bluuebunny/crossref_metadata_2025 huggingface dataset:
The N‐glycans of jack bean α‐mannosidase
Authors: Kimura Yoshinobu , Hess Daniel , Sturm Arnd
Published: March 2003

The acid hydrolase α‐mannosidase, which accumulates in plant vacuoles and probably is involved in the catabolism and turnover of N‐linked glycoproteins, is itself a glycoprotein with at least one high‐mannose‐type and one complex‐type N‐glycan. The puzzling finding that α‐mannosidase stably carries its own substrate suggests that the N‐glycans have unique topologies, and important functions in protein folding, oligomerization or enzyme activity. As a first step towards the elucidation of this enigma, we purified the N‐glycans of jack bean α‐mannosidase and determined their structures by sugar composition analysis, mass spectrometry and 1H‐NMR. The structures of two N‐glycans were identified in an approximate ratio of one‐to‐one: a glucose‐containing high‐mannose‐type glycan (Glc1Man9GlcNAc2) and a small xylose‐ and fucose‐containing complex‐type glycan (Xyl1Man1Fuc1GlcNAc2). Isolation and sequencing of glycopeptides strongly suggests that one high‐mannose‐type and one complex‐type glycan are linked to specific glycosylation sites of the large α‐mannosidase subunit. The high‐mannose‐type glycan, which is a good substrate of the endoglycosidase (endo‐H), can only be removed from the enzyme after denaturation and cleavage of disulfide bonds by a reducing agent, suggesting that this glycan is buried within the folded polypeptide and, thus, protected from its hydrolytic activity. Denaturation and reduction of the native enzyme led to a marked decrease in α‐mannosidase activity. However, the activity could largely be recovered by renaturation in an appropriate renaturation buffer. In contrast, recovery of α‐mannosidase activity failed when the high‐mannose‐type glycan was removed by endo‐H prior to renaturation, indicating that this glycan appears to be important for enzyme activity.
A fourth isoform of endothelin‐converting enzyme (ECE‐1) is generated from an additional promoter
Authors: Valdenaire Olivier , Lepailleur‐Enouf Delphine , Egidy Giorgia , Thouard Anne , Barret Alain , Vranckx Roger , Tougard Claude , Michel Jean‐Baptiste
Published: March 2003

Human endothelin‐converting enzyme (ECE‐1) has been shown to exist as three isoforms (ECE‐1a, ECE‐1b and ECE‐1c) diverging in their N‐terminal sequence and displaying different patterns of subcellular localization. We report here the cloning of ECE‐1d, a novel isoform of 767 amino acids, which is generated from the same gene via the existence of an additional promoter located upstream from the third exon of the ECE‐1 gene. ECE‐1d converting activity is comparable to that of the other three isoenzymes. In contrast to ECE‐1b, ECE‐1d is expressed at the cell surface, although less strongly than ECE‐1a. We have also shown, by identifying ECE‐1b and ECE‐1d in rat, that the ECE‐1 diversity is conserved between human and rodent, suggesting its physiological relevance. The mRNA levels of the four isoforms were assessed in the two species in various cell types, revealing some differences. In particular, the ECE‐1a isoform, strongly expressed at the plasma membrane, was found to be highly expressed in primary cultures of endothelial cells but absent from primary cultures of smooth muscle cells.
Snake venom α‐neurotoxins and other ‘three‐finger’ proteins
Authors: Tsetlin Victor
Published: March 2003

The review is mainly devoted to snake venom α‐neurotoxins which target different muscle‐type and neuronal nicotinic acetylcholine receptors. The primary and spatial structures of other snake venom proteins as well as mammalian proteins of the Ly‐6 family, which structurally resemble the ‘three‐finger’ snake proteins, are also briefly discussed. The main emphasis is placed on recent data characterizing the α‐neurotoxin interactions with nicotinic acetylcholine receptors.
Functional importance of Asp56 from the α‐polypeptide of Phaseolus vulgaris glutamine synthetase
Authors: Clemente M. Teresa , Márquez Antonio J.
Published: March 2003

Replacement of Asp56 by site‐directed mutagenesis of the α‐gene from Phaseolus vulgaris glutamine synthetase heterologously expressed in Escherichia coli produces a complete loss of transferase enzyme activity, thus revealing essentiality of the residue for this particular enzyme activity. This happens independent of Asp56 being replaced by Ala or Glu, suggesting that the essentiality of this residue cannot be attributed to its negative electrical charge. However, a high level of glutamine synthetase biosynthetic specific activity (referred to glutamine synthetase protein, as determined immunologically), is present in D56A and D56E mutants, suggesting that Asp56 is an example of a residue that has a different role in the catalytic mechanism of both enzyme activities of this protein. Km for ATP, glutamate and Mg2+, as well as energy of activation, can be altered as a consequence of the performed mutations. However, the Km and catalytic efficiency for ammonium remains unaffected. Therefore, the catalytic role of Asp56 in the α‐polypeptide of higher plant glutamine synthetase is quite different from the role proposed for its highly conserved homologue in bacteria (Asp50 in E. coli), which has been associated with binding and deprotonation of ammonium. On the other hand, we also show other results indicating that Asp56 is important in the spatial conformation of the active site and/or the protein, Asp56 being a crucial residue in the salting‐out aggregation properties of the enzyme.
Recombinant pheromone binding protein 1 from Mamestra brassicae (MbraPBP1)
Authors: Campanacci Valérie , Longhi Sonia , Nagnan‐Le Meillour Patricia , Cambillau Christian , Tegoni Mariella
Published: March 2003

Pheromone binding proteins (PBPs) are small proteins (17 kDa on average) present at high concentrations (≈ 10 mm) in the sensillum lymph of Lepidoptera antennae, where they play a key role in the perception of pheromones. By expression in Escherichia coli, we have obtained large quantities (2–3 mg·L−1) of pure, soluble, Mamestra brassicae PBP1 (MbraPBP1). These quantities are compatible with the requirements of X‐ray and NMR studies. The recombinant protein has been characterized by native‐polyacrylamide gel electrophoresis, Western blotting, N‐terminal sequencing, mass spectrometry, gel filtration, circular dichroism, and NMR. Moreover, the recombinant MbraPBP1 has been shown to be able to bind the specific pheromone and a structural analogue, Z11‐16:TFMK (cis‐11‐hexadecenyl trifluoromethyl ketone), in displacement experiments. Our results on MbraPBP1 confirm and extend previous findings on PBPs. MbraPBP1 and two PBPs from different species have been found to exist as dimers under nondenaturing conditions. The CD and structural prediction data confirm a markedly helical structure for insect PBPs rather than the β‐barrel fold found in vertebrates odorant binding proteins. We have tentatively identified the location of the helices and the short β‐strands with respect to the binding site. Currently we have obtained small diffracting crystals of the recombinant MbraPBP1 and determined their space group and molecular content.
Solution structure of plantaricin C, a novel lantibiotic
Authors: Turner David L. , Brennan Lorraine , Meyer Helmut E. , Lohaus Christiane , Siethoff Christoph , Costa Helena S. , Gonzalez Beatriz , Santos Helena , Suárez Juan E.
Published: March 2003

Plantaricin C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin, is a lantibiotic. One dehydroalanine, one lanthionine and three β‐methyl‐lanthionine residues were found in its 27 amino acid sequence. The plantaricin C structure has two parts: the first comprises the six NH2‐terminal residues, four of which are lysines, which confer a strong positive charge to this stretch. The amino acids in positions 7 and 27 form the lanthionine bridge, giving a globular conformation to the rest of the molecule. The β‐methyl‐lanthionine bridges are established between residues 12–15, 13–18 and 23–26. This central region has a charge distribution compatible with an amphipathic α‐helix, through which plantaricin C would become inserted into the membrane matrix of sensitive organisms, provoking the opening of pores and leakage of the cytoplasmic content.
Comparison of the chemical mechanisms of action of yeast and equine liver alcohol dehydrogenase
Authors: Leskovac Vladimir , Trivić Svetlana , Anderson Bruce M.
Published: March 2003

The pH‐dependence of the steady‐state kinetic parameters and the ligand‐binding parameters for competitive dead‐end inhibitors for the yeast alcohol dehydrogenase (EC 1.1.1.1, constitutive, cytoplasmic) reaction was studied in the pH range 6–10. These studies were designed in order to assign the appropriate pKa values to all dissociation forms of enzyme in the chemical mechanism of action for the yeast enzyme, previously proposed by Cook and Cleland [P. F. Cook & W. W. Cleland (1981) Biochemistry20, 1796–1816]. In addition, the chemical mechanism of action for the yeast enzyme, proposed in this work, was compared with a similar mechanism of action for the horse liver enzyme, proposed by Cook and Cleland. Substantial differences were found, especially in the binding of coenzymes and in the structure of enzyme–coenzyme complexes.
A coil–helix instead of a helix–coil motif can be induced in a chloroplast transit peptide from Chlamydomonas reinhardtii
Authors: Krimm Isabelle , Gans Pierre , Hernandez Jean‐François , Arlaud Gérard J. , Lancelin Jean‐Marc
Published: March 2003

A synthetic peptide MQVTMKSSAVSGQRVGGARVATRSVRRAQLQV corresponding to the 32 amino acid chloroplast transit sequence of the ribulose bisphosphatase carboxylase/oxygenase activase preprotein from Chlamydomonas reinhardtii, required for translocation through the envelope of the chloroplast, has been characterized structurally using CD and NMR under the same experimental conditions as used previously for the 32 amino acid presequence of preferredoxin from the same organism [Lancelin, J.‐M., Bally, I., Arlaud, G. J., Blackledge, M., Gans, P., Stein, M. & Jacquot, J.‐P. (1994) FEBS Lett.343, 261–266]. The peptide is found to undergo a conformational transition in aqueous 2,2,2‐trifluoroethanol, characterized by three turns of amphiphilic α‐helix in the C‐terminal region preceded by a disordered coil in the N‐terminal region. Compared with the preferredoxin transit peptide, the helical and coiled domains are arranged in the reverse order along the peptide sequence, but the positively charged groups are distributed analogously as well as the hydrophobic residues within the amphiphilic α‐helix. It is proposed that such coil–helix or helix–coil motifs, occasionally repeated, could be an intrinsic structural feature of chloroplastic transit peptides, adapted to the proper translocase and possibly to each nuclear‐encoded chloroplast preproteins. This feature may distinguish chloroplastic transit sequences from the other organelle‐targeting peptides in the eukaryotic green alga C. reinhardtii, particularly the mitochondrial transit sequences.
Effects of contractile activity and hypothyroidism on nuclear hormone receptor mRNA isoforms in rat skeletal muscle
Authors: Schuler Michael J. , Bühler Susanne , Pette Dirk
Published: March 2003

