bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Mitochondrial‐Protein Synthesis in Rat Small‐Intestinal Epithelium
Authors: de Jonge Hugo R. , HÜLSMANN Willem C.
Published: March 2005

Because of its very fast turnover, implying a rapid biogenesis of intestinal mitochondria, rat‐small‐intestinal epithelium was regarded as an excellent target for the action of chloramphenicol and oxytetracycline, specific inhibitors of mitochondrial protein synthesis. With constant blood levels of chloramphenicol and oxytetracycline above 10 μg/ml the levels of the mitochondrial cytochromes aa3, b and c1 in villous and crypt cells decreased gradually until a minimum level was reached after about 48 h of treatment. The rapid onset of the fall in cytochrome aa3 content within the villous cells indicated a further synthesis of this enzyme outside the proliferative compartment of the crypt. No significant change was observed in the cellular contents of numerous other mitochondrial and extramitochondrial enzymes, confirming the restricted function of the mitochondrial protein‐synthesizing machinery for the biosynthesis of mammalian mitochondria. Prolonged treatment with oxytetracycline, but not with chloramphenicol, caused a severe damage of the villous epithelium. This toxic effect was probably not related to the lowered cytochrome content of the mitochondria.
5‐S RNA: Investigation of the Different Extent of Phosphorylation at 5′ Terminus
Authors: Soave Carlo , NUCCA Roberto , Sala Elide , VIOTTI Angelo , Galante Ennio
Published: March 2005

5‐S RNA from wheat germ, homogeneous by gel filtration and polyacrylamide gel electrophoresis, yields three peaks by hydroxyapatite chromatography. Experiments are presented which firmly establish that the three peaks are constituted by 5‐S RNA molecules ending at their 5′ terminus with mono‐, di‐, or triphosphate groups. The isolation of 5‐S RNA by direct extraction of RNA from wheat embryos homogenized in different buffers and the inability of Escherichia coli alkaline phosphatase to dephosphorylate unmelted 5‐S RNA molecules suggests that the three species are not an artifact of extraction.

The peculiar ability of the hydroxyapatite chromatography to fractionate 5‐S RNA molecules on the basis of the extent of their 5′‐terminal phosphorylation was used to investigate the pattern of 5′‐phosphorylation of 5‐S RNA from various organisms. The most striking feature observed is that 5‐S RNAs from prokaryotic organisms contain only 5′‐monophosphate molecules. In contrast, the presence of 5′ mono‐, di‐ and triphosphate molecules was observed in all the eukaryotic organisms investigated. The significance of this finding in relation to the biosynthesis of 5‐S RNA is discussed.
Studies on Soybean Trypsin Inhibitors
Authors: Koide Takehiko , IKENAKA Tokuji
Published: March 2005

Soybean trypsin inhibitor (Kunitz) was divided into four peptide fragments by the combination of limited hydrolysis with trypsin at acidic pH, chemical cleavage of methionyl bonds with cyanogen bromide, and reduction and carboxymethylation of disulfides in protein or peptide. Each fragment was separated and purified by gel filtration on Sephadex G‐50, G‐75 or G‐100, and referred to as fragments A, B, C, and D from the amino‐ to the carboxyl‐terminal region of the inhibitor.

Soybean trypsin inhibitor (Kunitz) consisted of 181 amino acid residues and its molecular weight was proved to be 20100. Fragments A, B, C, and D consisted of 63, 21, 30, and 67 amino acid residues, respectively. One of the two disulfide bridges in the inhibitor was involved in fragment D, and the other linked fragment A with fragment C.
Purification and Properties of an Inhibitor of the Tryptophan‐Synthase‐Inactivating Enzymes in Yeast
Authors: Ferguson A. Ross , Katsunuma Tsunehiko , Betz Heinrich , Holzer Helmut
Published: March 2005

An inhibitor of the tryptophan‐synthase‐inactivating enzymes has been isolated from bakers' yeast and purified 27‐fold on a protein basis, by heat treatment, precipitation by trichloroacetic acid, ethanol fractionation and acid precipitation. The inhibitory activity of the purified material is not affected by incubation with RNAase, DNAase, or muramidase, but is destroyed by incubation with pronase or trypsin. Inhibitory activity depends, therefore, on the protein content of the material.

The extent to which the inactivating enzymes are inhibited is proportional to inhibitor concentration. Tryptophan‐synthase inactivases I and II are both inhibited. The inhibitor appears to be specific in its action: it is much more effective than soybean trypsin inhibitor in inhibiting the inactivases, but has comparatively little effect on the esterolytic activity of trypsin.

Inhibitory activity was measured in crude extracts of Saccharomyces cerevisiae growing in batch culture. In the exponential growth phase, the inhibitor was barely detectable, but its levels subsequently rose at the end of the exponential phase and in the stationary phase. The possible biological significance of these observations is discussed.
A Mutant of Escherichia coli Defective in Ribonucleosidediphosphate Reductase
Authors: Karlström H. Olle , Fuchs James A.
Published: March 2005

We have studied the ribonucleosidediphosphate reductase of a deoxyuridine‐requiring mutant LD195 of Escherichia coli K. This enzyme was purified 50fold from the mutant and a control strain. In all steps of the purification the mutant strain yielded normal activity of the B1 subunit of the enzyme but approximately 10% of the wild‐type activity of the B2 subunit. The activity is similarly reduced for all three substrates tested CDP, UDP, and GDP.

The mutation appears to affect the physical structure of the B2 subunit, since addition of high concentrations of sodium acetate can restore enzymatic activity to the partially purified B2 subunit from the mutant strain. In contrast, the same concentrations strongly inhibit the wild‐type B2 subunit.

Although the mutant has a requirement of deoxyuridine at 42°C but not at 30°C (as shown in an accompanying paper), mutant protein B2 is not more thermo‐labile than wild‐type protein B2. This requirement therefore probably reflects an increased demand of deoxyribonucleotides at higher temperatures.

Hydroxyurea inhibits mutant and wild‐type ribonucleosidediphosphate reductase to the same extent in vitro. In spite of this, hydroxyurea inhibits growth of the mutant strain at a 20fold lower concentration than that required for comparable inhibition of growth of the parent. This is explained by the greatly decreased protein B2 activity of the mutant.
Purification of a Phosphodiesterase from Bothrops atrox Venom by Affinity Chromatography
Authors: Frischauf Anna‐Maria , ECKSTEIN Fritz
Published: March 2005

The purification of a phosphodiesterase from Bothrops atrox venom by chromatography on phosphocellulose and affinity chromatography with O‐(4‐nitrophenyl)‐O′‐phenyl‐thiophosphate ester coupled to activated Sepharose is described. Phosphatases are removed by chromatography on hydroxyapatite. The enzyme is a single‐chain protein with a molecular weight of approx 130000 as judged by dodecylsulfate‐gel electrophoresis and ultracentrifugation. It has a specific activity of 3.3 μmol bis(4‐nitrophenyl)phosphate hydrolyzed min−1 mg−1 at 24°C.
Cytochrome c: A Thermodynamic Study of the Relationships among Oxidation State, Ion‐Binding and Structural Parameters
Authors: Margalit Rimona , SCHEJTER Abel
Published: March 2005

The standard redox potential at I= 0.01, 25°C and pH 7.0 has been determined for the following species of cytochrome c: horse heart, baker's yeast isoenzyme‐1, Candida species yeast, tuna heart and turkey heart. The thermodynamic parameters of the redox reaction were evaluated and found to be identical, within experimental error, in the five species investigated. The effect of pH on the standard redox potential of horse heart cytochrome c was studied in the pH range 7.0–11.2. A single heme‐linked ionization of the oxidized protein was observed in this pH range, with a dissociation constant of 3 × 10−9 at I= 0 and 25°C. The effects of the electrostatic media on the standard redox potential of horse heart cytochrome c depend on the nature of the anions employed. For non‐binding medium at 25°C, the observed potential dependence on the ionic strength is given by the equation: E0obs= 0.274–0.336 √I/(1 + 6√I). In binding medium, specific binding of ions to the protein takes place.
The Kinetics of the Conversion of Chicken Pepsinogen to Chicken Pepsin
Authors: Bohak Zvi
Published: March 2005

The kinetics of the conversion of chicken pepsinogen to chicken pepsin was studied at 25°C by following the appearance of peptic activity using a pH‐static method and by following the changes in the fluorescence emission of the protein. It was found that in pH range of 2–4 both processes were first‐order reactions, and that the change in fluorescence emission was faster than the appearance of activity. The results are interpreted in terms of a unimolecular mechanism comprising two steps. In this pH range the zymogen undergoes a rapid conformational change to form an intermediate, and the subsequent conversion of this intermediate to the active enzyme is the rate‐determining step. In this step the amino‐terminal peptide of the zymogen is split by intramolecular catalysis which requires the protonation of a group, presumably a carboxyl group, with a pK of 3.1 in the active site of the enzyme. Above pH 4 the conformational change becomes the rate‐deter mining step in the unimolecular activation process, and a bimolecular pathway in which chicken pepsinogen in its native conformation is activated by active chicken pepsin becomes apparent.

It was found that the peptide fragment formed during the conversion of chicken pepsinogen to chicken pepsin, though not an inhibitor of chicken pepsin activity, can combine with chicken pepsin and protect it against denaturation at pH 10.
Adsorption of Nerve Growth Factor onto Surfaces Implications for the Assay in Tissue Culture
Authors: Pearce Frederick L. , BANTHORPE Derek V. , Cook Janet M. , VERNON Charles A.
Published: March 2005

Apparently minor modifications to the basic method for the assay of nerve growth factor resulted in differences in measured biological activity of up to nine orders of magnitude. These differences were caused by the reversible adsorption of the material onto the apparatus used in the assay procedure. The adsorption was probably promoted by London dispersion forces since the effect persisted both over a range of pH and when glass, plastic and silicone‐coated glass apparatus were used.

Under conditions in which the adsorption was minimised, the test material showed a specific activity in tissue culture of between 10−2 and 10−3 [μg ml−1. Previous reports of highly active preparations of nerve growth factor, some having specific activities of about 10−10μg ml−1, can probably be attributed to similar artefacts in the assay method.
Purification of a Messenger‐Specific Initiation Factor from Ascites‐Cell Supernatant
Authors: Wigle Donald T.
Published: March 2005

A supernatant initiation factor from Krebs II ascites cells required for the initiation of translation of encephalomyocarditis virus RNA has been purified to homogeneity. The purification process involves (NH4)2SO4 fractionation followed by chromatography on hydroxyapatite, DEAE‐Sephadex A‐50, Sephadex G‐100 superfine and phosphocellulose. The molecular weight of the factor is about 53000 as determined by dodecylsulfate‐polyacrylamide‐disc‐gel electrophoresis.
Studies on Yeast Hemoglobin
Authors: Oshino Reiko , Oshino Nozomu , Chance Britton , Hagihara Bunji
Published: March 2005

