bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulated expression and phosphorylation of the 23–26‐kDa ras protein in the sponge Geodia cydonium
Authors: ROBITZKI Andrea , SCHRÖDER Heinz C. , UGARKOVIĆ Đurđica , KUCHINO Yoshiyuki , KURELEC Branko , GAMULIN Vera , MÜLLER Werner E. G.
Published: March 2005

We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N‐terminal region and a less conserved C‐terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c‐Ha‐ras‐1 and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln‐tRNA. The sponge ras protein was isolated and partially purified (23–26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous aggregation factor and insulin. Insulin‐mediated phosphorylation of the ras protein resulted in a decrease in its Kd with GTP from 2 μM to 80 nM. The activated ras protein displayed high GTPase activity if the partially purified protein was incubated with homologous lectin and lectin receptor molecules. These results suggest that in the sponge, ras is activated by the insulin/insulin(insulin‐like)‐receptor system. This transition enables the ras protein to interact with the lectin‐receptor/lectin complex, a process which may ultimately lead to an initiation of an intracellular signal‐transduction chain.
Stimulation of Ca2+ uptake into epididymal bull spermatozoa by analogues of amiloride
Authors: BREITBART Haim , CRAGOE Edward J. , LARDY Henry A.
Published: March 2005

Certain amiloride analogues 3′,4′‐dichlorobenzamil 2′,4′‐dimethylbenzamil and α′,2′‐benzobenzamil hydrochloride (ATBB) stimulate calcium accumulation and motility by epididymal bovine spermatozoa. This stimulation can be seen at a range of 0.1–0.4 mM, while at higher concentration there is inhibition of calcium uptake by these amiloride analogues. The amiloride derivative 5‐(4‐chlorobenzyl)‐2′,4′‐dimethylbenzamil (CBDMB), which bears a 4‐chlorobenzyl substituent on the 5‐amino nitrogen atom, did not stimulate calcium uptake. The amiloride analogue 3′,4′‐dichlorobenzamil inhibits the Na+/Ca2+ ‐exchange activity in isolated plasma membrane vesicles, and the stimulatory effect of 3′,4′‐dichlorobenzamil on calcium uptake into epididymal sperm could be seen in Na+ ‐free medium. Thus, the stimulation of Ca2+ accumulation in the cells caused by 3′,4′‐dichlorobenzamil is not a result of inhibiting the Na−‐dependent Ca2‐ clearance. There is no stimulation of Ca2+ uptake into ejaculated cells by adding 3′,4′‐dichlorobenzamil, which is not due to the presence of the calcium‐transport inhibitor (caltrin) in these cells [Rufo, G. A., Schoff, P. K. & Lardy, H. A. (1984) J. Biol. Chem. 259, 2547–2552].

The stimulatory effect of 3′,4′‐dichlorobenzamil on Ca2+ uptake is inhibited by the voltage‐dependent Ca2+‐channel blockers nifedipin and diltiazem. This indicates that the stimulation of Ca2+ uptake by the amiloride analogues is due to the activation of a voltage‐dependent Ca2+ channel of the plasma membrane.
Sorting pathways of mitochondrial inner membrane proteins
Authors: MAHLKE Kerstin , PFANNER Nikolaus , MARTIN Jörg , HORWICH Arthur L. , HARTL Franz‐Ulrich , NEUPERT Walter
Published: March 2005

Two distinct pathways of sorting and assembly of nuclear‐encoded mitochondrial inner membrane proteins are described. In the first pathway, precursor proteins that carry amino‐terminal targeting signals are initially translocated via contact sites between both mitochondrial membranes into the mitochondrial matrix. They become proteolytically processed, interact with the 60‐kDa heat‐shock protein hsp60 in the matrix and are retranslocated to the inner membrane. The sorting of subunit 9 of Neurospora crassa Fo‐ATPase has been studied as an example. Fo subunit 9 belongs to that class of nuclear‐encoded mitochondrial proteins which are evolutionarily derived from a prokaryotic ancestor according to the endosymbiont hypothesis. We suggest that after import into mitochondria, these proteins follow the ancestral sorting and assembly pathways established in prokäryotes (conservative sorting). On the other hand, ADP/ATP carrier was found not to require interaction with hsp60 for import and assembly. This agrees with previous findings that the ADP/ATP carrier possesses non‐amino‐terminal targeting signals and uses a different import receptor to other mitochondrial precursor proteins. It is proposed that the ADP/ATP carrier represents a class of mitochondrial inner membrane proteins which do not have a prokaryotic equivalent and thus appear to follow a non‐conservative sorting pathway.
Subunit constitution of carbonic anhydrase from Chlamydomonas reinhardtii
Authors: KAMO Tomoko , SHIMOGAWARA Kousuke , FUKUZAWA Hideya , MUTO Shoshi , MIYACHI Shigetoh
Published: March 2005

Carbonic anhydrase purified from the cell surface of Chlamydomonas reinhardtii was inactivated by treatment with dithiothreitol. This treatment caused dissociation of the holoenzyme into 35‐kDa (A) and 4‐kDa (B) subunits as revealed by SDS/PAGE. The 35‐kDa subunit was further separated into two components A1 (35 kDa) and A2 (36.5 kDa) by SDS/PAGE using a gradient gel. These two components have the same amino acid sequence up to at least the 10th amino acid from the N‐terminus. The molecular masses were estimated at 76 kDa and 35 kDa for the holoenzyme and the large subunit, respectively, and the molar ratio of the former to the latter at 1:2, by using the techniques of low‐angle laser light‐scattering photometry and precision differential refractometry combined with gel‐filtration HPLC. The molar ratio of the 35‐kDa/4‐kDa subunits was estimated at 1:1 the gelfiltration HPLC monitored with precision differential refractometry. Atomic‐absorption spectrophotometry revealed that the holoenzyme contains two atoms of zinc. These results suggest that the holoenzyme is a heterotetramer composed of two large subunits (A1 and A2) and two small subunits (B).
Techniques in plant molecular biology – progress and problems
Authors: WALDEN Richard , SCHELL Jeff
Published: March 2005

Progress in plant molecular biology has been dependent on efficient methods of introducing foreign DNA into plant cells. Gene transfer into plant cells can be achieved by either direct uptake of DNA or the natural process of gene transfer carried out by the soil bacterium Agrobacterium. Versatile gene‐transfer vectors have been developed for use with Agrobacterium and more recently vectors based on the genomes of plant viruses have become available. Using this technology the expression of foreign DNA, the functional analysis of plant DNA sequences, the investigation of the mechanism of viral DNA replication and cell to cell spread, as well as the study of transposition, can be carried out. In addition, the versatility of the gene‐transfer vectors is such that they may be used to isolate genes not amenable to isolation using conventional protocols. This review concentrates on these aspects of plant molecular biology and discusses the limitations of the experimental systems that are currently available.
Reconstitution of translocation activity for secretory proteins from solubilized components of Escherichia coli
Authors: TOKUDA Hajime , SHIOZUKA Kouichi , MIZUSHIMA Shoji
Published: March 2005

The protein translocation system of Escherichia coli was solubilized and reconstituted, using the octylglucoside dilution method, into liposomes prepared from E. coli phospholipids. SecA, ATP, phospholipids and membrane proteins were found to be essential for the translocation of a model secretory protein, uncleavable OmpF‐Lpp. Phospholipids were found to play roles not only in liposome formation but also in the stabilization of membrane proteins during the octylglucoside extraction. The effects of IgGs specific to five distinct regions of the SecY molecule on protein translocation into proteoliposomes were examined. IgGs specific to the amino‐ and carboxyl‐terminal regions of the SecY molecule strongly inhibited the translocation activity, indicating the participation of SecY in the translocation. Generation of a proton motive force due to the simultaneous reconstitution of F0F1‐ATPase was also observed in the presence of ATP. An ATP‐generating system consisting of creatine phosphate and creatine kinase significantly enhanced the formation of the proton motive force and the protein translocation activity of the proteoliposomes. Collapse of the proton motive force thus generated partially inhibited the translocation.
Participation of a non‐covalent phosphointermediate in ATP hydrolysis by the sarcoplasmic reticulum Ca2+‐transport ATPase
Authors: FASSOLD Elisabeth , HASSELBACH Wilhelm , KÜCHLER Bernd
Published: March 2005

With increasing SDS/protein ratios, covalent phosphorylation by ATP and Pi is abolished before ATP hydrolysis (Pi production) ceases. We have shown that the SDS‐dependent profiles of the decline in covalent phosphorylation by either substrate are virtually identical, reflecting a common mechanism of detergent interaction, while ATP can be hydrolysed via a non‐covalent phosphointermediate. Our studies support that the transfer of both terminal Pi from ATP, as well as Pi to its final binding site, is a multistep reaction involving electrostatic interaction with one or more amino acid side chains, including a Lys residue.
Thymosins: both nuclear and cytoplasmic proteins
Authors: WATTS Julian D. , CARY Peter D. , SAUTIERE Pierre , CRANE‐ROBINSON Colyn
Published: March 2005

A simple procedure based on perchloric acid extraction has been developed for the preparation and purification of bovine prothymosin α and thymosins β4 and β9 in high yields. Spectroscopic observations show these proteins to be non‐folding at neural pH. The cellular locations of human prothymosin α, rat parathymosin and calf thymosin β4, all so‐called ‘thymic hormones', have been studied by injection into the cytoplasm of Xenopus oocytes, followed by separate monitoring of nuclear and cytoplasmic concentrations. It is shown that human prothymosin α and rat parathymosin both migrate to the nucleus whilst thymosin β4 remains in the cytoplasm. The peptide (1–88) of calf prothymosin α is shown not to accumulate in the Xenopus nucleus, demonstrating that the C‐terminal 21 residues, which include a KKQK sequence, are required for nuclear migration. The present data, in association with existing evidence of wide tissue distribution and the lack of signal peptides, indicate that these proteins do not behave as hormones in the usual sense of the word. It is suggested that thymosin β4 should be grouped separately from the pro‐ and parathymosins.
Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment‐1
Authors: MUHLRAD Andras , CHAUSSEPIED Natalie
Published: March 2005

Myosin subfragment‐1 from rabbit skeletal muscle was digested by thermolysin at 25°, 12° and 0°C. Thermolysin cleaves subfragment‐1 heavy chain into two stable fragments, 28 kDa and 70 kDa, aligned in this order from the N‐terminus [Applegate, D. & Reisler, E. (1983) Proc. Natl Acad. Sci. USA 80, 7109–7112]. The rate of digestion at 25°C was significantly increased in the presence of MgATP and somewhat less in the presence of MgADP, or magnesium pyrophosphate. This activating effect of the nucleotides was decreased at 12°C and completely eliminated at 0°C. The results can be explained by assuming that there are two subfragment‐1 conformers [Shriver, J. W. & Sykes, B. D. (1981) Biochemistry 20, 2004–2012], and that both the addition of ATP or its analogs, and lowering the temperature, shift the conformational equilibrium in the direction that is more susceptible to thermolysin. Actin inhibited thermolysin digestion of subfragment‐1 at all three temperatures studied. Actin inhibition can be explained either by shifting the equilibrium of the conformers in the direction of the less susceptible form or by direct interference of actin with the binding of thermolysin to subfragment‐1. Actin inhibition of thermolysin digestion also prevailed when subfragment‐1 was in a ternary complex with nucleotide and actin, in both the strongly and weakly attached states. Similarly, actin inhibited the digestion of subfragment‐1 modified by 1,4‐phenylenediamine, which also forms a weakly attached complex with actin. No difference could be found in the accessibility of the thermolysin‐susceptible site of subfragment‐1 at the 28–70 kDa junction in either rigor, strongly or weakly attached states, which indicates the similarity of the structure proximal to this specific site in the three attached states.
Electron microscopic analysis of DNA forks generated by Escherichia coli DNA helicase II
Authors: WESSEL Rainer , MÜLLER Holger , HOFFMANN‐BERLING Hartmut
Published: March 2005