Absolute molecule numbers of thyroid hormone receptor isoforms T3Rα1, T3Rα2, T3Rβ1, and the 9‐cis retinoic acid receptor γ were measured in adult fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles of rat by competitive reverse transcriptase (RT)‐PCR. The nuclear hormone receptor corepressor (NCoR) mRNA was quantified by noncompetitive RT‐PCR in the same muscles. T3Rβ1 mRNA was the most abundant isoform in both muscle types. All nuclear hormone receptor (NHR) mRNAs were found at lower molecule numbers in fast than in slow muscle. No differences existed with regard to NCoR mRNA. With the exception of T3Rα1 in the EDL, hypothyroidism led to decreases in NHR mRNAs, especially in SOL, but did not significantly affect the level of NCoR mRNA. Enhanced neuromuscular activity of the fast EDL muscle, as induced by chronic low‐frequency stimulation, transiently increased NHR mRNAs, but decreased NCoR mRNA. These chronic‐low‐frequency‐stimulation‐induced changes were attenuated by hypothyroidism.
Isolation, characterization and cDNA cloning of nicotianamine synthase from barley
Authors: Herbik A. , Koch G. , Mock H.‐P. , Dushkov D. , Czihal A. , Thielmann J. , Stephan U. W. , H. Bäumlein
Published: March 2003

Basic cellular processes such as electron transport in photosynthesis and respiration require the precise control of iron homeostasis. To mobilize iron, plants have evolved at least two different strategies. The nonproteinogenous amino acid nicotianamine which is synthesized from three molecules of S‐adenosyl‐l‐methionine, is an essential component of both pathways. This compound is missing in the tomato mutant chloronerva, which exhibits severe defects in the regulation of iron metabolism. We report the purification and partial characterization of the nicotianamine synthase from barley roots as well as the cloning of two corresponding gene sequences. The function of the gene sequence has been verified by overexpression in Escherichia coli. Further confirmation comes from reduction of the nicotianamine content and the exhibition of a chloronerva‐like phenotype due to the expression of heterologous antisense constructs in transgenic tobacco plants. The native enzyme with an apparent Mr of ≈ 105 000 probably represents a trimer of S‐adenosyl‐l‐methionine‐binding subunits. A comparison with the recently cloned chloronerva gene of tomato reveals striking sequence homology, providing support for the suggestion that the destruction of the nicotianamine synthase encoding gene is the molecular basis of the tomato mutation.
Stage‐specific expression of α1,2‐fucosyltransferase and α1,3‐fucosyltransferase (FT) during mouse embryogenesis
Authors: Liu Ni , Jin Cheng , Zhu Zheng M. , Zhang Jianing , Tao Hua , Ge Changhui , Yang Shoujun , Zhang Shuzheng
Published: March 2003

Lex[Galβ1–4(Fucα1–3)GlcNAc] and Ley[Fucα1–2Galβ1–4(Fucα1–3)GlcNAc] are both stage‐specific embryonic antigens. Lex is first detected on the blastomeres of the 8‐cell stage embryo, which correlates with the onset of blastomere compaction. Ley is highly expressed on the surface of the blastocyst, which has been shown to be involved in blastocyst attachment in the mouse. In the present study, mouse α1,2‐FT (also known as FUT1) and α1,3‐FT (also known as Fuc‐TIV), which were responsible for Lex and Ley formation, were examined in preimplantation stage embryos by reverse transcription‐PCR and in situ hybridization. α1,3‐FT mRNA was detected in all embryos of preimplantation stage, while α1,2‐FT mRNA emerged in the later stage embryos from 8‐cell to 16‐cell to the blastocyst. These results indicated the expression of Ley was regulated by α1,2‐FT. In situ hybridization showed that these two enzyme mRNAs were detected only in morula and blastocyst stage embryos. The α1,2‐FT and α1,3‐FT mRNAs were located in both the inner cell mass and the trophoblast cells. 2‐Cell and 4‐cell embryos were isolated from the oviduct and cultured in vitro to the 8‐cell, morula and blastocyst stage. The expression of α1,2‐FT and α1,3‐FT were observed in these embryos developed in vitro; immunohistochemical analysis also showed that Ley expression was positive. These results suggested the stage‐specific expression of Ley on the embryos was synthesized by endogenous α1,2‐FT and α1,3‐FT rather than transfer from other sources. In addition, the expression of α1,2‐FT was differentially regulated and the uterine factor was not prerequisite of the expression of Ley.
The lipopolysaccharide of Moraxella catarrhalis
Authors: Holme Tord , Rahman Motiur , Jansson Per‐Erik , Widmalm Göran
Published: March 2003

Moraxella catarrhalis has recently been shown to be both widespread and pathogenic, in contrast to previous reports. Several factors have been suggested as virulence factors, lipopolysaccharide (LPS) being one. Recent studies have shown the LPS to be without the O‐chain, i.e. the polysaccharide part, and to have specific structural features corresponding to each of the three serogroups, A, B and C. The structures resemble in many respects those present in other Gram‐negative nonenteric bacteria, with a galabiosyl element as a prominent common denominator. The presence of such common structures suggests that the LPS of these bacteria might be a part of a mechanism of survival for bacteria colonizing the human host.
Host defence peptides from the skin glands of the Australian Blue Mountains tree‐frog Litoria citropa
Authors: Wegener Kate L. , Wabnitz Paul A. , Carver John A. , Bowie John H. , Chia Brian C. S. , Wallace John C. , Tyler Michael J.
Published: March 2003

Nineteen citropin peptides are present in the secretion from the granular dorsal glands of the Blue Mountains tree‐frog Litoria citropa; 15 of these peptides are also present in the secretion from the submental gland. Two major peptides, citropin 1.1 (GLFDVIKKVASVIGGL‐NH2), citropin 1.2 (GLFDIIKKVASVVGGL‐NH2) and a minor peptide, citropin 1.3 (GLFDIIKKVASVIGGL‐NH2) are wide‐spectrum antibacterial peptides. The amphibian has an endoprotease which deactivates these membrane‐active peptides by removing residues from the N‐terminal end: loss of three residues gives the most abundant degradation products. The solution structure of the basic peptide citropin 1.1 has been determined by NMR spectroscopy [in a solvent mixture of trifluoroethanol/water (1 : 1)] to be an amphipathic α‐helix with well‐defined hydrophobic and hydrophilic regions. The additional four peptides produced by the dorsal glands are structurally related to the antibacterial citropin 1 peptides but contain three more residues at their C‐terminus [e.g. citropin 1.1.3 (GLFDVIKKVASVIGLASP‐OH)]. These peptides show minimal antibacterial activity; their role in the amphibian skin is not known.
Molecular cloning, expression and characterization of the human serine/threonine kinase Akt‐3
Authors: Masure Stefan , Haefner Burkhard , Wesselink Jan‐Jaap , Hoefnagel Evert , Mortier Eva , Verhasselt Peter , Tuytelaars An , Gordon Robert , Richardson Alan
Published: March 2003

Akt (also known as PKB or RAC‐PK) is an intracellular serine/threonine kinase involved in regulating cell survival. Although this makes it a promising target for the discovery of drugs to treat human cancer, a complicating factor may be the role played by Akt in insulin signalling. Two human isoforms, Akt‐1 and Akt‐2, have been described previously and a third isoform has been identified in rats (here termed Akt‐3, but also called RAC‐PK‐γ or PKB‐γ). We describe the identification of the corresponding human isoform of Akt‐3. The gene encoding human Akt‐3 was localized to chromosome 1q43–44. The predicted protein sequence is 83% identical to human Akt‐1 and 78% identical to human Akt‐2, and contains a pleckstrin homology domain and a kinase domain. In contrast to the published rat Akt‐3 isoform, human and mouse Akt‐3 also possess a C‐terminal ‘tail’ that contains a phosphorylation site (Ser472) thought to be involved in the activation of Akt kinases. In addition to phosphorylation of Ser472, phosphorylation of Thr305 also appears to contribute to the activation of Akt‐3 because mutation of both these residues to aspartate increased the catalytic activity of Akt‐3, whereas mutation to alanine inhibited activation. Akt‐3 activity could be inhibited by the broad spectrum kinase inhibitor staurosporine and by the PKC inhibitor Ro 31‐8220, but not by other PKC or PKA inhibitors tested. Although Akt‐3 is expressed widely, it is not highly expressed in liver or skeletal muscle, suggesting that its principle function may not be in regulating insulin signalling. These observations suggest that Akt‐3 is a promising target for the discovery of novel chemotherapeutic agents which do not interfere with insulin signalling.
Veratryl alcohol‐mediated oxidation of isoeugenyl acetate by lignin peroxidase
Authors: ten Have Rimko , de Thouars Robbert G. , Swarts Henk J. , Field Jim A.
Published: March 2003

The mechanism of the veratryl alcohol (VA)‐mediated oxidation of isoeugenyl acetate (IEA) by lignin peroxidase, and the subsequent spontaneous Cα–Cβ cleavage of IEA to vanillyl acetate were studied. IEA oxidation only occurred in the presence of VA. It probably did not bind to lignin peroxidase as evidenced by an unaffected Km for VA in the presence of IEA, and by the fact that a 10‐fold molar excess of the unreactive IEA counterpart, eugenyl acetate, did not affect the IEA oxidation rate. IEA was very efficient in recycling VA. Up to 34 mol of IEA were oxidized per mol VA. Formation of the predominant VA oxidation product, veratraldehyde, was postponed until IEA was almost completely oxidized. Together these findings suggest that IEA was oxidized by VAz.rad;+ rather than directly by lignin peroxidase. Thus, VA functioned as a redox mediator during IEA oxidation which is remarkable considering the high calculated ionization potential of 8.81 eV.

 Regardless of the presence of O2, ≈ 2 mol of IEA were consumed per mol H2O2, which indicated that IEA was enzymatically oxidized by one electron to the putative radical cation (IEAz.rad;+). After formation of IEAz.rad;+, a series of O2‐dependent chemical reactions were responsible for Cα–Cβ cleavage to the major oxidation product vanillyl acetate, as evidenced by the observation that an N2 atmosphere did not inhibit IEA oxidation, but almost completely inhibited vanillyl acetate formation. GC‐MS analyses revealed that under an air atmosphere 1‐(4′‐acetoxy‐3′‐methoxyphenyl)‐2‐propanone, 1‐(4′‐acetoxy‐3′‐methoxyphenyl)‐1‐hydroxy‐2‐propanone, and 1‐(4′‐acetoxy‐3′‐methoxyphenyl)‐2‐hydroxy‐1‐propanone were also formed. Formation of the latter two was diminished under an N2 atmosphere.
Down‐regulation of mammalian 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase activity with highly purified liposomal cholesterol
Authors: Plemenitas Ana , Watson John A.
Published: March 2003

Chinese hamster ovary‐215 cells (CHO‐215) cannot synthesize C27 and C28 sterols because of a defect in the reaction that decarboxylates 4‐carboxysterols [Plemenitas, A., Havel, C.M. & Watson, J.A. (1990) J. Biol. Chem.265, 17012–17017]. Thus, CHO‐215 cell growth is dependent on an exogenous metabolically functional source of cholesterol. We used CHO‐215 cells to (a) determine whether highly purified (> 99.5%) cholesterol, in egg lecithin liposomes, could down‐regulate derepressed 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase activity and if so (b) determine whether the loss in reductase catalytic activity correlated kinetically with the synthesis and accumulation of detectable oxycholesterol derivatives.

Liposomal cholesterol (26–39 µm) supported maximum CHO‐215 growth and initiated suppression of HMG‐CoA reductase activity at concentrations greater than 50 µm. Maximum suppression (50–60%) of reductase activity was achieved with 181.3 µm liposomal cholesterol in 6 h. Also, regulatory concentrations of highly purified liposomal [3H]cholesterol were not converted (biologically or chemically) to detectable levels of oxy[3H]cholesterol derivatives during 3–6 h incubations. Lastly, a broad‐spectrum cytochrome P450 inhibitor (miconazole) had no effect on liposomal cholesterol‐mediated suppression of HMG‐CoA reductase activity.

These observations established that (a) highly purified cholesterol, incorporated into egg lecithin liposomes, can signal the down‐regulation of derepressed mammalian cell HMG‐CoA reductase activity and (b) if oxycholesterol synthesis was required for liposomal cholesterol‐mediated down‐regulation, the products had to be more potent than 24‐, 25‐, or 26‐/27‐hydroxycholesterol.
The pneumococcal common antigen C‐polysaccharide occurs in different forms
Authors: Karlsson Camilla , Jansson Per‐Erik , Skov Sørensen Uffe B.
Published: March 2003

The structure of the pneumococcal common antigen, C‐polysaccharide, from a noncapsulated pneumococcal strain, CSR SCS2, was studied using 1H‐NMR, 13C‐NMR and 31P‐NMR spectroscopy. The dependence of NMR chemical shifts on the variation in pD was also studied. It was established that the C‐polysaccharide is composed of a backbone of tetrasaccharide‐ribitol repeating units that are linked to each other by a phosphodiester linkage between position 5 of a d‐ribitol residue and position 6 of a β‐d‐glucopyranosyl residue. The polysaccharide is substituted with one residue of phosphocholine at position 6 of the 4‐substituted 2‐acetamido‐2‐deoxy‐α‐d‐galactopyranosyl residue. Both galactosamine residues in the polysaccharide are N‐acetylated.

                    O)‐P‐Cho

                      |

                    6

6)‐β‐d‐Glcp‐(1→3)‐α‐AATp‐(1→4)‐α‐d‐GalpNAc‐(1→3)‐β‐d‐GalpNAc‐(1→1)‐d‐ribitol‐5‐P‐(O→

where AAT is 2‐acetamido‐4‐amino‐2,4,6‐trideoxy‐d‐galactose and Cho is choline. This structure differs, concerning phosphocholine substituents and N‐acetylation, from those reported previously for pneumococcal C‐polysaccharide [Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Biochemistry19, 4712–4719; Fischer, W., Behr, T., Hartmann, R., Peter‐Katalinic, J. & Egge, H. (1993) Eur. J. Biochem.215, 851–857; Kulakowska, M., Brisson, J.‐R., Griffith, D.W., Young, N.M. & Jennings, H.J. (1993) Can. J. Chem.71, 644–648]. The structures of the C‐polysaccharides present in three pneumococcal types were also examined. They contain one (in 18B) or two (in 32F and 32A) phosphocholine residues in the repeating unit. The degree of substitution was not determined. The backbone of all examined C‐polysaccharides was identical and in all cases both galactosamine residues appeared to be N‐acetylated.
Characterization and analysis of a novel glycoprotein from snake venom using liquid chromatography‐electrospray mass spectrometry and Edman degradation
Authors: Zeng Rong , Xu Qiang , Shao Xiao‐Xia , Wang Ke‐Yi , Xia Qi‐Chang
Published: March 2003

An N‐linked glycosylation in a novel C‐lectin protein from snake venom was observed by Edman degradation and liquid chromatography‐electrospray mass spectrometry. The peptides obtained by trypsin cleavage were analyzed to confirm the amino acid sequence and Asn5 was found to be the N‐glycosylation site. The result was further confirmed by N‐glycosidase digestion. In addition, the protein and tryptic peptides with and without glycan chain were characterized by mass spectrometry according to the mass difference. The glycopeptide obtained from proteolytic digestion was analyzed and the glycoforms were identified as high‐mannose type by tandem MS coupled with α‐mannosidase digestion. An oxidized Met residue was detected and located in the protein by mass spectrometry.
Acquisition of species‐specific O‐linked carbohydrate chains from oviducal mucins inRana arvalis
Authors: Coppin Alexandra , Maes Emmanuel , Flahaut Christophe , Coddeville Bernadette , Strecker Gérard
Published: March 2003

The extracellular matrix surrounding amphibian eggs is composed of mucin‐type glycoproteins, highly O‐glycosylated and plays an important role in the fertilization process. Oligosaccharide‐alditols were released from the oviducal mucins of the anuranRana arvalisby alkali‐borohydride treatment in reduced conditions. Neutral and acidic oligosaccharides were fractionated by ion‐exchange chromatographies and purified by HPLC. Each compound was identified by matrix assisted laser desorption ionization‐time of flight (MALDI‐TOF) spectrometry, NMR spectroscopy, electrospray ionization‐tandem mass spectroscopy (ESI‐MS/MS) and permethylation analyses. This paper reports on the structures of 19 oligosaccharide‐alditols, 12 of which have novel structures. These structures range in size from disaccharide to octasaccharide. Some of them are acidic, containing either a glucuronic acid or, more frequently, a sulfate group, located either at the 6 position of GlcNAc or the 3 or 4 positions of Gal. This latter sulfation is novel and has only been characterized in the speciesR. arvalis. This structural analysis led to the establishment of several novel carbohydrate structures, demonstrating the structural diversity and species‐specificity of amphibian glycoconjugates.
Supramolecular organization of photosystem II and its light‐harvesting antenna in partially solubilized photosystem II membranes
Authors: Boekema Egbert J. , van Roon Henny , van Breemen Jan F. L. , Dekker Jan P.
Published: March 2003

