bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a Thyroliberin‐Degrading Serum Enzyme Catalyzing the Hydrolysis of Thyroliberin at the Pyroglutamyl‐Histidine Bond
Authors: BAUER Karl , NOWAK Peter
Published: March 2005

The enzymatic hydrolysis of thyroliberin (< Glu‐His‐Pro‐NH2) by serum enzymes can be preferentially inhibited by 1 mM diisopropylfluorophosphate. Under such conditions, pyroglutamic acid and His‐Pro‐NH2 are formed as the only enzymatic split products. As a secondary metabolite, histidylproline diketopiperazine is formed non‐enzymatically by intramolecular condensation of the dipeptide amide. Due to their differences in charge, the enzymatically formed split products can easily be separated from thyroliberin by ion‐exchange chromatography on cellulose‐phosphate paper and thus, by using radioactively labelled thyroliberin as tracer, highly sensitive tests could be developed for the quantitative determination of the thyroliberin‐degrading enzymatic activity.

By gel filtration, the thyroliberin‐degrading serum enzyme could be partially purified and separated from two enzymes which catalyze the hydrolysis of pyroglutamyl β‐naphthylamide, the substrate of the known pyroglutamate aminopeptidases. For the serum tested, the extremely low activities of the pyroglutamate aminopeptidases did not contribute to the observed rapid degradation of thyroliberin. On the other hand, pyroglutamyl β‐naphthylamide was not effectively hydrolyzed by the thyroliberin‐degrading serum enzyme. Furthermore, in contrast to the pyroglutamate aminopeptidases, which are activated by EDTA and dithiothreitol, the thyroliberin‐degrading serum enzyme was inhibited by these agents. This enzyme, having an approximate molecular weight of 260000, is also effectively inhibited by other chelating agents, by heavy metal ions and by p‐chloromercuribenzoate but not by 2–iodoacetamide and N‐ethylmaleimide or by serine protease inhibitors. When tested in vitro, the activity of the thyroliberin‐degrading serum enzyme was not affected by thyroid hormones, steroids or adenohypophyseal hormones.

These results demonstrate that the degradation of thyroliberin at the pyroglutamyl‐histidine bond is catalyzed by a serum enzyme which exhibits unique enzyme‐chemical characteristics distinctly different from those of the known pyroglutamate aminopeptidases catalyzing the same reaction.
Two‐Dimensional Analysis of Proteins Associated with Heterogenous Nuclear RNA in Various Animal Cell Lines
Authors: BRUNEL Claude , LELAY Marie‐Noëlle
Published: March 2005

The protein complement of heterogenous nuclear RNA · protein particles from human HeLA, mouse L and Chinese hamster (CHO) cells has been analysed by two‐dimensional gel electrophoresis using the two techniques described by O'Farrell [J. Biol. Chem. (1975) 250, 4007–4021 and Cell (1977) 72, 1133–1142].

Over a hundred individual spots have been reproducibly detected both in stained gel and in autoradiographs of proteins labeled with L‐[35S]methionine. Large similarities, especially in the 25000–40000 Mr cluster of basic protein, were found among these three mammalian species.

As far as phosphoproteins are concerned, it was observed that the bands already described by one‐dimensional gels [Eur. J. Biochem. (1978) 86, 301–310] with Mr values of 28000, 30000, 37000 and 52000 are resolved into about 15 individual spots, suggesting a corresponding number of distinct states of phosphorylation. It was also clearly demonstrated that phosphoproteins are unrelated to the major basic protein species.

Particles of different size classes were analysed with respect to their content of individual proteins, both non‐phosphorylated and phosphorylated. The most salient feature observed was that phosphoproteins become progressively more abundant with particles of increasing size. This raises the possibility that at least some of these phosphoproteins might belong to a nuclear structure to which hnRNA is normally bound.
Reaction of Rat Hepatic Glucokinase with Substrate‐Related and Other Alkylating Agents
Authors: CONNOLLY Bernard A. , TRAYER Ian P.
Published: March 2005

The reaction of rat hepatic glucokinase with a variety of alkylating agents has been investigated. The substrate‐related alkylating agents N‐bromoacetylglucosamine, N(‐N‐bromoacetyl)glycylglucosamine and N‐(N‐bromoacetyl)‐6‐aminohexanoylglucosamine are all good competitive inhibitors, with respect to glucose, of the glucokinase reaction. However, whereas N‐bromoacetylglucosamine is a true affinity label for rat muscle hexokinase type II [Connolly and Trayer (1979) Eur. J. Biochem. 93, 375–385] it inactivates the liver enzyme only very slowly. Time‐dependent irreversible inactivation of glucokinase is best achieved when the bromoacetyl group is made more “mobile” by interposing at least a three‐atom spacer molecule between it and the sugar ring. The substrate‐unrelated but chemically similar reagents, N‐bromoacetylgalactosamine and bromoacetic acid, do not inhibit glucokinase and only cause slight non‐specific inactivation. The kinetics of inactivation of glucokinase by N‐(N‐bromoacetyl)‐6‐aminohexanoylglucosamine are in accord with the reversible formation of an enzyme‐inhibitor complex prior to modification, indicating that the reagent is active site directed. Both glucose and N‐acetylglucosamine protect competitively against inactivation. Use of 14C‐labelled reagent showed that 1 mol reagent/mol protein caused complete activity loss, although an additional 1 mol/mol protein reacted without any effect on activity. The inactivation rate is relatively insensitive to pH variation over the range 6.5–9. Thus it would seem that the modified residue is not a cysteine unless it is unusually perturbed. This behaviour contrasts with that of yeast hexokinase, bovine brain hexokinase type I and rat muscle hexokinase type II with similar affinity reagents where a thiol is modified with complete activity loss.
The Binding of Chloride Ions to Ligated and Unligated Human Hemoglobin and Its Influence on the Bohr Effect
Authors: VAN BEEK Gerard G. M. , ZUIDERWEG Erik R. P. , DE BRUIN Simon H.
Published: March 2005

The contribution of the interaction of chloride ions with deoxy and oxyhemoglobin to the Bohr effect can be described by a simple binding model. Applying this model to experimental data reveals that at physiological pH and ionic strength about half of the release of Bohr protons is due to a difference in chloride ion binding to deoxy‐ and oxyhemoglobin. The chloride‐independent part of the Bohr effect corresponds with the shift in pK which His‐146β shows upon oxygenation. The proton absorption by hemoglobin observed upon oxygenation below pH 6 is apparently due to a chloride‐ion‐induced proton uptake, which is larger for oxyhemoglobin than for deoxyhemoglobin. The analysis of the experimental data indicates the existence of only two oxygen‐linked chloride ion binding sites in both deoxy and oxyhemoglobin. In deoxyhemoglobin the binding sites most likely consist of Val‐1α of one chain and Arg‐141α of the partner chain. The sites in oxyhemoglobin consist of groups with a pK value in the neutral pH range; they do not contain lysyl or arginyl residues.
The Effect of 12‐O‐Tetradecanoyl‐phorbol 13‐Acetate on the Ribormclease Activity of Circulating Human Lymphocytes
Authors: KYNER David , CHRISTMAN Judith K. , ACS George
Published: March 2005

12‐O‐Tetradecanoyl‐phorbol 13‐acetate is a very effective tumor promoter and inflammatory agent and can act as a mitogen for a subset of T lymphocytes. We report here that even short exposure of lymphocytes to 12‐O‐tetradecanoyl‐phorbol 13‐acetate changes the balance between the levels of neutral ribonuclease and ribonuclease inhibitor. The most dramatic change occurs in a B‐lymphocyte‐enriched population. We find that most, if not all, of the neutral ribonuclease activity in circulating lymphocytes is associated with this population and that this activity is lost with exposure to 12‐O‐tetradecanoyl‐phorbol 13‐acetate. Both 12‐O‐tetradecanoyl‐phorbol 13‐acetate and phytohaemagglutinin increase the level of ribonuclease inhibitor in T cells. However, phytohaemagglutinin has no effect on the ribonuclease or inhibitor level of the B‐cell‐enriched population.
The Control of Phosphoprotein Phosphatase by the Second‐Site Phosphorylation of a Substrate
Authors: USUI Hirofumi , NISHIMURA Noriko , IMAZU Michinori , IMAOKA Teiji , KINOHARA Nobuhisa , TAKEDA Masao
Published: March 2005

The phosphorylation of Ser‐32, in addition to Ser‐36 of H2B histone, stimulated the rate of Pi release from Ser‐36 by the small form (Mr 31000) of pig heart phosphoprotein phosphatase both in the absence and presence of 50 mM magnesium acetate. By phosphorylation at Ser‐32, the Km value for Ser‐36 phosphate in H2B histone was increased from 0.38 μM to 1.16 μM in the absence of magnesium acetate, but not significantly changed (from 37.4 μM to 26.2 μM) in the presence of magnesium acetate. With the large form (Mr 224000) of the phosphoprotein phosphatase, however, the phosphorylation at Ser‐32 suppressed the rate of Pi release from Ser‐36 both in the absence and presence of magnesium acetate. The Km value of the large form for Ser‐36 phosphate in H2B histone was nevertheless increased by phosphorylation at Ser‐32, from 1.2μM to 5.3 μM in the presence of magnesium acetate, but not changed (from 0.26 μM to 0.23 μM) in the absence of magnesium acetate.
A Membrane Glycoprotein of Aggregating Dictyostelium Cells with the Properties of Contact Sites
Authors: MÜLLER Kurt , GERISCH Gunther , FROMME Inge , MAYER Hubert , TSUGITA Akira
Published: March 2005

Aggregating cells of Dictyostelium discoideum form EDTA‐stable cell contacts which are blocked by a Fab (antigen‐binding fragment) preparation from antisera raised against membranes. The target site of the blocking Fab fragments has been identified as a specific glycoprotein. In this paper its purification, carbohydrate and amino acid composition are described. Purification was 800‐fold, starting with cells lysed by digitonin. The plasma membranes, preserved as ghosts by this treatment, were purified in a two‐phase system and extracted with butan‐1‐ol. The water phase contained predominantly concanavalin‐A‐binding glycoproteins and was particularly rich in contact sites A. These were further purified on DE‐cellulose and sucrose gradients. Sodium dodecylsulphate/polyacrylamide gel electrophoresis of the purified material revealed one major glycoprotein band in the molecular weight region of 80000 to 90000, depending on the acrylamide concentration. The sugars found in contact sites A were mannose, N‐acetylglucosamine, fucose, and possibly glucose. The protein moiety contained 8 % proline and was particularly rich in hydroxy amino acids.
Synthesis of Biologically Active Portions of an Intercistronic Region by Use of a New 3′‐Phosphate Incorporation Method to Protect 3′‐OH and Their Binding to Ribosomes
Authors: NEILSON Thomas , GREGOIRE Rene J. , FRASER Alan R. , KOFOID Eric C. , GANOZA M. Clelia
Published: March 2005

