bluuebunny/crossref_metadata_2025 huggingface dataset:
Mössbauer studies on the metal‐thiolate cluster formation in Fe(II)‐metallothionein
Authors: DING Xiaoqi , BILL Eckhard , GOOD Meinrad , TRAUTWEIN Alfred X. , VAŠÁK Milan
Published: March 2005

The stepwise 57Fe(II)‐thiolate cluster formation in rabbit liver metallothionein‐2 (MT) has been followed at pH 8.5 using Mössbauer spectroscopy. The zero‐field spectra recorded at 4.2 K exhibit at all stages of filling one virtually identical single quadrupole splitting ΔEQ and isomer shift δ as found for reduced rubredoxin (Rdred) or the model compound [Fe(II)(SPh)4]2−, thus indicating an Fe(II)‐tetrathiolate coordination. A similar conclusion was reached also in previous electronic absorption studies [M. Good and M. Vašák (1986) Biochemistry 25, 8353–8356]. The Mössbauer spectra obtained in the presence of a magnetic field were analyzed on the basis of a spin‐Hamiltonian formalism resulting in Mössbauer parameters similar to those for Rdred and the inorganic model compound [Fe(II)(SPh)4]2−. The identity of the Mössbauer parameters of partially and fully metal‐occupied MT suggests that a comparable distortion of the metal binding sites must exist. Simulation of the spectra revealed that the Fe(II) ions in the partially metal‐occupied 57Fe(II)4‐MT form appear to be magnetically isolated, whereas in the fully metal‐saturated 57Fe(II)7‐MT form a ratio of 3:4 of paramagnetic to diamagnetic subspectra was obtained. The latter result suggests the existence of three isolated metal binding sites and a metal‐thiolate cluster containing four metal ions. In the light of structure determinations of MT containing Zn(II) and/or Cd(II) [W. Braun et al. (1986) J. Mol. Biol. 187, 125–129, and W. F. Furrey et al. (1986) Science (Wash. DC) 231, 704–710], which revealed two metal‐thiolate clusters containing three and four metal ions, respectively, and involving all 20 cysteine residues in metal binding, the appearance of Mössbauer parameters characteristic of three isolated Fe(II) sites in 57Fe(II)7‐MT is peculiar and deserves further studies. It is concluded, moreover, that the four‐metal cluster is diamagnetic with the four Fe(II) ions being antiferromagnetically coupled. The appearance of magnetic coupling above four Fe(II) equivalents bound to apoMT indicates that the cluster formation occurs in a two‐step process.
The Ca2+‐pumping ATPase and the major substrates of the cGMP‐dependent protein kinase in smooth muscle sarcolemma are distinct entities
Authors: BALTENSPERGER Kurt , CARAFOLI Ernesto , CHIESI Michele
Published: March 2005

It has been proposed that the plasma membrane Ca2+ pump of smooth muscle tissues may be regulated by cGMP‐dependent phosphorylation [Popescu, L. M., Panoiu, C., Hinescu, M. & Nutu, O. (1985) Eur. J. Pharmacol. 107, 393–394; Furukawa, K. & Nakamura, H. (1987) J. Biochem. (Tokyo) 101, 287–290]. This hypothesis has been tested on a smooth muscle sarcolemma preparation from pig thoracic aorta. The actomyosin‐extracted membranes showed ATP‐dependent Ca2+ uptake as well as cGMP‐dependent protein kinase (G‐kinase) activity. The molecular masses of the major protein substrates of the G‐kinase (G1) and that of the Ca2+ pump were compared. Electrophoretic analysis of the phosphorylated intermediate of the sarcolemmal Ca2+‐ATPase and the G1 phosphoprotein showed that these two proteins are not identical. The results were confirmed by using a 125I‐calmodulin overlay technique and an antibody against human erythrocyte Ca2+‐ATPase. Ca2+‐uptake experiments with prephosphorylated membrane vesicles were carried out to elucidate possible effects of cGMP‐dependent phosphorylation of membrane proteins on the activity of the Ca2+ pump. The cGMP‐dependent phosphorylation was found to be extremely sensitive to temperature leading to very low steady‐state phosphorylation levels at 37°C. The difficulty was overcome by ATP[γS], which produced full and stable thiophosphorylation of G1 during the Ca2+‐uptake experiments at 37°C. However, the cGMP‐dependent thiophosphorylation failed to influence the Ca2+‐uptake properties of sarcolemmal vesicles. The results show that the Ca2+ pump of smooth muscle plasma membrane is not a direct target of the cGMP‐dependent protein kinase and is not regulated by the cGMP‐dependent phosphorylation of membrane proteins.
Secondary structure and expression in vivo and in vitro of messenger RNAs into which upstream AUG codons have been inserted
Authors: van Duijn Luuk P. , HOLSAPPEL Siger , KASPERAITIS Marcellé , BUNSCHOTEN Hans , KONINGS Danielle , VOORMA Harry O.
Published: March 2005

