bluuebunny/crossref_metadata_2025 huggingface dataset:
Fructose 2,6‐bisphosphate in germinating oat seeds
Authors: LARONDELLE Yvan , CORBINEAU Françoise , DETHIER Monique , COME Daniel , HERS Henry‐Géry
Published: March 2005

When dormant oat seeds were imbibed at the non‐permissive temperature of 30°C, the concentration of phosphoenolpyruvate and of glycerate 3‐phosphate, which are two inhibitors of phosphofructokinase 2, increased almost linearly during 30 h. By contrast, these metabolites increased only after a lag period of about 10 h in nondormant seeds imbibed at the same temperature. As a consequence of this, the concentration of the C3 derivatives remained always remarkably lower in non‐dormant than in dormant seeds. Accordingly, the concentration of fructose 2,6‐bisphosphate, which increased similarly in the two types of seeds during the first 8 h after the start of imbibition, then reached a plateau in dormant seeds but continued to increase for another 8 h in non‐dormant seeds, reaching a maximal value a few hours before the beginning of radicle protrusion. When the dormant seeds were imbibed at the permissive temperature of 10°C, the evolution of all metabolites was slowed down but behaved like that of non‐dormant seeds imbibed at 30°C. Experiments in which the dormant seeds were submitted to a jump from 10°C to 30°C and vice versa, always provoked reverse changes in the concentration of the C3 derivatives and of fructose 2,6‐bisphosphate, the latter being increased in all conditions that allowed germination. Dormant seeds were also allowed to germinate at 30°C by imbibition during 24 h in the presence of 3% ethanol. Again, this permissive treatment caused an arrest in the accumulation of C3 derivatives and an increase in fructose 2,6‐bisphosphate.

Another, apparently unrelated, biochemical difference between dormant and non‐dormant oat seeds was their inorganic pyrophosphate content, which was approximately five‐fold higher in non‐dormant than in dormant seeds. This difference was observed before and persisted during imbibition as long as measurement could be made and was not affected by the temperature jumps or by ethanol. In contrast to the phosphoric esters under investigation, pyrophosphate was not preferentially located in the embryo.
Trout sperm motility
Authors: CHRISTEN Richard , GATTI Jean‐Luc , BILLARD Roland
Published: March 2005

For freshwater fish the motile period of sperm is extremely brief, even after a dilution in isotonic media. This result is in contrast to most other animals (ranging from invertebrates to mammals), in which sperm are generally motile for at least several hours.

We have analyzed the reasons for the brevity of this movement by studying the relationships between the metabolism of trout sperm and the activation of their motility upon dilution. Sperm motility was not initiated when the dilution medium contained an elevated concentration of potassium (20–40 mM), but dilution in an isotonic solution of sodium chloride triggered an immediate activation of motility, and sperm swam vigorously. Motility of sperm decreased rapidly and 15 s after dilution sperm were moving slowly in small circles. Sperm became abruptly immotile at 20–30 s and flagella straightened. When millimolar concentrations of Ca2+ were also present in the dilution medium, movement did not not stop abruptly, flagella kept beating and stopped only after 1–2 min.

When sperm remained immotile they retained a high concentration of ATP. The activation of motility induced a rapid decrease of ATP. In the absence of calcium, and after the cessation of motility, ATP increased slowly back to its original concentration. In the presence of millimolar concentrations of calcium the concentration of ATP decreased to a very low level and remained low thereafter.

The progressive decrease of the flagellar beat frequency, that had been observed during the period of trout sperm movement, might be related to the rapid exhaustion of intraflagellar ATP. Motility could be reinduced in sperm that had recovered high concentrations of ATP, demonstrating the functional integrity of the motile apparatus even after flagellar arrest.

In conclusion we suggest that the maximum duration of trout sperm motility, at most 2 min (as a consequence of a depletion of ATP during the movement), is due to a low mitochondrial oxidative phosphorylation capacity.
The high‐resolution X‐ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis
Authors: BODE Wolfram , PAPAMOKOS Evangelos , MUSIL Djordje
Published: March 2005

Triclinic crystals of the complex formed by eglin with subtilisin Carlsberg were analyzed by X‐ray diffraction. The crystal and molecular structure of this complex was determined with data that extended to 0.12‐nm resolution by a combination of Patterson search methods and isomorphous replacement techniques. Its structure was refined to a crystallographic R value of 0.178 (1.0–0.12 nm) using an energy‐restraint least‐squares procedure. The complete subtilisin molecule could be traced without ambiguity in the refined electron density. The eglin component, from which an amino‐terminal segment is cleaved off, is only defined from Lys8I (i.e. the lysine residue 8 of the inhibitor) onwards. Per unit cell, 436 fixed solvent molecules and 2 calcium ions were located.

In spite of 84 amino acid replacements and one deletion, subtilisin Carlsberg exhibits a very similar polypeptide fold to subtilisin BPN. The root‐mean‐square deviations of all α‐carbon atoms (excluding those at the deletion (1971) Biochem. Biophys. Res. Commun. 45, 337–344] and subtilisin Novo [Drenth, J., Hol, W. G. J., Jansonius, J. N. & Kockoek, R. (1972) Eur. J. Biochem. 25, 177–181] are 0.077 nm and 0.103 nm. Most of these deviations result from global shifts rather than changes of the local geometry. The single‐residue deletion at position 56 affects only the surrounding conformation. Two sites of high electron density and close distances to surrounding oxygen ligands have been found in the Carlsberg enzyme which are probably occupied by calcium ions.