T7 phage DNA eroded with λ exonuclease (to create 3′‐protruding strands) or exonuclease III (to create 5′‐protruding strands) was treated under unwinding assay conditions with DNA helicase II. Single‐stranded DNA‐binding protein (of Escherichia coli or phage T4) was added to disentangle the denatured DNA and the complexes were examined in the electron microscope. DNA helicase II complexes filtered through a gel column before assay retain the ability to generate forks suggesting that DNA helicase II unwinds in a preformed complex by translocating along the bound DNA strand. The enzyme initiates preferentially at the ends of the λ‐exonuclease‐treated duplexes and is found at a fork on the initially protruding strand. It also initiates at the ends of the exonuclease‐III‐treated duplexes where, as with approximately 5% of the forks traceable back to a single‐stranded gap, it is found on the initially recessed strand. The results are consistent with the view that DNA helicase II unwinds in the 3′– 5′ direction relative to the bound strand. They also confirm that the enzyme can initiate at the end of a fully base‐paired strand. At a fork, DNA helicase II is bound as a tract of molecules of approximately 110 nm in length. Tracts of enzyme assemble from non‐cooperatively bound molecules in the presence of ATP. During unwinding, DNA helicase II apparently can translocate to the displaced strand which conceivably can deplete the leading strand of the enzyme. Continued adsorption of enzyme to DNA might replenish forks arrested by strand switch of the unwinding enzyme.
The complete primary structure of two distinct forms of human α(IX) collagen chains
Authors: MURAGAKI Yasuteru , KIMURA Tomoatsu , NINOMIYA Yoshifumi , OLSEN Bjorn Reino
Published: March 2005

Type IX collagen molecules contain three genetically distinct subunits. One of the subunits, α2(IX), contains a covalently attached glycosaminoglycan side chain. A second subunit, α1(IX), has been found to be synthesized in two forms. The two forms are generated by the alternative use of two transcription start sites and splice patterns. The two forms have been found in chicken, mouse and human but cDNAs encoding both forms have only been reported for chicken.

In the present report we describe the isolation of cDNA clones encoding the complete translated portion of both forms of human α1(IX) collagen chains. Nucleotide sequence analysis has permitted the determination of the primary structure of both forms. These probes and sequences should prove useful in future studies of chondrodysplasias involving type IX collagen.
The complete amino‐acid sequences of human ganglioside GM2 activator protein and cerebroside sulfate activator protein
Authors: FÜRST Werner , SCHUBERT Jörg , MACHLEIDT Werner , MEYER Helmut E. , SANDHOFF Konrad
Published: March 2005

The complete amino‐acid sequences of human ganglioside GM2 activator protein and cerebroside sulfate activator protein have been established by Edman degradation. The GM2 activator is composed of 162 amino acids, the first two serine residues being present in only 20% of the material. A single carbohydrate chain is N‐glycosidically linked to Asn32. Three hydrophobic α‐helices may contribute to its lipid‐binding site. Three amino acids differ from those found by cDNA sequencing which may be due to a polymorphism. The cerebroside sulfate activator consists of 80 amino acids and carries one N‐linked carbohydrate chain at Asn21. The C‐terminal valine residue is lacking in about 80% of the material. In spite their similar functions, both activator proteins show no sequence or structural similarities.
De‐ADP‐ribosylation actin by Clostridium perfringens iota‐toxin and Clostridium botulinum C2 toxin
Authors: JUST Ingo , GEIPEL Udo , WEGNER Albrecht , AKTORIES Klaus
Published: March 2005

The reverse reaction of the ADP‐ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota‐toxin was studied. In the presence of nicotinamide (30–50 mM) C2 toxin and iota‐toxin decreased the radioactive labeling of [32P]ADP‐ribosylated actin and catalyzed the formation of [32P]NAD. The pH optima for both reactions were 5.5 – 6.0. Concomitant with the removal of ADP–ribose, the ability of actin to polymerize was restored and actin ATPase activity increased. Neither ADP–ribosylation nor removal of ADP–ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions. ADP–ribosylation of platelet actin by C2 toxin was reversed by iota–toxin, confirming recent reports that both toxins modify the same amino acid in actin. However, C. botulinum C2 toxin was not able to cleave ADP–ribose from skeletal muscle actin which had been incorporated by iota–toxin, corroborating the different substrate specificities of both toxins.
A novel mycolic acid in a Mycobacterium sp. from the environment
Authors: LUQUIN Marina , ROUSSEL Jean , LOPEZ‐CALAHORRA Francisco , LANÉELLE Gilbert , AUSINA Vicente , LANÉELLE Marie‐Antoinette
Published: March 2005

A fast‐growing, non‐photochromogenic mycobacterium isolated from the environment exhibited, on thinlayer chromatograms, a characteristic pattern of mycolates composed of unsaturated mycolates and also an unknown more polar component. Spectroscopic analysis and chemical degradation showed that this latter component was a novel mycolic acid containing a methoxy group at the ω‐1 position (instead of ω–17 and ω–18 in known methoxymycolates), and two double bonds in the long mero aldehyde chain (instead of one as in known mycolates with additional oxygenated groups).
Membrane topography of the subunits of ubiquinol – cytochrome‐c oxidoreductase of Saccharomyces cerevisiae
Authors: HEMRIKA Wieger , BERDEN Jan A.
Published: March 2005

The topology of the subunits of ubiquinol–cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisiae has been determined using a digitonin/proteinase K assay. With this assay we were able selectively to disrupt the mitochondrial membranes and to identify the subunits which became proteinase‐K‐sensitive after disruption of either the outer or both outer and inner membranes. This approach confirmed previous indications for the localization of the core I protein, cytochrome c1, cytochrome b, the FeS protein and the 17‐kDa subunit, while it also provided direct evidence for the site of accessibility to proteinase K of the 14‐kDa and 11‐kDa subunits. The 14‐kDa subunit faces the mitochondrial matrix and the 11‐kDa subunit faces the intermembrane space.
Construction and characterization of a recombinant murine monoclonal antibody directed against human fibrin fragment‐D dimer
Authors: VANDAMME Anne‐Mieke , BULENS Frank , BERNAR Hilde , NELLES Luc , LIJNEN Roger H. , COLLEN Désiré
Published: March 2005

cDNA libraries in λ phage were generated from the murine hybridoma secreting mAb‐15C5, a monoclonal antibody directed against fragment‐D dimer of crosslinked human fibrin [Holvoet et al. (1989) Thromb. Haemo‐stasis 61, 307–313], and clones encoding fragments of the heavy (γ1) and the light (k) chain were isolated. The κ‐chain cDNA was reconstructed from two overlapping clones encoding 20 amino acids of signal sequence and the 214 amino acids of the mature protein chain. The γ1‐chain cDNA was reconstructed from the mAb‐15C5 κ‐chain signal sequence, the mAb‐15C5 γ1 variable‐domain coding sequence and murine γ1‐gene and γ1‐chain cDNA fragments encoding the constant domains. These cDNAs were expressed in Chinese hamster ovary cells, selected cell lines were scaled up in roller bottle culture, and recombinant mAb‐15C5 was purified from the conditoned medium by chromatography on Zn‐chelate–Sepharose, protein‐A–Sepharose and insolubilized fragment‐D dimer, with a yield of 50 μg/1 and a recovery of 20%. SDS‐gel electrophoresis without reduction revealed a homogeneous band, and after reduction a light‐chain band with identical and a heavy‐chain band with a somewhat slower mobility than that of the natural mAb‐15C5. Competitive binding revealed a comparable affinity of natural and recombinant mAb‐15C5, for fibrin fragment‐D dimer.

Thus recombinant mAb‐15C5, obtained by co‐expression of the reconstructed cDNAs of the k and γ1 chain in Chinese hamster ovary cells, has very similar properties to natural mAb‐15C5. These recombinant mAb‐15C5 cDNAs may be useful for the construction of a humanized monoclonal antibody for thrombus imaging, and for targeting of thrombolytic agents to fibrin.
Molecular symmetry and arrangement of subunits in extracellular hemoglobin from Caenestheria inopinata
Authors: TSFADIA Yossi , SHAKED Isabella , DANIEL Ezra
Published: March 2005

The subunit structure of extracellular hemoglobin from the clam shrimp Caenestheria inopinata was studied using the method of cross‐linking by bifunctional reagents followed by SDS/PAGE. Two phases were distinguished in the cross‐linking by glutardialdehyde: a fast phase characterized by the appearance of two bands in electrophoresis corresponding to single polypeptide chains and cross‐linked chain pairs, and a slow phase where five bands corresponding to even numbers, 2–10, of cross‐linked polypeptide chains are observed. Theoretical curves for the distribution of protein among the various cross‐linked species were calculated assuming allowed arrangements of ten identical subunits. Equally good descriptions of the cross‐linking were provided by two models with dihedral symmetry: a ring arrangement with two types of alternating interactions and a two‐layered eclipsed arrangement with one type of interaction between subunits from different layers and another between subunits within the same layer. A way out of the ambiguity was found by carrying out the fast phase of the cross‐linking reaction with dimethyl‐3,3′‐dithiobispropionimidate · 2HCl, a bifunctional reagent containing an S‐S bond that can be cleaved by 2‐mercaptoethanol, following up with glutardialdehyde in the slow phase. The observation in the presence of 2‐mercaptoethanol of electrophoretic bands corresponding to trimeric and higher cross‐linked polypeptide chain species rules out the alternating ring and confirms the two‐layered eclipsed model. The arrangement of subunits found in this work from consecutive cross‐linking can account satisfactorily for molecular profiles previously obtained from electron microscopy of Caenestheria hemoglobin [Ilan, E., David, M. M., & Daniel, E. (1981) Biochemistry 20, 6190–6194].
Characterization of an active, fluorescein‐labelled kinesin
Authors: MARYA Pankaj K. , FRAYLICH Paul E. , EAGLES Peter A. M.
Published: March 2005

Kinesin was isolated from bovine intradural nerve roots and conjugated with 5‐(iodoacetamido)fluorescein. The modified kinesin (AF‐kinesin) supports the movement of organelles along microtubules at rates comparable with those obtained using unmodified kinesin. AF‐kinesin was purified by high‐performance liquid chromatography. SDS/PAGE analysis of the purified fraction showed the presence of a fluorescent band at the position of the 125‐kDa kinesin heavy chain. This protein promoted microtubule gliding with MgATP and with MgGTP at rates comparable to those of unlabelled kinesin. AF‐kinesin had a fluorescein/protein ratio of one. Video microscopy at low light levels was used to monitor the interactions between the analogue and microtubules. AF‐kinesin binds to microtubules in the presence of adenosine 5′‐[β,γ‐imino]triphosphate or ADP. Brief incubation of the microtubule · AF‐kinesin complex with 10 mM ATP or GTP completely removes the labelled molecule. AF‐kinesin can be inactivated in its ability to cause microtubule gliding by irradiating it with light that bleaches the bound fluorophore. When the protein is damaged in this way it still binds to microtubules and does so in the presence of ATP.
Rapid equilibrium between monomeric, dimeric and tetrameric forms of the 46‐kDa mannose 6‐phosphate receptor at 37°C
Authors: WAHEED Abdul , von FIGURA Kurt
Published: March 2005

At 4°C, detergent‐solubilized 46‐kDa mannose 6‐phosphate receptor (MPR 46) exists as a mixture of dimeric and tetrameric forms [A. Waheed, A. Hille, U. Junghans & K. von Figura (1990) Biochemistry 29, 2449–2455]. At 37°C, MPR 46 exists as a mixture of monomeric, dimeric and tetrameric forms, which can be separated by sucrose density centrifugation or chromatography on a mannose‐6‐phosphate affinity matrix. Monomeric MPR 46 did not bind to the affinity matrix, while dimeric and tetrameric receptors were eluted with increasing concentrations of mannose 6‐phosphate. Depending on the incubation temperature, dimeric MPR 46 preferentially associates to tetramers (≤16°C) or dissociates to monomers (≥25°C). The presence of mannose 6‐phosphate shifts the equilibrium to higher quaternary structure, with a maximal effect at 37°C. Incubation of dimeric or tetrameric receptor at pH 4.0 induces a rapid dissociation of about a third of the receptor with t1/2 of < 2 min, followed by a phase of slow dissociation. Readjusting the pH to 7.5 induces a rapid formation of dimers and tetramers, which is promoted by the presence of mannose 6‐phosphate or an increase of the receptor concentration. These observations may indicate that the recycling of the receptor between the Golgi apparatus, where it binds ligands at near‐neutral pH, and the acidic prelysosomes, where it releases the ligands, is associated with a change of its quaternary structure, which in turn may affect the recycling kinetics. In keeping with this assumption, we observed in baby hamster kidney cells over‐expressing MPR 46 and in membrane prepared from these cells monomeric, dimeric and tetrameric forms of the receptor.
Protozoan myoglobin from Paramecium caudatum
Authors: TSUBAMOTO Yoshiharu , MATSUOKA Ariki , YUSA Keisuke , SHIKAMA Keiji
Published: March 2005

Native oxymyoglobin (MbO2) was isolated directly from the cells of Paramecium caudatum with complete separation from metmyoglobin (metMb) on a DEAE‐cellulose column. It was examined for its spectral and stability properties. When compared with sperm whale MbO2 used as a reference, Paramecium MbO2 was found to be much more susceptible to autoxidation over a wide range of pH (4–11) in 0.1 M buffer at 25°C. Kinetic analysis has revealed that a proton‐catalyzed displacement of O2− from MbO2 by an entering water molecule can play a dominant role in the autoxidation reaction of Paramecium MbO2 to metMb, as in the case of sperm whale MbO2 involving the distal histidine as its catalytic residue. At pH values higher than 9.5, however, Paramecium MbO2 was found to be oxidized to yield a hemichrome. The spontaneous formation of hemichromes is at variance with the other known myoglobins and is therefore discussed in relation to the unusual amino acid sequence of Paramecium myoglobin having a large number of deletion [Iwaasa, H. et al. (1989) J. Mol. Biol. 208, 355–358].
Heat‐shock and related stress enhance RNA polymerase II C‐terminal‐domain kinase activity in HeLa cell extracts
Authors: LEGAGNEUX Vincent , MORANGE Michel , BENSAUDE Olivier
Published: March 2005

Changes in protein kinase activities are thought to contribute to the alteration of gene expression after heat shock and related stresses. In an attempt to identify enzymes which might be involved in both chromatin structure modification and transcriptional switch in heat‐shocked cells, we have studied protein kinase activities in heat‐shocked cell lysates with two exogenous substrates: a tetramer of a heptapeptide (heptapeptide 4) corresponding to the RNA polymerase II C‐terminal domain (CTD), and the histone H1. Heat‐shock and arsenite stress were found to stimulate strongly CTD kinase activity. H1 kinase activity was also stimulated but more weakly. Stimulation of CTD and H1 kinases occurs mainly at the early phase of recovery and by a process which is independent of protein synthesis. The stress‐induced H1 kinase is shown to contain a molecule related to the mitotic‐promoting factor (MPF) Cdc2 component. On the other hand, though Cdc2‐related protein has also been reported to be part of a CTD kinase complex, we show that the stress‐induced CTD kinase activity corresponds to a distinct entity. It is proposed that stress activation of CTD kinase might be involved in changing the specificity of RNA polymerase II.
Conformation in solution of porcine brain natriuretic peptide determined by combined use of nuclear magnetic resonance and distance geometry
Authors: INOOKA Hiroshi , KIKUCHI Takashi , ENDO Satoshi , ISHIBASHI Yoshihiro , WAKIMASU Mitsuhiro , MIZUTA Eiji
Published: March 2005

The conformation in solution of porcine brain natriuretic peptide was determined by combined use of NMR spectroscopy and distance geometry. A set of 157 inter‐proton‐distance constraints was derived from the two‐dimensional NOE spectra, and further a set of three hydrogen bond constraints was obtained from analysis of the temperature dependence of labile protons. The five structures with minimal violations were selected after performing distance‐geometry calculations starting from 40 random initial conformations. The distance‐geometry structures were further refined by the use of restrained energy minimization and restrained molecular dynamics. This structure shows a compact conformation with the carboxy‐terminal region, Asn21–Tyr26, folded back to the disulfide‐linked loop region, Cys4–Cys20. The characteristics of the conformation determined are as follows: conformations of the three segments interposed by glycine residues, which are Arg7–Ile12, Ser14–Leu18 and Cys20–Arg25, were well defined and the segments Arg7–Ile12 and Cys20–Arg25 are rather close to each other and nearly parallel. The biological significance of these local conformations is discussed on the basis of comparisons with those of atrial natriuretic peptide reported by Kobayashi et al. [Kobayashi, Y., Ohkubo, T., Kyogoku, Y., Koyama, S., Kobayashi, M. & Go, N. (1988) J. Biochem. (Tokyo) 104, 322–325].
A natural‐abundance 13C‐NMR study of Dictyostelium discoideum metabolism
Authors: KLEIN Gérard , COTTER David A. , MARTIN Jean‐Baptiste , SATRE Michel
Published: March 2005

Amoebae and spores of the cellular slime mold Dictyostelium discoideum have been investigated by natural‐abundance proton‐decoupled 13C‐NMR spectroscopy. Axenically grown vegetative amoebae have been found to contain, as prominent metabolites, the polyamines 1,3‐diaminopropane (3.2 mM), putrescine (9.4 mM) and spermidine (1.7 mM). We also detected lactic acid (4.4 mM) and the following amino acids as free metabolites in concentrations ranging over 1–3 mM: glycine, alanine, glutamine and glutamate. The glycogen level is highly dependent upon growth state, being below the level of NMR detection in early‐exponential cells and reaching about 110 mM glucose equivalents in plateau‐phase cells. Dormant spores contained high amounts of trehalose (50 mM), glutamine (73 mM) and glutamate (20 mM). The latter two compounds were not reported previously to be present in such high concentrations in Dictyostelium spores. Germination induced by heat‐shock activation was monitored by 13C NMR. No change in the major components occurred during the activation step. The progressive disappearance of trehalose during germination correlated with the decrease of glutamine and glutamate. In general, the data suggest that germinated spores contain a composition of free metabolites very similar to that of starved vegetative amoebae.
Casein kinase II phosphorylates DNA‐polymerase‐α–DNA‐primase without affecting its basic enzymic properties
Authors: PODUST Vladimir , BIALEK Gabriele , STERNBACH Hans , GROSSE Frank
Published: March 2005

Immunoaffinity‐purified DNA‐polymerase‐α–DNA‐primase complex from calf thymus was phosphorylated in vitro by highly purified casein kinase II from the same tissue. Specific phosphorylation of the DNA‐polymerizing α subunit and the primase‐associated γ subunit was observed. About 1 mol phosphate/mol polymerase–primase was incorporated. Despite this effect, neither the DNA polymerase nor the DNA primase activity were changed after phosphorylation by casein kinase II. Furthermore, dephosphorylation of polymerase–primase with alkaline phosphatase did not change the polymerase or the primase activity to a significant extent. Moreover, both alkaline phosphatase and casein kinase II had no effect on the processivity of DNA synthesis and on the lengths and amounts of primers formed by the DNA primase. Because DNA polymerase α maintained all its basic properties even after extensive treatment with alkaline phosphatase, it is unlikely that phosphorylation has a direct influence on the activities of the DNA‐polymerase‐α–DNA‐primase complex. The possible influence of post‐translational phosphorylation on the formation of a complex of polymerase α and its accessory proteins is discussed.
Two of the three genetic variants of goat αs1‐casein which are synthesized at a reduced level have an internal deletion possibly due to altered RNA splicing
Authors: BRIGNON Ghislaine , MAHE Marie‐Françoise , RIBADEAU‐DUMAS Bruno , MERCIER Jean‐Claude , GROSCLAUDE François
Published: March 2005

This paper describes the elucidation of the primary structure of the three genetic variants of goat αs1‐casein, αs1‐Cn D, E and F, which have been found to be associated with reduced amounts of αs1‐casein in milk. Variant E has the same electrophoretic mobility as variant B, but differs from the latter by the substitutions of Arg for Lys and of Thr for Ala at positions 100 and 195. A genetically controlled event which does not affect the amino acid sequence of this variant might be responsible for its lower rate of synthesis compared to that of αs1‐casein B. The deletion of 11 amino acids at positions 59–69 and of 37 amino acids at positions 59–95 in variant B leads to variants D and F. In both cases the deletions, which start at the same position of the polypeptide chain, include the major phosphorylation site of the protein. On the basis of sequence data for casein genes and cDNAs, it was concluded that the deletions occurring in the D and F variants are due to the exclusion of one and several exons, respectively. The observed deletions in the proteins could thus be the consequence of splice site mutations which would induce altered RNA processing and hence reduce the rate of synthesis of the casein.
In vivo13C‐NMR studies on the metabolism of the lugworm Arenicola marina
Authors: JURETSCHKE Hans‐Paul , KAMP Günter
Published: March 2005

13C‐NMR natural‐abundance spectra of specimens of Arenicola marina obtained, showed seasonal changes in the concentration of some metabolites, with the osmolite alanine as well as triacylglyceride storage compounds present at high concentrations. Glycogen was sometimes only barely detectable due to the low natural abundance level of 13C.

Glycogenic metabolism of the lugworm A. marina was studied in vivo by 13C‐NMR spectroscopy using 13C‐labelled glucose. During recovery from a hypoxic period [1‐13C]glucose was incorporated into glycogen. [1‐13C]Glucose was injected 5 h after the end of hypoxia to guarantee sufficient and reliable 13C labelling of glycogen. An earlier injection of [1‐13C]glucose led to considerably diminished incorporation of 13C‐labelled glucosyl units into glycogen, probably due to the consumption of the available glucose as fuel for ATP production. No scrambling of 13C into the C6 position of glycogen was observed, indicating a lack of gluconeogenic activity.

13C was also incorporated into the C3 positions of alanine and alanopine. To assign correctly this last 13C‐NMR resonance, the compound was synthesized biochemically.

No labelling of glycogen was observed when [3‐13C]alanine was injected into the coelomic cavity with similar incubation conditions being used.

The 13C of [1‐13C]glucose, incorporated into glycogen, showed a very low turnover rate in normoxic lugworms as shown by two 13C{1H}‐NMR spectra, one obtained 48 h after the other. On the other hand, in hypoxia lugworms the signal due to 13C‐labelled glycogen decreased very rapidly proving a high turnover rate. The disappearance of 13C from glycogen during the first 24 h of hypoxia indicates that the last glycosyl units to be synthesized are the first to be utilized.

Lugworms were quite sensitive to the 1H‐decoupling field used for obtaining the 13C{1H}‐NMR spectra, especially at 11.7 T. Using bi‐level composite‐pulse decoupling and long relaxation delays, no tissue damage or stress‐dependent phosphagen mobilization, as judged by 31P‐NMR spectroscopy, was observed.
Comparison of three classes of human liver alcohol dehydrogenase
Authors: EKLUND Hans , MÜLLER‐WILLE Per , HORJALES Eduardo , FUTER Olga , HOLMQUIST Barton , VALLEE Bert L. , HÖÖG Jan‐Olov , KAISER Rudolf , JÖRNVALL Hans
Published: March 2005

Conformational models of the three characterized classes of mammalian liver alcohol dehydrogenase were constructed using computer graphics based on the known three‐dimensional structure of the E subunit of the horse enzyme (class I) and the primary structures of the three human enzyme classes. This correlates the substrate‐binding pockets of the class I subunits (α, β and γ in the human enzyme) with those of the class II and III subunits (π and χ, respectively) for three enzymes that differ in substrate specificity, inhibition pattern and many other properties. The substrate‐binding sites exhibit pronounced differences in both shape and properties. Comparing human class I subunits with those of class II and III subunits there are no less than 8 and 10 replacements, respectively, out of 11 residues in the substrate pocket, while in the human class I isozyme variants, only 1–3 of these 11 positions differ. A single residue, Va1294, is conserved throughout. The liver alcohol dehydrogenases, with different substrate‐specificity pockets, resemble the patterns of other enzyme families such as the pancreatic serine proteases.

The inner part of the substrate cleft in the class II and III enzymes is smaller than in the horse class I enzyme, because both Ser48 and Phe93 are replaced by larger residues, Thr and Tyr, respectively. In class II, the residues in the substrate pocket are larger in about half of the positions. It is rich in aromatic residues, four Phe and one Tyr, making the substrate site distinctly smaller than in the class I subunits. In class III, the inner part of the substrate cleft is narrow but the outer part considerably wider and more polar than in the class I and II enzymes. In addition, Ser (or Thr) and Tyr in class II and III instead of His51 may influence proton abstraction/donation at the active site.
Export and purification of a cytoplasmic dimeric protein by fusion to the maltose‐binding protein of Escherichia coli
Authors: BLONDEL Arnaud , BEDOUELLE Hugues
Published: March 2005

A hybrid between the maltose‐binding protein (MalE) of Escherichia coli and the gene 5 protein (G5P) of phage M13 was constructed at the genetic level. MalE is a monomeric and periplasmic protein while G5P is dimeric and cytoplasmic. The hybrid (MalE‐G5P) was synthesized in large amounts from a multicopy plasmid and efficiently exported into the periplasmic space of E. coli. The export was dependent on the integrity of the signal peptide. MalE‐G5P was purified from a periplasmic extract by affinity chromatography on cross‐linked amylose, with a yield larger than 50000 molecules/E. coli cell. The hybrid specifically bound denatured but not double‐stranded DNA cellulose, as native G5P. Sedimentation velocity and gel‐filtration experiments showed that MalE‐G5P exists as a dimer. Thus, it was possible to efficiently translocate through the membrane a normally cytoplasmic and dimeric protein, by fusion to MalE. Moreover, the passenger protein kept its activity, specificity and quaternary structure in the purified hybrid. MalE‐G5P will enable the study of mutant G5P that no longer binds single‐stranded DNA and therefore cannot be purified by DNA‐cellulose chromatography.
Physicochemical studies of human O6‐methylguanine‐DNA methyltransferase
Authors: BHATTACHARYYA Debasish , FOOTE Robert S. , BOULDEN Amy M. , MITRA Sankar
Published: March 2005

O
6‐Methylguanine‐DNA methyltransferase, present in most organisms, removes mutagenic and carcinogenic O6‐alkylguanine from DNA by accepting the alkyl group in a stoichiometric reaction. The protein has been partially purified from human placenta. It reacts with second‐order rate constants of 2.20 × 108 and 0.067 × 108 lmol‐1 min‐1 at 37°C for duplex and single‐stranded DNA substrates, respectively. The corresponding value for the alkylated base in synthetic poly(dC, dG, m6dG) is 0.02 × 108 lmol−1 min−1. The native protein is monomeric with a molecular mass of 22–24 kDa. Methylation of the protein does not lead to a gross change in its conformation but causes a slight reduction in its isoelectric point of 6.2. Although DNA protects the protein from heat inactivation, both duplex and single‐stranded DNAs inhibit its activity in a concentration‐dependent manner. The transferase reaction rate is also strongly inhibited by salt with about 20% of the maximum rate observed in physiological ionic strength. This inhibition is nonspecific with respect to the ions of univalent salts.
Distribution of the early light‐inducible protein in the thylakoids of developing pea chloroplasts
Authors: CRONSHAGEN Udo , HERZFELD Frank
Published: March 2005

The distribution of the early light‐inducible protein (ELIP) of pea (Pisum sativum) between grana and stroma thylakoids was studied. An antibody raised against a bacterial‐expressed fusion protein containing ELIP sequences was used.

Illumination of dark‐grown pea seedlings causes an accumulation of the ELIP in the thylakoid membranes with a maximum level at 16 h. During continuous illumination exceeding 16 h the level decreases again. The fractionation of thylakoid membranes of 48‐h‐illuminated pea seedlings in grana and stroma thylakoids reveals that there is no uniform distribution of ELIP in the thylakoids. Rather 60–70% of ELIP was found in the stroma thylakoids and 30–40% in the grana thylakoids. This distribution is in accordance with that of photosystem I but not with that of photosystem II. After Triton‐X‐100 solubilization almost all ELIP is found in the photosystem‐I‐containing fraction. This also supports an association of ELIP with photosystem I.
Diabetes‐induced changes in guanine‐nucleotide‐regulatory‐protein mRNA detected using synthetic oligonucleotide probes
Authors: GRIFFITHS Susanne L. , KNOWLER John T. , HOUSLAY Miles D.
Published: March 2005

Synthetic oligonucleotide probes were designed to detect the α‐subunits of the guanine‐nucleotide‐regulatory proteins (G‐proteins) Gi‐1, Gi‐2, Gi‐3 and Gs (Gi is inhibitory and Gs is stimulatory). Each probe detected a single major mRNA species in Northern blots of RNA extracted from a variety of tissues. A probe was designed to identify the two forms of G‐protein β‐subunits, β1 and β2. This probe hybridised with a single 1.8‐kb transcript (β2) in RNA from all tissues studied except for brain, where a less‐abundant 3.4‐kb transcript (β1) was also detected. These probes were used to assess whether the induction of diabetes, using streptozotocin, altered the levels of mRNA coding for specific G‐protein components. In hepatocytes, diabetes caused a significant reduction in the number of transcripts coding for α‐Gs, α‐Gi‐2 and α‐Gi‐3; mRNA for α‐Gi‐1 was undetectable. In adipocytes, diabetes increased dramatically the mRNA coding for α‐Gi‐1 and α‐Gi‐3, whilst no significant changes occurred in the fractions coding for α‐Gi‐2 and α‐Gs. No significant changes in the mRNA coding for G‐protein α‐subunits were observed in either brain, heart, skeletal muscle or kidney. Diabetes did not cause any significant changes in the mRNA coding for β2 in any tissue or cell population studied. Such results on the relative levels of mRNA encoding G‐protein components was obtained by comparing equal amounts of total RNA from tissues of control and diabetic animals. G‐protein mRNA levels were expressed relative to ribosomal 28S RNA levels and, in some instances, relative to transcripts for a structural protein called CHO‐B. The total cellular levels of both RNA and DNA were assessed in the various tissues and cells studied. Major falls in RNA levels cell appeared to occur in hepatocytes and to a lesser extent in adipocytes and skeletal muscle. Thus major reductions in G‐protein transcripts occurred in hepatocytes. The detected changes in G‐protein mRNA are discussed in relation to the available evidence on G‐protein expression. We suggest that diabetes causes tissue‐specific changes in the levels of mRNA for particular G‐protein species; this may have consequences for the functioning of cellular signal‐transduction mechanisms in the affected tissues.
Overexpression in Escherichia coli of a methionine‐free designed interleukin‐2 receptor (Tac protein) based on a chemically cleavable fusion protein
Authors: HÜSKEN Dieter , BECKERS Thomas , ENGELS Joachim W.
Published: March 2005

Several fusion proteins of our previously chemically synthesized gene encoding the interleukin‐2‐receptor α subunit (IL‐2Rα or Tac protein) were constructed. They were designed in order to be cleavable by cyanogen bromide. Thus, the original internal methionines of the IL‐2Rα were replaced by either alanine, valine, leucine or isoleucine, based on secondary structure predictions. Additionally, aspartate at position 6 was substituted for glutamate in order to stabilize the acid‐labile Asp‐Pro bond. Direct C‐terminal fusion of total β‐galactosidase and portions thereof did not result in substantial amounts of the expected construct. Ternary fusions consisting of β‐galactosidase domains N‐ and C‐terminally fused to the mutant synthetic methionine‐free interleukin‐2 receptor α subunit (synIL‐2Rα) yielded inclusion bodies amounting to 4–7% of the total protein. This first overexpression of a type I membrane receptor can be rationalized by the known β‐galactosidase structure models. The fusion protein can be cleaved with cyanogen bromide, isolated and the resulting synIL‐2Rα detected by Western blot analysis.