bluuebunny/crossref_metadata_2025 huggingface dataset:
On the role of the termination factor RF‐2 and the 16S RNA in protein synthesis
Authors: LANG Anne , FRIEMERT Constanze , GASSEN Hans Günter
Published: March 2005

In the translational termination step of protein synthesis the three termination codons UAA, UAG or UGA are recognized by so‐called release or termination factors. The release factor RF‐1 interacts with UAG and UAA whereas RF‐2 is specific for UGA and UAA. Two mechanisms concerning the termination event have been discussed so far: recognition of the termination codon by the protein in a tRNA‐like manner or double‐strand formation between the codon and the 3′ end of the 16S rRNA which is stabilized by the termination factor. Using equilibrium dialysis we show that 40% of the ribosomes can bind UGAA in an RF‐2‐dependent manner.

The stability with the correct combination RF‐2—UGA is tenfold higher as compared to the wrong termination codon UAG. We confirm prior findings that the termination factor RF‐2 is bound to the A‐site of the ribosome. In addition to the ribosomal proteins L2, L10, L7/L12 and L20 of the large subunit and S6 and S18 of the small subunit, the 16S rRNA became labelled when radioactive UGA was crosslinked to the ribosome in the presence of RF‐2. Our data support a mechanism of termination in which a double strand between the termination codon and the 3′ end of the 16S rRNA is formed as the starting event. The resulting RNA‐RNA double strand in turn may be recognized and stabilized by the termination factor.
Gene synthesis, expression in Escherichia coli and purification of immunoreactive human insulin‐like growth factors I and II
Authors: HUMMEL Michael , HERBST Hermann , STEIN Harald
Published: March 2005

We have expressed synthetic genes encoding human insulin‐like growth factors I and II in large quantities in Escherichia coli as fusion proteins with the 300 N‐terminal amino acids of the E. coli trpE gene product. The resulting hybrid proteins were purified from the insoluble fraction of crude bacterial lysates using a rapid HPLC separation procedure employing a C8 reversed‐phase column and a gradient of 2‐propanol in formic acid. With the procedure we obtained in volatile solvents highly purified fusion proteins that were used for further biochemical and immunological procedures. Here we describe biochemical characteristics of the bacterially expressed fusion proteins and demonstrate that these proteins share substantial antigenic properties with the native polypeptides allowing the establishment of highly specific monoclonal antibodies.
Mechanisms of flavoprotein‐catalyzed reactions
Authors: GHISLA Sandro , MASSEY Vincent
Published: March 2005

Flavoproteins are a class of enzymes catalyzing a very broad spectrum of redox processes by different chemical mechanisms. This review describes the best studied of these mechanisms and discusses factors possibly governing reactivity and specificity.
Purification of isoleucyl‐tRNA synthetase from Methanobacterium thermoautotrophicum by pseudomonic acid affinity chromatography
Authors: RECHSTEINER Thomas , LEISINGER Thomas
Published: March 2005

The isoleucyl‐tRNA synthetase of the archaebacterium Methanobacterium thermoautotrophicum was purified 1500‐fold to electrophoretic homogeneity by a procedure based on affinity chromatography on Sepharose‐bound pseudomonic acid, a strong competitive inhibitor of this enzyme. The purified enzyme is a monomer with a molecular mass of 120 kDa. In this respect and in its km values for the PPi‐ATP exchange, and aminoacylation reactions, it resembles the isoleucyl‐tRNA synthetases from eubacterial and eukaryotic sources. Its aminoacylation activity is optimal at pH 8.0 and at 55°C. Pseudomonic acid is a strong competitive inhibitor of the aminoacylation reaction with respect to both l‐isoleucine (KIlei 10 nM) and ATP (KATPi 20 nM).
The gene encoding dihydrolipoyl transacetylase from Azotobacter vinelandii
Authors: HANEMAAIJER Roeland , WESTPHAL Adrie H. , BERG Axel , DONGEN Walter VAN , de KOK Arie , VEEGER Cees
Published: March 2005

The gene encoding the dihydrolipoyl transacetylase (E2) component from Azotobacter vinelandii has been cloned in Escherichia coli. High expression of the gene was found when the cells were grown for more than 14 h. The E2 produced was partially active, varying 10 and 90% in different experiments. By limited proteolysis of the protein it was shown that the catalytic domain was incorrectly folded, caused by formation of intermolecular or intramolecular S‐S bridges. The enzyme was fully activated after unfolding in 2.5 M guanidine hydrochloride containing 2 mM dithiothreitol, followed by refolding by dialysis. Active E2 was isolated in a simple three‐step procedure. It possessed a specific activity in the same order as that found after isolation of E2 from purified pyruvate dehydrogenase complex from A. vinelandii. Active E2 comprises about 7% of the total soluble cellular protein in the E. coli clone.

By genetic manipulation, deletion mutants of E2 were created, one encoding the lipoyl domain and the N‐terminal half of the pyruvate‐dehydrogenase (E1)‐ and lipoamide‐dehydrogenase (E3)‐binding domain, the other encoding the catalytic domain and the C‐terminal half of the E1‐ and E3‐binding domain. In E. coli expression of both mutants was observed.
Isolation of heparin‐binding growth factors from bovine, porcine and canine hearts
Authors: QUINKLER Wolfgang , MAASBERG Michael , BERNOTAT‐DANIELOWSKI Sabine , LÜTHE Norbert , SHARMA Hari S. , SCHAPER Wolfgang
Published: March 2005

Fresh bovine, porcine and canine hearts were homogenized and mitogens for mesoderm‐derived cells were purified in three different steps. Extraction by two different ammonium sulfate precipitations was followed by cation‐exchange chromatography and by heparin‐Sepharose affinity chromatography. A heparin‐Sepharose fraction from heart (eluted at 1.1 M NaCl) increased mitotic activity in serum‐deprived cultures of porcine aortic endothelial and smooth muscle cells, and in human fibroblasts. This mitogenic activity is potentiated by heparin and inhibited by γ‐interferon. The heart mitogenic fraction showed one double peak on HPLC at A215 and one polypeptide‐band on SDS/PAGE. These peaks and bands were identical to those obtained from bovine brain. The heart acidic fibroblast growth factor (aFGF) showed a positive signal in Western blots using antibodies raised against brain aFGF. Gas‐phase amino acid sequencing established that the mitogens were identical to aFGF and the N‐terminally truncated aFGF. Extraction in the presence of a protease inhibitor (pepstatin A) produced a higher‐molecular mass form of aFGF with a blocked amino terminus.

