bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetics and equilibrium of enzymatic synthesis of peptides in aqueous/organic biphasic systems
Authors: NAKANISHI Kazuhiro , KIMURA Yukitaka , MATSUNO Ryuichi 
Published: March 2005

We studied kinetics and the equilibrium relationship for the thermolysin‐catalyzed synthesis of N‐(benzyloxy‐carbonyl)‐l‐aspartyl‐l‐phenylalanine methyl ester (Z‐Asp‐PheOMe) from N‐(benzyloxycarbonyl)‐l‐aspartic acid (Z‐Asp) and l‐phenylalanine methyl ester (PheOMe) in an aqueous‐organic biphasic system. This is a model reaction giving a condensation product with dissociating groups. The kinetics for the synthesis of Z‐Asp‐PheOMe in aqueous solution saturated with ethyl acetate was expressed by a rate equation for the rapid‐equilibrium random bireactant mechanism, and the reverse hydrolysis reaction was zero‐order with respect to Z‐Asp‐PheOMe concentration. The courses of synthesis of Z‐Asp‐PheOMe in the biphasic system were well explained, by the rate equations obtained for the aqueous solution and by the partition of substrate and condensation product between the both phases. The rate of synthesis in the biphasic system was much lower than in aqueous solution due to the unfavorable partition of PheOMe in the aqueous phase.The equation for the equilibrium yield of Z‐Asp‐PheOMe in the biphasic system was derived assuming that only the non‐ionized forms of the substrate and condensation product exist in the organic phase. It was found theoretically and experimentally that the yield of Z‐Asp‐PheOMe is maximum at the aqueous‐phase pH of around 5, lower than for synthesis in aqueous solution. The effect of the organic solvent on the rate and equilibrium for the synthesis of Z‐Asp‐PheOMe could be explained by the variation in the partition coefficient.The effect of the partitioning of substrate on the aqueous‐phase pH change was also shown.
Purification, subunit structure and pharmacological effects on cardiac and smooth muscle cells of a polypeptide toxin isolated from the marine snail Conus tessulatus
Authors: SCHWEITZ Hugues , RENAUD Jean‐François , RANDIMBIVOLOLONA Nicole , PRÉAU Catherine , SCHMID Annie , ROMEY Georges , RAKOTOVAO Lala , LAZDUNSKI Michel 
Published: March 2005

The most active component in smooth muscle contraction, isolated from the whole venom of the marine snail Conus tessulatus, has a molecular mass of about 55 kDa. The toxin protein, tessulatus toxin, appeared to be constituted by two distinct polypeptide bands of 26 kDa and 29 kDa. The pure toxin caused a marked contraction of both guinea‐pig ileum and rabbit aorta at nanomolar concentrations. Tessulatus‐toxin‐induced contraction was indirectly prevented by classical inhibitors of the voltage‐dependent Ca2+ channel. Tessulatus toxin caused a large increase in the initial rate of 45Ca2+ uptake by cardiac cells. This uptake was insensitive to Ca2+ channel blockers at concentrations 100–1000 fold higher than those known to block voltage‐dependent Ca2+ channels in these cells. Voltage clamp experiments have confirmed that tessulatus toxin was not directly active on the Ca2+ current. Tessulatus‐toxin‐stimulated 45Ca2+ influx was inhibited by dichlorobenzamil and suppressed when Na+ was substituted by Li+, indicating that the toxin acted via activation of the Na+/Ca2+ exchange system in cardiac cells. Activation by tessulatus toxin of the Na+/Ca2+ exchange system occurred via a toxin‐stimulated Na+ entry into cardiac cells and was observed in the same range of toxin concentration which produced 45Ca2+ entry. The Na+ entry system that was activated by tessulatus toxin was insensitive to classic inhibitors of known Na+ entry systems in cardiac cells. Possible mechanisms by which tessulatus toxin induced Na+ entry into cardiac cells and contractions in smooth muscles are discussed. Tessulatus toxin is cytotoxic when used at high concentrations.
Phosphorus‐31 nuclear‐magnetic‐resonance study of phosphorylated metabolites compartmentation, intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver
Authors: DESMOULIN Franck , COZZONE Patrick J. , CANIONI Paul 
Published: March 2005

A quantitative analysis of the phosphorus‐31 NMR spectra of excised perfused rat liver has been carried out at 80.9 MHz using a 30‐mm sample cell.

The results indicate that in liver from fed rats, all intracellular ATP is detected by NMR. In contrast, only the cytosolic fractions of Pi and ADP can be observed as indicated by careful analysis of spectra obtained from perchloric acid liver extracts and intact liver under valinomycin perfusion. In well‐oxygenated perfused liver the ATP concentration is 7.4 mM. Values of 5.3 mM and 0.9 mM are found respectively for Pi and ADP concentrations in the cytosolic compartment. Cytosolic pH value (pHi) is 7.25±0.05 and free magnesium concentration 0.5 mM.
Addition of 70 mM (0.4%) ethanol to the perfusate of a fed rat liver induces 25% and 38% reduction of ATP and Pi levels, respectively. A large amount of sn‐glycerol 3‐phosphate is synthesized (up to 11 mM) in the cytosol. After ethanol withdrawal, a large overshoot in cytosolic Pi is observed, which is indicative of a net uptake of Pi across the plasma membrane that occured during ethanol oxidation. No significant pH variation is observed during ethanol infusion.
In perfused liver ofrats subjected to 48‐h fasts, the concentrations of cytosolic phosphorylated metabolities are 5.3 mM, 0.8 mM and 11.5 mM for ATP, ADP and Pi, respectively. The perfusion of the liver with 70 mM ethanol does not change the adenine nucleotide levels, while the Pi content is decreased by 10%.
During a 4‐min hypoxia, induced by reducing the perfusion flow rate from 12 ml to 3 ml min−1 (100 g body weight)−1, ATP concentration decreases to 5.8 mM in the fed rat liver. Cytosolic Pi and ADP increase to 8.7 mM and 1.6 mM, respectively. The cytosolic pH evolves to more acidic values and reaches 7.02±0.05 at the end of the 4‐min hypoxic period.
Role of cytosolic free calcium and phospholipase C in leukotrien‐B4‐stimulated secretion in human neutrophils
Authors: LEW P. Daniel , MONOD Antoinette , WALDVOGEL Francis A. , POZZAN Tullio 
Published: March 2005

Various leukotriene analogues were tested for their capacity to raise the cytosolic free calcium concentration, [Ca2+]i, and to stimulate exocytosis in human neutrophils. Their order of potency for both parameters was LTB4 > the stereochemical isomer of LTB4, (5S, 12S)‐LTB4 ≫ the sulphidopeptides LTD4, LTC4. The correlation between [Ca2+]i and secretion indicates that an increase of [Ca2+]i above a threshold level of about 300 nM is necessary for stimulating secretion with LTB4. This threshold is about an order of magnitude higher than that required for the chemotactic peptide formyl‐methionyl‐leucyl‐phenylalanine (fMet‐Leu‐Phe).The increase in [Ca2+]i elicited by LTB4 was unaffected by increasing cellular cAMP, while secretion was completely inhibited. These results indicate, that similar to fMet‐Leu‐Phe, leukotrienes generate other signals in addition to [Ca2+]i elevations. Contrary to previous claims, leukotrienes stimulate polyphosphoinositide hydrolysis, as indicated by the increase in [3H]inositol trisphosphate, InsP3, observed upon stimulation of myo‐[3H]inositol‐labelled neutrophils with LTB4 or (5S‐12S)‐LTB4. The two InsP3 isomers [Ins(1,4,5)P3 and Ins(1,3,4P3] were separated by high‐pressure liquid chromatographed and, as reported for other cell types, the formation of Ins(1,4,5)P3 precedes that of Ins(1,3,4)P3. Maximal stimulatory doses of LTB4 or (5S, 12S)‐LTB4 produce about 50% the amount of InsP3 generated by equimolar concentrations of fMet‐Leu‐Phe.The present observation suggest that, though the transmembrane signalling systems activated by LTB4 and fMet‐Leu‐Phe are the same, the different efficacy of these two agonists at stimulating neutrophil functions is due, at least in part, to a different degree of activation of phospholipase C.
Purification and characterization of rat liver glutaminase
Authors: HEINI Hans G. , GEBHARDT Rolf , BRECHT Andreas , MECKE Dieter 
Published: March 2005

Phosphate‐dependent glutaminase (EC 3.5.1.2) from livers of starved rats was purified about 400‐fold to near homogeneity. The specific activity of the final pool was more than 30 U/mg protein.For the rapid quantification of the enzyme activity a simple and sensitive assay, based on the determination of the produced ammonia with an o‐phthalaldehyde reagent, was developed which avoids massive dilution of the samples. The enzyme preparation involved extraction of the enzyme from sonified isolated mitochondria after treatment with a brief hypotonic shock followed by ammonium sulphate precipitation, ion‐exchange and hydroxyapatite chromatography. A major improvement was the stabilization of the enzyme by chymostatin protecting it from degradation by a protease of presumably lysosomal origin. In the presence of chymostatin or leupeptin the half‐life of glutaminase in a crude mitochondrial preparation subsequent to mild treatment with digitonin could be increased to more than 200 h.The relative molecular mass of the protein (Mr170 500) was estimated by sucrose gradient ultracentrifugation. The molecular mass of the subunits (Mr 57000) was determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These results suggest a protein composed of three subunits of identical molecular mass. The molecular data clearly differentiate liver glutaminase from the phosphate‐dependent glutaminase present in kidney.
Subcellular location of enzymes involved in the N‐glycosylation and processing of asparagine‐linked oligosaccharides in Saccharomyces cerevisiae
Authors: TILLMANN Ulrich , GÜNTHER Roland , SCHWEDEN Jürgen , BAUSE Ernst 
Published: March 2005

A particulate translation system isolated from the yeast Saccharomyces cerevisiae was shown to translate faithfully in‐vitro‐transcribed mRNA coding for a mating hormone precursor (prepro‐α‐factor mRNA) and to N‐glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles. Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn‐Xaa‐Thr, supporting the view that the glycan chains are N‐glycosidically attached to the prepro‐α‐factor polypeptide. The accumulation in the presence of acceptor peptides of a membrane‐specific, unglycosylated translation product (pp‐α‐Fo) differing in molecular mass from a cytosolically located, protease‐K‐sensitive α‐factor polypeptide (pp‐α‐Fcyt) by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation. This conclusion is supported by the observation that the multiply glycosylated α‐factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product pp‐α‐Fcyt but larger than the membrane‐specific pp‐α‐Fo.Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors (e.g. 1‐deoxynojirimycin, N‐methyl‐1‐deoxynojirimyin and 1‐deoxymannojirimycin) indicate that the N‐linked oligosaccharide chains of the glycosylated prepro‐α‐factor species are extensively processed under the in vitro conditions of translation. From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation‐competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase II and a trimming mannosidase, which process the prepro‐α‐factor glycans down to the (Man)8(GlcNAc)2 stage. Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes.
Purification and biochemical characterization of the complete structure of a proteolytically modified β‐2‐microglobulin with biological activity
Authors: NISSEN Mogens Holst , THIM Lars , CHRISTENSEN Mogens 
Published: March 2005

