bluuebunny/crossref_metadata_2025 huggingface dataset:
d‐Alanine Carboxypeptidase Activity of Micrococcus lysodeikticus Released into the Protoplasting Medium
Authors: LINDER Regina , SALTON Milton R. J.
Published: March 2005

Conversion of whole cells of Micrococcus lysodeikticus to protoplasts allowed the release of a soluble form of a D‐alanine carboxypeptidase into the protoplasting medium. The enzyme cleaves the terminal D‐alanine from the radioactively labelled UDP‐N‐acetylmuramyl‐pentapeptide containing l‐lysine as the diamino acid. However, the enzyme is only minimally active in this fraction so that it had to be enriched and partially purified before its properties could be studied. Chromatography on carboxymethyl‐Sephadex removed the lysozyme used in the protoplasting of the cells. The material which was unadsorbed to the column was applied to an affinity chromatography column of Ampicillin‐Sepharose. Most of the contaminating protein was washed from the column while the D‐alanine carboxypeptidase adhered to the resin and could be eluted with 0.5 M Tris‐HCl buffer pH 8.6. Some of the properties of the enzymic activity were studied using this preparation. The enzyme was activated by Mg2+ ions with a broad optimum from 15–35 mM. It was maximally active when NaCl at a concentration of 0.06–0.08 M was added to the assay, and the pH curve was biphasic with an alkaline optimum. The Km for substrate was found to be 0.118 mM. Enzymic activity was completely inhibited by low concentrations of Ampicillin and penicillin G.
Protein Kinase of Bacteriophage T7
Authors: PAI Shou‐Hsiung , PONTA Helmut , RAHMSDORF Hans‐Jobst , HIRSCH‐KAUFFMANN Monica , HERRLICH Peter , SCHWEIGER Manfred
Published: March 2005

A protein kinase, ATP: protein phosphotransferase (EC 2.7.1.37) was detected in Escherichia coli after infection with bacteriophage T7. The enzyme was purified from the ribosomal wash fraction by conventional methods, affinity chromatography on Cibacron blue and on lysozyme coupled to Sepharose, and by cellogel electrophoresis. An approximately 5000‐fold purification was achieved.
Hydroxylation of p‐Coumaric Acid by Illuminated Chloroplasts
Authors: HALLIWELL Barry
Published: March 2005

Chloroplasts isolated from leaves of spinach‐beet (Beta vulgaris L. ssp. vulgaris) do not catalyse the hydroxylation of p‐coumaric acid in the dark unless a reductant (such as ascorbate, NADH or NADPH) is added. Superoxide dismutase has no effect on this reaction.

Illuminated chloroplasts catalyse the hydroxylation in the absence of added reductant. This reaction is completely inhibited by superoxide dismutase, but catalase has little effect.

Both hydroxylation in the light and hydroxylation in the dark in the presence of reductants are inhibited by diethyldithiocarbamate, EDTA, cyanide and 2‐mercaptoethanol.

It is proposed that O2.− generated by illuminated chloroplasts is involved in the provision of a reductant to the enzyme phenolase.
Solid‐State Protein Kinase
Authors: KINZEL Volker , KÜBLER Dieter , ALONSO Angel
Published: March 2005

The capacity of partially purified rat muscle protein kinase coupled to cyanogen‐bromide‐activated Sepharose 4B to (radio‐)phosphorylate proteins in vitro was evaluated using histones from calf thymus and rat liver and certain other proteins as substrates. Data are presented which point to a low substrate specificity of this enzyme. It is demonstrated that even within a short time period histones are efficiently phosphorylated without the introduction of contaminating (phospho‐)proteins. Thereby phosphoserine residues are formed. The phosphorylation reaction usually performed at 30°C is shown to function quite efficiently also at 4°C. It proceeds even at 30°C for several hours at pH values close to the physiological range without the release of proteins from the solid matrix. The phosphorus transfer can be largely increased with the use of high ATP concentrations. The stability of the substrates is sufficient to suggest a wide applicability of this solid‐state protein kinase in the phosphorylation of proteins either for labeling or as a tool to modify proteins post‐synthetically under gentle conditions. The solid enzyme seems to be suitable for radioactively labeling proteins of more complex biological structures, such as membrane surfaces.
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.
Transport of Amino Acids in Membrane Vesicles of Rhodopseudomonas spheroides Energized by Respiratory and Cyclic Electron Flow
Authors: HELLINGWERF Klaas J. , MICHELS Paul A. M. , DORPEMA Jan W. , KONINGS Wil N.
Published: March 2005

Active transport of amino acids in whole cells and membrane vesicles from the facultative photosynthetic bacterium Rhodopseudomonas spheroides is coupled to electron flow in the respiratory chain and in the cyclic electron transfer system. In vesicles made from cells grown aerobically in the dark transport of amino acids is most effectively energized by the oxidation of NADH and to a lesser extent by ascorbate or succinate in the presence of N,N,N′,N′‐tetramethyl‐1,4‐phenyldiamine dihydrochloride or by ascorbate ± phenazine methosulphate via the respiratory chain with oxygen as terminal electron acceptor. In membrane vesicles from cells grown anaerobically in the light the energy for active transport of amino acids is supplied upon illumination by photo‐oxidation of bacteriochlorophyll and subsequent electron flow through the cyclic electron transfer system. The initial rate of transport increases with the light intensity upto saturation levels. In both vesicle preparations, inhibitors of electron transfer flow inhibit amino acid uptake. In order to obtain light‐driven amino acid transport the isolation of membrane vesicles needs to be performed in a medium with a redox potential between 0 and 100 mV. Illumination of vesicles made from cells grown anaerobically in the light results in the generation of a membrane potential as is indicated by the uptake of the lipophylic cation triphenyl‐methylphosphonium.
Calcitonin Biosynthesis: Evidence for a Precursor
Authors: MOYA Fernando , NIETO Antonio , R‐CANDELA José L.
Published: March 2005

Calcitonin biosynthesis has been studied in chicken ultimobranchial glands incubated in vitro in the presence of radioactive amino acids. The results obtained suggest the existence of a biosynthetic precursor of higher molecular weight or procalcitonin. This precursor has been identified by pulsechase experiments, molecular weight determinations, biological activity measurements and analysis of tryptic peptides. Its molecular weight is about 13000 (calcitonin, about 3500) as determined by polyacrylamide gel electrophoresis. Procalcitonin is present in small amounts in chicken ultimobranchial glands and it is biologically active in rats.
Radioactive Labelling and Characterization of the Products of Activated Mouse Lymphocytes
Authors: SORG Clemens
Published: March 2005

For chemical characterization of the products of activated lymphocytes a radioactive doublelabel technique was developed which allows one to distinguish those products synthesized either de novo or in increased amounts by the stimulated culture. Spleen cells from Balb/c mice were cultured in serum‐free medium in the presence or absence of concanavalin A and simultaneously labelled with radioactive leucine. Optimal culture conditions were established by determining parameters such as cell density, mitogen concentration, and kinetics of protein synthesis following stimulation. Combined supernatants of stimulated and unstimulated cultures each labelled with either [3H]leucine or [14C]leucine were fractionated on Sephadex G‐75. Materials derived from control or stimulated supernatants both yielded a qualitatively similar radiolabelled profile. The isotope ratio of stimulated to nonstimulated culture, however, showed a broad peak at KD 0–0.35 (approx. mol. wt 75000–20000) which was further analyzed by isoelectric focusing. Pools of every two fractions were focused in polyacrylamide gels at pH 3.5–10. By determining the isotope ratio, the isoelectric point, and the KD (mol wt), it was possible to distinguish at least 24 molecules which had been produced only, or in greater degree, by the stimulated culture.
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.
Induction Potential for Glyoxylate Cycle Enzymes during the Cell Cycle of Euglena gracilis
Authors: WOODWARD John , MERRETT Michael J.
Published: March 2005

In light/dark synchronized cultures of Euglena gracilis Klebs Z the enzymes malate synthase, isocitrate lyase and acetate thiokinase were induced upon addition of acetate at all stages of the cell cycle. Cycloheximide and p‐fluorophenylalanine inhibited the development of enzyme activity, showing that induction was dependent on protein synthesis. The maximum rate of induction for all three enzymes was constant for much of the cell cycle but doubles in a single step during the period of DNA replication. Although these data indicate that enzyme potential was regulated by gene dosage and that the structural gene for each enzyme was continuously available for transcription during the cell‐cycle it was not possible by using inhibitors of RNA synthesis, to demonstrate concurrent transcription during enzyme induction.
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.
Effect of Substrate Structure on the Pre‐Steady‐State Kinetics of Oxidation by Liver Alcohol Dehydrogenase
Authors: BLACKWELL Leonard F. , HARDMAN Michael J.
Published: March 2005

The oxidation of a series of primary alcohols by liver alcohol dehydrogenase has been studied under conditions of [S]0≫ [E]0 using the stopped‐flow method. A biphasic process, with exponential rise to a steady state, was observed for most of the alcohols and the rate constants for the transient phase were determined. No transient phase could be detected for 2‐chloroethanol and 2‐nitroethanol and steady‐state measurements were made for these alcohols. The rate constants for the hydrogen transfer step were obtained from the pre‐steady‐state rate constants for the various alcohols and correlated with the Taft σ* constant. The ϱ* value obtained (−1.8) is consistent with rate‐limiting hydride transfer coupled with removal of the hydroxyl proton by a suitable basic group on the enzyme. A possible identity for this group is suggested.
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.
Occurrence and Properties of a Prostaglandin F2α Receptor in Bovine Corpora Lutea
Authors: POWELL William S. , HAMMARSTRÖM Sven , SAMUELSSON Bengt
Published: March 2005

Prostaglandin F2α was specifically bound by a particulate fraction from bovine corpora lutea. The rate constants for the association (7.5×103 M−1s−1) and dissociation (2.1×10−4s−1) reactions gave a dissociation constant of 2.8×10−8 M which is similar to that determined from a Scatchard plot of binding data at equilibrium (5×10−8 M). The receptor was stable for several hours at 23°C but was rapidly destroyed at 37°C. The pH optimum for the binding reaction was 6.3. The receptor had high specificity for prostaglandin F2α and had much lower affinities for other prostaglandins. Luteinizing and follicle‐stimulating hormones had no effect on the prostaglandin F2α‐receptor interaction.
Temperature‐Induced Transitions in the Conformation of Intermediates in the Hydrolytic Cycle of Myosin
Authors: WATTERSON John G. , SCHAUB Marcus C. , LOCHER Ruedi , DI PIERRI Susi , KUTZER Margrit
Published: March 2005

The conformations of the transitory intermediates of the myosin ATPase occurring during the hydrolytic cycle, enzyme without ligand, enzyme‐substrate complex and two different forms of enzyme‐product complex, have been characterized in terms of numbers and classes of reactive thiol groups based on incorporation of radioactively labeled alkylation reagent. The techniques employed allowed this to be done under steady‐state conditions in the presence of high ligand concentrations on intact myosin from rabbit fast skeletal muscles at low ionic strength where the protein is in the gel state as it is in muscle. The binding of a divalent cation (Mg2+ or Ca2+) nucleotide complex exposes thiol‐1 as well as thiol‐2 groups. The long‐lived ATPase intermediate occurring at temperatures above 10°C adopts the same conformation with Mg2+ and Ca2+ ions. This intermediate does not protect the thiol‐1 and thiol‐2 groups but exposes a number of thiol‐3 groups which seem to be located distant from the active site. The conformation of the intermediate prevailing in the presence of ATP changes with lowering temperature below 10°C and is identical with that found in the presence of ADP at 0°C indicating a change in the rate‐limiting step of the hydrolytic cycle. In the absence of divalent cations no such temperature‐dependent change in conformation was observed. Evaluation of the activation entropies shows that the structure of the long‐lived intermediate occurring above 10°C in the presence of Mg2+ ions goes through a transformation from low to high order at around 20°C. In the case of the monovalent‐cation‐stimulated ATPase a constant activation energy of around 70kJ/mol, typical of many enzyme reactions, was found over the entire temperature range from 0–35°C.
Rapidly Labelled RNA in Tetrahymena pyriformis
Authors: POUSADA Claudina R. , MARCAUD Lise , PORTIER Marie‐Madeleine , HAYES Donal H.
Published: March 2005

39‐S, 29‐S and 18‐S nuclear rRNA precursors of Tetrahymena pyriformis have been characterized by acrylamide gel electrophoresis. Evidence is presented for the presence of high‐molecular‐weight non‐methylated RNA species (28 – 39 S, Mr range 1.9 – 2.7 × 106) in the cytoplasm of this protozoa.
Activity of Polyribosomes from the Muscle of Normal and Dystrophic Mice in Cell‐Free Amino‐Acid Incorporation
Authors: NWAGWU Mark
Published: March 2005

Polyribosomes sedimenting in the manner characteristic of those from embryonic chick muscle, as described by Heywood et al. in 1967 (Proc. Natl Acad. Sci. U.S.A. 57, 1002–1009) were reproducibly obtained from normal mouse muscle by homogenization of the muscle with a Dounce homogenizer. The polyribosome profiles of dystrophic muscle were qualitatively similar to those of normal muscle except that the relative amount of ribosomes in polyribosome complexes was smaller (44%± 3 S.E.) in dystrophic muscle than in normal muscle (67%± 4 S.E.). In spite of this difference, polyribosomes from dystrophic muscle incorporated amino acids in vitro at a faster rate and produced a larger amount of polypeptide at the end of the reaction than polyribosomes from normal muscle.
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.
Isolation and Identification of 2‐Deoxy‐2,3‐dehydro‐N‐Acetylneuraminic Acid from the Urine of a Patient with Sialuria
Authors: KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. , SCHAUER Roland , STRECKER Gèrard , MOÖTREUIL Jean
Published: March 2005

N‐Acetylneuraminic acid preparations from the urine of a patient with sialuria contain 1—2% of 2‐deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid. This new human sialic acid was isolated by ion‐exchange and partition chromatography. The structure has been elucidated by mass spectrometry and confirmed by comparison with the synthetic compound. The properties of this unsaturated sialic acid in the orcinol/Fe3+/HCl and the periodic acid/thiobarbituric acid tests as well as in thin‐layer and gas‐liquid chromatography are described. It does not react with acylneuraminate pyruvate‐lyase. The origin of this new human sialic acid is discussed.
Proteolytic Enzymes of Neurospora crassa
Authors: SIEPEN Dietrich , YU Peter‐Hao , KULA Maria‐Regina
Published: March 2005

Five intracellular proteolytic enzymes from Neurospora crassa were isolated and partially characterized: an acidic and an alkaline endopeptidase, one carboxypeptidase and two aminopeptidases. All these proteinases were purified from the same crude extract to homogenity by heat treatment, precipitation with ammonium sulfate, chromatography on DEAE‐cellulose, CM‐cellulose, DEAE‐Sephadex, hydroxyapatite and by gel filtration. The acid proteinase hydrolysed acid‐denatured haemoglobin at pH 3.0. The alkaline proteinase and the carboxypeptidase are serine proteinases that require a sulfhydryl group for activity. The aminopeptidases are both metallo‐proteinases; one posseses broad specifity to the B‐chain of oxidized insulin, the other posseses only narrow specifity and can only split the N‐terminal basic amino acids of peptides.
Inactivation of Rhodanese by Pyridoxal 5′‐Phosphate
Authors: CANNELLA Carlo , PECCI Laura , COSTA Mara , PENSA Bernardo , CAVALLINI Doriano
Published: March 2005

Pyridoxal 5′‐phosphate and other aromatic aldehydes inactivate rhodanese. This inactivation reaches higher extents if the enzyme is in the sulfur‐free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5′‐phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5′‐phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate–rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5′‐phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship between loss of activity and pyridoxal 5′‐phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5′‐phosphate.
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.
Specific Determination of N‐Acetyl‐β‐d‐hexosaminidase Isozymes A and B by Radioimmunoassay and Radial Immunodiffusion
Authors: GEIGER Benjamin , BEN‐YOSEPH Yoav , ARNON Ruth , NAVON Ruth
Published: March 2005

The two major isozymes of N‐acetylhexosaminidase, namely hexosaminidases A and B were quantitatively determined in tissues and biological fluids of both normal individuals and Tay‐Sachs patients. The determination was carried out by two sensitive immunoassays: radial immunodiffusion, using chromogenic substrate, and radioimmunoassay, which were developed in this study. For this pupose we used either a cross‐reactive antiserum which reacts to a similar extent with both isozymes, or an antiserum reacting exclusively with hexosaminidase A (obtained by selective immunoadsorption). This enabled the quantitisation of the two isozymes separately, or in the presence of each other, in purified enzyme preparations or in tissue homogenates, affording a direct positive determination of hexosaminidase A. The results demonstrated that normal tissues contain the two isozymes in comparable amounts, whereas tissues of Tay‐Sachs patients lack hexosaminidase A or any material which carries the A‐specific antigenic determinants. The possible applications of these assays and their potential use in diagnosis are discussed.
On the Primary Structure of Human Plasminogen and Plasmin
Authors: WIMAN Björn , WALLÉN Per
Published: March 2005

Most of the cyanogen bromide fragments obtained from human plasminogen and plasmin have been purified using combinations of gel filtration and ion‐exchange chromatography. The purified fragments have been characterized by molecular weight determination (dodecyl sulphate electropho‐resis), amino acid analysis, carbohydrate analysis and direct NH2‐terminal amino acid sequence determination. Since some of the purified fragments were compounds with uncompletely cleaved methionyl bonds it was possible to clarify the organization of most of the cyanogen bromide fragments in the plasminogen molecule. The fragment containing the arginyl‐valyl bond cleaved during the second step of the activation process is further identified. It is also shown that the micro‐heterogeneity that normally exists in human plasminogen probably has its origin in several sites. One such site is situated in the light (B) chain of plasmin, while another is situated in the carboxy‐terminal part of the heavy (A) chain. Neither of these sites seems to contain sialic acid.
Amino‐Acid Composition of the Covalent Rigid‐Layer Lipoprotein in Cell Walls of Proteus mirabilis
Authors: GRUSS Peter , GMEINER Jobst , MARTIN Hans Herbert
Published: March 2005

Cell walls of Proteus mirabilis in the stationary phase of growth contain a lipoprotein in covalent linkage to peptidoglycan and probably also in free form in the outer membrane. The protein moiety of this lipoprotein is composed of about 50 amino acids and has an approximate molecular weight of 5500. The Proteus lipoprotein has glycine and phenylalanine as specific components which are not present in lipoproteins of other enteric bacteria. Treatment of the peptidoglycan‐lipoprotein complex of Proteus with trypsin leaves lysine as the only lipoprotein amino acid attached to the peptidoglycan. This suggests that in P. mirabilis, as in Escherichia coli, the lipoprotein is linked to the peptidoglycan by its C‐terminal lysine residue.
The Kinetics of the Exchange of G‐Actin‐Bound 1: N6‐Ethenoadenosine 5′‐Triphosphate with ATP as Followed by Fluorescence
Authors: WAECHTER Felix , ENGEL Juergen
Published: March 2005

1: N6‐Ethenoadenosine 5′‐triphosphate (ɛATP), a fluorescent analog of ATP, binds to monomeric actin with a binding constant which is only about 5 times smaller than that of ATP. The spectroscopic changes which occur when ɛATP binds to actin are studied and used to monitor the kinetics of nucleotide exchange. The first‐order rate constant which is measured at a large excess of ATP over ɛATP strongly depends on the ATP and Ca2+ concentrations. This finding is explained by a mechanism in which the nucleotide dissociates much more easily from Ca2+‐free than from Ca2+‐bound actin. Of special interest is the temperature dependence of the dissociation rate constant. The Arrhenius plot shows a sharp bend near 24 °C.
Isolation and Characterization of Active Ribosomal Subunits from Human Placenta
Authors: VANDUFFEL Luk , PEETERS Ben , ROMBAUTS Wilfried
Published: March 2005

We have developed a method for the large‐scale isolation of active ribosomal subunits from human placenta. The technique involves incubating crude ribosomes for 15 min at 37°C with 0.2 mM puromycin in 50 mM Tris‐HCl buffer, pH 7.6, 500 mM KCl and 3 mM MgCl2 followed by centrifugation at 5 °C in a BXV zonal rotor using an equivolumetric sucrose gradient in the same buffer, upon which 80–90% of all ribosomes are dissociated into subunits. The purified subunits differ in their chemical composition, the 60‐S particle containing no more than 36% protein whereas the 40‐S subunit consists of 43% protein. In poly(U)‐directed protein synthesis, tested in a completely homologous cell‐free system, one recombined couple polymerizes at 37°C 12 to 17 phenylalanine residues at an initial rate of 0.7 residues per minute. However, free 80‐S ribosomes obtained by puromycin treatment of the crude ribosomes and reassociation of the subunits without prior isolation, have an even higher incorporating activity (20–25 mol phenylalanine/mol of ribosome). At least 55% of the subunits were estimated to actively participate in the polyphenylalanine synthesis.
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.
Proteoglycans of Guinea‐Pig Costal Cartilage
Authors: LOHMANDER Stefan
Published: March 2005

Proteoglycans were extracted, in a yield of about 90%, from costal cartilage of young, growing guinea‐pigs. Three solvents were used in sequence: 0.4 M guanidine · HCl, pH 5.8, 4 M guanidine · HCl, pH 5.8, and 4 M guanidine · HCl/0.1 M EDTA, pH 5.8. The proteoglycans were purified and fractionated by cesium chloride density gradient ultracentrifugation under associative and dissociative conditions. Gel chromatography on Sepharose 2 B of proteoglycan fractions from associative centrifugations showed the presence of both aggregated and monomer proteoglycans. The ratio of aggregates to monomers was higher in the second extract than in the other two extracts. Dissociative gradient centrifugation gave a similar distribution for proteoglycans from all three extracts. Thus, with decreasing buoyant density there were decreasing ratios of polysaccharide to protein, and of chondroitin sulfate to keratan sulfate. In addition, there was with decreasing density an increasing ratio of chondroitin 4‐sulfate to chondroitin 6‐sulfate. Amino acid analyses of dissociative fractions were in accordance with previously published results. On comparing proteoglycan monomers of the three extracts, significant differences were found. Proteoglycans, extracted at low ionic strength, contained lower proportions of protein, keratan sulfate, chondroitin 6‐sulfate and basic amino acids than those of the second extract. The proteoglycans of the third extract also differed from those of the other extracts.

The results indicate that the proteoglycans of guinea‐pig costal cartilage exist as a very polydisperse and heterogenous population of molecules, exhibiting variations in aggregation capacity, molecular size, composition of protein core, degree of substitution of the protein core, as well as variability in the type of polysaccharides substituted.