To examine the influence of bases contiguous to a starter codon, a rapid means of assembling biologically active regions corresponding to portions, or to analogues of portions of intercistronic regions is desirable. To do this, a chemical method for the specific insertion of 3′‐monophosphate groups on to chemically synthesized trinucleotides, tetranucleotides and hexanucleotides of defined sequence bearing different bases at the 3′ terminus has been devised. The method involved phosphorylation of blocked oligoribonucleotides synthesized by a phosphotriester method. The deblocked oligoribonucleotides were phosphorylated at the 5′ end with T4‐induced polynucleotide kinase. The products of this kinase reaction served as“donors” in RNA ligase reactions. The [32P]pC‐A‐U‐A‐U‐Gp, [32P]pA‐U‐Gp, [32P]pU‐A‐A, [32P]pA‐G‐G‐Ap, [32P]pC‐U‐U‐Ap and [32P]pU‐C‐C‐Up “donors” were used to synthesize A‐G‐G‐A[32P]pC‐A‐U‐A‐U‐Gp, U‐C‐C‐U[32P]pC‐A‐U‐A‐U‐Gp, U‐A‐A[32P]pA‐U‐G, A‐U‐G[32P]pU‐A‐A, U‐A‐A‐G[32P]pA‐G‐G‐Ap, U‐C‐C‐U[32P]pC‐U‐U‐Ap and A‐U‐U‐C[32P]pU‐C‐C‐Up indicating that the method functions with all bases. A‐U‐Gp, PA‐U‐Gp and pC‐A‐U‐A‐U‐Gp were isolated free of reactants and, along with PA‐U‐G, were all shown to promote the formation of translational initiation complexes. A‐G‐G‐A[32P]pC‐A‐U‐A‐U‐Gp, which corresponds to the 5′‐terminal portion of the intracistronic region of the maturation protein of bacteriophage Qβ, bound more efficiently to Escherichia coli ribosomes than the U‐C‐C‐U[32P]pC‐A‐U‐A‐U‐Gp, [32P]pC‐A‐U‐A‐U‐Gp, [32P]pA‐U‐G‐U‐A‐A or [32P]pU‐A‐A‐A‐U‐G controls. The results suggested that the content and number of residues at the 5′ terminus attached to A‐U‐G affect binding of oligonucleotides to ribosomes; purine nucleosides appear to be more effective than pyrimidine nucleosides in this regard.
Evidence for a Second Desensitized State of β‐Adrenergic Receptor with Low Affinity for β‐Antagonists and Normal Reactivity towards β‐Agonists in Adipocyte Membranes Previously Exposed to β‐Antagonists
Authors: GIUDICELLI Yves , LACASA Daniètle , AGLl Brigitte
Published: March 2005

When adipocyte membranes are successively exposed to (−)‐propranolol or (±)‐alprenolo1 at 25 or 4°C, repeatedly washed and then assayed for (−)‐[3H]dihydroalprenolol binding, the apparent number of β‐adrenergic binding sites is markedly decreased. Induction of this peculiar type of receptor desensitization does not require prolonged exposure of the membranes to the β‐adrenergic antagonists (half‐time: 1 min), is stereospecific, concentration‐dependent and almost complete with high concentrations of antagonists. p[NH]ppG, which reduces the affinity of fat cell β‐adrenergic receptors for agonists, does not prevent the antagonist‐induced decrease in the receptor number. The magnitude of the desensitizating effect induced separately by (−)‐isoproterenol and (−)‐propranolol is not additive in membranes exposed to both drugs, suggesting that the receptors lost after exposure to agonists are the same sites as part of those lost after exposure to antagonists. However, contrary to the results found in membranes desensitized by agonists, adenylate cyclase activity remained fully responsive to catecholamines in membranes exposed to β‐antagonists. As shown by kinetic studies on (−)‐[3H]dihydroalprenolol binding, this β‐antagonist‐induced receptor desensitization is reversible after prolonged incubation. These data which have never yet been described in the other reported desensitizable β‐adrenergic systems, suggest that, when exposed to β‐antagonists, the fat cell β‐adrenergic receptors undergo a conformational change leading to a peculiar state which has low affinity for antagonists but behaves towards agonists as does the receptor in its resting state.
Postsynaptic Effects of Crotoxin and of Its Isolated Subunits
Authors: BON Cassian , CHANGEUX Jean‐Pierre , JENG Tzzy‐Wen , FRAENKEL‐CONRAT Heinz
Published: March 2005

Crotoxin is a potent neurotoxin from the venom of Crotalus durissus terrificus. It is composed of two subunits: a basic phospholipase A2 with low toxicity (component B) and an acidic protein seemingly devoid of intrinsic biological activity (component A). Crotoxin and its isolated phospholipase subunit block the depolarisation caused by cholinergic agonists on the isolated electroplaque from Electrophorus electricus. The other component, which is inactive when applied alone, enhances the pharmacological activity of the phospholipase when the two components are used together. Crotoxin also blocks the increase of 22 Na+ efflux caused by carbamylcholine from excitable microsacs prepared from Torpedo marmorata electric organ. Crotoxin therefore acts postsynaptically, but does not interfere with the binding of α‐toxin from Nuja nigricollis to the nicotinic cholinergic receptor site. Instead, like local anesthetics, it stabilizes a desensitized form of the acetylcholine receptor characterized by its high affinity for agonists. The phospholipase component B binds in a non‐saturable manner to receptor‐rich membranes. In contrast, component A does not bind to acetylcholine receptor‐rich membranes, but completely prevents the non‐saturable binding of component B. When the two components are applied together, a saturable binding of the latter is observed with the acetylcholine receptor‐rich membranes.
Heterogeneity of the Nonpolysomal Cytoplasmic (Free) mRNA · protein Complexes of Embryonic Chicken Muscle
Authors: BAG Jnanankur , SELLS Bruce H.
Published: March 2005

The distribution of pulse‐labeled poly(A)‐containing RNAs between the polysomal and nonpolysomal (free) cytoplasmic fractions of embryonic chicken muscle was examined by affinity chromatographys on oligo(dT)‐cellulose. Approximately 60% of the total cytoplasmic poly(A)‐containing RNAs were present in the polysome; the remainder were present outside the polysome, the 3H‐labeled ones being present in the form of ribonucleoprotein complexes. These free mRNA · protein complexes can be divided into two populations: one can bind to an oligo(dT)‐cellulose column as a ribonucleoprotein complex, suggesting that the 3′‐poly(A) region was available for interaction with the oligo(dT) residues; in the other, this region was presumably covered with proteins and can only bind to the oligo(dT)‐cellulose column following their removal. The mRNA · protein bound to oligo(dT)‐cellulose was subfractionated into three populations. The first fraction was eluted from the column by using low‐ionic‐strength buffer at 2°C, the major portion was eluted by a combination of low‐ionic‐strength buffer and a temperature of 37°C, while the remainder was eluted with 50% formamide. Each fraction displayed a distinctive buoyant density in a CsCl gradient, suggesting a difference in their protein content.

The nature of polypeptides associated with these fractions was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Polypeptides of Mr 36500, 41000, 45000, 47000, 51000 and 75000 were common to all three mRNA protein fractions. The third population (eluted with 50% formamide) contains five additional polypeptides of Mr 54000, 66000, 68000, 73000 and 105000.

The direct interaction of free proteins with oligo(dT)‐cellulose was studied by modification of the binding of mRNA · protein to oligo(dT) by saturation of oligo(dT) sequences with poly(A) or the poly(A) segment of mRNA. protein with poly(U). Two polypeptides of Mr 41 000 and 54000 showed independent binding to these columns; they were eluted with 50 formamide but not at 37°C in low‐salt buffer. Therefore, the source of the 41000‐Mr and 54000‐Mr polypeptides was considered as other than mRNA · protein.

The translation of these mRNA · protein fractions showed that all three can be translated either as protein‐free RNA or complexed with proteins.
Macromolecular Complexes of Aminoacyl‐tRNA Synthetases from Eukaryotes
Authors: BREVET Annie , KELLERMANN Odile , TONETTI Hubert , WALLER Jean‐Pierre
Published: March 2005

The gel‐filtration behaviour of a high‐molecular‐weight complex containing seven aminoacyl‐tRNA synthetases purified from sheep liver was examined on columns of 6% agarose (Bio‐Gel A‐5m). Evidence is provided for selective interaction of the complex with the agarose matrix.

The binding capacity of agarose for the complex is dictated by the ionic strength of the equilibrating buffer: it is low but significant in 25 mM potassium phosphate buffer at pH 7.5 (ionic strength 0.068 M) and is considerably enhanced in 50 mM Tris‐HCl buffer at the same pH. In either case, raising the salt concentration leads to nearly complete recovery of enzyme activities.

In a column equilibrated in 25 mM phosphate buffer, application of the complex at a protein concentration in large excess over the binding capacity of the gel leads to co‐elution of each of the seven aminoacyl‐tRNA synthetases as a unique, symmetrical peak of apparent molecular weight close to 106, with 80%, recovery of activities. Conversely, application of the complex at low protein concentration, in amounts equivalent to the binding capacity of the gel, leads to complete retention of the enzymes which may be recovered by raising the phosphate concentration to 0.2 M. It is further shown that application of the complex at low protein concentration on a column equilibrated with 0.2 M potassium phosphate buffer to prevent interaction with the matrix, leads to co‐elution of each of the aminoacyl‐tRNA synthetases as a unique peak of apparent molecular weight close to 106. This result attests to the remarkable stability of the complex, which fails to dissociate at high salt concentration, even in the diluted state.

The above results are best interpreted as the reflection of ion‐exchange‐mediated interactions between the residual charged group on the agarose gel and the components of the high‐molecular‐weight complex. The preferential retention of the complex on agarose in ionic conditions (i. e. 25 mM phosphate or 50 mM Tris‐HCl buffers at pH 7.5) which ensure normal gel filtration behaviour of several marker proteins, may be ascribed to tighter binding of the complex due to multiple‐site interactions on account of its larger size.
Biocalcification by the Marine Alga Emiliania huxleyi (Lohmann) Kamptner
Authors: DE JONG Elisabeth , VAN RENS Lisette , WESTBROEK Peter , BOSCH Leendert
Published: March 2005

Emiliania huxleyi (Lohmann) Kamptner, belonging to a group of marine unicellular algae, the Coccolithophoridae, possesses a cell wall containing calcified structures called coccoliths. These coccoliths contain a water‐soluble acid polysaccharide. The polysaccharide contains ester sulphate and uronic acid groups and binds Ca2+ preferentially from a medium also containing Na+ and Mg2+. It is thought to perform a regulatory function in the calcification process. In the present paper we describe a series of studies in vivo intended to give a preliminary characterization of a number of metabolic steps leading to coccolith formation. In view of the putative role of the acid polysaccharide in coccolith synthesis, we have concentrated on the incorporation of polysaccharide precursors by calcifying cells. As a second approach we investigated the differences between calcifying cells and cells of the same species that have lost the capability to produce coccoliths (non‐calcifying cells).

Calcifying cells were offered radioactive calcium, bicarbonate, galactose and sulphate in the light. Most of the calcium label and a small part of the label of galactose and bicarbonate was incorporated into extracellular coccoliths. The label of galactose was detected in at least seven of the constituent monosaccharides of the coccolith‐associated polysaccharide. Radioactive sulphate was also incorporated into the extracellular coccolith polysaccharide. In contrast to the label of bicarbonate and galactose that of calcium reached a steady‐state concentration intracellularly within 2 h. A technique was developed for the isolation of intracellular CaCO3, presumably coccoliths in statu nascendi. About 10% of the label of bicarbonate and galactose was incorporated into an intracellular acid polysaccharide, resembling the coccolith‐associated macromolecule. Non‐calcifying cells fail to take up Ca2+ and galactose. We demonstrated that non‐calcifying cells produce an acid polysaccharide, resembling the coccolith‐associated macromolecule. The former polysaccharide is shed into the surrounding medium and can be labelled by offering the non‐calcifying cells radioactive bicarbonate.
NAD‐Dependent Formate Dehydrogenase from Methylotrophic Bacterium, Strain 1
Authors: EGOROV Aleksey M. , AVILOVA Tatyana V. , DIKOV Michael M. , POPOV Vladimir O. , RODIONOV Yuriy V. , BEREZIN Ilia V.
Published: March 2005

1. NAD‐dependent formate dehydrogenase was isolated from gram‐negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion‐exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40 %.

2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultra‐centrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2‐mercaptoethanol.

3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80000.

4. Amino acid composition and some other physico‐chemical properties of the enzyme were studied.

5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37°C).

6. Formate dehydrogenase was rapidly inactivated in the absence of –SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2‐mercaptoethanol or EDTA.
Cleavage of Elongation Factor G into Compact Domains
Authors: ALAKHOV Yuli B. , STENGREVICS Olaf A. , FILIMONOV Vladimir V. , VENYAMINOV Sergei Yu.
Published: March 2005

A limited trypsinolysis of native elongation factor G results in the formation of two large fragments resistant to further proteolysis. The fragments were isolated in homogeneous state in conditions when their native structure is retained. According to circular dichroism and scanning calorimetry data the formed fragments retain the stability and compact structure that they had in the whole protein.
Electron Paramagnetic Resonance of the Copper in Dopamine β‐Monooxygenase
Authors: SKOTLAND Tore , PETERSSON Leif , BÄCKSTRÖM Dan , LJONES Torbjørn , FLATMARK Torgeir , EHRENBERG Anders
Published: March 2005

The water‐soluble form of dopamine β‐monooxygenase from bovine adrenal medulla was studied. Addition of excess CuSO4 to purified enzyme preparations followed by extensive ultrafiltration against copper‐free buffer at pH 7.0, gave preparations with about four copper atoms per enzyme tetramer of Mr 290000. The enzyme‐bound copper was shown by the rapid‐freeze technique and electron paramagnetic resonance (EPR) to be reduced by ascorbate at a rate which was faster than the overall catalytic rate; about 10% of the copper was oxidized to Cu(II) during steady‐state catalysis in the presence of excess ascorbate. These results support an electron‐transfer function of the enzyme‐bound copper during catalysis and indicate that the reduction by ascorbate is not the rate‐limiting step.

The enzyme‐bound copper was rapidly chelated by EDTA at pH 7.0, and the apoenzyme thus obtained after dialysis revealed no EPR‐detectable copper. Addition of CuSO4 to the apoenzyme gave an EPR spectrum similar to that of the native enzyme, and the apoenzyme was fully reactivated by the optimal concentration of CuSO4 in less than 2 s.
Translation in vitro of Rat Brain mRNA Coding for a Variety of Tubulin Forms
Authors: GOZES Illana , de BAETSELIER Annie , LITTAUER Uriel Z.
Published: March 2005

Prenatal rat brain tubulin was resolved by isoelectric focusing into five to six components (isotubulins 1–6) while in mature brain nine distinct forms were evident (isotubulins 1–9). Tubulin, isolated from various brain regions, displayed different proportions of these nine isotubulins which may result from the heterogeneity in brain cell population. Mature brain mRNA, when translated in vitro, in the reticulocyte lysate cell‐free system, directed the synthesis of five tubulin forms, namely isotubulins 1, 3, 4 (or 5), 6 and 7. The mRNA species coding for isotubulins 3 and 7 could be partially separated on formamide/sucrose gradients, while in the absence of formamide the mRNA species directing the synthesis of isotubulins 1, 4 (or 5) and 6 showed differences in mobility. It therefore appears that brain mRNA may consist of five different species coding for distinct tubulin forms. Moreover, a marked age‐dependent enhancement in the relative translation of the mRNA coding for isotubulin 7, which is not apparent among the translation products directed by the prenatal mRNA, was detected. Our results indicate that some of the age‐dependent variations in tubulin microheterogeneity may be controlled at the mRNA level.
Locust Collagen: Morphological and Biochemical Characterization
Authors: ASHHURST Doreen E. , BAILEY Allen J.
Published: March 2005

Segment‐long‐spacing crystallites and reconstituted fibrils have been made from collagen extracted from the ejaculatory duct of the adult male locust, Locusta migratoria. These show the same banding pattern after positive staining as segment‐long‐spacing crystallites and fibrils made from mammalian type I collagen. Native fibrils show the same periodic pattern as type I fibrils, but it is not so distinct.

Biochemical analysis of pepsin‐digested locust collagen shows that there are two collagenous components. The α chains of the major component are similar to mammalian α1 (I) chains, except that the number of hydroxylysine residues is elevated and the CNBr peptides differ. There are no α2 chains; hence this locust collagen molecule is an α1 trimer. The second component, which is present in only minute quantities, may be a type IV basement membrane collagen.