A modified form of β‐2‐microglobulin (β‐2‐m) has previously been described to be present in serum from patients suffering from autoimmune diseases, acquiried immune deficiency syndrome and small‐cell lung cancer [Plesner, T. and Wiik, A. (1979) Scand. J. Immunol. 9, 247–254; Bhalla et al. (1985) Clin. Chem. 31, 1411–1412; Nissen et al. (1984) Clin. Chim. Acta 141, 41–50]. In the present study we describe the purification and characterization of this modified human serum β‐2‐m from patients with small‐cell lung cancer. Purified urinary β‐2‐m was added to the serum samples incubated at 20°C for five days to obtain a higher yield of modified β‐2‐m (m‐β‐2‐m). m‐β‐2‐m was then purified from serum by gel filtration followed by chromatofocusing of the fractions containing β‐2‐m.m‐β‐2‐m was found to have an apparent molecular mass of 15 kDa and a pI of 5.3 when analyzed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and analytical isoelectric focusing respectively. Amino acid analysis of m‐β‐2‐m revealed that the protein is missing one lysine residue compared to the composition deduced from the cDNA sequence of β‐2‐m. Amino acid sequence analysis showed that m‐β‐2‐m consists of two polypeptide chains produced by a proteolytic cleavage of β‐2‐m in the disulphide loop. After reduction and alkylation of m‐β‐2‐m the two chains were separated by reverse‐phase high‐pressure liquid chromatography. By amino acid sequencing, amino acid residues 1–56 and 59–99 were identified in the A and B chains respectively. By comparision of the amino acid composition of m‐β‐2‐m with the known sequence of β‐2‐m it was possible to deduce the existence of a Ser‐57 in the A chain. Thus proteolytic clevage of β‐2‐m in the intrachain disulphide loop releases the amino acid Lys‐58, which results in a modified form of β‐2‐m with a molecular mass of 11 620 Da as determined by amino acid analysis.
Inactivation of dihydropteridine reductase (human brain) by platinum (II) complexes
Authors: ARMAREGO Wilfred L. F. , OHNISHI Akiko 
Published: March 2005

Potassium tetrachloroplatinate (K2PtCl4) inactivates dihydropteridine reductase from human brain in a time‐dependent and irreversible manner. The inactivation has been followed by measuring enzyme activity and fluorescence changes. The enzyme is completely protected from inactivation by NADH, the pterin cofactor [quinonoid 6‐methyl‐7,8‐dihydro(6H)pterin] and dithiothreitol. Evidence is presented that K2PtCl4 reacts at the active site and that (a) thiol group(s) is involved in, or is masked by, this reaction.K2PtCl4 is a stronger inhibitor of human brain dihydropteridine reductase than cis‐ and trans‐ diaminodichloroplatinum, cis‐dichloro[ethylenediamine]platinum and K4Fe(CN)6, whereas H2PtCl6 is considerably weaker and (Ph3P)3RhCl is inactive.
Free apolipoproteins A‐I and A‐IV present in human plasma displace high‐density lipoprotein on cultured bovine aortic endothelial cells
Authors: SAVION Naphtali , GAMLIEL Aviva , TAUBER Jean‐Pierre , GOSPODAROWICZ Denis 
Published: March 2005

Adult bovine aortic endothelial (ABAE) cells, exposed to serum‐free medium, specifically bind 125I‐labeled human high‐density lipoprotein (125I‐HDL). Addition of human lipoprotein‐deficient serum (LPDS) reduces the specific binding of 125I‐HDL in a concentration‐dependent manner, such that LPDS at a concentration of 6 mg protein/ml almost completely inhibits the specific binding of 125I‐HDL. ABAE cultures exposed to 125I‐labeled LPDS (125I‐LPDS) specifically bind two peptides, which appear as minor iodinated components in 125I‐LPDS. The binding of these two components is abolished in the presence of excess amounts of unlabeled LPDS or HDL. Preincubation of ABAE cells with 25‐hydroxycholesterol (25‐HC) results in an increase in the binding of the two 125I‐LPDS components, similar to the increase observed in 125I‐HDL binding in the presence of 25‐HC. These two LPDS components comigrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‐PAGE) with apolipoproteins A‐I and A‐IV of molecular masses 28 kDa and 43 kDa respectively. Furthermore, these two proteins were transfered from the SDS gel to nitrocellulose paper and interacted specifically with anti‐(A‐I) and anti‐(A‐IV) sera respectively.When ABAE cultures, pretreated with 25‐HC in the presence of LPDS, are subjected to cell‐surface iodination, the A‐IV appears as one of the major proteins on the cell surface accessible to iodination. The interaction of A‐IV with the cell surface of 25‐HC‐treated cells is not specific to ABAE cells and appears also in human skin fibroblasts.Analysis of the relative amounts of various apolipoproteins in the 125I‐HDL bound to ABAE cells demonstrates a decrease in the relative amount of iodinated A‐II concomitant with increase in the relative amounts of the other iodinated apolipoproteins, when compared to the composition of the native 125I‐HDL. These changes are similar whether the binding is done in the presence or absence of LPDS. It indicates that the decrease in 125I‐HDL binding in the presence of LPDS is not due to displacement of the iodinated apolipoproteins A‐I and A‐IV in the 125I‐HDL by unlabeled A‐I and A‐IV present in LPDS.The results indicate that free apolipoproteins A‐I and A‐IV, present in LPDS, can displace HDL on the cell surface of ABAE cells. Thus, free A‐I and A‐IV, present in plasma, control the binding of HDL to endothelial cells and may regulate the process of cholesterol removal from the cells performed by HDL.
Estrogen stimulates growth of mammary tumor cells ZR‐75 without activation of S6 kinase and S6 phosphorylation
Authors: NOVAK‐HOFER Ilse , KÜNG Willy , FABBRO Doriano , EPPENBERGER Urs 
Published: March 2005

Growth of the human mammary tumor cell line ZR‐75‐1 is stimulated by epidermal growth factor (EGF) and α‐type transforming growth factor (αTGF), as well as by estradiol (E2). The role of activation of S6 kinase and S6 phosphorylation in the EGF(αTGF)‐induced and E2‐induced growth was investigated. Maximal effects on growth are observed at 10nM EGF or αTGF. EGF as well as αTGF treatment of serum‐starved cells leads to rapid activation of S6 kinase; the activity is increased about tenfold after 30min of EGF treatment and declines with the time reaching about 25% of the maximal activity after 2 h of EGF treatment. Similar to the growth response, S6 kinase is activated at lower doses of EGF than αTGF and shows a maximal response at 10 nM for both growth factors. In contrast to this finding the incubation of serum‐starved cells with E2 over a concentration range between 1 pM and 10 nM and times from 30 min to 4 h does not lead to increased S6 kinase activity. On investigating whether this lack of response to E2 does not. When S6 phorphorylation is monitored over times from 1 h to 6 h, it is observed that EGF leads to increased S6 phorphorylation, whereas E2 does not. The rate of onset of protein synthesis in the first 2 h of stimulation, when EGF‐induced S6 phosphorylation is maximal, is more rapid with EGF than with E2.The results suggest that different pathway are involved in E2‐induced and EGF(αTGF)‐induced proliferation.
Proton translocation by the H+ ‐ATPase of mitochondria
Authors: ZANOTTI Franco , GUERRIERI Ferruccio , CHE Yan Wu , SCARFÒ Rosanna , PAPA Sergio 
Published: March 2005

A study is presented on the effect of chemical modification of thiol groups on proton conduction by the H+‐ATPase complex in ‘inside out’ submitochondrial particles, before and after removal of the F1 moiety, and by F0 liposomes.The results obtained show that modification with monofunctional reagents [N‐ethylmaleimide, 2,2′‐dithiobispyridine, mersalyl and N‐(7‐dimethylamino‐4‐methyl‐coumarinyl)‐maleimide] of thiol residues in membrane integral proteins of F0 results in inhibition of proton conduction. Comparison of the inhibitory effects with the binding of [14C]N‐ethylmaleimide to the various F0 polypeptides indicates that the inhibition of proton conduction by thiol reagents was correlated with modification of the 25‐kDa, 11‐kDa and 9‐kDa (N,N′‐dicyclohexylcarbodiimide‐binding protein) proteins. Involvement of the last component is supported by the observation that modification by thiol reagents depressed the binding of N,N′‐decyclo[14C]hexylcarbodiimide to the 9‐kDa protein.
A plasma‐membrane associated ATPase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius
Authors: LÜBBEN Mathias , SCHÄFER Günter 
Published: March 2005

