bluuebunny/crossref_metadata_2025 huggingface dataset:
Fatty‐acid‐induced activation of NADPH oxidase in plasma membranes of human neutrophils depends on neutrophil cytosol and is potentiated by stable guanine nucleotides
Authors: SEIFERT Roland , SCHULTZ Günter
Published: March 2005

Both cis and trans unsaturated fatty acids and sodium dodecyl sulfate activated NADPH oxidase in plasma membranes of human neutrophils in the presence of neutrophil cytosol. In contrast, 5,8,11,14‐icosatetraynoic acid, saturated fatty acids, esters, peroxides and 4β‐phorbol 12‐myristate 13‐acetate, a potent activator of protein kinase C, were inactive. 5,8,11,14‐icosatetraynoic acid inhibited superoxide formation elicited by fatty acids. Guanosine 5′[γ‐thio]triphosphate (GTP[γS]), a potent activator of guanine‐nucleotide‐binding proteins (N‐proteins) enhanced superoxide formation elicited by fatty acids up to fourfold, supporting our previous suggestion that NADPH oxidase is regulated by an N‐protein [Seifert, R. et al. (1986) FEBS Lett. 205, 161‐165]. Cytosols from various tissues, soybean lipoxygenase and protein kinase C, purified from chicken stomach, did not substitute neutrophil cytosol. The activity of neutrophil cytosol was destroyed by heating at 95°C. Superoxide formation was not affected by the inhibitor of protein kinase C 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H‐7). Removal of cytosolic ATP by preincubation with hexokinase and glucose, dialysis of neutrophil cytosol or chelation of calcium with EGTA did not abolish the stimulatory effect of arachidonic acid and GTP[γS]. Thus, the cytosolic cofactor appears to be a neutrophil‐specific and heat‐labile protein, which is neither a lipoxygenase nor protein kinase C.
Characterization of six toxins from the venom of the Moroccan scorpion Buthus occitanus mardochei
Authors: VARGAS Orietta , MARTIN Marie France , ROCHAT Hervé
Published: March 2005

When the venom of the Moroccan scorpion Buthus occitanus mardochei was submitted to a combination of several chromatographic steps (including gel‐filtration and ion‐exchange chromatographies), seven proteins were obtained, six being lethal to mice. These proteins have been characterized by their chemical, immunological and toxic properties. The amino acid sequence (66 residues) of Bom III, the most noteworthy toxin of the venom as for its amino acid composition, is proposed following automatic sequencing of the reduced and S‐methylated protein and of chymotryptic peptides. It was obvious that this sequence is somewhat different from those of toxins belonging to the same structural and immunological group (Bom III was found to be immunologically related to Buthus occitanus tunetanus toxins I and II which both share with it 56% of homology. Furthermore, Bom III was found to be unable to compete (as does Bot I) with toxin II of Androctonus australis Hector (an α‐type toxin) for neurotoxin binding site 3 on the sodium channel of rat brain synaptosomes. Bom III was also unable to compete with toxin II of Centruroīdes suffusus suffusus (a β‐type toxin) to neurotoxin binding site 4 of the same channel.
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.
Quasi‐irreversibility in the unfolding‐refolding transition of phosphoglycerate kinase induced by guanidine hydrochloride
Authors: MITRAKI Anna , BETTON Jean‐Michel , DESMADRIL Michel , YON Jeannine M.
Published: March 2005

The reversibility of the unfolding‐refolding transition of horse muscle phosphoglycerate kinase, induced by guanidine hydrochloride (Gdn · HCl), was studied using the regain of enzyme activity as a probe of the native structure. An irreversibility in the reactivation process was detected when the protein was incubated in a critical concentration of denaturant (0.7 ± 0.1 M Gdn · HCl). This apparent irreversibility was observed for the unfolding process (N → D) as well as for the refolding process (D → N). The formation of the trough followed biphasic kinetics at 23°C, the first phase obeying a first‐order reaction corresponded to an isomerization of an intermediate; the second phase, protein‐concentration‐dependent, was suppressed by lowering the temperature to 4°C. The structural properties of the inactive species were studied; all the β structures were recovered, but about 29% of the helical structures remained unfolded, and two SH groups were buried. Simulated kinetics were compared with the experimental results and were used to extend the minimum folding scheme previously proposed from equilibrium and kinetic studies [Betton et al. (1984) Biochemistry 23, 6654–6661; Betton et al. (1985) Biochemistry 24, 4570–4577]. The intermediates trapped under these conditions were structured but devoid of catalytic activity. Taking into account the structural properties of these species, the nature of the interactions involved in their formation and stabilization is discussed.
Reversible binding of Salmonella typhimurium lipopolysaccharides by immobilized protamine
Authors: HELANDER Ilkka M. , VAARA Martti
Published: March 2005