Another mitogen, eluted at 1.6 M NaCl from heparin‐Sepharose, reacted with polyclonal antiserum against human recombinant basic fibroblast growth factor (bFGF) and showed a 66% (12 from 18 amino acids determined by gas‐phase sequencing) similarity with bFGF. This polypeptide increased the mitotic activity of the same cell lines but was more potent than aFGF.
Resonance assignments in the proton‐NMR spectrum of carbonmonoxy hemoglobin by two‐dimensional methods
Authors: CRAESCU Constantin T. , MISPELTER Joël
Published: March 2005

Proton‐nuclear‐magnetic‐resonance spectroscopy is a powerful tool for investigating the solution structure of biopolymers provided that a substantial number of proton resonances are assigned in the spectrum. For large proteins the assignments have usually been made by the comparative one‐dimensional NMR investigations of the parent and derivative proteins in different physicochemical conditions. In this paper, we show that the more powerful two‐dimensional methods could be successfully applide to proteins of the size of human adult hemoglobin (Mr= 64500). J‐Correlated and NOE‐correlated spectroscopy, together with topological relationships in the known crystalline structure, enabled us to assign a large number of resonances. The majority of the assigned resonances correspond to the heme substituents and to amino acids in the heme pockets of the two subunits. These results thus provide an extensive set of intrinsic probes for mapping the conformation of the ligand‐binding site and its functional changes. Comparison of the observed ring‐current shifts of the assigned resonances with those calculated from the known crystallographic coordinates suggests a close similarity between the heme‐pocket tertiary conformation in solution and in the crystalline state. A significant difference was noted for Leu141 in β subunits which, in solution, appears to have stronger contacts with the heme groups than in the crystalline form. The present results also demonstrate that two‐dimensional‐NMR methods could be successfully applied to the investigation of the structure of large biomolecules in solution (Mr≤ 65000).
Growth stimulation of human epidermal cells by urokinase is restricted to the intact active enzyme
Authors: KIRCHHEIMER Johannes C. , CHRIST Guenter , BINDER Bernd R.
Published: March 2005

In this study we report on the effect of urokinase fragments on the proliferation of cells of the human epidermal cell line, CCL 20.2, which expresses high‐affinity receptors for urokinase and the growth of which is stimulated by intact active 54‐kDa urokinase. The 33‐kDa fragment containing only the active‐site domain, does not bind to the receptors and does not stimulate cell proliferation, while the 17‐kDa fragment, containing only the kringle and the growth‐factor domains binds to the receptor but does not stimulate growth of the human epidermal cell line. Growth promotion of this tumor cell line by urokinase is therefore restricted to the complete intact and active urokinase molecule.
Metabolism of cysteinyl leukotrienes in non‐recirculating rat liver perfusion
Authors: WETTSTEIN Matthias , GEROK Wolfgang , HÄUSSINGER Dieter
Published: March 2005

The uptake, metabolism and biliary excretion of the cysteinyl leukotrienes LTC4, LTD4 and LTE4, were studied in a non‐recirculating rat liver perfusion system at constant flow in both antegrade (from the portal to the caval vein) and retrograde (from the caval to the portal vein) perfusion directions. During a 5‐min infusion of [3H]LTC4,[3H]LTD4 and [3H]LTE4 (10 nmol/l each) in antegrade perfusions single‐pass extractions of radioactivity from the perfusate were 66%, 81% and 83%, respectively. Corresponding values for LTC4 and LTD4 in retrograde perfusions were 83% and 93%, respectively, indicating a more efficient uptake of cysteinyl leukotrienes in retrograde than in antegrade perfusions. The concentrations of unmetabolized leukotrienes in the effluent perfusate were 8–12% in antegrade and 2–4% in retrograde perfusions. [14C]Taurocholate extraction from the perfusate was inhibited by LTC4 by only 3%, suggesting that an opening of portal‐venous/hepatic‐venous shunts does not explain the effects of perfusion direction on hepatic LTC4 uptake.

Following infusion of [3H]LTC4 and [3H]LTD4, in the antegrade perfusion direction, about 80% and 87%, respectively, of the radiolabel taken up by the liver was excreted into bile. In retrograde perfusions, however, only 40% and 57%, respectively, was excreted into bile and the remainder was slowly redistributed into the perfusate, indicating that leukotrienes were taken up into a hepatic compartment with less effective biliary elimination or converted to metabolites escaping biliary excretion. The metabolite pattern found in bile was not affected by the direction of perfusion. Biliary products of LTC4 were polar metabolites (31–38%), LTD4 (27–30%), LTE4 (about 1%) and N‐acetyl‐LTE4 (3–4%) in addition to unmodified LTC4 (17–18%).

LTC4 was identified as a major metabolite of [3H]LTD4 in bile, amounting to about 20% of the total radioactivity excreted into bile. This is probably due to a γ‐glutamyltransferase‐catalyzed glutamyl transfer from glutathione in the biliary compartment, as demonstrated in in vitro experiments. The presence of sinusoidal γ‐glutamyltransferase activity in perfused rat liver was shown in experiments on the hydrolysis of infused γ‐glutamyl‐p‐nitroanilide. 90% inhibition of this enzyme activity by AT‐125 did not affect the metabolism of LTC4.

