bluuebunny/crossref_metadata_2025 huggingface dataset:
Relation between domain evolution, specificity, and taxonomy of the α‐amylase family members containing a C‐terminal starch‐binding domain
Authors: Janeček Štefan , Svensson Birte , MacGregor E. Ann
Published: March 2003

The α‐amylase family (glycoside hydrolase family 13; GH 13) contains enzymes with approximately 30 specificities. Six types of enzyme from the family can possess a C‐terminal starch‐binding domain (SBD): α‐amylase, maltotetraohydrolase, maltopentaohydrolase, maltogenic α‐amylase, acarviose transferase, and cyclodextrin glucanotransferase (CGTase). Such enzymes are multidomain proteins and those that contain an SBD consist of four or five domains, the former enzymes being mainly hydrolases and the latter mainly transglycosidases. The individual domains are labelled A [the catalytic (β/α)8‐barrel], B, C, D and E (SBD), but D is lacking from the four‐domain enzymes. Evolutionary trees were constructed for domains A, B, C and E and compared with the ‘complete‐sequence tree’. The trees for domains A and B and the complete‐sequence tree were very similar and contain two main groups of enzymes, an amylase group and a CGTase group. The tree for domain C changed substantially, the separation between the amylase and CGTase groups being shortened, and a new border line being suggested to include the Klebsiella and Nostoc CGTases (both four‐domain proteins) with the four‐domain amylases. In the ‘SBD tree’ the border between hydrolases (mainlyα‐amylases) and transglycosidases (principally CGTases) was not readily defined, because maltogenic α‐amylase, acarviose transferase, and the archaeal CGTase clustered together at a distance from the main CGTase cluster. Moreover the four‐domain CGTases were rooted in the amylase group, reflecting sequence relationships for the SBD. It appears that with respect to the SBD, evolution in GH 13 shows a transition in the segment of the proteins C‐terminal to the catalytic (β/α)8‐barrel(domain A).
Functional properties of the sex‐hormone‐binding globulin (SHBG)‐like domain of the anticoagulant protein S
Authors: Saposnik Béatrice , Borgel Delphine , Aiach Martine , Gandrille Sophie
Published: March 2003

Protein S (PS) possesses a sex‐hormone‐binding globulin (SHBG)‐like domain in place of the serine‐protease domain found in other vitamin K‐dependent plasma proteins. This SHBG‐like domain is able to bind a complement fraction, C4b‐binding protein (C4b‐BP). To establish whether the PS SHBG‐like domain can fold normally in the absence of other domains, and to obtain information on the specific functions of this region, we expressed the PS SHBG‐like domain alone or together with its adjacent domain EGF4. The folding of the two recombinant modules was studied by analyzing their binding to C4b‐BP. The apparent dissociation constants of this interaction indicated that both recombinant modules adopted the conformation of native PS, indicating that the PS SHBG‐like region is an independent folding unit. We also obtained the first direct evidence that the SHBG‐like domain alone is sufficient to support the interaction with C4b‐BP. In addition, both recombinant modules were able to bind Ca2+ directly, as shown by the migration shift in agarose gel electrophoresis in the presence of Ca2+, together with the results of equilibrium dialysis and the functional effect of Ca2+ on the C4b‐BP/PS interaction, confirming the presence of one Ca2+ binding site within the SHBG‐like domain. Neither recombinant module exhibited activated protein C (aPC) cofactor activity in a clotting assay, suggesting that the PS SHBG‐like region must be part of the intact molecule for it to contribute to aPC cofactor activity, possibly by constraining the different domains in a conformation that permits optimal interaction with aPC.
A comparative analysis of the time‐dependent antiproliferative effects of daunorubicin and WP631
Authors: Villamarín Silvia , Mansilla Sylvia , Ferrer‐Miralles Neus , Priebe Waldemar , Portugal José
Published: March 2003

Jurkat T lymphocytes were treated with daunorubicin and WP631, a daunorubicin‐based DNA binding agent, in experiments aimed to analyze cellular uptake of these drugs and their effect on cell viability. WP631 was taken up more slowly than daunorubicin, but laser confocal microscopy and spectrofluorometric quantification showed that the drug accumulated in the cells. Despite the slow uptake rate, the antiproliferative capacity of WP631 (measured as IC50 after a 72‐h continuous treatment) was greater than that of daunorubicin. The propensities of daunorubicin and WP631 to promote apoptosis were compared. Our results indicate that the major effect of WP631 was a G2/M arrest followed, after about 72 h of treatment, by polyploidy and mitotic (reproductive) death. In contrast, daunorubicin induced a rapid response with classic features of apoptosis.
Chromatin and transcription
Authors: Gregory Philip D. , Hörz Wolfram
Published: March 2003

The last year has seen much progress in our understanding of chromatin and transcription. Transcriptionally active chromatin has long been correlated with a higher level of histone acetlyation. The discovery of a nuclear histone acetyltransferase activity encoded by factors with a role in transcription raises the possibility that the cell is able to dynamically modulate the (local) level of histone acteylation, switching chromatin templates from inactive to transcriptionally active states. Furthermore, histone acetylation states have shown to play a role in determining the efficacy of transcriptionally silenced chromatin in both yeast and Drosophila. The advances in our knowledge regarding the role of the origin‐recognition complex in the establishment of silencing, and the requirement for a locally concentrated zone of the silence information regulator proteins in the nucleus has provided insights into the complex architecture of silenced chromatin. The goal of understanding the mechanisms by which the cell is able to ’open' repressive chromatin structures has prompted the discovery of multiple chromatin remodelling activites. These large protein complexes identified from organisms as diverse as yeast, mouse, fly and man demonstrate the ubiquity and fundamental importance of the ability to perturb the structure of chromatin allowing transcription of the desired genes. These data provide the latest and potentially most significant demonstration of the importance of the nucleosome in the regulation of transcription.
Structural characterisation of two hexasaccharides and an octasaccharide from chondroitin sulphate C containing the unusual sequence (4‐sulpho)‐N‐acetylgalactosamine−β1−4‐(2‐sulpho)‐glucuronic acid−β1−3‐(6‐sulpho)‐N‐acetylgalactosamine
Authors: Chai Wengang , Lawson Alexander M. , Gradwell Michael J. , Kogelberg Heide
Published: March 2003

