bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in Guinea‐Pig Dermal Collagen during Development
Authors: SHUTTLEWORTH C. Adrian , FORREST Leslie 
Published: March 2005

Guinea‐pig dermis was digested with pepsin and the solubilized collagen molecules separated by differential salt precipitation at pH 7.5. Differences in subunit composition and amino acid analysis were noted between type I and type III collagen.Incorporation of radioactive proline into the developing foetus enabled isolation of labelled type I and type III collagens. Comparison of the specific activity of the isolated collagen molecules showed that type III collagen had a high specific activity in the early stages of foetal development, which decreased dramatically during foetal development. The specific activity of pepsin‐solubilized type I collagen remained fairly constant during foetal development.
Altered α Subunits in Phenylalanyl‐tRNA Synthetases from p‐Fluorophenylalanine‐Resistant Strains of Escherichia coli
Authors: HENNECKE Hauke , BÖCK August 
Published: March 2005

Three different phenylalanyl‐tRNA synthetases have been purified to near homogeneity, one from a wild‐type strain of Escherichia coli and the others from two independently isolated p‐fluorophenylalanine‐resistant strains. The mutant enzymes were not able to use p‐fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild‐type enzyme by several electrophoretic and immunological criteria.The α and β subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl‐tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild‐type and mutant subunits indicate that the mutant α subunit is responsible for p‐fluorophenylalanine resistance and therefore seems to carry the phenylalanine‐binding site or to participate in its formation.
The Internal‐Alkaline pH Gradient, Sensitive to Uncoupler and ATPase Inhibitor, in Growing Clostridium pasteurianum
Authors: RIEBELING Volker , JUNGERMANN Kurt , THAUER Rudolf K. 
Published: March 2005

The intracellular pH was measured in growing Clostridium pasteurianum with an acid‐base equilibrium distribution method. [14C]Dimethyloxazolidinedione, [14C]methylamine and [14C]acetic acid were used as “ΔpH‐indicators”. During growth the extracellular pH decreased from 7.1 to 5.1; simultaneously the intracellular pH changed from 7.5 to 5.9. Thus the intracellular pH was more alkaline than the extracellular pH by 0.4 to 0.8 pH‐units.
This pH gradient (interior alkaline) was abolished by the proton conductor carbonylcyanide m‐chlorophenylhydrazone and the ATPase inhibitor N,N′‐dicyclohexylcarbodiimide. The pH gradient could not be demonstrated in cells depleted of an energy substrate. These results suggest that the pH gradient is formed by an ATPase‐driven extrusion of protons from the cells rather than by a Donnan potential.
Growth of the organism was inhibited by low concentrations of both carbonylcyanide m‐chlorophenylhydrazone (5 μM) and dicyclohexylcarbodiimide (5 μM). This finding suggests that the pH gradient is essential for the growing cell as it may be required for substrate accumulation and other types of transport processes.
A Kinetic Study of Mitochondrial Calcium Transport
Authors: REED Ken C. , BYGRAVE Fyfe L. 
Published: March 2005

This report describes a kinetic analysis of energy‐linked Ca2+ transport in rat liver mitochondria, in which a ruthenium red/EGTA [ethanedioxy‐bis(ethylamine)‐tetraacetic acid] quenching technique has been used to measure rates of45Ca2+ transport. Accurately known concentrations of free 45Ca2+ were generated with Ca2+/nitrilotriacetic acids buffers for the determination of substrate/velocity relationships.The results show that the initial velocity of transport is a sigmoidal function of Ca2+ concentration (Hill coefficient = 1.7), the Km being 4 μM Ca2+ at 0°C and pH 7.4. These values for the Hill coefficient and the Km remain constant in the presence of up to 2 mM phosphate, but with 10 mM acetate both parameters are increased slightly. Both permeant acids increase the maximum velocity to an extent dependent on their concentration.The Ca2+ ‐binding site(s) of the carrier contains a group ionizing at pH approximately 7.5 at 0°C, which is functional in the dissociated state. The stimulatory effect of permeant acids is ascribed to their facilitating the release of Ca2+ from the carrier to the internal phase, an interpretation which is strengthened by the lack of effect of the permeant anion SCN− on Ca2+ transport.Studies on the time‐course of Ca2+ uptake and of EGTA‐induced Ca2+ efflux from pre‐loaded mitochondria demonstrate the reversibility of the carrier in respiring mitochondria and the extent to which this property is influenced by permeant acids.These data are accommodated in a carrier mechanism based on electrophoretic transport of Ca2+ bound to pairs of interacting acidic sites.
Equivalent and Non‐equivalent Binding Sites for tRNA on Aminoacyl‐tRNA Synthetases
Authors: KRAUSS Gerhard , PINGOUD Alfred , BOEHME Detlev , RIESNER Detlev , PETERS Frens , MAASS Günter 
Published: March 2005