The ability of agarose‐linked protamine to bind Salmonella typhimurium lipopolysaccharides was investigated. Radioactively labelled lipopolysaccharides were isolated both from a smooth strain (SH6749, labelled with [14C]galactose) and from a rough strain (SH5014, lipopolysaccharide chemotype Rb2, labelled with [3H]acetate). From 50‐μg samples of the lipopolysaccharides, protamine‐agarose columns bound 99.5–99.9% of the input radioactivity. The binding efficacy was not affected by pH in the range from 3.7 to 10.5. Maximal binding capacity of protamine‐agarose for highly soluble (triethylamine form) lipopolysaccharide of SH5014 was estimated to be 13.5 mg/ml packed adsorbent. The bound lipopolysaccharides could be totally released from the columns and recovered by elution with the anionic detergent sodium deoxycholate, or with 0.5 M NaCl in the presence of the uncharged detergent Triton X‐100. By analysis in sodium dodecyl sulfate/polyacrylamide gels, the macromolecular quality of the recovered lipopolysaccharide was shown to be identical to that of the original. Protamine‐agarose chromatography can thus be applied to purify lipopolysaccharide preparations, and to separate as well as concentrate lipopolysaccharides from dilute solutions without altering their composition. This application was challenged with water as well as insulin solution experimentally contaminated with radiolabelled lipopolysaccharide. While the insulin protein did not bind to the protamine‐agarose, the contaminating lipopolysaccharide was effectively trapped.
Modulation of human neutrophil function by C‐reactive protein
Authors: BUCHTA Richard , FRIDKIN Mati , PONTET Michel , CONTESSI Elisa , SCAGGIANTE Bruna , ROMEO Domenico
Published: March 2005

Investigation of the effect of C‐reactive protein (CRP), an acute‐phase reactant, on the functional capacities of human neutrophils was carried out as the basis for elucidating the biological function of C‐reactive protein. An initial stimulation at low concentrations, followed by inhibition of superoxide production, and secretion of vitamin‐B12‐binding protein in the presence of two stimulants, phorbol myristate acetate and concanavalin A, and of neutrophil chemotaxis with increasing concentration of CRP was observed. Correlation between modulation of cell function, at least at relatively high CRP concentrations (> 50 μg/ml) and an increase in the intracellular level of cAMP is suggested. CRP was also found to enhance neutrophil phagocytosis of particles not containing phosphorylcholine, the native CRP ligand. The proposed role of CRP in neutrophil function is one of regulation and as a negative feedback for potential cytotoxic neutrophil functions.
Detection and quantification of peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidases
Authors: PLUMMER Thomas H. , PHELAN Arthur W. , TARENTINO Anthony L.
Published: March 2005

A detailed study of the oligosaccharide specificity of the almond enzyme, peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase A, was undertaken by comparing the rate of release of intact oligosaccharide chains from defined glycopeptides of all significant classes. The oligosaccharide of a trisialo‐triantennary pentaglycopeptide from fetuin was released at the highest rate. A procedure was developed for the isolation of this glycopeptide in high yield from 5 g fetuin. Sequence analysis established the structure as Leu‐Ala‐Asn(CHO)‐Cys‐Ser. The Cys(Cm) and the Cys(Ae) derivatives of the glycopeptide were reacted with 4‐(dimethylamino)‐azobenzene‐4′‐sulfonyl (dabsyl) chloride to yield a monosubstituted and a disubstituted glycopeptide respectively. This chromophore confers high sensitivity at 436 nm on a pentapeptide backbone having minimal bonds for protease cleavage. A procedure was developed wherein these dabsyl derivatives were used in a high‐performance liquid chromatography assay. The dabsyl‐pentapeptide was retarded significantly from the dabsyl‐glycopeptide and provided a sensitive method (1–2 nmol) of detection of peptide‐N4‐(N‐acetylglucosaminyl)asparagine amidase activity. Enzyme was detected in crude extracts of all eight séed sources surveyed. The enzyme from Pisum sativum was partially purified and its properties were compared with the correponding enzyme from almonds.
Biogenesis of very‐low‐density lipoproteins in rat liver
Authors: WONG Laurence , PINO Richard M.
Published: March 2005