The structures of two hexasaccharides and an octasaccharide, isolated from shark cartilage chondroitin sulphate C after partial chondroitin ABC lyase digestion, have been elucidated by disaccharide analysis, liquid secondary‐ion mass spectrometry, and 1H/13C NMR spectroscopy. In the case of the octasaccharide, the order of the internal disaccharide units was deduced from a novel one‐dimensional TOCSY/ROESY excitation sculpting experiment [Gradwell M. J., Kogelberg, H. & Frenkiel, T. A. (1997) J. Magn. Reson. 124, 267−270] which incorporates two double‐pulsed field‐gradient spin‐echo sequences. The oligosaccharides each contain the unusual motif GalNAc(4SO.es−.rb.ei3.rb)β1−4GlcA(2SO.es−.rb.ei3.rb)β1−3GalNAc(6SO.es−.rb.ei3.rb). From the adjoining sequences in the oligosaccharides, it is apparent that this motif occurs interspersed with typical chondroitin sulphate‐C disaccharides, GlcAβ1−3GalNAc(6SO.es−.rb.ei3.rb). Such differentially sulphated domains along the glycosaminoglycan chains are potential sites for specific recognition processes.
Ribosomal S6 kinase is activated as an early event in preemergence development of encysted embryos of Artemia salina
Authors: Malarkey Kevin , Coker Kenneth J. , Sturgill Thomas W.
Published: March 2003

Dormant Artemia salina cysts contain desiccated gastrulae that are metabolically inactive, and physiologically arrested. Following rehydration, embryos resume development via alterations in protein expression, in the complete absence of cell division. In mammals, activation of p70 ribosomal S6 kinase (p70S6k) has been implicated in translational control, in particular the selective up‐regulation of translation of mRNAs with polypyrimidine tracts at their 5′ start sites. We therefore investigated ribosomal S6 kinase activity in preemergence development. We demonstrate that an S6 kinase activity is rapidly stimulated (within < 15 min) following rehydration and coincides with the onset of ribosomal S6 subunit phosphorylation. This S6 kinase activity displays chromatographic and biochemical characteristics that are similar to those of mammalian p70S6k. Partially purified Artemia S6 kinase was inactivated by treatment with protein phosphatase 2A. Activation of S6 kinase activity was shown to be due to an enzymatic step(s), and not simply rehydration of stored, active enzyme. The temporal profile of activation of S6 kinase activity is compatible with a regulatory function for p70S6k in early preemergence development of encysted Artemia. These studies identify activated Artemia cysts as a system for biochemical studies of p70S6k regulation.
The prismane protein resolved
Authors: Kröckel Monika , Trautwein Alfred X. , Arendsen Alexander F. , Hagen Wilfred R.
Published: March 2003

The prismane protein of Desulfovibrio vulgaris, in its isolated, its one‐electron‐reduced and its oxidized states, was the subject of a detailed Mössbauer investigation. Measurements were recorded in the range 0.295−77 K and in the field range 0−6.2 T (parallel and perpendicular to the γ beam). The paramagnetic parts of the magnetically split Mössbauer spectra were analyzed with the spin‐Hamiltonian formalism, including the nuclear Hamiltonian; the diamagnetic parts result from the nuclear Hamiltonian only. The field‐dependent spectra at 295 mK and 4.2 K indicate that the paramagnetic part of the isolated protein represents a spin‐coupled 4Fe unit with the spin of one Fe site (5/2) oriented antiparallel to the spins of the other three Fe sites (5/2, 5/2 and 2), yielding a total cluster spin, Stot of 9/2. The Mössbauer parameters of the individual Fe sites indicated that this unit represents a 4Fe cluster with an unusual mixture of bridging and terminal ligands and metal coordinations (hybrid cluster). The diamagnetic part of the isolated protein represents an additional 4Fe unit, which, according to its Mössbauer parameters, is a [4Fe2.5+‐4S] cubane. The parameter changes upon one‐electron oxidation or reduction and the magnetic properties of the two clusters in the three oxidation states of the protein investigated here reveal that the redox behavior of the prismane protein is exclusively related with the hybrid cluster.

reduced
isolated
oxidized
Fe2+−O−Fe2+
Fe3+−O−Fe2+
Fe3+−O−Fe3+∣∣∣∣∣∣Fe.cf5.3+.cf5..cf1..nu@dR.dn@dU.ed.nu.mu3.S.md3..dn.md4.S.mu4..ed.nu@dU.dn@dR.edFe.cf5.3+.cf5..cf1.
Fe.cf5.3+.cf5..cf1..nu@dR.dn@dU.ed.nu.mu3.S.md3..dn.md4.S.mu4..ed.nu@dU.dn@dR.edFe.cf5.3+.cf5..cf1.
Fe.cf5.3+.cf5..cf1..nu@dR.dn@dU.ed.nu.mu3.S.md3..dn.md4.S.mu4..ed.nu@dU.dn@dR.edFe.cf5.3+.cf5..cf1.

These findings are contrary to the former hypothesis of one or two [6Fe‐6S] cluster(s) as the prosthetic group of this protein [Hagen, W. R., Pierik, A. J. & Veeger, C. (1989) J. Chem. Soc. Faraday Trans. 185, 4083−4090; Moura, I., Tavares, P., Moura, J. J. G., Ravi, N., Huynh, B. H., Liu, M.‐Y. & LeGall, J. (1992) J. Biol. Chem. 287, 4487−4496]. However, they are in full agreement with the crystal structure of the isolated protein, which, concurrent with our Mössbauer investigation, has been solved.
Ca2+‐dependent protein kinases of Paramecium
Authors: Kim Kwanghee , Messinger Lynn A. , Nelson David L.
Published: March 2003

Two genes for Ca2+‐dependent protein kinases, PCaPK‐α and PCaPK‐β, were isolated from a Paramecium genomic DNA library. The coding region of PCaPK‐α encoded 481 amino acids and that of PCaPK‐β encoded 493 amino acids, predicting molecular masses of 55 603 Da and 57 131 Da for each putative protein. The sequences of the protein kinase catalytic domains of PCaPK‐α and PCaPK‐β were closely related to those of the Ca2+‐dependent protein kinases (CDPKs) from Plasmodium, Eimeria, and several plants, and the catalytic region of the Ca2+/calmodulin‐dependent protein kinase family (35−48 % identity). In the junction region between the catalytic and regulatory regions, only 9 of 31 amino acid residues are the same in the two Paramecium genes, and the sequences encoded in the Paramecium genes differ from those in the plant CDPK genes in about 20 of 31 residues in the junction region. The C‐terminal region of the Paramecium kinases shared sequence similarity with Paramecium calmodulin (30−34 % identity). Two Ca2+‐dependent protein kinases previously characterized from Paramecium (52 kDa CaPK1, and 50 kDa CaPK‐2) are activated by Ca2+ in the micromolar concentration range and they directly bind Ca2+ in a 45Ca2+ overlay blot assay. The size predicted from the genes, the presence of four putative Ca2+‐binding motifs encoded in PCaPK‐α and PCaPK‐β, and the immunological cross‐reaction of expressed cloned fragments of these genes with CaPK‐2, suggest that they encode proteins of the same family.
Molecular cloning, expression and tissue distribution of glial‐cell‐line‐derived neurotrophic factor family receptor α‐3 (GFRα‐3)
Authors: Masure Stefan , Cik Miroslav , Pangalos Menelas N. , Bonaventure Pascal , Verhasselt Peter , Lesage Anne S. J. , Leysen Josee E. , Gordon Robert D.
Published: March 2003

Neurturin and glial‐cell‐line‐derived neurotrophic factor (GDNF) promote the survival and maintenance of different types of neuronal cells and signal through a receptor complex composed of a ligand binding subunit, either GDNF family receptor α‐1 (GFRα‐1) or α‐2 (GFRα‐2), together with the cRET membrane‐bound protein tyrosine kinase. We have cloned GFRα‐3, a novel receptor belonging to the GFRα family, that is 35 % identical by amino acid sequence to both GFRα‐1 and GFRα‐2. GFRα‐3 is a protein composed of 400 amino acid residues with three potential N‐linked glycosylation sites together with the features characteristic of a glycosyl‐phosphatidylinositol‐anchored membrane protein. The heterologous expression of a FLAG‐tagged GFRα‐3 in human embryonic kidney cells showed that the protein is bound to the cell surface via a glycosyl‐PtdIns anchor and is glycosylated, with different glycoforms migrating on SDS/PAGE with apparent molecular masses ranging over 43−62 kDa. The gene for GFRα‐3 was mapped to human chromosome 5 in a region (q31.1−q31.3) where several disease loci, growth factor and growth factor receptor genes have been localized. Using northern blot analysis or reverse‐transcription PCR, GFRα‐3 was shown to be expressed within the nervous system predominantly in the cerebellum and the spinal cord while in peripheral tissues GFRα‐3 was found to be expressed mostly in the colon, small intestine, pancreas, heart, testis and prostate. Using a GFRα‐3‐specific [35S]cRNA[γS] probe, in situ hybridization histochemistry experiments confirmed the expression in the cerebellum.
Biochemical characterization of Anabaena sp. strain PCC 7120 non‐specific nuclease NucA and its inhibitor NuiA
Authors: Meiss Gregor , Franke Ingo , Gimadutdinow Oleg , Urbanke Claus , Pingoud Alfred
Published: March 2003

