bluuebunny/crossref_metadata_2025 huggingface dataset:
Release of 14‐kDa group‐II phospholipase A2 from activated mast cells and its possible involvement in the regulation of the degranulation process
Authors: MURAKAMI Makoto , KUDO Ichiro , SUWA Yorimasa , INOUE Keizo
Published: March 2005

Group II phospholipase A2 was detected in appreciable amounts in rat peritoneal mast cells. The effect of several inhibitors specific to 14‐kDa group‐II phospholipase A2, including two proteinaceous inhibitors and a product of microorganisms with a low molecular mass, on mast‐cell activation was examined. When rat peritoneal mast cells were sensitized with IgE and then challenged with antigen, the specific phospholipase‐A2 inhibitors suppressed histamine release in a concentration‐dependent manner. By contrast, these inhibitors showed no effect on prostaglandin generation under the same conditions. Histamine release from rat peritoneal mast cells subjected to non‐immunochemical stimuli, such as concanavalin A, the Ca2+ ionophore A23187, compound 48/80 and substance P was also suppressed. When rat peritoneal mast cells were treated with 14‐kDa‐group‐II‐phospholipase‐A2‐specific inhibitors, washed and stimulated, histamine release was not affected appreciably. Similar suppressive effects of the inhibitors on histamine release were observed with mouse cultured bone‐marrow‐derived mast cells. When bone‐marrow‐derived mast cells were activated, they secreted both a soluble and an ecto‐enzyme form of 14‐kDa group‐II phospholipase A2, although appearance of the enzyme associated with the external surface of cells was observed transienty. An appreciable amount of membrane phospholipids was degraded during activation of mast cells, which was de‐creased by treatment with 14‐kDa‐group‐II‐phospholipase‐A2 inhibitor. These observations suggest that degranulation and eicosanoid generation in mast cells are regulated independently by discrete phospholipases A2 and that the 14‐kDa group‐II phospholipase A2 released from mast cells during activation may play an essential role in the progression of the degranulation process.
Altered plasma membrane H+‐ATPase from the Dio‐9‐resistant pmal‐2 mutant of Schizosaccharomyces pombe
Authors: GHISLAIN Michel , DE SADELEER Myriam , GOFFEAU André
Published: March 2005

The pmal‐2 mutation affecting the plasma membrane H+‐ATPase of Schizosaccharomyces pombe has been selected for resistance to the antibiotic Dio‐9. In membrane fractions purified from glucose‐starved cells, the mutant ATPase activity is reduced by 96%, is insensitive to inhibition by vanadate and has a pH profile displaced in the acidic pH range when compared to the wild type. The maximum velocity of the H+‐ATPase activity of plasma membranes from glucose‐activated pmal‐2 cells is activated 20‐fold. This is in striking contrast with the wild‐type ATPase activity, the maximal velocity of which is not affected by glucose. However, similar to the wild‐type enzyme, glucose activation of the pmal‐2 mutant H+‐ATPase reduces the Km for MgATP 9–2 mM and shifts the optimal pH from 4.8 to 6.0–6.5.

The pmal‐2 mutation modifies Lys250 to a threonine, which is highly conserved in fungal and plant H+‐ATPases. These results, compared to those reported for mutations of neighbour residues in yeast or mammalian P‐type ATPases, suggest that Lys250 could play a significant role, not only in phosphate binding and/or in the E1P‐E2P conformational isomerisation, but also in glucose activation of the H+‐ATPase.
Chicken Vigilin Gene organization and expression pattern
Authors: HENKEL Barbara , SCHMIDT Cornelia , ZORBAS Haralabos , PÖSCHL Ernst , GLOE Torsten R. , PURSCHKE Werner G. , MÜLLER Peter K.
Published: March 2005

Chicken vigilin was identified as a member of an evolutionary‐conserved protein family with a unique repetitive domain structure. 14 tandemly repeated domains are found in chicken vigilin, all of which consist of a conserved sequence motif (subdomain A) and a potential α‐helical region (subdomain B) [1]. We have established the physical structure of the chicken vigilin gene by restriction‐fragment analysis and DNA sequencing of overlapping clones isolated from a phage λ genomic DNA library. The chicken vigilin gene is a single‐copy gene with a total of 27 exons which are distributed over a region of some 22 kbp. Exon 1 codes for a portion of the 5′ untranslated region, exon 2 contains the translation start point and forms, along with exons 3 and 4, the N‐terminal non‐domain region. Exons 5–25 encode the vigilin domains 1–14 and the remaining exons 26 and 27 contain the non‐domain C‐terminal as well as the untranslated regions. The domain structure of the protein is reflected in the positioning of introns which demarcate individual domains. While domains 1–3 and 8–10 are each encoded by a single exon (5–7, 16–18); all other domains are contained in a set of two exons which are separated by introns interspersed at variable positions of the DNA segment coding for the conserved sequence motif. In conclusion, the data presented suggest that the chicken vigilin gene evolved by amplification of a primordial exon unit coding for the fundamental bipartite vigilin domain.
Purification and characterization of a novel lactonohydrolase, catalyzing the hydrolysis of aldonate lactones and aromatic lactones, from Fusarium oxysporum
Authors: SHIMIZU Sakayu , KATAOKA Michihiko , SHIMIZU Kentaro , HIRAKATA Masao , SAKAMOTO Keiji , YAMADA Hideaki
Published: March 2005

A novel lactonohydrolase, an enzyme that catalyzes the hydrolysis of aldonate lactones to the corresponding aldonic acids, was purified 10‐fold to apparent homogeneity, with a 61% overall recovery, from Fusarium oxysporum AKU 3702, through a purification procedure comprising DEAE‐Sephacel, octyl‐Sepharose CL‐4B and hydroxyapatite chromatographies and crystallization. The molecular mass of the native enzyme, as estimated by high‐performance gel‐permeation chromatography, is 125 kDa, and the subunit molecular mass is 60 kDa. The enzyme contains 15.4% (by mass) glucose equivalent of carbohydrate, and about 1 mol calcium/subunit. The enzyme hydrolyzes aldonate lactones, such as D‐galactono‐γ‐lactone and L‐mannono‐γ‐lactone, stereo‐specifically. Furthermore, it can catalyze the asymmetric hydrolysis of D‐pantoyl lactone, which is a promising chiral building block for the chemical synthesis of D‐pantothenate. These reactions are reversible, and the reaction equilibrium at pH 6.0 has a molar ratio of nearly 1:1 with D‐pantoyl lactone and D‐pantoic acid. The Km and Vmax for D‐galactono‐γ‐lactone are 3.6 mM and 1440 U/mg, respectively, and those for D‐galactonate are 52.6 mM and 216 U/mg, respectively. The enzyme also irreversibly hydrolyzes several aromatic lactones, such as dihydrocoumarin and homogentisic‐acid lactone.
Purification, cDNA cloning and Northern‐blot analysis of mitochondrial chaperonin 60 from pumpkin cotyledons
Authors: TSUGEKI Ryuji , MORI Hitoshi , NISHIMURA Mikio
Published: March 2005

Two different cDNA clones, pMCPN60‐1 and pMCPN60‐2, encoding the mitochondrial homologues of chaperonin 60 (Cpn60) were isolated from a cDNA library of germinating pumpkin cotyledons by use of mixtures of synthetic oligonucleotides based on the N‐terminal amino acid sequence of the protein. Determination of the complete nucleotide sequences of the two cDNA revealed that pMCPN60‐1 and pMCPN60‐2 each contain one open reading frame that encodes a protein of 575 amino acids with molecular masses of 61052 Da and 61127 Da, respectively. The deduced amino acid sequences of the two polypeptides include a 32‐residue N‐terminal putative mitochondrial presequence attached to the mature polypeptides, and they are 95.3% identical. From a comparison of deduced amino acid sequences with other Cpn60, it appears that the mature polypeptides of pumpkin mitochondrial Cpn60 are 44–59% identical to the other Cpn60, namely, GroEL of Escherichia coli, the 60‐kDa heat‐shock protein (Hsp60) of mitochondria in the yeast Saccharomyces cerevisiae, P1 protein of mammalian mitochondria and the Ribulose‐1,5‐bisphosphate carboxylase/oxygenase subunit‐binding proteins α and β of plastids in higher plants. Genomic Southern‐blot analysis identified at least two copies of the gene for mitochondrial Cpn60 in the pumpkin genome. The levels of mRNA for mitochondrial Cpn60 in cotyledons, hooks and hypocotyls of pumpkin seedlings increased in response to heat stress, as deduced from Northern‐blot analysis, indicating that pumpkin mitochondrial Cpn60 is a heat‐induced stress protein.
Structures and functions of the sugar chains of glycoproteins
Authors: KOBATA Akira
Published: March 2005

Most proteins within living organisms contain sugar chains. Recent advancements in cell biology have revealed that many of these sugar chains play important roles as signals for cell‐surface recognition phenomena in multi‐cellular organisms. In order to elucidate the biological information included in the sugar chains and link them with biology, a novel scientific field called ‘glycobiology’ has been established. This review will give an outline of the analytical techniques for the structural study of the sugar chains of glycoproteins, the structural characteristics of the sugar chains and the biosynthetic mechanism to produce such characteristics. Based on this knowledge, functional aspects of the sugar chains of glycohormones and of those in the immune system will be described to help others understand this new scientific field.
The asparaginyl‐tRNA synthetase gene encodes one of the complementing factors for thermosensitive translation in the Escherichia coli mutant strain, N4316
Authors: AOKI Hiroyuki , YAWORSKY Paul J. , PATEL Samir D. , MARGOLIN‐BRZEZINSKI Denise , PARK Kyung S. , GANOZA M. Clelia
Published: March 2005

Escherichia coli strain N4316 is a mutant that exhibits temperature‐sensitive growth at 43°C and temperature‐sensitive translation in vivo and in vitro. Extracts of the mutant produce an aberrant pattern of translation products of MS2 bacteriophage RNA. Previous work has shown that a protein, called ‘rescue’, isolated from the parental strain partly corrects the defective translation in vitro. Here we report the purification to homogeneity of a second factor from ribosomal eluates of the wild‐type parental strain; the purified protein is a homodimer of 54 kDa.