We present an extended analysis of the organization of green plant photosystem II and its associated light‐harvesting antenna using electron microscopy and image analysis. The analysis is based on a large dataset of 16 600 projections of negatively stained PSII–LHCII supercomplexes and megacomplexes prepared by means of three different pretreatments. In addition to our previous work on this system [Boekema, E.J., van Roon, H., Calkoen, F., Bassi, R. and Dekker, J.P. (1999) Biochemistry38, 2233–2239], the following results were obtained.

The rotational orientation of trimeric LHCII at the S, M and L binding positions was determined. It was found that compared to the S trimer, the M and L trimers are rotationally shifted by about −20° and −50°, respectively.

The number of projections with empty CP29, CP26 and CP24 binding sites was found to be about 0, 18 and 4%, respectively. We suggest that CP26 and CP24 are not required for the binding of trimeric LHCII at any of the three binding positions.

A new type of megacomplex was observed with a characteristic windmill‐like shape. This type III megacomplex consists of two C2S2 supercomplexes connected at their CP26 tips.

Structural variation in the region of the central dimeric photosystem II complex was found to occur at one specific position near the periphery of the complex. We attribute this variation to the partial absence of an extrinsic polypeptide or one or more small intrinsic membrane proteins.
The amyloid β‐protein precursor of Alzheimer's disease is degraded extracellularly by a Kunitz protease inhibitor domain‐sensitive trypsin‐like serine protease in cultures of chick sympathetic neurons
Authors: Caswell Mark D. , Mok Su San , Henry Anna , Cappai Roberto , Klug Genevieve , Beyreuther Konrad , Masters Colin L. , Small David H.
Published: March 2003

The amyloid β‐protein precursor (APP) of Alzheimer's disease (AD) is cleaved either by α‐secretase to generate an N‐terminally secreted fragment, or by β‐ and γ‐secretases to generate the β‐amyloid protein (Aβ). The accumulation of Aβ in the brain is an important step in the pathogenesis of AD. Alternative mRNA splicing can generate isoforms of APP which contain a Kunitz protease inhibitor (KPI) domain. However, little is known about the physiological function of this domain. In the present study, the metabolic turnover of APP was examined in cultured chick sympathetic neurons. APP was labelled by incubating neurons for 5 h with [35S]methionine and [35S]cysteine. Intracellular labelled APP decayed in a biphasic pattern suggesting that trafficking occurs through two metabolic compartments. The half‐lives for APP in each compartment were 1.5 and 5.7 h, respectively. A small fraction (10%) of the total APP was secreted into the culture medium where it was degraded with a half‐life of 9 h. Studies using specific protease inhibitors demonstrated that this extracellular breakdown was due to cleavage by a trypsin‐like serine protease that was secreted into the culture medium. Significantly, this protease was inhibited by a recombinant isoform of APP (sAPP751), which contains a region homologous to the Kunitz protease inhibitor (KPI) domain. These results suggest that KPI forms of APP regulate extracellular cleavage of secreted APP by inhibiting the activity of a secreted APP‐degrading protease.
Dipetalogastin, a potent thrombin inhibitor from the blood‐sucking insectDipetalogaster maximus
Authors: Mende Katrin , Petoukhova Olga , Koulitchkova Valentina , Schaub Günter A. , Lange Ute , Kaufmann Roland , Nowak Götz
Published: March 2003

A cDNA coding for the thrombin inhibitor dipetalogastin has been isolated from a stomach library of Dipetalogaster maximus, a blood‐sucking insect. The open reading frame of the cloned inhibitor cDNA codes for a protein of 344 amino‐acid residues. Sequence analysis reveals the existence of three repeated homologous main regions, indicating that the inhibitor consists of three domains. Each domain shows a double‐headed structure with an internal sequence homology like rhodniin, the thrombin inhibitor from the blood‐sucking insect Rhodnius prolixus. Peptide sequence comparisons of the deduced amino‐acid sequence exhibit a high homology of the domains I and II to the natural inhibitor dipetalogastin from the stomach content of D. maximus and to rhodniin, respectively. Significant sequence similarities to Kazal‐type inhibitors, like the conserved sequence CGXDXXTYXNXC and several cysteine residues, indicate that the thrombin inhibitor from D. maximus is a further blood‐sucking insect which belongs to the Kazal‐type family (besides rhodniin). A biologically active recombinant protein corresponding to domain II of the dipetalogastin cDNA was expressed in Escherichia coli. The isolated recombinant dipetalogastin with a molecular mass of 12.91 kDa has proved to be a specific thrombin inhibitor similar to its natural counterpart as well as rhodniin and hirudin. The Ki value of the recombinant dipetalogastin was determined to be 49.3 ± 22.28 fm.
Conformational stability of human erythrocyte transglutaminase
Authors: Bergamini Carlo M. , Dean Mariangela , Matteucci Gabriella , Hanau Stefania , Tanfani Fabio , Ferrari Carlo , Boggian Marisa , Scatturin Angelo
Published: March 2003

Tissue‐type transglutaminase is irreversibly inactivated during heat treatment. The rate of inactivation is low at pH 7.5; it increases slightly at acid pH (6.1) but much more at alkaline pH (9.0–9.5), suggesting that specific effects take place in the alkaline range, possibly in relation to decreased stability of the transition‐state intermediate as pH is raised above 9.0. Differential scanning calorimetry experiments indicate that thermal unfolding of the protein occurs with two separate transitions, involving independent regions of the enzyme. They are assigned to domains 1 and 2 and domains 3 and 4, respectively, by a combination of calorimetric and spectroscopic techniques. When considering the effects of pH, we noted that transglutaminase was unfolded via different pathways at the different pH values considered. At acid pH, the whole structure of the protein was lost irreversibly, with massive aggregation. At neutral and, even more so, at alkaline pH, aggregation was absent (or very limited at high protein concentration) and the loss of secondary structure was dependent on the ionization state of crucial lysine residues. Unfolding at pH 9.5 apparently chiefly involved the N‐terminal region, as testified by changes in protein intrinsic fluorescence. In addition, the C‐terminal region was destabilized at each pH value tested during thermal unfolding, as shown by digestion with V8 proteinase, which is inactive on the native protein. Evidence was obtained that the N‐terminal and C‐terminal regions interact with each other in determining the structure of the native protein.
Evolution of oligomeric proteins
Authors: D’Alessio Giuseppe
Published: March 2003

The model system made up of a monomeric and a dimeric ribonuclease of the pancreatic‐type superfamily has recently attracted the attention of investigators interested in the evolution of oligomeric proteins. In this system, bovine pancreatic ribonuclease (RNase A) is the monomeric prototype, and bovine seminal ribonuclease (BS‐RNase) is the dimeric counterpart. However, this evolutionary case is unusual, as BS‐RNase is the only dimeric member of the whole large superfamily comprising more than 100 identified members from amphibia, aves, reptilia and mammalia. Furthermore, although the seminal‐type RNase gene can be traced back to the divergence of the ruminants, it is expressed only in a single species (Bos taurus). These unusual findings are discussed, as well as previous hypotheses on the evolution of seminal RNase. Furthermore, a new ‘minimalist’ hypothesis is proposed, in line with basic principles of structural biology and molecular evolution.
Identification and characterization of a melanocyte‐specific novel 65‐kDa peripheral membrane protein
Authors: Samaraweera Preminda , Donatien Philippe D. , Qazi Sadia , Kobayashi Takeshi , Hearing Vincent J. , Panthier Jean‐Jacques , Orlow Seth J.
Published: March 2003

In order to study proteins of the melanosome, we developed a panel of antisera against various protein fractions of melanosomes from B16 melanoma cells. An antiserum raised against a Triton X‐100 insoluble fraction of melanosomes recognized a 65‐kDa protein in melanocytes from mice homozygous for the buff mutation, but not in their wild type counterparts. Further studies were conducted using a specific, second generation antiserum raised against the purified protein. The protein was also detected in melanocytes cultured from albino mice, but absent in cultured mouse cell lines not of melanocyte origin. Density gradient centrifugation of subcellular organelles and indirect immunofluorescent cell staining, indicated that the protein was associated with melanosomes and vesicles. The protein on intact organelles could be made soluble using sodium carbonate, and digested with proteases in the absence of detergent suggesting that it was a peripheral membrane protein localized on the cytosolic face of organelle membranes. Metabolic labelling of cells and N‐glycosidase F digestion of cell extracts indicated that the protein was not N‐glycosylated. Based on its intracellular localization and biochemical defects in the buff mouse, a potential role has been suggested for the 65‐kDa protein in intracellular membrane trafficking.
Impact of amino acids 22–27 of Rho‐subfamily GTPases on glucosylation by the large clostridial cytotoxins TcsL‐1522, TcdB‐1470 and TcdB‐8864
Authors: Müller Stefani , von Eichel‐Streiber Christoph , Moos Michael
Published: March 2003

Here we report data describing some principles of the interaction between small GTP‐binding proteins and large Clostridial cytotoxins (LCTs). Our investigation was based on the differential glucosylation of Rac1 versus RhoA by LCTs TcsL‐1522, TcdB‐1470 and TcdB‐8864. Chimeric RhoA/Rac1 proteins and GTPases mutated at defined regions or single amino acids were used as substrates. Starting with chimeric Rac/Rho proteins we demonstrated that proteins containing the N‐terminal 73 amino acids of Rac1 (but not those of RhoA) were efficiently glucosylated. Within this stretch, three regions differ significantly in Rac1 and RhoA. Regions containing amino acids 41–45 and 50–54 had no effect on toxin induced glucosylation, whereas amino acids 22–27 had a drastic impact on the potential of all three toxins to covalently modify the GTPases. Point mutations K25T of RhoA (numbering according to Rac1) and K27A of Cdc42 significantly increased glucosylation by the cytotoxins; introduction of lysines at the equivalent positions of Rac1 hindered modification. Our experiments demonstrate the influence of this charged residue on GTPase–LCT interactions. Amino acids 22–27 are part of the transition between the α1‐helix to the switch I region of small GTP‐binding proteins; both are known structures for specificity determination of the interactions with physiologic partners. Comparing these structures with data from our investigation we suggest that TcsL‐1522, TcdB‐1470 and TcdB‐8864 mimic aspects of the physiologic interactions of small GTP‐binding proteins.
Biogenesis of giant mitochondria during insect flight muscle development in the locust, Locusta migratoria (L.)
Authors: Sogl Bettina , Gellissen Gerd , Wiesner Rudolf J.
Published: March 2003