When [3H]LTE4 was infused in the antegrade perfusion direction, biliary metabolites comprised N‐acetyl‐LTE4 (24%) and polar components (60%).

The data show (a) that cysteinyl leukotrienes are effectively taken up and metabolized during a single liver passage, the metabolites being excreted into bile and released into perfusate; (b) zonal hepatocyte heterogeneities regarding leukotriene effector, uptake and biliary excretion sites are likely to exist; and (c) LTD4 in bile is converted to LTC4, suggesting also a biosynthetic function of canalicular or biliary γ‐glutamyltransferase, at least in the experimental system of perfused rat liver.
2‐(4′‐Chlorophenyl)benzothiazole is a potent inducer of cytochrome P450IA1 in a human and a mouse cell line
Authors: KÄENLAMPI Sirpa O. , TUOMI Katri , KORKALAINEN Merja , RAUNIO Hannu
Published: March 2005

Two benzothiazole derivatives, 2‐(4′‐chlorophenyl)benzothiazole (CPBT) and 2‐(4′‐formylphenyl)benzothiazole (FPBT) were studied for their ability to induce aryl hydrocarbon hydroxylase activity in a mouse and a human cell line. In both the mouse hepatoma cell line, Hepa‐1, and the human choriocarcinoma cell line, JEG‐3, a high aryl hydrocarbon hydroxylase activity was observed after treatment with CPBT. In contrast, FPBT had a very weak inducing capacity in both cell lines. The maximal induction by CPBT was several times (e.g. in Hepa‐1 about fourfold on average) greater than that observed with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). A specific cDNA probe for mouse cytochrome P450IA1 gene was used to quantify mRNA levels in Hepa‐1 cells. CPBT increased cytochrome P450IA1 mRNA to a level of 88% of that induced by TCDD. Immunoblot analysis with monoclonal antibody 1‐7‐1, directed against rat liver cytochrome P450IA1 and P450IA2, showed that the amount of P450IA1 is substantially increased in Hepa‐1 cells after treatment with CPBT. The observation that CPBT competed TCDD off its specific cytosolic binding site suggests a receptor‐mediated induction of cytochrome P450IA1 mRNA. An in vitro activation effect did not explain the exceptionally high hydroxylase activity. The results show that CPBT is a more efficient inducer of aryl hydrocarbon hydroxylase than TCDD in Hepa‐1 and JEG‐3 cells and that the induction is supported by P450IA1. The discordant effect of CPBT on mRNA and aryl hydrocarbon hydroxylase activity suggests that post‐translational modifications of P450IA1 account for a major part of the increased enzyme activity.
Characterization of an FMN‐containing cyclohexanone monooxygenase from a cyclohexane‐grown Xanthobacter sp.
Authors: TROWER Michael K. , BUCKLAND R. Martin , GRIFFIN Martin
Published: March 2005

A soluble cyclohexanone monooxygenase was purified 16.1‐fold to homogeneity from a Xanthobacter sp. grown upon cyclohexane as sole source of carbon and energy. The native enzyme is a 50‐kDa single polypeptide chain associated with FMN rather than FAD as flavin prosthetic group in a 1:1 stoichiometric relationship. The monooxygenase catalyses the transformation of cyclohexanone to the lactone 1‐oxa‐2‐oxocycloheptane in an oxygen ring insertion reaction. Only related cycloalkanone substrates are accepted for oxygenation, no activity is shown towards straight‐chain alkanones. Enzyme activity is strongly inhibited by sulphydryl‐reactive agents, but is relatively insensitive to metal chelators, electron transport inhibitors and the metal ions Fe3+ and Cu2+. Cyclohexanone monooxygenase has Km values for cyclohexanone and NADPH of <0.5 μM and 12.5 μM respectively. Kinetic investigations under steady‐state conditions demonstrate that the flavoprotein prosthetic group, FMN, is involved in the monooxygenase catalytic mechanism. The systematic name for the enzyme is cyclohexanone, NADPH:oxygen oxidoreductase (6‐hydroxylating, 1,2‐lactonizing) (EC 1.14.13.22).
Ultraviolet and magnetic‐circular‐diochroic spectroscopic studies of Gd(III) complexed with diethylenetriaminepentaacetic acid
Authors: BLIGH S. W. Annie , DRAKE Alex F. , SADLER Peter J.
Published: March 2005

Sensitive probes are required for studying the biochemistry of Gd(III) contrast agents used in magnetic resonance imaging. We show that complexation of Gd(III) by diethylenetriamine‐N,N,N′,N′,N”‐pentaacetic acid (DTPA) in aqueous solution can be readily determined from the sharp 4f‐f bands for free and bound Gd(III) in the range 270–282 nm, and, with greater sensitivity, from the associated magnetic‐circular‐dichroic spectra.
Hormone‐induced expression of the CHSl gene from Saccharomyces cerevisiae
Authors: APPELTAUER Ute , ACHSTETTER Tilman
Published: March 2005

When MATa cells of Saccharomyces cerevisiae have been treated with the mating hormone α‐factor an increase in chitin synthase zymogen, as well as chitin content in the cell‐wall fraction, have been reported. With a DNA probe derived from the cloned CHSl gene that codes for chitin synthase I [Bulawa, C. E., Slater, M., Cabib, E., Au‐Young, J., Sburlati, A., Adair, W. L. and Robbins, P. (1986) Cell 46, 213–225] a Northern analysis was conducted of CHSl‐specific transcripts. α‐Factor‐treated MATa cells revealed more than sixfold elevated steady‐state levels of CHSl mRNA as compared to control cells. MATα cells responded the same way when treated with a‐factor although induction rate was somewhat smaller. After hormone application a rapid increase in CHSl mRNA levels could be observed that occurred also in the absence of ongoing protein synthesis.