Thermoacidophilic archaebacteria have gained much interest because of their phylogenetic distance to eubacteria and eukaryotes and also because of their unique living conditions. Investigation of the energy‐converting system therefore offers a key for understanding the evolutionary position and environmental adaptation of these unusual bacteria.A plasma‐membrane‐associated adenosine triphosphatase with specific activities of 0.3–0.6 μmol min−1 (mg protein)−1 has been detected in the thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 639). The enzyme exhibits two optima at pH 5.5 and 8.0, sulfite activation leads to only one optimum at pH 6.25. In the presence of the divalent cations Mg2+ or Mn2+ it hydrolyzes ATP with highest reactivity and also other purine and pyrimidine nucleotides, but not ADP and pyrophosphate. A specific stimulation by monovalent cations is not observed. The ATPase activity is not inhibited by N, N′‐dicyclohexylcarbodiimide, azide or vanadate, but it is by the vacuolar ATPase inhibitor nitrate with an [I]50 of 8 mM. Linear Arrhenius plots up to 75°C reflect pronounced adaptation to the hot environment of the archaebacterium.The solubilized ATPase as localized by activity staining in non‐denaturating gels and further analyzed by sodium dodecyl sulfate electrophoresis is composed of two major polypeptides of 65 and 51 kDa reminiscent of the α and β subunits of eubacterial and eukaryotic F0F1‐ATPases. The ATPase is suggested as a probable candidates for a reversibly acting ATP synthase responsible for oxidative phosphorylation found in Sulfolobus acidocaldarius.
Biosynthesis of poly(galactosylglycerol phosphate) in Bacillus coagulans
Authors: YOKOYAMA Kohei , ARAKI Yoshio , ITO Eiji 
Published: March 2005

The pathway for the de novo synthesis of a teichoic acid, poly(galactosylglycerol phosphate), in Bacillus coagulans AHU 1366 was studied by means of characterization and stepwise conversion of lipid‐linked intermediates. Incubation of membranes with UDP‐N‐acetylglucosamine and UDP‐glucose yielded a disaccharidelinked polyprenylpyrophosphate, whose sugar moiety was characterized as glucosyl(β1→4)N‐acetylglucosamine (Glc‐GlcNAc). By incubation with membranes and CDP‐glycerol, Glc‐GlcNAc‐PP‐prenol was converted to a series of glycolipids characterized as (Gro‐P)1–6‐Glc‐GlcNAc‐PP‐prenol (Gro = glycerol).Glc‐[14C]GlcNAc‐PP‐prenol was converted to polymer by incubation with membranes, CDP‐glycerol and UDP‐galactose. Smith degradation of the polymer gave two radioactive fragments corresponding to (Gro‐P)3‐Glc‐GlcNAc and (Gro‐P)4‐Glc‐GlcNAc. These results, together with data on gel chromatography of radioactive polymer synthesized from UDP‐[3H]galactose, CDP‐glycerol and Glc‐[14C]GlcNAc‐PP‐prenol, led to the conclusion that in this strain poly(galactosylglycerol phosphate) is probably synthesized through the following pathway: GlcNAc‐PP‐prenol → Glc‐GlcNAc‐PP‐prenol → (Gro P)3–4‐Glc‐GlcNAc‐PP‐prenol → (Gro‐P‐Gal)n‐(Gro‐P)3–4‐Glc‐GlcNAc‐PP‐prenol → (Gro‐P‐Gal)n‐(Gro‐P) 3–4‐Glc‐GlcNAc‐P‐peptidoglycan complex.