bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of the ADP/ATP Translocator from Beef Heart Mitochondria as the Bongkrekate‐Protein Complex
Authors: AQUILA Heinrich , EIERMANN Wolfgang , BABEL Wilfried , KLINGENBERG Martin
Published: March 2005

The isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekate‐protein complex is described, using hydroxyapatite chromatography and gel filtration in Triton X‐100 solution.

The inhibitor is bound to the protein prior to solubilization with detergent for protection against denaturation. Only the intact bongkrekate‐protein passes easily through the hydroxyapatite column. Bongkrekate shields the protein in contrast to carboxyatractylate only partially against proteinases present in the crude extract.

The isolated bongkrekate protein shows the same molecular weights in dodecylsulfate and Triton X‐100, the same amino acid composition and the same isoelectric point as the earlier isolated carboxyatractylate‐protein complex. It differs by its higher sensitivity against trypsin and thermolysin.

The identity of both proteins is demonstrated by interconversion of the bongkrekate‐protein into the carboxyatractylate‐protein. The process requires the catalysis by ADP or ATP, the natural substrates of the protein.

The formation of the extractable [3H]bongkrekate‐protein complex in mitochondria requires the presence of ADP or ATP.

These data, the immunological studies presented earlier, and the differences in the reactivity of –SH groups of the isolated bongkrekate and carboxyatractylate complexes (to be published) indicate that both proteins represent different conformational states of the translocator protein (m‐state and c‐state).
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.
Bovine Lenticular γ‐Glutamylcysteine Synthetase: Reaction Sequence
Authors: VAN BUSKIRK Gale E. , GANDER John E. , RATHBUN William B.
Published: March 2005

The sequence of substrate addition and product release during the reaction catalyzed by γ‐glutamylcysteine synthetase was investigated with purified enzyme from bovine lens. Thermal inactivation and kinetic studies suggest that l‐glutamate is the first substrate to bind to the enzyme. l‐β‐Chloroalanine was used as the l‐cysteine analogue. Utilizing substrate activation and product inhibition studies, the following reaction sequence was determined: l‐glutamate binding, ATP binding, ADP release, l‐β‐chloroalanine binding, followed by inorganic phosphate and then dipeptide release. The implications of this mechanism with regard to control of the enzyme in situ and its importance in glutathione synthesis are discussed.
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.
Deoxyribonucleic Acid Polymerase from Physarum polycephalum
Authors: BAER Anton , SCHIEBEL Winfried
Published: March 2005

DNA polymerase was purified 1000‐fold from the cytoplasm of microplasmodia of the myxomycete Physarum polycephalum. The activity was found in two forms exhibiting molecular weights of 204000 and 116000 respectively. Both forms eluted together from DNA‐cellulose and DEAE‐Sephadex columns. The Stokes radii were 6.5 and 5.5 nm. The sedimentation coefficients were 7.6 and 5.2 S. The frictional ratios of 1.69 suggest a highly hydrated and/or an asymmetric structure of the molecule. The enzyme‐catalyzed reaction was sensitive to N‐ethylmaleimide (60% inhibition by 1 mM). Unlike DNA polymerase α from mammalian cells the Physarum enzyme was stimulated by 30 mM NaCl. Activated DNA was the preferred template. Poly(A) · (dT)12 was not accepted. The Km value for deoxynucleoside triphosphates was 3 μM, for activated DNA 50 μg/ml and for Mg2+ at the optimum [K+] of 150 mM about 0.6 mM.
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.
Formation of Complexes between Microsomal Cytochrome P‐450‐Fe(II) and Nitrosoarenes Obtained by Oxidation of Arylhydroxylamines or Reduction of Nitroarenes in situ
Authors: MANSUY Daniel , BEAUNE Philippe , CRESTEIL Thierry , BACOT Charles , CHOTTARD Jean‐Claude , GANS Pierre
Published: March 2005

A cytochrome P‐450 complex exhibiting a Soret peak at 454 nm is formed by direct interaction of nitrosobenzene with NADPH‐reduced rat liver microsomes in anaerobic conditions, by reaction of phenylhydroxylamine with aerobic microsomes or during nitrobenzene reduction by NADPH‐reduced or dithionite‐reduced microsomes. In the latter conditions, the complex formation is only transient as it is unstable to dithionite. Analogous reactions with myoglobin lead to the previously described myoglobin‐Fe(II) · nitrosobenzene complex which has similar properties to those of the 454‐nm‐absorbing cytochrome P‐450 complex. This analogy, together with the various conditions of its formation, strongly indicates that it is a cytochrome‐P‐450‐Fe(II) · nitrosobenzene complex. The corresponding complex with the 4‐chloro‐nitrosobenzene ligand is formed in similar conditions. Cytochrome P‐450‐Fe(II) complexes with nitrosoarenes seem less stable than the previously described complexes with nitrosoalkanes.
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.
The Purification and Properties of the Aspartate Aminotransferase and Aromatic‐Amino‐Acid Aminotransferase from Escherichia coli
Authors: POWELL Janet T. , MORRISON John F.
Published: March 2005

A simple and convenient procedure is described for the isolation in good yield of two amionotransferases from various strains of Escherichia coli. On the basis of thier substrate specificities on of the enzymes has been classified as an aromatic amino acid aminotransferase and the other as an aspartate aminotransferate. But both act on a wide range of substrates Pyridioxal phosphate is bound more strongly to the aspartate aminotransferate than to the aromatic amino transferase which cannot be fully re‐activated afer removal of the prosthetic group. Both enzymes are composed of two subunits which appear to be identical.
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.
Stability of Polyadenylated RNA in Differentiating Myogenic Cells
Authors: SINGER Robert H. , KESSLER‐ICEKSON Gania
Published: March 2005

Three independent methods of measurement showed that cytoplasmic polyadenylated RNA from the differentiating myogenic cell line L8 consists of two main populations with regard to stability, one with a half‐life of less than 4 h and the other with a half‐life of 17–54 h. Similar results were obtained in the presence and absence of actinomycin D. During the fusion of mononucleated myoblasts into multinucleated fibers, there was an increase in both the steady‐state pool of the more stable polyadenylated RNA and the proportion of stable polyadenylated RNA synthesized in pulse labelling.
The Purification and Properties of Pig‐Liver Catechol‐O‐Methyl Transferase
Authors: GULLIVER Peter A. , TIPTON Keith F.
Published: March 2005

A procedure utilising affinity chromatography is described for the large‐scale purification of pig‐liver catechol‐O‐methyl transferase. The enzyme prepared by this method appears to be homogeneous by polyacrylamide gel electrophoretic criteria and gel chromatography. It is stable for prolonged periods when stored at ‐5 °C in 20% (v/v) glycerol. The enzyme has a molecular weight of about 23000 and does not appear to be a compound of subunits, or to associate to any appreciable degree. The pH optimum of the enzyme's activity is approximately pH 7.1–7.4, it does not catalyse the methylation of benzimidazole and has a Km of 0.64 mM and 0.056 mM towards 3,4‐dihydroxyphenylacetic acid and S‐adenosyl‐l‐methionine, respectively. Amino acid analysis showed the presence of five cysteine residues.
Immobilization of Reticulocyte Elongation Factor EF‐2
Authors: LAM Kin Sing , HEINTZ Roger L.
Published: March 2005

Conditions are described whereby the ADP‐ribosylation (from NAD+) of reticulocyte elongation factor EF‐2, catalyzed by diphtheria toxin, is essentially complete and whereby the reverse of this process may be carried out with recovery of 60–70% of the original EF‐2 activity. Both reactions proceed well at room temperature. The reverse reaction is much slower than the ADP‐ribosylation process and requires high nicotinamide concentrations. For the reverse reaction to occur at a significant rate it is necessary to lower the pH to 6.5 (from the 7.5 used for the forward reaction). NAD+ covalently linked to agarose may replace NAD+ in the diphtheria toxin reaction. The characteristics of this reaction are similar to those of the reaction employing free NAD+ except that the velocity is reduced and the concentration of NAD+ moieties greatly increased. NAD+ immobilized on agarose through the C‐8 of the adenine ring is a superior substrate compared with NAD+ linked to agarose via its periodate‐oxidized ribose moieties. Preliminary experiments indicate that reversal of this latter reaction with recovery of biological activity may be possible.
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.
High‐Resolution Proton‐Magnetic‐Resonance Studies of Chromatin Core Particles
Authors: CARY Peter D. , MOSS Tom , BRADBURY E. Morton
Published: March 2005

The binding of histones in chromatin core particles and in core particles depleted of histones H2A and H2B has been studied by high‐resolution proton nuclear magnetic resonance (NMR) at 270 MHz. At low ionic strengths it is shown that histones H3 and H4 are bound in the core particle. Further, whereas the apolar regions of H2A and H2B are also bound to the core particle, the basic N‐terminal and C‐terminal regions are more mobile and give rise to sharp resonances in the NMR spectrum of the core particle. Between 0.3 and 0.6 M NaCl there is further release of basic regions of histones H3 and H4 from the complex. The dissociation of the core particle between 0.6 and 2.0 M NaCl is accompanied by the release of the structured apolar regions of the histones as evidenced by the appearance of a complex aromatic spectrum and perturbed upfield ring‐current‐shifted methyl resonances. Arginine residues are implicated in the binding between histones and DNA and 69% of these residues are found in the apolar regions of the histones. The interactions between histones and DNA in the core particle thus involves H3 and H4 and the apolar regions of H2A and H2B. It is suggested that these basic regions of H2A and H2B have binding sites outside the core particle.
A Study of the Thermal Unfolding of Escherichia coli Phenylalanine Transfer RNA by Chemical Modification at Elevated Temperatures
Authors: GODDARD John P. , LOWDON Margaret
Published: March 2005

Escherichia coli tRNAPhe was modified by 3 M sodium bisulphite pH 6.0 for 24 h in the temperature range 25 °C (× 5 °C) to 55 °C and in the absence of added magnesium ions. The sites and extents of conversion of cytidines to uridine occurring at each temperature were determined by fingerprinting. The new sites of cytidine modification found at higher reaction temperatures were assumed to arise from breakage of secondary and tertiary structure hydrogen bonds involving cytidine residues. From these data, we conclude that hydrogen bonds within the ‘complex core’ of the tRNA (including the base pairs G‐10 · C‐25, C‐11 · G‐24 and C‐13 · G‐21 within the dihydrouridine stem and the tertiary structure base pair G‐15 · C‐48 melt at a lower temperature than the tertiary structure hydrogen bonds between G‐19 in the dihydrouridine loop and C‐56 in the TΨC loop.
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.
Type‐I Trimer and Type‐I Collagen in Neutral‐Salt‐Soluble Lathyritic‐Rat Dentine
Authors: WOHLLEBE Marissa , CARMICHAEL David J.
Published: March 2005

Triple‐helical collagen molecules have been obtained from EDTA‐demineralized lathyritic rat incisors by neutral buffer extraction. Component α chains, isolated by sequential ion‐exchange and gel‐filtration chromatography, were shown to be α1I and α2 chains by cyanogen bromide peptide analysis. The α1I:α2 chain ratio was approximately 3:1, which is greater than expected for type I collagen. The excess of α1I chains over that required for type I collagen was due to the presence of type I trimer molecules. Fractional salt precipitation separated type I collagen from type I trimer. It is not known at present if type I trimer synthesis also occurs in normal rat tissues.
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.
Processing in vitro of Precursor Periplasmic Proteins from Escherichia coli
Authors: RANDALL Linda L. , JOSEFSSON Lars‐Göran , HARDY Simon J. S.
Published: March 2005

Precursors of two secreted periplasmic proteins in Escherichia coli, arabinose‐binding protein and maltose‐binding protein, were synthesized in vitro on membrane‐bound polysomes. Addition of Triton X‐100 to the system resulted in processing of the precursors to mature forms.
The Rate of Release of ATP from Its Complex with Myosin
Authors: CARDON Jeffrey W. , BOYER Paul D.
Published: March 2005

An approach previously published from this laboratory for measurement of the rate of dissociation of ATP from its complex with myosin has been carefully evaluated. The procedure has been found valid, and the off constant (21 °C, I= 0.21 M, pH 7.0) is 1 × 10−4 s−1. Other data for the rate of ATP binding give a Kd for myosin ATP of 6 × 10−11 M. Reasons for the apparent discrepancy between this value and thatreported by others have been examined. When various factors are appropriately taken into account, this discrepancy is eliminated.
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.
Structure and Synthesis of a Lipid‐Containing Bacteriophage
Authors: SCHÄFER Rolf , FRANKLIN Richard M.
Published: March 2005

The lipid‐containing bacteriophage PM2 was reconstituted stepwise from its purified denatured subunits. In the first step the nucleocapsid was reconstituted from the DNA and the two nucleocapsid proteins. Slight biochemical differences between reconstituted nucleocapsids and those isolated from native virus were seen. Combination of reconstituted nucleocapsid or nucleocapsid from virions with the coat and spike proteins in the presence of the viral lipids resulted in the formation of infectious virus in both cases. The reconstituted particles contained amounts of viral components similar to those in native virus, except for the lipid content. The amount of lipids present in the reconstituted particles was twice as high as the lipid content of native bacteriophage.
N‐Acetylphenylalanyl‐tRNA Hydrolase from Yeast
Authors: COLOMA Antonio , HEREDIA Claudio F.
Published: March 2005

This paper describes the purification and properties of an enzyme present in yeast which splits N‐acetylphenylalanyl‐tRNA to N‐acetylphenylalanine and tRNA. The enzyme has been extensively purified, as shown by gel electrophoresis. The hydrolase has a molecular weight of 35000 as estimated by filtration on Sephadex G‐150, is maximally active in the presence of a divalent cation (Mg2+, Mn2+ or Ca2+) and has a pH optimum at around neutrality. The enzyme is highly specific in hydrolyzing N‐acetylphenylalanyl‐tRNA (Km=0.4 μM). Phenylalanyl‐tRNA is hydrolyzed with a similar apparent affinity but with an efficiency of 40% of that found for N‐acetylphenylalanyl‐tRNA. Other free or N‐substituted aminoacyl‐tRNAs are not substrates of this hydrolase. Neither of the two reaction products are effective inhibitors of this enzyme. Based on its substrate specificity, the trivial name of N‐acetylphenylalanyl‐tRNA hydrolase is proposed for this enzyme.
The 5‐S RNA Binding Protein from Yeast (Saccharomyces cerevisiae) Ribosomes
Authors: NAZAR Ross N. , YAGUCHI Makoto , WILLICK Gordon E. , ROLLIN C. Fernand , ROY Camille
Published: March 2005

The ribonucleoprotein complex between 5‐S RNA and its binding protein (5‐S RNA · protein complex) of yeast ribosomes was released from 60‐S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE‐cellulose. This protein, designated YL3 (Mr= 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4–9) and migrated as one of four acidic 60‐S subunit proteins when analyzed by the Kaltschmidt and Wittman two‐dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5‐S RNA binding proteins, EL 18 from Escherichia coli and HL13 from Halobacterium cutirubrum:Ala1‐Phe2‐Gln3‐Lys4‐Asp5‐Ala6‐Lys7‐Ser8‐Ser9‐Ala10‐Tyr11‐Ser12‐Ser13‐Arg14‐Phe15‐Gln16‐Tyr17‐Pro18‐Phe19‐Arg20‐Arg21‐Arg22‐Arg23‐Glu24‐Gly25‐Lys26‐Thr27‐Asp28‐Tyr29‐Tyr3s;of particular interest was homology in the cluster of basic residues (18–23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (Mr= 24700) and CN2 (Mr= 11300) by CNBr treatment; the larger fragment originated from the N terminus. The N‐terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5‐S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5‐S RNAs, a model for the evolution of the eukaryotic 5‐S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5‐S RNA · protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5‐S RNA‐protein interaction.
13C Nuclear Magnetic Resonance of Protamines. The Three Main Components of Clupeine
Authors: BONORA Gian Maria , FERRARA Luciano , PAOLILLO Livio , TONIOLO Claudio , TRIVELLONE Enrico
Published: March 2005

The three main components (called YI, YII and Z) of clupeine a protamine from herring have been isolated and characterized by 13C nuclear magnetic resonance. Resonances belonging to the many different amino acid carbon atoms are observable and assigned. The effect of urea on the clupeine fractions points to the molecules being essentially in extended form in 2H2O solutions. Unusual properties of clupeine fractions can be observed in the presence of phosphate. Two of the fractions, i.e. YI and Z, show that a partial folding occurs in some well‐defined sections whereas fraction YII remains essentially unaltered.
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.
The Effect of Mg2+ on Some Properties of Nucleotide‐Free Elongation Factor Tu from Bacillus stearothermophilus
Authors: WITTINGHOFER Alfred , LEBERMAN Reuben
Published: March 2005

The nucleotide‐free elongation factor from Bacillus stearothermophilus provides a means to study the effect of Mg2+ ions on various reactions of the protein. The binding of GDP to the protein is stimulated by Mg2+. From comparative studies with other metal ions, particularly Mn2+, it appears that this stimulation is due to the formation of a metal · GDP complex which is bound to the protein. Protection against proteolysis by trypsin is afforded by both Mg2+ and Mg · GDP, but not by GDP alone. The rate of substitution of the sulphydryl group associated with aminoacyl‐tRNA binding, by either 5,5′‐dithio‐bis(2‐nitrobenzoic acid) or N‐ethylmaleimide is reduced in the presence of Mg2+. All these observations show that Mg2+ not only is involved in GDP binding but also has a direct effect on the tertiary structure of the protein.
Nonrandom Segregation of Histones during Chromatin Replication
Authors: RUSSEV George , TSANEV Roumen
Published: March 2005

Exponentially growing Ehrlich ascites tumor cells were labelled in vivo during roughly two cell cycles by three different methods: (a) DNA was labelled during the first cell cycle with [14C]thymidine, and during the second cycle with bromodeoxyuridine + [3H]thymidine; (b) proteins were labelled during the first cell cycle with L‐[14C]valine and DNA during the second cycle with bromodeoxyuridine + [3H]thymidine; (c) both proteins and DNA were labelled during the same cell cycle with L‐(14C]valine and bromodeoxyuridine + [3H]thymidine respectively. DNA isolated from cells labelled according to method (a) was also resolved on a CsCl density gradient to show that the two successive labels are incorporated in the same DNA molecules.

Chromatin was isolated by salt extraction of nuclei obtained with Nonidet P40 from Ehrlich ascites tumor cells labelled according to methods (b) and (c); 4–8% of the total protein (10–15% of the histones) were chemically bound to DNA by the procedure of Levina and Mirzabekov [Dokl. Akad. Nauk. SSSR, 221, 1222–1225 (1975)]. The unbound proteins were removed by centrifugation in a Cs2SO4/sarcosyl density gradient. The chemically linked nucleohistone was alkali‐denatured and the two DNA strands, together with the bound histones, were separated on a shallow alkaline metrizamide density gradient. The newly synthesized histones were found to be associated with the newly synthesized DNA strand while the old histones remained with the parantal DNA strand.
Acyl‐Coenzyme‐A Synthetase I from Candida lipolytica
Authors: HOSAKA Kohei , MISHINA Masayoshi , TANAKA Takao , KAMIRYO Tatsuyuki , NUMA Shosaku
Published: March 2005

Acyl‐coenzyme‐A synthetase I from Candida lipolytica has been purified to homogeneity as evidenced by polyacrylamide gel electrophoresis in the presence and absence of dodecylsulfate as well as by Ouchterlony double‐diffusion analysis. The purification procedure involves resolution of cellular particles with Triton X‐100 and chromatography on phosphocellulose, Blue‐Sepharose and Sephadex G‐100. The purified enzyme exhibits a specific activity of 20–24 U/mg protein at 25°C, which is about 100‐fold higher than those of long‐chain acyl‐CoA synthetases hitherto reported. The molecular weight of the enzyme has been estimated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate to be approximately 84000. The enzyme is specific for fatty acids with 14–18 carbon atoms regardless of the degree of unsaturation. Studies with the use of specific antibody to acyl‐CoA synthetase I have indicated that this enzyme is immunochemically distinguishable from acyl‐CoA synthetase II.
Mechanism of Carbamoyl‐Phosphate Synthetase
Authors: RUBIO Vicente , BRITTON Hubert Greenslade , GRISOLIA Santiago
Published: March 2005

This paper demonstrates, by pulse‐chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its γ‐phosphoryl group to carbamoyl phosphate. This bound ATPB can react with NH3, HCO3− and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N‐acetylglutamate, but not NH3 or HCO3−, are required for this binding; the amount bound depends on the concentration of ATP (Kapp= 10–30 μM ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer.

Binding of ATPB follows a slow exponential time course (t1/2 8–16 s, 22°C), independent of ATP concentration and little affected by NH3, HCO3− or by incubation of the enzyme with unlabelled ATP prior to the pulse of [γ‐32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO3− when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate (‘Cbm‐P precursor’) in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm‐P precursor when the enzyme reverts to the inaccessible conformation.

Pulse‐chase experiments in the absence of NH3 and HCO3− (< 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a ‘burst’ in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO3− in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 μM) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5′)pentaphospho(5′)adenosine (Ap5A) fails to inhibit the enzyme significantly.

A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321–329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent the conformational change postulated by others from steady‐state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO3−‐dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.