We wanted to discover whether the conformation of the mRNA leader sequence is involved in translational fidelity. For this purpose we constructed several mutants of Semliki Forest virus 26s mRNA and inserted AUG codons into the leader sequence. We then analyzed the results of in vitro and in vivo translation of these mRNAs, probed enzymatically the secondary structure and performed minimal energy folding of the transcripts. Our results indicate that the position of a hairpin in the leader sequence determines at which AUG codon downstream from that hairpin translation is initiated.
Molecular cloning and expression of salmon prolactin cDNA
Authors: SONG Shiduo , TRINH Khiet‐Yen , HEW Choy L. , HWANG Shinn‐Jia , BELKHODE Suresh , IDLER David R.
Published: March 2005

Prolactin was purified from chum salmon pituitaries. It was resolved into two variants by reverse‐phase high‐performance liquid chromatography. A cDNA library was prepared from Pacific chinook salmon pituitaries. Salmon prolactin gene was screened using a synthetic oligonucleotide based on partial protein sequence. A positive clone (PRL‐10) was identified and sequenced. It is a full‐size clone containing 1.1 kb and coding for a preprolactin of 211 amino acids. A modified prolactin plasmid (PRL‐10A), in which the 5′ untranslated sequence and the nucleotide sequence coding for the signal peptide of prolactin were deleted, was reconstructed into an expression vector using the heat‐inducible λpL promotor. Mature prolactin, a single polypeptide of 22 kDa, was efficiently expressed in the bacteria at an elevated temperature.
Inhibition of ornithine decarboxylase induction of interferon (α+β) and its reversal by dibutyryl adenosine 3′, 5′‐monophosphate
Authors: NISHIGUCHI Shuhei , OTANI Shuzo , MATSUI‐YUASA Isao , MORISAWA Seiji , MONNA Takeyuki , KUROKI Tetsuo , KOBAYASHI Kenzo
Published: March 2005

Interferon (α+β) given to C3H/HeN mice intraperitoneally inhibited increases in the activities of adenylate cyclase and ornithine decarboxylase after partial hepatectomy. The inhibition of ornithine decarboxylase was prevented by administration of dibutyryl CAMP. Core (2′‐5′)oligo(adenylate), i.e. A2′p5′A2′p5′A or (A2′P)2A, as well as interferon inhibited the increases in these two enzymes caused by partial hepatectomy. The inhibition by (A2′p)2A of ornithine decarboxylase activity was reversed by dibutyryl CAMP. These results suggested that the activity of interferon was similar to that of (A2′p)2A and that the inhibition of ornithine decarboxylase induction caused by these agents resulted from the inhibition of adenylate cyclase activity.
Pertussis toxin abolishes the inhibitory effects of prostaglandins E1, E2,I2 and F2α on hormone‐induced cAMP accumulation in cultured hepatocytes
Authors: MELIEN Øyvind , WINSNES Randi , REFSNES Magne , GLADHAUG Ivar P. , CHRISTOFFERSEN Thoralf
Published: March 2005

Several prostaglandins inhibit the cAMP response to glucagon and β‐adrenergic stimulation in hepatocytes. To probe the mechanism of this inhibition, we have examined in primary hepatocyte cultures how pretreatment with pertussis toxin (islet‐activating protein) influences the ability of the cells to respond to hormones and prostaglandins. Pertussis toxin augmented the effects of glucagon, epinephrine and isoproterenol, and also markedly enhanced the cAMP response to prostaglandin E1 (PGE1). Furthermore, whereas PGE1, PGE2, PGI2 and PGF2α, attenuated the cAMP responses to glucagon in control cultures, this inhibition was abolished in cells pretreated with pertussis toxin. A more detailed comparison was made of the effects of PGE1 and PGF2α. In cells not treated with pertussis toxin, both these prostaglandins at high concentrations reduced the cAMP response to glucagon and isoproterenol by approximately 50%, but dose‐effect curves showed that PGE1 was about 100‐fold more potent as an inhibitor than PGF2α. Pertussis toxin abolished the inhibitory effects of PGE1 and PGF2α with almost identical time and dose requirements. The results obtained with PGE1, PGE2, PG1, and PGF2α suggest that prostaglandins of different series attenuate hormone‐activable adenylate cyclase in hepatocytes through a common mechanism, dependent on the inhibitory GTP‐binding protein.
Site‐site interactions in EF‐hand calcium‐binding proteins
Authors: HOFMANN Theo , ENG Susan , LILJA Hans , DRAKENBERG Torbjörn , VOGEL Hans J. , FORSÉN Sture
Published: March 2005

Europium(III) binding to 9‐kDa calbindin from pig intestines was studied by direct excitation of the 7Fo→5Do transition of the ion and by near‐ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C‐terminal metal‐binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N‐terminal site (site I) by europium gives excitation spectra that are pH‐dependent and show a peak at 579.4 nm (peak 1 a) at pH 5 which shifts to 578.7 nm (peak 1 b) over the pH range 5–7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration.

There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1‐calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.
Expression of a constitutive form of cytochrome P450 during rat‐liver development and sexual maturation
Authors: BARROSO Margarida , DARGOUGE Olivier , LECHNER Maria Celeste
Published: March 2005

Cytochrome P450 cDNA clone designated pP450 IGC 1 has been previously isolated from a phenobarbital‐induced rat liver cDNA library, characterized and proven to correspond to a constitutive cytochrome P450 sensitive to phenobarbital stimulation. IGC 1 pDNA, as well as a unique synthetic oligonucleotide probe, were used to investigate the mRNA levels at different developmental stages (fetal, male and female rats of various ages) in parallel with the study of the expression of the liver development and male phenotype markers: albumin, α‐fetoprotein and α2u globulin mRNAs.

