bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis of wall tiechoic acids in Staphylococcus aureus H, Micrococcus varians and Bacillus subtilis W23
Authors: HARRINGTON Charles R. , BADDILEY James
Published: March 2005

The precursors for linkage unit (LU) synthesis in Staphylococcus aureus H were UDP‐GIcNAc, UDP‐N‐acetylmannosamine (ManNAc) and CDP‐glycerol and synthesis was stimulated by ATP. Moraprenol‐PP‐GlcNAc,‐ManNAc‐(glycerol phosphate)1–3 was formed from chemically synthesised moraprenol‐PP‐GlcNAc, UDP‐ManNAc and CDP‐glycerol in the presence of Triton X‐100. LU intermediates formed under both conditions served as acceptors for ribitol phosphate residues, from CDP‐ribitol, which comprise the main chain. The intial transfer of GlcNAc‐1‐phosphate from UDP‐GlcNAc was very sensitive to tunicamycin whereas the subsequent transfer of ManNAc from UDP‐ManNAc was not.

Poly(GlcNAc‐1‐phosphate) and LU synthesis in Micrococcus varians, with endogenous lipid acceptor, UDP‐GlcNAc and CDP‐glycerol, was stimulated by UDP‐ManNAc. Synthesis of LU on exogenous moraprenol‐PP‐GlcNAc, with Triton X‐100, was dependent on UDP‐ManNAc and CDP‐glycerol and the intermediates formed served as substrates for polymer synthesis. Membranes from Bacillus subtilis W23 had much lower levels of LU synthesis, but UDP‐ManNAc was again required for optimal synthesis in the presence of UDP‐GlcNAc and CDP‐glycerol. Conditions for LU synthesis on exogenous moraprenol‐PP‐GlcNAc were not found in this organism.

LU synthesis on endogenous acceptor in the absence of UDP‐ManNAc was explained by contamination of membranes with UDP‐GlcNAc 2‐epimerase. Under appropriate conditions, low levels of this enzyme were sufficient to convert UDP‐GlcNAc into a mixture of UDP‐Glc‐NAc and UDP‐ManNAc and account for LU synthesis. The results indicate the formation of prenol‐PP‐GlcNAc‐ManNac‐(glycerol phosphate)1–3 which is involved in the synthesis of wall teichoic acids in S. aureus H, M. varians and B. subtilis W23 and their attachment to peptidoglycan.
Nucleotide sequence of clonedcDNA specific for rat ribosomal protein L35a
Authors: TANAKA Tatsuo , WAKASUGI Kazuyoshi , KUWANO Yuh , ISHIKAWA Kiichi , OGATA Kikuo
Published: March 2005

A cDNA clone specific for rat ribosomal protein L35a, which is known to be a tRNA‐binding protein, was isolated by hybrid‐selected translation from a cDNA library made for 8‐9‐S poly(A)‐rich RNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. It consists of one base pair from the 5′ leading sequence, the entire coding sequence of 333 base pairs and 14 base pairs from the 3′ trailing sequence. The primary structure of protein L35a was deduced from the nucleotide sequence. It consists of 109 amino acids with a molecular mass of 12422. The calculated amino acid composition is consistent with that reported for the hydrolysate of L35a. The amino acid sequence showed marked homology with the reported partial sequence ofXenopus leavisribosomal protein L32, but not significant homology with Escherichia coliribosomal proteins that bind to tRNA.
Effects of putrescine on synthesis and degradation of ornithine decarboxylase in primary cultured hepatocytes
Authors: KANAMOTO Ryuhei , UTSUNOMIYA Kazunori , KAMEJI Takaaki , HAYASHI Shin‐ichi
Published: March 2005

Changes in both synthesis rate and degradation rate of ornithine decarboxylase (ODC) were pursued in primary cultures of adult rat hepatocytes during the process of ODC induction caused by asparagine and glucagon and also during the process of rapid ODC decay caused by putrescine. The synthesis rate of ODC was determined by [35S]methionine incorporation into the enzyme, which was separated afterwards by immunoprecipitation and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. The degradation rate of ODC was determined by following the decay of prelabeled ODC. The enzyme induction caused by asparagine (10 mM) and glucagon (1 μM) was due both to an increase in the synthesis rate and to a decrease in the degradation rate. Addition of 10 mM putrescine caused a rapid decay of ODC activity, which was faster than ODC decay in the presence of cycloheximide. This rapid decay in ODC activity was accompanied by slightly slower decay in ODC protein, which was due both to partial suppression of ODC synthesis and to several fold acceleration‐of ODC degradation.
Rhodamine 6G inhibits the matrix‐catalyzed processing of precursors of rat‐liver mitochondrial proteins
Authors: KUZELA Stefan , JOSTE Vigg , NELSON B. Dean
Published: March 2005

Several inner membrane proteins from rat liver mitochondria have been translated for the first time in rabbit reticulocyte lysates. These include the Rieske iron‐sulfur protein, cytochrome c1 and core protein I of the cytochrome bc1 complex, the α and β subunits of F1 ATPase, and subunit IV of cytochrome oxidase. All were translated from free polysomes as larger‐molecular‐mass precursors, and were processed to their mature forms by isolated liver mitochondria or by the isolated mitochondrial matrix fraction.