It is concluded that the fibrous collagen molecule of the locust is very similar to that of mammalian type I trimers and to those of other invertebrates which have been examined.
Inhibitory Effects of ‘Cap’ Analogues on Globin mRNA and Encephalomyocarditis RNA Translation in a Reticulocyte Cell‐Free System
Authors: FRESNO Manuel , VÁZQUEZ David
Published: March 2005

The cap analogues 7‐methylguanosine 5′‐phosphate [m7G(5′)p], 7‐methylguanosine 5′‐triphos‐phate [m7G(5′)ppp] and 2′‐O‐methylguanosine 5′‐triphosphate [Gm(5′)ppp] inhibit the translation of capped globin mRNA and encephalomyocarditis (EMC) RNA (a naturally uncapped mRNA) in a reticulocyte cell‐free system. This inhibition occurs at the level of protein synthesis initiation and is of a competitive type since it can be overcome by increasing the mRNA concentration. However, the translation of globin mRNA is more sensitive to the inhibitory effects of the cap analogues m7G(5′)p and m7G(5′)ppp than translation of EMC RNA. The same spectra of specific inhibition is also observed with some other initiation inhibitors such as aurintricarboxylic acid, which inhibits mRNA binding, but not with pactamycin which does not affect mRNA interaction. A model is presented suggesting that this preferential inhibition by cap analogues could be explained mainly by the different affinities of globin mRNA and EMC RNA for the initiation complexes between 40‐S subunits and Met‐tRNAf. Moreover Gm(5′)ppp cannot be considered simply as a cap analogue since it also affects some step prior to mRNA binding.
Biosynthèse de cellulose bactérienne à partir de d‐glucose uniformément enrichi en 13C
Authors: GAGNAIRE Didier Y. , TARAVEL François R.
Published: March 2005

Biosynthesis of Bacterial Cellulose from d‐Glucose Uniformly Enriched in 13C

As a typical illustration of the use of a statistically‐enriched 13C precursor for nuclear‐magnetic‐resonance (NMR) studies of biosynthetic mechanisms, the following example is described.

A sample of cellulose was synthesized by Acetobacter xylinum from a 50–50 mixture of d‐glucose and statistically enriched d‐[13C]glucose (87%13C). Each carbon atom of the cellulose is equally enriched in 13C (35%), as seen from the NMR spectrum of the acetate. A precise analysis of this product was performed by chemical transformation into a small molecule (3‐O‐acetyl‐1,2;5,6‐di‐O‐isopropylidene‐α‐d‐glucofuranose) whose 1J(13C‐13C) coupling constants provide for a direct determination of the four populations of molecules with a 13C at the Q position (i= 2, 3, 4, 5) having as neighbours (Ci−1 and Ci+1)12C or 13C.

A general statistical treatment of such problems is proposed, with the determination of two kinds of parameters.

For two consecutive carbons, the terms Pi,i+1 give the probability of both carbons Ci and Ci+1 in the final product coming from the same precursor molecule. The experimental values are: P1,2= 0.89, P2,3= 0.60, P3,4= 0.26, P4,5= 0.95, and P5,6= 0.93.

For three consecutive carbons, in some favourable cases, a correction term Ci,i+2 is introduced, to explain experimental deviations of populations from those calculated with two independant probabilities Pi,i+1 and Pi+1,i+2 In the example described here, two terms can be determined: C1,3= 0.066, C2,3= 0.44.

Seven values of P and C are obtained in a single experiment. These values allow for a correct interpretation of other experiments that are described, such as biosynthesis where pure statistically 13C‐enriched glucose is used as precursor; experiments with specifically labelled glucose ([1‐13C] and [6‐13C]); and the global isotopic composition of all products, as measured by mass spectrometry.

The biosynthetic interpretation is done in terms of the following pathways: (i) direct incorporation, (ii) the pentose phosphate cycle (giving CO2, glyceraldehyde 3‐phosphate and glucose 6‐phosphate), (iii) the Entner‐Doudoroff pathway (giving pyruvate and glyceraldehyde 3‐phosphate), (iv) triose recombination (giving pyruvate and glucose 6‐phosphate).

The interpretation of the seven experimental terms P and C does not allow for constant percentages for these pathways during the biosynthesis, but indicates the following values for the whole process: (i) 23% of the glucose molecules in cellulose come from an unmodified molecule of glucose; (ii) 26% have been involved at least once in the pentose cycle, and 10 at least twice; (iii) 30% of the glucose molecules come from triose recombination.
Troponin C from Rabbit Slow Skeletal and Cardiac Muscle Is the Product of a Single Gene
Authors: WILKINSON J. Michael
Published: March 2005

Troponin C has been isolated from rabbit slow skeletal and cardiac muscle and the complete amino acid sequence of the slow muscle protein determined. Amino acid analysis and peptide mapping of the cardiac protein showed it to be very similar to, if not identical with, the slow muscle protein. This identity has been proved by the isolation and characterisation of tryptic peptides from the cardiac protein. It seems very likely that troponin C from these two tissues is the product of a single gene, in contrast to troponin I and troponin T which are the products of different genes. The amino acid sequence shows only one difference from that of bovine cardiac troponin C, the highly conservative replacement of an aspartic for a glutamic acid at position 115. No differences were found in the N‐terminal region where these proteins appear to have lost one of the Ca2+ binding sites found in fast skeletal muscle troponin C. The possible significance of this finding in relation to the binding of troponin C to the different types of troponin I is discussed.
The Interaction of Chromatin with Alkylating Agents
Authors: YERUSHALMI Aharon , YAGIL Gad
Published: March 2005

The reaction of L5178Y lymphoblast cell chromatin with the alkylating agent bis(2‐chloroethyl)‐methylamine has been studied as a function of time, pH and reagent concentration. The reaction with DNA of chromatin from which the proteins were dissociated, as well as with purified calf thymus DNA, was studied in parallel. The extent of alkylation of DNA in intact chromatin was 4–5 times as much as in parallel free DNA samples; up to 4% of nucleotide base pairs were substituted. The extent of monofunctional substitution of the proteins was similar, on a weight basis, to that of DNA. Chromatographic analysis of the depurinated products showed that in chromatin, as in DNA, position N‐7 of guanine is the major site of reaction. Up to 25% of the reaction products were guanines cross‐linked as bis(2‐guanin‐7‐yl‐ethyl)methylamine, indicating a considerable degree of DNA‐DNA cross linking. Column analysis shows that up to 40% of the nuclear proteins are cross‐linked to DNA at 10 mM bis(2‐chloroethyl)methylamine. The increased reactivity of intact chromatin is interpreted in terms of a conformational change in the position of the DNA bases when in the organized nucleohistone complex.
Sequence Organisation in Nuclear DNA from Physarum polycephalum
Authors: HARDMAN Norman , JACK Peter L. , FERGIE Roy C. , GERRIE Linda M.
Published: March 2005

Nuclear DNA from Physarum polycephalum is shown to contain three sequence components by reassociation kinetic analysis; a foldback component consisting of 6% of the DNA, a component with the properties of repetitive sequences comprising 31% of the DNA, and a majority component containing 63% of the DNA which reassociates with the kinetics characteristic of single‐copy sequences. The complement of repetitive sequences is comprised of about 80 families of repeated elements, each containing approximately 1800 repeats per family. On average, these sequences are 6.4% richer in guanine and cytosine than total Physarum nuclear DNA. The repetitive sequences within a single family appear not to be identical, since on denaturation and annealing they give rise to collections of heteroduplexes less stable than native DNA. It is calculated that these duplexes are about 10% mismatched on average. Hydroxyapatite binding of DNA fragments of different sizes containing reassociated repeated elements demonstrates that these sequences are interspersed with single‐copy sequences in a large portion of the Physarum genome. These observations are confirmed by direct examination of reassociated DNA using the electron microscope. In this manner it is shown that repetitive sequence elements possess a wide spectrum of lengths averaging 590 nucleotide residues, and they are separated by intervening segments of DNA about 930 residues in length.
Modification of Rat Hemopexin Properties upon Heme Binding
Authors: BERNARD Nicole , LOMBART Christian , WAKS Marcel
Published: March 2005

Rat hemopexin and its complex with heme were found to have the same Stokes' radius, 3.9 nm, as determined by gel filtration. Therefore no polymerisation occurs as a result of heme binding. The conformational parameters calculated from circular dichroism spectra indicate that hemopexin and its complex consist of 20%β sheet and mainly of disordered structure. No change of the secondary structure is therefore observed upon heme binding. Hemopexin reveals five bands by analytical electrofocusing with pI ranging from 5.5 to 5.95. This microheterogeneity is not due to sialic acid differences between the variants. Upon heme binding the pI of the variants decrease to lower values (from 4.8 to 5.25). This decrease in the pI value of hemopexin is thought to modify the tertiary structure through charge effects and may allow the binding of the heme‐hemopexin complex to the hepatocytes.
Escherichia coli Murein Transglycosylase
Authors: KUSSER Wolfgang , SCHWARZ Uli
Published: March 2005

Escherichia coli murein transglycosylase, a potential autolysin which splits the sugar chains of the murein sacculus, was rapidly purified from a crude cell extract by sequential chromatography on columns of blue Sepharose and poly(U)‐Sepharose. In accordance with the binding to blue Sepharose and poly(U)‐Sepharose, the transglycosylase is inhibited by Cibacron blue F3G‐A, the affinity ligand of blue Sepharose, and also by polynucleotides, the latter, however, with varying efficiency. Among the polynucleotides tested, single‐stranded DNA was found to be one of the most potent inhibitors.

When bound to a blue Sepharose column, transglycosylase could be displaced from the column with single‐stranded DNA. Taken together, these results point to a polynucleotide binding area on the transglycosylase molecule.

Some aspects of the blue Sepharose affinity chromatography and the possible biological significance of the transglycosylase are discussed.
Binding of Manno‐oligosaccharides to Concanavalin A
Authors: VAN LANDSCHOOT Anita , LOONTIENS Frank G. , DE BRUYNE Clement K.
Published: March 2005

The association constants for binding of methyl α‐d‐mannopyranoside (I), mannobiose (II) and mannotriose (III) to concanavalin A were determined in the temperature range 285–313 K by a substitution titration, using 4‐methylumbelliferyl α‐d‐mannopyranoside as a carbohydrate‐specific and fluorescent indicator. All binding equilibria are simple, but establish extremely slowly with II and III. At 298.3 K, K increases moderately from I to III: (6.4 ± 0.5) × 103, (1.2 ± 0.1) × 104 and (1.10 ± 0.05) × 105 M−1. For binding of I, II and III, the –ΔH° values are constant (36 ± 2 kJ mol−1) and equal to the average value (36.1 ± 0.6 kJ mol−1) obtained for the three corresponding 4‐methylumbelliferyl α‐d‐manno‐oligosaccharides [Van Landschoot, A., Loontiens, F. G., and De Bruyne, C. K. (1978) Eur. J. Biochem. 83, 277–285]. The data are interpreted as arising from specific binding to a single mannopyranosyl residue in (α 1 → 2)‐linked manno‐oligosaccharides.
Binding Kinetics of 4‐Methylumbelliferyl α‐Mannobioside to Concanavalin A by Fluorescence Stopped‐Flow Measurements
Authors: VAN LANDSCHOOT Anita , LOONTIENS Frank G. , CLEGG Robert M. , JOVIN Thomas M.
Published: March 2005

The stopped‐flow binding kinetics of concanavalin A with fluorescently labelled (α 1 → 2)‐linked manno‐oligosaccharides are characterized by signal changes which are monophasic for 4‐methyl‐umbelliferyl α‐d‐mannopyranoside (I), biphasic for 4‐methylumbelliferyl α‐mannobioside(II) and triphasic for 4‐methylumbelliferyl α‐mannotrioside (III). The results indicate that the carbohydrate‐binding site interacts specifically with a single mannopyranosyl group, resulting in as many possible complexes as there are mannopyranosyl groups in the oligosaccharide chain.

Pseudo‐first‐order kinetics with II and concanavalin A in the range 6–334 μM yield a faster time constant (110–37 ms) with a saturable amplitude and a slower one (3.9–0.29s) with an amplitude which reaches a peak at 30 μM. The concentration dependencies of the two apparent rate constants and the corresponding amplitudes are compatible with two equivalent mechanisms for which the reaction‐rate parameters are calculated. Each involves two complexes with spectroscopic parameters which are dependent upon the mechanism. The first mechanism consists of binding followed by an isomerisation, interpreted as sliding of the internal mannopyranosyl residue to bind preferentially via the terminal residue. In the second mechanism, these two binding modes would alternate; here, the binding via the terminal residue is preferred at equilibrium by a factor of 68; both association‐rate parameters are comparable and similar to those for I or simple glycosides. Consistent results were obtained by temperature‐jump relaxation kinetics.
Studies on Energy Supply for Genetic Processes
Authors: CHAUSTOVA Larisa P. , GRINIUS Leonas L. , GRINIUVIENĖ Bronė B. , JASAITIS Antanas A. , KADZIAUSKAS Jurgis P. , KIAUŠINYTĖ Rita J.
Published: March 2005

In this study the hypothesis considering the involvement of the transmembraneous electrochemical gradient of protons () in the transport of DNA through the membrane of Bacillus subtilis cell during the initial stages of genetic transformation was verified experimentally. The value of the membrane potential of 100–140 mV (minus inside) was calculated from the data of K+distribution across the valinomycin‐treated cell membrane. The treatment of cells with protono‐phoreous uncouplers of oxidative phosphorylation during the exposure of transforming DNA to competent cells led to inhibition of the initial stages of genetic transformation without a detectable effect on the intracellular ATP concentration. The effects of uncouplers on the genetic transformation correlated with their effects on the value of the membrane potential. The dissipation of by K+ and H+ fluxes catalyzed by a mixture of valinomycin and nigericin led to the inhibition of the intial stages of transformation. The inhibitory effect of valinomycin was potentiated by nigericin at low concentration and vice versa. The same efficiency of genetic transformation in the presence of either phosphate or arsenate was observed in a modified Spizizen's medium. Genetic transformation in arsenate‐containing medium preceded despite the decrease of the ATP level in the cell population. This pattern of inhibitory effects supports the concept of being involved in the process of DNA transport in the initial stages of genetic transformation.
Purification and Characterization of Multiplication‐Stimulating Activity
Authors: MOSES Alan C. , NISSLEY S. Peter , SHORT Patricia A. , RECHLER Matthew M. , PODSKALNY Judy M.
Published: March 2005

Multiplication‐stimulating activity (MSA) refers to a family of insulin‐like growth factors that have been purified from serum‐free medium conditioned by a Buffalo rat liver cell line (BRL‐3A). Using Dowex ion‐exchange chromatography, gel chromatography on Sephadex G‐75, and preparative disc acrylamide gel electrophoresis, several polypeptides with the full biological multiplication‐stimulating activity have been isolated. One of these polypeptides, designated MSA II‐1, previously has been used to study the relationship of the activity to the insulin‐like growth factors (somato‐medins) purified from human plasma. Polypeptide II‐1 is a single chain polypeptide of molecular weight 8700. Glycine is the COOH‐terminal amino acid. Edman degradation of carboxymethylated MSA II‐l did not reveal a free NH2‐terminus. A polypeptide of lower molecular weight than MSA II‐1 has also been purified. This polypeptide (MSA III‐2) has been shown to be more potent than MSA II‐1 in the rat‐liver‐membrane radioreceptor assay and in a competitive binding assay utilizing the rat‐serum somatomedin‐binding protein(s). The relationship of these various polypeptides has been investigated by gel filtration in guanidine hydrochloride and by acrylamide gel electrophoresis of the reduced and native polypeptides.
Interaction of the lamB Protein with the Peptidoglycan Layer in Escherichia coli K12
Authors: GABAY Joëlle , YASUNAKA Kakuko
Published: March 2005

In Escherichia coli K12 the product of gene lamB is an outer membrane protein involved in the transport of maltose and maltodextrins and serving as a receptor for several bacteriophages including λ. About 30 to 40% of this protein can be recovered associated to peptidoglycan when the cells are dissolved in sodium dodecyl sulfate in the presence of 2 mM Mg2+ ions. The bound protein can then be quantitatively eluted from peptidoglycan by incubating the complex in Triton X‐100 and EDTA, or sodium dodecyl sulfate and NaCl. The protein eluted in such ways is still totally active in its phage‐neutralizing activity. Two other outer membrane proteins known to behave similarly to the lamB protein are proteins Ia and Ib. However the binding of these proteins to peptidoglycan appears tighter, in several respects, than that of the lamB protein. The lamB protein may span the outer membrane since it appears to interact with the peptidoglycan on the inner side of this membrane while it is known to be accessible to both phages and antibodies at the cell surface.