The biogenesis of giant mitochondria in flight muscle of Locusta migratoria (L.) was analyzed at the molecular level. During the 2 weeks between the beginning of the last larval stage and the imago capable of sustained flight, individual mitochondria have been shown to enlarge 30‐fold and the fractional mitochondrial volume of muscle cells increases fourfold [Brosemer, R.W., Vogell, W. and Bücher, Th. (1963) Biochem. Z. 338, 854–910]. Within the same period, the activity of cytochrome c oxidase, containing subunits encoded on mitochondrial DNA, increased twofold. However, no significant change in mitochondrial DNA copy number, and even a threefold decrease in mitochondrial transcripts, was observed. Mitochondrial translation rate, measured in isolated organelles, was twofold higher in larval muscle, which can be explained only partly by the higher content of mitochondrial RNAs. Thus, rather unusually, in this system of mitochondrial differentiation, the mitochondrial biosynthetic capacity correlates with the rate of organelle biogenesis rather than the steady‐state concentration of a marker enzyme. The copy number of mitochondrial DNA does not seem to play a major role in determining either mitochondrial transcript levels or functional mass.
Oriented channels reveal asymmetric energy barriers for sugar translocation through maltoporin of Escherichia coli
Authors: Van Gelder Patrick , Dumas Fabrice , Rosenbusch Jürg P. , Winterhalter Mathias
Published: March 2003

Sugar transport through maltoporin of Escherichia coli was investigated. This protein facilitates maltooligosaccharide translocation via a binding site in the channel. Because incorporation of the protein into the bilayer results in randomly orientated channels, we re‐examined the postulated symmetric translocation model by reconstitution of maltoporin under an externally applied field. Upon binding of bacteriophage λ, which exploit surface‐exposed loops of maltoporin as the receptor, sugar permeation, but not the ion current, was blocked. Thus using the phage‐to‐probe orientation we were able to show that the channels were ≈ 80% directionally inserted into the bilayer. Moreover, asymmetry of the channel was revealed because sugar entrance through the ‘open’ periplasmic side of maltoporin was similarly reduced. Here a new asymmetrical two‐barrier model is presented. Based on liposome‐swelling assays and current‐fluctuation analysis we conclude that the periplasmic side of the porin shows a two‐ to threefold higher energy barrier than the extracellular loop‐side of the channels.
The structure of the membrane‐binding 38 C‐terminal residues from bovine PP3 determined by liquid‐ and solid‐state NMR spectroscopy
Authors: Bak Mads , Sørensen Morten D. , Sørensen Esben S. , Rasmussen Lone K. , Sørensen Ole W. , Petersen Torben E. , Nielsen Niels C.
Published: March 2003

The secondary structure and membrane‐associated conformation of a synthetic peptide corresponding to the putative membrane‐binding C‐terminal 38 residues of the bovine milk component PP3 was determined using 1H NMR in methanol, CD in methanol and SDS micelles, and 15N solid‐state NMR in planar phospholipid bilayers. The solution NMR and CD spectra reveal that the PP3 peptide in methanol and SDS predominantly adopts an α‐helical conformation extending over its entire length with a potential bend around residue 19. 15N solid‐state NMR of two PP3 peptides 15N‐labelled at the Gly7 and Ala32 positions, respectively, and dissolved in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol phospholipid bilayers shows that the peptide is associated to the membrane surface with the amphipathic helix axis oriented parallel to the bilayer surface.
Peptide phosphorylation by calcium‐dependent protein kinase from maize seedlings
Authors: Loog Mart , Toomik Reet , Sak Katrin , Muszynska Grazyna , Järv Jaak , Ek Pia
Published: March 2003

Ca2+‐dependent protein kinase (CDPK‐1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cβ (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose‐membrane‐attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK‐1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L‐5X‐4R‐3X‐2X‐1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA.
Release of the cyano moiety in the crystal structure of N‐cyanomethyl‐N‐(2‐methoxyethyl)‐daunomycin complexed with d(CGATCG)
Authors: Saminadin Pascale , Dautant Alain , Mondon Martine , Langlois d’Estaintot Béatrice , Courseille Christian , Précigoux Gilles
Published: March 2003

Doxorubicin is among the most widely used anthracycline in cancer chemotherapy. In an attempt to avoid the cardiotoxicity and drug resistance of doxorubicin therapy, several analogues were synthesized. The cyanomorpholinyl derivative is the most cytotoxic. They differ greatly from their parent compound in their biological and pharmacological properties, inducing cross‐links in drug DNA complexes.

The present study concerns N‐cyanomethyl‐N‐(2‐methoxyethyl)‐daunomycin (CMDa), a synthetic analogue of cyanomorpholino‐daunomycin. Compared to doxorubicin, CMDa displays a cytotoxic activity on L1210 leukemia cells at higher concentration but is effective on doxorubicin resistant cells. The results of fluorescence quenching experiments as well as the melting temperature (ΔTm = 7.5 °C) studies are consistent with a drug molecule which intercalates between the DNA base pairs and stabilizes the DNA double helix. The crystal structure of CMDa complexed to the hexanucleotide d(CGATCG) has been determined at 1.5 Å resolution. The complex crystallizes in the space group P41212 and is similar to other anthracycline‐hexanucleotide complexes. In the crystal state, the observed densities indicate the formation of N‐hydroxymethyl‐N‐(2‐methoxyethyl)‐daunomycin (HMDa) with the release of the cyano moiety without DNA alkylation. The formation of this degradation compound is discussed in relation with other drug modifications when binding to DNA. Comparison with two other drug‐DNA crystal structures suggests a correlation between a slight change in DNA conformation and the nature of the amino sugar substituents at the N3′ position located in the minor groove.