The hepatic subcellular distribution of apolipoprotein B (apo B) was studied quantitatively by using an enzyme immunoassay developed for apo B and by immunoadsorption‐precipitation of [3H]leucine‐labelled apo B. Over 50% (of 0.59 μg/mg protein) of the apo B was located in the microsomal fraction. Further subfractionation of the microsomes revealed that 47% of the microsomal apo B was in the Golgi apparatus, while another 43% was associated with the rough endoplasmic reticulum. The smooth endoplasmic reticulum accounted for only 4% of the total. When rat livers were labelled with [3H]leucine for 10 min, the rough endoplasmic reticulum accounted for 80% of the total immunoadsorbed precipitable apo B radioactivity while the smooth accounted for 20%, with no contribution from the Golgi. However, only 8.7% of the total radioactive immunoadsorbed precipitable apo B was lipoprotein‐associated, the remainder being membrane‐bound. Lipoprotein‐associated apo B radioactivity in the smooth endoplasmic reticulum accounted for 40% with the rough contribution attributed at 50% and the Golgi at 9%. We concluded that (a) there are two major pools of apo B in rat liver microsomes; (b) although the apo B mass may be negligible in the smooth endoplasmic reticulum, the latter does play a role in lipoprotein biogenesis. The possible function of apo B associated with membranes of the microsomes is also discussed.
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.
A nuclear mutation affecting mitochondrial transcription in Saccharomyces cerevisiae
Authors: LISOWSKY Thomas , SCHWEIZER Eckhart , MICHAELIS Georg
Published: March 2005

Mitochondrial transcription was studied in a nuclear temperature‐sensitive pet mutant of Saccharomyces cerevisiae. The mitochondrial RNA levels in vivo and the in vitro transcriptional activities of isolated mitochondria were analysed. In comparison to the wild‐type an overall reduction of mitochondrial gene expression together with a changed expression pattern was observed for the mutant, indicating a defect in mitochondrial RNA synthesis. These findings were supported by studies with a purified DNA‐protein complex from yeast mitochondria. This complex was able to synthesize ribosomal and messenger RNAs in an in vitro system. Proteins from wild‐type and mutant transcription complexes were tested for their DNA‐binding abilities; one of the proteins identified in the wild type had either lost this ability or was absent in the mutant.
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.
Isomeric equilibria in complexes of adenosine 5′‐triphosphate with divalent metal ions
Authors: SIGEL Helmut
Published: March 2005

Solution structures of M(ATP)2− complexes are reviewed. First the self‐stacking properties of ATP4− and M(ATP)2− are shortly described. It is emphasized that for an evaluation of solution structures of M(ATP)2− complexes only results from diluted solutions (below 1 mM) should be used. Next, a comprehensive set of stability data obtained under such conditions from potentiometric pH titrations is summarized for the complexes of Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+ with ATP, and for comparison also with pyrimidine nucleoside 5′‐triphosphates (YTPs), i.e. CTP, UTP and TTP. The stabilities for the M(ATP)2− complexes are mostly larger than those for the corresponding M(YTP)2− species; this increased stability results from the metal ion back‐binding to the base residue in M(ATP)2−, i.e. macrochelates are formed. Detailed analysis of the stability data allows calculation of the percentage of the closed form for the several M(ATP)2− complexes: back‐binding is most pronounced in Cu(ATP)2− (67 ± 2%), remarkable in Zn(ATP)2− (28 ± 7%), and not observable for Ca(ATP)2− (2 ± 6%). Comparison of these results with those from 1H‐NMR and ultraviolet spectrophotometric studies allows the conclusion that two types of base back‐bound macrochelates are formed: one with a direct, i.e. innersphere, M2+/N‐7 coordination, and one with a water molecule between the metal ion and N‐7, i.e. an outersphere interaction occurs [e.g. to about 10% in Mg(ATP)2−] through hydrogen bonding of a coordinated water to N‐7. The formation degree of both forms of these closed isomers is quantified. The biological implications of these results are indicated and the versatility of ATP as a ligand is discussed by summarizing pertinent examples.
Interaction of phalloidin with chemically modified actin
Authors: MIKI Masao , BARDEN Julian A. , dos REMEDIOS Cristobal G. , PHILLIPS Leonidas , HAMBLY Brett D.
Published: March 2005