Eglin consists of a twisted four‐stranded β‐sheet flanked by an α‐helix and by an exposed proteinase binding loop on opposite sides. Around the reactive site, Leu45I‐Asp46I, this loop is mainly stabilized by electrostatic/hydrogen bond interactions with the side chains of two arginine residues which project from the hydrophobic core [Bode, W., Papamokos, E., Musil, D., Seemüller, W. & Fritz, H. (1986) EMBO J. 5, 813–818]. The reactivesite loop conformation resembles that found in other ‘small’ proteinase inhibitors. The scissile peptide bond is not cleaved but its carbonyl group is slightly distorted from planar geometry. Most of the intermolecular contacts are contributed by the nine residues of the reactive‐site loop Gly40I–Arg48I. The hydrophobic side chains of Pro42I (P4), Leu45I (P1) and Leu47I (P2′), which are exposed to solvent in the free inhibitor, are only partially buried in the complex and are still in van der Waals contact with several solvent molecules.
Type VI collagen represents a major fraction of connective tissue collagens
Authors: TRÜEB Beat , SCHREIER Thomas , BRUCKNER Peter , WINTERHALTER Kaspar H.
Published: March 2005

A new method for the isolation of type VI collagen from peptic tissue digests is presented which gives tenfold higher yields than methods previously reported. From the amounts of purified protein obtained from human placenta, bovine uterus, chicken gizzard and entire mouse bodies we conclude that type VI collagen represents a major fraction of connective tissue collagens.
pH‐dependent interconvertible allosteric forms of murine melanoma tyrosinase
Authors: DEVI Chintamaneni Chaya , RAMAIAH Abburi , TRIPATHI Ram Kumar
Published: March 2005

Murine melanoma melanosomal tyrosinase, solubilised at pH 6.8 and 1% Igepal, exhibits a lag in cresolase activity which increases with increasing concentration of tyrosine.

The enzyme, solubilised at pH 5.0 and assayed at pH 5.0, does not exhibit lag even at inhibitory concentrations of tyrosine while the same enzyme when assayed at pH 6.8 exhibits characteristic lag.

When the enzyme was solubilised from a melanosomal fraction with detergent/water without any buffer, significant linear activity for 2 h was seen at an inhibitory concentration of tyrosine, indicating for the first time the presence of a form of tyrosinase without lag and inhibition by excess tyrosine.

Exposure of the enzyme solubilised in buffer/detergent at pH 6.8 to rapid decrease in pH to 5.0 or 4.7 makes the enzyme remain irreversibly in the form without characteristic lag, even at an inhibitory concentration of tyrosine and at pH 6.8.

These results may be interpreted as follows. The enzyme at pH 6.8 exists in the E form with an allosteric site for tyrosine. Decrease of the pH of the enzyme solution from 6.8 to 5.0 or 4.7 by dialysis results in the reversible protonation of the enzyme, which no longer binds tyrosine at its allosteric site and consequently inhibition by excess tyrosine and lag were not observed at acidic pH. However, if the enzyme was rapidly brought to pH 5.0 from 6.8 it remains irreversibly in the protonated form even at pH 6.8.

Ascorbic acid acts as an effective reductant for the hydroxylation of tyrosine by tyrosinase, while 3,4‐dihydroxyphenylalanine is both an effective reductant and counteracts the inhibition by tyrosine at pH 6.8.
Oscillation of ion fluxes in mammalian erythrocytes
Authors: HOLMUHAMEDOV Ehson L. , TEPLOVA Vera V. , SADYKOV Yuri H.
Published: March 2005

The dependence of ionophore‐induced oscillations in rat erythrocytes on various concentrations of A23187, FCCP and Ca2+ was analysed using ion‐selective electrodes. The oscillations were shown to be independent of the extracellular concentration of carbonylcyanide p‐trifluoromethoxyphenylhydrazone and Ca2+. The dependence of oscillations on the concentration A23187 was shown to be a threshold characteristic and represented by a bellshaped curve. In the course of oscillations the redistribution of A23187 between cells and the incubation medium was demonstrated using high‐speed centrifugation. A hypothesis for oscillatory‐state generation in erythrocytes was suggested on the basis of pH‐dependent changes of the Ca2+ ionophore A23187 content in cells. According to this hypothesis the H+ concentration within the external membrane‐adjacent layer serves as a causative factor for induction of cyclic desorption of A23187 molecules from the cell membrane.
Lysosomal and non‐lysosomal peptidyl hydrolases of the bloodstream forms of Trypanosoma brucei brucei
Authors: LONSDALE‐ECCLES John D. , GRAB Dennis J.
Published: March 2005

African trypanosomes have thiol‐dependent proteolytic activity that resembles some of the cathepsin‐like activity found in mammalian lysosomes [Lonsdale‐Eccles, J. D. & Mpimbaza, G. W. N. (1986) Eur. J. Biochem. 155, 469–473]. Here we show that this activity is found in lysosome‐like organelles which we have isolated (density = 1.082 g/cm3 in Percoll) from bloodstream forms of Trypanosoma brucei brucei. They are approximately 250 nm in diameter, are bounded by a single limiting membrane, and contain acid phosphatase. The predominant proteolytic and peptidolytic activity of these organelles has a pH optimum about 6.0, exhibits latency, and has the characteristics of mammalian cathepsin L (and possibly cathepsin H) with respect to its hydrolysis of small fluorogenic peptidyl substrates such as benzyloxycarbonyl‐phenylalanyl‐arginyl‐7‐amido‐4‐methylcoumarin. This substrate appears to be a good marker for trypanosomal lysosomes. The cathepsin‐L‐like activity is inhibited by the thiol‐protease inhibitors, E‐64, cystatin, leupeptin and mercurial compounds.

The proteolytic activity of the lysosome‐like fraction is observed as a single band of activity with an approximate molecular mass of 27 kDa when measured after electrophoresis in the fibrinogen‐containing sodium dodecyl sulphate/polyacrylamide gels. The addition of mammalian serum to this purified fraction, or to whole trypansosome homogenates, results in the appearance of additional bands of activity, with a concomitant increase in the total observed proteolytic activity. The serum of some species of animal (e.g. goat and guinea pig) appear to lack the ability to generate this new and increased activity, while rat, rabbit, human and bovine sera exhibit varying capacities to generate the new activity, the cow being the most effective. The apparent molecular masses of the new bands of activity are different for each mammalian species, suggesting that the activator is a species‐specific molecule or class of molecules.

We also show that Trypansosoma brucei contains soluble peptidolytic activity with an alkaline pH optimum. It is inhibited by the serine‐protease inhibitor diisopropylfluorophosphate, but not by inhibitors such as phenylmethylsulphonyl fluoride, α1‐antitrypsin, or aprotinin. Nor is it inhibited by the thiol‐protease‐specific inhibitors E‐64 or cystatin, although it is susceptible to inhibition by tosyllysylchloromethane, leupeptin, HgCl2 and p‐chloromercuribenzoate. This enzymic activity has a preference for arginyl residues in the primary binding site (the P1 position), as also does the activity from the lysosomes. However, the subsite specificity of the soluble enzymic activity is distinct from that of the lysosomes. The soluble enzyme(s) prefers small neutral molecules in the P2 and P3 subsites whereas the lysosomal enzyme(s) prefer hydrophobic molecules in these positions.
Orientation of the brush‐border membranal proteinase which specifically splits the catalytic subunit of cAMP‐dependent protein kinase
Authors: DE JONGE Hugo , SCHMEEDA Hilary , SHALTIEL Shmuel
Published: March 2005

The active site of the rat intestinal brush‐border membranal proteinase [Alhanaty E. and Shaltiel, S. (1979) Biochem. Biophys. Res. Commun. 89, 323–332], which splits the catalytic subunit (C) of cAMP‐dependent protein kinase with a remarkable specificity [Alhanaty E., Tauber‐Finkelstein, M., Schmeeda, H. and Shaltiel, S. (1985) Curr. Topics Cell. Regul. 27, 267–277], is shown to face predominantly the cell exterior; vesicles prepared from these brush‐borders (mostly sealed and right‐side‐out) fully express the proteinase activity as judged by the fact that there is no increase in activity upon rupture or solubilization of the vesicles.

Although the brush‐border vesicles contain a cAMP‐dependent protein kinase, this membrane‐bound kinase is not likely to be the physiological target of the proteinase, since it appears to have an intracellular orientation and, at least in the vesicles, to be inaccessible to the proteinase. It is, therefore, suggested that the physiological substrate of the proteinase might be either an extracellular cAMP‐dependent protein kinase, which is lost (e.g. removed, inactivated or degraded) in the course of vesicle isolation, or a kinase domain in one of the family of proteins recently shown to have a considerable structural and conformational homology with C. Alternatively the physiological site of action of this kinase‐splitting proteinase might be an intracellular organelle to which it is translocated by endocytosis.
Primary structure and differential expression of β‐amylase in normal and mutant barleys
Authors: KREIS Martin , WILLIAMSON Martin , BUXTON Bernadette , PYWELL Jackie , HEJGAARD Jørn , SVENDSEN Ib
Published: March 2005

The primary structure of barley endosperm β‐amylase, an enzyme which catalyses the liberation of maltose from 1,4‐α‐D‐glucans, has been deduced from the nucleotide sequence of a cloned full‐length cDNA. The mRNA is 1754 nucleotides long [excluding the poly(A) tail] and codes for a polypeptide of 535 amino acids with a relative molecular mass of 59663. The deduced amino acid sequence was compared with the sequences of ten peptides obtained from the purified enzyme and unambiguous identification was obtained. The N‐terminal region of the deduced sequence was identical to a 12‐residue cyanogen‐bromide‐peptide sequence, indicating that β‐amylase is synthesized as the mature protein. A graphic matrix homology plot shows four glycine‐rich repeats, each of 11 residues, preceding the C‐terminus. Southern blotting of genomic DNA demonstrates that β‐amylase is encoded by a small gene family, while cDNA sequence analysis indicates the presence of at least two types of mRNA in the endosperm. Dot and northern blot analysis show that Hiproly barley contains greatly increased levels of β‐amylase mRNA compared to the normal cultivar Sundance, whereas Risø mutant 1508 contains only trace amounts. These results correlate well with the deposition of β‐amylase during endosperm development in these lines. Low but similar amounts of β‐amylase mRNAs sequences were detected in leaves and shoots from normal and mutant barleys, demonstrating that the mutant lys3a (1508) and lysl (Hiproly) genes do not affect the expression of β‐amylase in these tissues.
Thromboxane synthase catalyses hydroxylations of prostaglandin H2 analogs in the presence of iodosylbenzene
Authors: HECKER Markus , BAADER Wilhelm J. , WEBER Peter , ULLRICH Volker
Published: March 2005