Complexes between tRNAPhe (yeast), tRNASer (yeast) and tRNATyr (Escherichia coli) and their cognate aminoacyl‐tRNA synthetases have been studied by sedimentation velocity runs in an analytical ultracentrifuge. The amount of complex formation was determined by the absorption and the sedimentation coefficients of the fast‐moving boundary in the presence of excess tRNA or excess synthetase respectively. The same method has been applied to unspecific combinations of tRNAs and synthetases. Inactive material of tRNA or synthetase does not influence the results.

Two moles of tRNAPhe can be bound to one mole of phenylalanyl‐tRNA synthetase with a binding constant ≫ 106 M−1. The binding constants for both tRNAs are very similar; the binding sites are independent of each other. Omission of Mg2+ does not prevent binding.
Two moles of tRNASer can be bound to one mole of Seryl‐tRNA synthetase; the binding of the first and second tRNA is non‐equivalent. K1≫ 106 M−1, K2 is determined to be 1.3×105 M−1 at pH 7.2. Omission of Mg2+ prevents complex formation.
Tyrosyl‐tRNA synthetase behaves very similarly to seryl‐tRNA synthetase. The binding constant for the weakly bound tRNA is 2.3×105 M−1 at pH 7.2, and 2.5×106 M−1 at pH 6.0. No complexes are observed in the absence of Mg2+.
Unspecific binding was only obtained with phenylalanyl‐tRNA synthetase. It binds tRNASer (yeast), tRNAAla (yeast) and tRNATyr (E. coli) with a binding constant about 100 times lower compared to its cognate tRNA.

The binding data are discussed with respect to the tertiary structure of the tRNAs, the subunit structure of the synthetases and the possible physical basis for the non‐equivalence of binding sites.
The Distribution of J Blood‐Group Activity on Lipoprotein and Protein Fractions of Bovine Serum
Authors: RADAŠ Antonín , THIELE Otto Wolfgang 
Published: March 2005

There is a diversity of carriers of the J blood‐group activity of bovine serum. The qualitative and quantitative distribution of the J activity on different carriers was studied, using various fractionation procedures.Approximately one third of J activity was found in the total lipids extracted from serum, two thirds in the lipid‐free residue precipitated by lipid extraction. One third of the lipid J substance was found to be bound to the very low density lipoprotein, two thirds to the low density lipoprotein, while the high density lipoprotein was completely free of J activity. All non‐lipidic J substance was present in the lipid‐free protein. There was no J activity in the low molecular weight mucoproteins of serum and in the apoproteins of the lipoprotein fractions.The lipoprotein fractions were prepared by ultracentrifugation at different solvent densities. The lipoprotein fractions were characterized by chemical analyses and physical properties. The lower total cholesterol concentration of bovine serum, as compared to human serum, is reflected in a lower concentration of low density lipoprotein.The results obtained by ultracentrifugation coincide with the results obtained by precipitation of “β‐lipoproteins” with dextran sulfate and calcium chloride and with results obtained by gel filtration of bovine serum. The “β‐lipoprotein” fraction contains lipoproteins of very low and low density, and probably chylomicrons and a variety of other proteins, however no high density lipoprotein.
Evidence for Cooperative Effects in the Exchange Reaction Catalysed by the Oxoglutarate Translocator of Rat‐Heart Mitochondria
Authors: SLUSE Francis E. , SLUSE‐GOFFART Claudine M. , DUYCKAERTS Claire , LIÉBECQ Claude 
Published: March 2005

The initial rates of the exchange external oxoglutarate/internal malate through the inner membrane of rat‐heart mitochondria, for various concentrations of the two substrates, have been reinvestigated for an extended range of concentrations of the external oxoglutarate. This has been made possible by use of the inhibitor‐stop technique that allows 100 times smaller incubation times than the centrifugation‐stop technique used previously.Under the experimental conditions the uptake of the external‐labelled oxoglutarate into the mitochondrial‐matrix space is mediated by the oxoglutarate translocator performing a ono‐to‐one exchange of the anions oxoglutarate (external) and malate (internal).Two intermediary‐plateau regions are observed in the kinetic saturation curve of the translocator by the external oxoglutarate, revealing a complex rate equation which is found to be the product of two one‐substrate functions. Analysing these features it is shown that the model, proposed earlier, of a “double carrier” as catalyst in a rapid‐equilibrium random bi‐bi mechanism, is still applicable but that several external binding sites have to be considered. As already noticed the external and the internal substrates bind to their respective sites independently of each other.Furthermore, some additional requirements imposed by the observed kinetics suggest that the exchange reaction is performed by only one translocator species made of identical interacting subunits.The anion exchange is tentatively viewed as a rotation of a subunit around an axis situated in the plane of the membrane after two independent local configuration changes induced by the binding of the two substrates on this subunit.
Hydrogen‐Exchange Study of the Conformational Stability of Human Carbonic‐Anhydrase B and Its Metallocomplexes
Authors: ZÁVODSZKY Péter , JOHANSEN Jack T. , HVIDT Aase 
Published: March 2005

In the range of pH 4.6–8.8, 25°C, the apoenzyme of carbonic anhydrase B shows no evidence of any gross conformational changes, as studied by the hydrogen‐deuterium exchange method.At pH 4.6 the addition of Co(II), Cd(II) or Mn(II) to the apoenzyme results in a destabilization of the native protein conformation, but in the range of pH 5.5–8.8 these metal ions, and Zn(II), slightly increase the conformational stability of the protein, in so far as they reduce the probability ϱ of solvent exposure of the peptide groups.In comparison with other proteins studied, native carbonic anhydrase is characterized by a rather compact conformation; for half of the peptide groups the probability of solvent exposure is less than 10−4, corresponding to changes in standard free energy larger than 5.5 kcal mol−1 (23 kJ mol−1) following the conformational transitions by which these groups are exposed to the solvent.
Chlorinating Ability of Human Phagocytosing Leucocytes
Authors: ZGLICZYŃSKI Jan Maciej , STELMASZYŃSKA Teresa 
Published: March 2005

The course of chlorination in neutrophilic granulocytes has been shown. The process of 36CI incorporation occurs during and after the engulfment of bacteria by granulocytes. Incorporated radioactivity was found in insoluble fractions.The myeloperoxidase obtained from neutrophils catalyzes chlorination of protein (bovine serum albumin) and bacteria (Staphylococcus epidermidis) in the presence of hydrogen peroxide and chloride. The products of chlorination are insoluble.Chlorination in neutrophils is inhibited by the iodide and myeloperoxidase inhibitors azide and cyanide.A quantitative method of determination of biological chlorination in cells has been devised.
Isolation and Identification of the Urinary Pigment Uroerythrin
Authors: BERÜTER Josef , COLOMBO Jean‐Pierre , SCHLUNEGGER Urs Peter 
Published: March 2005

The red pigment uroerythrin, a chromophore known to be adsorbed by the amorphous urate sediments (sedimentum lateritium), has been isolated from human urine and further purified as its trimethyl derivative.Urine was applied to a column of Amberlite XAD‐2 resin on which uroerythrin and other pigments were adsorbed. The pigments were eluted with methanol and uroerythrin was further purified by extraction with ether at pH 4.0, repeated chromatography on lipophilic Sephadex LH‐20 and thin‐layer chromatography on silica gel. For optimal purification uroerythrin was converted into the trimethyl derivative and chromatographed on silica gel thin‐layer plates.The structure of the pigment has been studied by chromate degradation followed by identification of the imide products by thin‐layer chromatography. From these results and from infrared, mass spectral and nuclear magnetic resonance data a tripyrrole structure for uroerythrin is concluded. The proposed structure for the chromophore is related to that of the bile pigment biliverdin consisting, however, only of the rings A, B and C.
Prostaglandin 15‐Hydroxy Dehydrogenase from Human Placenta
Authors: SCHLEGEL Werner , GREEP Roy O. 
Published: March 2005

The enzyme system prostaglandin 15‐hydroxy dehydrogenase, which catalyzes the inactivation of all biologically active prostaglandins, has been purified 1270‐fold from human placenta. Kinetic studies on the enzyme have provided information on a well‐organized control mechanism to avoid prostaglandin accumulation and for a fast prostaglandin degradation. 15‐Ketoprosta‐glandin E2 and 13,14‐dihydro‐15‐ketoprostaglandin E2 inhibit prostaglandin 15‐hydroxy dehydrogenase non‐competitively with respect to prostaglandin E2.The rate equation of enzyme reaction for two substrates was used for determination of the equilibrium constant and Michaelis constants of the enzyme.The following kinetic constants for prostaglandin 15‐hydroxy dehydrogenase have been found.The equilibrium constant with repect to prostaglandin E2 is 18 μ, the Michaelis constant Km for prostaglandin E2 is 1 μ for NAD+ 44 μ. The inhibition constants for 15‐ketoprostaglandin E2 are Ki(slope)= 70 μ, Ki(intercept)= 150 μ, and for 13,14‐dihydro‐ 15‐ketoprostaglandin E2Ki(slope)= 80 μ, and Ki(intercept)= 150 μ. The maximal velocity for the forward reaction is V1= 0.45 μmol/min.These kinetic data exclude a random or ping‐pong mechanism, and also a Theorell‐Chance type as suggested by Braithwaite and Jarabak. We propose, therefore, a sequential ordered mechanism.The isoelectric point for prostaglandin 15‐hydroxy dehydrogenase is at pH 5.35, judged by isoelectric focusing.
Characterization of Oxytocin Receptors on Isolated Rat Fat Cells
Authors: BONNE Dominique , COHEN Paul 
Published: March 2005

The binding of the 3H‐labelled neurohypophyseal nonapeptide hormone, oxytocin, to isolated rat fat cells has been measured under conditions where this compound elicits the known activation of glucose oxidation by these cells, called “insulin‐like” action. Uptake by the cells of the [3H]peptide as a function of various concentrations of the hormone in the medium indicated the presence of two classes of binding sites with different apparent affinities and capacities. The sites of the first type exhibit a rather high affinity, but low capacity, for oxytocin (5 nM; 3 × 104 sites per cell) and appear to be saturable under a reversible process. Evaluation of dose‐response relationships suggest that they may be directly related to the measured biological response (i.e. activation of the glucose to 14CO2 conversion). Competition experiments show that [3H]oxytocin binding to the cells remains constant within a large range of insulin concentrations. The apparent capacity of different hormone analogs to compete with oxytocin for binding to this class of receptors has been evaluated and compared with the measured insulin‐like activity of these different compounds. The sites of the second category have significantly lower affinity, but higher capacity for oxytocin, and were found to be not saturable under the experimental conditions.[3H]Oxytocin uptake by ghosts prepared from the isolated fat cells showed striking similarities to the binding process described for whole cells, although the affinity and total capacity of the former were found to be slightly lower. The basal and adrenalin‐stimulated adenylate cyclase of these fractions appeared to be unaffected by various concentrations of oxytocin.It is concluded that there may exist on the rat fat cell membranes a discrete number of oxytocin receptors possessing high specificity for oxytocin and exhibiting affinities and kinetic behaviour similar to those of other characterized oxytocin receptors. They are believed to be independent of the other hormonal receptors of the rat fat cells.
Labile Sulfur in Human Superoxide Dismutase
Authors: CALABRESE Lilia , FEDERICI Giorgio , ROTILIO Giuseppe , FINAZZI‐AGRÒ Alessandro , BANNISTER William H. , BANNISTER Joseph V. 
Published: March 2005

The nature of the intense absorption band at 320 nm of the copper and zinc‐containing enzyme superoxide dismutase, from human red blood cells, has been investigated. The band does not depend on the metal prosthetic groups of the enzyme, as it is still present in the apo protein. When, however, copper alone is removed from the enzyme with a treatment involving the use of cyanide, the band is also lost. Nevertheless the copper‐free protein is able to recover both the enzyme activity and the native electron paramagnetic resonance spectrum as easily as the apo protein.
A number of other treatments are able to abolish the band. They include reaction with reducing agents such as dithiothreitol, sulfite, borohydride, exposure to denaturants such as guanidine HCl and sodium dodecyl sulfate, and exposure to pH values below pH 3 or above pH 13.
Four sulfur atoms per protein molecule were found to be associated to the 320‐nm chromophore on the basis of quantitative determinations following reaction with cyanide or sodium borohydride.
A molar absorption coefficient of 1150 M−1 cm−1 was determined per each chromophoric group. In spite of this relatively high value and unusual stability, a persulfide group, R‐S‐SH, seems to be the most likely structure for this chromophore.
Bovine and equine superoxide dismutase do not show spectral or chemical evidence for such a group. This, and the recovery of activity and spectral properties of copper in the cyanide‐treated human enzyme, indicate that labile sulfur is not associated with the superoxide dismutase activity of this protein.
The Joining of the 30‐S Initiation Complex with the 50‐S Subunit, the Main Target for Thiostrepton
Authors: NAAKTGEBOREN Nico , VERMAAS Anita , VOORMA Harry O. 
Published: March 2005

The study undertaken in this paper on the mode of action of thiostrepton provides data which permit a more precise localization of the main target of thiostrepton.There is severe impairment of the joining of the 50‐S subunit, probably carrying thiostrepton, with either the 30‐S subunit or the 30‐S initiation complex.The degree of impairment of this coupling is temperature dependent, being almost completely inhibited at 0°C, whereas at 37°C the effect is much less marked, provided that natural messenger RNA is present. The inhibition of initiation by thiostrepton is more severe in the presence of IF‐1, a factor, which similar to thiostrepton, is able to shift the dynamic equilibrium of 70‐S ⇌ 50‐S + 30‐S more towards dissociation.By means of 14C‐labeled IF‐2 it is demonstrated that the binding of IF‐2 into the 70‐S initiation complex is prevented by thiostrepton, which seems to be the main cause for non‐coupling.
Oestrogen Receptors in Chick Oviduct
Authors: BEST‐BELPOMME Martin , MEŠTER Ján , WEINTRAUB Hadassa , BAULIEU Etienne‐Emile 
Published: March 2005

Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2–4 weeks, untreated by hormone) chicken oviduct.In the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA= 4 · 109 M−1 at 4°C. The binding of [3H] oestradiol is abolished by 1 μM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low‐ionic‐strength medium. The estimated number of binding sites per nucleus is about 5000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two‐step mechanism. The half‐life time for the faster dissociation step is 18 h at 0 °C, 25 min at 20°C and 10 min at 30°C, and for the slower one is 180 h, 115 min and 60 min, respectively.In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor.After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA= 7 · 108 M−1 at 37 °C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken.In the high‐speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (≤ 100 sites/cell) and at the limits of sensitivity of the methods used.In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell.