bluuebunny/crossref_metadata_2025_split huggingface dataset:
Specificity and Interactions at the Cationic Sites of the Axonal (Na+, K+)‐Activated Adenosinetriphosphatase
Authors: ROSSI Bernard , GACHE Christian , LAZDUNSKI Michel 
Published: March 2005

Specificity at the Mg2+ site has been investigated with P‐nitrophenylphosphate and dinitrophenylphosphate as substrates. The interaction parameters between the enzyme and its effectors at the Mg2+ site have been determined. Ni2+ and Cd2+ can be added to the series of known Mg2+ analogues. Tb3+ also inhibits the enzymatic activity and appears as a promising tool for further studies since it interacts with the (Na+,K+)‐ATPase in a relatively high affinity process.
Interactions between the Mg2+ and Na+ sites have been studied by the P‐nitrophenylphosphatase activity. Na+ decreases the apparent affinity of the enzyme for Mg2+ without strong modification of the cooperativity index.
Kinetic parameters for ATPase, P‐nitrophenylphosphatase and dinitrophenylphosphatase activations by K+ analogues have been determined and the pH dependence of the apparent affinity studied.
Heterotropic interactions between K+ and Na+ sites have been studied with K+, Tl+ and NH+4. Three substrates have been used. Firstly, with dinitrophenylphosphate, increasing Na+ concentration changes the positive cooperativity for K+ to a negative one, then back to a positive cooperativity. Simultaneously, the apparent affinity increases then decreases. Secondly, with ATP, an increase of the Na+ concentration decreases the apparent affinity for K+ and changes the cooperativity from a negative to a positive one. Thirdly, with P‐nitrophenylphosphate, interactions resemble those obtained with dinitrophenylphosphate. When added at concentrations which stimulate the activity, ATP imposes its type of heterotropic interactions between Na+ and K+ sites.
Calcium Metabolism and Enzyme Secretion in Guinea Pig Pancreas
Authors: LUCAS Miguel , SCHMID Gerhard , KROMAS Rosa , LÖFFLER Georg 
Published: March 2005

In isolated pancreatic acinar cells from the guinea pig stimulation of enzyme secretion by carbamoylcholine is slightly diminished in the absence of extracellular Ca. LaCl3in a concentration, which does not influence the secretory response to carbamoylcholine, nearly completely abolishes45Ca uptake by cells, indicating that Ca uptake is not necessary for secretion.In cells preloaded with45CaCl2, addition of carbamoylcholine leads to an immediate release of45Ca, Which can be blocked by atropine or 8‐(N,N‐diethylamino)‐octyl 3,4,5‐trimethoxybenzoate and is not influenced by LaCl3in concentrations, which do not inhibit secretion.A similar release of45CaCl2from preloaded cells is obtained by addition of the mitochondrial inhibitors antimycin A, carbonylcyanideptrifluoromethoxyphenylhydrazone (FCCP), and oligomycin. Possibly due to markedly diminished ATP levels, neither antimycin A nor FCCP act as secretagogues, both compounds being inhibitors of secretion. Oligomycin, which decreases ATP levels only to 20%, stimulates secretion.Mitochondria and microsomes from pancreatic tissue are able to accumulate45Ca. Mitochondrial45Ca uptake can be driven by ATP or active respiration and is inhibited by NaN3, oligomycin, antimycin A or FCCP. Microsomal45Ca uptake is ATP‐dependent. NaN3and mitochondrial inhibitors have no influence on microsomal45Ca uptake, which is stimulated several‐fold by oxalate.The results support the assumption, that in the guinea pig pancreas Ca mobilization from intracellular stores is necessary to initiate secretion. Due to their ability for an active accumulation of45Ca both mitochondria and microsomes could serve as intracellular calcium stores.
Regulation of Turnover GTPase Activity of Elongation Factor G: the 30‐S‐Coupled and 30‐S‐Uncoupled Reactions
Authors: SANDER Gernot , PARLATO Giuseppe , CRECHET Jean‐Bernard , NAGEL Klaus , PARMEGGIANI Andrea 
Published: March 2005

The elongation factor G (EF‐G) GTPase activity exists in two differently regulated forms: one, dependent on both ribosomal subunits, is primarily expressed at high monovalent cation concentration; the other, dependent on the presence of the 50‐S subunit alone, becomes predominant at low concentrations of monovalent cations (30‐S‐uncoupled EF‐G GTPase). Both reactions show comparable Km values and responses to changes in [Mg2+] and pH, the 30‐S subunit increasing the affinity of EF‐G for the ribosome at both high and low salt concentration. At saturating [EF‐G], this causes an inhibition by the 30‐S subunit at low salt, whereas at higher ionic strengths the 30‐S subunit favors the turnover GTPase activity, apparently by counteracting the weakening effect of monovalent cations. By contrast, the EF‐G GTPase activity uncoupled from the 30‐S subunit steadily decreases with rising salt concentration.The action of the 30‐S subunit follows the predictions of the polyelectrolyte theory [Douzou, P. and Maurel, P. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 1013]. As the pH is increased, the bell‐shaped K+ curve normally found with the 50‐S plus 30‐S subunits approaches the steadily descending curve observed with the 50‐S subunit alone, implicating an accumulation of negative charges in the vicinity of the binding site for the factor and/or the catalytic center. Thus these results suggest that the 30‐S subunit may expose a critical region of the ribosomal RNA, leading to a greater affinity for EF‐G.The 30‐S subunit exerts its effect at temperatures ranging from 20–65°C, and between pH 5 and 10.2. The pH optimum for association of the subunits and GTPase activity was the same (pH 8). Low salt concentration encourages formation of 70‐S ribosomes, while 50‐S and 30‐S subunits alone form multimers.NH4+ was the most efficient monovalent cation in 30‐S‐coupled EF‐G GTPase activity, followed by K+, Cs+, Li+ and Na+. The order of stimulation by divalent cations was Ba2+ > Sr2+ > Mg2−≥ Ca2+ > Mn2+.Treatment with the polyamine, spermidine, renders the GTPase activity dependent on both ribosomal subunits, insensitive to changes in monovalent cation concentration while suppressing the 30‐S‐uncoupled activity. This may represent a vital function in suboptimal growth conditions, when cells are unable to maintain an even intracellular K+ concentration.
Insulin‐Like Growth Factors I and II: Some Biological Actions and Receptor Binding Characteristics of Two Purified Constituents of Nonsuppressible Insulin‐Like Activity of Human Serum
Authors: ZAPF Jürgen , SCHOENLE Eugen , FROESCH E. Rudolf 
Published: March 2005

The insulin‐like growth factors I and II, two purified constituents of nonsuppressible insulinlike activity opf human serum, have nbeen compared with regard to some bniological actions in vitro and some receptor‐binding characteristics.

In the presence of an excess of insulin‐antibodies the insulin‐like growth factors I and II (a) stimulate the net gas exchange in rat adipose tissue (fat pad assay) to a similar extent; (b) enhance 3‐O‐methylglucose transport, glucose oxidation and lipogenesis from glucose in rat adipocytes, with slight but significant differences in potency; (c) inhibit lipolysis in adipose tissue from fastedrefed rats, again with slight potency differences.
In adipose toissue and adipocytes the biological activity of insulin‐like growth factors I and II is between 1/35 and 1/125 of that of insulin on a molar basis.
Insulin‐like growth factors I and II are equally potent mitogens (stimulation of DNA‐synthesis and cell multiplication in cultured chick embryo fibroblasts).
Insulin‐like growth factors I and II stimulate 35SO42‐ incorporation into rat costal cartilage with minor differences in potency.
In rat adipocytes, insulin‐like growth factors I and II compete with insulin for binding to thje insulin receptor. However, their potencies in ‘displacing’125I‐labeled insulin are between 75 (for factor II) and 290 times (for factor I) lower than that of insulin. In addition, the factors I and II bind to binding sites specific for the factors and for which insulin does not compete. Considerable differences between factors I and II exist regarding their specific binding and their potencies in competing for binding of labeled factors I and II.
In chick embryo fibroblasts and isolated chick embryo chondrocytes the insulin‐like growth factors I and II compete more or less equally for binding to a binding site specific for the factors and which appears to mediate their effects on cell growth and sulfation.
Indsulin‐like growth factors I and II bind specifically to a partially purified carrier protein of human serum. Pronounced differences in specific binding and their potencies of competition are observed.