Among the Candida species, C. mycoderma and C. roubsta possess significant amounts of a hemoglobin‐like pigment. This pigment is identified as yeast hemoglobin by its spectroscopic properties and its ability of reversible binding with oxygen. The oxygen affinity of yeast hemoglobin in the respiring and nonrespiring cells is 0.02 μM which is identical to the value observed with an isolated yeast hemoglobin. Yeast hemoglobin in the cell can be decomposed specifically by pretreatment of the cells with ethyl hydrogen peroxide. Comparison of the hemoglobin‐free cells with the intact cells indicates that a lack of yeast hemoglobin does not cause any significant delay of cell multiplication. Neither the rate of cell respiration nor the behaviors of the redox states of mirochondrial cytochromes responding to the changes of oxygen concentration between 1 and 0.01 μM is affected by the presence or absence of yeast hemoglobin.
Tyrosyl‐tRNA Synthetase of Escherichia coli B
Authors: Chousterman Suzanne , Chapeville François
Published: March 2005

Using EDTA‐dialyzed tyrosyl‐tRNA synthetase and tRNA, which retain 0.8 and 0.25 atom of Mg2+ per molecule, respectively, tyrosine activation as well as tyrosyl‐tRNA formation were investigated in the absence of added magnesium ions. It is shown that the tyrosyladenylate · enzyme complex is still formed, but the rate of the reaction is reduced by a factor of 104. The rate of the ATP‐PPi exchange reaction, under these conditions, is reduced by a factor of 4 · 104. In the presence of pyrophosphate the predominant reaction is not the pyrophosphorolysis of the enzyme‐bound tyrosyladenylate but its hydrolysis.

The rate of tyrosyl‐tRNA formation, starting with free ATP and tyrosine and in the presence of spermidine, was only 15 times lower than in the presence of Mg2+. Spermidine has no effect on the rate of the ATP‐PPi exchange reaction.
Properties of Purified Malic Enzyme in Relation to Crassulacean Acid Metabolism
Authors: Brandon Pieter C. , van Boekel‐mol Titie N.
Published: March 2005

Malic enzyme from leaves of the Crassulacean species Bryophyllum tubiflorum was purified more than 1200‐fold by a rather simple procedure involving repeated fractionation with ammonium sulphate and gel‐filtration. A specific activity (international standards) of 68 was obtained. The molecular weight of the enzyme, measured by the gel‐filtration method, was 237000.

Thiol activation is required for catalytic activity. The activity is completely dependent on divalent cations; greatest activity was obtained with Mn2+, an equimolar amount of Mg2+ was 86% as effective. Maximum activity was obtained at about pH 7.2. At 30 °C and pH 7.2 the Km for Mn2+ was 0.46 mM, that for NADP was 14 μM, and that for malate amounted to 0.37 mM. The Km for malate was temperature‐dependent: it remained constant in the temperature range from 17 to 39 °C, but below 17 °C and beyond 39 °C the affinity for malate decreased and a Km‐value of 0.94 mM was recorded. The enzyme was completely NADP‐specific and was stable at temperatures up to 55 °C; maximum activity was recorded at 50 °C.

No effects of monovalent cations such as Na+, K+, Li+, or NH4+ could be recorded. The enzyme was inhibited by coenzyme A and thiamine pyrophosphate, but not by acetyl‐coenzyme A. The effects of temperature as well as the inhibition by coenzyme A and thiamine pyrophosphate fit the physiology of Crassulacean acid‐metabolism.
Fingerprinting Double‐Stranded Non‐Radioactive RNA from a Fungal Virus
Authors: Loviny Thérèse , Székely Maria
Published: March 2005

The fingerprinting technique has been applied to double‐stranded viral RNA. Under appropriate conditions pancreatic and T1 ribonuclease digests were obtained which upon two‐dimensional paper electrophoresis yielded specific patterns, similar to those obtained from single‐stranded RNAs. The structure of some simpler nucleotides on the fingerprints have been determined. The method seems suitable to characterize and sequence all kinds of double‐stranded RNA molecules.

Labelling of the virus in vivo yielded RNA preparations of rather low radioactivity. Polynucleotide kinase was used therefore to label the digest of non‐radioactive RNA for fingerprinting. The possibilities and limitations of this technique for sequencing non‐radioactive nucleic acids is discussed.
Etude de l'acide polyxanthylique et de ses complexes
Authors: Bachner Lucien , Massoulié Jean
Published: March 2005

Poly(X) displays two kinds of thermal dissociations, corresponding to two different states of the polymer. At low temperatures, and for values of pH which are not too high, the polymer is organized in a multi‐stranded structure, in which the xanthylic residues are partially ionized, i.e.carry negative charges; this structure is called poly(Xα−)n, where α is the degree of ionization, and n the number of strands. α is a variable parameter. The thermal dissociation of this structure is cooperative, and its transition temperature (TmX) is maximum at pHmax. pHmax depends upon the saline concentration.

In alkaline solutions, the xanthylic residues are totally ionized: poly(X−) shows another type of thermal transition. Even though this transition is not cooperative, it differs from purely non‐cooperative transitions in that its melting temperature is dependent upon salt concentration (ΔTm= 13 °C for a factor of 10), and the ΔH value is extremely high (109 kJ/mol, or 26 kcal/mol).

Poly(X) forms a double‐stranded complex with poly(U) in which the xanthylic residues are negatively charged, poly(X−) · poly(U). With poly(I) we find two complexes. The pH range of stability of these complexes implies that the degree of ionisation of the xanthylic groups is variable, as we found in the structure poly(Xα−)n: These two‐ and three‐stranded complexes, poly(Xα−) · poly(I) and poly(Xα−) · 2 poly(I) are differently affected by ionic strength. We describe the stability diagrams (temperature, pH, ionic strenght) for all these structures, and we present possible hydrogen bonding schemes. It appears that the negative charges of the xanthylic residues help strengthen the hydrogen bonds, so that the stability of these structures depends upon this ionization (α): the balance between this factor and electrostatic repulsion would then explain the occurrence of an optimum pH value for the stability of the structure.
On the Mechanism of Oligonucleotide‐Primed DNA Synthesis
Authors: Oertel Wolfgang , Schaller Heinz
Published: March 2005

Oligodeoxyribonucleotides from various sources and of defined chain length were examined for their capacity to prime DNA synthesis on base complementary and non‐complementary single‐stranded DNA templates. Using Escherichia coli DNA polymerase I as enzyme, primer activity could be detected at chain lengths as few as five to six nucleotides. The activity was higher on base complementary templates and increased progressively with chain length from 0.07% priming efficiency to about 20% at chain length 16. On non‐complementary templates primer activity was low with maximal efficiencies around 0.1% at chain lengths of 7–10. Polypyrimidine nucleotides and homooligonucleotides of dT, dA and dC showed very low primer efficiencies (0.0001%–0.03%). The high priming activity of the complementary oligonucleotides strongly depended on the preincubation with the template and was paralleled by the stable annealing of primer molecules to the template DNA. It is concluded that the annealing reaction of template and primer is the rate‐limiting step in chain initiation and that the stability of the base‐paired primer template duplex determines the primer efficiency of an oligonucleotide.
Studies on the Fluorescence of 1‐Anilino‐8‐naphthalenesulfonate by the Membranes of the Sarcoplasmic Reticulum
Authors: Augustin Jan , Hasselbach Wilhelm
Published: March 2005

The quantum yield of 1‐anilino‐8‐naphthalenesulfonate associated with native and lipid‐depleted sarcoplasmic vesicles is nearly identical. The slight increase of the average apparent dissociation constant of the interaction of this dye with lipid‐depleted vesicles is accompanied by a decrease in the number of binding sites for the dye.

Oleic acid is a noncompetitive inhibitor of the anilino‐naphthalenesulfonate binding to native and lipid‐depleted sarcoplasmic vesicles.

Rising temperatures decrease the quantum yield and increase the average apparent dissociation constant.

The polarisation of the fluorescence of membrane‐bound anilino‐naphthalenesulfonate reaches relatively high values with a significant increment for lipid‐depleted sarcoplasmic vesicles. There is only a slight temperature dependence and no viscosity dependence for both native and lipid‐depleted vesicles. The polarisation is reduced by rising concentrations of anilino‐naphthalenesulfonate.

Energy transfer measurements indicate that the dye is located very closely to the membrane‐bound tryptophan, suggesting a donor‐acceptor distance of 2 nm.
Interactions of Substrates with a Purified 4‐Methoxybenzoate Monooxygenase System (O‐Demethylating) from Pseudomonas putida
Authors: Bernhardt Frithjof‐Hans , Erdin Nazmiye , Staudinger Hansjürgen , Ullrich Volker
Published: March 2005

The interaction of substrates with a 4‐methoxybenzoate O‐demethylating enzyme system was studied by use of crude cell‐free extracts and also by the purified enzyme system. The two components of the enzyme system, an iron‐containing flavoprotein and an iron‐sulphur protein, were obtained in pure state from Pseudomonas putida grown on 4‐methoxybenzoate as the sole carbon source. The purified enzyme system requires NADH and oxygen as cofactors and acts on various substrates. The highest affinity is found for 4‐methoxybenzoate, but also N‐methyl‐4‐aminobenzoate is demethylated and 4‐alkylbenzoates are hydroxylated at the side chain. The enzyme is rather specific for para‐substituted benzoic acid derivatives whereas 3‐methoxybenzoate and 4‐hydroxy‐3‐methoxybenzoate are demethylated slowly. The enzyme is also able to hydroxylate the aromatic ring. This is shown by the isolation of 3,4‐dihydroxybenzoate as the hydroxylation product of 3‐hydroxy‐ or 4‐hydroxy‐benzoate, respectively. Studies on substrate binding and oxygen consumption with substrate analogues showed an absolute requirement for the carboxy group at the aromatic ring. Benzoic acid derivatives without a suitable CH‐bond uncouple oxygen uptake with a concomitant formation of hydrogen peroxide. Measurements of oxygen consumption indicate that the affinity towards oxygen is substrate dependent, probably due to steric alterations as a consequence of substrate binding.
Biosynthesis of Cartilage Procollagen
Authors: Dehm Peter , Prockop Darwin J.
Published: March 2005

Cells were isolated by controlled enzymic digestion of sternal cartilages from chick embryos and the cells were studied immediately after isolation for up to 4 h in vitro. The cells isolated under the conditions employed had a high degree of viability, they remained in suspension for 2 to 4 h in vitro and during this time they synthesized and secreted collagen in the precursor form which has been called “transport form” or “procollagen”. The cells also incorporated [35S]sulfate into macromolecules which were secreted into the medium.

Continuous labeling of the cartilage cells demonstrated a lag of about 33 min between the time at which the rate of synthesis of collagen [14C]hydroxyproline became linear and the time at which the secretion of collagen [14C]hydroxyproline into the medium became linear.

Gel filtration and polyacrylamide‐gel electrophoresis in sodium dodecylsulfate indicated that all the secreted collagen was in the precursor form. The procollagen differed from the procollagen previously isolated from bone, tendon and cultured fibroblasts in that it apparently consisted of a single type of polypeptide chain. Isotopic studies suggested that the NH2‐terminal extension on the cartilage procollagen contained cystine and tryptophan and appeared therefore to be similar in this respect to that of other procollagens.
Characterization of Five Neurotoxins Isolated from the Venoms of Two Elapidae Snakes Naja haje and Naja nigricollis
Authors: Kopeyan Charles , van Rietschoten Jurphaas , Martinez Gérard , Rochat Hervé , Miranda François , Lissitzky Serge
Published: March 2005

Two neurotoxins were purified from Naja nigricollis venom using equilibrium chromatography. This method was shown to produce the most resolution to purify proteins having very close physico‐chemical properties. The toxins obtained were characterized, as well as three neurotoxins previously isolated from Naja haje venom, by the determination of N‐terminal (35 to 39 residues) and C‐terminal sequences.

The usefulness of N‐terminal automatic sequencing for rapid identification of proteins is discussed.
Role of DNA‐RNA Hybrids in Eukaryotes
Authors: Wyers Françoise , Sentenac André , Fromageot Pierre
Published: March 2005

A ribonuclease H activity which degrades RNA‐DNA hybrids has been partially purified from extracts of Saccharomyces cerevisiae. The activity is easily detected in the crude extracts of exponentially growing cells. The purified enzyme is a small, basic, considerably heat resistant protein which has the following properties: (a) it shows no activity with single‐stranded RNA or synthetic double‐stranded RNA but rapidly hydrolyses the RNA moiety of RNA‐DNA hybrids; (b) the action of the enzyme is predominantly and, may be exclusively, endonucleolytic in character: it will, therefore, remove hybridized RNA covalently bound to a DNA chain; (c) extensive degradation of the hybrid yields a limit digest of oligoribonucleotides which dissociate from the DNA strand and become resistant to further cleavage and (d) the enzyme is most active at alkaline pH and requires Mg2+ and thiol groups for optimum activity. Reagents for sulfhydryl groups, ethidium bromide and some rifampicin analogues are potent inhibitors of the reaction. An RNA‐dependent DNA polymerase activity co‐purifies with ribonuclease H but the polymerase activity can be selectively heat inactivated.
Hydrogen‐Ion‐Titration Curve of Rabbit Immunoglobulin against Ovalbumin
Authors: Ansari Aftab A. , Salahuddin A.
Published: March 2005

The hydrogen ion equilibria of rabbit immunoglobulin raised against purified ovalbumin, was studied in order to see how its titration behaviour compares with that of non‐specific normal immunoglobulin. Potentiometric as well as spectrophotometric titration of the antiovalbumin was performed at three temperatures, i.e. 15, 25 and 35 °C and at an ionic strength of 0.15. From the nature of the titration curve, it was possible to count and characterise the ionizable groups in the antibody. Thus, the number of different dissociable groups per 160000g antiovalbumin were: 128 carboxyl groups (pKint. = 4.62), 16 imidazole groups (pKint. = 6.3), one α‐amino group (pKint. = 7.5, 71 ɛ‐amino groups (pKint. = 9.72), 56 phenoxyl groups (pKint. = 9.75), and 45 guanidinium groups (pKint. = 11.6) where pKint is the intrinsic pK. Using these values, the titration curve could be generated farely well up to pH 10. The ionization behaviour of carboxyl as well as imidazole groups in the specific immunoglobulin was found to be the same as that for similar groups in normal immunoglobulin. Unlike phenoxyl groups in normal immunoglobulin, 39 phenoxyl groups in antiovalbumin titrated with normal P pKint. and the remaining 17 groups became available for titration only upon alkali‐induced unfolding of the protein molecule at pH 13. The abnormally low value of the electro‐static interaction factor could be attributed to the absence of electrostatic interactions in the molecule and to the possible inadequacy in the theoretical treatment of hydrogen ion equilibria of proteins like immunoglobulin.
Biochemical and Immunological Characterization of Threonyl‐tRNA Synthetase of Two Borrelidin‐Resistant Mutants of Escherichia coli K12
Authors: Paetz Wilfried , Nass Gisela
Published: March 2005

The threonyl‐tRNA synthetase of two wild‐type Escherichia coli K12 strains and of two borrelidin‐resistant mutants thereof has been purified about 100‐fold. A neutralizing antiserum has been prepared against one of the wild‐type threonyl‐tRNA synthetases. The comparative characterization of the threonyl‐tRNA synthetases of the two wild strains and the two borrelidin‐resistant mutants revealed that the threonyl‐tRNA synthetase of one borrelidin‐resistant mutant (K12B borr 2) exhibits a lowered Km value for threonine and ATP and an increased K1 value for borrelidin with respect to threonine. Furthermore the activity of the threonyl‐tRNA synthetase of this mutant k more sensitive to higher pH values, is more rapidly heat‐inactivated and shows a different shape of its antiserum‐neutralization curve than the threonyl‐tRNA synthetase of the parental strain (K12B). The threonyl‐tRNA synthetase of the other borrelidin‐resistant mutant (K12B borr 3) was investigated in the same way and it did not show any difference to the threonyl‐tRNA synthetase of the parental strain, except that the specific activity of the enzyme in crude extracts of this mutant is 5 times as high as the corresponding value in crude extracts of K12B.

From these data it is concluded that borrelidin resistance in K12B borr‐2 is due to a structurally altered threonyl‐tRNA synthetase (structural mutant) whereas in K12B borr‐3 it is due to the constitutive increase of the threonyl‐tRNA synthetase level by a factor of five (regulatory mutant).

The inhibition of the threonyl‐tRNA synthetase by borrelidin with respect to threonine was noncompetitive in all four cases. In order to observe a considerable heat inactivation of the threonyl‐tRNA synthetase within 10 min a temperature of 60°C was necessary. The heat‐inactivation process was not affected by the presence of threonine, but it was hastened by the presence of ATP or tRNA.
Effects of Actinomycin D, Toyocamycin and Cycloheximide on the Synthesis of Low‐Molecular‐Weight Nuclear RNAs in HeLa Cells
Authors: Hamelin Richard , Larsen Christian J. , Tavitian Armand
Published: March 2005

The effects of three antibiotics on the synthesis of the small‐molecular‐weight nuclear RNA (called small nRNA) have been investigated. These antibiotics have been chosen for their different mechanism of action on RNA synthesis. From these investigations the following observations can be made.

a) Low concentrations of actinomycin D, whose effect on the synthesis of nuclear‐precursor ribosomal RNA is well known, abolish the synthesis of the 7‐S rRNA or E peak (or 28‐S‐associated rRNA). Amounts ten times higher are not sufficient to inhibit completely the synthesis of other small nRNAs although the A, C and D peaks and the methylated species of the G and G' peaks are much more inhibited than the 5‐S rRNA.

b) Toyocamycin also depresses the synthesis of the 7‐S rRNA or E peak. It does not inhibit however the synthesis of the other nucleoplasmic peaks such as the C, D and G‐G' species. It seems to exert an indirect effect on the nucleolar A, B and H species of small nRNAs at the same time as it blocks the conversion of the 45‐S precursor rRNA into the mature 28‐S and 18‐S rRNA.

c) The mechanism of action of cycloheximide on small nRNAs synthesis was investigated both in monolayers and suspension cultures. Although protein synthesis was completely inhibited in both cases, effects on small nRNAs were different: a much greater degradation of various classes of RNA was observed when suspension cultures were used. It is suggested that this degradation was due to an instability of some species of newly synthesized RNA upon cycloheximide treatment.
Purification and Partial Characterization of α‐Factor, a Mating‐Type Specific Inhibitor of Cell Reproduction from Saccharomyces cerevisiae
Authors: Duntze Wolfgang , Stötzler Dieter , Bücking‐Throm Elizabeth , Kalbitzer Sigrid
Published: March 2005

Cells of Saccharomyces cerevisiae exhibiting the α mating type excrete into the culture medium a low‐molecular‐weight substance, termed α factor. This factor, which specifically inhibits DNA replication in cells of the opposite mating type a, has been purified more than 100000‐fold from culture filtrates of α cells. Purified α factor appears to be homogeneous as judged from thin‐layer chromatography and thin‐layer electrophoresis in different systems. It shows a positive‐ninhydrin reaction and, upon hydrolysis, gives rise to several ninhydrin‐positive substances of which the amino acids leucine, glycine, proline, glutamic acid, tyrosine, tryptophan and possibly histidine have been identified. The presence of tryptophan and tyrosine is also confirmed by the ultraviolet absorption spectrum of the factor. In addition, purified α factor contains cupric ions which can be separated from the ninhydrin‐positive material by thin‐layer electrophoresis at pH 3.6. Gel filtration on Sephadex G‐25 in 8 M urea indicates a molecular weight in the range of 1400. The properties of the purified α factor are consistent with those of a low‐molecular‐ weight peptide.
Metal‐Combining Properties of Human Lactoferrin
Authors: Teuwissen Bruno , Masson Pierre L. , Osinski Pierre , Heremans Joseph F.
Published: March 2005

When iron · lactoferrin and apolactoferrin were submitted to nitration with tetranitromethane, the number of unmodified tyrosine residues recovered upon acid hydrolysis was 4–5 in the former case against 0–1 in the latter. The iron‐binding capacity of lactoferrin appeared to remain intact following nitration of the iron‐containing form of the protein, whereas metal‐combining properties were destroyed when apolactoferrin was treated similarly.

Tetranitromethane was found to cause changes in lactoferrin that went far beyond the mere nitration of a few tyrosine residues. Much of the tyrosine was destroyed and the same applied to tryptophan, part of which was converted to unidentified reaction products.
The Mechanism of Activation of Mutant β‐Galactosidase by Specific Antibodies
Authors: Melchers Fritz , Messer Walter
Published: March 2005

Measurements of the rates of activation of mutant β‐galactosidases of Escherichia coli by specific antibodies to enzyme activity suggest that the rate‐limiting step of this activation reaction is the mono‐molecular reaction, with which a mutant enzyme population changes between two conformations. Four mutant β‐galactosidases produced by one group of mutants all have an activation energy for this monomolecular reaction of 23.2 kcal/mol. Mutant enzyme‐activating antibodies appear to change the equilibrium between a wild‐type‐enzyme and a mutant‐enzyme conformation to more active molecules by stabilizing the wild‐type conformation. Different antisera activate one mutant enzyme population to different specific activities, all below that of the wild‐type enzyme.

Activating antibodies can be enriched from antibodies with specificities against determinants on the enzyme by passage of antiserum over Sepharose‐mutant enzyme immunosorbent columns.
Murex trunculus Haemocyanin
Authors: Wood Edward J. , Dalgleish Douglas G.
Published: March 2005

The oxygenation of Murex trunculus haemocyanin was studied over a range of pH values and Mg2+ concentrations by following the increase in absorption at 346 nm as oxygen was bound. In the absence of Mg2+ the oxygen‐binding curves appeared to be hyperbolic, n in the Hill plots was approximately unity, and there was no appreciable Bohr effect. In the presence of Mg2+ the Hill plots showed a number of slopes and there was a marked, reverse Bohr effect. Deoxygenation of a solution haemocyanin at pH 9.9 in the presence of 0.01 M Mg2+, consisting of about 95%, wholes and 5%, halves, resulted in dissociation to 63.2% wholes and 36.8%, halves, and this dissociation was slowly reversible upon re‐oxygenation. Changes in oxygenation state were accompanied by changes in the circular dichroism spectrum. Upon deoxygenation, the bands at 346, 480 and 560 nm of the copper · oxygen complex were abolished but there was little change in the doublet at 210–222 nm. However, the positive band at ∼250 nm approximately doubled in intensity.
Affinité comparée des sérums embryonnaires et maternels pour la corticostérone et la progestérone chez le rat
Authors: Savu Lia , Crépy Odette , Nunez Emmanuel , Jayle Max‐Fernand
Published: March 2005

The binding parameters of corticosterone and progesterone have been measured comparatively in the blood sera from rat, embryos and pregnant rats, at the 19th day of pregnancy.

The level of corticosterone binding is significantly higher in the sera of foetal rats than in in the sera of pregnant rats. In contrast, the fixation of progesterone appears quantitatively similar in the embryo and in the pregnant rat.

Scatchard analysis shows that a single protein species, with high specific affinity for corticosterone is present in the foetal as well as in the pregnant serum. The association constants have similar values, i.e.Kass= 3 to 5 × 108 M‐1 at 25 ° C; these values are characteristic for the corticosterone‐binding globulin of the rat. However, the binding capacity [concentration of binding sites (n) × number of moles of binding protein (M)] is 2 to 3 times higher in the foetal serum. The results are interpreted as indicating that the higher levels of fixation of this steroid in the embryo, compared to the mother, are due to a quantitative rather than qualitative difference between their binding proteins.

The systems involved in the binding of progesterone are more complex in the embryo as well as in the pregnant rat. The values of the association constants and the results of competition and heat‐inactivation experiments suggest a probable contribution of the corticosterone binding globulin to these systems. However, the progesterone seems to bind mainly to one or several proteins with low affinity (Kass= 6 × 106 M‐1 at 25 °C) and great binding capacity (n×M= 215 × 10‐8/g of serum proteins). The foetal and pregnant rat sera appear to behave similarly in the process of progesterone binding.
Outer Membrane of Salmonella
Authors: Mühlradt Peter F. , Menzel Johannes , Golecki Jochen R. , Speth Volker
Published: March 2005

The UDP‐galactose‐4‐epimeraseless strain 1195 of Salmonella typhimurium bears incomplete lipopolysaccharide, and only after addition of galactose to the growth medium it produces wild‐type lipopolysaccharide. It was demonstrated in a double‐label experiment that already existent, incomplete lipopolysaccharide is not completed after galactose addition but that only the newly synthesised lipopolysaccharide carries the wild‐type specificity.

It was thus possible to locate newly synthesised lipopolysaccharide with specific anti‐wild‐type, ferritin‐conjugated antibodies in freeze‐etch preparations and ultra‐thin sections.

With the freeze‐etch technique new lipopolysaccharide was found to appear in about 220 patches per cell, 30 s after 50 mM galactose had been added to the medium. After growth for 2–3 min under these conditions new lipopolysaccharide was found to be evenly spread over the entire cell surface.

In ultra‐thin sections of plasmolysed cells 86% of the ferritin patches, indicating newly synthesised lipopolysaccharide, was found located over sites where the cytoplasmic and the outer membrane adhere to one another. It is discussed that new lipopolysaccharide emerges from these sites and that lateral movement of lipopolysaccharide in the outer membrane results in an even distribution on the bacterial cell surface.
Different Effects of Streptomycin on the Ribosomes from Sensitive and Resistant Mutants of Chlamydomonas reinhardi
Authors: BOSCHETTI Arminio , BOGDANOV Stefan
Published: March 2005

Ribosomes from three mutant strains of Chlamydomonas reinhardi were analysed in the analytical ultracentrifuge. The mutants were streptomycin sensitive and streptomycin resistant with a medelian and a uniparental pattern of inheritance of streptomycin resistance. At low ionic strength, the dissociation of the 70‐S ribosomes from the sensitive and the mendelianresistant strain is greater and their dimerisation smaller than in the uniparental mutant. On the other side the ultracentrifugation pattern of the ribosomal fraction of the sensitive strain is different from that of the two resistant strains. In experiments in vivo, streptomycin prevents the dissociation of the 70‐S ribosomes and induces the formation of 100‐S dimers. These “stick‐ ing effects” are greatest in the sensitive strain and smallest in the uniparental resistant mutant. It is suggested that in the two resistant mutants two different proteins of the 70‐S ribosomes are altered and that the streptomycin resistance of the uniparental mutant is localized in the chloroplast ribosomes. This assumption is strengthend by the finding that in the uniparental mutant the chloroplast ribosomes are streptomycin resistant, as measured by the ribulose 1,5‐bisphos‐ phate activity, whereas in the chromosomal‐resistant mutant they are streptomycin sensitive.
Fixation of a Salmonella minnesota R‐Form Glycolipid on the Membrane of Normal and Transformed Rat‐Embryo Fibroblasts
Authors: Bara Jacques , Lallier Réal , Brailovsky Carlos , Nigam Vijai N.
Published: March 2005

Binding of Salmonella minnesota R form glycolipid mR595 to cultured rat embryo fibroblasts has been demonstrated by three methods: by uptake of labelled glycolipid, by passive hemagglutination and by immunofluorescence. The results by the three methods are correlative. Normal rat embryo fibroblasts exhibit lower fixation capacity than SV40 transformed rat embryo fibroblasts. Trypsinization of the two types of cells results in increased fixation of the bacterial glycolipid. Glycolipid mR595 binding is localized at the cell surface.
Molecular Models on the Insertion of a Salmonella minnesota R‐Form Glycolipid into the Cell Membrane of Normal and Transformed Cells
Authors: Bara Jacques , Lallier Réal , Trudel Michel , Brailovsky Carlos , Nigam Vijai N.
Published: March 2005

Based on chemical alterations in the cell surface of normal cells following transformation and the fixation of bacterial glycolipid mR595 by normal, transformed and trypsinized cells, theoretical models for the insertion of glycolipid mR595 in the lipid bilayer of normal, trypsinized and transformed cells are presented.
Inactivation of α‐Chymotrypsin by a Bifunctional Reagent, 3,4‐Dihydro‐3,4‐dibromo‐6‐bromomethylcoumarin
Authors: Béchet Jean‐Jacques , Dupaix Alain , Yon Jeannine , Wakselman Michel , Robert Jean‐Claude , Vilkas Michel
Published: March 2005

α‐Chymotrypsin is rapidly and irreversibly inactivated by 3,4‐dihydro‐3,4‐dibromo‐6‐bromo‐methylcoumarin (VII) at neutral pH. The inactivation is pH dependent between pH 5 and 7 and is delayed when the active site of the enzyme is protected by acetylation of the active serine‐195 or by binding of a competitive inhibitor.

This dihydrocoumarin VII and its 6‐methyl analogue are substrates for α‐chymotrypsin, but when the ester bond in VII is broken during formation of the acyl‐enzyme, a reactive p‐hydroxybenzylbromide group is generated in situ within the active site. This newly formed group then alkylates one histidine residue (probably histidine‐57), as shown by amino acid analysis of the modified enzyme on acid hydrolysis. A model non‐enzymic reaction shows that the dihydrocoumarin VII is able to alkylate imidazole in aqueous solution at pH 7, yielding the N‐substituted imidazole, 3‐bromo‐6‐(imidazol‐1‐yl‐methyl)coumarin (XI).

The modified enzyme has practically no activity against a specific substrate and seems no longer to have its intact active site. However its binding site is at least partly free for it is still able to bind proflavin.

α‐Chymotrypsin is also inactivated by 6‐bromomethylcoumarin, the ester bond of which is stable, but the mode of inactivation and the properties of the modified enzyme are different from those found in the study with the dihydrocoumarin VII.
A Rapid Assay for the Initiation of Protein Synthesis in Extracts of Animal Cells
Authors: Smith Alan E. , Wigle Donald T.
Published: March 2005

A rapid method for the quantitative and qualitative measurement of the initiation of protein synthesis in mammalian cell‐free extracts is described. Use of the antibiotic sparsomycin, which inhibits chain elongation but has little effect on initiation, results in the accumulation of short initiation peptides during protein synthesis in vitro. The initiation peptides remain bound to tRNA but are not attacked by the putative aminopeptidase responsible for the removal of N‐terminal methionine from nascent polypeptides. After electrophoresis and autoradiography such initiation peptides labelled with [35S] methionine yield a specific fingerprint for each site at which initiation has occurred. The optimal conditions for cell‐free incubation, isolation and characterisation of initiation peptides are described.
A Specific Ribonucleoprotein Fragment from Escherichia coli 30‐S Ribosomes
Authors: Székely Maria , Brimacombe Richard , Morgan Joan
Published: March 2005

32P‐labelled ribosomal sub‐particles were hydrolysed with ribonuclease T1, under conditions which have previously been shown to yield a specific fragment of RNA · protein (“band III”), containing only four of the 21 30‐S ribosomal proteins. The hydrolysate was fractionated on a polyacrylamide gel using the methods already published, and gel fractions containing the specific RNA · protein fragment were subjected to electrophoresis on a second polyacrylamide gel, in the presence of sodium dodecylsulphate to dissociate RNA and protein. Two bands of [32P]RNA were reproducibly observed, containing about 320 and 360 nucleotides, respectively.

Two‐dimensional fingerprints of ribonuclease T1, hydrolysates from both these RNA species showed that the two fragments were almost identical. Oligonucleotides from the fingerprints were digested with pancreatic ribonuclease, and the secondary digestion products analysed and compared with the known sequence of 16‐S ribosomal RNA. Several characteristic oligonucleotides from regions D, E and P of the RNA were identified unambiguously. Other oligonucleotides were found which could have arisen from more than one part of the 16‐S RNA, but in each case one of the possibilities was in regions D, E, E′ or P.

It was concluded that the band III proteins are associated with these contiguous regions of the 16‐S RNA, i.e. near to its 3′‐end.
The Translation in vitro of mRNA for Immunoglobulin Heavy Chains
Authors: Cowan Nicholas Justin , Milstein Cesar
Published: March 2005

The addition of mRNA preparations to a number of heterologous cell‐free systems has been shown to result in the synthesis of immunoglobulin light chains. This paper reports the results of experiments in which an mRNA fraction prepared from MOPC 21 mouse myeloma cells has been used to stimulate the synthesis of immunoglobulin heavy chains in rabbit reticulocyte lysates. The synthesis of heavy chains in a homologous cell‐free system containing either microsomes or membrane‐derived polysomes is also described. The synthetic product has been characterised by finger‐printing in each case. The results reveal the presence of a unique methionine‐containing peptide in digests of the product of the rabbit reticulocyte cell‐free system. This peptide is not observed in corresponding digests of heavy chains synthesized in vivo or in the homologous cell‐free system in vitro.
Reduction of C19‐, C21‐, C24‐, and C27,‐3‐Oxosteroids by Rat‐Liver Microsomes
Authors: BjÖRKHEM Ingemar , Danielsson Henry , Wikvall Kjell
Published: March 2005

The reduction of various C19, C21, C24, and C27‐3‐oxosteroids by rat liver microsomes was studied. Large variations were observed in the relative rates of 3α and 3β reduction of the 3‐oxo‐steroids. Variations were also observed in the degree of stimulation by NADH and NADPH. Except for C27‐3‐oxosteroids, the ratio between the 3α‐ and 3β‐hydroxy derivative formed was higher with microsomes from female rats than than with those from male rats. The 3α‐ and 3β‐hydroxysteroid dehydrogenase activities showed a specificity towards the 4B‐hydrogen of NADH. Experiments with different inhibitors, with partially inactivated microsomes and with partially purified Triton extracts of microsomes indicate the presence of several different 3α‐ and 3β‐hydroxysteroid dehydrogenases in rat liver microsomes with different specificities towards NADH and NADPH and with overlapping substrate specificities.