In order to minimize possible side effects of CHSl‐coding sequences on expression and mRNA stability a CHSl::SUC2 chimaeric gene was constructed where 730 bp of the CHSl promoter region (+ 20 bp of the coding region) were fused in frame to a fragment of the SUC2 coding region. The fusion protein exhibits invertase activity that has been used to monitor CHSl promoter activity. By analysis of shortened versions of the CHSl promoter a 94‐bp DNA fragment has been identified that confers hormone inducibility to the CHSl promoter. According to the published sequence of the CHSl gene, this fragment contains four repeats of a TGAAACA consensus sequence previously identified in the α‐factor‐inducible BARl promoter [Kronstad, J. W., Holly, J. A. and MacKay, V. L. (1987) Cell 50, 369–377]. This heptamer may represent the cis‐acting element involved in mating‐hormone‐mediated gene expression in yeast.
Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins
Authors: SEIFERT Roland , WENZEL Katharina , ECKSTEIN Fritz , SCHULTZ Günter
Published: March 2005

Whereas the chemotactic peptide, N‐formyl‐l‐methionyl‐l‐leucyl‐l‐Phenylalanine (fMet‐Leu‐Phe), induced NADPH‐oxidase‐catalyzed superoxide (O2−) formation in human neutrophils, purine and pyrimidine nucleotides per se did not stimulate NADPH oxidase but enhanced O2− formation induced by submaximally and maximally stimulatory concentrations of fMet‐Leu‐Phe up to fivefold. On the other hand, fMet‐Leu‐Phe primed neutrophils to generate O2− upon exposure to nucleotides. At a concentration of 100 μM, purine nucleotides enhanced O2− formation in the effectiveness order adenosine 5′‐O‐[3‐thio]triphosphate (ATP[γS]) > ITP > guanosine 5′‐O‐[3‐thio]triphosphate (GTP[γS]) > ATP = adenosine 5′‐O, ‐[2‐thio]triphosphate (Sp‐diastereomer) = GTP = guanosine 5′‐O, [2‐thio]diphosphate (GDP[βS] = ADP > adenosine 5′‐[β,γ‐imido]triphosphate = adenosine 5′‐O‐[2‐thio]triphosphate] (Rp‐diastereomer). Pyrimidine nucleotides stimulated fMet‐Leu‐Phe‐induced O2− formation in the effectiveness order uridine 5′‐O‐[3‐thio]triphosphate (UTP[γS]) > uridine 5′‐O‐[2‐thio]diphosphate (UDP[βS]) = uridine 5′‐O[2‐thio]triphosphate (Rp‐diastereomer) (Rp)‐UTP[βS]) = UTP > CTP. Uracil nucleotides were similarly effective potentiators of O2− formation as the corresponding adenine nucleotides. GDP[βS] and UDP[βS] synergistically enhanced the stimulatory effects of ATP[γS], GTP[γS] and UTP[γS]. Purine and pyrimidine nucleotides did not induce degranulation in neutrophils but potentiated fMet‐Leu‐Phe‐induced release of β‐glucuronidase with similar nucleotide specificities as for O2− formation. In contrast, nucleotides per se induced aggregation of neutrophils. Treatment with pertussis toxin prevented aggregation induced by both nucleotides and fMet‐Leu‐Phe. Our results suggest that purine and pyrimidine nucleotides act via nucleotide receptors, the nucleotide specificity of which is different from nucleotide receptors in other cell types. Neutrophil nucleotide receptors are coupled to guanine‐nucleotide‐binding proteins. As nucleotides are released from cells under physiological and pathological conditions, they may play roles as intercellular signal molecules in neutrophil activation.
Soluble and particulate Ins(1,4,5)P3/Ins(1,3,4,5)P4 5‐phosphatase in bovine brain
Authors: ERNEUX Christophe , LEMOS Manuela , VERJANS Benoît , VANDERHAEGHEN Pierre , DELVAUX Anne , DUMONT Jacques E.
Published: March 2005

Ins(1,4,5)P3 5‐phosphatase catalyses the dephosphorylation of Ins(1,4,5)P3 in the 5 Position. At 1 μM Ins(1,4,5)P3, 10–15% of total activity of a bovine brain homogenate was measured in the soluble fraction, whereas 85–90% was in the particulate fraction. Particulate activity could be solubilized by cholate or, to a lower extent, by 2 M KCl. Two soluble enzymes (type I and type II) could be fractionated by DEAE‐Sephacel chromatography. Soluble activities have been further purified by blue‐Sepharose, Sephacryl S‐200 and phosphocellulose chromatography. Specific activities reached 10–30 μmol · min−1 mg protein−1 for type I and were 10–20 times lower for type II. Type I and type II Ins(1,4,5)P3 5‐phosphatase displayed different Km values and molecular masses, as estimated by gel filtration. Type I dephosphorylated both Ins(1,4,5)P3 and Ins(1,3,4,5)P4; in contrast, type II specifically dephosphorylated Ins(1,4,5)P3 but not Ins(1,3,4,5)P4. Type I Ins(1,4,5)P3 5‐phosphatase eluted as a single peak of activity with an apparent molecular mass of 51 kDa when gel filtration was performed in the presence of cholate. This molecular mass is identical to the molecular mass estimated for the particulate Ins(1,4,5)P3 5‐phosphatase that was solubilized by cholate. Km values for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 obtained with type I Ins(1,4,5)P3 5‐phosphatase were 11 μM and 1 μM, respectively. Similar values were obtained with particulate Ins(1,4,5)P3 5‐phosphatase. In conclusion, the catalytic domains of type I and particulate Ins(1,4,5)P3 5‐phosphatase activity may be very similar, if not identical, but different from type II phosphatase.
Purification of granulocyte chemotactic peptide/interleukin‐8 reveals N‐terminal sequence heterogeneity similar to that of β‐thromboglobulin
Authors: van DAMME Jo , BEEUMEN Jozef , CONINGS Rene , DECOCK Benny , BILLIAU Alfons
Published: March 2005