Thus, the insulin‐like growth factors I and II, two structurally closely related polypeptides, display more or less similar activities in various biological systems in vitro. In all of these, their biological potency can be reasonably correlated with their potency of competition either for the insulin receptor (fat cells) or for a specific binding site (fibroblasts and cyhondrocytes). In some of the radioactive ligand systems, where the biological function of binding sites for insulin‐like growth factor is still unknown (such bindings sites are found in fat cells and on the serum carrier protein), clear cut differences between factors I and II are apparent.
A Spectroscopic Study of Nitric‐Oxide‐Treated Ceruloplasmin
Authors: VAN LEEUWEN François X. R. , VAN GELDER Bob F. 
Published: March 2005

An analysis of the absorbance difference (oxidized minus reduced) and circular dichroic (CD) spectrum of ceruloplasmin in Gaussian curves is presented. Above 330 nm, both spectra are composed of the same set of six Gaussian bands, Attributed to typr‐1 copper. An Additional band in the absorbance difference spectrum at 320 nm is ascribed to typr‐3 copper.The time dependence of thje effect of nitric oxide on the absorbance difference, circular dichroism and electron paramagnetic resonance (EPR) spectrum of ceruloplasmin has been studied.Upon incubation with nitric oxide for 5 min, the CD spectrum of ceruloplasmin shows a slight decrease of the ellipticity around 600 nm, and a marked decrease at 456 nm, indicative of the non‐equivalence of the type‐1 coppers in ceruloplasmin, Longer incubation (2 h) with nitric oxide leads to a decrease of the CD and absorbance spectrum of the ceruloplasmin‐NO compound, due to reduction o0f the type‐1 copper.Upon prolonged incubation with NO, the type‐2 copper EPR spectrum (93 K) changes and a distinct superhyperfine pattern appears in the g, suggesting an interaction of the type‐2 copper with nitrogen atoms from the protein moiety.At 15 K the EPR spectrum of oxidized ceruloplasmin exhibits a small signal at g=4. Upon incubsation with nitric oxide this signal increases in intensity, and shows a well‐resolved hyperfine pattern of seven lines. In addintion ot hte type‐2 copper signal, a broad signal is observed at g=2. It is proposed that these signals in NO‐treated ceruloplasmin stem from magnetically coupled type‐3 coppers.
Effect of pH on the Process of Ternary‐Complex Interconversion in the liver‐Alcohol‐Dehydrogenase Reaction
Authors: KVASSMAN Jan , PETTERSSON Gösta 
Published: March 2005

Kinetic relationships referring to multiple‐turnover conditions have been derivedf for the slowest exponential transient appearing in two‐substrate enzyme reactions proceeding by an ordered ternary‐complex mechanism. The validity of these and previously derived theoretical reltionships for his mechanism has been tested by application to the liver alcohol dehydrogenase reaction.
All essential features of the transient‐state kinetics of the alcohol oxidation by NAD− in the liver alcohol dehydrogenase system can be qulitatively and quantitativley explained in view of the compulsory‐order mechanism in the proposed scheme. There is no kinetic evidence for any half‐of‐the‐site reactivity of the enzyme. A consistent set of rate constants is reported for the enzymic oxidation of benzyl alcohol at pH 8.75.
Transient‐state rate parameters for benzyl alcohol/benzaldehyde catalysis by liver alcohol dehydrogenase have been dtermined at different pH. The interpretation of usch rate parameters is critically discussed with reference to their informative value for the purpose of determination of rate constants (k and k′) for the process of ternary‐complex intercovnersion in th proposed scheme. It is concluded that the apparent rate constant (k′) for hydride transfer form benzyl alcohol to NAD+ is dependent on a proton dissociation step with a PKa of 6.4, wherase the rate constant (k) for hydride transfer from NADH to benzaldehyde exhibits no corresponding dependence on proton association.
The asymmetric pH dependence of the forward and reverse rat of ternary‐complex inter‐conversion during liver alcohol dehydrogenase catalysis appears to reflect an obligaytory step of alcohol/alcoholate ion equlibratio occurring at the ternary‐complex level. It is suggested that the observed pKa 6.4 dependence of the transient rat of alcohol oxidation can be attributed to a coupled acid‐base system involving minimally the enzyme‐bound alcohol and the protein residues Ser‐48 and His‐51.
d‐Glucose Transport in Trypanosoma brucei
Authors: GRUENBERG Jean , SHARMA Pukh Raj , DESHUSSES Jacques 
Published: March 2005