We have established overexpression systems and purification protocols for NucA and NuiA, a sugar non‐specific nuclease and its protein inhibitor from Anabaena sp. strain PCC 7120, in order to characterize these proteins in detail. CD spectroscopy revealed that NucA has a similar secondary‐structure composition, 13 %α helix and 20 %β sheet, to the related Serratia nuclease, while NuiA represents a protein with a higher α‐helical (29 %) and β‐sheet (24 %) content than NucA. Denaturation experiments showed that the stabilities of NucA and NuiA are in the typical range for proteins of mesophilic organisms, NuiA with Δ.cf2.G.cf2..cf1..es0.rb.eiH.ei2.dbO.rb= 63.4 J mol.es−1.rb.eiresidue.rb, being slightly more stable than its target NucA with ΔΔ.cf2.G.cf2..cf1..es0.rb.eiH.ei2.dbO.rb = 46.3 J mol.es−1.rb.eiresidue.rb. The nuclease requires divalent metal ions as cofactors, the optimum concentration being around 5 mM for Mn2+ or Mg2+. The order of effectiveness of various divalent cations to function as cofactors for the hydrolytic activity of NucA is Mn2+ = Co2+ > Mg2+ Ni2+ @WR Ca2+ = Cd2+ at a concentration of 5 mM. Nuclease activity decreases with increasing concentration of monovalent salt. The activity of NucA shows a pH optimum at pH 5.5−7.5. The temperature optimum is around 35 +C, the activation energy was calculated to be 53 kJ mol−1. The specific activity of the nuclease towards high molecular‐mass DNA is 8.4×106 Kunitz‐units mg−1, which means that NucA is one of the most active nucleases known. Kinetic constants for the cleavage of various DNA and RNA substrates by NucA are all in the range Km  0.1 mg ml−1 and kcat ≅ 1000 s−1. As other non‐specific nucleases, NucA exhibits sequence preferences, similar to the related Serratia nuclease, NucA avoids cleavage of d(A) d(T) tracts. The nucleolytic activity of NucA is completely inhibited at equimolar concentrations of nuclease and inhibitor. An ultracentrifugation analysis showed that NucA and NuiA form a 1 : 1 complex. The interaction of NucA with NuiA was also investigated by CD spectroscopy and revealed no major conformational changes upon complex formation of the two proteins.
Purification and preliminary characterization of an Fcε‐receptor‐activated protein‐tyrosine phosphatase from mast cells
Authors: Hampe Christiane Susanne , Pecht Israel
Published: March 2003

Immunological stimulation of rat, mucosal type, mast cells (line RBL‐2H3) by clustering the type I Fcε receptor (FcεRI) causes a fast yet transient tyrosyl phosphorylation of several proteins. We report here the characterization of a protein‐tyrosine phosphatase (PTP) involved in the very early steps coupling the FcεRI stimulus to the cell secretory response. We have observed earlier that a PTP activity present in one of the cells' solubilized particulate fractions is enhanced 2−3‐fold upon cell stimulation by FcεRI clustering [Hampe, C. S. & Pecht, I. (1994) FEBS Lett. 346, 194−198]. This PTP (MpII PTP) was now isolated and purified to homogeneity and appears as a 45‐kDa protein. Sequence comparison of two peptide stretches of MpII PTP with those of proteins present in the Swiss‐Prot and EMBL data banks revealed no significant similarity to a known protein. Hence, we assume that the MpII PTP is a novel protein. The possible involvement of MpII PTP in the stimulus‐secretion coupling cascade was investigated. Abrogation of the antigen‐induced FcεRI clustering by an excess of monovalent hapten suppressed the FcεRI‐mediated MpII PTP activity enhancement to the levels observed in resting cells. The activity enhancement required the presence of extracellular Ca2+ ions and was also induced by an artificial increase of the free intracellular concentration of these ions. It is apparently mediated by protein kinase C (PKC) since phorbol myristate induced an additive increase in the MpII PTP activity to that induced by antigen. Also, treatment with the PKC‐specific inhibitor bisindolylmaleimide suppressed the antigen‐induced MpII PTP activity enhancement. PKC involvement was further supported by the finding that the MpII PTP 45‐kDa protein underwent seryl phosphorylation following antigen stimulation. This modification was found to be further enhanced by pretreatment with phorbol myristate prior to antigen stimulation.
13C‐NMR spectroscopy of keratan sulphates
Authors: Huckerby Thomas N. , Brown Gavin M. , Nieduszynski Ian A.
Published: March 2003

Skeletal keratan sulphate has been fragmented using the enzyme keratanase II, and 13C chemical‐shift data are reported for five reduced sialylated pentasaccharides that derived from the non‐reducing chain terminal region. They have the structures : NeuAc(α2‐6)Gal(β1‐4)GlcNAc6S(β1‐3)Gal(β1‐4)GlcNAc6S‐ol, NeuAc(α2‐3)Gal(β1‐4)GlcNAc6S(β1‐3)Gal(β1‐4)GlcNAc6S‐ol, NeuAc(α2‐6)Gal(β1‐4)GlcNAc6S(β1‐3)Gal6S(β1‐4)GlcNAc6S‐ol, NeuAc(α2‐3)Gal(β1‐4)GlcNAc6S(β1‐3)Gal(6S)(β1‐4)GlcNAc6S‐ol, and NeuAc(α2‐3)Gal(6S)(β1‐4)GlcNAc6S(β1‐3)Gal(6S)(β1‐4)GlcNAc6S‐ol, where GlcNAc6S‐ol represents N‐acetyl‐glucosaminitol 6‐O‐sulphate and NeuAc represents N‐acetylneuraminic acid. The use of these 13C‐NMR spectroscopy data for the recognition of specific chain‐capping structures within native keratan sulphates is discussed. In addition, examination of the data derived from the NeuAc(α2‐6) capping structures strongly suggests that sulphation three residues away from the neuraminic acid cap has a profound effect upon the conformation of the capping region.
Kinetics and specificity of reductive acylation of wild‐type and mutated lipoyl domains of 2‐oxo‐acid dehydrogenase complexes from Azotobacter vinelandii
Authors: Berg Axel , Westphal Adrie H. , Bosma Hans J. , De Kok Aart
Published: March 2003

The kinetics and specificity of reductive acylation of lipoyl domains derived from Azotobacter vinelandii 2‐oxo‐acid dehydrogenase complexes, catalysed by A. vinelandii and Escherichia coli complexes, have been investigated. With the wild‐type pyruvate dehydrogenase complex from A. vinelandii the rate of reductive acetylation and deacetylation was studied by rapid mixing methods. The rate of reductive acetylation, 126 s−1, corresponds well with the turnover rate derived from steady‐state measurements. Deacetylation was rapid and specific for coenzyme A. No deacetylation was observed with reduced or oxidised lipoamide or with dithiothreitol. The rate of reductive acetylation of complex‐bound lipoyl domains by pyruvate dehydrogenase (E1p) is at least 60 times higher than of free lipoyl domains under comparable conditions. This gain in catalytic rate indicates a large diffusion limitation of lipoyl domains when attached via the flexible linker segments to the complex, and illustrates the efficiency of substrate channeling in the multienzyme complex.

The 2‐oxo‐acid dehydrogenases exhibit specificity for lipoyl domains in the reductive acylation reaction. The A. vinelandii lipoyl domain derived from the pyruvate dehydrogenase complex is a good substrate for A. vinelandii E1p, but not for A. vinelandii 2‐oxoglutarate dehydrogenase (E1o), and vice versa. The A. vinelandii lipoyl domain of the pyruvate dehydrogenase complex is also, although at a lower rate, reductively acetylated by E. coli E1p and reductively succinylated by E. coli E1o. Likewise, the A. vinelandii lipoyl domain derived from the 2‐oxoglutarate dehydrogenase complex is recognised by E. coli E1o, but not by E. coli E1p. This suggests that common determinants of the lipoyl domains exist that are responsible for recognition by the E1 components. On the basis of the observed specificity and lipoyl domain sequences and structures, an exposed loop of the A. vinelandii 2‐oxoglutarate dehydrogenase complex lipoyl domain was subjected to mutagenesis. Although the reductive acylation experiments of mutants of the lipoyl domain indicate the importance of this loop for recognition, it is probably not the single determinant for specificity.
Semenogelin I and semenogelin II, the major gel‐forming proteins in human semen, are substrates for transglutaminase
Authors: Peter Anders , Lilja Hans , Lundwall Åke , Malm Johan
Published: March 2003

The major seminal vesicle secreted proteins in human semen, semenogelin I and semenogelin II, interact non‐covalently and via disulphide bridges to instantly form a coagulum upon ejaculation. The coagulum is liquefied after a few minutes due to the action of a prostatic serine protease, prostate‐specific antigen (PSA). In contrast to rat semen, which forms an insoluble plug within minutes of expulsion, no transglutaminase‐mediated cross‐linking has been demonstrated in ejaculated human semen. However, we here show that semenogelin I and semenogelin II, both in seminal vesicle fluid and purified from semen, are substrates for factor XIIIa, the fibrin cross‐linking transglutaminase. The cross‐linking of the semenogelins, which was conformation‐dependent, and the incorporation of a fluorescence‐labelled amine, were visualised by SDS/PAGE and Western blot. Purified semenogelin I and semenogelin II could be cross‐linked separately into complexes. Moreover, digestion of semenogelin with PSA produced fragments, some of which were cross‐linked into complexes by factor XIIIa. We also found that PSA was unable to release any semenogelin fragments during exposure of the high molecular‐mass complexes of cross‐linked semenogelin to active PSA.
Ferric citrate uptake by cultured rat hepatocytes is inhibited in the presence of transferrin
Authors: Graham Ross M. , Morgan Evan H. , Baker Erica
Published: March 2003

Diseases associated with iron overload occur worldwide. In subjects suffering from these conditions, transferrin is likely to be fully saturated and excess plasma iron must be complexed to other molecules. Consequently, the liver, which is the major site of iron storage, will be presented with iron in both transferrin‐bound and non‐transferrin‐bound forms and these forms may compete for uptake by hepatocytes. The endogenous low‐molecular‐mass iron chelator, citrate, is considered to be a major contributing molecule to non‐transferrin iron transport. This study was conducted to investigate the effects of transferrin on the uptake of citrate and iron citrate by hepatocytes in culture. Rat hepatocytes were incubated with 100 μM [14C]citrate and 1.0 μM 55Fe in the presence or absence of various forms of transferrin. Binding and internalisation of both citrate and iron were inhibited in a dose‐dependent manner with increasing concentration of diferric transferrin, with iron uptake decreasing to less than 5 % of control values. Apotransferrin was markedly more effective in blocking citrate and iron uptake, reaching the same levels of inhibition at a 15‐fold lower concentration of protein. The binding of citrate to the cell membrane was not affected significantly by changing the iron saturation of transferrin but internalisation decreased with decreasing saturation. In contrast, both the binding and internalisation of iron decreased with decreasing saturation. Incubations carried out using 55Fe‐labelled citrate in the presence of 59Fe‐labelled diferric transferrin indicated that citrate‐mediated iron binding by the cells decreased with increasing diferric transferrin concentrations but the citrate iron was not replaced by iron from transferrin during the 15‐min incubation period. Instead, total iron uptake decreased. These data suggest that citrate‐mediated iron uptake by hepatocytes shares at least one common pathway with transferrin‐mediated iron uptake.
Growth‐blocking peptide expressed in the insect nervous system
Authors: Hayakawa Yoichi , Noguchi Hirofumi
Published: March 2003

Growth‐blocking peptide (GBP) is an insect biogenic peptide that retards larval growth and, consequently, delays the onset of pupation of lepidopteran larvae. Transcription of the GBP gene has been proven to be regulated in a tissue‐dependent manner by an approximately 0.8‐kb mRNA transcript in fat body and 2.5‐kb transcript in the brain central nervous system (CNS) [Hayakawa, Y., Ohnishi, A., Yamanaka, A., Izumi, S. & Tomino, S. (1995) FEBS Lett. 367, 185−189]. The GBP cDNA in the brain CNS was isolated, sequenced and found to be almost identical with that in the fat body, except that it encodes a long 5′‐untranslated region (approximately 1.1 kb), thus indicating that the brain CNS is synthesized as a polyprotein precursor and released through posttranslational processing in the same manner as that predicted in the fat body. Sequence analyses of the genomic GBP gene fragments demonstrated that the 5′‐untranslated region contains an intron encoding a sequence element, TGATAA, which has been reported to be important for expression in the insect larval fat body, suggesting that this element contributes to a tissue‐specific expression of the shorter GBP mRNA in fat body. Ribonuclease protection assays showed that parasitization by a parasitic wasp elevated the expression of GBP mRNA in brain CNS. The parasitization‐induced increase of GBP precursor and GBP concentrations was also demonstrated in brain CNS. Brain CNS dopamine was increased eightfold by incubating the tissue with GBP for 8 h through the elevation of dopa decarboxylase activity by a transcriptional enhancement perhaps due to GBP‐induced increase of cytoplasmic Ca2+ concentration. Dopamine elevation reduced in vitro incorporation of methionine into proteins synthesized in brain CNS.
Changes in glycosaminoglycan structure and composition of the main heparan sulphate proteoglycan from human colon carcinoma cells (perlecan) during cell differentiation
Authors: Molist Anna , Romarís Manuel , Lindahl Ulf , Villena Juan , Touab Malika , Bassols Anna
Published: March 2003

Colon carcinoma cells provide a useful model to study the biochemical processes associated with cell differentiation. Undifferentiated HT29, differentiated HT29MTX−3 and HT29MTX−6, and Caco2 human colon carcinoma cells have been used to study the production of proteoglycans and to characterize the glycosaminoglycan structure of the heparan sulphate chains. All the cell lines produce mainly a heparan sulphate proteoglycan that is found partly in the extracellular medium and associated to the cell membrane. The heparan sulphate proteoglycans from the media were purified by ion‐exchange chromatography and subjected to structural analysis. The heparan sulphate proteoglycan from differentiated cells is larger and more homogeneous in size than the heparan sulphate proteoglycan from undifferentiated HT29 cells. No differences in protein core structure were observed when cells were labeled with [35S]methionine and the protein cores visualized by gel electrophoresis. Nevertheless, differences in glycosaminoglycan composition were found correlated with the degree of differentiation. The heparan sulphate chains from differentiated HT29MTX−3 and HT29MTX−6 cells have a higher sulphation degree than those from undifferentiated HT29 cells. The heparan sulphate from Caco2 cells is the most highly sulphated species. The differences are mainly attributed to O‐sulphate groups. The increase in O‐sulphation was more pronounced for
D‐glucosamine 6‐O‐sulphate than for
L‐iduronic acid 2‐O‐sulphate groups.
CD44 variant exon v5 encodes a tyrosine that is sulphated
Authors: Sleeman Jonathan P. , Rahmsdorf Ursula , Steffen Anja , Ponta Helmut , Herrlich Peter
Published: March 2003

Functional differences between members of the CD44 family of cell surface glycoproteins is mediated in part by differential post‐translational modification of these proteins and by alternative splicing. Tyrosine sulphation is a secondary modification of the primary amino acid structure of a number of secreted, transmembrane and lysosomal proteins, which is associated with promotion of protein‐protein interactions. Here we identify a cannonical tyrosine‐sulphation motif within rat and mouse CD44 exon v5. We show that inorganic sulphate is incorporated into the metastasis‐associated rat CD44v4−v7 splice variant. The sulphate is not incorporated into sulphated glycosaminoglycan or other sugar modifications of CD44v4−v7. A point mutation of the exon v5 tyrosine to phenylalanine destroys inorganic sulphate incorporation into CD44v4−v7. These results demonstrate that the tyrosine‐sulphation motif within rat CD44 exon v5 is used in vivo, and suggest that exon v5 may be involved in mediating CD44 ligand binding‐activity by means of its sulphated tyrosine.
Disulphide bonds assignment in the inter‐α‐inhibitor heavy chains
Authors: Flahaut Christophe , Mizon Charlotte , Aumercier‐Maes Pierrette , Colson Pierre , Bailly Christian , Sautiere Pierre , Mizon Jacques
Published: March 2003

Human inter‐α‐inhibitor (IαI) is a plasma serine‐proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan : a light one named bikunin, carrying the antiproteinase activity and two heavy chains H1 and H2. The amino acid sequences of these heavy chains are highly similar; however when IαI is digested by neutrophil proteinases, their proteolytic susceptibility strongly differs [Balduyck, M., Piva, F., Mizon, C., Maes, P., Malki, N., Gressier, B., Michalski, C. & Mizon, J. (1993) Human leucocyte elastase (HLE) preferentially cleaves the heavy chain H2 of inter‐α‐trypsin inhibitor (ITI), Biol. Chem. Hoppe‐Seyler 374, 895−901].

We mapped the disulphide topology of the IαI heavy chains in order to investigate whether or not disulphide bonds might be responsible for their differential susceptibility to proteolysis. Using amino acid sequencing and mass spectrometry analysis, we demonstrate that the H1 heavy chain contains one free thiol group and two disulphide bridges of which one links two largely spaced cysteine residues (Cys239 and Cys511). Thus H1 is clearly different from H2 which contains two disulphide bonds between closely located cysteine residues. However, using immunoprint analysis, we show that, when IαI is subjected to a limited digestion by Staphylococcus aureus V‐8 proteinase, the two polypeptide chains are similarly susceptible to proteolysis. This enzyme preferentially cleaves the IαI heavy chains from their N‐terminal extremity. These results are consistent with the circular dichroism (CD) analysis, suggesting that the conformation of the polypeptide backbone of H1 is not very different from that of H2, with calculated α‐helicities of 24 % and 28 %, respectively. The CD measurements reveal that the aromatic amino acids of H1 and H2 are in a different asymmetrical environment.

Inside the IαI molecule, the heavy chains are linked to the glycosaminoglycan chain via their C‐terminal aspartic acid residue. Thus we suggest that the affinity of cationic neutrophil proteinases for the anionic glycosaminoglycan is responsible for the cleavage of the heavy chains (mainly H2) near their C‐terminal end and the high susceptibility of IαI to these proteinases.
Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit
Authors: Sargent Frank , Ballantine Stuart P. , Rugman Paul A. , Palmer Tracy , Boxer David H.
Published: March 2003

An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N‐terminally sequenced. The large subunit is encoded by the hybC gene and shows no N‐terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N‐terminal and the subsequent internal tryptic‐fragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416−4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40 % identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N‐terminal signal sequence containing a twin‐arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.
The influence of nickel and cobalt on putative members of the oxygen‐sensing pathway of erythropoietin‐producing HepG2 cells
Authors: Porwol Torsten , Ehleben Wilhelm , Zierold Karl , Fandrey Joachim , Acker Helmut
Published: March 2003

Cobalt and nickel stimulate, as does hypoxia, the production of erythropoietin (EPO) in HepG2 cells. Under hypoxic conditions, a decrease in the level of intracellular reactive oxygen species (ROS) is thought to stimulate EPO expression. Cobalt and nickel may interact with the putative oxygen sensor by changing the redox state of the central iron atom of heme proteins, similar to the effects of hypoxia. It was investigated, therefore, whether cobalt and nickel interact with hemeproteins or ROS scavenging systems in the control of intracellular ROS level. Cobalt chloride (100 μM, 24 h) oxidized non respiratory as well respiratory hemeproteins and increased the oxygen consumption. In contrast, nickel chloride (300 μM, 24 h) primarily reduced respiratory hemeproteins and decreased the oxygen consumption. In HepG2 cells treated with CoCl2, iron and cobalt were localized in cytosolic granules close to the cell nucleus and in mitochondria at concentrations up to 12 mM or 41 mM, respectively. Intracellular nickel was not measurable. Three‐dimensional reconstruction of confocal laser microscopy images revealed hot spots of hydroxyl radical generation by a Fenton reaction at the sites of cytosolic iron accumulation. The ˙OH levels decreased in cobalt‐treated (to 81 %) as well as in nickel‐treated (to 67 %) HepG2 cells, accompanied by an increase of EPO expression to 167 % and 150 %, respectively. Our results underline the importance of ˙OH formed by a Fenton reaction for triggerimg EPO production. Identification of the primary hemeprotein being the oxygen sensor was not possible due to the antagonistic effects of cobalt and nickel on the redox state of detectable hemeproteins.
β‐Amyrin synthase
Authors: Kushiro Tetsuo , Shibuya Masaaki , Ebizuka Yutaka
Published: March 2003

β‐amyrin, a typical pentacyclic triterpene having an oleanane skeleton, is one of the most commonly occuring triterpenes in nature and is biosynthesized from (3S)‐2,3‐oxidosqualene. The enzyme, β‐amyrin synthase, catalyzing the cyclization of oxidosqualene into β‐amyrin, generates five rings and eight asymmetric centers in a single transformation. A homology‐based PCR method was attempted to obtain the cDNA of this enzyme from the hairy root of Panax ginseng which produces oleanane saponins together with dammarane‐type saponins. Two sets of degenerate oligonucleotide primers were designed at the regions which are highly conserved among known oxidosqualene cyclases (OSCs). Nested PCRs using these primers successfully amplified the core fragment which revealed the presence of two OSC clones PNX and PNY. Specific amplification of each clone by 3′‐RACE and 5′‐RACE was carried out to obtain the whole sequences. The two clones exhibited 60 % amino acid identity to each other. A full‐length clone of PNY was ligated into the yeast expression vector pYES2 under the GAL1 promoter to give pOSCPNY . β‐amyrin production was observed with the mutant yeast lacking lanosterol synthase, transformed by this plasmid. The sequence of pOSCPNY contains an open reading frame of 2289 nucleotides which codes for 763 amino acids with a predicted molecular mass of 88 kDa. Sequence comparison with other OSCs showed a high level of similarity with lanosterol, cycloartenol and lupeol synthases. The other clone, pOSCPNX, was shown to be cycloartenol synthase by similar expression in yeast. The present studies have revealed that distinct OSC exists for triterpene formation in higher plants, and the high level of similarity with cycloartenol synthase indicates close evolutional relationship between sterol and triterpene biosynthesis.
Assignment of disulfide bridges in bovine CD36
Authors: Rasmussen Jan T. , Berglund Lars , Rasmussen Margit S. , Petersen Torben E.
Published: March 2003