In vitro processing, catalyzed by the isolated matrix fraction, is inhibited by rhodamine 6G. The latter is a fluorescent probe, which accumulates specifically in mitochondria of whole cells and which is used extensively to visualize mitochondrial morphology. The concentration of rhodamine 6G required for inhibition in vitro is similar to that of o‐phenanthroline. Rhodamine 6G inhibits matrix‐catalyzed processing of all precursors tested, indicating that the mechanism of inhibition is common for a variety of functionally unrelated precursors. The novel action of rhodamine 6G reported here can form the basis for its inhibition of precursor processing in intact hepatoma cells [Kolarov, J. & Nelson, B. D. (1984) Eur. J. Biochem. 144, 387–392].
Nucleotide sequence and transcription of the fbc operon from Rhodopseudomonas sphaeroides
Authors: GABELLINI Nadia , SEBALD Walter
Published: March 2005

The fbc operon from Rhodopseudomonas sphaeroides encodes the three redox carriers of the ubiquinol‐cytochrome‐c reductase (b/c1 complex): FeS protein, cytochrome b and cytochrome c1 [Gabellini, N. et al. (1985) EMBO J. 2, 549‐553]. The nucleotide sequence of 3874 bp of cloned R. sphaeroides chromosomal DNA, including the three structural genes fbcF, fbcB and fbcC has been determined. The reading frames of the fbc genes could be identified readily since the encoded amino acid sequences are highly homologous with the sequences of the corresponding mitochondrial polypeptides.

Initiation and termination points for transcription have been investigated by S1 nuclease protection analysis. The transcription of the fbc operon starts approximately 240 base pairs upstream from the start codon of the fbcF gene and terminates 120 base pairs downstream from the stop codon of the fbcC gene. Nucleotide sequences resembling recognition signals for the binding and release of the RNA polymerase were identified.

The N‐terminal amino acid sequence of the mature cytochrome c1 was obtained by automated Edman degradation of the isolated subunit, confirming the fbcC reading frame and indicating that the bacterial pre‐apocytochrome c1 has a transient leader sequence including 21 residues. The N‐terminal sequence of one hydrophilic peptide of the FeS protein has been also obtained confirming the fbcF reading frame.

The deduced amino acid sequences are discussed in relation to the known primary structures of the homologous proteins from mitochondria and chloroplasts. The primary structures of the polypeptides are evaluated with respect to (a) their topology in the membrane, (b) their biogenesis, (c) the structure of the catalytic sites and (d) subunit interactions.
Purification and characterization of an inhibitor of plasminogen activator released by rat mammary adenocarcinoma cells
Authors: GRANT Adrienne J. , RAMSHAW Ian A. , BADENOCH‐JONES Peter , EICHNER Ronald D. , HUNT Nicholas H.
Published: March 2005

An inhibitor of plasminogen activator (PA) secreted by a tumorigenic, but non‐metastatic, rat mammary adenocarcinoma cell line has been purified to apparent homogeneity and characterized. It strongly inhibited human urokinase, but was 100 times less potent in inhibiting bovine trypsin and had no effect on plasmin or thrombin. A secreted, urokinase‐type PA (Mr 48000) and a cell‐associated PA from a metastatic rat adenocarcinoma cell line were also strongly inhibited. In contrast, a tissue‐type PA (Mr 66000), secreted by human melanoma cells, was only slightly inhibited. Purified inhibitor showed a band of Mr 66000 in sodium dodecyl sulphate/polyacrylamide gel electrophoresis and an isoelectric point of 4.5 after chromatofocusing. The inhibition of human urokinase was non‐competitive.
Isolation of the low‐molecular‐mass form of catechol O‐methyltransferase from rat liver and immunocytochemical localization of the enzyme in the glycogen compartment
Authors: VESER Joseph , MARTIN Rainer
Published: March 2005

The low‐molecular‐mass form of two distinct catechol O‐methyltransferase activities (S‐adenosyl‐L‐methionine:catechol O‐methyltransferase, COMT, EC 2.1.1.6) has been purified to homogeneity from rat liver using 40–70% ammonium sulfate precipitation, gel filtration on Sephadex G‐100, adsorption on hydroxyapatite C and ion‐exchange chromatography on DEAE‐Sepharose CL‐6B. The relative molecular mass Mr, determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis is 22400 ± 500. Irradiation of the enzyme in the presence‐of 8‐azido‐[methyl‐3H]AdoMet results in the specific labeling of the catalytic site of the enzyme. Photolabeling was successful with crude COMT preparations and with the isolated enzyme. Immunocytochemical studies present new information about the localization of the low‐molecular‐mass form in the liver parenchyma. Subcellularly COMT immunoreactivity could be attributed exclusively to the compartment with glycogen granules. Nucleus, mitochondria and endoplasmic reticulum showed no immunostaining.
Study of the plasma clearance of antibody – ricin‐A‐chain immunotoxins
Authors: BOURRIE Bernard J. P. , CASELLAS Pierre , BLYTHMAN Hildur E. , JANSEN Franz K.
Published: March 2005

In recent years, antibody–ricin‐A‐chain immunotoxins have been investigated as anti‐neoplastic agents. To achieve in vivo therapy it is necessay that the immunotoxin remains in circulation at a sufficiently high level for a sufficiently long time to allow binding to tumor cells to occur. Therefore, examination of the pharmacology of immunotoxins may elucidate the reasons for the poor in vivo tumoricidal effect of immunotoxin described before. In this study the plasma clearance of antibody–ricin‐A‐chain immunotoxins, after intravenous injection in animals of different species, has been examined. Sensitive and reproducible techniques were developed to monitor the level of immunotoxin and its constituents in the blood. It is shown that immunotoxins are rapidly eliminated from the bloodstrem. Neither the properties of the antibody moiety nor the nature of the linkage binding ricin A‐chain to antibody account for the disappearance of immunotoxin from the plasma. On the other hand, we found that the rapid clearance of immunotoxin is due to the mannose residues on the ricin A‐chain moiety which are specifically recognized by liver cells. When immunotoxin is administrated together with yeast mannan, which enhances the level of active immunotoxin in circulation by inhibition of liver uptake, the anti‐cancer efficacy of immunotoxin in vivo is drastically improved.
Characterization of three different flavodoxins from Azotobacter vinelandii
Authors: KLUGKIST Jan , VOORBERG Jan , HAAKER Huub , VEEGER Cees
Published: March 2005

The flavodoxins from Azotobacter vinelandii cells grown N2‐fixing and from cells grown NH4OAc have been purified and characterized. The purified flavodoxins from these cells are a mixture of three different flavodoxins (Fld I, II, III) with different primary structures. The three proteins were separated by fast protein liquid chromatography; Fld I eluted at 0.38 M K Cl, Fld II at 0.43 M K Cl and Fld III at 0.45 M K Cl. The most striking difference between the three flavodoxins was the midpoint potential (pH 7.0, 25°C) of the semiquinone/hydroquinone couple, which was −320 mV for Fld I and −500 mV for the other two flavodoxins (Fld II and Fld III).