Cytochrome P450 IGC 1 mRNA responsiveness to phenobarbital administration was studied in animals of both sexes at different developmental stages. P450 IGC 1 mRNA was not detectable in fetuses nor in male or female rat liver before sexual maturation, becoming elevated at 45 days age, increasing up to 3 months and reaching relatively higher levels in the female liver than in the male. The concentrations of P450 IGC 1 mRNA closely paralleld the increases in α2u globulin mRNA in male liver, as analyzed by dot and Northern blot hybridization. However, P450 IGC 1 was markedly induced by phenobarbital already at 20 days age both in males and females while α2u globulin mRNA was not inducible before sexual maturation.

It is concluded that the expression of cytochrome P450 IGC 1 mRNA is associated with the sexual maturation being differently modulated in the male and in the female rat liver. This cytochrome P450 isoenzyme may play an important physiological role in sex differential steroid metabolism and susceptibility to cytotoxic and genotoxic agents.
Circular dichroic spectra of elapid cardiotoxins
Authors: GROGNET Jean‐Marc , MÉNEZ André , DRAKE Alex , HAYASHI Kyozo , MORRISON Ian E. G. , HIDER Robert C.
Published: March 2005

Cardiotoxins isolated from elapid snake venoms constitute a chemically homogeneous family of molecules. Within this group several biologically different subclasses exist. We report a comparative analysis of the structure of 20 cardiotoxins using circular dichroism, immunological methods and secondary‐structure prediction. It is shown that cardiotoxins fall within two structural subclasses. Toxins of group I are characterized by (a) CD spectra having an intense positive band close to 192.5 nm and a negative trough at 225 nm with no positive band around 230 nm, (b) strong cross‐reactivity with a polyclonal antiserum specific for Naja nigricollis toxin γ and (c) a high tendency to form a reverse turn in the region of position 11. Toxins of group II are characterized by (a) CD spectra displaying a much weaker positive band at 192.5 nm, a negative band around 210 nm and a positive band at 230 nm, (b) little cross‐reactivity with the aforementioned antiserum and (c) a high reverse‐turn potential at position 31. It is suggested that the observed differences result from differing curvatures in the antiparallel β sheet which constitutes the main secondary structure of cardiotoxins.
Purification and partial characterization of a calcium‐stimulated protease from the cyanobacterium, Anabaena variabilis
Authors: LOCKAU Wolfgang , MASSALSKY Brigitte , DIRMEIER Andrea
Published: March 2005

A calcium‐stimulated protease was purified to apparent homogeneity from the heterocyst‐forming cyanobacterium Anabaena variabilis ATCC 29413. As judged from experiments with inhibitors and chromogenic peptide substrates, the enzyme is a serine protease with a substrate specificity like trypsin. Its apparent relative molecular mass is 52000. Calcium depletion inhibits the enzymic activity by 92%. Half‐maximal activity requires about 0.5 μM free Ca2+. The enzyme binds to a hydrophobic column in a calcium‐dependent manner, indicating calcium‐induced exposure of a hydrophobic domain. The possible role of the protease in heterocyst differentiation is discussed.
Substrate specificity, regiospecificity and stereospecificity of halogenation reactions catalyzed by non‐heme‐type bromoperoxidase of Corallina pilulifera
Authors: ITOH Nobuya , HASAN A. K. M. Quamrul , IZUMI Yoshikazu , YAMADA Hideaki
Published: March 2005

Many organic compounds were found to be substrates for halogenation reactions catalyzed by the non‐heme‐type bromoperoxidase found in the red alga Corallina pilulifera. Anisole, 1‐methoxynaphthalene and thiophene were converted to o and p‐bromoanisoles, 1‐methoxy‐4‐bromonaphthalene and 2‐bromothiophene respectively. Regiospecificity of the enzymatic bromination of anisole was tested and found to be the same as in the chemical reaction with NaOBr. The enzyme also acted on substituted alkenes such as styrene, cyclohexene, trans‐cinnamic acid, trans‐cinnamyl alcohol and cis‐propenylphosphonic acid, to give the respective bromohydrin compounds or decarboxylated bromo compound. These bromohydrin compounds were always mixtures of stereoisomers. In the light of the above findings together with the previous studies concerning the halogenation mechanism, the bromoperoxidase of C. pilulifera was considered to have no specific restriction site for these substrates.
The transmembrane electrochemical gradient of Na+ as driving force for methanol oxidation in Methanosarcina barkeri
Authors: MÜLLER Volker , BLAUT Michael , GOTTSCHALK Gerhard
Published: March 2005

A sodium ion gradient (inside low) across the cytoplasmic membrane of Methanosarcina barkeri was required for methanogenesis from methanol. This could be concluded from the following results. (a) Inhibition of the Na+/H+ antiporter by K+ or amiloride led to an inhibition of methanogenesis from methanol. (b) Upon addition of the sodium ionophore monensin the Na+ gradient was abolished and at the same time methanogenesis from methanol was inhibited. (c) Methanogenesis was impaired when the Na+ gradient had the opposite orientation (inside high). All these inhibitory effects were not observed when H2 was present in addition to methanol indicating that the oxidation of methanol to CO2 was driven by a sodium‐motive force. In accordance with this, a methanol‐dependent influx of Na+ and a corresponding decrease of the membrane potential could be observed, when the Na+/H+ antiporter was inhibited by amiloride. This influx was indicative of the presence of a Na+ transport system which was functional when the oxidation of methanol had to be driven, but was not functional when H2 was present for reduction of methanol to methane.
Identification of [1‐14C]pantothenic‐acid‐mediated modified mitochondrial proteins
Authors: HUTH Walter , ARVAND Maryam , MÖLLER Ulrike
Published: March 2005

The in vivo administration of [1‐14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2‐mercaptoethanol. Two mitochondrial matrix proteins acetyl‐CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3‐oxoacyl‐CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl‐CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 1 ± 0.15 (n= 10).

After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl‐CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.
NH2‐terminal sequence of the isolated yeast ATP synthase subunit 6 reveals post‐translational cleavage
Authors: MICHON Thierry , GALANTE Monique , VELOURS Jean
Published: March 2005

The three mitochondrially translated ATP synthase subunits of Saccharomyces cerevisiae were extracted from the enzyme and from whole mitochondria using an organic solvent mixture and then purified by reverse‐phase HPLC. The amino acid composition of subunit 6 is close to the one predicted from the oli2 gene. The partial amino terminal sequence of subunit 6 reveals a post‐translational cleavage site between the Thr‐10 and Ser‐11 residues of the precursor. Thus, mature subunit 6 contains 249 amino acid residues and displays a molecular mass of 27943 Da.
Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3‐oxo, 4‐oxo and 5‐oxo esters in baker's yeast
Authors: HEIDLAS Jürgen , ENGEL Karl‐Heinz , TRESSL Roland
Published: March 2005

Two NADPH‐dependent oxidoreductases catalyzing the enantioselective reduction of 3‐oxo esters to (S)‐ and (R)‐3‐hydroxy acid esters, [hereafter called (S)‐ and (R)‐enzymes] have been purified 121‐ and 332‐fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G‐25 filtration, DEAE‐Sepharose CL‐6B chromatography, Sephadex G‐150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography.

The relative molecular mass Mr, of the (S)‐enzyme was estimated to be 48000–50000 on Sephadex G‐150 column chromatography and 48000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3‐oxo esters, 4‐oxo and 5‐oxo acids and esters enantioselectively to (S)‐hydroxy compounds in the presence of NADPH. The Km, values for ethyl 3‐oxobutyrate, ethyl 3‐oxohexanoate, 4‐oxopentanoic and 5‐oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively.

The Mr of the (R)‐enzyme, estimated by means of column chromatography on Sepharose 6B, was 800 000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200000 and 210000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3‐oxo esters to (R)‐hydroxy esters, using NADPH for coenzyme. Km, values for ethyl 3‐oxobutyrate and ethyl 3‐oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively.

Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)‐enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr, 2.4 × 106) showed no activity in catalyzing the reduction of 3‐oxo esters.
tRNA‐mediated labelling of proteins with biotin
Authors: KURZCHALIA Teymuras V. , WIEDMANN Martin , BRETER Holger , ZIMMERMANN Wolfgang , BAUSCHKE Eberhardt , RAPOPORT Tom A.
Published: March 2005

We have developed a new method for the rapid and sensitive detection of cell‐free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl‐tRNA that is modified in the ɛ‐position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non‐radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell‐free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues.

The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from ∼0.8 nm to ∼2 nm after modification).
The final step in methane formation
Authors: ELLERMANN Joachim , HEDDERICH Reiner , BÖCHER Reinhard , THAUER Rudolf K.
Published: March 2005

Methyl‐coenzyme M reductase (= component C) from Methanobacterium thermoautotrophicum (strain Marburg) was highly purified via anaerobic fast protein liquid chromatography on columns of Mono Q and Superose 6. The enzyme was found to catalyze the reduction of methylcoenzyme M (CH3‐S‐CoM) with N‐7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP = component B) to CH4. The mixed disulfide of H‐S‐CoM and H‐S‐HTP (CoM‐S‐S‐HTP) was the other major product formed. The specific activity was up to 75 nmol min−1 mg protein−1. In the presence of dithiothreitol and of reduced corrinoids or titanium(III) citrate the specific rate of CH3‐S‐CoM reduction to CH4 with H‐S‐HTP increased to 0.5–2 μmol min−1 mg protein−1. Under these conditions the CoM‐S‐S‐HTP formed from CH3‐S‐CoM and H‐S‐HTP was completely reduced to H‐S‐CoM and H‐S‐HTP. Methyl‐CoM reductase was specific for H‐S‐HTP as electron donor. Neither N‐6‐mercaptohexanoylthreonine phosphate (H‐S‐HxoTP) nor N‐8‐mercaptooctanoylthreonine phosphate (H‐S‐OcoTP) nor any other thiol compound could substitute for H‐S‐HTP. On the contrary, H‐S‐HxoTP (apparent Ki=0.1 μM) and H‐S‐OcoTP (apparent Ki= 15 μM) were found to be effectives inhibitors of methyl‐CoM reductase, inhibition being non‐competitive with CH3‐S‐CoM and competitive with H‐S‐HTP.
A homologous in vitro system to analyze transcription of a mouse immunoglobulin μ heavy‐chain gene
Authors: GILLER Thomas , BRUNNER Luzia , PICK Leslie , BRACK Christine
Published: March 2005

In order to investigate the molecular mechanisms of the regulation of immunoglobulin (Ig) gene transcription, a cell‐free system was developed in which a cloned mouse Ig μ heavy‐chain gene was transcribed using nuclear extracts prepared from a mouse B cell hybridoma line. To monitor transcription, an RNA · RNA hybridization assay was developed in which a 32P‐labeled, SP6‐synthesized RNA probe complementary to Ig μ RNA was hybridized to unlabeled RNA transcribed in the nuclear extract. Accurate initiation of transcription, which resulted in the protection of the RNA probe from digestion with nuclease S1, was detected by the separation of the products on denaturing polyacrylamide gels, followed by autoradiography. Using this assay, an in‐vitro‐synthesized RNA was detected. The 5′ end of the in‐vitro‐transcribed Ig μ RNA maps exactly to the same position as the 5′ end of the corresponding in vivo mRNA and its formation was sensitive to the addition of low levels of α‐amanitin (1 μg/ml), indicating transcription by RNA polymerase II.

It was shown by competition experiments with oligonucleotides containing the ‘decamer recognition site’ that this sequence interacts with (a) decamer‐binding factor(s) and plays a positive role in transcription. The competition effects of the decamer‐containing oligonucleotide appeared to be restricted to the decamer motif present in the promoter region. No effects of the enhancer region were detectable in vitro. Little or no transcriptional activity was found in transcription experiments using the Ig μ promoter and nuclear extracts prepared from HeLa cells. This suggests that tissue‐specific factors involved in Ig μ heavy‐chain gene transcription are present in the mouse B cell extracts.
Cell‐cycle‐related metabolic and enzymatic events in proliferating rat thymocytes*
Authors: BRAND Karl , AICHINGER Sabine , FORSTER Susanne , KUPPER Stefan , NEUMANN Bernhard , NÜRNBERG Wolf , OHRISCH Günther
Published: March 2005

Cell‐cycle progression of rat thymocytes stimulated with concanavalin A and interleukin 2 was monitored at 12‐h intervals by pulse labeling aliquots of the cell culture with [3H]thymidine, by measuring cellular DNA and protein content and by counting the number of cells in the cultures. The cell cycle was completed after 96 h of culture with the S phase peaking at 48 h. Early events in thymocyte activation were enhanced phosphatidylinositol turnover and the induction of ornithine decarboxylase. Concomitant changes were observed in the rates of DNA synthesis and glycolysis accompanied by a 20‐fold increase in glucose uptake 48 h after stimulation. However, the maximal increment in the glycolytic rate preceded that of DNA synthesis by 12 h. Apart from the quantitative changes which occurred during the cell‐cycle progression, there was also a change from partial aerobic glucose degradation to CO2 (26%) to almost complete anaerobic conversion of glucose to lactate (85%) and less than 3% to CO2. Glycolytic enzyme levels increased fourfold to tenfold and reached their maxima 48 h after mitogenic stimulation. Maximal increments of glycolytic enzyme activities preceded or coincided with the maximal increments of the glycolytic rate. Actinomycin D (1.5 ng/ml) completely inhibited DNA and RNA synthesis but did not show any inhibitory effect either on glycolytic enzyme induction or on enhanced glycolysis. During mitosis and return of the cells to the non‐proliferative state, all of the enhanced metabolic rates returned to their initial levels and the elevated enzyme activities were decreased also. The marked changes of metabolic rates and enzyme activities observed at the various phases of the cell cycle suggest that these biochemical events may also serve as suitable parameters for evaluating the response of lymphocytes towards mitogens and lymphokines.
New thiol inhibitors of Clostridium histolyticum collagenase
Authors: YIOTAKIS Athanasios , HATGIYANNACOU Athina , DIVE Vincent , TOMA Flavio
Published: March 2005

An extensive series of synthetic mercaptotripeptides (HS‐CH2‐CH2‐CO‐Pro‐Yaa) was prepared, and the inhibitory constants were determined on the Clostridium histolyticum collagenase. Among the factors which control the optimal binding of these inhibitors, we found that the presence of a free C‐terminal carboxylate group in the position P3′ of the compounds is of primary importance. In general, the esterification of this carboxylate group decreased the potency of the inhibitors by two orders of magnitude. We observed also that the enzyme favored the inhibitors having a long linear apolar or basic side‐chain at the position P3′. The present data suggest a large S3′ subsite of the C. histolyticum collagenase. The compound which contains a homoarginine residue at the P3′ position with a Ki of 0.2 μM proved to be the most potent synthetic inhibitor known to date for the C. histolyticum collagenase.
Topological and functional characterization of the F0I subunit of the membrane moiety of the mitochondrial H+‐ATP synthase
Authors: HOUŠTĚK Josef , KOPECKÝ Jan , ZANOTTI Franco , GUERRIERI Ferruccio , JIRILLO Emilio , CAPOZZA Giuseppe , PAPA Sergio
Published: March 2005

Using isolated polypeptides of the F0 sector of bovine heart mitochondrial H+‐ATPase, antisera were developed detecting specifically two components of F0. These two components were identified as F0I and oligomycin‐sensitivity‐conferring protein (OSCP) respectively. Both F0I and OSCP were digested by mild trypsin treatment of submitochondrial particles depleted of the catalytic part of H+‐ATPase (USMP). Proteolysis was largely prevented by binding of F1 to F0. Proteolysis of F0I resulted in the formation of three immunoreactive. membrane‐bound fragments of apparently 26 kDa, 25.5 kDa and 18 kDa, respectively, indicating that F0I contains trypsin‐accessible Arg or Lys residues located close to the end and the middle part of the protein, respectively, which are in intimate contact with F1. Digestion of USMP with trypsin resulted in depression of passive H+ conduction through F0 which could be ascribed to proteolysis of F0I.
Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera
Authors: CHUNGJATUPORNCHAI Wipa , HÖFTE Herman , SEURINCK Jozef , ANGSUTHANASOMBAT Chanan , VAECK Mark
Published: March 2005

The complete nucleotide sequence of a cloned gene encoding a 130‐kDa crystal protein of Bacillus thuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito‐specific toxin with a LC50 value of 43 ng/ml to third‐instar larvae of Aedes aegypti. Bt8 is processed by proteases or midgut extracts of mosquito larvae into toxic fragments of 68–78 kDa. Deletion mapping indicated that the active fragment of Bt8 is localized in the N‐terminal half of the protoxin molecule. The deduced amino acid sequence of Bt8 has been compared with that of Bt2, a Lepidoptera‐specific toxin, previously cloned from Bacillus thuringiensis berliner. Highly homologous amino acid stretches are present in the C‐terminal half of the proteins. The N‐terminal parts show much less sequence homology but they display a strikingly similar distribution of hydrophilic and hydrophobic amino acids. In addition, Bt8 and Bt2 show a significant immunological cross‐reaction. The data indicate that although these B.t. delta endotoxins exhibit a different insect‐host specificity, they are structurally related and might use a similar mechanism to interact with insect cell membranes.
Does rat liver 6‐phosphofructo‐1‐kinase exhibit ‘Half‐of‐the‐sites‐phosphorylation’?
Authors: DOMENECH Carlos E. , MIESKES Gottfried , SÖLING Hans‐Dieter
Published: March 2005

6‐Phosphofructo‐1‐kinase (PFK‐1) from a variety of species and organs can undergo phosphorylation by cAMP‐dependent protein kinase. In most studies the stoichiometry of the phosphorylation reaction was far below the expected minimum value of 4 mol phosphate/mol PFK‐1 tetramer. The present study with rat liver PFK‐1 and purified catalytic subunit of CAMP‐dependent protein kinase was undertaken in order to find the maximum phosphorylation stoichiometry under well‐defined conditions. Irrespective of whether PFK‐1 had been first treated with purified protein phosphatase 2C or not, no more than 1.66±0.22 mol phosphate/mol PFK‐1 tetramer was incorporated, the highest single value being 2 mol phosphate/PFK‐1 tetramer. This stoichiometry was found to be independent from the method of protein evaluation (gel dye‐binding assay or amino acid analysis) and from the concentration of PFK‐1 in the phosphorylation system (15.6 nM‐0.53 pM). The stoichiometry was not affected by the presence of allosteric ligands, fructose‐1,6–bisphosphatase or the PFK‐1‐inactivating protein. The possibility could be excluded that partial proteolysis was responsible for the incomplete phosphorylation. Two‐dimensional polyacrylamide gel electrophoresis gave no indication of the existence of two different subunits in rat liver PFK‐1. Possible reasons why rat liver PFK‐1 undergoes ‘half‐of‐the‐sites’ phosphorylation are discussed.
Binding of the delta endotoxin from Bacillus thuringiensis to brush‐border membrane vesicles of the cabbage butterfly (Pieris brassicae)
Authors: HOFMANN Christina , LÜTHY Peter , HÜTTER Ralf , PLISKA Vladimir
Published: March 2005

The insecticidal delta endotoxin of Bacillus thuringiensis was labeled with iodine‐125. Brush‐border membrane vesicles, prepared from the midgut epithelium of Pieris brassicae larvae, known to be highly susceptible to the toxin, and from a non‐target tissue: the small intestine of rat, were examined for binding of 125I‐toxin. The toxin was bound specifically only to insect vesicles. Its binding to the insect membrane system was competitively inhibited by 127I‐toxin and non‐iodinated toxin, whereas the binding of the 125I‐toxin to the mammalian membrane system was not affected by unlabeled toxin. Vesicles of P. brassicae possess two individual binding‐site populations for iodinated toxin with dissociation constants of 46 nM and 490 nM. The Hill coefficients of both sites were approximately 1 and the binding capacities were 0.2 pmol and 30 pmol/mg vesicle protein for the high and the low‐affinity sites respectively. The estimation of the dissociation constant for non‐iodinated toxin, using a competition experiment, revealed only one binding‐site population which possessed a dissociation constant of 235 nM. It is concluded that this is the binding site for the native toxin. This site was sensitive towards treatment with proteases or mixed glycosidases. It is suggested that it is a protein or a glycoprotein.
Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4
Authors: CLAESSON Hans‐Erik , HAEGGSTRÖM Jesper
Published: March 2005

Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl‐containing leukotrienes (364%, mean, n= 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4.

The increased biosynthesis of cysteinyl‐containing leukotrienes and leukotriene B4 in coincubations of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5‐lipoxygenase pathway, induced by a factor contributed by endothelial cells.

Secondly, when endothelial cells prelabeled with [35S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A4, the specific activity of the isolated cysteinyl‐containing leukotrienes were similar. Thus, transfer of leukotriene A4 from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl‐containing leukotrienes in mixed cultures of leukocytes and endothelial cells.

In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.
Fusion between vesicles from the pathway of receptor‐mediated endocytosis in a cell‐free system
Authors: WOODMAN Philip G. , WARREN Graham
Published: March 2005

Fusion between endocytic vesicles containing 125I‐transferrin and ones containing anti‐transferrin antibody was monitored by the formation of an immune complex. Fusion required ATP and only occurred between specific populations of vesicles. Cytosol was also required. Trypsinisation of the preparations or pre‐treatment with N‐ethylmaleimide abolished the fusion reaction.
Reprogrammed expression of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae
Authors: FARRELL Leigh B. , GEARING David P. , NAGLEY Phillip
Published: March 2005

A synthetic gene has been designed and constructed by total chemical synthesis as a first step in the functional relocation from the mitochondrion to the nucleus of a gene encoding subunit 9 of the yeast mitochondrial ATPase complex. This gene (NAP9) incorporates codons frequently used in nuclear genes of Saccharomyces cerevisiae and additionally includes a series of unique restriction enzyme cleavage sites to facilitate future systematic manipulations of the gene and its protein product. Following the expression of the NAP9 gene by transcription and translation in vitro, a radiolabelled protein was produced which displayed a gel electrophoretic mobility and solubility in chloroform/methanol characteristic of the authentic subunit 9 proteolipid encoded in vivo by the mitochondrial olil gene. In order to achieve import into mitochondria of yeast subunit 9, a fusion was made between the NAP9 gene and DNA encoding the cleavable presequence of the nuclearly encoded precursor to subunit 9 from Neurospora crassa. Following expression in vitro, the resultant fusion protein was imported and appropriately processed by isolated yeast mitochondria. The import of yeast subunit 9 was less efficient than that observed in parallel import experiments with yeast subunit 8 attached to the same presequence or with the naturally occurring intact N. crassa subunit 9 precursor. Yeast subunit 9 lacking a leader sequence is not imported into mitochondria but, unlike subunit 8, it does not embed itself into the outer membrane, in spite of its highly hydrophobic character.
Genomic and cDNA cloning of the human C1 inhibitor
Authors: CARTER Philip E. , DUNBAR Bryan , FOTHERGILL John E.
Published: March 2005

Amino acid sequencing of trypsin fragments of C1 inhibitor gave regions of low codon degeneracy that were used for oligonucleotide probes. Human liver cDNA libraries gave clones containing most of the protein sequence, showing that the inhibitory domain belongs to the ‘serpin’ class of protein inhibitors. Fragments of these cDNA clones were used to probe human genomic cosmid libraries. The genomic sequence was found to be about 17 × 103 base pairs, with a coding sequence of approximately 1800 base pairs containing introns at amino acid positions –6, 162, 207, 275, 321, 395, and one in the 5′ non‐coding region. There is very little similarity of intron position amongst the serpin genes. All but one of the intron positions in the C1 inhibitor structural gene correspond to surface residues if C1 inhibitor is considered to have a structure similar to the cleaved form of α1‐antiproteinase. The serine and threonine residues in the N‐terminal 100 amino acids of the sequence thought to carry complex carbohydrates are found in a single exon.
Electrostatic potential of macromolecules measured by pKa shift of a fluorophore
Authors: FRIEDRICH Klaus , WOOLLEY Paul , STEINHÄUSER Klaus G.
Published: March 2005

The procedures developed earlier (Friedrich and Woolley, preceding paper in this journal) for probing electrostatic potential with the fluorescein label were applied to transfer RNA. By using tRNA species that contain chemically reactive bases we were able to label these bases with fluorescein derivatives and thus to ‘map’ the electrostatic potential around the molecule. Both the electrostatic potential and the fluorescence emission an‐isotropy data that were obtained at the same time could be understood in terms of the well‐known, paradigmatic crystal structure of tRNAPhe. However, within the distribution of the various tRNA species, tRNAfMet appeared to occupy an extreme position, which suggests a relation between the conformation in solution and the initiation function of this molecule. Comparison with theoretical predictions by others of the electrostatic potential map of tRNA showed agreement in respect of trends, but the values of the potentials measured were orders of magnitude lower than predicted. This we attribute primarily to solvation.
Influence of uracil on the conformational behaviour of RNA oligonucleotides in solution
Authors: van den HOOGEN Yvonne Th. , ERKELENS Cornelis , de VROOM Erik , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

NMR studies were carried out on some alternating pyrimidine‐purine sequences: the single‐stranded tetramers CACA and UGUG and the self‐complementary octamer CACAUGUG. Assignments, based upon COSY, homonuclear Hartmann‐Hahn, and NOESY experiments, are given for the resonances of all base protons and of several sugar protons. Chemical shift vs temperature profiles were used to obtain thermodynamic parameters for the single‐stranded stack ⇆ random coil and the duplex ⇆ random coil equilibria. The populations of N‐type conformer of the ribose rings were estimated from the observed J1′2′. Comparisons with another alternating pyrimidine‐purine sequence Um62AUm62A and with the deoxyribose counterparts d(CACA), d(TGTG) and d(CACATGTG) are given.