The partial sequence of the second protein was determined, and a recombinant plasmid was isolated based on its ability to complement the temperature‐sensitive growth phenotype of the mutant at the non‐permissive temperatures. The cloned gene was sequenced, mapped to the 20.9‐min region of the E. coli chromosome and shown to code for a 466‐amino‐acid protein with a molecular mass of 52 kDa. Analysis of the DNA sequence and the correspondence to that of the partial protein sequence has identified the complementing factor as asparaginyl‐tRNA synthetase. Marker rescue experiments indicate that the asnS mutation in N4316 resides within the motif 2 domain of the synthetase. A potential role of this synthetase in restoring normal protein synthesis with respect to ribosomal frameshifting, read‐through of nonsense codons and protein copy number is discussed.
Characterization of the reverse transcriptase of 1731, a Drosophila melanogaster retrotransposon
Authors: CHAMPION Serge , MAISONHAUTE Claude , KIM Myeong Hee , BEST‐BELPOMME Martin
Published: March 2005

The nucleotide sequence of 1731, a retrotransposon cloned from the genome of Drosophila melanogaster, reveals a structural similarity with the proviral form of the retroviruses including a pol‐like gene containing a putative reverse‐transcriptase(RT)‐coding sequence. Diverse parts of that sequence were subcloned and expressed in Escherichia coli. It has been demonstrated that the expression of the RT‐like sequence, when translated, gives rise to peptides displaying enzyme activity characteristic of a true RT enzyme. In addition, rabbit antisera directed against such recombinant proteins allowed us to detect an immunoreactive protein of around 110 kDa, which was only present in D. melanogaster cell lines, but not in cells derived from Drosophila virilis or Drosophila hydei, whose genomes do not bear the 1731 element. This protein is expected to correspond to a non‐processed pol‐gene translated product and cosediments with virus‐like particles exhibiting RT activity.
Ovohemerythrin, a major 14‐kDa yolk protein distinct from vitellogenin in leech
Authors: BAERT Jean‐Luc , BRITEL Mostafa , SAUTIERE Pierre , MALECHA Jean
Published: March 2005

A 14‐kDa protein was identified as a major component of mature oocytes of the leech Theromyzon tessulatum. This protein was, like vitellin, detected in the content of yolk granules and was purified by gel‐permeation and ion‐exchange chromatography. The yolk protein corresponded to an iron‐binding protein which exists in a monomeric unglycosylated form and had no similarities to vitellin. However, a strong resemblance between this protein and sipunculid hemerythrin, a non‐heme iron‐binding protein, was observed on the basis of its characteristics including molecular mass, iron content, ultraviolet/visible spectrum, amino acid composition and N‐terminal sequence. These similarities with hemerythrin and the accumulation of the protein in the oocyte justify the name ovohemerythrin given to the molecule.

A coelomic‐fluid protein immunologically related to ovohemerythrin was detected in vitellogenic animals. The protein was purified with the chromatographic procedure used to isolate ovohemerythrin from oocytes and was found to be similar to the oocyte protein. This circulating ovohemerythrin was present in large amounts in the coelomic fluid while gametogenesis is in progress, i. e. after the third and last blood meal of the animal (stage 3), except at the time of oocyte enlargement when its concentration decreases dramatically. However, in contrast to vitellogenin, which is detected specifically in the coelomic fluid of leeches at stage 3, circulating ovohemerythrin is also observed after the first (stage 1) and second (stage 2) blood meal. This observation suggests a more complex function for ovohemerythrin than being merely a yolk nutrient for the embryo.
Proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins
Authors: SCOCCHI Marco , SKERLAVAJ Barbara , ROMEO Domenico , GENNARO Renato
Published: March 2005

Bac5 and Bac7, antibiotics of the bactenecin (proline/arginine‐rich peptide) family, are stored as proforms in the large granules of bovine neutrophils [Zanetti, M., Litteri, L., Gennaro, R., Horstmann, H. and Romeo, D. (1990) J. Cell Biol. 111, 1363–1371]. These proforms have been purified to homogeneity from granule extracts by immunoaffinity and reverse‐phase chromatography. While mature bactenecins efficiently kill Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium with minimal inhibitory concentrations of 6–12 μg/ml, proBac5 and proBac7 do not affect the growth of the same microorganisms, even at 500 μg/ml. Previous investigations have suggested that the conversion of probactenecins into mature antimicrobial peptides is catalyzed by a neutral serine protease stored in the azurophil granules. Purified proBac5 and proBac7 were thus treated with elastase, cathepsin G or proteinase 3, which constitute the pool of neutral serine proteases of the azurophils, and the reaction products were identified by Western blot analysis, mass spectrometry, and N‐terminal sequence analysis. Of the three proteases, only elastase is able to catalyze the stepwise cleavage of probactenecins into the corresponding mature peptides, which have the same mass, N‐terminal sequence and antibiotic activity of authentic Bac5 and Bac7. These results point to the importance of cooperation between azurophils and large granules in mounting a defense reaction.
Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane‐bound malic enzyme
Authors: KENDRICK Andrew , RATLEDGE Colin
Published: March 2005

The component fatty acids of the endogenous phospholipids of microsomal preparations of Mucor, when shaken at 30°C, increased in both chain length and in degree of unsaturation. The net effect was the production of γ‐linolenic acid which, over 2 h, increased from 17% to 32% of total fatty acids present. No further significant changes occurred after this time.

The major site for desaturation/elongation reactions was at the sn‐2 position of Ptdlns. PtdCho and PtdEtn were not implicated.

Of numerous metabolites and cofactors added to the microsomes, only malate could prolong the elongation/desaturation reactions for up to 6 h. This effect was shown to be due to a membrane‐associated malic enzyme [malate dehydrogenase (decarboxylating) NADP+] with the NADPH produced being used in fatty‐acid desaturation.

Kinetic analysis of cytosolic and microsomal enzymes [both in 0.1% (mass/vol.) Chaps] could not distinguish between them. However, when the microsomal malic enzyme was dialysed to remove Chaps, it lost 90% of activity, although the cytosolic malic enzyme lost only 20% activity.

The structural analogue of malate, tartronic acid, which is an inhibitor of malic enzyme, also inhibited the malate‐induced stimulation of fatty‐acyl group desaturation and elongation in the microsomal membranes.

It is concluded that two distinct malic enzymes exist, one soluble and one membrane bound, with similar active sites. Both have different roles in the production of NADPH, for lipid metabolism. The former will produce NADPH for fatty‐acid biosynthesis whilst the latter produces NADPH for fatty‐acid desaturation.
β‐Adrenergic stimulation of C6 glioma cells: effects of cAMP overproduction on cellular metabolites
Authors: PIANET Isabelle , CANIONI Paul , LABOUESSE Julie , MERLE Michel
Published: March 2005

We used 31P‐NMR spectroscopy to investigate the response of living C6 glioma cells to stimulation by a β‐adrenergic agonist, isoproterenol. In the presence of 3‐isobutyl‐1‐methylxanthine, stimulation induced an accumulation of cAMP, making possible the NMR detection of the second messenger in living cells grown on microcarrier beads and perfused in the NMR tube. The cAMP signal rose to a maximum level within 20–25 min of stimulation; thereafter it decreased to the detection threshold within 60 min. At the same time, 40% increases of phosphomonoester and diphosphodiester signals were observed, whereas no significant change in phosphocreatine and nucleotide signals was detected. The kinetics of changes of the cellular content in phosphorylated metabolites were analyzed after recording 31P‐NMR spectra of cell perchloric acid extracts as a function of time of stimulation. cAMP accumulation in stimulated cells was evidenced by a near linear increase of its NMR signal as a function of incubation time (from 0 to 60 min). Concomitantly with the production of cAMP, the data showed 30% decreases of phosphocreatine and ATP levels within 60 min of stimulation, and an unexpected redistribution of pyrimidine and purine nucleoside triphosphates. At the same time, levels of phosphomonoesters (phosphorylcholine and phosphorylethanolamine) and phosphodiesters (glycerophosphorylcholine and glycerophosphorylethanolamine) rose (50% increase).

13C‐NMR spectra of cell perchloric acid extracts prepared after isoproterenol stimulation of cells incubated in the presence of [1‐13C]glucose indicated a higher glucose content in stimulated cells, whereas the resonance of ribose C1 was diminished. Moreover, the resonances of C1 of ethanolamine and choline (and their derivatives) were increased in spectra of stimulated cells, whereas that of C3 of serine was decreased. In addition, the 13C‐NMR data indicated that neither the pattern of glutamate carbon enrichment nor the glutamate/glutamine ratio was modified in stimulated cells. On the other hand, the heteronuclear coupling pattern of the lactate (methyl group) resonance in 1H‐NMR spectra of cell incubation media indicated that no change occurred in the carbon flux through the pentosephosphate shunt under stimulation. The results of this multinuclear NMR approach are discussed in terms of metabolic responses of C6 cells to β‐adrenergic stimulation and cAMP overproduction.
Purification of human leukotriene C4 synthase from dimethylsulfoxide‐differentiated U937 cells
Authors: NICHOLSON Donald W. , KLEMBA Michael W. , RASPER Dita M. , METTERS Kathleen M. , ZAMBONI Robert J. , FORD‐HUTCHINSON Anthony W.
Published: March 2005

Human leukotriene C4 (LTC4) synthase was purified > 10000‐fold from dimethylsulfoxide‐differentiated U937 cells. Steps included: (a) solubilization of membrane‐bound LTC4 synthase from microsomal membranes by the anionic detergent taurocholate; (b) successive anion‐exchange chromatography steps in the presence of taurocholate plus Triton X‐100 (primary anion exchange) then taurocholate plus n‐octyl glucoside (secondary anion exchange); and (c) LTC2‐affinity chromatography on a matrix that was constructed by first biotinylating synthetic LTC2 then immobilizing the biotinylated LTC2 on streptavidin agarose. The purification of human LTC4 synthase was enabled by the finding that LTC4 synthase activity in preparations enriched > 500‐fold was absolutely dependent on the presence in LTC4 synthase incubation mixtures of divalent cations (specifically Mg2+) and phospholipids (specifically phosphatidylcholine), and that reduced glutathione, which was required at 2‐4mM for stabilization of LTC4 synthase, irreversibly inactivated the enzyme when present at ≥ 5 mM during freeze/thaw cycles. The > 10000‐fold purified LTC4 synthase preparation was comprised of three polypeptides having molecular masses of 37.1, 24.5 and 18.0 kDa. An 18‐kDa polypeptide in both microsomal membranes and in the LTC4‐affinity purified fraction was specifically labelled by a radioiodinated LTC4 photoaffinity probe (azido 125I‐LTC4). The Km values in the LTC2‐affinity purified preparation for reduced glutathione and LTA4 were 1.83mM and 19.6 μM (respectively), closely resembling the Km values in isolated human blood monocytes. The Vmax of LTC2‐affinity purified LTC4 synthase was 2–4 μmol LTC4 formed · min−1· mg−1.
Cytosolic and intranuclear calcium signals in rat basophilic leukemia cells as revealed by a confocal fluorescence Microscope
Authors: NAKATO Kazuhiro , FURUNO Tadahide , INAGAKI Kenji , TESHIMA Reiko , TERAO Tadao , NAKANISHI Mamoru
Published: March 2005

A confocal fluorescence microscope with an argon‐ion laser (488 nm) and a He‐Cd laser (325 nm) was used to study spatial heterogeneity of the calcium signals in rat basophilic leukemia 2H3 cloned cell line (RBL‐2H3). After stimulation with antigen (2,4‐dinitrophenol‐conjugated bovine serum albumin), fluo‐3‐fluorescence intensities increased in individual RBL‐2H3 cells with different lag times. Time‐dependent profiles of the fluo‐3‐fluorescence intensities resembled closely the patterns of the sequential fluorescence‐ratio images of fura‐2, which were used to measure the intracellular free‐calcium concentration ([Ca2+]i) in individual RBL‐2H3 cells using a conventional fluorescence microscope. The present results obtained using the confocal fluorescence microscope showed spatial heterogeneities of fluo‐3‐fluorescence intensities, suggesting the existence of spatial heterogeneity of [Ca2+]i in RBL‐2H3 cells. That is, the results showed that calcium signals first occurred transiently at pseudopodia in RBL‐2H3 cells, then the signals transferred to the central parts of the cells. In addition, from the fluorescence images of co‐loaded Hoechst 33342 (bisbenzimide H 33342, a DNA‐specific probe) which were produced by excitation with a He‐Cd laser, it was found that the fluorescence images of the nucleus were quite similar to those of the calcium signals mentioned above. This suggested that the receptor‐mediated calcium signals were transferred not only to the cytoplasm but also to the nucleus.
Molecular basis of low‐temperature sensitivity in pig hemoglobins
Authors: CONDÒ Saverio G. , CORDA Marcella , SANNA Maria T. , PELLEGRINI Maria G. , RUIZ Maite Prats , CASTAGNOLA Massimo , GIARDINA Bruno
Published: March 2005

It has been generally assumed that mammals have blood with a greater temperature sensitivity than ectothermic organisms. Recent results have shown that in some species of mammals, Hb displays a value of overall oxygenation enthalpy (δH) much less exothermic than that observed for most mammalian hemoglobins, including human adult Hb. In this respect, a very interesting case is represented by porcine blood which shows a modest effect of temperature, the temperature coefficient of its oxygen‐dissociation curve being significantly lower than that of human blood.

Here we report a detailed functional characterization of pig Hb, which, interpreted on the basis of the amino acid sequence of the α and β chains of the molecule, sheds some light on the molecular basis of the phenomenon.
Stopped‐flow fluorescence and steady‐state kinetic studies of ligand‐binding reactions of glucoamylase from Aspergillus niger
Authors: OLSEN Karsten , SVENSSON Birte , CHRISTENSEN Ulla
Published: March 2005

The presteady‐state and steady‐state kinetics of the binding and hydrolysis of substrates, maltose and isomaltose, and the transition‐state analogue, gluconolactone, by glucoamylase from Aspergillus niger were investigated using initial‐rate, stopped‐flow and steady‐state methods. The change in the intrinsic fluorescence of the enzyme was monitored. Distinct mechanistic differences were observed in the interaction of the enzyme with maltose compared to isomaltose. Hydrolysis of maltose requires a three‐step mechanism, whereas that of isomaltose involves at least one additional step. The rates of an observed conformational change, which is the second discernible step of the reactions, clearly show a tighter binding of maltose compared to isomaltose, probably because the reverse rate constants differ. Compared to the non‐enzymic hydrolysis the transition‐state stabilization energy of glucoamylase is approximately −66 kJ/mol with maltose and only −14 kJ/mol with isomaltose.

Kinetic analysis of the binding of the inhibitor, gluconolactone, implies that independent interactions of two molecules occur. One of these, apparently, is a simple, fast association reaction in which gluconolactone is weakly bound. The other resembles binding of maltose, involving a fast association followed by a conformational change. Based on the results obtained, we propose new reaction mechanisms for Aspergillus glucoamylase.
Stable expression of chicken type‐VI collagen α1, α2 and α3 cDNAs in murine NIH/3T3 cells
Authors: COLOMBATTI Alfonso , BONALDO Paolo , BUCCIOTTI Francesco
Published: March 2005

As a component of an extensive network of microfibrils interwoven with large collagen fibers and in close contact with cell surfaces, type VI collagen plays an important role in cell‐matrix interactions. To investigate the behaviour of chicken type VI collagen chains in heterologous host cells as a means to understanding the pattern of assembly of this collagen, we transfected murine NIH/3T3 cells with cDNAs encoding chicken α1(VI), α2(VI) and α3(VI) chains. Cell lines that constitutively expressed the individual chains were analyzed by metabolic labeling and immunoprecipitation with specific antibodies. No self‐association was observed for either α1(VI) or α2(VI) chains which were secreted as monomeric polypeptides. Furthermore, neither the chicken α1(VI) nor α2(VI) chains associated with the endogenous murine chains to form chimeric chicken/murine heterotrimers. In contrast, chimeric chicken/murine heterotrimers were detected in cell lines transfected with chicken α3(VI) cDNA. These chimeric forms appeared to be properly aligned since their triple helices were stable to pepsin digestion. In addition, the chimeric heterotrimers coassembled and gave rise to disulfide‐linked type VI collagen molecules.
Alteration of translation and stability of mRNA for the poly(A)‐binding protein during myogenesis
Authors: ADAMOU John , BAG Jnanankur
Published: March 2005

The regulation of synthesis of various factors involved in mRNA translation during differentiation of muscle cells was examined. The steady‐state levels of mRNAs coding for eukaryotic initiation factor (eIF) 2α, 2β and elongation factor (eEF)‐1α were measured in both proliferating rat L6 myoblast and differentiated myotubes. The steady‐state levels of these mRNAs were not altered during myogenesis. Furthermore, the distribution of these mRNAs between repressed and translated populations remained unchanged. Recent studies suggest a role for poly(A)‐binding protein (PABP) in translation initiation. Therefore, we also examined the expression of PABP mRNA during myogenesis. The PABP mRNA was less abundant in myotubes compared to myoblasts. However, the synthesis of PABP remained unchanged. In myoblasts, approximately 50–60% of the total mRNA was associated with polyribosomes, whereas in myotubes more than 80% of the mRNA was associated with polyribosomes. These results, therefore, suggest that the PABP mRNA was more efficiently translated in differentiated myotubes than in the proliferating myoblasts. Measurement of the stability and transcription of PABP mRNA showed that, while transcription was not affected during myogenesis, the stability of the mRNA was reduced in differentiated cells. The t1/2 of PABP mRNA in myoblasts was 13 h compared to 7.5 h in myotubes. This observation suggests that the reduced steady‐state level of PABP mRNA in myotube were largely due to the change in stability of this mRNA during myogenesis.
Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1
Authors: LEVITSKY Dmitrii I. , SHNYROV Valery L. , KHVOROV Nikolai V. , BUKATINA Anna E. , VEDENKINA Natalia S. , PERMYAKOV Eugene A. , NIKOLAEVA Olga P. , POGLAZOV Boris F.
Published: March 2005

The thermal unfolding and domain structure of myosin subfragment i (S1) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2°C), while the binding of the non‐hydrolysable analogue of ATP, adenosine 5′‐[β,γ‐imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (Vi), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.1°C, respectively. Such an increase of S1 thermostability in the complexes S1‐AdoPP[NH]P and S1‐ADP‐Vi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes S1‐AdoPP[NH]P and S1‐ADP‐Vi is similar and differs radically from that of nucleotide‐free S1 and S1 in the S1‐ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis: one of them corresponds to nucleotide‐free S1 and to the complex S1‐ADP, and the other corresponds to the intermediate complexes S1‐ATP and S1‐ADP‐Pi. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p‐phenylene‐N, N′‐dimaleimide (p PDM) thiol cross‐linking almost does not differ from that of the nucleotide‐free S1. This means that pPDM‐cross‐linked S1 in contrast to S1‐AdoPP[NH]P and S1‐ADP‐Vi can not be considered a structural analogue of the intermediate complexes S1‐ATP and S1‐ADP‐Pi.
Corn Kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin
Authors: ABE Makoto , ABE Keiko , KURODA Masaharu , ARAI Soichi
Published: March 2005

A full‐length cDNA clone for a cysteine proteinase inhibitor (cystatin) was isolated from a λgt10 cDNA library of immature corn kernels by screening with a mixture of cDNA inserts for oryzacystatins I and II. The cDNA clone spans 960 base pairs, encoding a 135‐amino‐acid protein containing a signal peptide fragment. The protein, named corn cystatin I, is considered to be a member of the cystatin superfamily, since it contains the commonly conserved Gln‐Val‐Val‐Ala‐Gly region that exists in most known cystatins as a probable binding site and is significantly similar to other cystatins in its overall amino acid sequence. Corn cystatin I expressed in Escherichia coli showed a strong papain‐inhibitory activity. Northern blot analysis showed that the amount of mRNA for corn cystatin I reaches a maximum 2 weeks after flowering and then decreases gradually.
Purification and characterization of a hemocyte proteinase of Sarcophaga, possibly participating in elimination of foreign substances
Authors: SAITO Hiromi , KURATA Shoichiro , NATORI Shunji
Published: March 2005

We have reported that foreign protein injected into the abdominal cavity of Sarcophaga peregrina (flesh fly) larvae is degraded in the hemolymph by a proteinase secreted by hemocytes [Suzuki, T. and Natori, S. (1985) Comp. Biochem. Physiol. 81A, 191–193]. Here we report the purification and characterization of a proteinase from larval hemocytes. This enzyme is a cysteine proteinase consisting of 26‐kDa and 29‐kDa subunits with similar substrate specificity to mammalian cathepsin B. This enzyme was shown to be released from hemocytes into the hemolymph of larvae following injection of sheep red blood cells into the larvae, suggesting that it participates, at least in part, in elimination of foreign substances introduced into the body cavity.
Specific phosphorylation of the acidic central region of the N‐myc protein by casein kinase II
Authors: HAGIWARA Tamio , NAKAYA Kazuyasu , NAKAMURA Yasuharu , NAKAJIMA Hidenori , NISHIMURA Susumu , TAYA Yoichi
Published: March 2005

The central region of the N‐myc protein has a characteristic amino acid sequence EDTLSDSDDE‐DD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c‐myc and L‐myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor‐suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the β‐galactosidase and N‐myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N‐myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wildtype N‐myc. However, no phosphorylation of β‐galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N‐myc protein containing most of the C‐terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two‐dimensional tryptic mapping of phosphorylated N‐myc proteins, major spots of in vitro‐labeled and in‐vivo‐labeled N‐myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N‐myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed.
Germin isoforms are discrete temporal markers of wheat development
Authors: LANE Byron G. , CUMING Andrew C. , FRÉGEAU Judith , CARPITA Nicholas C. , HURKMAN William J. , BERNIER François , DRATEWKA‐KOS Ella , KENNEDY Theresa D.
Published: March 2005

Nascent synthesis and accumulation of germin and its mRNA mark the onset of renewed growth when wheat embryos are germinated in water. Germin is a water‐soluble, pepsin‐resistant protein that is not found in immature embryos, or in mature embryos before their germination. An antiserum was raised by injecting rabbits with germin that was freed of other proteins by pepsinization and gel filtration. The antiserum has been used to detect, in extracts of mature embryos from dry, ungerminated wheat grains, a protein that is antigenically related to germin. The antigenically related protein has been named pseudogermin. Pseudogermin accumulates, maximally, between 20–25‐days postanthesis, then declines appreciably in amount by 30‐days postanthesis, in soluble extracts of immature embryos from several wheat varieties. The antiserum was also used to identify germin and pseudogermin among the proteins extracted from cell walls and to bind immunogold to cell walls preparatory to visualizing freeze‐cleaved embryos by scanning electron microscopy. Wall‐associated germin accounts for about 40% of the total germin in germinating wheat embryos. Appearance of germin in the apoplast is the most conspicuous germination‐related change in the distribution of cell‐wall proteins. It seems that germin may act at the level of the apoplast and that pseudogermin may subsume the role of germin at low water potentials during embryogenesis. The N‐terminal eicosapeptide sequences in germin and pseudogermin are very similar but SDS/PAGE analysis detects discrete differences between the mobilities of their constituent monomers as well as gross differences between the stabilities of the parent oligomers. Like germin, pseudogermin is a water‐soluble, pepsin‐resistant protein, but pseudogermin has unprecedented disulphide‐independent thermostability properties that have never been previously reported for a water‐soluble oligomeric protein. Polysaccharides that co‐purify with otherwise pure specimens of germin (and pseudogermin) have been isolated for analysis and shown to be highly substituted glucuronogalactoarabinoxylans. The possible biological significance of selective and tenacious association between germin and glucuronogalactoarabinoxylans is discussed in relation to cell expansion during embryogenic and germinative development of wheat, as are some peculiarities of amino‐acid sequence that suggest a possible relation between germin and a proton‐specific ion pump: gastric ATPase.
Dissociation of F‐actin induced by hydrostatic pressure
Authors: GARCIA Celia R. S. , AMARAL J. Adalberto , ABRAHAMSOHN Paulo , VERJOVSKI‐ALMEIDA Sergio
Published: March 2005

F‐actin purified from rabbit skeletal muscle undergoes reversible dissociation when subjected to hydrostatic pressures up to 240 MPa. Dissociation and reversibility were detected by the following procedures: fluorescence spectral changes observed under pressure, when either intrinsic tryptophan or pyrenyl emission of N‐(1‐pyrenyl)iodoacetamide‐labeled actin were monitored; electron microscopy of samples fixed under pressure; size‐exclusion HPLC of pressurized actin. The effect of pressure upon F‐actin that had been polymerized in the presence of either Mg2+, Ca2+ or K+ was studied. The standard volume changes for the association of actin subunits, calculated from pressure/dissociation curves were 74 ± 14 ml/mol for Mg‐F‐actin, 79 ± 12 ml/mol for Ca‐F‐actin and 328 ± 63 ml/mol for K‐F‐actin, indicating that actin subunits are packed differently in the polymer depending on which cation is present. All pressure/dissociation data could be fitted by a model for dissociation of a dimer, which suggests that in the F‐actin filament there is a predominant intersubunit interaction interface, most likely the head‐to‐tail intrastrand interaction between two subunits which repeats itself along the polymer. A tenfold change in total protein concentration from 20 μg to 200 μg/ml Mg‐F‐actin did not cause a change in the pressure required for half‐maximal dissociation. This indicates a heterogeneity of free energy of association among actin monomers in the Mg‐F‐actin polymer, suggesting that, in addition to the predominant intersubunit interaction, the disordered interactions in the filament significantly contribute to the heterogeneity of microenvironments in the interface between the subunits.
A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei
Authors: SCHMITZ Ruth A. , ALBRACHT Simon P. J. , THAUER Rudolf K.
Published: March 2005

We have recently reported that the thermophilic archaeon Methanobacterium wolfei contains two formylmethanofuran dehydrogenases, I and II. Formylmethanofuran dehydrogenase II, which is preferentially expressed in tungsten‐grown cells, has been purified and shown to be a tungsten‐iron‐sulfur protein. We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum grown cells and shown that it is a molybdenum‐iron‐sulfur protein. The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per mol 146‐kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non‐heme iron but no tungsten (<0.01 mol). The molybdenum enzyme differed from the tungsten enzyme (8 U/mg) in that it catalyzed the oxidation of N‐furfurylformamide and formate and was inactivated by cyanide. The two enzymes also differed significantly in the pH optimum, in the apparent Km for the electron acceptor, and in the chromatographic behaviour. The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N‐terminal amino acid sequences determined for the α and β subunits were identical up to residue 23, indicating that the two proteins are isoenzymes.

The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution.
Kinetic characterization of the perturbation by dodecylmaltoside of sarcoplasmic reticulum Ca2+ ‐ATPase
Authors: de FORESTA Béatrice , HENAO Fernando , CHAMPEIL Philippe
Published: March 2005

We investigated the functional aspects of the interaction between the sarcoplasmic reticulum (SR) membranous Ca2+‐ATPase and the non‐ionic detergent dodecylmaltoside, using detergent concentrations allowing perturbation of the membrane but not its solubilization. At pH 7.5, the effects of dodecylmaltoside on ATPase activity and delipidation had previously been shown to resemble, in some respects, those of octa(ethylene glycol) monododecylether (C12E8), an appropriate detergent for ATPase studies. Our aim here was to explore the specific effects of dodecylmaltoside on the different steps in the ATPase catalytic cycle, which may owe their specificity to the difference between the polar head groups of dodecylmaltoside and C12E8. This was done at 20 °C, both at pH 6 in the absence of KCl and at pH 7.5 in the presence of 100 mM KCl, two conditions under which the characteristics of unperturbed ATPase have already been well defined. Preliminary estimation of dodecylmaltoside partition between water and SR membranes at pH 6 yielded a partition coefficient K close to 4 × 105 (ratio of the molar fraction of dodecylmaltoside in the lipid to that in the aqueous phase at a low detergent concentration, assuming that most of this detergent was present in the lipid phase). At near saturation of SR membranes, bound dodecylmaltoside was roughly equimolar with the constituent phospholipids.

Non‐solubilizing concentrations of dodecylmaltoside inhibited SR ATPase activity by up to 65–70% at pH 7.5, but not at pH 6, unlike the results of similar experiments with C12E8. The rates of the four main steps in the ATPase catalytic cycle were measured by fast kinetic techniques; they were similarly modified at both pH. Dodecylmaltoside slowed down both the rate of calcium‐saturated ATPase phosphorylation and the rate of ATPase isomerization after phosphorylation, two steps which were not targets of perturbation by C12E8. The slowing down of the isomerization step by dodecylmaltoside might well explain why it inhibited overall ATPase activity at pH 7.5. In contrast to C12E8, dodecylmaltoside did not affect the dephosphorylation step, which was the main target of inhibition by C12E8 and the main rate‐limiting step at pH 6. However, like C12E8, dodecylmaltoside accelerated the calcium binding‐induced transition of nonphosphorylated ATPase.

Another striking feature of the perturbation induced by dodecylmaltoside was that it significantly altered the binding of 45Ca2+ to the ATPase and the corresponding conformational changes. At pCa 5–5.5, it almost halved calcium binding to the ATPase but ATPase phosphorylation was unimpaired. These results establish dodecylmaltoside as a potentially useful tool for studying the interaction between the two Ca2+ binding sites of membranous SR ATPase and their coupling with the catalytic site of ATPase.
A point mutation of low‐density‐lipoprotein receptor causing rapid degradation of the receptor
Authors: MIYAKE Yasuko , TAJIMA Shoji , FUNAHASHI Tohru , YAMAMURA Taku , YAMAMOTO Akira
Published: March 2005

The exons of the low‐density‐lipoprotein‐(LDL)‐receptor gene from a Japanese patient with homozygous familial hypercholesterolemia were amplified by the polymerase chain reaction (PCR), and their nucleotide sequences were determined. A point mutation from G to C was found in exon 9, which was expected to change Asp at position 412 to His. This amino acid change occurred within the epidermal‐growth‐factor‐precursor homology domain of the LDL receptor, slightly impairing the processing from the precursor to the mature form and causing rapid degradation of the mature form in the fibroblasts of the patient. The mutant LDL‐receptor gene transfected into COS‐1 cells expressed a LDL‐receptor protein with the same properties as the protein expressed in the fibroblasts of the patient; impaired processing and rapid degradation of the synthesized receptor protein. The mutation was identified in family members of the patient by dot‐blot hybridization of PCR‐amplified DNA with the mutant oligonucleotide. The family members carrying the mutant gene showed higher serum cholesterol levels than the others. However, their cholesterol levels were also greatly influenced by the apolipoprotein‐E phenotype.
Improvement in the heterogeneous N‐termini and the defective N‐glycosylation of human interleukin‐6 by genetic engineering
Authors: HASEGAWA Masakazu , ORITA Tetsuro , KOJIMA Tetsuo , TOMONOH Kikuo , HIRATA Yuichi , OCHI Norimichi
Published: March 2005

Recombinant human interleukin‐6 (IL‐6), expressed in Chinese hamster ovary cells, has heterogeneous N‐termini of Ala1 and Va13, as does naturally occurring IL‐6. This heterogeneity is thought to be caused by difficulty in cleavage of the signal sequence. To obtain homogeneous IL‐6, Pro at ‐1 was exchanged for A1a by site‐directed mutagenesis. Alternatively, the signal sequence was replaced with that of human granulocyte‐colony‐stimulating factor. In both cases, the IL‐6 designed to start with Ala1 was still heterogeneous, while the IL‐6 designed to start with Val3 showed a homogeneous N‐terminus. It is suggested that the heterogeneity of the N‐terminus is caused not only by the signal sequence, but also by the succeeding sequences of the mature protein. Only a portion of recombinant human IL‐6 is N‐glycosylated. Asn46, being exchanged for Gln by site‐directed mutagenesis, was confirmed to be partially N‐glycosylated. The defective N‐glycosylation was assumed to be caused by interference or tension from a disulfide bond near the N‐glycosylation site. To verify this hypothesis, the Cys45 and Cys51 forming the disulfide bond were exchanged for Ser. The N‐glycosylated species became predominant upon this substitution, suggesting that formation of the disulfide bond is a cause of the defective N‐glycosylation.