All three flavodoxins were present in cells grown on NH4OAc. In cells grown on N2 as N source only Fld I and Fld II were found. The concentration of Fld II was 10‐fold higher in N2‐fixing cells than in cells grown on NH4OAc. Evidence has been obtained that Fld II is involved in electron transport to nitrogenase.

As will be discussed, our observation that preparations of Azotobacter flavodoxin are heterogeneous, has consequences for the published data.
Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii
Authors: KLUGKIST Jan , HAAKER Huub , VEEGER Cees
Published: March 2005

The involvement of the cytoplasmic membrane in electron transport to nitrogenase has been studied. Evidence shows that nitrogenase activity in Azotobacter vinelandii is coupled to the flux of electrons through the respiratory chain.

To obtain information about proteins involved, the changes occurring in A. vinelandii cells transferred to nitrogen‐free medium after growth on NH4Cl (derepression of nitrogenase activity) were studied. Synthesis of the nitrogenase polypeptides was detectable 5 min after transfer to nitrogen‐free medium. No nitrogenase activity could be detected until t= 20 min, whereupon a linear increase of nitrogenase activity with time was observed. Synthesis of nitrogenase was accompanied by synthesis of flavodoxin II and two membrane‐bound polypeptides of Mr 29000 and 30000. Analysis with respect to changes in membrane‐bound NAD(P)H dehydrogenase activities revealed the induction of an NADPH dehydrogenase activity, which was not detectable in membranes isolated from cells grown in the presence of NH4OAc. This induced activity was associated with the appearance of a polypeptide of Mr 29000 in the NADPH dehydrogenase complex.
Nucleotide binding by myosin in glycerol‐extracted rabbit skeletal muscle fibres at temperatures between −35°C and 10°C
Authors: TREGEAR Richard , KELLAM Stephen
Published: March 2005

Glycerol‐extracted rabbit psoas fibres were incubated at temperatures between −35°C and +10°C in a lowionic‐strength relaxing solution containing 50% ethyleneglycol, 100 μM [3H]MgATP, 1 mM [14C]mannitol and < 0.01 μM Ca2+. The fibres were then rinsed in a solution containing 1 mM ATP and the bound nucleotide eluted in trichloroacetic acid; all these operations were carried out at the cold temperature. Residual bound nucleotide was eluted with trichloroacetic acid at room temperature. The fibres were found to bind approximately 180 μM nucleotide, which is consistent with binding to the enzymatic site of myosin. The eluate, obtained in the cold, was analysed on poly(ethyleneimine)‐cellulose for its ATP and ADP content. At temperatures down to – 22°C most of the bound nucleotide was ADP and there was little variation of this fraction with temperature. As the temperature was lowered below −22°C the ATP fraction rose sharply; by −35°C it predominated. These results are similar in type to those found by Biosca et al. [(1984) Biochemistry 23, 1947–1953] on isolated subgragment 1, but are displaced to a much lower temperature range. Thus in a muscle fibre only a low thermal energy is needed for myosin to hold its nucleotide in a constant balance between ATP and ADP.
An essential requirement of cardiolipin for mitochondrial carnitine acylcarnitine translocase activity
Authors: NOËL Henri , PANDE Shri V.
Published: March 2005

The phospholipid requirement for the optimal solubilization of carnitine acylcarnitine translocase from the inner membrane vesicles of rat liver mitochondria and for its reconstitution in liposomes was investigated. At the octylglucoside‐solubilization step, the presence of cardiolipin proved superior to the other lipids tested. For reconstitution, a mixture having phosphatidylcholine, phosphatidylethanolamine and cardiolipin was found to be particularly effective. The requirement of cardiolipin at this step was met less effectively by other anionic phospholipids. Moreover, in intact mitochondria of rat liver and heart, the translocase activity was markedly inhibited by micromolar concentrations of doxorubicin, a specific cardiolipin‐binding agent.
Formation of an aspartyl phosphate intermediate in the reactions of nucleoside phosphotransferase from carrots
Authors: STELTE Beatrix , WITZEL Herbert
Published: March 2005

The nucleoside phosphotransferase from carrots forms N‐phosphorylhydroxylamine when substrates are hydrolysed in the presence of hydroxylamine. Denaturation of the enzyme after short incubation with the substrates leads to a protein, in which, after reduction with [3H]NaCNBH3 and completer hydrolysis with 6 M HCl, labelled homoserine can be detected.

The first experiment provides evidence for an activated phosphorylenzyme, the second experiment shows that the intermediate is an acyl phosphate formed by a nucleophilic attack of an aspartate β‐carboxylate group.
Topology of the binding site of blood‐clotting factors in model membranes
Authors: PRIGENT‐DACHARY Jeanne , FAUCON Jean‐Francois , BOISSEAU Michel‐René , DUFOURCQ Jean
Published: March 2005

Factors II, X and IX are blood‐clotting proteins which bind to phospholipid interfaces in the presence of Ca2+ to activate coagulation. The topology of their binding site on the membrane was investigated in two ways.

First, the transition temperature changes of equimolar mixtures of dipalmitoylglycerophosphocholine/phosphatidylserine and dimyristoylglycerophosphocholine/dipalmitoylglycerophosphoserine were examined by the fluorescence polarization of 1,6‐diphenylhexatriene. Results show that Ca2+ triggers a shift of about 3–4°C and that blood‐clotting factors further increase this shift by about 1.5 °C. This suggests that in the gel phase, Ca2+ induces some aggregation of the phosphatidylserine molecules which is reinforced by blood proteins.

Second, isothermal energy transfer experiments were performed with natural lipids in their fluid phase. The tryptophan residues of the factors were the energy donors, and pyrene covalently bound to a fatty acid chain of either phosphatidylcholine or phosphatidic acid was the energy acceptor. These pyrene‐phospholipids probe either the neutral or the acidic component of phospholipid mixtures.

It is concluded that the binding sites of the factors are constituted by both types of lipids and that their composition depends on the membrane. Factor II exhibits some specificity for acidic phospholipids and seems to be surrounded by non‐interacting zwitterionic lipids. Factor IX appears to be surrounded by statistically the same amount of charged and zwitterionic lipids.

We also demonstrate that binding can also occur without Ca2+. This Ca2+‐independent binding probably involves electrostatic and hydrophobic forces but its physiological significance remains to be elucidated.
The primary structure of the α subunit of human elongation factor 1
Authors: BRANDS Jolanda H. G. M. , MAASSEN J. Antonie , VAN HEMERT Formijn J. , AMONS Reinout , MÖLLER Wim
Published: March 2005

The primary structure of the α subunit of elongation factor 1 (EF‐ 1α) from human MOLT 4 cells was determined by cDNA sequencing. The data show that the conservation of the amino acid sequence is more than 80% when compared with yeast and Artemia EF‐1α. An inventory of amino acid sequences around the guanine‐nucleotide‐binding site in elongation factor Tu from Escherichia coli and homologous amino acid sequences in G proteins, initiation and elongation factors and proteins from the RAS family shows two regions containing conserved seqence elements.

Region I has the sequence apolar‐Xaa‐Xaa‐Xaa‐Gly‐Xaa‐Xaa‐Yaa‐Xaa‐Gly‐Lys‐Thr(Ser)‐Xaa‐Xaa‐Xaa‐Xaa‐X‐apolar. Except for RAS proteins, Yaa is always an acidic amino acid residue. Region II is characterized by the invariant sequence apolar‐apolar‐Xaa‐Xaa‐Asn‐Lys‐Xaa‐Asp. In order to facilitate sequence comparison we have used a graphic display, which is based on the hydrophilicity values of individual amino acids in a sequence.
Fractionation of DNA restriction fragments with ion‐exchangers for high‐performance liquid chromatography
Authors: MÜLLER Werner
Published: March 2005

The fractionation abilities of several ion‐exchangers of the high‐performance liquid chromatography type for two sets of DNA restriction fragments, ranging from 7 base pairs (bp) to about 650 bp and differing in their mean base composition, have been studied. The ion‐exchangers tested comprise the RPC‐5, the 5‐PW DEAE and the Mono Q as polymer‐based resins, and the Nucleogens 500 and 4000, both prepared from silica beads. The results indicate that all the ion‐exchangers except the 5‐PW DEAE perfectly separate fragment sizes up to about 90 bp, the 5‐PW DEAE separating to 45 bp only. Above 200 bp only the Mono Q resin works in a satisfactory way provided that about 100 μg DNA mixture, containing less than 25 fragments within the given size range, is loaded per milliliter of packed resin. Appreciable base‐pair specificities were detected for most of the resins which cause substantial retardations of the d(A + T)‐rich fragments with respect to the eluting salt concentration. If the latter dominate in the DNA sample, acceptable results were only obtained with the Mono Q resin when the column was operated at elevated temperature.
Ah receptor for 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin
Authors: DENISON Michael S. , HARPER Patricia A. , OKEY Allan B.
Published: March 2005

In early experiments Ah receptor appeared to be localized in cytosol when in its unoccupied state and it was thought that the receptor translocated into nuclei only when occupied by its ligands. However, a recent report [Whitlock and Galeazzi (1984) J. Biol. Chem. 259, 980–985] concluded that unoccupied Ah receptor in the intact cell was primarily located within the nucleus and that apparent ‘cytosolic’Ah receptor was a redistribution artifact caused by fractionation of cells in large volumes of buffer. We examined the effect of buffer volume and ionic strength on apparent ‘cytosolic’ versus ‘nuclear’ distribution of unoccupied Ah receptor in liver from C57BL/6J mice and Sprague‐Dawley rats as well as Hepa‐1c1c9 cells in culture. In all three systems the Ah receptor appears to shift out of the nuclear fraction and into the cytosolic fraction as the volume of buffer is increased or when the ionic strength of the buffer is increased. In each system, however, the distribution of the Ah receptor was identical to the distribution of each of three standard cytosolic marker enzymes: aldolase, glucose‐6‐phosphate dehydrogenase and lactate dehydrogenase. Co‐distribution of unoccupied Ah receptor with these cytosolic marker enzymes during fractionation at varied buffer volumes and ionic strengths makes it seem unlikely that the unoccupied receptor is predominantly a ‘nuclear’ component in intact cells. Marker enzyme data favor an interpretation that unoccupied Ah receptor is primarily cytoplasmic or that this soluble protein is in equilibrium between cytoplasm and nucleus.
L16, a bifunctional ribosomal protein and the enhancing effect of L6 and L11
Authors: BAXTER Ross Macpherson , ZAHID Nasir
Published: March 2005

L16 exhibits both peptide bond and transesterification activities when reconstituted into 2 M LiCl core particles. L6 and L11, when reconstituted in a similar manner in the absence of L16, manifest significant transesterification activity. Both L6 and L11 enhance the transesterification activity of L16; L11 being more active than L6 in this respect. However, both L6 and L11 have minimal effect on peptide bond formation when reconstituted with L16 at concentrations more than 2.5 M equivalents. Both L6 and L11 exhibit a differential effect on transesterification.

The affinity‐labelling agents, like PhCH2SO2F, diisopropylfluorophosphate and ethoxyformic anhydride, have been used to explore the role of residues in peptide bond formation and transesterification. It is proposed that the Ser‐Phe combination present in L16, L11 and L6 is involved in transesterification in addition to the single histidine in L16. The single histidine in L16 appears to be important in the catalysis of peptide bond formation and transesterification.
Enzymic synthesis of isoflavones
Authors: KOCHS Georg , GRISEBACH Hans
Published: March 2005

The NADPH and oxygen‐dependent conversion of (2S)‐naringenin to genistein catalyzed by a microsomal preparation from elicitor‐treated soybean cell suspension cultures has been resolved into two steps. In the first step (2S)‐naringenin is converted to a product (P‐2) which yields genistein in a second step. The chemical behaviour of P‐2 and its ultraviolet and mass spectral data are consistent with a 2‐hydroxyisoflavanone structure. The conversion of (2S)‐naringenin to P‐2 requires NADPH, oxygen and cytochrome P‐450. The participation of cytochrome P‐450 was demonstrated by CO inhibition of the reaction and its partial reversal by light, and by inhibition with typical cytochrome P‐450 inhibitors. On a Percoll gradient the membrane fraction which catalyzes P‐2 formation coincides with marker enzymes for the endoplasmic reticulum and with the position of cytochrome P‐450. Enzymatic activity for conversion of P‐2 to genistein is mainly present in the supernatant of the 160000 x g fraction. This reaction, formally a dehydration, does not require NADPH or oxygen.
A model for the architecture of the hemocyanin from the arthropod Squilla mantis (Crustacea, Stomatopoda)
Authors: BIJLHOLT Martha , van BRUGGEN Ernst F. J.
Published: March 2005

Squilla mantis hemocyanin is composed of two hexameric subunits but has electron microscopic profiles different from other bis‐hexameric hemocyanins, e.g. Astacus and Homarus. We distinguished three different electron microscopic profiles of S. mantis hemocyanin: two sideviews and a topview. These profiles were studied using computer image alignment and correspondence analysis [Van Heel, M. and Frank, J. (1981) Ultramicroscopy 6, 187–194]. With the results of this analysis we were able to build a three‐dimensional model for the quaternary structure of this hemocyanin. In this model the two hexamers are stacked in such a way that their hexagonal surfaces overlap to about 60% of their width. In the overlap area four subunits are arranged in two different interhexameric pairs, each forming a bridging area between the two hexamers.
Specific cleavage of the fibroblast receptor for platelet‐derived growth factor by an endogenous Ca2+‐dependent thiol protease
Authors: EK Bo , HELDIN Carl‐Henrik
Published: March 2005

Previous studies have shown that platelet‐derived growth factor (PDGF) stimulates the phosphorylation of two components in membranes prepared from human fibroblasts in the presence of Ca2+. One of these represents the 185‐kDa PDGF receptor, which undergoes autophosphorylation, and the other has an Mr of 130000. We show in this communication that the 130‐kDa component is derived from the 185‐kDa receptor via proteolysis by an endogeneous Ca2+‐dependent protease, which is dependent on a reduced ‐SH group for its activity. The 130‐kDa fragment contains several of the characteristics of the receptor, such as the PDGF‐binding site and the major autophosphorylation sites. Furthermore, the cleaved receptor retains tyrosine kinase activity.
Covalent linking of poly(ethyleneglycol)‐bound NAD with Thermus thermophilus malate dehydrogenase
Authors: EGUCHI Tamotsu , IIZUKA Takashi , KAGOTANI Tadashi , LEE Joung Hee , URABE Itaru , OKADA Hirosuke
Published: March 2005

Poly(ethyleneglycol)‐bound NAD (PEG‐NAD) was covalently linked to Thermus thermophilus malate dehydrogenase with a bifunctional reagent, 3,3′‐(1,6‐dioxo‐1,6‐hexanediyl)bis‐2‐thiazolidinethione. The covalently linked malate‐dehydrogenase–PEG–NAD complex (MDH‐PEG‐NAD) was purified by DEAE‐Sephadex column chromatography to remove unbound PEG‐NAD, and fractionated by blue‐Sepharose column chromatography into four preparations: MDH‐PEG‐NAD I, MDH‐PEG‐NAD II, MDH‐PEG‐NAD III and MDH‐PEG‐NAD IV. The average numbers of NAD moieties covalently bound per subunit of MDH‐PEG‐NAD I, MDH‐PEG‐NAD II, MDH‐PEG‐NAD III and MDH‐PEG‐NAD IV were 1.2, 1.2, 0.8 and 0.5, respectively, and the values were confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. 60–80% bound NAD moiety of these preparations of MDH‐PEG‐NAD was reduced by the enzyme moiety in the presence of L‐malate, and the specific activity of the enzyme moiety of the preparations was more than 80% that of the native enzyme.

MDH‐PEG‐NAD I has the following properties. The Km value for exogeneous NAD is three times that of the native enzyme. The coenzyme activity of its NAD moiety is 20–40% that of native NAD for alcohol and lactate dehydrogenases. The complex catalyzes the oxidation of L‐malate in the presence of the redox system of 5‐ethylphenazinium ethyl sulfate and a tetrazolium salt with a rate constant of 0.11 s−1. The coenzyme moiety of the complex can also be recycled by coupled reactions of the active site of the same complex and alcohol dehydrogenase. These results indicate that MDH‐PEG‐NAD works as an NAD(H)‐regeneration unit for coupled reactions.
Micellar enzymology
Authors: MARTINEK Karel , LEVASHOV Andrey V. , KLYACHKO Natalya , KHMELNITSKI Yuri L. , BEREZIN Ilya V.
Published: March 2005

Experimental approaches to modelling the enzymatic function of biological membranes are discussed. Emphasis is given to pseudohomogeneous systems such as proteolipid complexes and enzymes in organic solvents; the latter are solubilized with phospholipids or synthetic surfactants. Methods for producing and studying such micellar systems are considered. The key research problems of micellar enzymology are formulated and its relation to enzyme membranology is discussed. Finally, the new potentialities are noted of applied enzymology (biotechnology) offered by application of a colloidal solution of water in organic solvents as a microheterogeneous medium for enzymatic reactions.
Genes coding for the elongation factor EF‐1α in Artemia
Authors: LENSTRA Johannes A. , Van VLIET Anja , ARNBERG Annika C. , Van HEMERT Formijn J. , MÖLLER Wim
Published: March 2005

The elongation factor EF‐1α is one of the most abundant proteins in eukaryotic cells, where it catalyzes the binding of aminoacyl‐tRNA to ribosomes. The genes coding for this protein in the brine shrimp Artemia were analyzed by gene cloning, electron microscopy and chromosomal blot hybridization. There are only a few (about four) copies of one type of gene per haploid genome. These genes contain five exons divided over 104 base pairs. Local rearrangements give rise to a number of gene variants. Cross‐hybridizations of Artemia cDNA probes with yeast and Drosophila DNA revealed two different yeast EF‐1α genes and one or two different Drosophila genes, respectively.

Nucleotide sequencing revealed signals for synthesis and processing of EF‐1α transcripts as well as the exact location of exons. One interruption in the coding sequence corresponds closely to a splice junction in the gene coding for the homologous chloroplast protein EF‐Tu from Euglena gracilis, presumably of prokaryotic origin. The first exon in the chloroplast gene codes for the region of EF‐Tu that is homologous to regions of the elongation factor EF‐G and of the initiation factor IF2, respectively.
Complete structural analysis of globoseries glycolipids by two‐dimensional nuclear magnetic resonance
Authors: GASA Shinsei , NAKAMURA Mitsuru , MAKITA Akira , IKURA Mitsuhiko , HIKICHI Kunio
Published: March 2005

Combined two‐dimensional proton nuclear magnetic resonance allowed the determination of complete oligosaccharide structures of glycolipids belonging to the globo series, without any other analytical methods. Although a chemical modification by peracetylation was required for the above purpose, the derivatization permitted facile assignment of the pyranose ring proton resonances of the oligosaccharide moiety. Two‐dimensional chemical‐shift‐correlated spectroscopy of the acetylated glycolipid enabled us to elucidate the glycosidic positions from the chemical shifts of the protons at the substituted sites. The monosaccharide species were also identified from the characteristic splitting patterns of the methine protons on individual pyranose rings. The sequence of the monosaccharides was inferred from the interresidue connectivity across glycosidic linkages shown by two‐dimensional nuclear Overhauser effect spectroscopy, which also gave intraresidue interaction on the pyranose rings. The linkage sites of long oligosaccharide chains having more than five monosaccharides, such as globopentaosylceramide, were analyzed by two‐dimensional J‐relayed coherence transfer, which yielded 1,3 interactions along with 1,2 interactions.
Differential features of ribosomes and of poly(U)‐programmed cell‐free systems derived from sulphur‐dependent archaebacterial species
Authors: LONDEI Paola , ALTAMURA Sergio , CAMMARANO Piero , PETRUCCI Lavinia
Published: March 2005

The properties of poly(U)‐directed cell‐free systems developed from the sulphur‐dependent, thermophilic archaebacteria Desulfurococcus mobilis, Thermoproteus tenax, Sulfolobus solfataricus, Thermococcus celer and Thermoplasma acidophilum have been compared. All systems are truly thermophilic in requiring incubation at temperatures close to the physiological optimum for cell growth. Under optimized conditions the error frequency in tRNA selection is less than 0.4% at 80°C, and synthetic efficiencies (Phe residues polymerized per ribosome in 40 min) span from 4 for Tp. tenax, to 10 for Tc. celer, to 20–25 for D. mobilis and T. acidophilum and to 40 for S. solfataricus. According to requirements for polypeptide synthesis and to degree of stability of the ribosomal subunits' association, sulphur‐dependent thermophiles cluster into two groups. Group I organisms (D. mobilis, Tp. tenax, S. solfataricus) harbour 70‐S monomers composed of weakly associated subunits, whose poly(Phe)‐synthesizing capacity is totally dependent on added spermine while being drastically inhibited by monovalent cations. Group II organisms (Tc. celer and T. acidophilum) contain 70‐S particles composed of tightly bonded subunits, whose synthetic capacity is independent of spermine while being totally dependent on monovalent cations. Spermine promotes poly(Phe) synthesis on ribosomes of group I organisms by converting the peptidyltransferase center into an active conformation, while monovalent cations are inhibitory by preventing the interaction between the free ribosomal subunits. The closeness between Tc. celer and T. acidophilum ribosomes provides new insight on the phylogenetic placement of Thermococcaceae.
A functional link between the dark Mg‐ATPase activity and the light‐induced enzymatic cascade in rod outer segments
Authors: BENNETT Nelly
Published: March 2005

The characterization of a light‐induced scattering change in suspensions of rod fragments, which requires previous swelling of the disks by the dark Mg‐ATPase described by Uhl et al. [FEBS Lett. 107, 317–322 (1979)] is reported here. Reconstitution experiments demonstrate that this signal is dependent on the presence of G‐protein, GTP and cGMP phosphodiesterase. Fast reversal associated with regenerability requires in addition the presence of some protein(s) of the cytoplasm (probably the rhodopsin kinase) and ATP. The amount of excited rhodopsin which saturates the signal is the same as that which saturates the previously described ‘dissociation signal’ [Kühn et al. (1981) Proc. Natl Acad. Sci. USA 78, 6873–6877] associated with the formation of the phosphodiesterase activator Gαgtp (α subunit of the G‐protein with GTP bound). The kinetics of the signal is slightly slower than that of the dissociation signal and its amplitude is proportional to the extent of swelling of the disks. These results suggest that the interaction between Gαgtp and the phosphodiesterase modifies some structural feature of the disks and provide evidence for the existence of a functional link between the dark Mg‐ATPase and the light‐induced enzymatic cascade.
Characterization of monoclonal antibodies to histone 2B
Authors: WHITFIELD William G. F. , FELLOWS Gillian , TURNER Bryan M.
Published: March 2005

Two mouse monoclonal IgM antibodies have been isolated which bind to histone 2B (H2B), as shown by protein blotting and immunostaining and by solid‐phase radioimmunoassay (RIA). One of these (HBC‐7) was specific for H2B by both techniques whereas the other (2F8) cross‐reacted with histone H1 by RIA. Both antibodies failed to recognize H2B limit peptides from trypsin‐digested chromatin and did not bind to Drosophila H2B, which differs extensively from vertebrate H2B only in the N‐terminal region. These findings indicate that both antibodies recognize epitopes within the trypsin‐sensitive, N‐terminal region comprising residues 1–20. Binding of antibody HBC‐7 was inhibited by in vitro ADP‐ribosylation of H2B at glutamic acid residue 2. This strongly suggests that the epitope recognized by HBC‐7 is located at the N‐terminus of H2B, probably between residues 1 and 8. We have used solid‐phase radioimmunoassay to investigate factors which influence the accessibility of this epitope in chromatin. Removal of H1 (‘stripping’) from high‐molecular‐mass chromatin had no effect on HBC‐7 binding, nor was any difference observed between binding to stripped chromatin and to 146‐base‐pair (bp) core particles derived from it by nuclease digestion. These results suggest that accessibility of the N‐terminal region of H2B is not influenced by H1 itself or by the size or conformation of linker DNA. In contrast, binding of antibody HBC‐7 to 146‐bp core particles derived from unstripped chromatin was reduced by up to 70%. Binding was restored by exposure of these core particles to the conditions used for stripping. Analysis of the protein content of core particle preparations from stripped and unstripped chromatin suggests that these findings may be attributable to redistribution of non‐histone proteins during nuclease digestion. Pre‐treatment of high‐molecular‐mass chromatin or 146‐bp core particles with the intercalating dye ethidium bromide resulted in a severalfold increase in binding of HBC‐7. The major changes in nucleosome morphology induced by ethidium are therefore accompanied by an increase in accessibility of the N‐terminal region of H2B, possibly as a direct result of changes in the spatial relationship between H2B and core DNA.
Characterization of phage‐Xp10‐coded RNA polymerase
Authors: LIAO You‐Di , TU Jenn , FENG Teng‐Yung , KUO Tsong‐Teh
Published: March 2005

A bacteriophage‐coded RNA polymerase was isolated from bacteriophage‐Xp10‐infected Xanthomonas campestris pv. oryzae. The enzyme was purified to homogeneity through precipitation by poly(ethylene glycol) and chromatography on DEAE‐cellulose, heparin—Sepharose 4B and blue‐dextran–Sepharose 4B. It is composed of a single polypeptide of Mr 96000. The enzyme preferred denatured Xp10 DNA, calf thymus DNA, host bacterium DNA and poly[d(A‐T)] as templates. The optimal concentration of MgCl2 is 16 mM. The optimal temperature and pH are 37°C and 8.0, respectively. The Km of ATP is 26 μM. DNA, MgCl2 and four ribonucleotides were required for enzyme activity. If ATP alone was present, half of the Xp10 RNA polymerase activity was retained. The enzyme activity was inhibited by KCl, spermidine, actinomycin D, heparin, blue dextran and ethidium bromide; it was resistant to rifampicin and streptovaricin. N‐Ethylmaleimide did not affect the enzyme activity. The transcription site and product of Xp10 RNA polymerase upon Xp10 DNA were analyzed by DNA/RNA hybridization and polyacrylamide‐agarose composite gel electrophoresis. The enzyme could specifically transcribe the late region of Xp10 genome and produce two RNA bands.
Mechanism of the control of dioxygen binding in a dimeric cobalt‐substituted insect hemoglobin
Authors: YU Nai‐Teng , MACKIN THOMPSON Helen , ZEPKE Diethardt , GERSONDE Klaus
Published: March 2005

Resonance Raman spectroscopy has been used to investigate the allosteric control mechanism for O2 binding in a cobalt‐substituted dimeric insect hemoglobin (CTT II), which exhibits a large Bohr effect due to a pH‐induced transition between two ligand affinity states. Substitution of cobalt for iron in CTT II does not modify the Bohr effect, but permits the resonance enhancement (hence the detection) of Raman lines corresponding to the vibrations of the axial ligand‐cobalt bonds.

Using 16O2/18O2 isotope substitution the O‐O and Co‐O2 stretching and the Co‐O‐O bending mode have been assigned to the two affinity states of this hemoglobin: v (O‐O) changes from 1152 cm −1 (pH 5.5; t conformation) to about 1125 cm−1 (pH 9.5, r conformation), v(Co‐O2) from 512 cm−1 (pH 5.5) to 537 cm−1 (pH 9.5) and δ (Co‐O‐O) from 378 cm−1 (pH 5.5) to 390 cm−1 (pH 9.5). The Co‐Nɛ(His) stretching mode has also been detected changing from 313 cm−1 (pH 5.5) to 307 cm−1 (pH 9.5). For the first time, reciprocal behaviour between the Co‐Nɛ and Co‐O2 bonds and between the Co‐O2 and the O‐O bonds in an allosteric hemoglobin are demonstrated. Furthermore, the pH sensitivity of a vinyl bending mode in the range of 411–415 cm−1 has been investigated and shown also to reflect the t⇌r conformation transition.
Exposure of major neutral glycolipids in red cells to galactose oxidase
Authors: LAMPIO Anja , RAUVALA Heikki , GAHMBERG Carl G.
Published: March 2005

The exposure of several major red‐cell glycolipids to galactose oxidase was studied by oxidizing the cells with the enzyme and reducing them with NaB2H4. After isolation, the deuterium label was detected by mass fragmentography. 60–70% globoside in human and porcine erythrocytes was exposed as measured by this method. In contrast, asialo‐GM2 in guinea‐pig erythrocytes and Forssman glycolipid in sheep erythrocytes were mainly in a cryptic state. Neuraminidase treatment increased the incorporation of deuterium label to asialo‐GM2 4–8‐fold. A similar effect was seen in Forssman glycolipid when sheep red cells were labeled with the neuraminidase/galactose oxidase/NaB3H4 method. In contrast, the increase in labeling was only about 10–40% in porcine and human globosides, which were efficiently exposed to galactose oxidase already in native red cells.
Control of urea production, glutamine release and ammonia uptake in the perfused rat liver by the sympathetic innervation
Authors: BALLÉ Christoph , JUNGERMANN Kurt
Published: March 2005

The nervous control of hepatic urea and glutamine release and of ammonia uptake was studied in the rat liver perfused in situ.

Electrical stimulation of the nerve bundles around the hepatic artery and the portal vein resulted in a reduction of urea release, of glutamine output and of ammonia uptake. At the same time, as observed before [Hartmann et al. (1982) Eur. J. Biochem. 123, 521–526], nerve stimulation led to a decrease of portal flow as well as to an increase of glucose release and a shift of lactate uptake to output.

Noradrenaline infusion mimicked the nerve‐dependent metabolic and hemodynamic changes in a first approximation only at the highly unphysiological concentration of 0.1 μM. It was without effect at 0.01 μM, which might be reached in the sinusoids as a result of overflow from the vasculature.

In the presence of sodium nitroprusside nerve stimulation no longer reduced urea output, glutamine release and ammonia uptake or portal flow, yet it still increased glucose and lactate release.

Phentolamine clearly reduced the alterations after nervous stimulation of urea output, ammonia uptake and portal flow, while propranolol was essentially not effective. The nerve‐stimulation‐dependent reduction of glutamine release was almost abolished in the presence of phentolamine and lowered to 50% by propranolol.

Glucagon stimulated urea output but had no influence on glutamine release, ammonia uptake and portal flow. Nerve stimulation antagonized the glucagon‐stimulated urea release.

The present results suggest that in the perfused liver α‐sympathetic hepatic nerves regulate urea release, glutamine output and ammonia uptake predominantly by an indirect mechanism via hemodynamic alterations, but glucose release by a direct mechanism also in the absence of circulatory changes.
Tissue‐specific heterogeneity of the 3′‐untranslated region of L‐type pyruvate kinase mRNAs
Authors: MARIE Joëlle , SIMON Marie‐Pierre , LONE Yu‐Chun , COGNET Mireille , KAHN Axel
Published: March 2005

A single L‐type pyruvate kinase (PK) gene seems to exist per haploid genome. It is expressed in the liver, kidney and small intestine in the form of three mRNA species of 2, 2.2 and 3.2 × 103 bases (kb). All three species are polyadenylated and translatable into the same L‐type subunit. Primer extension experiments demonstrate that all three PK mRNAs have the same 5′ ends. Nuclease S1 protection experiments with various cDNA and 3′ genomic probes indicate that the different mRNA species only differ by the length of their 3′ noncoding region. The mechanism responsible for the production of the three transcripts seems to be the use of alternative unusual polyadenylation sites. Run‐on assays with specific probes recognizing only the 3.2‐kb or all three mRNA species show that the transcription proceeds across the gene with similar rate. This means that the process involved in generation of the three transcripts is a posttranscriptional event, probably due to different sites of endonucleolytic cleavage of primary transcripts extending 3′ from the gene region encoding the mature mRNAs. The ratio between the different PK mRNA species is, to a certain extent, tissue‐specific and changes with development. The role of an ‘identifier sequence’ located in the 3′ noncoding sequence of the 3.2‐kb species in such a tissue‐specific use of alternative polyadenylation sites is discussed.
Biochemical comparison of the Neurospora crassa wild‐type and the temperature‐sensitive leucine‐auxotroph mutant leu‐5
Authors: AIRAS R. Kalervo , SCHISCHKOFF Jörg , CRAMER Friedrich
Published: March 2005

The cytoplasmic leucyl‐tRNA synthetases were purified from a wild‐type Neurospora crassa and from a temperature‐sensitive leucine‐auxotroph (leu‐5) mutant. A detailed steady‐state kinetic study of the aminoacylation of the tRNALeu from N. crassa by the purified synthetases was carried out.

These enzymes need preincubation with dithioerythritol and spermine before the assay in order to become fully active. The Kappm value for leucine was lowered by high ATP concentrations and correspondingly the Kappm, ATP was lowered by high leucine concentrations. The Kappm, Leu was lowered by high pH, a pK value of 6.7 (at 30°C) was calculated for the ionizable group affecting the Km. At the concentrations of 2 mM ATP, 20 μM leucine, 0.3 μM tRNALeu, and pH 7 the apparent Km values were Kappm, ATP= 1,3 mM, Kappm, Leu= 49 μM and Kappm, tRNA= 0.15 μM. No essentially altered cytoplasmic leucyl‐tRNA synthetase was produced by the temperature‐sensitive mutant strain when kept at 37°C.

In none of these experiments could we find any difference between the wild‐type enzyme and the enzyme from the mutant strain (wheter grown at permissive temperature, 28°C, or grown at permissive temperture for 24 h followed by growth at 37°C). We therefore think that the small difference in the Km value for leucine of the wild‐type and mutant enzyme, established in some earlier investigations, is not due to a difference in the kinetic properties of the enzyme molecules but to an external influence. The almost total lack of the mitochondrial leucyl‐tRNA synthetase in the mutant strain besides the leucine autotrophy remains the only difference between the wild‐type and mutant strains.