Human platelet microsomes supplemented with iodosylbenzene converted the stable prostaglandin H2 analog 15(S)‐hydroxy‐11α,9α‐epoxymethano‐5(Z),13(E)‐prostadienoic acid (U46619) into three metabolites (17.5% yield) which were not formed in the presence of specific thromboxane synthase inhibitors. The same three products were also formed among others by incubation of U46619 with liver microsomes from phenobarbital‐pretreated rats with NADPH/O2 or with iodosylbenzene. The NADPH‐supported metabolism of U46619 was suppressed in the presence of carbon monoxide. Combined gas chromatography/negative‐ion chemical ionization mass spectrometry analysis revealed for all three compounds the incorporation of one oxygen atom which according to the electron impact fragmentation pattern had to be introduced either at the 9‐methylene group or at the cyclopentane ring. The identification of these metabolites as 9β,15(S)‐dihydroxy‐11α,9α‐epoxymethano‐5(Z),13(E)‐prostadienoic acid and the R and S isomer of 15(S)‐hydroxy‐11α,9α‐(C‐hydroxy‐epoxymethano)‐5(Z),13(E)‐prostadienoic acid is only tentative since no reference compounds were available, but clearly thromboxane synthase was acting as an oxene transferase in this reaction. In contrast to U46619, its 9,11‐epoxymethano isomer U44069 was found to be only a poor substrate for the oxene transferase activity of thromboxane synthase (1% yield) which indicates a preference for the 9‐methylene group of U46619 which is orientated close to the heme iron of thromboxane synthase as evidenced by spectroscopic studies. Low‐level chemiluminescence detected following incubation of iodosylbenzene with partly purified thromboxane synthase is in agreement with the formation of an activated (FeO)3 oxygen species. In summary, these results point to a common role of the thiolate ligand in the oxygen activation mechanism by thromboxane and prostacyclin synthase and liver cytochrome P‐450 monooxygenases.
Purification and characterization of an 8‐hydroxy‐5‐deazaflavin‐reducing hydrogenase from the archaebacterium Methanococcus voltae
Authors: MUTH Erika , MÖRSCHEL Erhard , KLEIN Albrecht
Published: March 2005

A methylviologen and 8‐hydroxy‐5‐deazaflavin(F420)‐reducing hydrogenase was purified over 800‐fold to near homogeneity from the archaebacterium Methanococcus voltae with 10 U mg−1 F420‐reducing activity. It is the only hydrogenase in this organism. The enzyme showed Km values of 16 μM for F420 and 1.2 mM for methylviologen. A turnover number of 1050 min−1 was calculated for the minimal active unit. The protein tends to aggregate. The molecular mass of the minimal active unit is 105 kDa. Larger molecules of 745 kDa were regularly observed. The enzyme was resolved into subunits with molecular masses of 55 kDa, 45 kDa, 37 kDa and 27 kDa by SDS/polyacrylamide gel electrophoresis. Reversible conversion of an anionic into an uncharged form was observed by DEAE‐cellulose chromatography with concomitant changes in substrate specifities. The methylviologen‐reducing activity was heat‐resistant up to 65°C and was not affected by antiserum raised against the native enzyme, while F420 reduction was inactivated by both treatments. Nickel and selenium contents were determined as 0.6–0.7 mol each, FAD content as 1 mol and iron as 4.5 mol/mol protein (105 kDa), respectively.

Electron micrographs taken from the purified enzyme show ring‐shaped molecules of 18 nm diameter, which represent the high‐molecular‐mass species of the enzyme.
Enzyme‐catalysed peptide amidation
Authors: BRADBURY Alan F. , SMYTH Derek G.
Published: March 2005

A series of hydrazones and semicarbazones of glyoxylic acid were shown to have a potent inhibitory effect on the enzyme‐catalysed conversion of D‐Tyr‐Val‐Gly to D‐Tyr‐Val‐NH2. Among the derivatives tested, the inhibitory activity was increased by the presence of hydrophobic substituents and decreased by polar substituents. The inhibition produced by glyoxylic acid phenylhydrazone was shown to be competitive. No inhibition was obtained with pyruvic acid phenylhydrazone, which possesses a methyl group in place of the α‐H of glyoxylic acid phenylhydrazone. The inhibitory potencies of these non‐peptide substances are in accord with the specificity exhibited by the amidating enzyme in its reaction with peptide substrates.

The inhibition produced by the glyoxylic acid derivatives was shown to be due to their ability to act as substrates for the peptide‐amidating enzyme. The product formed from [14C]glyoxylic acid phenylhydrazone was identified as oxalic acid phenylhydrazide by co‐chromatography in three chromatographic systems. The results demonstrate that the enzyme catalysed oxidation of glyoxylic acid phenylhydrazone takes place by a mechanism involving hydroxylation. It is implicit that peptide amidation catalysed by the same enzyme proceeds by a similar mechanism.
Demonstration of the existence of an organo‐specific NADH dehydrogenase in heart mitochondria
Authors: NOHL Hans
Published: March 2005

Experimental evidence is presented showing the existence of an NADH‐consuming enzyme in heart mitochondria, in addition to the NADH – ubiquinone oxidase of complex I. In contrast to the latter, the novel enzyme is accessible from the extramitochondrial space. Removal of the outer membranes from intact mitochondria had no influence on exogenous NADH consumption, indicating its location at the cytosolic face of the inner membrane. The enzyme could be solubilized from this membrane and purified by sedimentation through preformed sucrose gradients. Liver mitochondria exhibited no oxidation of external NADH, suggesting that the enzyme is organo‐specific. The “exogenous NADH dehydrogenase” of heart mitochondria was found to introduce reducing equivalents into the respiratory chain before the rotenone block, indicating that the enzyme is associated with complex I. The enzyme was also demonstrated to be involved in electron flow from the respiratory chain to exogenous electron acceptors, including NAD+. This permitted us to elicit the existence of an energy‐dependent reversed electron flow from complex II to complex I. The redox shuttle established by the novel enzyme could be of significance for the regulation of cellular NADH and the metabolic activation of foreign compounds such as adriamycin.
Phosphorylase phosphatase from skeletal muscle membranes
Authors: VILLA‐MORUZZI Emma , HEILMEYER Ludwig M. G.
Published: March 2005

Microsomes containing 12–15 U/mg phosphorylase phosphatase were obtained from skeletal muscle glycogen particles following glycogen digestion and differential centrifugation. The phosphatase associated with the membranes is in an inhibited state; dilution induces dissociation and deinhibition of the enzyme. Phosphatase‐depleted membranes can rebind purified phosphatase catalytic subunit but not the complex between catalytic subunit and inhibitor 2. Binding involves a receptor, deduced from saturation phenomena, which is responsible for inhibition of the bound enzyme and which is a protein, since trypsin treatment releases all bound enzyme and prevents rebinding. The phosphatase extracted from the membranes is of type 1 and is a mixture of complexes, the major ones displaying a Mr of 300000 and 70000. From these complexes the 35‐kDa catalytic subunit can be obtained either by trypsin treatment or by acetone precipitation Purification to homogeneity involves chromatography on polylysine and FPLC chromatography on Mono Q and Polyanion SI columns. The purified enzyme exhibits a specific activity of 26800 U/mg (27900 U/mg after trypsin treatment) and consists of a major protein of 38 kDa (SDS gel electrophoresis). A minor component of 33 kDa, which may represent either a proteolytic product or an isozyme, can be separated. Both 38‐kDa and 33‐kDa catalytic subunits form a 70‐kDa inactive complex with inhibitor 2 and upon incubation of the complexes the catalytic subunit is slowly converted to the inactive conformation which can then be reactivated by either the kinase FA or trypsin and Mn2+. Alternatively the inactive catalytic subunit is reactivated by Mn2+ alone once it has been isolated by FPLC chromatography on SI. The observation that the same catalytic subunit is present at various cell locations (namely cytosol, glycogen particles and microsomes), though in different conformations, is in favour of the hypothesis that displacement of the catalytic subunit from one cell site to the other may represent a new mechanism for phosphatase regulation in skeletal muscle.
Modes of attachment of acetylcholinesterase to the surface membrane
Authors: SILMAN Israel , FUTERMAN Anthony H.
Published: March 2005

Acetylcholinesterase (AChE) occurs in multiple molecular forms differing in their quaternary structure and mode of anchoring to the surface membrane. Attachment is achieved by post‐translational modification of the catalytic subunits. Two such mechanisms are described. One involves attachment to catalytic subunit tetramers, via disulfide bridges, of a collagen‐like fibrous tail. This, in turn, interacts, primarily via ionic forces, with a heparin‐like proteoglycan in the extracellular matrix. A second such modification involve the covalent attachment of a single phosphatidylinositol molecule at the carboxyl‐terminus of each catalytic subunit polypeptide; the diacylglycerol moiety of the phospholipid serves to anchor the modified enzyme hydrophobically to the lipid bilayer of the plasma membrane. The detailed molecular structure of these two classes of acetylcholinesterase are discussed, as well as their biosynthesis and mode of anchoring.
Translation of an mRNA in rat L6 muscle cells is regulated within the cell cycle
Authors: PRAMANIK Saroj K. , BAG Jnanankur
Published: March 2005

In muscle cells two populations of mRNA are present in the cytoplasm. The majority of mRNA is associated with ribosomes and active in protein synthesis. A small population of cytoplasmic mRNA occur as free mRNA‐protein complex and is not associated with ribosomes. This apparently repressed population of mRNA from rat L6 myoblast cells was used to construct a cDNA library. Radioactively labeled cDNA preparations of polysomal and free (or repressed) mRNA populations showed that at least ten recombinant clones preferentially annealed to the cDNA from repressed mRNA. One of these clones was extensively studied. The DNA from a recombinant plasmid D12 hybridized to a 1.3‐kb poly(A)‐rich mRNA. In proliferating myoblast cells, the 1.3‐kb mRNA was more abundant in the polysomal fraction and mostly free in the non‐dividing myotubes. In contrast to this mRNA, 90% of α and β actin mRNAs were translated in both myoblasts and myotubes. Further analysis of distribution of the 1.3‐kb RNA in the polysomal (active) and free (repressed) fractions in fusion‐arrested post‐mitotic myotubes suggested that fusion of myoblasts was not necessary for the control of translation of this mRNA. Withdrawal of muscle cells from the cell cycle appeared to be involved in regulating translation of this mRNA. The presence of this mRNA was not, however, limited to muscle cells. This mRNA was also present in the repressed state in rat liver and kidney cells. These results, therefore, suggest that the 1.3‐kb mRNA is probably translated during a particular phase of the cell cycle and is not translated in terminally differentiated non‐dividing cells. Messenger RNA homologous to the 600‐base‐pair insert of the recombinant plasmid D12 was isolated by hybrid selection procedure from both polysomal mRNA of myoblasts and free mRNA of myotubes. Translation of the hybrid selected mRNAs from both myoblasts and myotubes in rabbit reticulocyte lysate cell‐free system synthesized a 40‐kDa polypeptide. These results suggest that the repressed population of 1.3‐kb mRNA can be translated in vitro. The hybridization pattern of DNA from the recombinant plasmid D12 with rat genomic DNA suggested that the 1.3‐kb mRNA is derived from moderately repetitive rat DNA with a repetition frequency of approximately 100 copies per haploid genome.
Secretion of biologically active porcine prophospholipase A2 by Saccharomyces cerevisiae
Authors: VAN DEN BERGH Carel J. , BEKKERS August C. A. P. A. , DE GEUS Pieter , VERHEIJ Hubertus M. , DE HAAS Gerard H.
Published: March 2005

The cDNA coding for pocine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in Saccharomyces cerevisiae. Expression and secretion of proPLA could only be obtained after fusing the proPLA to the prepro sequence of the yeast α‐mating factor. Upon secretion, the fusion protein was cleaved by the KEX2 protease yielding a 140‐amino‐acid zymogen‐like form of the phospholipase A2. This protein was purified in high yield by ion‐exchange chromatography. Limited proteolysis with trypsin cleaved the ‘zymogen’ to yield active phospholipase A2, which was indistinguishable from the authentic porcine pancreatic enzyme. These results show that a protein with a disulphide bridge content as high as 7 per 124 amino acid residues can be correctly processed by the yeast secretory apparatus.
Tyrocidine‐induced modulation of the DNA conformation in Bacillus brevis
Authors: BOHG Anne , RISTOW Hansjürgen
Published: March 2005

Using the [3H]trimethylpsoralen photobinding method [Sinden, R. R., Carlson, J. O. & Pettijohn, D. E. (1980) Cell 21,773–783], a decrease in unrestrained torsional tension of DNA was detected in Bacillus brevis cells when they had entered the sporulation phase. This decrease in superhelicity was found in cells which synthesized the peptide antibiotic tyrocidine and which were stimulated to sporulate. Fluctuations in superhelicity probably reflect a highly complicated picture of tension‐relaxing and tension‐inducing activities. Addition of tyrocidine to vegetative cells reduced by one‐half the torsional tension from DNA, whereas ethidium bromide relaxes DNA completely. Cross‐links between DNA and tyrocidine were introduced with ultraviolet light in vitro and in vivo indicating that the modulation of the DNA conformation in the cell may in fact be due to a DNA‐tyrocidine interaction. In a growing B. brevis culture exogenous [3H]tyrocidine could only be photobound to DNA after the cells had entered the sporulation phase. Our results could mean that the peptide antibiotic tyrocidine is active in B. brevis on the DNA level as one regulatory factor controlling DNA functions.
Purification and comparison of two forms of S‐adenosyl‐l‐methionine synthetase from rat liver
Authors: CABRERO Carmen , PUERTA Jose , ALEMANY Susana
Published: March 2005

Only two S‐adenosyl‐l‐methionine synthetase forms exist in rat liver: high‐MrS‐adenosyl‐l‐methionine synthetase and low‐MrS‐adenosyl‐l‐methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High‐MrS‐adenosyl‐l‐methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48.5‐kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low‐MrS‐adenosyl‐l‐methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low‐MrS‐adenosyl‐l‐methionine synthetase cross‐reacted with the two forms. Reverse‐phase HPLC runs of tryptic digestions of high‐Mr and low‐MrS‐adenosyl‐l‐methionine synthetase showed that the peptide maps of the two forms were very similar, if not identical. High‐MrS‐adenosyl‐l‐methionine synthetase activity was inhibited by S‐adenosyl‐l‐methionine and pyrophosphate. Depending on the dose used, S‐adenosyl‐l‐methionine activated or inhibited low‐MrS‐adenosyl‐l‐methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction.
Isolation and characterisation of a human calcitonin‐gene‐related‐peptide receptor
Authors: FOORD Steven M. , CRAIG Roger K.
Published: March 2005

We describe the identification and purification of a receptor for calcitonin‐gene‐related peptide (CGRP) from human term placenta, using lectin and β‐CGRP–Affigel affinity chromatography. The membrane‐bound receptor has an estimated Mr of 240000, as determined by cross‐linking 125I‐labelled α‐CGRP (125I‐α‐CGRP) using discuccinimidyl suberate and SDS/polyacrylamide gel electrophoresis, or of 263000, as judged by sucrose gradient centrifugation of the soluble partially purified native receptor preparation. Cross‐linking studies with disuccinimidyl suberate and N‐hydroxysuccinimidyl‐4‐azidobenzoate using membrane‐solubilised, partially purified and CGRP‐affinity‐purified preparations, show a number of 125I‐α‐CGRP binding subunit(s) of Mr 62000–68000. Silver staining of the purified CGRP receptor preparation showed two distinct doublets in this plus a number of minor doublets of lower Mr. The receptor binds human β‐CGRP with greater affinity than α‐CGRP, and showed little affinity for human calcitonin. Adsorption isotherms and Scatchard analysis of 125I‐α‐CGRP binding to the membrane‐bound or soluble purified receptor are consistent, under the conditions used, with a single binding site of high affinity. Molecular cloning at present in progress should define the amino acid sequence and subunit composition of the human placental CGRP receptor, since at present the observed heterogeneity of CGRP‐binding proteins may be interpreted in a number of ways, for instance: receptor heterogeneity, variable glycosylation of one of two subunits, or limited proteolysis of a single subunit during purification.
Protein kinase C potentiates isoproterenol‐mediated cyclic AMP production without modifying the homologous desensitization process in J774 cells
Authors: CHAMBAUT‐GUERIN Anne‐Marie , THOMOPOULOS Pierre
Published: March 2005

