bluuebunny/crossref_metadata_2025 huggingface dataset:
The Receptor of Bacteriophage Lambda: Evidence for a Biosynthesis Dependent on Lipid Synthesis
Authors: PAGES Catherine , LAZDUNSKI Claude , LAZDUNSKI Andrée
Published: March 2005

Inhibition of lipid synthesis in cerulenin‐treated cells or in a mutant strain defective in sn‐glycerol‐3‐phosphate acyltransferase after glycerol deprivation, results in a marked decrease of insertion of LamB protein into the outer membrane. No lambda receptor was found in any other cell compartment or in the medium under these conditions. The LamB protein synthesis was inhibited by about 70% in the absence of lipid synthesis. The residual 30% protein produced during inhibition of fatty‐acid or phospholipid synthesis, was probably incorporated into the outer membrane since no further incorporation was observed after resumption of these syntheses. Besides OmpF and OmpC protein [Bocquet‐Pagés, C., Lazdunski, C., and Lazdunski, A. (1981) Eur. J. Biochem. 118, 105‐111], at least four other proteins of the outer membrane are also subject to alteration of levels in the absence of lipid synthesis. Under these conditions the uptake of maltose, like the uptake of 5′AMP [Bocquet‐Pagés, C., Lazdunski, C., and Lazdunski, A. (1981) Eur. J. Biochem. 118, 105‐111], was inhibited as much as 60%. These results are discussed with regard to the biosynthesis and assembly of the outer membrane proteins.
Effect of Thyroid Hormones and Cyclic AMP on Some Lipogenic Enzymes of the Fat Cell
Authors: CORREZE Claude , BERRICHE Salahedine , TAMAYO Libardo , NUNEZ Jacques
Published: March 2005

Thyroid hormones exert two types of effect on the lipogenic pathway of the fat cell of the young rat: one which is independent of cyclic AMP, and another which is under cyclic nucleotide control:

Thyroidectomy leads to an enhanced conversion of glucose to glucose 6‐phosphate by the hexokinase system. The activity of hexokinase I is increased twofold in the fat cell from hypothyroid rats. The oxidation of glucose 6‐phosphate by the pentose phosphate pathway is also increased after thyroidectomy. The V of both glucose 6‐phosphate and gluconate‐6‐phosphate dehydrogenases is doubled (56 mU × min−1× mg−1 to 114 mU × min−1× mg−1) whereas the Km, is unchanged (19 μM). The activities of ATP citrate lyase and of acetyl‐CoA carboxylase are higher in the adipocytes from hypothyroid rats. The activity of fatty acid synthetase is also stimulated, but to a lesser extent. The apparent Km of acetyl‐CoA carboxylase is 0.14 mM and 0.06 mM and the V is 17.8 nmol xmin−1 xmg‐′ and 27.8 nmolxmin−1 xmg protein−1 in the adipocytes from normal and hypothyroid rats respectively. In contrast, for ATP citrate lyase the V only is doubled (62 mU × min−1× mg−1 to 111 mUxmin−1xmg−1).

When fat cells from normal or hypothyroid rat were incubated in the presence of methylxanthines, dibutyryladenosine 3′,5′‐monophosphate, isoproterenol or epinephrine the activities of the hexokinase system and of glucose‐6‐phosphate and gluconate‐6‐phosphate dehydrogenases remained unchanged. In contrast, dibutyryl‐ adenosine 3′,5′‐monophosphate inhibited, by 60 ‐ 80%, the activities of ATP citrate lyase and acetyl‐CoA carboxylase in both cell types. In adipocytes prepared from normal rats, isoproterenol and epinephrine inhibited the activities of these two latter enzymes by 30 ‐ 60% while isobutylmethylxanthine potentiated their effect. In contrast, with fat cells from hypothyroid rats, isoproterenol and epinephrine had no effect upon the activities of ATP citrate lyase and acetyl‐CoA carboxylase, whereas a combination of epinephrine and ibobutylmethylxanthine was clearly inhibitory.

In conclusion, thyroid hormones regulate lipogenesis in the fat cell of the young rat firstly because they control the accumulation of the substrates of this pathway, glucose 6‐phosphate and NADPH, by changing the concentrations of the hexokinase system and of the rate‐limiting enzymes of the pentose phosphate pathway. Secondly, because they have a specific effect on the amount of the enzymes (ATP citrate lyase and acetyl‐CoA carboxylase) which channel acetyl‐CoA towards long chain fatty acid synthesis; and thirdly because they allow, in response to the lipolytic hormones, the accumulation of cyclic AMP which is responsible for the inactivation of these two latter enzymes.
A New Mechanism for the Reabsorption of Thyroid Iodoproteins: Selective Fluid Pinocytosis
Authors: VAN DEN HOVE Marie‐France , COUVREUR Marianne , DE VISSCHER Michel , SALVATORE Gaetano
Published: March 2005

Rat thyroid hemilobes were incubated in presence of exogeneous, heterologous and homologous thyroglobulins. The median density of the thyroglobulin molecules originally added to the medium was compared with that of the molecules remaining at the end of a three‐hour incubation period (37°C). A certain degree of specificity in the reabsorption process of thyroglobulin was found: the uptake of homologous molecules (rat) was higher than heterologous (hog) molecules. The median density of the iodoproteins remaining after the incubation did not change for the heterologous whereas it shifted towards lower density for the homologous thyroglobulins (equilibrium labelled, 35 iodine atoms/molecule). In addition, rat follicular cells display selectivity in the endocytosis of homologous thyroglobulin. Among the rat molecules, the normally iodinated are taken up more actively than the lowly iodinated or newly synthesized ones. Dissimilarity in the median density of the thyroglobulin molecules before and after endocytosis was only evident for the equilibrium‐labelled, normally iodinated rat preparation: the disappearence from the medium of 25‐ 30% of exogenous iodoproteins was sufficient to lower significantly the median density of the remaining molecules. This means that the thyroglobulin molecules having higher density are taken up preferentially by the tissue. A mechanism involving specific interactions between the iodoproteins and the surFace of thyroid cell is suggested.
Metal‐Binding Properties of Calmodulin
Authors: WANG Chih‐Lueh A. , AQUARON Robert R. , LEAVIS Paul C. , GERGELY John
Published: March 2005

