bluuebunny/crossref_metadata_2025 huggingface dataset:
The solution structure of human transforming growth factor α
Authors: HARVEY Timothy S. , WILKINSON Anthony J. , TAPPIN Michael J. , COOKE Robert M. , CAMPBELL Iain D.
Published: March 2005

The solution structure of transforming growth factor α has been determined by a combination of highresolution 1H‐nuclear magnetic resonance and distance geometry and restrained molecular dynamics. The 382 restraints derived from the NMR experiments were used to calculate many distance geometry structures, which were then refined by restrained molecular mechanics. Five of these structures were further refined using a variety of methods.

Comparison of independently measured parameters, such as calculated hydrogen bonding patterns and experimental amide exchange rates, have been used to evaluate the accuracy of the structures. Also, possible mechanisms to explain the pH‐dependent conformational interconversion observed are suggested.

Finally comparisons between this work and others on this topic have been made.
Structural organization of the multifunctional animal fatty‐acid synthase
Authors: WITKOWSKI Andrzej , RANGAN Vangipuram S. , RANDHAWA Zafar I. , AMY Christopher M. , SMITH Stuart
Published: March 2005

The amino acid sequence of the multifunctional fatty‐acid synthase has been examined to investigate the exact location of the seven functional domains. Good agreement in predicting the location of interdomain boundaries was obtained using three independent methods. First, the sites of limited proteolytic attack that give rise to relatively stable, large polypeptide fragments were identified; cryptic sites for protease attack at the subunit interface were unmasked by first dissociating the dimer into its component subunits. Second, polypeptide regions exhibiting higher‐than‐average rates of non‐conservative mutation were identified. Third, the sizes of putative functional domains were compared with those of related monofunctional proteins that exhibit similar primary or secondary structure. Residues 1–406 were assigned to the oxoacyl synthase, residues 430–802 to the malonyl/acetyl transferase, residues 1630–1850 to the enoyl reductase, residues 1870–2100 to the oxyreductase, residues 2114–2190 to the acyl‐carrier protein and residues 2200–2505 to the thioesterase. The 47‐kDa transferase and 8‐kDa acyl‐carrier‐protein domains, which are situated at opposite ends of the multifunctional subunit, were nevertheless isolated from tryptic digests as a non‐covalently associated complex. Furthermore, a centrally located domain encompassing residues 1160–1545 was isolated as a nicked dimer. These findings, indicating that interactions between the head‐to‐tail juxtaposed subunits occur in both the polar and equatorial regions, are consistent with previously derived electron‐micrograph images that show subunit contacts in these areas. The data permit refinement of the model for the fatty‐acid synthase dimer and suggest that the malonyl/acetyl transferase and oxoacyl synthase of one subunit cooperate with the reductases, acyl carrier protein and thioesterase of the companion subunit in the formation of a center for fatty‐acid synthesis.
Structural and functional differences between carbonic anhydrase isoenzymes I and II as studied by site‐directed mutagenesis
Authors: BEHRAVAN Gity , JONASSON Per , JONSSON Bengt‐Harald , LINDSKOG Sven
Published: March 2005

Site‐specific mutagenesis has been used to replace amino acid residues in the active site of human carbonic anhydrase II with residues characterizing carbonic anhydrases I. Previous studies of [Thr200 → His]isoenzyme II [Behravan, G., Jonsson, B.‐H. & Lindskog, S. (1990) Eur. J. Biochem. 190, 351–357] showed that His200 is important for the specific catalytic properties of isoenzymes I. In this paper some properties of two single mutants, Asn62 → Val and Asn67 → His, as well as a double mutant, Asn67 → His/Thr200 → His, are described. The results show that neither Val62 nor His67 give rise to isoenzyme‐I‐like properties, while the double mutant behaves like the single mutant with His200. At pH 8.9, the variant with Val62 has a higher value of kcat/Km for CO2 hydration than unmodified isoenzyme II, whereas the variant with His67 has an enhanced kcat value. The replacement of Asn62 with Val results in a 20% increase of the 4‐nitrophenyl acetate hydrolase activity. For the double mutant, the esterase activity is quite close to that calculated on the assumption that the effects of the two single mutations on the free energy of activation are additive.
Identification of possible two‐reactant sources of oscillations in the Calvin photosynthesis cycle and ancillary pathways
Authors: RYDE‐PETTERSSON Ulf
Published: March 2005

A systematic search for possible sources of experimentally observed oscillations in the photosynthetic reaction system has been performed by application of recent theoretical results characterizing the transient‐state rate behaviour of metabolic reactions involving two independent concentration variables. All subsystems involving two independent reactants in metabolically fundamental parts of the Calvin cycle and the ancillary pathways of starch and sucrose synthesis have been examined in order to decide on basis of their kinetic and stoichiometric structure whether or not they may trigger oscillations.

The results show that no less than 20 possible oscillators can be identified in the examined reaction system, only three of which have been previously considered as potential sources of experimentally observed oscillations. This illustrates the superiority of the method now applied over those previously used to identify possible two‐reactant sources of metabolic oscillations and indicates that there should be no difficulty in complex metabolic pathways to point to a multitude of interactions that may trigger an oscillatory rate behaviour of the system.
A study of the control of NADP+‐dependent isocitrate dehydrogenase activity during gonadotropin‐induced development of the rat ovary
Authors: JENNINGS Gary Thomas , STEVENSON Patricia Margaret
Published: March 2005

The concentration of cytoplasmic NADP+‐dependent isocitrate dehydrogenase increased 20.2‐fold during gonadotropin‐induced development of the immature rat ovary. Measurement was by protein (Western) blotting using polyclonal antibodies raised against purified enzyme from the porcine corpus luteum. The increase in enzyme concentration during development correlated well with the 18.5‐fold increase observed for the specific activity of the enzyme in the cytosolic fraction.

An immunochemical similarity was demonstrated between the cytoplasmic enzyme from the ovary, testes, placenta, skeletal muscle, brain, liver, kidney, mammary and adrenal gland. However the mitochondrial NADP+‐dependent isocitrate dehydrogenase from these tissues was found to be immunochemically distinct from the cytoplasmic enzyme.

The concentration of the substrate d(±)‐threo‐isocitrate in the ovaries was measured by fluorometry and found to increase 3.1‐fold during hormone‐induced development. The intracellular concentration of substrate was estimated to be of the same order of magnitude as the enzyme concentration.

We conclude that the increase in cytoplasmic NADP+‐dependent isocitrate dehydrogenase activity observed during the gonadotropin‐stimulated development of the rat ovary is due to increased concentration of enzyme rather than to an activation of the enzyme. The activity of the enzyme in vivo appears to be regulated by the availability of the substrate d(±)‐threo‐isocitrate.
Amino acid sequence analysis of the annexin super‐gene family of proteins
Authors: BARTON Geoffrey J. , NEWMAN Richard H. , FREEMONT Paul S. , CRUMPTON Michael J.
Published: March 2005

The annexins are a widespread family of calcium‐dependent membrane‐binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat‐specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried salt‐bridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one‐repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re‐evaluated in the Appendix, in the light of the recently published X‐ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family.
The neutrophil respiratory burst
Authors: HARDY Stephen J. , ROBINSON Brenton S. , POULOS Alf , HARVEY Dianne P. , FERRANTE Antonio , MURRAY Andrew W.
Published: March 2005

The oxygen‐dependent respiratory burst is a key neutrophil function required for the killing of bacteria. However, despite intensive investigation, the molecular events which initiate the respiratory burst remain unclear. Recent reports have suggested the agonist‐induced hydrolysis of cellular phosphatidylcholine (PtdCho) by phospholipase D may be an essential requirement for initiating or mediating the respiratory burst. We have investigated the effects of the chemotactic peptide N‐formylmethionylleucylphenylalanine (fMLF), the phorbol ester 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) and the polyunsaturated fatty acids arachidonic [20:4 (n‐6)] and docosahexaenoic [22:6 (n‐3)] acids in light of this hypothesis. Ethanol‐inhibited superoxide production in response to 20:4, 22:6 and fMLF, in a dose‐dependent fashion, suggesting an involvement of phospholipase D. The phosphatidic‐acid phosphohydrolase inhibitor DL‐propranolol completely inhibited superoxide production induced by both 20:4 and 22:6, and partially inhibited the response to TPA. In contrast, superoxide production in response to fMLF was increased by propranolol. fMLF and TPA, but not the fatty acids, stimulated phospholipase D as indicated by the accumulation of phosphatidic acid and, in the presence of ethanol, phosphatidylethanol derived from PtdCho. Extracellular Ca2+ was found to be an essential requirement for fMLF‐induced superoxide production. However, responses to the fatty acids were dramatically enhanced under Ca2+.‐free conditions. Responses to TPA were independent of the extracellular Ca2+ concentration. Both fatty acids and fMLF, but not TPA, mobilised Ca2+ from intracellular stores, a response insensitive to the effects of both ethanol and propranolol. These results show that, unlike fMLF and TPA, the fatty acids do not cause hydrolysis of PtdCho by phospholipase D. However, the data indirectly suggests that the fatty acids may initiate the phospholipase‐d‐catalysed hydrolysis of phospholipids other than PtdCho.
Purification and characterization of aryl acylamidase from Nocardia globerula
Authors: YOSHIOKA Hideki , NAGASAWA Toru , YAMADA Hideaki
Published: March 2005