The J774 murine macrophage cells possess a β2‐adrenergic receptor coupled to adenylate cyclase, which can be regulated by homologous desensitization. Stimulation of protein kinase C by phorbol esters or oleoyl acetyl glycerol potentiates two‐to‐threefold the isoproterenol‐induced cyclic AMP accumulation. These promoters act at a post‐receptor level, since the number and affinity of the β‐adrenergic receptors, measured by use of the hydrophilic ligand [3H]CGP‐12177, are not modified. In addition, the effect of cholera toxin is similarly increased and pretreatment of the cells with pertussis toxin prevents the action of phorbol esters. On the other hand, these promoters are ineffective on isoproterenol‐induced desensitization and the rates of receptor segregation and recovery remain unchanged. Therefore, protein kinase C modulates the isoproterenol‐stimulated adenylate cyclase, whereas it is inactive on the homologous desensitization process.
Iodide‐induced inhibition of adenylate cyclase activity in horse and dog thyroid
Authors: COCHAUX Pascale , VAN SANDE Jacqueline , SWILLENS Stéphane , DUMONT Jacques E.
Published: March 2005

The characteristics of the iodide‐induced inhibition of cyclic AMP accumulation in dog thyroid slices have been previously described [Van Sande, J., Cochaux, P. and Dumont, J. E. (1985) Mol. Cell. Endocrinol. 40, 181–192]. In the present study we investigated the characteristics of the iodide‐induced inhibition of adenylate cyclase activity in dog and horse thyroid. The inhibition of cyclic AMP accumulation by iodide in stimulated horse thyroid slices was similar to that observed in dog thyroid slices. The inhibition was observed in slices stimulated by thyroid‐stimulating hormone, cholera toxin and forskolin. Increasing the concentration of the stimulators did not overcome the iodide‐induced inhibition. Adenylate cyclase activity, assayed in crude homogenates or in plasma‐membrane‐containing particulates (100000 ×g pellets), was lower in homogenates or in particulates prepared from iodide‐treated slices than from control slices. This inhibition was observed on the cyclase activity stimulated by forskolin, fluoride or guanosine 5′‐[β, γ‐imino]triphosphate, but also on the basal activity. It was relieved when the homogenate was prepared from slices incubated with iodide and methimazole. Similar results were obtained with dog thyroid. The inhibition persisted when the particulate fraction was washed three times during 1 h at 100000 ×g, in the presence of bovine serum albumin or increasing concentrations of KCl. It was similar whatever the duration of the cyclase assay, in a large range of protein concentration. These results indicate that a stable modification of adenylate cyclase activity, closely related to the plasma membrane, was induced when slices were incubated with iodide. Iodide inhibition did not modify the affinity of adenylate cyclase for its substrate (MgATP), but induced a decrease of the maximal velocity of the enzyme. The percentage inhibition was slightly decreased when Mg2+ concentration increased, and markedly decreased when Mn2+ concentration increased. A detectable adenylate cyclase activity was demonstrated when intact slices were incubated in the presence of [α‐32P]ATP, probably because of the presence of broken cells produced during the slicing. Iodide had no direct effect on this cyclase system, which confirms that iodide needs the integrity of the cell to induce the inhibition and suggests that the inhibition is not transmitted between cells.
cDNAs for canavalin and concanavalin A from Canavalia gladiata seeds
Authors: YAMAUCHI Daisuke , NAKAMURA Kenzo , ASAHI Tadashi , MINAMIKAWA Takao
Published: March 2005

By a method of Escherichia coli expression‐vector‐primed cDNA synthesis, a cDNA expression library was constructed from total poly(A)‐rich RNA that was prepared from immature embryos of Canavalia gladiata. Essentially full‐length cDNA clones for two seed proteins, canavalin and concanavalin A, were selected from the library by immunological screening of the colonies and in vitro RNA synthesis and translation. The complete amino acid sequence of canavalin was determined from the nucleotide sequence of the corresponding cDNA and was found to be very homologous to 7S seed proteins of other legumes. The nucleotide sequence of the cDNA predicts a 26‐amino‐acid extension in the precursor at the amino terminus of the mature canavalin. Canavalin mRNA and concanavalin A mRNA levels at successive stages of the seed development were estimated by RNA blot hybridization and results indicated that the two mRNA levels are differently regulated.
NMR analysis of the interdomain region of yeast phosphoglycerate kinase
Authors: WILSON Henry R. , WILLIAMS Robert J. P. , LITTLECHILD Jennifer A. , WATSON Herman C.
Published: March 2005

Previous proton and phosphorus nuclear magnetic resonance studies with yeast phosphoglycerate kinase have been extended using a higher‐resolution spectrometer and a greater variety of binding agents. The new study shows that, apart from a few isolated mobile side chains distributed over the protein surface, there is a mobile section of phosphoglycerate kinase associated with the inter‐domain region of the molecule. This region gives relatively well resolved resonances which are quite distinct from those originating from the remainder of the protein. This suggests that the molecule fluctuates between many states including several open or substrate binding forms in addition to the closed and supposedly catalytically competent form of the enzyme. The occupancy of these states appears to be affected by several anions including sulphate, phosphate and cobalticyanide, as well as substrates and their analogues.
Interactions of fatty acids with neutral fatty‐acid‐binding protein from bovine liver
Authors: SCHULENBERG‐SCHELL Helmut , SCHÄFER Petra , KEUPER Hermann J. K. , STANISLAWSKI Bernd , HOFFMANN Eberhard , RÜTERJANS Heinz , SPENER Friedrich
Published: March 2005

Hepatic‐type fatty‐acid‐binding protein (hFABP) from the cytosol of bovine liver is a 14.4‐kDa neutral protein with a blocked N‐terminus and a disulfide system located on the surface of the protein. It binds two molecules of fatty acid in one binding site, apparent dissociation constants of the oleic acid/hFABP complex are 0.24 μM and 2.15 μM. Computer analysis of circular dichroic spectra predicts that hFABP contains about 12%α‐helix, 45%β‐structure, 15%β‐turn and 27% unordered structure. Ellipticities indicative of secondary structure are not affected by fatty acid binding.