d‐Glucose and 2‐deoxyglucose enter the ‘long‐slender’ bloodstream from of Trypanosoma brucei only by means of a carrier‐mediated process; no free diffusion can be observed. Permeation is not energy‐dependent. The uptake is driven by the downhill concentration gradient of free substrate. The latter is maintained by the continual removal of sugar, due to the extremely high activities of the glycolytic enzymes. The permeation process is the rate‐limiting step of glucose consumption, because permeation proceeds at a rate slower than metabolism.The inhibition of sugar uptake by glycerol was tested. Interactions at the carrier site can be ruled out since glucose and its 2‐deoxy analog exhibit different inhibition kinetics.
Influence of Mitochondrial Radical Formation on Energy‐Linked Respiration
Authors: NOHL Hans , BREUNINGER Volkert , HEGNER Dietmar 
Published: March 2005

The earlier observation that aging promotes peroxidation of lipids within inner mitochondrial membranes by a radical‐generating mechanism has provided a model for studying the relationship of membrane alterations to energy‐linked respiration.
Electron transport was impaired as a function of age only in the presence of substrates oxidized within the inner mitochondrial membrane. Respiratory control ratios and P:O ratios decreased regardless of substrate identity.
Arrhenius kinetic data revealed age‐dependent shifts of temperature breaks only for membrane‐bound enzymes including oligomycin‐sensitive ATPase. Specific activities decreased for all enzymes studied except cytochrome oxidase and the glutamate oxidase system.
Molecular motion data of spin‐labelled fatty acid analogues associated with phospholipid bilayer from inner mitochondrial membranes exhibited a decrease in lipid fluidity in membranes from aged rats.
The exposure of isolated mitochondria to a O2−‐radical generating system brought about identical changes in energy‐linked respiration and oxidative phosphorylation as did aging in the animal. The effect was completely inhibited only in the presence of both superoxide dismutase and catalase, indicating the participation of O2−‐radicals and H2O2 in causing the observed structural and functional changes.
The results support the concept of an active mechanism in mitochondria of living tissues which can contribute to pathophysiological processes by a sequential reaction involving radical‐induced changes in the hydrophobic bonding of lipid‐dependent enzyme systems.
Preferential Charging of tRNAMetf in Escherichia coli K12
Authors: RON Eliora Z. , FALK Avshalom , HELBERG Dvora , HOROWITZ Sarah , ZEEVI Menachem 
Published: March 2005

The charging of tRNAMetm and in vitro and in vitro and initaition of polysomes during methionine limitation were studied in two strains of Escherichia coli K12.In the wild‐type strain the distribution of polysomes as well as teh kinetic parameters of methionyl‐tRNA synthetase indicate preferential acylation of tRNAMetf. This preferential charging of tRNAMetf does not take place in a mutant strain which is also defective in initiation of polysomes during methionine limitation.
Mutants (ompA) Affecting a Major Outer Membrane Protein of Escherichia coli K12
Authors: HENNING Ulf , SONNTAG Ingeborg , HINDENNACH Ingrid 
Published: March 2005

Seventy independent mutants have been analyzed affecting a major protein, polypeptide II*, of the outer cell envelope membrane from Escherichia coli K12. They were classified as nonsense mutants of the amber type (20%), mutants most likely of the missense type possessing the prtein at normal concentrations (9%), and mutants either missing the protein or harboring it at much reduced concentrations for unknown reasons (71%). Forty of the mutants were analyzed genetically and all were found to map at or near ompA, the structural gene for protein II*.Two‐dimensional electrophoretic analyses of envelopes from such mutants revealed an unusual heterogeneity of the protein which on such patterns appeared as at least 12 well separated spots, and the majority of these is due to artifacts of the method but apparently specific for this protein. In no case was a polypeptide fragment found in envelopes from the nonsense mutants.The results are discussed regarding two different phages which use the protein as a receptor and concerning the biosynthetic incorporation of the protein into the outer membrane.
Difference in the Effect of Glucagon and Starvation upon l‐Type Pyruvate Kinase from Rat Liver
Authors: VAN BERKEL Theo J. C. , KRUIJT Johan K. , VAN DEN BERG Gerard B. , KOSTER Johan F. 
Published: March 2005

The mechanism of the glucagon action upon pyruvate kinase in isolated hepatocytes from fed or starved rats has been investigated. Glucagon incubation of hepatocytes increases the reaction responsible for pyruvate kinase inactivation. This inactivation reaction is completely dependent upon the presence of adenosine 3′: 5′‐monophosphate (cAMP), (half maximal activation at 0.1 μM cAMP) with cells incubated without glucagon, while with cells incubated with glucagon cAMP addition only has a marginal effect. It is concluded that glucagon acts by activation of the protein kinase responsible for pyruvate kinase inactivation.
Glucagon addition to isolated hepatocytes from fed and starved rats causes a similar decrease in pyruvate kinase activity of 40.7 ± 2.5% and 36.6 ± 3.6%, respectively, when measured at suboptimal phosphoenolpyruvate concentrations. Although optimal concentrations. of glucagon (1 μM) are used during the cell incubations, the pyruvate kinase activity can be inactivated further in vitro by incubation under conditions for optimal protein kinase activity. From the kinetic curves for the substrate phosphoenolpyruvate, obtained before and after further incubation in vitro, it can be concluded that pyruvate kinase by its presence inside the cell is protected against a complete inactivation by glucagon.
The difference in apparent affinity of pyruvate kinase for phosphoenolpyruvate from glucagonincubated cells versus cells incubated without glucagon disappears after further incubation of the supernatants of these cells in vitro under optimal conditions for protein kinase activity (plus 10 μM cAMP) and an enzyme with a low affinity for phosphoenolpyruvate is obtained (K0.5= 4.4 mM). These results indicate that the glucagon‐induced change in kinetic properties of pyruvate kinase can be adequately explained by an increased phosphorylation state of pyruvate kinase.
The difference in apparent affinity of pyruvate kinase for phosphoenolpyruvate from fed liver versus starved liver (P < 0.001) still persists after further incubation of the liver supernatants under optimal conditions for protein kinase activity in vitro (P < 0.001). For the completely inactivated enzyme from livers of fed and starved rats K0.5 values for phosphoenolpyruvate of respectively 2.8 and 4.4 mM are obtained. These results indicate that the nature of the effect of starvation differs from that of glucagon. It is suggested that the effect of starvation might be caused by partial proteolysis of the enzyme or oxidation of essential thiol groups.
Complexity of Polysomal Polyadenylated RNA in Undifferentiated and Differentiated Neuroblastoma Cells
Authors: FELSANI Armando , BERTHELOT Francis , GROS François , CROIZAT Bernard 
Published: March 2005

Using cDNA probes, we have analysed the sequence complexity and the frequency distribution of the polysomal poly(A)‐containing RNA from neuroblastoma cells at two different developmental states: either as round, immature neuroblasts, or as differentiated cells exhibiting the morphological properties of mature neurons. The total complexities measured for mRNA from undifferentiated and differentiated cells are identical and correspond to approximately 7000 average‐sized sequences of 1750 nucleotides distributed in the same three abundance classes.We have determined the homology between the mRNA populations corresponding to the two developmental states by heterologous cross‐hybridization: all the sequences from differentiated cells are present in the polysomes of undifferentiated cells. Conversely, the mRNA from differentiated cells fails to hybridize with about 15% of hybridizable cDNA corresponding to undifferentiated cells. This difference probably results from the disappearance of some mRNA species and may be related to the terminal differentiation of neuroblastoma cells.
Cell‐Free Transcription and Translation of Total Spinach Chloroplast DNA
Authors: BOTTOMLEY Warwick , WHITFELD Paul R. 
Published: March 2005

A highly efficient cell‐free Escherichia coli system for the coupled transcription/translation of chloroplast DNA is described. Evidence for the synthesis of more than 20 discrete size classes of [35S]‐methionine‐labelled polypeptides has been obtained by dodecylsulphate/polyacrylamide gel electrophoresis of the products formed in a reaction programmed with spinach chloroplast DNA. One of the major radioactive products co‐electrophoresed with the large subunit (Mr 55000) of ribulosebisphosphate carboxylase (fraction 1 protein). Its identity as the large subunit was established by comparing its partial proteolytic digestion products with those of purified large subunit protein. Because the level of radioactivity in many of the other polypeptides synthesized in vitro is so high, their identification likewise should be possible by comparing them with known chloroplast proteins.When spinach chloroplast DNA that had been digested with certain restriction endonucleases was used to programme the transcription/translation system, some of the polypeptides, which were observed using undigested DNA, were not detectable among the products. These results are discussed with regard to the distribution of restriction enzyme sites in the operons for some of the polypeptides, and with regard to the information they can provide on the location of genes on a restriction enzyme cleavage map of chloroplast DNA.