Metal‐binding properties of calmodulin have been studied by using trivalent lanthanide ions as analogs of Ca2+. In agreement with a report published as this work was in progress [Kilhoffer, M.‐C., Demaille,J.G.,and Gerald, D. (1980) FEBS Lett. 116, 269–2721 we found that sites I and II are the high‐affinity sites, while sites III and IV are the low‐affinity sites for Tb3+. Competition experiments suggest the same preference in binding also applies to Ca2+. With calmodulin selectively nitrated at either of the two tyrosine residues we found that, although both tyrosine groups can transfer energy to the bound Tb3+, the fluorescence of only Tyr‐138 is sensitive to metal binding. Direct excitation of bound Eu3+ ions using a laser indicates that all four sites possess very similar microenvironments. These studies demonstrate that the binding properties of calmodulin are different from those of the homologous protein troponir C.
Isolation and Characterization of New Collagens from Chick Cartilage
Authors: VON DER MARK Klaus , VAN MENXEL Marion , WIEDEMANN Hanna
Published: March 2005

Three unique collagen chains were isolated from chick sternal cartilage following pepsin solubilization of total cartilage collagens and removal of the predominant type II collagen by fractional salt precipitation. Native molecules containing 1α,2α and 3α chains precipitated between 0.7 M and 1.2 M NaCl at acidic pH and could be purified by chromatography on carboxymethyl‐cellulose and agarose columns. Although similar to mammalian 1α, 2α and 3α chains, differences in the mobilities on sodium dodecylsulfate gel electrophoresis, CNBr peptidc profiles and amino acid composition were found. The lα and 2α chains resemble, but are structurally distinct from, the chick αl(V) and α2(V) chains. The 3α chain appears to be closely related to the αl(II) chain, although some differences in the cyanogen bromide peptides suggest that they might be different gene products.

In addition, two collagenous fragments of Mr140000(M1)and 35000 (M2) were found which precipitated at 2.0 M NaCl at acidic pH. Both fragments contain interchain disulfide bonds. The larger fragment was reducible to subunits of approximate M, 120000,48000, 28000 and 11000. The smaller fragment gave rise to peptides of Mr about 12000 and 10000 after reduction. By the technique of rotary shadowing the native, unreduced larger fragment M1 appeared as a slender rod‐like molecule with a distinct bend approximately 40 nm from one end. We interpret this finding as indicative of a focal amino acid sequence irregularity, disrupting the triple‐helical conformation.
The Distribution of Ubiquinone‐10 in Phospholipid Bilayers
Authors: KATSIKAS Haralambos , QUINN Peter J.
Published: March 2005

The thermal behaviour of mixed aqueous dispersions of ubiquinone‐10 with dipalmitoylglycerophospho‐ choline, dimyristoylglycerophosphocholine or egg phosphatidylcholine has been examined by differential scanning calorimetry. Ubiquinone‐10 was found to undergo a series of thermal transitions in the heating process in the temperature range 290–300 K prior to a major melting endotherm at 317 K. The temperature of this endotherm was unaltered when the coenzyme was codispersed with phospholipid but enthalpy determinations showed that up to about 5 (mol/mol) and 20% (mol/mol) of ubiquinone‐10 was removed from the transition in mixtures with dimyristoyl and dipalmitoyl derivatives of phosphatidylcholine respectively. Likewise the pre‐transition and main endothermic transitions of the saturated phospholipids were largely unperturbed by the presence of ubiquinone‐10 but up to 22% (mol/mol) and 8 (mol/mol) of the dimyristoyl and dipalmitoyl derivatives of phosphatidylcholine respectively were removed from the endotherm. These data suggest that at molar percentages less than 20% ubiquinone‐10 a high proportion of the coenzyme is molecularly mixed within the phospholipid bilayer but as the proportion increases there is a greater tendency for the coenzyme to form aggregates that display typical melting and crystallization behaviour.
A New Polyacrylamide Gel Electrophoresis System
Authors: TSUGITA Akira , SASADA Shingo , van den BROEK Rudolf , SCHEFFLER Jean Jacques
Published: March 2005

Polyacrylamide gel electrophoresis is one of the most efficient methods for separation and identification of proteins. A new gel electrophoresis system has been developed in order to facilitate both separation and extraction of peptides and proteins ranging in molecular weight from approximately 200 up to 100000. This system involves the use of a volatile buffer, triethylamine/formic acid pH 11.7, and a reaction with a covalently binding NH2 reagent, 1,3,6‐trisuIfonylpyrene 8‐isothiocyanate. Under these conditions, the addition of strongly negative charges to proteins is achieved which allowa migration according to molecular weight. The modified proteins being fluorescent require neither fixation nor staining for their detection. This is an absolute requirement especially when dealing with small peptides. An additional advantage of such a modification is the increased solubility of the proteins which makes their extraction easier and more efficient. The extracted proteins are easily freed from salts and other small molecules simply by evaporation and they are readily available for sequencing analysis using Edman degradation, carboxypeptidase digestion or amino acid analysis.
Na+ Channels with Binding Sites of High and Low Affinity for Tetrodotoxin in Different Excitable and Non‐excitable Cells
Authors: LOMBET Alain , FRELIN Christian , RENAUD Jean‐François , LAZDUNSKI Michel
Published: March 2005

The properties of interaction of tetrodotoxin with its receptor site on the voltage‐sensitive Na+ channel were analysed in two ways: (a) by titrating Na+ channels with a tetrodotoxin derivative, [3H]ethylenediamine‐ tetrodotoxin; (b) by studying the physiological properties of interaction of the toxin with its receptor from 22Na flux measurements.

Using a variety of cell types in culture, three different kinds of situations were observed.

1. Cells like N1E 115 neuroblastoma, CCI 39 fibroblasts, embryonic chick cardiomyocytes and chick skeletal myotubes only have one family of Na+ channels with high‐affinity binding sites (in the nanomolar range) for tetrodotoxin. These Na+ channels are the same ones as those that are activated by the alkaloid and polypeptide toxins that accelerate 22Na+ influx.

2. C9 cells have Na+ channels with low‐affinity binding sites for tetrodotoxin. These Na+ channels are also he ones that are activated by alkaloid and polypeptide toxins (the median inhibitory concentration for tetrodotoxin inhibition of 22Na+ influx through these Na+ channels is 300 nM).

3. Rat myotubes that have differentiated in culture in the absence of neuronal influence have both high‐affinity binding sites (in the nanomolar range) detected with the tritiated tetrodotoxin derivative and low‐affinity binding sites (in the micromolar range) detected by 22Na+ flux experiments. Only low‐affinity binding sites correspond to Na+ channels that can be activated with alkaloid and polypeptide toxins.
The Interaction of Sulfate with Carbonic Anhydrase
Authors: SIMONSSON Ingvar , LINDSKOG Sven
Published: March 2005

In the absence of sulfate the pH/rate profile for the 4‐nitrophenyl acetate hydrolase activity of bovine carbonic anhydrase is complex. The results fit with a microscopic ionization scheme involving two electrostatically interacting groups. The activity depends on the concentration of the basic form of one of these groups. Proton NMR spectra show that the active site residue, His‐64, has titration behaviour corresponding to that of the second group of the ionization scheme.

The addition of increasing concentrations of Na2SO4 gradually converts the pH/rate profile to that of a simple titration curve. A pKa of 6.9 is found at 50 mM Na2SO4. Concomitantly the titration curve of His‐64 changes. The results fit with the microscopic ionization scheme if it is assumed that significant SO2−4binding occurs only when both His‐64 and the activity‐linked group are protonated.

At constant pH, sulfate behaves as if it inhibits the enzyme only partially. However, data are presented suggesting that the enzyme‐sulfate complex is inactive, but the binding of SO2−4depends strongly on ionic strength. Thus, above about 25 mM sulfate any further increase of the sulfate concentration is nearly compensated by a decrease of the binding affinity. The binding of a monovalent anion, C1−, also depends on ionic strength, but this dependence is much less prominent than for SO2‐4.

Sulfate shows non‐competitive behaviour with respect to CO2 and appears to compete with HCO−3. Iodide and sulfate appear to be mutually competitive. The low pH limit of Ki for I−is 3 mM and about 0.1 mM in the presence of 50 mM Na2SO4 and in the absence of sulfate respectively.

In the absence of sulfate the Co(II)‐substituted bovine enzyme shows a complex pH/rate profile similar to that of the native enzyme. Changes of the optical spectrum parallel those of the activity. Relaxation rates (T−11)of water protons in the presence of Co(II) enzyme indicate that the Co(II)ion might have a H2O ligand at low pH in sulfate‐free solutions.

The effects on sulfate of human carbonic anhydrase II (or C) are similar to those on the bovine enzyme. The effects of sulfate on the human 1 (or B) enzyme are smaller.
Studies on the Specificity of the Binding Site of Vicia graminea Anti‐N Lectin
Authors: DUK Maria , LISOWSKA Elwira , KORDOWICZ Maria , WASNIOWSKA Kazimiera
Published: March 2005

Incubation of polyacrylamide gels after electrophoresis of glycoproteins with Vicia graminea lectin, labeled with radioactive iodine, showed that the lectin was bound strongly to blood‐group N and Ss human sialoglycoproteins, weakly to the major sialoglycoprotein of horse red cell membranes, and was not bound to blood‐group M sialoglycoprotein.

Inhibition of binding of the labeled lectin to blood‐group NN erythrocytes by untreated and modified human and horse erythrocyte glycoproteins and their structurally defined glycopeptide fragments was studied. The glycoproteins and glycopeptides were modified by desialylation, N‐acetylation, Edman degradation or by a combination of two or more of these procedures.

Desialylation and N‐acetylation incrcased thc inhibitory activity of N and M glycoproteins and glycopeptideb. The lectin was inhibited by the NH2‐terminal M and N glycopeptides of various length of the polypeptide chain, and was not inhibited by the internal tryptic glycopeptide. All modified N glycopeptides (terminated with a leucine residue) were distinctly stronger inhibitors of the lectin than respective M glycopeptides (terminated with a berine residue). In each of these two groups the most active were tryptic and pronase asialoglycopeptides with blocked amino groups. The M and N tryptic asialoglycopeptides were inactivated by Edman degradation and reactivated by the subsequent N‐acetylation.

Horse erythrocyte glycoprotein and its pronase glycopeptide were relatively weak inhibitors of V. graminea lectin, their activity was increased by desialylation, but was not affected by N‐acetylation.

Comparison of the structures of glycopeptides reacting and not reacting with V.graminea lectin suggested the following tentative conclusions. (a) V.graminea lectin can react with glycopeptides which have clusters of unsubdiluted or sialylated Gal(βl‐3)GalNAc‐ chains located at adjacent amino acid residues. (b) Reaction of glycopeptides with the lectin is also dependent on other (e.g. amino acid) residues present at the lectin‐binding site, namely, it is enhanced by a hydrophobic residue and is weakened by a free amino group.
The Ribosomal Serine Proteinase, Cathepsin R
Authors: LANGNER Jüurgen , KIRSCHKE Heidrun , BOHLEY Peter , WIEDERANDERS Bernd , KORANT Bruce D.
Published: March 2005

Ribosomes have been shown to contain a proteolytic activity, characterized as an endopeptidase with serine in the active center. The enzyme has been given the name cathepsin R, following the recommendations of Barrett et al. (in a publication from the Cold Spring Harbor Laboratory, New York) for naming new pro‐ teinases. The present paper contains evidence that cathepsin R in rat liver ribosomes is present in a cryptic form. Upon dissociation of ribosomes to subunits (and to minor extent also by 0.5 M KCl washes). the cryptic proteinase is released. Activation of the released cathepsin R is effected by equilibration with 2 MNaCl/O.05 M sodium acetate, pH 4.8. The molecular weight of free cathepsin R is 25000–30000.
Gluconeogenesis in vitro
Authors: MÖRIKOFER‐ZWEZ Stéphanie , STOECKLIN Franziska B. , WALTER Paul
Published: March 2005

A cell‐free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors at near physiological concentrations was shown to form glucose 6‐phosphate from malate + 3‐phosphoglycerate at a rate of 1.11± 0.09 μmol. min‐1. g liver−1 (mean ± SEM, n= 9, 30°C). At least 70% of glucose 6‐phos‐ phate formed was derived from malate as calculated from experiments with [U‐14C]malate. The indicated rates were measured between 10 min and 30 min incubation time when the system was near steady state with respect to the lactate/pyruvate ratio and to most of the gluconeogenic intermediates.

In the absence of mitochondria, the rate of formation of glucose 6‐phosphate from malate was about seven times lower than in their presence. A comparison between incubations carried out in presence or absence of mitochondria revealed that mitochondria decreased the lactate/pyruvate ratio and increased the ratio of (ATP + ITP)/(ADP + IDP). It could be shown that under the present incubation conditions, formation of glucose 6‐phosphate was closely linked to the ratio of (ATP + ITP)/(ADP + IDP) whereas changing redox ratios had little influence on the gluconeogenic rate.
The Role of the Components δ and y of the DNA‐Dependent RNA Polymerase of Lactobacillus curvatus in Promotor Selection
Authors: GIERL Alfons , ZILLIG Wolfram , STETTER Karl Otto
Published: March 2005

An average of 0.44 molecule each of the initiation factor δ and the RNA polymerase binding protein y and 0.54 molecule of the subunit y per molecule of Lactobacillus curvatus DNA‐dependent RNA polymerase have been found in the cell.

Free factor y displaces δ from free holo enzyme, Eδ. The formation of a binary complex from Ey, free δ, and poly[d(A‐T)], leads to immediate release of factor y. The release of the δ factor occurs upon the transition of the binary to a ternary complex.

A mixture of E and δ forms binary complexes with all T7 DNA HpaII restriction fragments. In contrast a mix‐ ture of Ey and δ binds selectively to promoter‐containing DNA fragments, indicating that the stimulatory effect of y on transcription is due to an increase in the rate of promoter selection.

The same RNA products are synthesised by Eδ and by Ey plus δ with T7 DNA as template. Thus the non‐ specific complexes formed by Eδ and T7 DNA are nonproductive.

On the basis of these findings we propose a model for the transcription cycle in Lactobacillus curvatus.
Identification by Affinity Labeling of Potential Sites in 23‐S rRNA Interacting with the 3′ End of tRNA
Authors: LEITNER Moshe , WILCHEK Meir , ZAMIR Ada
Published: March 2005

A derivative of tRNAPhe carrying at its 3′ end a photolyzable group was chemically synthesized by coupling p‐azidobenzoylglycylhydrazide to periodate‐oxidized tRNA. The reaction converts the 3′‐terminal ribofuranoside residue into a six‐membered ring. The binding ofp‐azidobenzoylglycylhydrazide‐t RNA to 70‐S ribosomes resembles the binding of Phe‐tRNA in its requirement for poly(U) and Mg2+dependence. Irradiation at wavelengths < 300 nm of complexes of p‐azidobenzoylglycylhydrazide‐tRNA and 70‐S ribosomes results in the covalent binding of approximately 20% of the reversibly bound tRNA to the ribosomes. The labeling occurs predominantly at a single site in 23‐S rRNA within the sequence included in the 18‐S fragment.

To identify the modified sequence, an RNase A digest of 23‐S rRNA was fractionated on a hydrophobic matrix (Porapak Q) and adsorbed and non‐adsorbed fractions were compared by fingerprint analysis. A relatively intense spot was observed in the expected region in the fingerprint of the adosrbed fraction. The presence of an unusual nucleotide in the sequence of this oligonucleotide was demonstrated by a mobility‐shift analysis of a partial nuclease P1 digest. The sequence derived is G★‐A‐A‐G‐C (the asterisk denoting the site of modification). Sequences at six positions in th e 23‐S rRNA are compatible with the sequence of the oligonucleotide determined. Of the six positions five are located within nucleotides 1467‐ 1887. This region of approximately 400 nucleotides is likely to include the sequence interacting with the 3′ end of tRNA and possibly forming part of the peptidyl‐ transferase center.
Covalent Addition of H2O, Enzyme Substrates and Activators to Pyrrolo‐quinoline Quinone, the Coenzyme of Quinoproteins
Authors: DEKKER Robert H. , DUINE Johannis A. , FRANK Johannes , VERWIEL Jzn. P. Eugéne J. , WESTERLING Jaap
Published: March 2005

The fluorescence excitation and the absorption spectrum of 2,7,9‐tricarboxy‐1H‐pyrrolo [2,3‐f]quinoline‐ 4,s‐dione (pyrrolo‐quinoline quinone, PQQ), measured in the pH range 7.0‐ 10.0, are quite different. However. when the temperature of the solution is lowered, the shape and maxima of these spectra become more similar. 1H‐NMR in 2H2/0 revealed a temperature‐dependent equilibrium between PQQ and a hydrated form. Evidence is presented that low temperature favours the formation of the fluorescing species which is PQQ, hydrated at the C‐5 position.

Even further hydration is possible since absorption, fluorescence and NMR spectroscopy of PQQ in borate buffer pH 10.0 reveal additional hydration at the C‐4 position, pointing to a dihydrate.

PQQ also reacts with quinoprotein enzyme substrates and activators. Spectroscopic measurements showed the existence of 5‐alkoxy‐5‐hydroxy‐PQQ and 5‐amino‐5‐hydroxy‐PQQ in the presence of alcohols and 2 M NH4CI, pH 9.0, respectively. In the latter case, the existence of 5‐imino‐PQQ could also be demonstrated. Addition compounds with amines appear to be unstable. The amines become probably oxidized because pyrrolo‐quinoline quinol (PQQH2) was found as the reaction product. On the other hand, an addition compound containing an imino bond could be isolated after addition of urea to a PQQ solution.

Spectral characteristics of PQQ and its addition compounds are presented since these data are necessary for the spectral analysis of quinoproteins and the quantitative estimation of coenzyme.
Isolation and Properties of Proteoglycans from Bovine Aorta
Authors: SCHMIDT Annette , PRAGER Martin , SELMKE Peter , BUDDECKE Eckhart
Published: March 2005

Calf arterial tissue, preincubated under organ culture conditions for 24 h in the presence of [35S]sulfate in combination with either [3H]glucosamine or [3H]mannose, yielded 81.8% of total uronic acids, 83% of incor‐ porated [35S]sulfate and 66.8 % of incorporated [3H]glucosamine on extraction with 4 M guanidinium chloride in the presence of protease inhibitors.

Two proteoglycans (A and B) differing in amino acid and glycosaminoglycan composition were purified by sequential fractionation of the guanidinium chloride extract on Sepharose 4B CL, caesium chloride density gradient centrifugation under dissociative conditions and ion‐exchange chromatography.

Gel filtration of the guanidinium chloride extract resulted in the separation of two [35S]proteoglycan‐con‐ taining fractions, eluted with the void volume (fraction A) and at Kav= 0.33 (fraction B).

Proteoglycan A was obtained from the Sepharose fraction A as a proteoglycan complex by a dissociative gradient. On ion‐exchange chromatography in the presence of 6 M urea the proteoglycan complex dissociated into proteoglycan A (Mr 813000) and hyaluronate (M, 81 000). Proteoglycan A contained 3‐4 chondroitin 4/6‐sulfate side chains (Mr 35000) and 10.4% protein.

Proteoglycan B was purified from the Sepharose fraction B by a dissociative gradient and subsequent ion‐ exchange chromatography of the bottom fraction. Proteoglycan B was characterized as a proteoglycan monomer (M r 190000) containing 20.3 % protein with 3 ‐ 4 chondroitin sulfate/dermatan sulfate chains (chondroitin sulfate 53 %, dermatan sulfate 46 %) attached. The specific 35S radioactivity of proteoglycan B was three‐times higher than that of proteoglycan A.

[3H]Mannose‐labelling of the isolated proteoglycans A and B indicated that both proteoglycans contained glycoprotein‐type oligosaccharides.

Pulse‐chase experiments with [35S]sulfate as label suggested that both proteoglycans A and B are primarily produced as monomers retarded by the Sepharose gel and that proteoglycan A is successively converted to a proteoglycan complex on interaction with hyaluronate.
Characterization of Cation‐Binding Sites on Panulirus interruptus Hemocyanin by 43Ca and 23Na NMR
Authors: ANDERSSON Thomas , CHIANCONE Emilia , FORSEN Sture
Published: March 2005

The binding of Na+ and Ca2+ ions to Panulirus interruptus hemocyanin has been studied by nuclear magnetic resonance. The combined use of 43Ca and 23Na NMR has allowed two different classes of sites to be distinguished. The sites are characterized by a relatively fast chemical exchange, local mobility and binding constants of the order of 103−4 M−1 for Ca2+ and Mg2+ and ∼ 102 M−1 for Na+. These features and the observed pH dependence of the Ca2+ binding are consistent with the protein ligands being carboxylate groups, some of which may occur in clusters.
Involvement of Prostaglandin E and Adenosine 3′ 5′‐Monophosphate in Lipopolysaccharide‐Stimulated Collagenase Release by Rat Kupffer Cells
Authors: BHATNAGAR Rakesh , SCHADE Ulrich , RIETSCHEL Ernst Th. , DECKER Karl
Published: March 2005

Kupffer cells exposed to bacterial lipopolysaccharide in vitro synthesized collagenase and released the major portion of it into the extracellular space while the intracellular level of enzyme was not altered significantly. Cycloheximide prevented the appearance of collagenase in the medium indicating de novo synthesis. Indo‐ methacin, an inhibitor of cyclooxygenase, also blocked collagenase synthesis. In line with this observation, Kupffer cells were found to synthesize substantial amounts of prostaglandin E2 when exposed to lipopoly‐ saccharide; concomitantly, cellular cAMP levels were increased. Indomethacin was shown to abolish the stimu‐ lated cAMP formation. Addition to the culture medium of cAMP or dibutyryladenosine 3′, 5′‐monophosphate as well as of prostaglandin E2 or, to a lesser extent, prostaglandin El allowed indomethacin‐inhibited cells to resume the production of collagenase. It is proposed that in rat Kupffer cells lipopolysaccharide‐elicited col‐ lagenase synthesis and excretion is mediated sequentially by stimulated production of prostaglandin E2, enhanced adenylate cyclase activity and increased intracellular cAMP levels.
Relationship between the Pharmacological Action of Crotoxin and Its Phospholipase Activity
Authors: MARLAS Guy , BON Cassian
Published: March 2005

Crotoxin, the major neurotoxin from Crotalus durissus terrificus venom, blocks the postsynaptic response of Electrophorus electroplaque to bath‐applied carbamoylcholine. Crotoxin is composed of two non‐covalently linked subunits: an acidic non‐enzymatic component, A, and a basic phospholipase component, B. According to our previous studies, component A restricts the binding of component B to high‐affinity sites which occur in Torpedo membrane fragments in approximatively equivalent amount to the cholinergic receptor sites. We demonstrate here that the catalytic activity of component B is actually required for the postsynaptic blocking action of the toxin.

Irreversible inactivation of component B was achieved by alkylation with p‐bromophenylacyl bromide of a histidine residue in the catalytic site. Although the native crotoxin complex was insensitive to the inhibitor, suggesting that the catalytic center is protected by component A, we obtained an enzymatically inactive crotoxin by recombination of the alkylated component B with component A. This modified crotoxin was devoid of enzymic activity, toxicity and pharmacological activity on the electroplaque.

The phospholipase activity of component B may be reversibly inhibited by replacing Ca2+ with Sr2+ ions in the physiological solution. This allowed us to identify several steps in the action of the toxin: a quasi‐irreversible binding step which occurs either in the presence of Ca2+ or Sr2+ ions, and a catalytic step which requires Ca2+ ions and is responsible for the irreversible blockade of the electroplaque response to cholinergic agonists.

Finally, we examined the effect of various toxic and non‐toxic phospholipases on the electroplaque response. The data exclude that the postsynaptic blockade results from indiscriminate hydrolysis of phospholipidb. They rather suggest that the pharmacological efficiency of crotoxin is due to the fact that component B exerts its catalytic activity at critical postsynaptic sites. These conclusions are in excellent agreement with the explanation of the synergistic effect of the two subunits which we derived from our binding data. The postsynaptic sensitive sites for crotoxin action might be closely related to the acetylcholine receptor sites.
Direct Measurement of Proton Transfer as a Probing Reaction for the Microenvironment of the Apomyoglobin Heme‐Binding Site
Authors: GUTMAN Menachem , NACHLIEL Esther , HUPPERT Dan
Published: March 2005

Aromatic alcohols fluoresce at different wavelengths in their neutral (øOH*) and anionic (øO*) excited states. Consequently, time‐resolved fluorescence measurements, at the respective wavelengths, can be used for measuring the rates of proton dissociation and recombination of the excited molecule. As the lifetime of the excited state is very short (a few nanoseconds), the measured reaction is that which takes place in a volume corresponding to the diffusion distance of the proton during the lifetime of the excited state.

8‐Hydroxypyrene 1,3,6‐trisulfonate (pK°= 7.7, pK*= 0.5) is bound to apomyoglobin with a stoichiometry of 1 : 1. In the bound state its neutral form fluorescence increase 20‐fold.

The binding affinity is pH‐dependent. Two protonatable groups, with pK= 6.5, participate in the stabilization of the negatively charged ligand in the binding site.

The ligand is bound only to the apoprotein and is displaced from its site by hemin. Thus we suggest that the ligand is bound to the heme binding site of apomyoglobin.

Time‐resolved fluorescence of the bound ligand yields the rate constants of proton dissociation and recombination as taking place within the heme binding cavity of apomyoglobin.

The rate of proton dissociation is slowed to 7 %, of the rate measured for the free ligand. Such a slow disso‐ ciation indicates a strong interaction of the water in the cavity with the walls [Gutman, M., Huppert, D., and Nachliel, E. (1982) Eur. J. Biochem. 121, 637‐642]. The water activity in the site is equivalent to a (H2O) = 0.67.
Binding of Substrates to Aspartate Aminotransferase
Authors: ARRIO‐DUPONT Martine , VERGÉ Daniel
Published: March 2005

The binding of substrates l‐glutamate and 2‐oxoglutarate to aspartate aminotransferase was studied by spectrophotometric titration according to Jenkins and D'Ari [J. Biol. Chem. 241 (1966) 2845 ‐28541. Our data, obtained over a wide range of substrate concentrations, were not consistent with the hypothesis of independent and equivalent binding sites. Two other possibilities were considered: (a) the sites are independent but not equivalent; (b) a negative cooperativity occurs between the sites. It is possible to distinguish between these two hypotheses because the substrate binding is complex, with a couple of substrates and two forms of enzyme in equilibrium with the enzyme‐substrate complexes. Distinct equations δA / E, =f[substrates], where δA= change in absorbance upon addition of substrates to the enzyme and Et= total site concentration, were derived for each case. The data were directly fitted to these equations by an iterative multilinear regression analysis, and the equilibrium constants were computed. This analysis showed that the binding sites are independent and not equivalent, with dissociation constants for 2‐oxoglutarate of 2 μM and 280 pM and dissociation constants for glutamate of 1.7 mM and 22 mM. The molar absorption coefficient of the binary complexes is 2250 M−1 cm−.
5‐Isothiocyanato‐1‐naphthalene Azide and p‐Azidophenylisothiocyanate
Authors: SIGRIST Hans , ALLEGRINI Peter Roland , KEMPF Christoph , SCHNIPPERING Christinc , ZAHLER Peter
Published: March 2005

Two hydrophobic, heterobifunctional azidoarylisothiocyanates have been synthesized. The reagents are applicable for group‐directed modification of membrane‐integrated protein segments and subsequent photo‐ induced cross‐linking with neighbouring membrane constituents. Both 5‐isothiocyanato‐I‐naphthalene azide and p‐azidophenylisothiocyanate are chemically characterized with respect to structure and photosensitivity. The reagents compete for the documented phenylisothiocyanate binding site(s) in bacteriorhodopsin and to the anion‐exchange protein (band 3) of human erythrocytes. Photoactivation of azidoarylisothiocyanate‐labeled membrane proteins leads to homopolymers when cross‐linked in purple membranes or in vesicles enriched in the anion‐exchange protein.
Interactions of Ellipticine with Model or Natural Membranes
Authors: TERCE Francois , TOCANNE Jean‐Francois , LANEELLE Gilbert
Published: March 2005