Stimulated human peripheral blood leukocytes produce a chemotactic factor for granulocytes (granulocyte chemotactic peptide/interleukin‐8; GCP/IL‐8), which is structurally related to platelet‐derived β‐thromboglobulin. Analytically pure GCP/IL‐8 and β‐thromboglobulin could be obtained after three purification steps, comprising adsorption to silicic acid, heparin‐Sepharose chromatography and ion‐exchange chromatography. Although GCP/IL‐8 and β‐thromboglobulin had a similar affinity for heparin, they could be separated on a cation‐exchange column. Both molecules were heterogeneous in that 6–7‐kDa protein doublets were detected upon SDS/PAGE. N‐terminal amino acid sequence analysis revealed the presence of six immunologically related but differently truncated polypeptides of β‐thromboglobulin, of which only two corresponded to previously described forms. Similarly, apart from a major polypeptide, five minor species of GCP/IL‐8 were detected that also differed by N‐terminus in that region of the primary structure where a significant similarity between the two molecules starts. GCP/IL‐8 was found to be chemotactic for granulocytes with a specific activity of 105 units/mg, whereas none of the β‐thromboglobulin species possessed detectable chemotactic activity.
Amino acid sequence of a crystalline seed albumin (winged bean albumin‐1) from Psophocarpus tetragonolobus (L.) DC
Authors: KORTT Alexander A. , STRIKE Philip M. , de JERSEY John
Published: March 2005

The complete amino acid sequence of winged bean albumin‐1 (WBA‐1) of Psophocarpus tetragonolobus (L.) DC has been determined. The protein consists of a single polypeptide chain of 175 amino acid residues, with one disulfide bond, corresponding to a molecular mass of 19333 Da. WBA‐1 was found to be homologous with the Kunitz‐type seed trypsin inhibitors. The similarity between WBA‐1 and the trypsin inhibitors from soybean and winged bean was 38% and 28%, respectively; similarity was most marked in the C‐terminal third of the sequence with identities of 47% and 37%, respectively. Significant similarity was found also between the 2S Kunitz‐type proteins and the carboxy‐terminal region of the 7S storage globulins, suggesting that these two groups of proteins are related and may have evolved from a common ancestral precursor. Circular dichroism measurements suggest a high content of β sheet (52%) while secondary structure predictions based on amino acid sequence indicate a similar content and distribution of β sheet to that found for soybean trypsin inhibitor by X‐ray diffraction studies.
Purification and properties of mouse liver coproporphyrinogen oxidase
Authors: BOGARD Marc , CAMADRO Jean‐Michel , NORDMANN Yves , LABBE Pierre
Published: March 2005

Coproporphyrinogen oxidase was purified to homogeneity from mouse liver. The specific activity of the pure enzyme was 3500 nmol · h−1· mg−1; its apparent molecular mass (35 kDa) was confirmed by immunological characterization of the enzyme in a trichloroacetic‐acid‐precipitated total‐liver‐protein extract. The native enzyme appeared to be a dimer of 70 kDa as determined by gel filtration under nondenaturating conditions. The Km value for coproporphyrinogen III was 0.3 μM. The purified enzyme was activated by neutral detergents and phospholipids (affecting both Vmax and Km) but inhibited by ionic detergents. Reactivity toward sulfhydryl agents suggested the possible involvement of (an) SH group(s) for the activity. When compared to the previously purified coproporphyrinogen oxidases (from bovine liver and yeast), the mouse liver coproporphyrinogen oxidase appears to share many common catalytic properties with both enzymes. However, its apparent molecular mass is very different from that of the bovine liver enzyme (71.6 kDa) but identical to that found for the yeast (Saccharomyces cerevisiae) enzyme.
Purification and characterization of an oxygen‐stable carbon monoxide dehydrogenase of Methanothrix soehngenii
Authors: JETTEN Mike S. M. , STAMS Alfons J. M. , ZEHNDER Alexander J. B.
Published: March 2005

Carbon monoxide dehydrogenase was purified to apparent homogeneity from Methanothrix soehngenii. In contrast with the carbon monoxide dehydrogenases from most other anaerobic bacteria, the purified enzyme of Methanothrix soehngenii was remarkably stable towards oxygen and it was only slightly inhibited by cyanide.

The native molecular mass of the carbon monoxide dehydrogenase of Methanothrix soehngenii determined by gel filtration was 190 kDa. The enzyme is composed of subunits with molecular mass of 79.4 kDa and 19.4 kDa in an α2β2 oligomeric structure. The enzyme contains 1.9 ± 0.2 (n= 3) mol Ni/mol and 19 ± 3 (n= 3) mol Fe/mol and it constitutes 4% of the soluble cell protein. Analysis of enzyme kinetic properties revealed a Km of 0.7 mM for CO and of 65 μM for methyl viologen. At the optimum pH of 9.0 the Vmax was 140 μmol of CO oxidized min−1 mg protein−1. The enzyme showed a high degree of thermostability.
Characterization of a cysteine‐containing peptide after affinity labelling of Ca2+‐ATPase of sarcoplasmic reticulum with the disulfide of 3′(2′)‐O‐biotinyl‐thioinosine triphosphate
Authors: KISON Ralf , MEYER Helmut E. , SCHONER Wilhelm
Published: March 2005

3′(2′)‐O‐Biotinyl‐thioinosine triphosphate is a substrate of the Ca2+ pump of sarcoplasmic reticulum. Its disulfide inactivates the Ca2+‐ATPase with two different velocities. The rapidly inactivated sulfhydryl group cannot be protected by ATP and is therefore considered to be outside the ATP binding site. The slowly reacting sulfhydryl group interacts with the disulfide of 3′(2′)‐O‐biotinyl‐thioinosine triphosphate with a dissociation constant of Kd== 137 μM and an inactivation velocity constant of 1.7×10−3 s−1. It is protected by ATP with two different dissociation constants of the enzyme‐ATP complex of Kd== 221 μM and 1130 μM. The slowly reacting sulfhydryl group is therefore considered to be part of the ATP binding site.

Since it was impossible to isolate a tryptic peptide by affinity purification on matrix‐bound avidin after affinity labelling with the disulfide of 3′(2′)‐O‐biotinyl‐thioinosine triphosphate, differential labelling with iodo[2‐14C]acetic acid after affinity labelling with the disulfide of 3′(2′)‐O‐biotinyl‐thioinosine triphosphate was carried out. Tryptic digestion and FPLC purification led to the isolation of a radioactive carboxymethyl derivative of the cysteine‐containing peptide ANACNSVIR.

This peptide is equivalent to the cDNA‐derived sequence 468–476 of Ca2+‐ATPase [Brandl et al. (1986) Cell 44, 597–607] and is located between the phosphorylation site, Asp351, and Lys515, a part of the putative purine binding subsite of ATP. Although the carboxymethylation of Cys471 is hindered by (biotinyl‐s6ITP)2, the strong dilution of the specific radioactivity of iodo[2‐14C]acetic acid in the isolated peptide 468–476 argues against its direct interaction with the ATP analogue. It is therefore proposed that Cys471 undergoes ATP‐dependent conformational changes.
Characterization of four different mammalian‐cell‐derived recombinant human interferon‐β1s
Authors: UTSUMI Jun , MIZUNO Yasuko , HOSOI Kazuo , OKANO Kiyoshi , SAWADA Ritsuko , KAJITANI Masayuki , SAKAI Ikuo , NARUTO Masanobu , SHIMIZU Hirohiko
Published: March 2005

In order to evaluate the structural identification of various recombinant human interferon‐β1s, the recombinant proteins were produced in four different mammalian cells (human PC12 and PC8 lung adenocarcinoma cells, Chinese hamster ovary cells and mouse C127 cells) and characterized. Each mammalian‐cell‐derived recombinant human interferon‐β1 represented a single band of 23 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, the same molecular mass as fibroblast‐derived natural human interferon‐β1. Specific activities, amino acid compositions, amino‐terminal sequences, peptide maps on C18 reversed‐phase high‐performance liquid chromatography and circular dichroic spectra of recombinant proteins were in good agreement with natural ones.

On the other hand, the patterns of isoelectric focusing were different between mammalian‐cell‐derived recombinant human interferon‐β1s and natural human interferon‐β1. Sugar composition analysis revealed that the recombinant protein from Chinese hamster ovary cells has a similar sugar composition to that of natural protein and the other recombinant proteins have increased amounts of galactose and glucosamine in comparison to the natural protein. Furthermore, there is no galactosamine in the natural protein, while small amounts of galactosamine were detected in the oligosaccharides released from PC8‐ and C127‐derived recombinant proteins by N‐glycanase.

These results indicate that mammalian‐cell‐derived recombinant human interferon‐β1s have identical polypeptides to those of natural human interferon‐β1 but their carbohydrate moieties, including unusual N‐linked oligosaccharides, are individually different from natural ones and depend on the host cell.
Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in Escherichia coli
Authors: IKEHATA Osamu , NISHIYAMA Makoto , HORINOUCHI Sueharu , BEPPU Teruhiko
Published: March 2005

The nitrile hydratase (NHase) of Rhodococcus species N‐774, which is composed of two subunits, α and β, catalyzes the hydration of various nitrile compounds to the corresponding amides. The amino acid sequences of the NH2 termini and the fragments obtained by digesting each of the two subunits with lysyl endopeptidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 4.4‐kb SphI fragment which contained DNA sequences hybridizing to several of the probes was cloned in pBR322 in Escherichia coli. The nucleotide sequences together with the determined amino acid sequences indicated that the α and β subunits of NHase consisted of 207 amino acids (Mr, 22918) and 212 amino acids (Mr, 23428), respectively. The open reading frame for the α subunit includes that for the β subunit with a short interval of only 26 base pairs; the two genes are probably translated in a polycistronic manner. Although large amounts of the α‐ and β‐subunit proteins were produced as insoluble forms in E. coli when the cloned genes were placed under the control of the lac promoter, no enzymatic activity was detected. The activity of the enzyme was restored, to some extent, by solubilization of the proteins with 8 M urea and subsequent dialysis for refolding at pH 10 in the presence of Fe2+ and pyrroloquinoline quinone.
The effect of caldesmon on assembly and dynamic properties of actin
Authors: GAŁA̧ZKIEWICZ Barbara , BELAGYI Joseph , DA̧BROWSKA Renata
Published: March 2005

Our earlier fluorescence measurements using N‐(1‐pyrenyl)iodoacetamide‐labeled actin revealed that caldesmon interacts with G‐actin accelerating its nucleation at low salt concentration and causing polymerization in the absence of salt [Galązkiewicz, B., Mossakowska, M., Osińska, H. & Dąbrowska, R. (1985) FEBS Lett. 184, 144–149]. In this work the caldesmon‐induced process of actin polymerization as well as the dynamic properties of the polymers formed have been investigated with the use of fluorescence, electron paramagnetic resonance (EPR) and electron microscopy techniques.

Fluorescence titration of N‐(1‐pyrenyl)iodoacetamide‐labeled actin with caldesmon showed saturation of the polymerization at a 1:3 molar ratio of caldesmon/actin monomer. Parallel pelleting experiments revealed, however, that the process of polymer formation is biphasic and only at higher concentrations of caldesmon does the copolymer contain around one caldesmon/three actin monomers. At low concentration of caldesmon a complex of one caldesmon/nine actin monomers is formed. EPR spectroscopy, using maleimide spin label bound at Cys374 of actin, also indicated that one caldesmon molecule polymerizes nine actin monomers. Taken together, these results might suggest the existence of weak and strong forms of actin binding to caldesmon and detection of only the latter by the fluorescence method.

Copolymers of actin and caldesmon are indistinguishable from actin polymerized by salt with respect to their appearance in the electron microscope and their ability to interact with heavy meromyosin, although they are characterized by lower torsional flexibility as indicated by immobilization of spin labels attached to actin.
Preferential induction of the rat hepatic P450 I proteins by the food carcinogen 2‐amino‐3‐methyl‐imidazo[4,5‐f]quinoline
Authors: RODRIGUES A. David , AYRTON Andrew D. , WILLIAMS Elisabeth J. , LEWIS David F. V. , WALKER Ronald , IOANNIDES Costas
Published: March 2005

Administration of the food carcinogen, 2‐amino‐3‐methyl‐imidazo[4,5‐f]quinoline (IQ) to rats gave rise to significant dose‐dependent increases in the microsomal O‐deethylations of ethoxycoumarin and ethoxyresorufin but had no effect on the O‐dealkylation of pentoxyresorufin and the NADPH‐dependent reduction of cytochrome c, and decreased the N‐demethylation of dimethylnitrosamine. Microsomal cytochrome b5 and total cytochrome P‐450 levels decreased following the administration of the carcinogen.

Hepatic microsomal preparations from IQ‐treated animals were much more efficient than control in activating the premutagen 2‐amino‐6‐methyldipyrido[1,2‐a:3′,2′‐d]imidazole to mutagenic intermediates in the Ames test.

Immunoquantification of two of the major families of cytochrome P‐450, namely P450 I and P450 II B, using ELISA techniques showed that treatment with IQ induced the apoprotein levels of the P450 I family but not of P450 II B.

Immunoblot analysis employing polyclonal antibodies against P450 I revealed that IQ induced both isoenzymes of this family, namely P450 I A1 and A2.

It is concluded that IQ is an inducer of the rat hepatic monooxygenases, selectively inducing the P450 I family as predicted by a computer‐graphic analysis of its dimensions which showed that it is a large, essentially planer, molecule.
Cholinesterases from flounder muscle
Authors: STIEGER Susi , GENTINETTA René , BRODBECK Urs
Published: March 2005

Flounder (Platichthys flesus) muscle contains two types of cholinesterases, that differ in molecular form and in substrate specificity. Both enzymes were purified by affinity chromatography. About 8% of cholinesterase activity could be attributed to collagen‐tailed asymmetric acetylcholinesterase sedimenting at 17S, 13S and 9S, which showed catalytic properties of a true acetylcholinesterase. 92% of cholinesterase activity corresponded to an amphiphilic dimeric enzyme sedimenting at 6S in the presence of Triton X‐100. Treatment with phospholipase C yielded a hydrophilic form and uncovered an epitope called the cross‐reacting determinant, which is found in the hydrophilic form of a number of glycosyl‐phosphatidylinositol‐anchored proteins. This enzyme showed catalytic properties intermediate to those of acetylcholinesterase and butyrylcholinesterase. It hydrolyzed acetylthiocholine, propionylthiocholine, butyrylthiocholine and benzoylthiocholine. The Km and the maximal velocity decreased with the length and hydrophobicity of the acyl chain. At high substrate concentrations the enzyme was inhibited. The p(IC50) values for BW284C51 and ethopropazine were between those found for acetylcholinesterase and butylcholinesterase. For purified detergent‐soluble cholinesterase a specific activity of 8000 IU/mg protein, a turnover number of 2.8 × 107 h−1, and 1 active site/subunit were determined.
Purification of GDP mannose: dolichyl‐phosphate O‐β‐D‐mannosyltransferase from Saccharomyces cerevisiae
Authors: HASELBECK Anton
Published: March 2005

The enzyme GDP mannose: dolichyl‐phosphate O‐β‐d‐mannosyltransferase (GDP‐Man: DolP mannosyltransferase) catalyzing the reaction: GDP‐man + DolP⇌ DolP‐Man + GDP has been purified from Saccharomyces cerevisiae to homogeneity. The purification was achieved using a combination of column chromatographic methods with preparative gel electrophoresis.

The enzyme has an apparent molecular mass of 30 kDa on SDS/polyacrylamide gels. Enzymatic activity could be correlated directly with this band.

Antibodies against the transferase were raised in rabbits. The immune serum obtained removed enzymatic activity from a detergent extract of yeast membranes and reacted specifically with the 30‐kDa band on immunoblots. Experiments addressing the orientation of this enzyme in the endoplasmic reticulum membrane are presented by using selective trypsin and N‐ethylmaleimide treatment.
Isolation and partial characterization of a cytochrome‐o complex from chromatophores of the photosynthetic bacterium Rhodospirillum rubrum FR1
Authors: SCHRATTENHOLZ André S. , NAWROTH Thomas , DOSE Klaus
Published: March 2005

A cytochrome‐o complex was isolated from chromatophores of photoheterotrophically grown Rhodospirillum rubrum FR1. The enzyme was extracted with the non‐denaturating detergent taurodeoxycholate and subsequently purified by sucrose‐density‐gradient centrifugation and gel‐permeation HPLC. The complex contains two types of cytochromes, one of them cytochrome o, and two copper atoms. It catalyzes the reduction of molecular oxygen, when N,N,N′,N′‐tetramethyl‐p‐phenylendiamine or ubiquinol 10 are offered as electron donors. The oxidase activity is inhibited by cyanide, carbon monoxide and 2‐heptyl‐2‐hydroxyquinoline N‐oxide. The molecular mass of the protein is 136 ± 15 kDa. The subunit analysis, by SDS continuous and gradient gels, revealed four subunits with molecular mass 66 kDa (subunit I), 39 kDa (subunit II), 20 kDa (subunit III) and 11 kDa (subunit IV).
Localization of adriamycin in model and natural membranes
Authors: DUPOU‐CÉZANNE Laurence , SAUFEREAU Anne‐Marie , TOCANNE Jean‐François
Published: March 2005

The interaction of adriamycin with lipids was studied in model (monolayers, small unilamellar vesicles, large multilamellar vesicles) and natural (chinese hamster overy cell) membranes by measurement of fluorescence energy transfer and fluorescence quenching. 2‐APam, 7‐ASte, 12‐ASte and anthracene‐phosphatidylcholine were used as fluorescent probes in which the anthracene group is well located at graded depths in the membrane. Eggyolk phosphatidylcholine and a 1/1 mixture of it with bovine brain phosphatidylserine were used in model membrane systems.