Modification of Tyr‐69 with tetranitromethane impairs the polymerizability of actin in accordance with the previous report [Lehrer, S. S. and Elzinga, M. (1972) Fed. Proc. 31, 502]. Phalloidin induces this chemically modified actin to form the same characteristic helical thread‐like structure as normal F‐actin. The filaments bind myosin heads and activate the myosin ATPase activity as effectively as normal F‐actin. When a dansyl group is introduced at the same point [Chantler, P. D. and Gratzer, W. B. (1975) Eur. J. Biochem. 60, 67–72], phalloidin still induces the polymerization. The filaments bind myosin heads and activate the myosin ATPase activity. These results indicate that Tyr‐69 is not directly involved in either an actin‐actin binding site or the myosin binding site on actin. Moreover, the results suggest that phalloidin binds to actin monomer in the presence of salt and its binding induces a conformational change in actin which is essential for polymerization, or that actin monomer fluctuates between in unpolymerizable and polymerizable form while phalloidin binds to actin only in the polymerizable form and its binding locks the conformation which causes the irreversible polymerization of actin.

Modification of Tyr‐53 with 5‐diazonium‐(1H)tetrazole blocks actin polymerization [Bender, N., Fasold, H., Kenmoku, A., Middelhoff, G. and Volk, K. E. (1976) Eur. J. Biochem. 64, 215–218]. Phalloidin is unable to induce the polymerization of this modified actin nor does it bind to it. Phalloidin does not induce the polymerization of the trypsin‐digested actin core. These results indicate that the site at which phalloidin binds is involved in polymerization and the probable conformational change involved in polymerization may be modulated through this site.
Coreconstitution of bacterial ATP synthase with monomeric bacteriorhodopsin into liposomes
Authors: WAGNER Norbert , GUTWEILER Matthias , PABST Rainer , DOSE Klaus
Published: March 2005

The conditions for coreconstitution of a bacterial ATP synthase and bacteriorhodopsin into lecithin liposomes and for light driven ATP synthesis have been optimized. A rate of maximally 280 nmol ATP min−1 mg ATP synthase−1 was achieved with monomerized bacteriorhodopsin compared with a rate of up to 45 nmol ATP min−1 mg−1 found for proteoliposomes containing bacteriorhodopsin in the form of purple membrane patches. The different rates are explained by the finding that monomeric bacteriorhodopsin is more homogeneously distributed among the liposomes than the purple membrane patches. The final activities depended on both the purification method for the two proteins and the coreconstitution procedure. Furthermore, the ratio (lipid to bacteriorhodopsin to ATP synthase) could be optimized. Light‐driven ATP synthesis depends also on the type of detergent used. The best result was obtained by deoxycholate. Also the relationship between proton translocation (by bacteriorhodopsin) and ATP synthesis activity was measured. A constant H+/ATP ratio was found at higher light intensities. This ratio increased strongly at lower light intensities.
Glucose‐6‐phosphate oxidation pathway in rat‐liver microsomal vesicles
Authors: HINO Yukinobu , ISHIO Sonoko , MINAKAMI Shigeki
Published: March 2005

The glucose‐6‐phosphate oxidation pathway present in microsomes was studied using intact microsomal membranes. The oxidation activity, which was measured by monitoring the formation of 14CO2 from [1‐14C]glucose 6‐phosphate, was greatly stimulated when azodicarboxylic acid bis(dimethylamide), methylene blue or cumene hydroperoxide was added to the assay mixture. Glutathione peroxidase and glutathione reductase are suggested to be involved in the oxidation reaction induced by these oxidizing reagents. We detected a significant activity of the glutathione reductase inherent to microsomes. The microsomal glutathione reductase is latent and requires detergent to reveal its activity. 4,4′‐Diisothiocyanostilbene 2,2′‐disulfonic acid (DIDS) inhibited the 14CO2 formation, but the inhibition was released by the addition of a detergent. Moreover, the inhibitory effect of DIDS was reversed by glucose 6‐phosphate but not by mannose 6‐phosphate. We conclude that the glucose‐6‐phosphate oxidation pathway in intact microsomes starts working under oxidative stress and that a transporter specific for glucose 6‐phosphate is involved in the reaction.
Chemical modification of charged amino acid moieties alters the electrophoretic mobilities of neurofilament subunits on SDS/polyacrylamide gels
Authors: GEORGES Elias , MUSHYNSKI Walter E.
Published: March 2005

The increase in the mobilities of neurofilament subunits on SDS‐PAGE after dephosphorylation was reversed upon boiling in urea or trifluoroacetylation of lysine ɛ‐amino groups. Trifluoroacetylation of native and dephosphorylated neurofilaments also resulted in an overall increase in the phosphorylation of the three subunits by the catalytic subunit of cyclic‐AMP‐dependent protein kinase. The gel‐electrophoretic mobility of neurofilament subunits was also shown to be influenced by carboxylic amino acid residues, as neutralization of these moieties by glycinamidation increased the mobilities of all three subunits on SDS‐PAGE. Neurofilament subunits that were both glycinamidated and dephosphorylated had apparent molecular masses of approximately 60 kDa, 112 kDa and 138 kDa. The major sites of these changes in the two largest subunits were shown to be the carboxy‐terminal tail domains, which are known to contain high percentages of glutamate. Since interspecies differences in the apparent molecular masses of neurofilament subunits were shown to persist after glycinamidation and dephosphorylation, they appear to be due to differences in polypeptide chain length, rather than glutamate content.
Separation, amino‐terminal sequence and cell‐free synthesis of the smallest subunit of sweet potato cytochrome c oxidase
Authors: NAKAGAWA Tsuyoshi , MAESHIMA Masayoshi , MUTO Hideyo , KAJIURA Hiroko , HATTORI Hiroyuki , ASAHI Tadashi
Published: March 2005

The smallest subunit (V) of sweet potato cytochrome c oxidase was separated into three polypeptides, Va, Vb and Vc with different molecular masses (7.4 kDa, 6.8 kDa and 6.2 kDa respectively) by highly resolving sodium dodecylsulfate polyacrylamide gel electrophoresis. Antibody against subunit V reacted specifically with the polypeptide Vc. When polyadenylated mRNA from sweet potato root tissue was translated in a wheat germ cell‐free system, the smallest subunit (Vc) of the polypeptides was synthesized to the same size as the mature form, which suggests that the mature subunit retains the signal for import into mitochondria. Within the N‐terminal first 25 amino acids there is a stretch of 16 non‐polar residues, periodically linked by basic residues, which might form an amphiphilic helix as the targeting signal.
Purification and characterization of a β‐glucosidase from Trichoderma reesei
Authors: CHIRICO William J. , BROWN Ross D.
Published: March 2005

A β‐glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The β‐glucosidase was purified using two successive DEAE‐Sephadex anion‐exchange chromatography steps, followed by SP‐Sephadex cation‐exchange chromatography and concanavalin‐A–agarose chromatography. Evidence for homogeneity is provided by polyacrylamide disc gel electrophoretic patterns, which show a single protein band.

Sedimentation equilibrium analysis yielded a molecular mass of 74.6 ± 2.4 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a single protein band with a molecular mass of 81.6 kDa. Thus, the enzyme appears to be a single, monomeric polypeptide. The β‐glucosidase is isoelectric at pH 8.5. The enzyme is rich in basic amino acids and contains few half‐cystine and methionine residues. The purified β‐glucosidase contains less than 1% by weight of neutral carbohydrate.

The β‐glucosidase catalyzes the hydrolysis of cellobiose, p‐nitrophenyl β‐d‐glucopyranoside and 4‐methyl‐umbelliferyl β‐d‐glucopyranoside; the values of V/Km for each substrate were determined to be 2.3 × 104, 6.9 × 105 and 2.9 × 106 M−1 s−1 respectively. The enzyme is optimally active from pH 4.5 to 5.0 and is labile at higher hydrogen ion concentrations. The β‐glucosidase has an unusually high affinity for d‐glucose (Ki= 700 μM). Comparison of inhibition constants for cello‐oligosaccharides suggests that the substrate‐binding region of the β‐glucosidase comprises multiple subsites.
Conformational transitions in the myosin head induced by temperature, nucleotide and actin
Authors: RȨDOWICZ Maria J. , SZILÁGYI Laszlo , STRZELECKA‐GOLASZEWSKA Hanna
Published: March 2005

Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment‐1 (S1) to that established for rabbit S1. The 97‐kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27‐kDa, 50‐kDa and 20‐kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49‐kDa and 20‐kDa peptides, and by a nucleotide‐promoted cleavage of the 29‐kDa peptide to a 24‐kDa fragment and the 49‐kDa peptide to a 44‐kDa fragment, analogous to the nucleotide‐promoted cleavage of the 27‐kDa and 50‐kDa fragments of rabbit S1 to the 22‐kDa and 45‐kDa peptides.

The same changes in the digestion patterns as those produced by the presence of nucleotide (ATP or its β, γ‐imido analog AdoPP[NH]P) at 25 °C were observed when the digestion was carried out at 0 °C in the absence of nucleotide. The low‐temperature‐induced changes were particularly well seen in the preparations from frog myosin. The presence of ATP or AdoPP[NH]P at 0 °C enhanced, whereas the complex formation with actin prevented, the low‐temperature‐induced changes. The results are consistent with there being two fundamental conformational states of the myosin head in an equilibrium that is dependent on the temperature, the nucleotide bound at the active site, and the presence or absence of actin.
A dual chromatin organization in the sperm of the bivalve mollusc Spisula solidissima
Authors: AUSIO J. , VAN HOLDE K. E.
Published: March 2005

Most of the DNA in the sperm of the bivalve mollusc, Spisula solidissima, is found to be associated with a specific high‐molecular‐mass, protamine‐like component, sharing features common both to protamines and to histones. We have found that this component coexists, in the mature sperm nucleus, with a complete set of histones, including an H1‐like histone. Such histones account for approximately 20% of the whole protein content in the sperm chromatin, the overall protein/DNA ratio (w/w) being 0.87. These data, together with micrococcal nuclease digestions in combination with salt fractionation, have allowed us to propose a structural model for this chromatin in which short nucleosomal domains are interspersed in a highly saturated protamine‐DNA complex.
Conformational analysis and the fine structure of cross peaks in phase‐sensitive homonuclear two‐dimensional correlated NMR spectra of oligosaccharides
Authors: BERMAN Elisha
Published: March 2005

Useful structural and conformational information was obtained from a detailed analysis of the fine structure of cross peaks that appears in the phase‐sensitive homonuclear correlated two‐dimensional spectra of complex large glycopeptides. The small chemical shift range observed for the oligosaccharide part results in a variety of cross‐peak patterns. A catalogue of cross‐peak fine structures and patterns expected for common carbohydrates are presented which can help in making some, all‐important and crucial, proton resonance assignments.

Analysis of the vicinal coupling constants extracted from the cross peaks corresponding to the hydroxymethyl protons of the inner core carbohydrates of glycopeptides derived from calf fetuin, has yielded information regarding the time‐averaged solution conformation of this triantennary structure. It was shown that in solution the Man(α1–6) arm of the core pentasaccharide unit, which carries a single antenna, adopts for an appreciable part of the time a conformation which brings it close to the arm that is attached to the peptide part. A similar conformation for the Man(α1–6) arm was previously observed for the biantennary oligosaccharide chain. Therefore, we concluded that the presence of an additional antenna on the Man(α1–3) arm of the core unit in the triantennary structure does not alter the basic biantennary conformation.
Molecular cloning and nucleotide sequence of full‐length cDNA for sweet potato catalase mRNA
Authors: SAKAJO Shigeru , NAKAMURA Kenzo , ASAHI Tadashi
Published: March 2005

A nearly full‐length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size‐fractionated poly(A)‐rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector‐primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5′‐termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900‐base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313–317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.
Possible involvement of eicosanoids in the zymosan and arachidonic‐acid‐induced oxygen uptake, glycogenolysis and Ca2+ mobilization in the perfused rat liver
Authors: DIETER Peter , ALTIN Joseph G. , DECKER Karl , BYGRAVE Fyfe L.
Published: March 2005

Exposure of perfused rat livers to zymosan, arachidonic acid and phenylephrine, but not to latex particles, induces pronounced oxygen uptake, glycogenolysis and Ca2+ mobilization. The oxygen uptake induced by arachidonic acid and by zymosan remains elevated even after the agents have been removed. NaN3 was found to be much more effective in inhibiting the oxygen uptake induced by phenylephrine than that induced by zymosan or arachidonic acid.

Glucose release induced by zymosan and by arachidonic acid reaches a maximum after about 2 min and then declines very rapidly even while the agents are still being infused. In contrast, glucose release induced by phenylephrine remains elevated for the duration of the infusion.

Ca2+ fluxes induced by arachidonic acid are similar to those induced by phenylephrine in that efflux occurs when the agent is administered and influx occurs only when the agent is removed. This contrasts to the Ca2+ flux changes induced by zymosan where both Ca2+ efflux and Ca2+ influx occur even while zymosan is still being infused.

Glucose release induced by zymosan is inhibited by bromophenacylbromide and nordihydroguaiaretic acid, but not by indomethacin. Indomethacin, however inhibits the arachidonic‐acid‐induced glucose release which is also inhibited by nordihydroguaiaretic acid but not by bromophenacylbromide. Indomethacin inhibits also the arachidonic‐acid‐induced Ca2+ flux changes whereas the zymosan‐ and the phenylephrine‐induced Ca2+ flux changes are not inhibited by the cyclooxygenase inhibitor.

The data presented in this paper suggest that in the perfused rat liver the zymosan‐induced glycogenolysis, as well as the Ca2+ flux changes and glycogenolysis induced by arachidonic acid, are mediated by eicosanoids.
Structural organization of the proopiomelanocortin gene in Xenopus laevis
Authors: MARTENS Gerard J. M.
Published: March 2005

This study reports the isolation and characterization of the entire proopiomelanocortin (POMC) gene of the amphibian Xenopus laevis. The Xenopus POMC gene consists of three exons of which the main exon 3 codes for all of the bioactive domains of the precursor protein. Intron A (2.6 kb) separates the segments encoding the 5′‐untranslated mRNA region and intron B (2.5 kb) interrupts the protein‐coding sequence near the signal peptide coding region. In that this structural organization of the Xenopus POMC gene is similar to those of the mammalian genes, apparently the POMC gene has been remarkably stable during 350 million years of vertebrate evolution. A comparative analysis of the 5′‐flanking sequences of the Xenopus and mammalian POMC genes reveals the presence of several conserved regions. One of these regions is homologous with sequences located upstream of the capping sites of other glucocorticoid‐regulated genes and another region contains a segment reminiscent of a viral enhancer consensus sequence.
Nickel‐[iron‐sulfur]‐selenium‐containing hydrogenases from Desulfovibrio baculatus (DSM 1743)
Authors: TEIXEIRA Miguel , FAUQUE Guy , MOURA Isabel , LESPINAT Paul A. , BERLIER Yves , PRICKRIL Ben , PECK Harry D. , XAVIER António V. , Le GALL Jean , MOURA José J. G.
Published: March 2005

The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane‐bound (detergent solubilization). Plasma‐emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non‐identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g∼ 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane‐bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(III). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane‐bound preparations could be decomposed into two sets with g at 2.34, 2.16 and ∼ 2.0 (component I) and at 2.33, 2.24, and ∼ 2.0 (component II). In the reduced state, after exposure to an H2 atmosphere, all the hydrogenase fractions gave identical EPR spectra.

EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron‐sulfur centers: center I (2.03, 1.89 and 1.86) detectable below 10 K, and center II (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni‐signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 8942]. During the course of a redox titration at pH 7.6 using H2 gas as reductant, this signal attained a maximal intensity around ‐320 mV. Low‐temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fast‐relaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time‐dependent activation but the membrane‐bound form required such a step in order to express full activity. This indicates that the redox state of the isolated enzyme is important for the full expression of enzymatic activity. The catalytic properties were also followed by the proton‐deuterium exchange reaction. The isolated hydrogenases produced H2/HD ratios higher than those observed for non‐selenium‐containing hydrogenases.