bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunological and biosynthetic studies on the mammalian 2‐oxoglutarate dehydrogenase multienzyme complex
Authors: HUNTER Anne , LINDSAY John Gordon 
Published: March 2005

High‐titre, monospecific, polyclonal antisera have been raised against purified mitochondrial 2‐oxoglutarate dehydrogenase complex (OGDC) from ox heart and two of its three constituent enzymes, 2‐oxoglutarate dehydrogenase (E1) and lipoyl succinyltransferase (E2). These specific antisera have been employed to monitor molecular events in the biosynthesis, import and maturation of this multimeric assembly. Lipoamide dehydrogenase (E3) elicits a poor antibody response in comparison to the other polypeptides of the complex.In cultured pig kidney cells (PK‐15), incubated with [35S]methionine in the presence of uncouplers of oxidative phosphorylation, appearance of stable higher‐Mr forms of the individual enzymes can be detected by specific immunoprecipitation and fluorographic analysis. In the case of 2‐oxoglutarate dehydrogenase, E1, the initial cytoplasmic translation product has a subunit Mr value of 1500–3000 greater than in the mature enzyme while the precursor of the lipoyl succinyltransferase, E2, contains an additional sequence of Mr 6000–8000. Competition studies have revealed the immunological similarity of the precursor molecules to the native subunits.On removal of uncouplers, processing of accumulated precursors is rapidly initiated and is complete within 40 min. Interestingly, antiserum to native 2‐oxoglutarate dehydrogenase complex fails to recognise E2 precursor molecules (pre‐E2), which can be immunoprecipitated, however, by antibodies raised against the denatured E2 subunit. It is concluded that pre‐E2 is conformationally dissimilar to native E2, which exists normally as a highly ordered, multimolecular aggregate in the native complex.
The complete amino acid sequence of adenylate kinase from baker's yeast
Authors: TOMASSELLI Alfredo G. , MAST Edeltraud , JANES Wolfgang , SCHILTZ Emile 
Published: March 2005

The complete amino acid sequence of cytosolic adenylate kinase (MgATP + AMP ⇄ MgADP + ADP) from baker's yeast has been determined.

Tryptic and clostripaic cleavage of the protein yielded 27 and 10 fragments, respectively. They were sequenced with either a solid‐phase sequencer or a gas‐phase sequencer.
Alignment of the clostripaic fragments was deduced from the sequence of peptides obtained by endoproteinase Lys‐C and cyanogen bromide cleavages.
The N‐terminus is blocked by an acetyl group as shown by proton magnetic resonance.
Carboxypeptidase A digestion of the whole protein showed that the C‐terminal sequence is ‐Lys‐Asn, in agreement with the sequence of peptides from tryptic, clostripaic and 2‐iodosobenzoic acid cleavages.
The enzyme is a monomer of 220 amino acids with Mr 24077. Comparison of the sequence of the cytosolic adenylate kinases from yeast and pig shows 25% identity with highly conserved segments in the putative active‐site region of the enzyme. After position 111, however, there is an insertion of 32 residues in the yeast species, similar to the adenylate kinase and the GTP:AMP phosphotransferase from beef heart mitochondria.
Purification and characterization of calcitonin receptors in rat kidney membranes by covalent cross‐linking techniques
Authors: BOUIZAR Zhor , FOUCHEREAU‐PERON Martine , TABOULET Jacqueline , MOUKHTAR Mohsen S. , MILHAUD Gérard 
Published: March 2005

We have characterized the binding parameters of renal receptors (Scatchard analysis revealed the presence of two binding sites: site I, Ka1= 1.29×109 M−1, number of binding sites = 9.9 × 106/μg protein; site II, Ka2= 0.93 × 108M−1, number of binding sites = 4.27×108/μg protein) and studied the effect of solubilization. The high‐affinity sites are preserved during affinity chromatography and the process results in a 6080‐fold purification of those sites. The lower‐affinity sites are also preserved but the overall purification factor is about 40% lower than that obtained using molecular sieving. The purification of the renal calcitonin receptor by molecular sieving (Sephacryl S‐200) is accompanied by total loss of the high‐affinity site; however, the low‐affinity site is enriched over 1642‐fold. Binding parameters were obtained for the purified fractions. Synthetic salmon calcitonin was also bound to renal membranes using the bifunctional reagent disuccinimidyl suberate and photo affinity cross‐linking using hydroxysuccinimidyl azidobenzonate reagent. Cross‐linked receptor eluted in the same volume as solubilized membranes specifically binding salmon calcitonin (S‐200 chromatography).Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of purified fractions showed several protein bands with apparent molecular masses ranging from 18000 Da to 100000 Da in the presence or absence of a reducing agent (2‐mercaptoethanol). Autoradiography of polyacrylamide gels of cross‐linked calcitonin receptor showed only three protein bands specifically binding salmon calcitonin. Their molecular masses were 70000 Da, 40 000 Da and 33 000 Da respectively. The 40 000‐Da molecule represents a major band (47% total binding species). This suggests that these three proteins are the principal components of the calcitonin receptor and that S‐S bonds are not involved in the assembly of the receptor subunits.
Amino‐acid‐transfer reactions in isolated nuclei of Ehrlich ascites tumor cells
Authors: ROTHBARTH Karsten , WERNER Dieter 
Published: March 2005

Nuclear enzymatic activities incorporating amino acids into acid‐insoluble material were investigated with respect to their differentiation from protein biosynthesis, reaction optima, requisites and localization. The product of the reaction was analyzed with respect to its localization and nature.(1)The nuclear activities are not inhibited by a number of inhibitors for protein biosynthesis. (2) The reaction optima found are similar to those of other residual nuclear syntheses including the stringent dependence on ATP. (3) All naturally occurring amino acids are utilized with different efficiencies. Their incorporation is neither cooperative nor competitive which points to individual incorporation mechanisms. Aminoacylation of tRNA may be involved because the incorporation is RNase‐sensitive and aminoacylation of tRNA can be shown under the reaction conditions. (4) The enzymatic activities are exclusively nuclear. Significant activity with unchanged characteristics is released by sonication. (5) 70% of the radiolabel incorporated is exported across the nuclear envelope during the incubation. The residual 30% of the radiolabel is distributed without enrichment in any nuclear subfraction. (6) The products are exclusively of polypeptide nature. Since distinct nuclear proteins (e.g. histones) which are definitely preformed in the cytoplasm by protein biosynthesis become radiolabelled by the incorporation of radiolabelled amino acids, it is evident that the incorporation takes place at preformed polypeptides. This is unequivocally proven by the incorporation of radiolabelled amino acids into exogenous proteins by means of the solubilized nuclear activities.The results indicate that the nuclear activity under investigation reflects a nuclear modification system for polypeptides which may be of similar importance as other post‐translational modification systems.
Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells
Authors: RICARD Jacques , NOAT Georges 
Published: March 2005

A theory is proposed to explain the physical bases of the ionic control of the activity of an enzyme system, located on a plant cell surface, and probably involved in cell‐wall synthesis and extension. The model, which is based on various previously published experimental results, involves several assumptions: a cell‐wall pectin methyl esterase de‐esterifies pectins and thus creates the fixed negative charges of the cell wall; various enzymes incorporate uncharged carbohydrates in cell‐wall material; cell‐wall extension implies the sliding of cellulose microfibrils; the enzymes responsible for carbohydrate incorporation are activated by protons in the pH range 4–8 and have very similar pH dependencies: the cell‐wall pectin methyl esterase is inhibited by protons in the same pH range.The mathematical derivation of this model, written in the form of a hypercycle, indicates that it is equivalent to a set of two antagonistic enzyme reactions: an enzyme reaction conditioned by pectin methyl esterase which results in the increase of fixed charge density of the cell wall; a number of ‘growth enzymes’, which produce extension and building up of the cell wall and therefore a decrease of charge density. The mathematical study of this model shows it may display a very high co‐operativity of its response to slight changes of pH. This co‐operativity means that the cell wall charge density may dramatically increase or decrease, within a very narrow pH range. The steep response of this system appears to be the direct consequence of different pH sensitivities of pectin methyl esterase and of the other cell‐wall enzymes involved in cell growth. Calcium, which tightly binds to the cell wall, may diminish or even suppress this abrupt charge transition.This model suggests a novel theory of the ionic control of cell‐wall expansion. The very basis of this theory is the existence of an electrostatic potential difference, ΔΨ, between the inside and the outside of the cell wall. When this ΔΨ value is large, the local proton concentration is high. Therefore the enzymes involved in cell wall extension and building up are active, but pectin methyl esterase is not. Therefore, the cell wall extends and the charge density decreases. The ΔΨ value then declines, as well as the local proton concentration. Under these conditions, the pectin methyl esterase becomes activated, whereas the ‘growth enzymes’ are not. This activation of pectin methyl esterase restores the initial, or an even higher, electrostatic potential difference, which in turn results in a decrease of local pH.In the following papers the basic ideas of this model are subjected to experimental control.
Labeling of individual amino acid residues in the membrane‐embedded F0 part of the F1 F0 ATP synthase from Neurospora crassa
Authors: HOPPE Jürgen , GATTI Domenico , WEBER Hans , SEBALD Walter 
Published: March 2005

Three F0 subunits and the F1 subunit β of the ATP synthase from Neurospora crassa were labeled with the lipophilic photoactivatable reagent 3‐(trifluoromethyl)‐3‐(m‐[125I]iodophenyl)diazirine ([125I]TID). In the proteolipid subunit which was the most heavily labeled polypeptide labeling was confined to five residues at the NH2‐terminus and five residues at the C‐terminus of the protein. Labeling occurred at similar positions compared with the homologous protein (subunit c) in the ATP synthase from Escherichia coli, indicating a similar structure of the proteolipid subunits in their respective organisms.The inhibitors oligomycin and dicyclohexylcarbodiimide did not change the pattern of accessible surface residues in the proteolipid, suggesting that neither inhibitor induces gross conformational changes. However, in the presence of oligomycin, the extent of labeling in some residues was reduced. Apparently, these residues provide part of the binding site for the inhibitor. After reaction with dicyclohexylcarbodiimide an additional labeled amino acid was found at position 65 corresponding to the invariant carbodiimide‐binding glutamic acid. These results and previous observations indicate that the carboxyl side chain of Glu‐65 is located at the protein‐lipid interphase. The idea is discussed that proton translocation occurs at the interphase between different types if F0 subunits. Dicyclohexylcarbodiimide or oligomycin might disturb this essential interaction between the F0 subunits.
Differential protein synthesis in the induction of thyroid cell proliferation by thyrotropin, epidermal growth factor or serum
Authors: LAMY Françoise , ROGER Pierre P. , LECOCQ Raymond , DUMONT Jacques E. 
Published: March 2005

Protein synthesis in the G1 period of the cell cycle has been investigated using two‐dimensional gel electrophoresis in primary cultures of dog quiescent thyroid cells, incubated in defined medium and induced to proliferate by the combined action of thyrotropin (TSH), epidermal growth factor (EGF) and serum or by each of these agents, acting alone.1. The analysis of the proteins, pulse‐labeled for 3 h with [35S]methionine, in quiescent cells deprived of serum and in cells that had been stimulated for various periods of time by the addition of TSH, EGF and serum showed maximal modifications before entry into S phase: the labeling of at least ten proteins was enhanced while that of at least six proteins was decreased. The synthesis of one of these proteins (protein 1; Mr∼ 81000) was maximal 9–12 h after stimulation by the proliferative agents but began to decrease at 15–18 h and was still decreased at 29–32 h.2. The study of the effect of each of the proliferation agents alone on the labeling of these sixteen proteins showed that TSH specifically stimulated the labeling of eight polypeptides (proteins 2–9) and that, in contrast, EGF and serum specifically increased the labeling of two other proteins (proteins 1 and 10). The labeling of one protein was decreased by each of the different agents (protein 6′) while TSH specifically decreased the labeling of four polypeptides (proteins 1′– 4′) and increased the labeling of one polypeptide (protein 5′) whose synthesis was decreased by EGF and serum.3. The specific effect of TSH on one protein labeling (protein 7; Mr∼ 39000) was potentiated by EGF and serum while the specific effect of EGF and serum on another protein labeling (protein 1) was potentiated by TSH. There is thus a correlation between the level of synthesis of these two proteins and the proliferative state of the cells, which is much greater when the stimulating agents are acting together.4. The induction of protein 1 synthesis by EGF was no longer observed when the cells were no longer proliferating. In the same way, TSH no longer stimulated the synthesis of protein 7 in thyroid cells at confluence.In conclusion, the present study has identified some proteins (proteins 1 and 7) which, as judged by the peculiar stimulation and the kinetics of their synthesis, could be part of the final key events triggering DNA replication in thyroid cells. Our findings are discussed within the framework of the previously described coexistence of cyclic‐AMP‐dependent (stimulated by TSH) and cyclic‐AMP‐independent (stimulated by serum or EGF) regulatory pathways leading to the initiation of DNA synthesis.
Differential effects of defined chemical modifications on antigenic and pharmacological activities of scorpion α and β toxins
Authors: AYEB Mohamed EL , DARBON Hervé , BAHRAOUI El Mostafa , VARGAS Orietta , ROCHAT Hervé 
Published: March 2005

Specific chemical modifications of scorpion α and β toxins have been used to study the involvement of particular residues in both the pharmacological and the antigenic sites of these toxins. Modification by 1,2‐cyclohexanedione of arginine‐27 of a β toxin, Centruroides suffusus suffusus toxin II, drastically decrease the antigenic activity without any influence on the pharmacological activity. Conversely, modification by the same reagent of arginine‐2 of an α toxin, Androctonus australis Hector toxin III, led to a 100‐times less pharmacologically potent derivative and did not induce a significant loss of antigenic activity. Excision of the N‐terminal pentapeptide of another α toxin, Buthus occitanus mardochei toxin III, by pepsin digestion led to a non‐toxic derivative retaining full antigenic activity. Thus, the N‐terminal part of the conserved hydrophobic surface of the toxin is highly implicated in the pharmacological activity, whereas the region of arginine‐27, located in the α helix situated on the back surface, opposite the conserved hydrophobic region, is fully implicated in the antigenic activity and is far from the pharmacological site.These results are good arguments in favor of the idea that in scorpion toxins the surfaces implicated in the pharmacological and the antigenic activities do not overlap. Since the antigenic sites are present in highly variable sequence the development of an efficient polyvalent serotherapy is questionable.
Temperature dependence of dihydropteridine reductase activity
Authors: RANDLES David 
Published: March 2005

The kinetic parameters Km and kcat of dihydropteridine reductase with a mixture of 6R and 6S quinonoid 7,8(6H)‐dihydrobiopterin were determined at several temperatures in the range 20–37°C. Both Km and kcat increased with temperature. Thermodynamic activation parameters were calculated and compared with those for the non‐enzymic reduction of quinonoid 7,8(6H)‐dihydrobiopterin by NADH. The temperature coefficients of the enzyme catalysed and uncatalysed reactions are 3.3 and 1.67 respectively.The results are consistent with an ordered bi‐bi enzyme mechanism, in which the rate‐determining step is an isomerisation of the ternary complex. This isomerisation involves a positive entropy of activation, which overcomes an enthalpy of activation that is significantly higher for the enzymic than for the non‐enzymic reaction.
Stimulation of the adenylate cyclase activity of rabbit bone marrow immature erythroblasts by erythropoietin and haemin
Authors: BONANOU‐TZEDAKI Sophia A. , SETCHENSKA Milka S. , ARNSTEIN Henry R. V. 
Published: March 2005

The effect of two agents of erythroid cell differentiation on the adenylate cyclase activity of fractionated rabbit bone marrow erythroblasts has been investigated.1. Addition of 0.2 U/ml erythropoietin to cell cultures causes a transient increase in the activity of plasma membrane adenylate cyclase, which is maximal by 20 min and disappears within 4 h. The magnitude of the response to hormonal stimulation depends on the stage of erythroid cell development and is greater in the more immature cells.2. Addition of 50 μM haemin to cultures of erythroblasts also causes an increase in the activity of adenylate cyclase, which differs from the effect of erythropoietin in kinetics and specificity of target cells. With immature cells the haemin‐induced stimulation starts after the first hour and continues to increase up to 20 h of culture.3. Erythropoietin but not haemin can stimulate the basal activity of adenylate cyclase in an in vitro assay containing plasma membranes of immature erythroid cells. The degree of activation depends on the concentration of erythropoietin and is maximal with 0.2–0.5 U/ml hormone (5–12 nM). In the presence of guanine nucleotides the activation of adenylate cyclase by erythropoietin is increased further but the effect is not additive. With respect to the basal and the guanine‐nucleotide‐stimulated activities of adenylate cyclase erythropoietin acts differently from the β‐agonist l‐isoprenaline. The in vitro effect of erythropoietin is abolished by the β‐thio analogue of GDP, GDP[βS], and extensive washing of membranes makes hormone action GTP‐dependent.4. The stimulation of adenylate cyclase by the addition of erythropoietin to the reaction mixture is inversely related to the extent of previous hormonal stimulation of the cells from which the membranes were prepared. This loss of hormonal responsiveness is due to desensitization or receptor down‐regulation and persists for up to 20 h.5. We conclude that in immature erythroblasts erythropoietin acts via a receptor and a guanine nucleotide‐binding protein with high affinity for GTP (EC50 < 10 nM), whereas haemin appears to activate adenylate cyclase indirectly, as a consequence of progressive perturbations of the plasma membrane.
Binding of ligands to horse liver alcohol dehydrogenase lacking zinc ions at the active sites
Authors: KOVÁŇ Jan , MATYSKA Luděk , ZEPPEZAUER Michael , MARET Wolfgang 
Published: March 2005

1. The zinc‐deficient enzyme binds the fluorescence probes for the enzyme substrate pocket (auramine O, 13‐ethylberberine, chlorprothixene and acridine orange) more tightly than the native enzyme, whereas 1‐anilinonaphthalene 8‐sulphonic acid is bound with comparable affinity.2. The use of fluorescence probes as reporter ligands revealed that the formation of binary complexes between the zinc‐deficient enzyme and aldehydes is possible (as with the native enzyme) and confirmed an increased affinity of coenzymes to the modified enzyme.3. The absence of catalytic zinc ions brings about a loss of the essential stabilization effect in simultaneous NADH and aldehyde binding to liver alcohol dehydrogenase.4. 2,2′‐Bipyridine, which chelates the active‐site zinc ion in the native enzyme, is bound rather loosely to the same site as aldehydes, auramine O and ethylberberine in the case of the zinc‐depleted enzyme.5. The stopped‐flow measurements showed that the pH dependence of auramine O and ethylberberine binding to native and zinc‐depleted enzyme is essentially similar. These data are compatible with the presence of ionizable groups in the surroundings of the bound probes. This group might be either His‐67, bound to the zinc ion, or the zinc‐liganding water molecule in the case of the native enzyme (pK close to 9), or the free His‐67 residue in the case of the zinc‐deficient enzyme (pK about 8).
Studies on polypeptide composition, hydrolytic activity and proton conduction of mitochondrial F0F1H+ATPase in regenerating rat liver
Authors: BUCKLE Malcolm , GUERRIERI Ferruccio , PAZIENZA Angela , PAPA Sergio 
Published: March 2005

A study of the F0F1ATPase complex of mitochondria isolated from regenerating rat liver following partial (70%) hepatectomy is presented. As we have previously reported, ATPase activity in submitochondrial particles prepared from regenerating rat liver 24 h following partial hepatectomy was depressed by 75% with respect to controls (submitochondrial particles from sham‐operated animals). Polyacrylamide gel electrophoresis and immunodecoration using an antibody raised against isolated bovine heart F1sector of the F0F1ATPase indicated a substantial decrease in F1content in the mitochondrial membrane from regenerating rat liver. Proton conduction by the F0F1ATPase complex was studied by following the anaerobic relaxation of the transmembrane proton gradient (ΔμH+) generated by succinate‐driven respiration. In control rat‐liver submitochondrial particles containing the F0F1moiety of the ATPase complex, anaerobic relaxation of ΔμH+showed biphasic kinetics, whilst the same process in particles derived from regenerating rat liver exhibited monophasic kinetics and was significantly more rapid. Oligomycin andN,N‐dicyclohexyl carbodiimide [(cHxN)2C] inhibited proton conductance by the F1‐F0ATPase complex in submitochondrial particles from both control and regenerating rat liver. Binding of [14C](cHxN)2C and immunodecoration using an antibody raised against bovine heart oligomycin‐sensitivity‐conferring protein (OSCP) indicated no difference in the content of either the (cHxN)2C binding protein or OSCP between control and regenerating rat‐liver mitochondrial membranes.The results reported show that the structural and functional integrity of the F0‐F1ATPase of rat liver is severely perturbed during regeneration.