Large fluorescence energy transfer was observed between these molecules as donors and the drug as acceptor. With liposomes, at pH 7.4 and over an adriamycin concentration range of 0–100 μM, the efficiency of energy transfer was 12‐ASte > 7‐ASte > 2‐APam, with 100% energy transfer for 12‐ASte above a drug concentration of 30 μM. At pH 5, where the fatty acids are buried deeper (0.45 nm) in the lipid bilayer due to protonation of the carboxyl group, the order of energy transfer 7‐ASte > 12‐ASte = 2‐APam was observed. Measurements of fluorescence quenching using the non‐permeant Cu2+ ion as quencher and spectrophotometric assays indicated that around 40% of the adriamycin molecules were deeply embedded in the lipid bilayer.

Adriamycin molecules thus appear to penetrate the lipid bilayer, with the aminoglycosyl group interacting with the lipid phosphate groups and the dihydroanthraquinone residue in contact with the lipid fatty acid chains.

In contrast, fluorescence energy transfer and quenching studies on CHO cells showed that adriamycin penetrated the plasma membrane of theese cells to a much more limited extent than in the model membrane systems. This can be related to the squeezing out of the drug from a film of phosphatidylcholine which was observed in monolayers by means of surface pressure, potential and fluorescence experiments.

These observations indicated that the penetration of adriamycin into lipid bilayers strongly depends on the molecular packing of the lipid.
Identification of a new pore in the mitochondrial outer membrane of a porin‐deficient yeast mutant
Authors: DIHANICH Melitta , SCHMID Angela , OPPLIGER Wolfgang , BENZ Roland
Published: March 2005

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent‐solubilized mitochondrial outer membranes of a porin‐free yeast mutant and of its parent strain. The addition of the detergent‐solubilized material resulted in a strong increase in the membrane conductance which was not observed if only the detergent was added to the aqueous phase. Surprisingly, the membrane conductance induced by the detergent extracts of the mutant membrane was only a factor of 20 less than that caused by the outer membrane of the parent strain under otherwise identical conditions. Single‐channel recordings of lipid bilayer membranes in the presence of mitochondrial outer membranes of the yeast mutant suggested the presence of a transient pore. The reconstituted pores had a single‐channel conductance of 0.21 nS in 0.1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. The pores present in the mitochondrial outer membranes of the yeast mutant shared some similarities with the pores formed by mitochondrial and bacterial porins although their effective diameter is much smaller than those of the ‘normal’ mitochondrial porins which have a single‐channel conductance of about 0.4 nS in 0.1 M KCl, corresponding to an effective diameter of 1.7 nm. Zero‐current membrane‐potential measurements suggested that the second mitochondrial porin is slightly cation‐selective. Its possible role in the metabolism of mitochondria is discussed.
Regulation of low‐density‐lipoprotein‐receptro mRNA by insulin in human hepatoma Hep G2 cells
Authors: WADE David P. , KNIGHT Brian L. , SOUTAR Anne K.
Published: March 2005

The low‐density‐lipoprotein(LDL)‐receptor mRNA content of human hepatoma (Hep G2) cells has been estimated from densitometric scans of autoradiograms obtained following the hybridisation of Northern blots of a poly(A)‐rich RNA fraction with a 32P‐labelled cDNA probe for the LDL‐receptor gene. The recovery of β‐actin mRNA was used to correct for losses occurring during the preparation of the poly(A)‐rich RNA. The content of LDL‐receptor mRNA was reduced when the cells were pre‐incubated in medium containing foetal calf serum, 25‐hydroxycholesterol, or LDL, compared to that measured in cells which had been pre‐incubated in medium containing lipoprotein‐deficient serum (LPDS). When insulin (100 mU/ml) was included in pre‐incubation medium containing LPDS, the amount of LDL‐receptor mRNA increased approximately twofold. The level of β‐actin mRNA was not significantly increased by insulin treatment. Addition of insulin to incubation medium containing LPDS also overcame the suppressive effect of exogenous LDL on the cellular content of mRNA for the LDL receptor. These findings suggest that one action of insulin in these cells may be to promote transcription of the LDL‐receptor gene by a mechanism that can override the sterol regulatory pathway.
Staphylococcal α‐toxin increases the permeability of lipid vesicles by cholesterol‐ and pH‐dependent assembly of oligomeric channels
Authors: FORTI Stefano , MENESTRINA Gianfranco
Published: March 2005

α‐Toxin, a lethal hemolytic toxin secreted by Staphylococcus aureus, forms ionic channels of large size in lipid membranes. To investigate the mechanism of channel assembly we have studied the kinetics of pore formation on small unilamellar vesicles. We have used two assays of vesicle permeabilization: one is the release of a fluorescent molecule trapped in their inner compartment; the other is the dissipation of an imposed potential.

Both methods indicate that the kinetics are complex consisting of an initial delay followed by a non‐linear relaxation. The dependence of the pore formation rate and the extent of permeabilization on the toxin/vesicle ratio indicates that aggregation of 4–10 preinserted toxin monomers underlies channel assembly.

The pH dependence of permeabilization suggests that protonation of an acidic group of the toxin is a prerequisite to channel formation. Inclusion of cholesterol in the target vesicles potentiates α‐toxin effects, in a dose‐dependent way, possibly by facilitating its protonation.

The location of the proton‐binding site on the two adjacent aspartic acid residues in positions 127 and 128 of the toxin monomer is proposed.