Previous 1H‐NMR investigations of Um62Um62A revealed that the population of bulge‐out structure diminishes compared to m62AUm62A due to the U(1)‐m62A(2) stacking interaction. In CACA a strong stacking proclivity (Tm= 310 K) together with a clear preference for N‐type ribose is observed. However, the stacking interactions in UGUG are relatively less stable (Tm= 288 K) and a bias towards S‐type sugar is present. Besides a small amount of stack, a significant contribution of bulge‐out structure is proposed for UGUG. We conclude that the nature of the pyrimidine base mainly determines the formation of bulge‐out structures. The poor stacking properties of uracil now appear to be mainly responsible for this phenomenon. Comparison with the deoxyribose counterparts shows a reasonable agreement between the Tm values of CACA and d(CACA), whereas the Tm, of UGUG (288 K) is much lower than the Tm, of d(TGTG) (315 K). It is suggested that the absence of bulge‐out structures in DNA purine‐pyrimidine‐purine sequences is related to the relatively strong stacking proclivity of dT residues compared to that of U residues.

The Tm values (average 341 K) for the duplex ⇆ random coil transition obtained for each residue of CACAUGUG appear very similar. All ribose rings, except the G(8), adopt a pure N conformer in the duplex. This is taken to mean that the differences in conformational behaviour of the constituent tetramers disappear upon duplex formation.
Stereoselectivity of the interaction of E‐ and Z‐2‐phoshoenolbutyrate with maize leaf phosphoenolpyruvate carboxylase
Authors: GONZALEZ Daniel H. , ANDREO Carlos S.
Published: March 2005

The aim of this work was to investigate the stereoselectivity of maize leaf phosphoenolpyruvate carboxylase with E‐ and Z‐2‐phosphoenolbutyrate as inhibitors and substrates. In addition, a procedure is presented for the separation of the isomers of 2‐phosphoenolbutyrate. The method is based on the different interaction of those compounds with a strong anion‐exchange high‐pressure liquid chromatography column using 50 mM potassium phosphate (pH 3) as elution buffer, and allows the obtention of pure E‐ and Z‐P‐enolbutyrate with high yield. The same system was used to identify Z‐P‐enolbutyrate as the product of the phosphorylation of 2‐oxobutyrate by rabbit muscle pyruvate kinase. In the presence of 5 mM Mg2+, both isomers of P‐enolbutyrate inhibited C4plant P‐enolpyruvate carboxylase; the values of Ki were 15–20 μM and 100–110 μM for Z‐and E‐P‐enolbutyrate, respectively. With 0.5 mM Mn2+, the Z isomer was also effective as inhibitor (Ki= 35–40 μM), while the E isomer produced activation of the carboxylase probably due to its binding at an allosteric site. Both compounds were substrates of the enzyme with similar V/Km values; however, V and Km for the two isomers were significantly different (i.e. Km= 110 μM for Z‐P‐enolbutyrate and 220 μM for E‐P‐enolbutyrate). The results indicate the existence of stereoselectivity for the binding of P‐enolbutyrate to the active site of P‐enolpyruvate carboxylase. However, this fact does not affect the use of the isomers as substrates by the plant carboxylase.
Nucleotide sequence of small chromatin‐associated RNA (fr3 RNA)
Authors: BRAJANOVIĆ‐UROŠEVIĆ Nadežda , NAORA Hiroto , MILLS Gordon C. , HOLOUBEK Viktor
Published: March 2005

A small RNA found in the fraction on non‐histone chromosomal proteins or rat liver and chicken reticulocytes [Holoubek, V., Deacon, N. J., Buckle, D. W. and Naora, H. (1983) Eur. J. Biochem. 137, 249–256] has been isolated from rat liver and then sequenced. The RNA is 30 nucleotides long and has the following composition: 5′AGUGGGGGACUGCGUUCGCGCUCUCCCCUG3′. This sequene is identical with the sequence of the last 30 nucleotides at the 3′end of small nuclear U1 RNA.
Subunits of Panulirus japonicus hemocyanin
Authors: MAKINO Nobuo , KIMURA Sadao
Published: March 2005

Structural and functional diversities of the subunits of Panulirus japonicus (spiny lobster) hemocyanin were investigated. The hemocyanin mostly exists as a hexamer in the native state. It was found that the hemocyanin is composed of three major subunits (Ib, II and III) and one minor subunit (Ia), which differ in N‐terminal sequence. In the dissociated state, the major subunits (Ib, II and III) showed no or very small Bohr effects. The O2 affinity of the subunit III was about three times as high as those of the other two.

The subunits could be reassociated into homogeneous and heterogeneous hexamers, which exhibited the cooperativity in O2 binding. The homohexamers were similar to each other in O2 affinity and the Bohr effect, though some differences were observed in the magnitude of the cooperativity. In particular, the subunit II homohexamer exhibited a high cooperativity, which was comparable to that of the native protein. The heterohexamers showed slightly higher O2 affinities and slightly lower cooperativity, as compared with the parent homohexamers.

It was concluded that there is no essential difference among the three major subunits of P. japonicus hemocyanin in the O2 binding and assembly properties.