The multifunctional membrane protein CD36 is expressed on platelets, mature monocytes and macrophages, microvascular endothelial cells and mammary epithelial cells. The exact physiological function of this glycoprotein is unclear. In order to determine the number and pattern of disulfide bridges, CD36 was purified from bovine milk fat globule membranes. The purification procedure involved Triton X‐114 extraction, DEAE‐Sepharose ion‐exchange chromatography and reverse‐phase chromatography on a Resource RPC column. The CD36 preparation was used for characterization of the disulfide bridge pattern, which was determined by peptide mapping, amino acid sequence analysis, and matrix‐assisted laser‐desorption ionization/time of flight mass spectrometry. We have found that there are no free cysteines in CD36 and that the six centrally clustered cysteines are linked by disulfide bonds, Cys242−Cys310, Cys271−Cys332 and Cys312−Cys321, resulting in a 1‐3, 2‐6 and 4‐5 arrangement of the disulfide bridges. These data are in agreement with a model where the protein is oriented so that it has two short intracellular segments (residues 1−6 and 461−471) and two transmembrane domains (residues 7−28 and 439−460), and with four cysteines expected to be acylated placed near the intracellular side of the membrane. The remaining part of CD36 is extracellular, comprising eight glycosylations and three disulfide bridges. In the CD36 family of membrane proteins, it is likely that a similar pattern of disulfide bridges can be found in the sensory neuron membrane protein‐1 from the silk moth Antheraea polyphemus and the mammalian scavenger receptor class B type I, whereas lysosome membrane protein II, and epithelial membrane protein from Drosophila melanogaster are both lacking one cysteine in the area of interest.
Relationship between structure and substrate‐chain‐length specificity of mitochondrial very‐long‐chain acyl‐coenzyme A dehydrogenase
Authors: Souri Masayoshi , Aoyama Toshifumi , Yamaguchi Seiji , Hashimoto Takashi
Published: March 2003

Very‐long‐chain acyl‐coenzyme A dehydrogenase (VLCAD) is one of four enzymes which catalyze the initial step of the mitochondrial β‐oxidation with different but overlapping substrate‐chain‐length specificities. A450P, a variant of VLCAD identified in a patient with VLCAD deficiency, showed abnormal substrate‐chain‐length specificity. Based on this mutation, we studied the relationship between the structure and substrate‐chain‐length specificity of VLCAD. When VLCAD was treated with trypsin, a homodimer protein of a 48‐kDa polypeptide deprived of both the amino‐terminal 22 amino acids and the carboxyl‐terminal 145 amino acids of VLCAD was obtained. Six Ala450 variants and tryptic‐VLCAD exhibited similar substrate specificities. Effects of long‐chain acyl‐CoA on the tryptic cleavage and changes in the catalytic properties by deprivation of the carboxyl‐terminal region suggest that this region interacts with the fatty acyl moiety of long‐chain acyl‐CoA. Thus, both Ala450 and the carboxyl‐terminal region, which are not shared by other acyl‐CoA dehydrogenases, are likely to be the determinating factors in the substrate‐chain‐length specificity of VLCAD.
Widespread presence in mammals and high binding specificity of a nuclear protein that recognises the single‐stranded telomeric motif (CCCTAA)n
Authors: Marsich Eleonora , Xodo Luigi E. , Manzini Giorgio
Published: March 2003

We have recently identified a protein in HeLa nuclear extracts which recognises the single‐stranded telomeric sequence (CCCTAA)n in vertebrates [Marsich, E., Piccini, A., Xodo, L. E. & Manzini, G. (1996) Nucleic Acids Res. 24, 4029−4033]. In this paper we provide further experimental evidence, using electrophoretic mobility shift assays, SDS/PAGE after ultraviolet cross‐linking, and gel permeation chromatography techniques, that : (a) this protein displays remarkably stringent requirements for the telomeric motif sequence, as (CCCTAAA)n, (CCCCAA)n and (TCCCAA)n are tightly bound, but (CCTAA)n is not; (b) it requires at least four CCC‐block repeats properly spaced to bind strongly to DNA, e.g. the polypurine stretch of the murine Ki‐ras promoter d(CTCCCTCCCTCCCTCCTTCCCTCCCTCCC), the CarG‐motif‐containing sequence d(CCATTTCCTAATTAGGTAAAAG), and d(C)22 are not recognised by this protein; (c) it is present in nuclear extracts from several vertebrate sources including human, rat, pig, hamster and chicken; (d) its molecular mass is about 40 kDa, as determined by SDS/PAGE and non‐denaturing gel permeation chromatography, suggesting that this protein is monomeric under native conditions.
Site‐directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design
Authors: Ghate Anita A. , Air Gillian M.
Published: March 2003

The neuraminidase of influenza virus is a surface glycoprotein that catalyzes the hydrolysis of glycosidic linkages between terminal sialic acids and adjacent sugar moieties. Neuraminidase function is critical for the spread of virus to new cells, and if the enzyme activity is inhibited, then virus infection is abrogated. The neuraminidase active site is conserved in all influenza type‐A and type‐B isolates, which makes it an excellent target for drug design. To determine the potential for resistance to develop against neuraminidase inhibitors, we have constructed mutations in seven of the conserved active‐site residues of a type B (B/Lee/40) neuraminidase and analyzed the effect of the altered side chains on enzyme activity. There is a reduction in kcat in all our mutants. A transition‐state analogue inhibitor shows variation in Ki with the mutant neuraminidases, allowing interpretation of the effects of mutation in terms of transition‐state binding and product release. The results show that Tyr409 is the most critical residue for enzyme activity, but that Asp149, Arg223, Glu275 and Arg374 also play important roles in enzyme catalysis. Based on the pH profile of neuraminidase activity of the D149E mutant protein, we conclude that Asp149 is not a proton donor, but is involved in stabilizing the transition state. If designed inhibitors are targeted to these residues where mutations are highly deleterious, particularly Tyr409, Glu275 and Asp149, the virus is unlikely to generate resistance to the drug.