Ellipticine is the type of a group of substances used against human cancer. It was shown that ellipticine and 9‐methoxyellipticine strongly interact with monolayers of negatively charged phospholipids. In the present paper it is shown by spectrophotometric methods that ellipticine interacts with suspensions of a model membrane with a 1/1 charge neutralization, and with a natural membrane, both negatively charged. At a physiological pH, ellipticine undergoes protonation in the presence of these membranes. Its apparent pK is increased by 1, and its spectral behaviour, when the pH is changed, indicates that the drug is in a less polar environment, suggesting that hydrophobic bonds link the drug and the lipids. The existence of such bonds is suggested by the fact that the drug is not significantly released from anionic membranes when the pH is increased to 10, at which value ellipticine is not ionized. The consequences of these ionic and hydrophobic interactions are briefly discussed.
Somatic Antigens of Pseudomonas aeruginosa
Authors: KNIREL Yuriy A. , VINOGRADOV Evgeny V. , SHASHKOV Alexander S. , DMITRIEV Boris A. , KOCHETKOV Nikolay K. , STANISLAVSKY Evgeny S. , MASHILOVA Galina M.
Published: March 2005

Structural and immunochemical studies have been performed on the 0‐specific antigens of two serologically related types of Pseudomonas aeruginosa 0 : 2a,b and 0 : 2a,c (Lanyi's classification). The 0‐specific polysaccharide chains of the two serotypes were shown to be acidic polysaccharides composed of repeating trisaccharide units consisting of l‐rhamnose, N‐acetyl‐d‐quinovosamine and N‐acetyl‐L‐galactosaminuronic acid residues. Based on 1 H and 13C nuclear magnetic resonance spectroscopy, data of methylation analysis and selective solvolysis with hydrogen fluoride, the repeating unit of the 0 : 2a,c 0‐specific polysaccharide was assigned the following structure →4)l‐GalNAcA(α1→ 3)d QuiNAc(αl→ 3)LRha(α1, where GalNAcA =N‐acetylgalactosaminuronic acid, QuiNAc= acetylquinovosamine and Rha = rhamnose.

The 0:2a,b 0‐specific polysaccharide had the same structure of the trisaccharide repeating unit but was distinguished by the presence of 0‐acetyl groups on 70–80% of the rhamnose residues in position 2. The 0‐acetyl groups were located both by methylation with methyltrifluoromethane sulphonate and by comparison of the 13C nuclear magnetic resonance spectra of the native and 0‐deacetylated polysaccharides.

Serological studies revealed the determinant role of the 0‐acetyl groups in the 0‐antigen 0 : 2a,b and suggested the 0‐factor 2b to be related to the 2‐0‐acetyl‐l‐rhamnose residue, whereas the 0‐factor 2c was probably deter‐ mined by the nonacetylated rhamnose residue. The dominant moiety of the determinant 2a common to the two antigens was obviously presented by the N‐acetyl‐l‐galactosaminuronic acid residue.
Direct Evidence that Cholesterol Ester Hydrolase from Adrenal Cortex is the Same Enzyme as Hormone‐Sensitive Lipase from Adipose Tissue
Authors: COOK Kenneth G. , YEAMAN Stephen J. , STRALFORS Peter , FREDRIKSON Gudrun , BELFRAGE Per
Published: March 2005

Several properties of the cytosolic cholesterol ester hydrolase from bovine adrenal cortex were investigated and those properties were compared directly with those of the well‐characterised hormone‐sensitive lipase, the rate‐limiting enzyme in adipose tissue lipolysis. Properties examined included : (a) activity against different substrates; (b) susceptibility to inhibition by NaF, Hg2+ ions and diisopropyl fluorophosphonate; (c) subunit molecular weight as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate; (d) ability to serve as a substrate for cyclic AMP‐dependent protein kinase; (e) effect of phosphorylation on enzyme activity: and (f) degradation pattern of polypeptides following limited proteolysis.

In all respects the two enzymes exhibited essentially identical characteristics. It is therefore concluded that the same protein, or two very similar proteins, catalyses the hydrolysis of cholesterol esters in adrenal cortex and lipolysis in adipose tissue.

The implication of this finding is discussed in relation to the hormonal control of steroidogenesis in adrenal cortex and of lipolysis in adipose tissue.
Alterations of the α or β Subunits of the Mitochondria1 ATPase in Yeast Mutants
Authors: BOUTRY Marc , GOFFEAU Andrè
Published: March 2005

Among 979 non‐glycerol growers of the yeast Schizosaccharomyces pombe, 40 strains were found to be deficient in the mitochondrial ATPase activity. Three of them exhibited an alteration in either the α or β subunits of the F1ATPase. The α subunit was not immunodetected in the A23/13 mutant. The β subunit was not immuno‐ detected in the B59/1 mutant. The existence of these two mutants shows that the α and β subunits can be present independently of each other in the inner mitochondrial membrane. The β subunit of the mutant F25/28 had a slower electrophoretic mobility than that of the wild‐type β subunit. This phenotype indicates abnormal processing or specific modification of the β subunit. All mutants showed reduced activities of the NADH–cytochrome c reductase and of the cytochrome oxidase and a decreased synthesis of cytochrome aa3 and cytochrome b. This pleiotropic phenotype appears to result from specific modifications in the mitochondrial protein synthesis. The mitochondrial synthesis of four polypeptides (three cytochrome oxidase and one cytochrome b subunits) was markedly decreased or absent while three new polypeptides (Mr= 54000, 20000 and 15000) were detected in all the mutants analysed. This observation suggests that a functional F1ATPase is necessary for the correct synthesis and/or assembly of the mitochondrially made complexes.
Properties of Catalase Purified from Whole Cells and Peroxisomes of n‐Alkane‐Grown Candida tropicalis
Authors: YAMADA Takao , TANAKA Atsuo , FUKUI Saburo
Published: March 2005

Peroxisomes appear profusely, in harmony with a marked enhancement of catalase activity level, in yeast cells growing on n‐alkanes or higher fatty acids as the sole carbon source. Catalase (H2O2 : H2O2 oxidoreductase, EC 1.11.1.6) was purified to homogeneity from the crude extract and from the peroxisome‐containing particulate fraction of alkane‐grown Candida tropicalis cells. The purified enzyme from each source was a similar protein of molecular weight 210000 composed of four identical subunits of molecular weight 54000, namely a kind of homotetramer. The enzyme contained one molecule of heme per subunit, giving the absorption spectrum characteristic of hemoprotein. β‐(3,4‐Dihydroxyphenyl)‐l‐alanine served as a substrate for the peroxidatic reaction by the enzyme. Ouchterlony double‐diffusion analysis and immunochemical titration with rabbit antiserum against peroxisomal catalase of n‐alkane‐grown C. tropicalis have indicated that cytoplasmic catalase of the yeast is immunologically indistinguishable with peroxisomal catalase.
Procollagen N‐Proteinase
Authors: TUDERMAN Leena , PROCKOP Darwin J.
Published: March 2005

Procollagen N‐proteinase was purified about 3700‐fold from chick embryo tendons. Electrophoresis of the protein after iodination and denaturation suggested it was homogeneous. However, the native enzyme could not be examined by gel electrophoresis, and therefore homogeneity of the preparation was not conclusively established. Antibodies to the enzyme completely inhibited activity and gave a single precipitant line by double immuno‐ diffusion. The Km for a native procollagen substrate was 0.3‐0.5 μM. The same protein after denaturation inhibited activity. The enzyme did not cleave type IV procollagen from human fibroblasts or a type IV procollagen from a mouse sarcoma. Ca2+ was required for maximal enzymic activity. The data suggested a second metal requirement, but this was not identified. Reducing agents and metal chelators inhibited activity, but there was little if any inhibition from several inhibitors of other neutral metalloproteinases.
Glutathione S‐Conjugate Transport Using Inside‐Out Vesicles from Human Erythrocytes
Authors: KONDO Takahito , MURAO Makoto , TANIGUCHI Naoyuki
Published: March 2005

Previous studies [Kondo, T., Dale, G. L. and Beutler, E. (1981) Biochim. Biophys. Acta, 645, 132‐1361 have shown evidence for the existence of two different active‐transport processes for glutathione disulphide (GSSG) in human erythrocytes (the high‐Km and low‐Km processes). In the present investigation adenosine‐triphosphate‐ dependent transport of glutathione S‐conjugate was characterized in comparison with active glutathione transport using inside‐out vesicles from human erythrocytes. Incubation of the vesicles with glutathione S‐conjugate (S‐2,4‐dinitrophenylglutathione) was found to inhibit competitively the high‐Km process of GSSG transport but not significantly affect the low‐Km process. The glutathione S‐conjugate transport required ATP. A Lineweaver‐Burk plot of the transport rate as a function of the conjugate concentration gave an apparent Km value of 0.94 mM. The Km value of ATP‐Mg was 0.76 mM. The transport of glutathione S‐conjugate was dependent on temperature. Preincubation of vesicles with dithiothreitol resulted in an increase of the transport rate while thiol reagents, such as iodoacetamide, N‐ethylmaleimide andp‐chloromercuribenzoate inhibited the transport. Addition of nucleotides, such as CTP, UTP or GTP had no effect on the transport. These findings suggest that glutathione S‐conjugate formed by the catalytic reaction of glutathione S‐transferase in erythrocytes under the exposure to electrophilic compounds, is eliminated via the same transport process for GSSG elevated under oxidative stress.
Incorporation of Labelled Leucine into Alkaline Phosphatase in Response to 1,25‐Dihydroxycholecalciferol in Chick Intestinal Brush Borders
Authors: WILSON Peter W. , LAWSON D. Eric M.
Published: March 2005

1,25‐Dihydroxycholecalciferol increases the incorporation of leucine into a protein component (Mr= 84000) of the chick intestinal brush‐border membrane. Evidence has now been obtained showing this protein to be sub‐unit of alkaline phosphatase. Pure chick intestinal alkaline phosphatase was prepared and antibodies raised against it in rabbits. This enzyme and the brush‐border protein behaved identically on gel electrophoresis, and in terms of their phosphorylating activity and enzyme activity whether as the enzymically active dimeric form or when reduced to the sub‐unit form. The anti‐(alkaline phosphatase) reacted with the protein into which the incorporation of leucine was stimulated by 1,25‐dihydroxycholecalciferol.
Biosynthesis of Escherichia coli Braun's Lipoprotein Precursors in vitro and Binding to Membrane Vesicles
Authors: PIOVANT Michel , LAZDUNSKI Claude
Published: March 2005

A cell‐free system only programmed by endogenous mRNA present in a 30000 × g supernatant fraction is described in which 15‐40 % of the material produced was constituted by two lipoprotein precursors. These two polypeptides (a) are immunologically related, (b) contain the signal peptide and (c) apparently differ by the presence in one of them of a substituent on the‐SH group of the single cysteine residue. The nature of this substituent could not be determined but no evidence was obtained that it could be a glyceride. No processing of any of these prolipoprotein forms could be demonstrated by addition of either Triton X‐100 or membrane vesicles. A similar binding of both precursors to inverted membrane vesicles was observed when they were provided either co‐translationally or post‐translationally and this reaction occurred without the help of major membrane compounds.