bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectroscopic Studies of the C‐terminal Secretion Signal of the Serratia marcescens Haem Acquisition Protein (HasA) in Various Membrane‐Mimetic Environments
Authors: Wolff Nicolas , Delepelaire Philippe , Ghigo Jean‐Marc , Delepierre Muriel
Published: July 2004

The structure of a peptide comprising the last 56 C‐terminal residues of the Serratia marcescens haem acquisition protein (HasA) secreted by an ATP‐binding cassette exporter was examined by 1H‐NMR, circular dichroic and fluorescence spectroscopies. The peptide, which contains the secretion signal of HasA, is efficiently secreted by the HasA transporter. It is largely unstructured and flexible in aqueous buffer solution, but its helical content increases upon addition of trifluoroethanol, detergents and lipids. By circular dichroism, a stable helical conformation is observed between 20% and 70% (by vol.) trifluoroethanol. The 1H‐NMR spectrum was analysed at these two trifluoroethanol concentrations; residues 7–15, 21–30 and 40–50 were shown to form relatively stable helices. In the presence of neutral detergent, α‐helix is induced to a similar extent upon micelle formation; in this case, fluorescence data indicate that at least the N‐terminus of the peptide interacts with the micelle. In the presence of negatively charged detergent, α‐helix is induced before micelle formation and the N‐terminus of the peptide seems not to be involved in this interaction. In the presence of negatively charged liposomes, the peptide interacts with the vesicle, again inducing a helical conformation. However, the helical content remains lower than upon addition of trifluoroethanol or neutral micelles. These results are compared to those previously obtained with the secretion signal of one of the Erwinia chrysanthemi metalloproteases which are transported efficiently by the HasA transporter. Both signals exhibit similar conformational features, despite their low sequence similarity.
A Small‐Angle Neutron Scattering Study of γ‐Crystallins Near Their Isoelectric Point
Authors: Petitt Paul , Edwards Michael Eugene , Forciniti Daniel
Published: July 2004

In this paper, a small‐angle neutron scattering study of γII‐crystallins near their isoelectric point is presented. The experiments were carried out using protein concentrations of 5.7–85.7 mg/ml at temperatures in the range 11–60°C. The experimental data were analyzed using an ellipsoidal model for intra‐particle correlations and the mean spherical approximation for interparticle correlations. Our studies revealed that γII‐crystallins have a thick hydration layer, which is possibly due to the special arrangement of polar and ionic groups on their surface. The temperature scan shows that, as a result of relatively strong attractive forces, clusters of two, three, or higher oligomers are present below 20°C. Our results suggest that protein clusters, with a distinctive hydration layer, form a protein‐rich phase that separates from a protein‐lean phase as the temperature is decreased below some threshold value.
Isolation and Biochemical Characterization of Monomeric and Dimeric Photosystem II Complexes from Spinach and Their Relevance to the Organisation of Photosystem II In vivo
Authors: Hankamer Ben , Nield Jon , Zheleva Daniella , Boekema Egbert , Jansson Stefan , Barber James
Published: July 2004

Membranes enriched in photosystem II were isolated from spinach and further solubilised using n‐octyl β‐d‐glucopyranoside (OctGlc) and n‐dodecyl β‐d‐maltoside (DodCl2). The OctGlc preparation had high rates of oxygen evolution and when subjected to size‐exclusion HPLC and sucrose density gradient centrifugation, in the presence of DodGlc2, separated into dimeric (430 kDa), monomeric (236 kDa) photosystem II cores and a fraction containing photosystem II light‐harvesting complex (Lhcb) proteins. The dimeric core fraction was more stable, contained higher levels of chlorophyll, β‐carotene and plastoquinone per photosystem II reaction centre and had a higher oxygen‐evolving activity than the monomeric cores. Their subunit composition was similar (CP43, CP47, DI, D2, cytochrome b 559 and several lower‐molecular‐mass components) except that the level of 33‐kDa extrinsic protein was lower in the monomeric fraction. Direct solubilisation of photosystem‐II‐enriched membranes with DodGlc2, followed by sucrose density gradient centrifugation, yielded a super complex (700 kDa) containing the dimeric form of the photosystem II core and Lhcb proteins: Lhcbl, Lhcb2, Lhcb4 (CP29), and LhcbS (CP26). Like the dimeric and monomeric photosystem II core complexes, the photosystem II‐LHCII complex had lost the 23‐kDa and 17‐kDa extrinsic proteins, but maintained the 33‐kDa protein and the ability to evolve oxygen. It is suggested. with a proposed model, that the isolated photosystem II‐LHCII super complex represents an in vivo organisation that can sometimes form a lattice in granal membranes of the type detected by freeze‐etch electron microscopy [Seibert, M., DeWit, M. & Staehelin, L. A. (1987) J. Cell Biol. 105, 2257–22651.
Energy‐Filtered Electron Microscopy Reveals that Talin is a Highly Flexible Protein Composed of a Series of Globular Domains
Authors: Winkler Jörg , Lünsdorf Heinrich , Jockusch Brigitte M.
Published: July 2004

Talin is a multidoinain cytoskeletal protein containing discrete binding sites for acidic phospholipids, β‐integrin, actin and vinculin. Hence, it is thought to link microfilaments to the cytoplasmic membrane in cell‐matrix adhesion sites, and this should critically depend on talin structure. To obtain more information on the latter, we used energy‐filtered transmission electron microscopy of negatively stained talin purified from chicken smooth muscle. We show that in buffers of physiological ionic strength. talin adopts an elongated shape (56 ± 7 nm in length), consisting of a series of globular masses. While these compact elements, arranged like beads on a string, were of rather uniform dimensions (3.8 nm in diameter), their center‐to‐center spacings varied, indicating the flexibility of the connecting strands. The ends of the elongated molecules frequently formed loops. The images obtained are consistent with the assumption that, under the conditions used, the majority of the talin molecules are monomeric. A minor fraction appeared as dimers, composed of two chains only partially intertwined, thus giving rise to Y‐shaped particles. Electron micrographs revealed that the biochemically defined 50‐kDa N‐terminal talin head domain is composed of two globular subunits, while chemical cross‐linking provided evidence that the C‐terminal 220‐kDa fragment is solely responsible for dimerization. These results imply that in the dimeric molecules, the polypeptide chains are arranged in parallel, in contrast to what has been described for human‐platelet talin. In buffers of low ionic strength (0.02 M instead of 0.15 M KCI), the molecules collapsed into a compact shape. By showing the high flexibility and versatility of its morphology, our data favour the concept of talin as an important resilient link in microfilament–plasma‐membrane attachment.
Primary Structure of EPV20, a Secretory Glycoprotein Containing a Previously Uncharacterized Type of Domain
Authors: Larsen Lotte B. , Ravn Peter , Boisen Anni , Berglund Lars , Petersen Torben E.
Published: July 2004

A 20‐kDa glycoprotein, EPV20, was isolated from bovine milk and characterized. The primary structure was determined by cDNA and protein sequencing combined with mass spectrometry. EPV20 is a 130‐residue polypeptide synthesized with a 19‐residue signal peptide. The function of EPV20 is unknown, but it displays 79% sequence similarity to a putative protein deduced from a human testis cDNA sequence designated HE1 (human epididymis clone 1) (Kirchhoff, C., 1992. EMBL/GeneBank/DDBJ Databases, accession number X6769X). Northern blot analysis showed the bovine EPV20 to be expressed in kidney, spleen, liver and mammary gland, but remarkably not in bovine testis. The six Cys residues of EPV20 were found to be disulfide‐linked in a 1–6, 2–3 and 4–5 pattern. This disulfide arrangement has been observed in other proteins, e.g. in human prostatic acid phosphatase, but the spacing between the cystines differs. Therefore, EPV20 represents a new structure among the large group of proteins containing domains with three disulfide bonds.
Cloning, Sequencing and Developmental Expression of Phosphofructokinase from Dictyostelium discoideum
Authors: Estévez Antonio M. , Martínez‐Costa Oscar H. , Sanchez Valentina , Aragón Juan J.
Published: July 2004

Phosphofructokinase (PFK) from Dictyostelium discoideuin is a non‐allosteric enzyme that lacks any of the characteristic regulatory mechanisms of PFK from other cells. We have determined the DNA sequence and analyzed the amino acid sequence of D. discoideum PFK, as an initial step toward understanding the peculiar properties of this enzyme. Three overlapping fragments, two of cDNA and one of genomic DNA, were isolated, which together could encode the complete sequence of D. discoideum PFK. The constructed full‐length cDNA coded for a protein of 834 amino acids, with a calculated molecular mass of 92.4 kDa, which was similar to other eukaryotic and prokaryotic PFK. Alignments of the amino acid sequence with other isozymes revealed that many of the amino acid residues assigned to binding sites of substrates and allosteric effectors are conserved in this enzyme, but changes were also found that may contribute to the absence of allosteric mechanisms. A phylogenetic tree for the eukaryotic PFK family was constructed and showed that the N‐terminal domain clustered with those of yeast subunits, whereas the C‐terminal domain was more related to PFK from metazoa. Southern blotting indicated that D. discoideum PFK is encoded by a single gene. The enzyme is present throughout the life cycle of D. discoideum, with a gradual decrease of its expression during development.
Expression and Intracellular Localization of Catechol O‐methyltransferase in Transfected Mammalian Cells
Authors: Ulmanen Ismo , Peränen Johan , Tenhunen Jukka , Tilgmann Carola , Karhunen Tuula , Panula Pertti , Bernasconi Lilia , Aubry Jean‐Piere , Lundström Kenneth
Published: July 2004

The intracellular localization of soluble and membrane‐bound isoforms of rat and human catechol O‐methyltransferase (COMT) was studied by expressing the recombinant COMT proteins either separately or together in mammalian cell lines (HeLa and COS‐7 cells) and in rat primary neurons. The distribution of soluble and membrane‐bound COMT enzyme was visualized by immunocytochemistry. For comparison, the localization of native COMT was studied in rat C6 glioma cells by immunoelectron microscopy. Staining of cells expressing membrane‐bound COMT with a COMT‐specific antiserum revealed an immuno fluorescence signal in intracellular reticular structures and in the nuclear membrane. Double‐staining of the cells with antisera against proteins specific for the rough endoplasmic reticulum indicated that they colocalized with membrane‐bound COMT, suggesting that it resided in the endoplasmic reticulum. Notably, no COMT‐specific fluorescence of plasma membranes was detected. The signal in the endoplasmic reticulum was also evident in the cells expressing both recombinant COMT forms. Intracellular native COMT reaction was detected by immunoelectron microscopy in rat C6 glioma cells and an intense cytoplasmic signal was seen in the primary neurons infected with the recombinant Semliki Forest virus. The cells expressing recombinant soluble COMT revealed intense nuclear staining together with diffuse cytoplasmic immunoreactivity, suggesting that a part of soluble COMT is transported to nuclei. Western blotting from rat liver and brain revealed soluble COMT in the nuclei. Enzyme activity measurements from liver cytoplasmic and nuclear fractions suggested that about 5% of the soluble COMT resided in nuclei. The intracellular localization of both COMT forms implies that COMT acts in the cytoplasm and possibly also in the nuclear compartment, and that the physiological substrates of COMT enzymes may have to be internalized before their methylation by COMT.
Muscle Fibre Stress in Response to Exercise
Authors: Hernando Raquel , Manso Rafael
Published: July 2004

Heat‐shock or stress proteins (HSPs) are considered to play an essential role in protecting cells from stress and preparing them to survive new environmental challenges. This study investigates the induction kinetics of synthesis and accumulation of 70‐kDa stress proteins in the soleus and extensor digitorum longus (EDL) muscles of the rat following exercise, as well as the isoform transitions that take place during the post‐exercise period. Relative synthesis rates (referred to constitutively expressed stress protein HSP73) of the 70‐kDa heat‐shock proteins were greatly enhanced after a single bout of exercise in both muscles. They peaked early in the post‐exercise period and returned to resting levels after approximately 5–6 h. The levels of the inducible stress protein HSP72 in the EDL rose only transiently following exercise, while its accumulation in the soleus was more continuous and stable. The amount of HSP73 increased only transiently in both muscle types after exercise. The constitutive expression of the stress protein HSP72 in the soleus muscle was much higher than in the EDL and other tissues, while that of HSP73 was relatively constant among tissues. Rat skeletal muscle HSP72 and HSP73 were made up of at least three isoforms of the same molecular mass and very close isoelectric points, although only one radiolabelled isoform was detected. The relative proportion of the most abundant isoforms of HSP72, isoforms 1 and 2, as well as their ratio (isoform 2/isoform 1), increased during the post‐exercise period. Since isoform 2 of HSP72 partially disappeared after incubating soleus muscle extracts of exercised rats with alkaline phosphatase, these data indicate that phosphorylation of HSP72 is an early event in the stress response of skeletal muscle to exercise stress.
Properties of Truncated Forms of the Elongation Factor 1α from the Archaeon Sulfolobus solfataricus
Authors: Masullo Mariorosario , Ianniciello Giuseppe , Arcari Paolo , Bocchini Vincenzo
Published: July 2004

Two truncated forms of the Sulfolohus solfutaricus elongation factor 1α (SsEF‐1α), corresponding to the putative domains G+M, Ss (GM)EF‐1α, and G, Ss (G)EF‐1α, have been constructed by gene engineering, produced in Escherichia coli and purified. Neither truncated form was able to sustain poly(Phe) synthesis but they were able to bind guanine nucleotides with an affinity much higher with respect to that of the intact factor. However, the difference in the affinity for GDP and GTP became progressively reduced with the extent of the truncation. The values of kcat and Km for GTP of the intrinsic GTPase of Ss EF‐1α triggered by 3.6 M NaCl were not affected by the deletions. In contrast, both Ss (GM)EF‐1α and Ss (G)EF‐1α were less thermostable than the intact factor; the region of the factor most responsible for the loss of resistance against heat inactivation was the C‐terminal domain. On the other hand the domain M was the regulator of the thermophilicity of Ss EF‐1α since only Ss (G)EF‐1α showed a reduced thermo‐philicity. Remarkably, both Ss (GM)EF‐1α and Ss (G)EF‐1α were able to exchange [3]HGDP for GTP at a very high rate so that they were no more sensitive to the stimulatory effect of Ss EF‐1β, which is the nucleotide exchange factor of Ss EF‐1α.
Assignment of the Ligand Geometry and Redox Potentials of the Trihaem Ferricytochrome c3 from Desulfuromonas acetoxidans
Authors: Turner David L. , Costa Helena S. , Coutinho Isabel B. , Legall Jean , Xavier Antcinio V.
Published: July 2004

Cytochrome c551.5 is a trihaem cytochrome of the cytochrome c3 family isolated from Desulfurornonas acetoxidans. Although several X‐ray structures are available for tetrahaem cytochromes of this family, there is no X‐ray structure for trihaem cytochromes. Cytochrome c551.5 was studied in the oxidized form by means of two‐dimensional NMR. The pattern of observed interhaem NOESY connectivities is in agreement with the haem core structure previously determined by NMR for the reduced protein [Coutinho, I. B., Turner, D. L., Liu, M. Y., LeGall, J. & Xavier, A. V. (1996) J. Biol. Inorg. Chem. 1, 305–311]. The similarities found between the haem core structure and the amino acid sequence of cytochrome c551.5 and those of tetrahaem cytochromes c3 allows each of the haems to be specifically assigned in the polypeptide sequence, and the attribution of the midpoint redox potentials to the individual haems. This also allows individual redox potentials to be assigned to each haem in the NMR spectrum. The paramagnetic shifts of the 13C resonances of the haem substituents were analyzed in terms of π molecular orbitals with perturbed D4h symmetry. The parameters of this analysis have been shown to be controlled by the orientation of the axial ligands in several other bis‐His–coordinated haems and hence the ligand geometry was deduced for cytochrome c551.5. The structural analogy between the relative haem plane orientations in cytochrome c551.5 and the tetrahaem cytochromes c3 is found to extend to the axial ligands with the largest differences being in the vicinity of the deleted fourth haem, using the numbering of cytochrome c3 haems.
Effects of Minor and Major Groove‐Binding Drugs and Intercalators on the DNA Association of Minor Groove‐Binding Proteins RecA and Deoxyribonuclease I Detected by Flow Linear Dichroism
Authors: Tuite Eimer , Sehlstedt Ulrica , Hagmar Per , Nordén Bengt , Takahashi Masayuki
Published: July 2004

Linear and circular dichroic spectroscopies have been employed to investigate the effects of small DNA ligands on the interactions of two proteins which bind to the minor groove of DNA, viz. RecA protein from Escherichia coli and deoxyribonuclease 1 (bovine pancreas). Ligands representing three specific non‐covalent binding modes were investigated: 4′,6‐diamidino‐2‐phenylindole and distamycin A (minor groove binders), methyl green (major groove binder), and methylene blue, ethidium bromide and ethidium dimer (intercalators). Linear dichroism was demonstrated to be an excellent detector, in real time, of DNA double‐strand cleavage by deoxyribonuclease I. Ligands bound in all three modes interfered with the deoxyribonuclease I digestion of dsDNA, although the level of interference varied in a manner which could be related to the ligand binding site, the ligand charge appearing to be less important. In particular, the retardation of deoxyribonuclease I cleavage by the major groove binder methyl green demonstrates that accessibility to the minor groove can be affected by occupancy of the opposite groove. Binding of all three types of ligand also had marked effects on the interaction of RecA with dsDNA in the presence of non‐hydrolyzable cofactor adenosine 5′‐O‐3‐thiotriphosphate, decreasing the association rate to varying extents but with the strongest effects from ligands having some minor groove occupancy. Finally, each ligand was displaced from its DNA binding site upon completion of RecA association, again demonstrating that modification of either groove can affect the properties and behaviour of the other. The conclusions are discussed against the background of previous work on the use of small DNA ligands to probe DNA‐protein interactions.
Functional Characterization of the N‐glycosylation Sites of Human Acid Sphingomyelinase by Site‐Directed Mutagenesis
Authors: Ferlinz Klaus , Hurwitz Robert , Moczall Heidi , Lansmann Stephanie , Schuchman Edward H. , Sandhoff Konrad
Published: July 2004

Most soluble lysosomal enzymes require a mannose‐6‐phosphate recognition marker present on asparagine‐linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N‐linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N‐glycosylation site was modified by site‐directed mutagenesis and subsequently expressed in COS‐1 cells. Evidence is presented that five of these sites are used. Elimination of the four N‐terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C‐terminal N‐glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticulum/Golgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticulum/Golgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.
Characterization of the Correlation Between ATP‐Dependent Aminophospholipid Translocation and Mg2+‐ATPase Activity in Red Blood Cell Membranes
Authors: Beleznay Zsuzsa , Zachowski Alain , Devaux Philippe F. , Ott Peter
Published: July 2004

Pseudosubstrates and inhibitors of ATPases were studied with respect to their capability to modulate the kinetic behavior of Mg2+‐ATPase and aminophospholipid translocation in red blood cell ghosts. ATP was substituted by the pseudosubstrates of P‐type ATPases acetyl phosphate and p‐nitrophenyl phosphate. With both pseudosubstrates, aminophospholipid translocation from the outer to the inner leaflets of re‐sealed erythrocyte ghosts could be observed, although with a significantly decreased velocity compared to that in presence of ATP, both with respect to phosphate hydrolysis and translocation. Similarly, the apparent affinities for the pseudosubstrates were much lower than for ATP. Among the inhibitors studied, suramin acted as a competitive inhibitor of ATP towards both Mg2+‐ATPase activity and aminophospholipid translocation. However, the inhibition of translocation occurred at a higher inhibitor concentration than the inhibition of Mg2+‐ATPase activity. With elaiophylin, only a partial inhibition of Mg2+‐ATPase activity could be detected, but translocation of labeled phosphatidylserine was almost completely abolished. With eosin Y, an almost complete inhibition of both Mg2+‐ATPase activity and translocation could be achieved. The observed responses of aminophospholipid translocation to ATPase inhibitors strongly suggest that a P‐type ATPase, part of which displays a Mg2+‐ATPase activity, is involved in aminophospholipid translocation.
Identification and Purification of Translation Initiation Factor 2 (IF2) from Thermus thermophilus
Authors: Vornlocher Hans‐Peter , Scheible Wolf‐Rüdiger , Faulhammer Heinz G. , Sprinzl Mathias
Published: July 2004

Translation initiation factor 2 (IF2) is one of three protein factors required for initiation of protein synthesis in eubacteria. The protein is responsible for binding the initiator tRNA to the ribosomal P site. IF2 is a member of the GTP/GDP‐binding protein superfamily. In the extreme thermophilic bacterium Thermus thermophilus, IF2 was identified as a 66‐kDa protein by affinity labeling and immunoblotting. The protein was purified to homogeneity. The specific activity indicates a stoichiometric IF2‐mediated binding of formylmethionine‐tRNA to 70s ribosomes. The N‐terminal amino acid sequences of the intact protein and of two proteolytic fragments of 25kDa and 40kDa were determined. Comparison with other bacterial IF2 sequences indicates a similar domain architecture in all bacterial IF2 proteins.
Quaternary Structure of the Extracellular Haemoglobin of the Lugworm Arenicola marina
Authors: Zal Franck , Green Brian N , Lallier François H , Vinogradov Serge N , Toulmond André
Published: July 2004

To elucidate the quaternary structure of the extracellular haemoglobin (Hb) of the marine polychaete Arenicola marina (lugworm) it was subjected to multi‐angle laser‐light scattering (MALLS) and to electrospray‐ionisation mass spectrometry (ESI‐MS). It was also subjected to SDS/PAGE analysis for comparative purposes. MALLS analysis gave a molecular mass of 3648 ± 24 kDa and a gyration radius of 11.3±1.7 nm. Maximum entropy analysis of the multiply charged electrospray spectra of the native, dehaemed, reduced and carbamidomethylated Hb forms, provided its complete polypeptide chain and subunit composition. We found, in the reduced condition, eight globin chains of molecular masses 15952.5 Da (a1), 15974.8 Da (a2), 15920.9 Da (b1), 16020.1 Da (b2), 16036.2 Da (b3), 16664.8 Da (c), 16983.2 Da (d1), 17033.1 Da (d2) and two linker chains L1, 25174.1 Da, and L2, 26829.7 Da. In the native Hb, chains b, c, d occur as five disulphide‐bonded trimer subunits T with masses of 49560.4 Da (T1), 49613.9 Da (T2), 49658.6 Da (T3), 49706.8 Da (T4), 49724.5 Da (T5). Linker chains L1 and L2 occur as one disulphide‐bonded homodimer 2L1 (D1) of 50323.1 Da and one disulphide‐bonded hetero‐dimer L1‐L2 (D2) of 51981.5 Da. Polypeptide chains a and d possess one free cysteine residue and chains d possess an unusual total of five cysteine residues. Semi‐quantitative analysis of ESI‐MS data allowed us to propose the following model for the one‐twelfth protomer: [(3a1)(3a2)2,T] (T corresponding to either T3, T4 or T5). From electron micrograph data T1 and T2 are probably located at the centre of the molecule as mentioned in previous studies. The Hb would thus be composed of 198 polypeptide chains with 156 globin chains and 42 linker chains, each twelfth being in contact with 3.5 linker subunits, providing a total mass of 3682 kDa including haems in agreement with the experimental molecular mass determined by MALLS. From ESI‐MS relative intensities and the model proposed above, the globid/linker ratio gave 0.71:0.29 and 0.73:0.27, respectively. The estimation of haem content by pyridine haemochromogen and by cyanmethaemoglobin (HiCN) methods also support the globin chain number provided by ESI‐MS.
Biochemical and Pharmacological Characterization of a Depressant Insect Toxin from the Venom of the Scorpion Buthacus arenicola
Authors: Cestèle Sandrine , Kopeyan Charles , Oughideni Razika , Mansuelle Pascal , Granier Claude , Rochat Hervé
Published: July 2004

A depressant toxin active on insects, Buthacus arenicola IT2, was isolated from the venom of the North African scorpion B. arenicola and its structural and pharmacological properties were investigated. B. arenicola IT2 is a single polypeptide of 61 amino acid residues, including 8 half‐cystines but no methionine and histidine, with a molecular mass of 6835 Da. Its amino acid sequence is 79–95% identical to other depressant toxins from scorpions. When injected into the cockroach Blatella germanica, B. arenicola IT2 induced a slow depressant flaccid paralysis with a LD50., of 175 ng. B. arenicola IT2 has two non‐interacting binding sites in cockroach neuronal membranes: one of high affinity (Kd1= 0.11±0.04 nM) and low capacity (Bmax1= 2.2 ±20.6 pmol/mg), and one of low affinity (Kd2= 24±27 nM) and high capacity (BmaxP = 226±292 pmol/mg). Its binding to these two sites was completely inhibited by Leiurus quinquestriatus quinquestriatus IT2, a depressant toxin from L. quinquestriatus quinquestriatus. Reciprocal‐binding experiments between B. arenicola IT2 and the excitatory insect‐toxin A. australis Hector IT revealed competition between the two toxins for the high‐affinity sites of B. arenicola IT2. B. arenicola IT2 has a higher affinity than L. quinquestriatus hebraeus IT2, a depressant toxin from L. quinquestriatus hebraeus. Thus, B. arenicola IT2 represents an interesting tool to study the receptor site for depressant toxins on insect sodium channels.
Cell Signalling Through Guanine‐Nucleotide‐Binding Regulatory Proteins (G Proteins) and Phospholipases
Authors: Exton John H.
Published: July 2004

Phospholipases are important enzymes in cell signal transduction since they hydrolyze membrane phospholipids to generate signalling molecules. Heterotrimeric guanine‐nucleotide‐binding regulatory proteins (G proteins) play a major role in their regulation by a variety of agonists that activate receptors with seven membrane‐spanning domains. Phospholipases of the C type, which hydrolyze inositol phospholipids to yield inositol trisphosphate and diacylglycerol, are regulated by the α and βγ subunits of certain heterotrimeric G proteins as well as by receptor‐associated and non‐receptor‐associated tyrosine kinases. Phospholipases of the D type, which hydrolyze phosphatidylcholine to phosphatidic acid, are regulated by members of the ADP‐ribosylation factor and Rho subfamilies of small G proteins, and by protein kinase C and other factors. This review presents recent information concerning the molecular details of G protein regulation of these phospholipases.
The Structural Organisation of LAMA4, the Gene Encoding Laminin αL4
Authors: Richards Allan , Luccarini Craig , Pope F. Michael
Published: July 2004

