (MbCO) has been photodissociated at low temperatures. The photoproduct Mb* has been identified by Mössbauer spectroscopy as a ferrous high‐spin complex with Mössbauer parameters which are different from those of deoxy‐myoglobin. From the time‐dependent change of the linewidth of the Mb* Mössbauer spectrum at 5 K over a time interval of 8 h, we conclude that there exist several slightly different Mb* conformations with different recombination characteristics. In order to obtain a convenient time resolution of the recombination behavior, we have investigated the time‐dependence of the Mb* absorption within a time interval of 102–5 · 104 s after the photo‐dissociation, with a Mössbauer drive of constant velocity. The resulting recombination data have then been analyzed with various approximation steps. It is shown that the interpretation of experimental data on the basis of two independent exponentials leads to pre‐exponential frequency factors which are of the order of A1= 10s−1 and A2= 105± 5 · 102s−1. The attempt to interpret the experimental data on the basis of a distribution of energies seems more plausible and is in agreement with the existence of several slightly different Mb* conformations. The corresponding frequency factor A is about 4 s−1 and is therefore in disagreement with the value of A (of about 107 s−1) which was derived from optical recombination data by Austin et al. within a time interval of 10−6–103 s after the photodissociation. Typical activation energies for the recombination process Mb*+ CO → MbCO are 8 kJ/mol. At low temperatures (T < 46 K) the recombination behavior is explained by quantum mechanical tunnelling.