Cationic amino acid residues of hFABP (1 His, 15 Lys, 2 Arg) were screened for ionic fatty acid/protein interactions. His was excluded, as 1H‐NMR analysis of His‐C2 and His‐C4 protons indicated that binding of oleic acid shifts the pK of His from 6.9 to 7.1 only in hFABP with the disulfide system in the oxidized state; acylation of His with diethylpyrocarbonate does not affect the binding of the fatty acid. Acetylation of Lys reduces binding marginally, whereas modification of Arg with phenylglyoxal lowers the binding activity by 65%.

From 1H‐NMR investigations, conformational changes within the protein, due to a sort of disaggregation of hFABP upon fatty acid binding, were derived. Most of the proton resonances sharpen up with ligand binding, and some of the methyl resonances shift positions, possibly because they are directly involved in the fatty acid/protein interaction.
Characterization of the actin polymerization‐inhibiting protein from chicken gizzard smooth muscle
Authors: RUHNAU Klaus , SCHRÖER Elke , WEGNER Albrecht
Published: March 2005

An actin polymerization‐inhibiting protein, occurring in crude preparations of vinculin from chicken gizzard, has been found to be heterogeneous. The molecular masses of the polymerization‐inhibiting peptides have been reported to range from 20 kDa to 80 kDa [Schröer, E. & Wegner, A (1985) Eur. J. Biochem. 153, 515–520], In this paper, a 21‐kDa peptide was isolated from the bulk of the other peptides by gel chromatography. The 21‐kDa peptide was identified as a polymerization‐inhibiting peptide by its ability to retard nucleated actin polymerization and to bind polymeric actin when it was blotted onto nitrocellulose. Antiserum raised to the 21‐kDa peptide was found to react with almost all peptides of the blotted heterogeneous polymerization‐inhibiting protein. The same peptides which reacted with antiserum cosedimented with polymeric actin. The major peptides of the blotted polymerization‐inhibiting protein bound polymeric actin. The largest peptide which reacted with antiserum and cosedimented with polymeric actin had a molecular mass of 85 kDa. The results suggest that the preparation of polymerization‐inhibiting protein contains mainly polymerization‐inhibiting peptides and only some contaminants, and that all the polymerization‐inhibiting peptides are proteolytic fragments stemming from a common precursor.
Reconstitution of myoglobin from apoprotein and heme, monitored by stopped‐flow absorption, fluorescence and circular dichroism
Authors: KAWAMURA‐KONISHI Yasuko , KIHARA Hiroshi , SUZUKI Haruo
Published: March 2005

The reconstitution reaction of ferric cyanomyoglobin from apomyoglobin and hemin dicyanide was investigated with a stopped‐flow apparatus by the use of five kinds of probes; (a) Soret absorption, (b) fluorescence quenching of tryptophan, (c) far‐ultraviolet CD, (d) near‐ultraviolet CD, and (e) Soret CD.

After mixing of apomyoglobulin with equimolar amounts of hemin dicyanide, the Soret absorption band was shifted to longer wavelengths within 10 ms. The shifted band kept its shape for a few seconds, and then gradually shifted to shorter wavelengths. A rate constant of the slow reaction was 1.1 × 10−2 s−1. Time courses of fluorescence quenching followed a second‐order reaction with a rate constant of 9 × 107 M−1 s−1. Far‐ultraviolet CD recovered to the level of native state within the response time of an apparatus (= 64 ms). Near‐ultraviolet CD and Soret CD changed with first‐order rate constants of 5–30 s−1 and 5 × 10−3 s−1 respectively.

On the basis of the kinetic results we propose the following reconstitution pathway of myoglobin. Apomyoglobin has essentially a highly folded structure similar to myoglobin, but there are some differences in the secondary structure between them. In the first step, heme enters the pocket‐like site of apomyoglobin and interacts with surrounding hydrophobic residues in the pocket, and then the interaction may give a complete ordered structure to the protein. Second, the tertiary structure of the heme pocket is partly constructed. Third, the iron‐proximal His bond occurs, followed by the attainment of the final conformation. This sequence of the events shows that the polypeptide chain is entirely folded before the completion of three‐dimensional structure of the heme pocket. The reconstitution pathway is fairly different from that of the α subunit of hemoglobin reported by Leutzinger and Beychok [Proc. Natl Acad. Sci. USA (1981) 78, 780–784], which described how a drastic recovery in helicity was observed on the heme‐binding, and that the recovery is introduced by the formation of the heme pocket structure. The difference in the results found for the α subunit and myoglobin suggests a difference in conformation: in apomyoglobin most of the helices are arranged and folded around a helix core to form a compact structure as a whole, while in apo‐α‐subunit some helices are not folded around the helix core. Helix D, which is absent in the α subunit, may play an important role in folding of the helices.