We have determined the complete structural arrangement of LAMA4, the gene encoding the laminin α4 chain. Using both yeast artificial chromosome clones and total human genomic DNA and primers derived from the cDNA sequence, regions of the gene were amplified and sequenced to determine the splice donor and acceptor sites. The introns were sized by agarose gel electrophoresis of the PCR products. The gene consisted of 39 exons spanning 122 kb. All of the splice sites conformed to the GTIAG rule, except intron 7 which possessed a GC dinucleotide at the donor splice site. The introdexon ratio was large at 17.8:1, mainly due to large introns at the 5' end of the gene. Regions at both the 5' and 3' end of the gene were subcloned from the yeast artificial chromosomes to enable untranscribed DNA to be sequenced. The gene represents the second of the laminin A gene family to be characterised and its structural organisation is similar to the equivalent regions of the LAMA2 gene.
Neutral Glycosphingolipids Induce Cell‐Cell Aggregation of a Variety of Hematopoietic Cell Lines
Authors: Yamaji Toshiyuki , Miyake Yurika , Kozutsumi Yasunori , Kawasaki Toshisuke
Published: July 2004

Exogenous neutral glycosphingolipids induced homotypic cell‐cell aggregation of a variety of hematopoietic cell lines. A mouse cytotoxic T cell line, CTLL‐2, was chosen to study the neutral glycosphingo‐lipid‐induced aggregation. Among neutral glycosphingolipids tested, galactosylceramide (GalCer) and glucosylceramide (GlcCer) were potent inducers, followed by lactosylceramide (LacCer); globotriaosyl‐ceramide (Gb3Cer) and neolactotetraosylceramide (nLc4Cer) were less effective. GalCer that contained a non‐hydroxy fatty acid was more efficient than GalCer containing an α‐hydroxy fatty acid. The minimum concentration of GalCer containing a non‐hydroxy fatty acid that induced aggregation was 1 μM, and maximum aggregation occurred at 10–20 μM within 24 h. Cytochalasin B and a mixture of sodium azide and 2‐deoxyglucose inhibited the aggregation, whereas cycloheximide, actinomycin D, and colchicine did not. Aggregated and dispersed cells, which were designated as competent cells, re‐aggregated in the absence of neutral glycosphingolipids. Anti‐GalCer polyclonal antibody inhibited GalCer‐induced re‐aggregation. Furthermore, competent cells bound and aggregated non‐competent cells in the absence of neutral glycosphingolipids. Cell‐cell aggregatign was similar for CTLL‐2 cells and the other hematopoietic cells that were tested. These findings suggest that the neutral glycosphingolipid‐induced cell‐cell aggregation of CTLL‐2 cells was mediated by heterophilic interaction(s) between glycosphingolipids and other cell surface components. These properties are shared by a variety of hematopoietic cell lines.
Molecular Localisation of Ferrochelatase in Higher Plant Chloroplasts
Authors: Roper Jennifer M. , Smith Alison G.
Published: July 2004

Within the chloroplast of higher plants, a crucial branchpoint of the tetrapyrrole synthesis pathway is the chelation of either Fe2+ to make haem, or Mg2+ for chlorophyll, catalysed by ferrochelatase or magnesium chelatase, respectively. One model that has been proposed for the control of this branchpoint, based on biochemical studies, is that the two enzymes are spatially separated within the chloroplast, ferrochelatase being exclusively in the thylakoids, while magnesium chelatase is associated with the envelope [Matringe, M., Camadro, J.‐M., Joyard, J. & Douce, R. (1994)J. Biol. Chem. 269, 15010–15015]. We have used a sensitive molecular method to investigate this possibility. Radiolabelled precursor proteins for ferrochelatase from Arabidopsis have been imported into isolated chloroplasts. Their distribution in the different subchloroplastic fractions have then been determined, and compared with that for light‐harvesting chlorophyll protein, which is exclusively thylakoidal, and the envelope‐located phosphate translocator. Clear evidence for the specific association of ferrochelatase protein with both thylakoid and envelope membranes has been obtained, thus suggesting strongly that the control of the branchpoint cannot be by spatial separation of the two chelatases.
Interactions Between Protein Disulphide Isomerase and Peptides
Authors: Klappa Peter , Hawkins Hilary C. , Freedman Robert B.
Published: July 2004

There is growing evidence that protein disulphide isomerase (PDI) has a common chaperone function in the endoplasmic reticulum. To characterise this function, we investigated the interaction of purified PDI with radiolabelled model peptides, somatostatin and mastoparan, by cross‐linking. The interaction between the peptides and PDI was specific, for it showed saturation and was abolished by denaturation of PDI. The interaction between a hydrophobic peptide without cysteine residues was much more sensitive to Triton X‐100 than the interaction between PDI and a more hydrophilic peptide with or without cysteine residues. We therefore propose that hydrophobic interactions between protein disulphide isomerase and peptides play an important role in the binding process. The interaction between PDI and the bound peptide therefore is enhanced by the formation of mixed disulphide bonds.
Isolation and Characterization of Niphatevirin, a Human‐Immunodeficiency‐Virus‐Inhibitory Glycoprotein from the Marine Sponge Niphates Erecta
Authors: O'Keefe Barry R. , Beutler John A. , Cardellina John H. , Gulakowski Robert J. , Krepps Benjamin L. , Mcmahon James B. , Sowder Raymond C. , Henderson Louis E. , Pannell Lewis K. , Pomponi Shirley A. , Boyd Michael R.
Published: July 2004

Anti‐human immunodeficiency virus (HIV)‐bioassay‐guided fractionation of aqueous extracts of the Caribbean sponge Niphates erecta led to isolation of a novel anti‐HIV protein, named niphatevirin. The protein was purified to homogeneity by ethanol precipitation, ammonium sulfate precipitation, gel‐permeation chromatography and concanavalin‐A–Sepharose affinity chromatography. Niphatevirin potently inhibited the cytopathic effects of HIV‐1 infection in cultured human lymphoblastoid (CEM‐SS) cells; the effective concentration of drug that results in 50% protection of the cells through inhibition of cell lethality, cell‐cell fusion and syncytium formation was approximately 10 nM. Delay of addition of niphatevirin to infected cultures by two hours markedly decreased (≈50%) cytoprotection; delay of addition by eight hours resulted in no antiviral activity. Niphatevirin bound to CD4 in a manner that prevented the binding of gp120, but did not directly bind gp120. Niphatevirin (6.5 μM) was inactive in both hemagglutination and hemolysis assays. Niphatevirin had a molecular mass of about 19 kDa by matrix‐assisted laser‐desorption ionization‐time of flight (MALDI‐TOF) mass spectrometry, and a native molecular mass of approximately 18 kDa by gel‐filtration chromatography. The protein had an acidic isoelectric point of 4.2–4.6, and was shown by periodate acid Schiff's staining to be glycosylated.
The Role of His113 and His114 in Pyruvate Decarboxylase from Zymomonas Mobilis
Authors: Schenk Gerhard , Leeper Finian J. , England Renee , Nixon Peter F. , Duggleby Ronald G.
Published: July 2004

Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a divalent cation as essential cofactors. Recently, the three‐dimensional structures of the enzyme from two yeasts have been determined. While these structures shed light on the binding of the cofactors and the reaction mechanism, the interactions between the substrate pyruvate and the enzyme remain unclear. We have used PDC from Zymomonas mobilis as a model for these enzymes in order to study substrate binding. The recombinant enzyme was expressed in Escherichia coli. High yield, simplicity of purification, high stability and simple kinetics make this model well suited for these studies. Activity measurements in the pH range between 5.8 and 8.5 indicated that a His residue may be involved in substrate binding. Analysis of an alignment of all known PDC protein sequences showed two invariant His residues (His113 and His114) which, according to the crystal structure of yeast PDC, are in the vicinity of the active site. Here we demonstrate that replacement of His114 by Gin does not have a great effect on cofactor and substrate binding. However, the kcat is decreased indicating that His114 may assist in catalysis. In contrast, substitution of His113 by Gln renders the enzyme completely inactive. This mutant has decreased affinity for both cofactors, as revealed by measurements of tryptophan fluorescence quenching. However, this decreased affinity is insufficient to account for the complete loss of activity. Despite its inability to support overall catalysis, this [Gln113]PDC mutant is capable of releasing acetaldehyde from 2‐(1‐hydroxyethyl)thiamin diphosphate supplied exogenously. It is proposed that upon substrate binding, His113 is placed close to C2 of the thiazole ring. Subsequent deprotonation of this atom leads to a conformational change that allows a flexible loop (residues 105–112) that precedes His113 to close over the active site. Hence, replacement of His113 by another residue interferes with this closure of the active site and thus disrupts the catalytic process.
Characterization and Purification from Bovine Neutrophils of a Soluble Guanine‐Nucleotide‐Binding Protein that Mediates Isozyme‐Specific Stimulation of Phospholipase C β2
Authors: Illenberger Daria , Schwald Frieder , Gierschik Peter
Published: July 2004

Members of the β isozyme subfamily of phosphatidylinositol‐specific phospholipase C (PLC) are stimulated by α subunits and βγ dimers of heterotrimeric guanine‐nucleotide‐binding proteins (G proteins). Myeloid differentiated human HL‐60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5′→O)‐3‐thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCβ2. The resolved factor, which behaved as 48‐kDa protein upon gel filtration, stimulated PLCβ2 but not PLCβ1 or PLCδ1. Activation of phosphatidylinositol 4‐phosphate 5‐kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCβ2 that lacked a carboxyl‐terminal region necessary for stimulation by members of the αq subfamily of the G‐protein α subunits. The results of this study suggest that a GTP‐binding protein distinct from αq subunits, probably a low‐molecular‐mass GTP‐binding protein associated with a regulatory protein, is involved in isozyme‐specific activation of PLCβ2.
Inactivation of the Regulatory Protein B of Soluble Methane Monooxygenase from Methylococcus Capsulatus (Bath) by Proteolysis can be Overcome by a Gly to Gin Modification
Authors: Lloyd John S. , Bhambra Amarbalita , Murrell J. Colin , Dalton Howard
Published: July 2004

The regulatory protein B of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), exists as a mixture of the full‐length active form and truncated forms, B′ and B″. Electrospray ionisation mass spectrometry (ESI‐MS) was used to identify a cleavage site between Met12 and Gly13, such that 12 amino acids were lost from the N‐terminus of protein B. This truncate was designated B′ and molecular masses were assigned to proteins B and B′ of 15 852.6 ± 0.4 Da and 14629.5 ± 0.3 Da, respectively. A cleavage site between Gln29 and Val30 was also identified such that 29 amino acids were lost from the N‐terminus of protein B. This truncate was designated B″ and had a molecular mass of 12 709.93 ± 0.02 Da. Proteins B′ and B″ were found to be inactive in the sMMO system. Addition of protease inhibitors or the heterologous expression of protein B in various strains of lon‐deficient or ompT‐deficient Escherichia coli, did not inhibit B′ formation. Expression of protein B as a glutathione S‐transferase fusion protein and subsequent purification of protein B from E. coli using affinity chromatography resulted in preparations of protein B with higher enzyme activities than that of wild‐type protein B. However, ESI‐MS confirmed that protein B′ was still present. Alteration of the Met12‐Gly13 cleavage site to Met12‐Gln13 revealed that the stability of G13Q at 20°C and 37°C was higher than that of wild‐type preparations. ESI‐MS indicated that protein B′ was absent and could only be identified after prolonged incubation at room temperature. The amount of active protein B present in the cell may be controlled by protein B cleavage, thereby regulating electron transfer. Alternatively, it may allow protein B to maintain a certain conformation necessary for enzyme activity and this may control the activity of sMMO in response to the supply of methane to the cell.
The Nuclear ABC1 Gene is Essential for the Correct Conformation and Functioning of the Cytochrome bc1 Complex and the Neighbouring Complexes II and IV in the Mitochondrial Respiratory Chain
Authors: Brasseur Gaël , Tron Pascale , Dujardin Geneviève , Slonimski Piotr P. , Brivet‐Chevillotte Paule
Published: July 2004

The nuclear ABC1 gene was isolated as a multicopy suppressor of a cytochrome b mRNA translation defect. Its inactivation leads to a respiratory deficiency suggesting a block in the bc1 segment of the respiratory chain [Bousquet, I., Dujardin, G. & Slonimski, P. P. (1991)EMBO J. 1O, 2023–2031]. In the present study, we established that deleting the ABC1 chromosomal gene from Saccharomyces cerevisiae does not prevent the assembly of the bc1 complex (complex III) but markedly impairs the kinetics of its high‐potential electron transfer pathway occurring on the positive, outer, side of the membrane, which results in reduced activity of the bc1 complex. In addition, the activity of complex II and its cytochrome b560 decrease drastically and complex IV activity is halved. It is also observed that the binding of the quinol to the bc1 complex ubiquinol oxidation site is affected and that adding exogenous quinones partially compensates for the respiratory deficiency in vitro, although the quinone content of mutant and wild‐type mitochondria are similar. Lastly, complexes II, III and IV are found to be thermosensitive and the bc1 complex exhibits greater sensitivity than the wild‐type strain to center N and P inhibitors, suggesting that the three multisubunit complexes have undergone structural modifications. The data suggest that the ABC1 gene product acts as a chaperone‐like protein essential for the proper conformation and efficient functioning of the bc1 complex and the effects of the Abe1 protein on the complexes II and IV might result from interactions with the modified bc1 complex.
cDNA Cloning and Expression of Secreted Xenopus Laevis Dipeptidyl Aminopeptidase IV
Authors: Vlasak Reinhard , Vilas Ulrike , Strobl Birgit , Kreil Günther
Published: July 2004

From a Xenopus laevis skin library a cDNA coding for dipeptidyl aminopeptidase IV (DPP IV) was isolated. The ORF codes for a protein with sequence similarity to DPP‐IV‐like proteins, including mammalian DPP IV and X. laevis fibroblast activation factor. In contrast to the membrane‐bound mammalian enzymes, mature X. laevis DPP IV is a soluble secreted polypeptide. The frog enzyme possesses a cleavable signal sequence; the mature protein starts at Thr30 of the polypeptide predicted from the cDNA sequence. Expression of the cloned cDNA by recombinant vaccinia virus resulted in the formation of a protein with the expected molecular mass and substrate specificity. Recombinant DPP IV was present in high concentration in the supernatant of infected cells and exhibited enzymatic activity towards the synthetic substrate alanyl‐prolyl‐p‐nitroanilide.
Deletion Analysis of the Schistosoma Mansoni 28‐kDa Glutathione S‐transferase Gene Promoter in Mammalian Cells
Authors: Serra Esteban , Zemzoumi Khalid , Dissous Colette
Published: July 2004

The 1241‐bp promoter region of the Schistosoma mansoni 28‐kDa glutathione S‐transferase gene (Sm28GST) was sequentially deleted and analyzed using the luciferase reporter gene system in different cell lines. The activator protein‐1 (AP‐1) site located at –231 seems to be responsible for the major part of the promoter activity. The 1241‐bp Sm28GST promoter was not, in transient transfection experiments, activated by reagents generating reactive oxygen species, such as hydrogen peroxide (H2O2), 3‐methyl‐
Characterization of Recombinant Perlecan Domain I and Its Substitution by Glycosaminoglycans and Oligosaccharides
Authors: Costell Mercedes , Manni Karlheinz , Yamada Yoshihiko , Timpl Rupert
Published: July 2004

Recombinant mouse perlecan domain I (173 residues) was produced in transfected embryonic kidney cells and purified from the culture medium on DEAE‐cellulose. It was shown to be modified by glycosaminoglycans and could be partially separated into two protein pools which were either substituted with heparan sulfate (fragment 1 A) or, to a smaller extent (20%), with chondroitin/dermatan sulfate or a mixture of both glycosaminoglycans (fragment IB). The average molecular mass of the glycosaminoglycans was about 8–10kDa and, thus, smaller than in tissue‐derived perlecans. Sequence and carbohydrate analyses localized the heparan sulfate attachment site to three Ser residues within SGD consensus sequences. Furthermore, the N‐terminal part of fragment IA contained six Thr/Ser residues substituted by branched galactosamine‐containing oligosaccharides and an N‐substituted Asn residue. Fragment I was also shown to contain unique immunological epitopes which are not dependent on glycosaminoglycans and are shared by tissue‐derived perlecan. Circular dichroism demonstrated a distinct a helix (20%) and β structure (60%) in fragment IA, consistent with predictions of a novel SEA protein module located in the C‐terminal part of domain I.