Aryl acylamidase was purified from an extract of N‐acetyl‐o‐toluidine‐induced cells of Nocardia globerula IFO 13510 in ten steps. The purified enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of approximately 126 kDa and consists of two subunits which are identical in molecular mass. The purified enzyme catalyzed the hydrolysis of N‐acetyl‐o‐toluidine to o‐toluidine and acetic acid at a rate of 47.7 μmol · min−1· mg−1 at 35°C. It also catalyzed the hydrolysis of various anilide derivatives and esters, as well as the transfer of an acetyl group to aniline as an acetyl acceptor. The purified enzyme was sensitive to thiol reagents such as HgCl2 and p‐chloromercuribenzoate. The amino‐terminal sequence (28 amino acid residues) of the enzyme was determined. Based on the substrate specificity of this enzyme, the pathway intermediates involved in the conversion of n‐acetyl‐o‐toluidine to 4′‐hydroxy‐N‐acetyl‐o‐toluidine are discussed.
Properties of recombinant NADP–malate dehydrogenases from Sorghum vulgare leaves expressed in Escherichia coli cells
Authors: JACQUOT Jean‐Pierre , KERYER Eliane , ISSAKIDIS Emmanuelle , DECOTTIGNIES Paulette , MIGINIAC‐MASLOW Myroslawa , SCHMITTER Jean‐Marie , CRÉTIN Claude
Published: March 2005

In this study, a cDNA clone coding for sorghum leaf NADP‐malate dehydrogenase [Crétin, C., Luchetta, P., Joly, C., Decottignies, P., Lepiniec, L., Gadal, P., Sallantin, M., Huet, J. C. & Pernollet, J. C. (1990) Eur. J. Biochem. 192, 299–303] was used either in the full‐length form or in a shorter form deprived of the 5′ end coding for the transit peptide. Both cDNA fragments were cloned into the expression vector pKK233‐2 and the resulting constructions were used to transform E. coli cells. The bacterial cells which do not contain any NADP‐dependent malate dehydrogenase before transformation were able to express the protein after transformation and induction, as detected both by activity measurements and by immunoblot. The recombinant proteins could be purified to homogeneity and their biochemical characteristics studied. They were identical to those of the enzyme isolated from corn or sorghum leaves, including the well known redox regulatory properties. The NADP–malate dehydrogenases derived from both constructions had a similar subunit size and the analysis of their N‐terminal sequences revealed that E. coli cells were able to recognize the processing signal of the precursor polypeptide and to mature and assemble the protein in a manner similar to higher plants.
Evidence for a direct interaction of Rev protein with nuclear envelope mRNA–translocation system
Authors: PFEIFER Karin , WEILER Barbara E. , UGARKOVIC Durdica , BACHMANN Michael , SCHRÖDER Heinz C. , MÜLLER Werner E. G.
Published: March 2005

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV‐1) with the nucleocytoplasmic mRNA‐transport system was investigated. In gel‐shift assay, the recombinant Rev protein used in this study selectively bound to the Rev‐responsive element (RRE) region of HIV‐1 env‐specific RNA. Nitrocellulose‐filter‐binding studies and Northern/Western‐blotting experiments revealed an association constant of η 1 × 1010 M−1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)‐binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)‐rich mRNA. Binding of 125I‐Rev to a 110‐kDa nuclear‐envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)‐rich RNA, such as actin RNA [but not efflux of poly(A)‐free RNA] from isolated nuclei and the nuclear‐envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev‐responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear‐envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down‐regulates the NTPase‐driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE‐free RNA, ATP‐dependent efflux of RRE‐containing RNA from resealed nuclear‐envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE‐containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear‐envelope pore complex.
Assembly of the surfactant protein SP‐A
Authors: SPISSINGER Thomas , SCHÄFER Klaus P. , VOSS Tilman
Published: March 2005

The C‐terminal non‐collagenous domain of the surfactant glycoprotein SP‐A was shown to be essential for its correct folding and assembly, as judged by the secretion of various deletion mutants transiently expressed in COS cells. A deletion mutant coding for this domain was successfully secreted while the expression of the collagenous domain only did not lead to any detectable secretion. Deletion mutants lacking small parts of the non‐collagenous domain interfered more or less with the correct folding and assembly of the molecule, thus either reducing or inhibiting the secretion. These data suggest that three prefolded non‐collagenous domains register and act as a nucleation center for the folding of the collagenous triple helix which proceeds in a zipper‐like fashion towards the N‐terminus.
Direct electrochemistry of the enzyme, methylamine dehydrogenase, from bacterium W3A1
Authors: BURROWS Alison L. , Allen H. , HILL O. , LEESE Troy A. , MCINTIRE William S. , NAKAYAMA Hiroaki , SANGHERA Gurdial S.
Published: March 2005

The electrochemical response of methylamine dehydrogenase from bacterium W3A1 at edge‐plane‐oriented pyrolytic graphite (epg) and modified gold electrodes has been investigated. Quasi‐reversible electron transfer has been observed. Variations in concentration of different cations and anions gave rise to both promotion and inhibition of the direct response. A catalytic response of the enzyme in the presence of methylamine has been observed at both an epg electrode and a 2,2′‐dithiodiglycolic‐acid‐modified gold electrode surface, and the effects of various cations and anions on the catalytic peak current have been investigated. The spectroelectrochemical results obtained at an optically transparent thin‐layer electrode, modified with 2,2′‐dithiodiglycolic acid, are also reported. In the presence of 1,1′‐dimethylferrocene‐3‐(1‐ethanol‐2‐amine) (14.8 μM), the results reveal a midpoint potential of –148 mV for methylamine dehydrogenase from bacterium W3A1. This is in very close agreement to the value obtained in the cyclic voltammetric investigations of –140 mV.
Purification and properties of an iron‐sulfur‐containing and pyridoxal‐phosphate‐independent l‐Serine dehydratase from Peptostreptococcus asaccharolyticus
Authors: GRABOWSKI Reiner , BUCKEL Wolfgang
Published: March 2005

l‐Serine dehydratase with a specific activity of 15 nkat/mg protein was present in the anaerobic eubacterium Peptostreptococcus asaccharolyticus grown either on l‐glutamate or l‐Serine. The enzyme was highly specific for l‐Serine with the lowest Km= 0.8 mM ever reported for an l‐Serine dehydratase. l‐Threonine (Km= 22 mM) was the only other substrate. V/Km for l‐serine was 500 times higher than that for l‐threonine. l‐Cysteine was the best inhibitor (Ki= 0.3 mM, competitive towards l‐serine). The enzyme was purified 400‐fold to homogeneity under anaerobic conditions (specific activity 6 μkat/mg). PAGE in the presence of SDS revealed two subunits with similar intensities (α, 30 kDa; β, 25 kDa). The molecular mass of the native enzyme was estimated as 200 ± 20 kDa (gel filtration) and 180 kDa (gradient PAGE). In the absence of oxygen the enzyme was moderately stable even in the presence of sodium borohydride or phenylhydrazine (5 mM each). However, by exposure to air the activity was lost, especially when the latter agent was added. The enzyme was reactivated by ferrous ion under anaerobic conditions. The inability of several nucleophilic agents to inactivate the enzyme indicated the absence of pyridoxal phosphate. This was confirmed by a microbiological determination of pyridoxal phosphate. However, the enzyme contained 3.8 ± 0.2 mol Fe and 5.6 ± 0.3 mol inorganic sulfur/mol heterodimer (55 kDa) indicating the presence of an [Fe‐S] center. The enzyme was successfully applied to measure l‐serine concentrations in bacterial media and in human sera.
Product inhibition of α‐chymotrypsin in reverse micelles
Authors: BRU Roque , WALDE Peter
Published: March 2005

The α‐chymotrypsin‐catalyzed hydrolysis of succinyl‐L‐alanyl‐L‐alanyl‐L‐prolyl‐L‐phenylalanyl p‐nitroanilide has been studied in reverse micelles of sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) in isooctane. It has been found that α‐chymotrypsin is strongly inhibited competitively by the acidic peptide product which is formed during the course of the reaction. It has also been shown that the application of the integrated form of the Michaelis‐Menten equation can be useful to detect possible inhibition effects and abnormal kinetic behavior of enzymes in reverse micelles. Furthermore, it has been shown that the turnover number (kcat) at low water content is lower than in water and increases as the water content in the system (wo= [H2O]/[AOT]) increases, kcat reaching the value in water at high wo. If however, initial velocity data, as obtained under conditions where the enzyme is not saturated with substrate, are plotted against wo, the curves are bell‐shaped, with a maximum around wo= 15.
Petunia peroxidase a: isolation, purification and characteristics
Authors: HENDRIKS Theo , WIJSMAN Henri J. W. , van LOON Leendert C.
Published: March 2005

The fast‐moving anionic peroxidase isoenzyme variant PRXa was purified from leaves of petunia (Petunia hybrida). Over 1300‐fold purification was achieved by subjecting extracellular extracts to two sequential acetone precipitations and resuspending the pellets at pH 5.0 and pH 8.0, respectively, followed by gel filtration and chromatofocusing. The purified enzyme had an absorbance ratio (A405 nm/A280 nm) of 3.6, a molecular mass of about 37 kDa and a pI of 3.8. Three molecular forms with slightly different molecular masses were separated by concanavalin‐A–Sepharose affinity chromatography, indicating that these three forms differ in their carbohydrate moieties.

The absorption spectrum of PRXa had maxima at 496 and 636 nm and a Soret band at 405 nm. Spectra of compounds I and IV were obtained by titrating a batch of PRXa stored for several months at ‐20°C with H2O2. The addition of 1 mol H2O2/mol freshly purified PRXa caused the formation of compound II, indicating that freshly isolated PRXa contains a bound hydrogen donor which is lost upon storage. Compound III was obtained from both preparations in the presence of excess H2O2.

The pH optimum of PRXa for the reaction with H2O2 and guaiacol was 5.0 and its specific activity 61 mkat/g protein. Among various aromatic compounds, coniferyl alcohol was polymerized by PRXa to presumed lignin‐like material. The extracellular localization and high affinity of PRXa for the cinnamic acid derivatives suggest that this isoenzyme functions in the polymerization or cross‐linking of lignin in the plant cell wall.
Heterogeneity of the complex N‐linked oligosaccharides at specific glycosylation sites of two secreted carrot glycoproteins
Authors: STURM Arnd
Published: March 2005

The N‐linked glycans from the 52/54‐kDa medium protein and cell wall β‐fructosidase, two glycoproteins secreted by carrot suspension culture cells, were characterized. Carrot cells were labelled with [3H]glucosamine or [3H]fucose. The 52/54‐kDa medium protein was isolated form the culture medium and β‐fructosidase from cell walls. The purified proteins were digested with trypsin and glycopeptides were isolated and sequenced. Glycans obtained from individual glycopeptides were separated by gel filtration chromatography and characterized by concanavalin A chromatography, by treatments with exoglycosidases and by sugar composition analysis.

The 52/54‐kDa medium protein and cell wall β‐fructosidase have one high‐mannose‐type glycan similar to those from yeast and animal glycoproteins. In addition, the 52/54‐kDa medium protein has three complex‐type and cell wall β‐fructosidase two complex‐type glycans per polypeptide. The complex‐type glycans isolated from individual glycosylation sites are fairly large and very heterogeneous. The smallest of these glycans has the structure [Xyl](Man)3[Fuc](GlcNAc]2Asn (square brackets indicating branching) whereas the larger ones carry additional sugars like terminal N‐acetylglucosamine and possibly rhamnose and arabinose in the case of the 52/54‐kDa medium protein and only arabinose in the case of cell wall β‐fructosidase. These terminal sugars are linked to the α‐mannose residues of the glycan cores. The 52/54‐kDa medium protein is secreted with large and homogeneous complex glycans, their heterogeneity originates from slow processing after secretion. The complex glycans from cell wall β‐fructosidase are processed before the enzyme is integrated into the cell wall.
Uptake of iron by apoferritin from a ferric dihydrolipoate complex
Authors: BONOMI Francesco , PAGANI Silvia
Published: March 2005

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin‐bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin.

Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin‐bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron.

Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25–40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50–60 mol iron/mol ferritin after a 48‐h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.
Epitope mapping by amino‐acid‐sequence‐specific antibodies reveals that both ends of the α subunit of Na+/K+‐ATPase are located on the cytoplasmic side of the membrane
Authors: ANTOLOVIC Roberto , BRÜLLER Hans‐Joachim , BUNK Sabine , LINDER Dietmar , SCHONER Wilhelm
Published: March 2005

Right‐side‐out vesicles of pig kidney microsomes and amino‐acid‐sequence‐specific antibodies were used to probe the sidedness of the C‐terminus and the N‐terminus of the catalytic α subunit of Na+/K+‐ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N‐terminal sequence GRDKYEPAAVSE (peptide 1–12) and against peptides corresponding to the C‐terminal sequences IFVYDEVRKLIIRRR (peptide 991–1005) and RPGGWVEKETYY (peptide 1005–1016). These antibodies were purified by affinity chromatography on the respective peptide‐Sepharose columns. Moreover, antibodies against the N‐terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the α subunit of pork kidney Na+/K+‐ATPase. These antibodies reacted with native as well as SDS‐denaturated Na+/K+‐ATPase. When the antibodies were used to probe the sidedness of the sequences in right‐side‐out vesicles of pig kidney microsomes, the N‐terminal peptide 1–12 as well as the C‐terminal peptides 991–1005 and 1005–1016 were found on the cytosolic side. Concanavalin A, however, which interacts with the β subunit, a glycoprotein, reacted with the outside of right‐side‐out vesicles.
Production and identification of natural monocyte chemotactic protein from virally infected murine fibroblasts
Authors: Van DAMME Jo , DECOCK Benny , BERTINI Riccardo , CONINGS René , LENAERTS Jean‐Pierre , PUT Willy , OPDENAKKER Ghislain , MANTOVANI Alberto
Published: March 2005

Primary cultures of mouse embryonic fibroblasts and confluent monolayers of mouse fibroblastoid cells (L929) were found to secrete a chemotactic factor specific for monocytes. Its biological activity was deduced from both the migration distance under agarose and the number of migrated monocytes in the micropore filter method. The monocyte chemotactic protein (MCP) was inducible in these cells by double‐stranded RNA and by infection with virus. In embryonic fibroblasts MCP was also produced in response to the cytokines interleukin‐1 (IL‐1) and tumor necrosis factor (TNF). Under all conditions for induction of MCP tested no production of chemotactic activity for granulocytes could be detected. MCP activity from virally infected L929 cells was concentrated and purified by sequential adsorption to controlled pore glass, heparin‐Sepharose chromatography, ion‐exchange FPLC and reversed‐phase HPLC. Pure MCP was found to occur mainly as a 7–8‐kDa protein. Although the mature protein possessed a blocked NH2‐terminus, it was identified by enzymatic cleavage and sequence analysis of an internal fragment. The sequence obtained corresponded to a part of the cDNA‐derived protein sequence of the murine ‘competence’ (JE) gene, inducible in fibroblasts by cytokines and virus. In all probability the 7–8‐kDa MCP form represents the natural product of the mouse gene JE. Murine MCP can thus be classified in the novel family of small inducible inflammatory proteins.
Probing the catalytic sites of triosephosphate isomerase by 31P‐NMR with reversibly and irreversibly binding substrate analogues
Authors: SCHNACKERZ Klaus D. , GRACY Robert W.
Published: March 2005

We have explored the degree of independence of the two catalytic centers, interactions between the catalytic centers and the subunit‐subunit contact sites, and different conformations of triosephosphate isomerase (TPI), by simultaneously employing irreversibly (covalent) and reversibly binding substrate analogues and monitoring their 31P‐NMR resonances. 3‐Chloroacetol phosphate (CAP) was bound to the active site by reaction with Glu165. The resulting, inactive (CAP‐TPI)2 complex exhibited two distinct 31P‐NMR resonances which were independent of pH and represent two conformational forms of the enzyme. Dissociation in guanidine hydrochloride followed by redimerization resulted in a single conformation. This was observed with the enzyme from chicken, rabbit and yeast. The inactive (CAP‐TPI)2 dimer was mixed with native TPI, and dissociated/reassociated to form heterodimers (CAP‐TPI)(TPI) in which one subunit contained the CAP label and the other subunit was unmodified. This hybrid migrated intermediate between the native and CAP‐modified enzyme on nondenaturing PAGE. The heterodimer exhibited 50% the activity of the native dimer, but kinetic properties were otherwise indistinguishable. The reversibly binding transition state analogue, 2‐phosphoglycolate (PGA), was used to probe the remaining vacant active site of the heterodimer. Bound PGA exhibited a pH‐independent 31P‐NMR resonance which was readily distinguishable from resonances of CAP‐TPI and free PGA. No differences were observed in the binding of PGA to the vacant subunit of the heterodimer or the native dimer, further pointing to the independent nature of the two catalytic centers. However, the (CAP‐TPI)(TPI) heterodimer was more susceptible to subunit dissociation in guanidine hydrochloride than the native dimer. Thus, it appears that the two active sites function completely independently of each other, but that the binding of CAP at the active center loosens the subunit‐subunit contact. In addition, the two forms of the enzyme‐inhibitor complex trapped by reaction with CAP may represent conformations with the hinged lid or flexible loop (residues 166–176) in the open and closed positions.
Effect of a high‐protein diet on the gene expression of a trypsin‐sensitive, cholecystokinin‐releasing peptide (monitor peptide) in the pancreas
Authors: TSUZUKI Satoshi , FUSHIKI Tohru , KONDO Atsuko , MURAYAMA Hidemichi , SUGIMOTO Etsuro
Published: March 2005

The adaptation to a high protein diet of the concentration and mRNA level of a trypsin‐sensitive, cholecystokinin‐releasing peptide (monitor peptide), which was proposed to be the mediator of the cholecystokinin release in response to protein intake, was investigated in the rat pancreas. Adult rats were placed on one of two isocaloric diets. One group was fed a 22% casein diet (control diet) and the other a 64% casein diet (high‐protein diet) for 14 days. In order to quantify the monitor peptide separately from pancreatic secretory trypsin inhibitor (PSTI‐II), which is highly similar in its amino acid and mRNA nucleotide sequences to the monitor peptide but has less cholecystokinin‐releasing activity, we used specific assay methods: HPLC was used for determining the monitor peptide concentration in zymogen granules and a synthetic oligonucleotide probe for determining the mRNA of the monitor peptide in the pancreas. The concentrations in the zymogen granules and the mRNA levels in the pancreas of the two peptides increased in parallel during the adaptation to the high protein diet, indicating that these two peptides were under the same control during the adaptation. The concentration and mRNA level of the monitor peptide, which were measured after 0, 3, and 14 days, increased throughout the experiment period, as did the concentration of trypsin. This suggested that the monitor peptide and trypsin may respond to similar signals during the adaptation to a high protein diet and that this apparent coordination may facilitate the adaptation of the pancreas to the diet.
Computer analysis of DNA and protein sequences
Authors: von HEIJNE Gunnar
Published: March 2005

Some recent trends in the development of theoretical methods for DNA and protein sequence analysis are reviewed, with particular emphasis on the design of new databases, motif searches, sequence alignment algorithms and applications of neural networks.
Three‐dimensional structure of fatty‐acid‐binding protein from bovine heart
Authors: MÜLLER‐FAHRNOW Anke , EGNER Ursula , JONES T. Alwyn , RÜDEL Heinz , SPENER Friedrich , SAENGER Wolfram
Published: March 2005

The complex of fatty‐acid‐binding protein (FABP) from bovine heart (cFABP, pI 4.9) with endogenous lipid was crystallized in the presence of ammonium sulfate as precipitant. The needle‐shaped crystals belong to the monoclinic space group C2, with unit‐cell constants a= 5.262(6) nm, b= 7.631(8) nm, c= 3.945(5) nm and β= 94.47(9)°. A native data set to 0.35 nm resolution was collected using synchrotron radiation and film methods.

An initial model for the three‐dimensional structure of the protein was constructed based on the crystal structure of the related bovine P2 myelin protein [Jones, T. A., Bergfors, T., Sedzik, J. & Unge, T. (1988) EMBO J. 7, 1597–1604] to which the amino acid sequence of bovine cFABP was adapted. Energy minimizations were carried out under different conditions using both an all‐atom and a united‐atom force field.

The optimized models were used to determine the crystal structure of cFABP by molecular‐replacement techniques. The structure was refined by simulated annealing to R= 0.267. As the bound lipid is heterogeneous, it could not be located in the electron‐density map and/or the attained resolution was not sufficient. Bovine cFABP is composed of ten antiparallel β strands forming a β barrel, and by two α helices. The structural features are similar to those of other members of the superfamily of hydrophobic molecule transporters.
Evidence for glycosylphosphatidylinositol anchoring of intralumenal alkaline phosphatase of the calf intestine
Authors: HOFFMANN‐BLUME Eva , GARCIA MARENCO Marvin B. , EHLE Heidrun , BUBLITZ Renate , SCHULZE Margarete , HORN Anton
Published: March 2005

Considerable amounts of intestinal alkaline phosphatase (AP) were found intralumenally in all animal species investigated, i.e. calf, pig, goat, rat, mouse, guinea pig, hen and carp. The ratios between the total activity of AP found intralumenally and the total intestinal activity vary considerably. Calves and pigs show the highest, i.e. 0.77 and 0.44, respectively, while rodents have much lower ratios. Only 20–34% of the intralumenal alkaline phosphatase (IAP) of the calf and pig is soluble and not within the sediment after centrifugation at 135000 ×g for 60 min, whereas the IAP of rodents is soluble in the range of 60–72% of the total IAP.

For the IAP of the mucosa and chyme of calf, all criteria were found which are generally used, indicating a glycosylphosphatidylinositol (GlcPtdIns) anchor as proved by strong hydrophobicity using Triton X‐114 phase partitioning, phenyl‐Sepharose binding and enzyme aggregation, and the susceptibility to phosphatidylinositol‐specific phospholipase C (PtdIns‐PLC) and papain digestion.

More than 80% of the mucosa alkaline phosphatase (MAP) of the proximal part of the intestine and of the particulate fraction of IAP exhibit these criteria indicating the presence of the GlcPtdIns‐anchor structure, whereas the anchor content of the soluble intralumenal enzyme decreases from the pylorus to the ileocecal junction.

MAP partially purified to a specific activity of 1747 IU/mg retains the anchor structure.

The results presented indicate that the release of large amounts of AP into the chyme is realized without splitting the GlcPtdIns anchor. The possible intralumenal function of this form of AP is discussed.
Structural features of cell wall teichoic acid and peptidoglycan of Actinomadura cremea INA 292
Authors: POTEKHINA Natalia V. , NAUMOVA Irina B. , SHASHKOV Alexander S. , TEREKHOVA Larisa P.
Published: March 2005

The teichoic acid from the cell wall of Actinomadura cremea INA 292 has an unusual structure, being a poly(galactosylglycerol phosphate) chain with glycerol phosphate groups. Monomeric units of 1‐O,β‐D‐galactopyranosylglycerol monophosphate are joined in the polymer by phosphodiester links involving the glycerol C3 and the galactose C6 atoms. Approximately every second galactosyl substituent has a glycerol phosphate residue at its C3 atom. The teichoic acid structure was established by chemical analysis and 13C‐NMR spectroscopy. There also is a peptidoglycan belonging to the A1γ type; as well as meso‐2,6‐diaminopimelic acid it contains small amounts of the LL form and glycine.
Recombinant human tyrosine hydroxylase isozymes
Authors: HAAVIK Jan , LE BOURDELLES Beatrice , MARTINEZ Aurora , FLATMARK Torgeir , MALLET Jacques
Published: March 2005

Human tyrosine 3‐monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1–TH4), generated by alternative splicing of pre‐mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04–0.1 atom iron and 0.02–0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13–40‐fold) by incubation with Fe(II) salts (concentration of iron at half‐maximal activation = 6–14 μM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd= 0.2–3 μM at pH 5.4–6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.
S‐Adenosylmethionine decarboxylase from the thermophilic archaebacterium Sulfolobus solfataricus
Authors: CACCIAPUOTI Giovanna , PORCELLI Marina , DE ROSA Mario , GAMBACORTA Agata , BERTOLDO Costanzo , ZAPPIA Vincenzo
Published: March 2005

S‐Adenosylmethionine decarboxylase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium optimally growing at 87°C, has been purified to homogeneity. The specific activity of the homogeneous enzyme is 12 nmol CO2 formed min−1 (mg protein)−1 and the overall yield 8%. The enzyme is thermophilic with an optimum at 75°C, is thermostable, and does not require divalent cations or putrescine for activity. It has a molecular mass of 32 kDa, and appears to be a monomeric protein.

S‐Adenosylmethionine decarboxylase from S. solfataricus contains covalently linked pyruvate as prosthetic group and is inactivated in a time‐dependent process by NaCNBH3, in the presence of both the substrate and the product. Incubation with decarboxylated S‐adenosyl[Me‐3H]methionine and NaCNBH3 resulted in the labeling of the protein at the active site.
Molecular cloning of the human α6 integrin subunit
Authors: HOGERVORST Frans , KUIKMAN Ingrid , VAN KESSEL Ad Geurts , SONNENBERG Arnoud
Published: March 2005

We have isolated cDNAs encoding the α6 subunit from a γ gt11 expression library from human keratinocytes by combined screening with a rabbit polyclonal anti‐α6 antibody and the polymerase chain reaction. The α6 subunit encoded by this cDNA consists of 1050 amino acids with a 991‐amino‐acid extracellular, a 23‐amino‐acid transmembrane and a 36‐amino‐acid cytoplasmic domain. The extracellular domain contains three putative divalent cation‐binding sites and nine potential N‐linked glycosylation sites. From a cDNA library from normal human mammary gland cells two different cDNAs for α6 were isolated, one of which is identical to the above cDNA. The two α6 subunits, called α6A and α6B, encoded by the two cDNAs each have a unique cytoplasmic domain, that of α6B being 18 amino acids longer than that of α6A.

Different carcinoma cell lines contain transcripts for both α6 subunits. K 562 leukemic cells have little α6A or α6B mRNAs. The overall level of expression varies in the carcinoma cell lines, but reflects α6 cell surface expression. In A375 melanoma cells, however, cell surface expression of α6 was low in spite of a high level of mRNA. This suggests that other mechanisms may be involved in regulating the expression of α6 on the surface of these cells. The mRNA for both α6 subunits is around 6 kb.

The α6 subunits are similar to other α subunits (26–31% identity with cleaved α subunits) of the integrin family but they are more similar to the α3 subunit (40% identity). This high degree of similarity may be the basis for their functional resemblance since both α3 and α6 subunits, when associated with β1, function as laminin receptors and bind to the long arm of laminin. The genes for α6 and β4, the alternative β subunit with which α6 combines on certain epithelial cells, were mapped to chromosome 2 and 17q11‐qter, respectively.
Expression and post‐translational processing of preprocecropin A using a baculovirus vector
Authors: HELLERS Marianne , GUNNE Hans , STEINER Håkan
Published: March 2005

A cDNA fragment encoding preprocecropin A was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in recombinant‐infected last instar larvae of Trichoplusia ni and in diapausing pupae of Hyalophora cecropia. The identity of the recombinant product was established by electrophoresis with detection of antibacterial activity and mass spectrometry. The prepropeptide had been correctly processed including removal of signal peptide and pro‐part. Biologically active and amidated cecropin A was exported to the hemolymph. The yield of recombinant protein in H. cecropia reached a level of 600 μg/ml hemolymph and about 70% of the material was amidated.
Conserved amino acid sequences among plant proteins sorted to protein bodies and plant vacuoles
Authors: SEBASTIANI Federico L. , FARRELL Leigh B. , VASQUEZ Max , BEACHY Roger N.
Published: March 2005

Amino acid sequence comparisons were made between the soybean α subunit of β‐conglycinin and 34 members of different plant protein families targeted to seed protein bodies or vacuoles. A number of short conserved amino acid sequences were identified in seed storage proteins, plant protease inhibitors and lectins, and the probable functions of these sequences are discussed. For proteins of known tertiary structure, these sequences map to the surface of the respective molecules. It is postulated that these regions produce a common secondary structure which could interact with other molecules involved in the sorting process. One of these regions, region A, is similar to the yeast carboxypeptidase Y (CPY) vacuolar targeting signal, and is present in both storage proteins and lectins. Computer modeling based upon the tertiary structure of concanavalin A (ConA) was used to generate models representing the structure of two highly related lectins from Dolichos biflorus, one of which is targeted to protein bodies and the other secreted. A different glycosylation pattern together with amino acid sequences upstream of the identified conserved amino acid sequences are predicted to modulate the presentation of the sorting domains in the lectins and be the determinant in the sorting of these lectins.
Molecular species of cardiolipin in relation to other mitochondrial phospholipids
Authors: SCHLAME Michael , BEYER Klaus , HAYER‐HARTL Manajit , KLINGENBERG Martin
Published: March 2005

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18–C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid–protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemstry 24, 3821–3826]. This association seems to be very strong as protein‐bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl‐monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier‐bound cardiolipin does not result in release of cardiolipin as judged by 31P‐NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin‐labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin‐labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent‐solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment.