bluuebunny/crossref_metadata_2025 huggingface dataset:
Action of Rheumatoid Synovial Collagenase on Cartilage Caollagen
Authors: WOOLLEY David E. , GLANVILLE Robert W. , EVANSON John M. , LINDERG Kenneth A.
Published: March 2005

The action of purified rheumatoid synovial collagenase on purified cartilage collagen, α1(II)3, in solution at 25°C has been characterised. The enzyme attacked cartilage collagen in solution producing a 58% reduction in specific viscosity and resulting in the appearance of two reaction products which represented approximately three‐quarter and one‐quarter fragments of the intact molecule as shown by disc electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. The α‐chain fragments which comprised each of these components corresponded to molecular weights of approximately 74000 and 21000.

Electron microscopy of segment‐long‐spacing crystallites of the reaction products revealed three‐quarter (TCA) and one‐quarter (TCB) length fragments, and permitted accurate localization of the cleavage locus between bands 41 and 42 (I‐41). This cleavage site and the formation of TCA and TCB reaction products are very similar to those found for type‐I collagen substrates.

Cartilage collagen in solution was found to be more resistant to collagenase attack than tendon collagen, the rate of cartilage collagen degradation being six times slower than that for tendon collagen, as judged by viscometry. The mid‐point melting temperatures (Tm) for lathyritic cartilage and tendon collagen were 40.5 and 41.5°C, and for the collagenase‐produced reaction products 38.5 and 37.5 °C, respectively. The significance of these findings is discussed in relation to the structure of type I and II collagens.
Purification des caséines χ de brebis
Authors: SOULIER Solange , RIBADEAU‐DUMAS Bruno , DENAMUR Robert
Published: March 2005

Purification of χ‐Caseins from Sheep. Analysis of the Glycan and Peptide Components

Starting from whole individual ovine casein prepared according to the method of Shahani, K. M. & Sommer, H. H. [J. Dairy Sci. 34, 1003–1009 (1951)], χ‐casein was isolated and purified by successive steps of chromatography on columns of dextran gel and hydroxyapatite.

On filtration through Sephadex G‐150 in a buffer containing urea, the bulk of the χ‐casein behaved as aggregates appearing in the void volume. Dissociation of these aggregates by reductive cleavage of disulfide bonds with 2‐mercaptoethanol, followed by a second filtration step on Sephadex G‐150 in the presence of both urea and 2‐mercaptoethanol, resulted in retardation of the χ‐casein, with separation from a contaminant representing 10–12% of the material applied. Further purification was achieved by chromatography on hydroxyapatite which eliminated the χs‐ and β‐caseins.

The purified χ‐casein had a molecular weight of about 20000, an absorption coefficient A1%1cm at 280 nm of 10.85 and a sialic acid and phosphorous content of 0.3% (w/w) each. The sugar fraction liberated on acid hydrolysis of the caseinomacropeptide showed the presence of N‐acetyl‐galactosamine, galactose and neuraminic acid in equimolar ratio. Neuraminic acid existed mainly as the N‐glycolyl derivative.

The polypeptide chain of the ovine χ‐casein was composed of about 170 amino‐acids residues. Compared to bovine χ‐caseins, the most notable difference was the presence of one additional cysteinyl and four additional aspartyl residues.

Starch‐gel and polyacrylamide‐gel electrophoresis clearly revealed the heterogeneity of ovine χ‐casein. Chromatographic fractionation of whole χ‐casein on DEAE‐cellulose also led to the separation of several fractions, the main characteristics of which are presented. Analysis of these fractions indicated that only those components which were firmly bound to DEAE‐cellulose were glycosylated.
Fluorimetric Study of Conformational Changes of Various α‐Lactalbumins on Agarose Carriers
Authors: BAREL André O. , PRIEELS Jean‐Paul
Published: March 2005

Various insoluble α‐lactalbumins (bovine, bovine glyco‐α‐lactalbumin, human and human nitrated) have been prepared by coupling these proteins on to an agarose gel with use of cyanogen bromide.

Some intrinsic fluorescence properties, such as fluorescence maximum and pH dependence, were considered in order to study conformational changes of the α‐lactalbumins covalently bound to an insoluble matrix. Examination of the pH–fluorescence profiles as well as the position of the maximum in the emission spectrum indicates that the Sepharose matrix does not appreciably modify the conformation of human and bovine glyco‐α‐lactalbumins. Some change in the fluorescence spectrum (peak shifting towards longer wavelength) was observed for bovine α‐lactalbumin and appeared to be due to alteration of the environment of the tryptophan side‐chains in the protein upon coupling to the agarose gel. The emission spectrum of the insolubilized human nitrated α‐lactalbumin indicates that the polypeptide chain of this protein gained some native conformation when covalently bound to the carrier.

The extrinsic fluorescence of a bound dye, such as 2‐p‐toluidinylnaphthalene‐6‐sulfonate, was used to study and to compare the hydrophobic sites on the surface of insoluble α‐lactalbumins with the same proteins in solution. Considering the fluorescence properties of the protein · dye complexes it was found that both states of α‐lactalbumins (insoluble and free in solution) bind the dye with similar association constants. However, the positions of the maxima in the emission spectra are all somewhat shifted towards longer wavelengths, suggesting that the dye binding site is located in a more polar environment when the proteins are bound to agarose. The human nitrated α‐lactalbumin retains about equal possibility of binding this fluorescent dye.

As shown for bovine α‐lactalbumin in solution, the binding of 2‐p‐toluidinylnaphthalene‐6‐sulfonate towards the various insoluble α‐lactalbumins was not appreciably modified by the presence of various small compounds such as sugars and UDP, which are effectors in the lactose synthetase function.
The Metabolism of Cyclohexanol by Acinetobacter NCIB 9871
Authors: DONOGHUE Nuala A. , TRUDGILL Peter W.
Published: March 2005

Acinetobacter NCIB 9871 was isolated by elective culture on cyclohexanol and grows with this compound as sole source of carbon. It displays a restricted growth spectrum, being unable to grow on a wide range of alternative alicyclic alcohols and ketones. Cyclohexanol‐grown cells oxidize the growth substrate at a rate of 230 μl of O2/h per mg dry wt with the consumption of 5.65 μmot of O2/μmol substrate. Cyclohexanone is oxidized at a similar rate with the consumption of 4.85 μmol of O2/μmol. 1 ‐Oxa‐2‐oxocycloheptane and 6‐hydroxyhexanoate are both oxidized at the same slow rate of 44 μl of O2/h per mg dry wt and adipate is not oxidized.

Studies with cell extracts reveal the presence of inducible dehydrogenases for cyclohexanol, 6‐hydroxyhexanoate and 6‐oxohexanoate and a monooxygenase, that in conjunction with a lactonase converts cyclohexanone to 6‐hydroxyhexanoate. The monooxygenase is therefore presumed to be of the lactone‐forming type and the pathway for conversion of cyclohexanol to adipate; cyclohexanol → cyclohexanone → 1‐oxa‐2‐oxocycloheptane → 6‐hydroxyhexanoate → 6‐oxohexanoate → adipate; for which key intermediates have been identified chromatographically, is identical with the route for the oxidation of cyclohexanol by Nocardia globerula CL1.
Dihydrofolate Reductase from Bovine Liver
Authors: BAUMANN Heinz , WILSON Kenneth J.
Published: March 2005

Dihydrofolate reductase from bovine liver has been purified 5000‐fold employing conventional techniques and methotrexate/aminohexyl/Sepharose affinity chromatography. Electrophoresis of the isolated enzyme on polyacrylamide gels resulted in the separation of two enzymatically active protein components which were not interconvertible by treatment with dihydrofolate and/or the coenzyme. The two forms, present in a ratio of 20: 1, were found by isoelectric focusing to have isoelectric points of 7.15 and 5.94. They had identical specific activities towards dihydrofolate (26.1–27.0 U/mg) and folate (1.3–2.2 U/mg), and had identical molecular weights (23500) and amino acid compositions.

Due to the small quantity of the acidic form and the similarity of the two forms, the amino‐terminal sequence (19 residues) was determined on a mixture of carboxymethylated reductase.

The single sulfhydryl group of the enzyme can be modified by several sulfhydryl reagents in the native enzyme without loss of activity. Modification of the same residue occurs in the denaturated state and partially inhibits renaturation to the fully active enzyme. One disulfide bridge was detected by reduction and alkylation. The cleavage of this bond did not effect the enzymatic activity.
Hydrophobic Interaction Determined by Partition in Aqueous Two‐Phase Systems
Authors: SHANBHAG Vithaldas P. , AXELSSON Claes‐Göran
Published: March 2005

In this report we describe a new method which is useful for measuring hydrophobic interactions between aliphatic hydrocarbon chains and proteins in aqueous environment. The method is based on partition of proteins in an aqueous two‐phase system containing dextran and poly(ethylene glycol) and different fatty acid esters of poly(ethylene glycol). The partition is measured under conditions where contributions from electrostatic interactions are eliminated. The difference in partition of proteins in phase systems with and without hydrocarbon groups bound to poly(ethylene glycol), Δlog K, where K is the partition coefficient, is taken as a measure of hydrophobic interaction. Δlog K varies with size of hydrocarbon chain and type of protein. The length of the aliphatic chain should be greater than 8 carbon atoms in order to get a measurable effect in terms of Δlog K. Bovine serum albumin, β‐lactoglobulin, hemoglobin and myoglobin have been shown to have different affinities for palmitic acid ester of poly(ethylene glycol). No hydrophobic effect could be observed for ovalbumin, cytochrome c or α‐chymotrypsinogen A.
Characterization of Cytoplasmic DNA of Mouse‐Myeloma Cells
Authors: SOLAGE Adina , LASKOV Reuven
Published: March 2005

Cytoplasmic DNA from mouse myeloma cells comprised between 1 % and 2 % of the total cellular DNA. Detergent‐prepared cytoplasmic lysate consisted mainly of 8‐S and 22‐S species. While these DNA species were present in the 13000×g pellet of the detergent‐prepared cytoplasmic lysate. only the light DNA species was present in the 13000×g supernatant fraction. In neutral CsCI gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA.

The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed‐circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent‐prepared cytoplasmic lysate. It was found that poly(A)‐containing mRNA and 28‐S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.
Effects of Specific Chemical Modification of Actin
Authors: CHANTLER Peter D. , GRATZER Walter B.
Published: March 2005

G‐actin has been nitrated with tetranitromethane in conditions that lead to the modification of one tyrosine residue. The reactive residue was found by earlier workers to be Tyr‐69. The nitrated actin is conformationally similar to native G‐actin, as judged by sedimentation velocity and circular dichroism analysis. A small proportion only is in the form of covalently linked dimers and trimers. The nitrated G‐actin will polymerise to form filaments, indistinguishable in the electron microscope from those of native F‐actin, but the polymerisation process is slower. Reduction of the nitrophenol group to the corresponding aminophenol leaves the properties of the protein in respect of polymerisation unchanged. When a dansyl group is introduced at the same point, however, the ability of the actin to polymerise is lost. The nitrated actin and its reduced counterpart will also bind heavy meromyosin, and the characteristic arrowhead formation of the bound molecules along the filaments can be seen in the electron microscope. Neither of the modified F‐actins, however, significantly activates or inhibits the myosin ATPase activity. The fluorescence of nitrated actin is strongly quenched through the presence of the nitrophenol chromophore. In soluble complexes with heavy meromyosin the fluorescence is indistinguishable from the sum of the separate contributions of the two protein components. There is thus no measurable excitation transfer between any tryptophan residues on the myosin heads, such as that inferred to be present in the ATPase site, and the nitrotyrosine in position 69 of the actin sequence. Implications of this observation are considered in relation to the different interaction sites in myosin and in actin. The activation of heavy meromyosin ATPase by copolymers containing actin and nitroactin in different proportions has been measured, and is not proportional to the fraction of native actin. The results are consistent with the view that the function of actomyosin depends on the interaction of the myosin heads with more than one actin subunit.
Polyadenylated RNA from Vicia faba Meristematic Root Cells
Authors: ESNAULT Robert , TRAPY Gerard , VAN HUYSTEE Robert B.
Published: March 2005

After incubating root apices from two‐day‐old bean seedlings with [3H]adenine the RNA was extracted from whole cells or polysomes, and the poly(A) sequences were isolated by nuclease digestion followed by poly(U)‐Sepharose chromatography. The alterations of the RNA molecules due to the various treatments were monitored by sucrose density gradients. It was found that sequential extraction first at pH 7.6 then at pH 9.0 did not result in a separation between RNA poor in poly(A) sequences and poly(A)‐rich RNA. Furthermore chromatography analysis of hydrolysates from nuclease‐resistant RNA extracted either at pH 7.6 or at pH 9.0 revealed that AMP constituted nearly 95% of the bases and that the poly(A) sequences, about 200 bases, were located at the 3′ terminus of the polyadenylated RNA. No size difference was found for the poly(A) segment between the pH‐7.6‐extracted RNA and that extracted at pH 9.0.
Depolymerisation of F‐Actin to G‐Actin and Its Repolymerisation in the Presence of Analogs of Adenosine Triphosphate
Authors: MANNHERZ Hans G. , BREHME Helga , LAMP Uli
Published: March 2005

A number of ATP analogs were tested for their ability to depolymerize F‐actin and interact with it. It was found that those analogues which are hydrolysable are incorporated into the F‐actin polymer whereas the non‐hydrolysable ones are displaced from the nucleotide binding site during the polymerization process
Biochemistry of Development in Insects
Authors: MUNICIO Angel M. , GARCIA Resurrección , PEREZ‐ALBARSANZ Miguel A.
Published: March 2005

Previous experiments showed that fatty acids were incorporated into triacylglycerols by homogenates of Ceratitis capitata larvae far more efficiently than by pharate adult homogenates. This metabolic behaviour of both stages of development of the insect has been interpreted throughout the existence of a different acyltransferase activity. To obtain new data on the acyltransferase mechanism, a time‐course of the stereospecific incorporation of labelled myristic, palmitic, oleic and linoleic acids into the sn‐positions of triacylglycerols has been followed.

Studies on the stereospecific incorporation of labelled fatty acids confirmed previous results. Palmitic acid was mainly incorporated into sn‐1 and sn‐3 positions whereas position 2 exhibited a low incorporation. Myristic acid acylated sn‐3 position at a higher rate than it acylated the other sn‐positions. Oleic acid was more specifically distributed than palmitic acid and linoleic acid was more efficiently incorporated than the monounsaturated acid. All these data reflect substrate differences in the acyltransferase activity of larval homogenates.

Pharate adult homogenates incorporated fatty acids very scarcely and mainly into positions (1 + 3).

Kinetics of incorporation of labelled fatty acids into the sn‐positions points to a non‐random distribution with respect to the major saturated and unsaturated fatty acids in triacylglycerols of larvae of Ceratitis capitata.
Steroid‐Induced Early Protein Synthesis in Rat Uterus and Prostate
Authors: PENNEQUIN Pierre , ROBEL Paul , BAULIEU Etienne‐Emile
Published: March 2005

An early ‘induced protein’, after exposure of the rat uterus to estradiol, is detected among the soluble proteins with a double‐labelling technique and electrophoretic fractionation. Efforts have been directed to establish the subcellular distribution of the induced protein, since such a protein, observable 1 h after hormone administration, may play an important role in the subsequent amplified responses, especially in terms of RNA synthesis. Moreover such an early discrete induced protein was sought in a comparable system responding to another hormone, namely prostate and seminal vesicles under androgens. The induced protein was not found in uterine nuclei of 21‐day‐old rats after 1 h of estradiol action in vivo and 1 h of tissue incubation with labelled leucine. This negative result summarizes a search among different nuclear protein fractions using various procedures; nor was induced protein observed in mitochondrial and microsomal pellets. Contrary to these negative findings, slight changes of histone labelling were observed under the experimental conditions used to demonstrate induced protein. In addition histone acetylation was increased after 1 h of estradiol action in vivo and 15 min tissue labelling in vitro with radioactive acetate.

Furthermore, an increase in total protein synthesis between 0 and 2 h after estradiol action was observed, the relative increase of incorporation of radioactive leucine into protein of estradiol‐treated vs non‐stimulated uteri being corrected for variations of the acid‐soluble radioactive leucine pool.

Attempts to obtain an early and discrete induced protein with androgens in prostate and seminal vesicles of immature or castrated rats after different times of exposure to testosterone, androstanolone and estradiol have been unsuccessful.

The contribution of both negative and positive findings in steroid‐induced early protein synthesis is discussed in the context of the current knowledge of hormone action.
Compartmentation of Acetyl‐CoA in Rat‐Liver Mitochondria
Authors: VON GLUTZ Gaston , WALTER Paul
Published: March 2005

The ratio of the specific radioactivities of 3‐hydroxybutyrate: citrate was determined in rat liver mitochondria which were incubated in the presence of [1‐14C]palmitate, pyruvate, bicarbonate, ATP, phosphate and' malonate. Without compartmentation this ratio would maximally be 2, however, under our conditions values of 2.5 ‐ 3.7 were observed.

In further experiments with mitochondria, the sensitivity of pyruvate carboxylase for acetylCoA produced from various precursors was tested. It was found that acetyl‐CoA produced from L‐acetylcarnitine or by oxidation from either pyruvate, octanoate or palmitylcarnitine but not from leucine led to a stimulation of pyruvate carboxylation.

These results demonstrate a compartmentation of acetyl‐CoA in liver mitochondria. The further finding that different mitochondrial fractions showed varying ratios of specific radioactivities of 3‐hydroxybutyrate: citrate indicates that the observed compartmentation may be explained by the existence of different types of mitochondria with varying enzyme patterns and acetyl‐CoA pools
Studies of the Distribution of Proteins Bound to CNBr‐Activated Sepharose 6B at the Electron‐Microscopic Level
Authors: LASCH Jürgen , IWIG Martin , KOELSCH Regine , DAVID Heinz , MARX Ingrid
Published: March 2005

Proteins were covalently attached to Sepharose by the CNBr method. Their distribution across the carrier beads was studied at the electron microscopic level. The approach has been to ferritinstain and to section the gel beads. Ferritin was either coupled directly to the polysaccharide backbone of the carrier or conjugated with pure rabbit anti‐aminopeptidase in order to visualize covalently bound leucine aminopeptidase by the immunferritin technique. The results corroborate earlier fluorescence microscopic findings of a uniform protein distribution, provided that a number of conditions are fulfilled
RNA Polymerase B from Drosophila melanogaster Larvae
Authors: GREENLEAF Arno L. , BAUTZ Ekkehard K. F.
Published: March 2005

A purification procedure is described by which we obtained DNA‐dependent RNA polyrnerase B (or II) from third‐instar larvae of Drosophila melanogaster in essentially pure form. The enzyme is similar to the analogous polymerases from other eukaryotes in its enzymic and structural properties. lt preferentially transcribes DNAs containing single‐stranded regions, and it is inhibited by low amounts of the toxin α‐amanitin; 50 % inhibition occurs at an α‐amanitin concentration of 0.03 μ/m1. Dodecylsulfate‐polyacrylamide gel electrophoresis resolves the purified Drosophila polymerase B into ten polypeptides with molecular weights as follows: 1 (174000), 2 (137000), 3 (34000). 4 (22000). 5 (18000), 6 and 7 (16000), 8 (15000), and 9 and 10 (less than 15000). The relative amounts of polypeptides 1–4 were constant at molar ratlos of approximately 1:1:1:2 in different preparations of the enzyme, while the amounts of polypeptides 5–10 showed more variation.

An antiserum directed against the Drosophila RNA polymerase B inhibited the activity in vitro of the B enzymes from Drosophila, yeast, and calf thymus. However, only the Drosophila enzyme gave a precipitin reaction with the antiserum. When the antiserum was added to Drosophila RNA polymerase B at different stages of the purification, the resulting precipitates were found to contain nearly constant proportions of seven of the ten polypeptides present in the purified enzyme
Determination in vitro of the Rate of Protein Synthesis and Degradation in Human‐Skeletal‐Muscle Tissue
Authors: LUNDHOLM Kent , SCHERSTÉN Tore
Published: March 2005

The present studies were aimed to evaluate the possibility to use a system for estimation in vitro of the biosynthesis and degradation rates of human skeletal muscle protein. A previously characterized human skeletal muscle preparation was used.

Amino acids and insulin stimulated significantly the incorporation rate of leucine into proteins. The effect of amino acids was more pronounced than that of insulin. The stimulatory effect of insulin could be decreased by amino acids. Insulin did not influence the tissue uptake or the oxidation rate of leucine.

The release of [14C]leucine deriving from degradation of prelabelled skeletal muscle fibre proteins was linear for at least 2.5 h of incubation and optimal with leucine at concentrations beyond 12.5 mmol/1 or in the presence of puromycin in the incubation medium. The rate of the release of radioactivity was significantly inhibited by amino acids and at borderline significance by insulin but not by puromycin. The specific radioactivity in prelabelled proteins decreased significantly in the presence of puromycin suggesting that leucine derived from protein degradation was reutilized in vitro. This reutilization was found to be 9 ± 1 % of leucine released from degradation of proteins in 30 subjects.

A statistically significant positive correlation between the cathepsin D activity in human skeletal muscle tissue and the degradative rate of prelabelled muscle proteins in vitro was observed.

The results indicate that biosynthesis and degradatiön of skeletal muscle proteins in this system in vitro were subjected to control mechanisms. It is suggested that the release of radioactivity from prelabelled muscle fibre proteins during incubation probably only reflects the degradation of some rapidly‐turning‐over proteins.
Spin‐Labelled Phosphofructokinase
Authors: JONES Robert , DWEK Raymond A. , WALKER Ian O.
Published: March 2005

Rabbit muscle phosphofructokinase, spin‐Labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors. The spectral changes have been interpreted in terms of a concerted allosteric transition between two conformational states with non‐exclusive binding of effectors. Ort this basis MgATP, fructose 6‐phosphate plus ATP, and NH4+ ions behave as potent positive effectors, inorganic phosphate, sulphate, AMP, fructose 6‐phosphate and fructose 1,6‐bisphosphate are less potent activators, and free ATP and H+ ions are negative effectors, in agreement with the kinetic behaviour, but citrate behaves anomalously. In addition, the allosteric equilibrium can be displaced towards the inhibited state by selectively modifying two further thiol groups.

Strong positive cooperativity occurs under suitable conditions with ATP, metal‐ATP and fructose 6‐phosphate. Biphasic changes of conformation, attributed to binding at the catalytic and inhibitory sites, have been observed in titrations with ATP. The differentiation of the two ATP binding sites arises in the presence of fructose 6‐phosphate because of a distinct concerted effect an conformation between the two substrates at the active site. A similar effect occurs between ATP and citrate. Other heterotropic effects are more consistent with simple modelst phosphates favour the binding, and reduce the cooperativity, of fructose 6‐phosphate and metal‐ATP, whereas excess ATP and H+ ions antagonise the binding and increase the cooperativity of fructose 6‐phosphate.

The observations are related to existing kinetic and binding studies where possible. Anomalous features of the behaviour suggest that the model should be regarded only as a first approximation
Cytochrome c Interaction with Membranes
Authors: VANDERKOOI Jane M. , ERECINSKA Maria
Published: March 2005

A cytochrome c derivative from which iron is removed has been prepared and characterized. Several lines of evidente indicate that native and porphyrin cytochrome c have similar conformations: they have similar elution characteristics an Sephadex gel chromatography; in both proteins the tryptophan fluorescence is quenched and the pK values of protonation of the porphyrin are identical. Porphyrin cytochrome c does not substitute for native cytochrome c in Bither the oxidase reaction or in restoring electron transport in cytochrome‐c‐depleted mitochondria. It does however competitively inhibit native cytochrome c in these reactions, the Ki for inhibition being larger than the Km for reaction. The absorption and emission spectra, and the polarized excitation spectrum of the porphyrin cytochrome c are characteristic of free base porphyrin. The absente of fluorescence quenching of porphyrin cytochrome c when the protein is bound to cytochrome oxidase suggests that heure to heure distance between these proteins is larger than 0.5 to 0.9 nm depending upon orientation. Binding of the porphyrin cytochrome c to phospholipids or to mitochondria increases the fluorescence polarization of a positively polarized absorption band, which indicates that the bound form of the protein does not rotate freely within the time scale of relaxation from the excited state
Decreased Dissociation of the 3‐Methylcrotonyl‐CoA Carboxylase Complex from Achromobacter in the Presence of 3‐Methylcrotonyl‐CoA
Authors: SCHIELE Ulrich , STÜRZER Magdalena
Published: March 2005

By inactivation of different concentrations of 3‐methylcrotonyl‐CoA carboxylase from Achromobacter IVS with a fixed concentration of iodoacetamide, it was demonstrated that the degree of dissociation of the complex is considerably lower in the presence of 3‐methylcrotonyl‐CoA. ATP did not produce this effect. This property could serve to regulate the intracellular degradation of the enzyme, if the dissociated subunits were attacked preferentially.
Limited Trypsinolysis of β‐Haemocyanin of Helix pomatia
Authors: GIELENS Constant , PRÉAUX Gisèle , LONTIE René
Published: March 2005

A limited trypsinolysis of the tenths of β‐haemocyanin of Helix pomatia was performed at pH 8.2. The absorbance at 346 nm remained constant, indicating a preservation of the oxygen‐binding sites. The five tryptic fragments were separated by chromatography on Sephadex G‐100 and on DEAEcellulose. They contained 2 Cu per 50000 daltons and showed different mobilities in agar electrophoresis. The molecular weights indicated that one fragment was constituted of three functional domains of about 50000 daltons, that two fragments were constituted of two domains, and two others of one domain. Twentieths of β‐haemocyanin seemed thus to be made up of 9 domains. The circular dichroic spectra of the fragments indicated the presence of two classes of copper groups according to their positive maximum at 455 or at 500 nm. The circular dichroic spectra also showed that no fragment could have originated from a larger one, confirming the presence of nine domains in the twentieths.
Ribosome Inactivation by the Toxic Lectins Abrin and Ricin
Authors: OLSNES Sjur , FERNANDEZ‐PUENTES Carmen , CARRASCO Luis , VAZQUEZ David
Published: March 2005

A sensitive test system for toxin‐treated ribosomes was worked out by treating rabbit reticulocyte ribosomes with abrin A‐chain, ricin A‐chain or ricinus agglutinin A‐chain, adding neutralizing amounts of specific antitoxins and testing for polyphenylalanine‐synthesizing activity in a system where the concentration of elongation factors and ribosomes were varied. The strongest inhibition was obtained in the presence of low concentrations of elongation factor 2 (EF‐2).

The activity of the ribosomes decreased with time of incubation with the toxin A‐chains. Addition of anti‐toxins stopped further inactivation. In systems containing untreated and toxin‐treated ribosomes the ability to polymerize phenylalanine was proportional to the concentration of untreated ribosomes.

There was a linear relationship between toxin A‐chain concentration and the number of ribosomes inactivated per minute. The inactivation rate increased with temperature, and the estimated activation energy was 10.6 kcal (44.3 kJ). Linewaver‐Burk plots of the data obtained by incubating various ribosome concentrations with toxins indicated a molecular activity of about 1500 ribosomes/minute for abrin and ricin A‐chains and 100 ribosomes/minute for ricinus agglutinin A‐chain. The apparent Michaelis constant was 0.1‐0.2 μM for all three A‐chains. The activity of the A‐chains in the intact cell is discussed.
Effect of Nucleic‐Acid‐Binding Compounds on the Hydrolytic Activity of Various Ribonucleases
Authors: HOLBROOK David J. , WHICHARD Leona P. , WASHINGTON Mildred E.
Published: March 2005

Studies were conducted on the stimulatory effect that various nucleic‐acid‐binding compounds have on the hydrolysis of RNA and polyribonucleotides by pancreatic ribonuclease A and by other ribonucleases. The stimulatory activity of chloroquine on tRNA hydrolysis by pancreatic ribonuclease was due to the formation of oligonucleotides of a wide range of sizes and was not due to the formation of very short (n > 5) oligonucleotide fragments of tRNA. The dextrorotatory and levorotatory isomers of chloroquine did not differ in their ability to stimulate the hydrolysis of tRNA by pancreatic ribonuclease A. In addition to chloroquine and primaquine, other nucleicacid‐binding compounds (e.g., quinacrine, lucanthone, and proflavin) stimulated the hydrolysis of tRNA by pancreatic ribonuclease A. Chloroquine did not alter the rate of hydrolysis by pancreatic ribonuclease A of low‐molecular‐weight substrates (cytidine cyclic 2′:3′‐monophosphate, uridine cyclic 2′:3′‐monophosphate, cytidylyl‐adenosine, or uridylyl‐uridine). Furthermore, chloroquine and primaquine did not affect the hydrolysis of poly(A) by high concentrations of pancreatic ribonuclease A. In studies on the hydrolysis of tRNA by other endoribonucleases, several of the nucleic‐acid‐binding compounds (e.g., quinacrine and ethidium) exhibited appreciable inhibition of both ribonuclease N1 and ribonuclease T1. None of the compounds tested stimulated the activity of ribonuclease T1, and only chloroquine, and perhaps lucanthone, stimulated the hydrolysis of tRNA by ribonuclease N1.
Evidence for an Aminoendopeptidase Localized Near the Cell Surface of Escherichia coli
Authors: LAZDUNSKI Andrée , MURGIER Maryse , LAZDUNSKI Claude
Published: March 2005

An enzyme capable of hydrolyzing the substrate L‐alanine p‐nitroanilide has been found in the various Escherichia coil strains tested. This enzyme has been called aminoendopeptidase since it shows both activities (see accompanying paper). It is released from the cells by osmotic shock and by lysozyme – EDTA spheroplasting treatment, and 50% of the total activity is directly detectable with suspensions of intact cells. However, the release by osmotic shock or spheroplasting is not as efficient as it is for alkaline phosphatase. This periplasmic aminoendopeptidase is constitutively produced but the differential rate of synthesis is increased 4‐fold when the cell growth is limited by Pi. The occurrence of this ‘derepression’ is simultaneous with that of alkaline phosphatase. Increasing the concentration of inorganic phosphate in the medium has no effect on the constitutive aminoendopeptidase synthesis. The effect of phosphate starvation is specific since starvation for neither nitrogen nor carbon and energy source are effective in derepressing aminoendopeptidase.
Regulation of Escherichia coli K10 Aminoendopeptidase Synthesis
Authors: LAZDUNSKI Andrée , PELLISSIER Corinne , LAZDUNSKI Claude
Published: March 2005

The synthesis of alkaline phosphatase in Escherichia coil is controlled by the action of at least four genes denoted phoS, phoT, phoR and phoB. The effect of mutations in the first three of these genes on the synthesis of periplasmic aminoendopeptidase of E. coil K10 have been investigated. phoR gene product does not seem to be involved either in the constitutive or in the derepressed synthesis of this enzyme. Mutations in phoS or phoT influence the intracellular level of Pi in much the same way as depletion of Pi from the growth medium, and only as a consequence influence the synthesis of aminoendopeptidase and alkaline phosphatase. Point, amber or deletion mutations in the alkaline phosphatase structural gene do not affect aminoendopeptidase synthesis. Thus, alkaline phosphatase and ‘derepressed’ aminoendopeptidase synthesis are co‐regulated by the endogenous level of inorganic phosphate. The way by which this regulation operates is discussed.
The First Enzyme of the gal Operon in Inducible and Operator‐Constitutive Strains of Escherichia coli
Authors: DEELEY Roger , BLACKBURN Peter , FERDINAND William , BRIDGEN John
Published: March 2005

UDPgalactose 4‐epimerase, the product of the first structural gene of the gal operon in Escherichia coli K‐12, has been purified from strain HfrH and from its gal operator‐constitutive mutant, HfrH 81‐2.

The two enzymes are purified by the same procedure, behaving identically throughout. They are identical in sedimentation coefficient, isoelectric point, electrophoretic mobility at pH 8.8 and amino‐terminal sequence for the first 30 residues.

Slight but reproducible differences were observed between the amino acid compositions and peptide maps of the two proteins. These differences may indicate real differences in the primary sequences of the proteins, but if so they are unlikely to be due to the operator‐constitutive mutation because of the identity of the sequences for the first 30 residues.

No evidence was found to indicate that the gal operator might overlap with the first structural gene.
Biosynthesis of a Specific Lipoprotein of the Escherichia coli Outer Membrane on Polyribosomes
Authors: HIRASHIMA Akikazu , INOUYE Masayori
Published: March 2005

With use of polyribosomes isolated from Escherichia coli, biosynthesis of a specific lipoprotein of the E. coli outer membrane was investigated. The products of the cell‐free system were analyzed by sodium dodecylsulfate‐polyacrylamide gel electrophoresis and immunoprecipitation with anti‐lipoprotein serum, followed by sodium dodecylsulfate‐polyacrylamide gel electrophoresis. It was found that one of the major products was the lipoprotein of the E. coli outer membrane. The lipoprotein was also found to be preferentially synthesized on smaller polyribosomes.
Radioimmunoassay for Human and Chick Prolyl Hydroxylases
Authors: TUDERMAN Leena , KUUTTI Eeva‐Riitta , KIVIRIKKO Kari I.
Published: March 2005

A radioimmunoassay is reported for measuring prolyl hydroxylase. The assay is based on the displacement of radioactively‐labelled prolyl hydroxylase from its antibody by the non‐labelled enzyme, and on the subsequent precipitation of the enzyme‐antibody complex by a cellulose‐bound second antibody. Pure prolyl hydroxylase was isolated from foetal human or chick embryo tissues by an affinity column procedure using poly(l‐proline). The enzyme was labelled with tritium using a technique of reductive alkylation with formaldehyde and sodium [3H]borohydride. No conversion of the enzyme tetramer to its monomers was found to take place during the tritiation reaction. Experiments on the dissociation of the non‐labelled enzyme indicated that the degree of displacement of the labelled enzyme was similar regardless of whether the non‐labelled enzyme was in the tetramer form or in that of the subunit monomers.

The sensitivity of the radioimmunoassay is of the order of 5‐10 ng immunoreactive prolyl hydroxylase. The concentrations of the immunoreactive prolyl hydroxylase assayed with the present method in human serum and skin and in several chick embryo tissues are reported.
On the Control of Arginine Metabolism in Chicken Kidney and Liver
Authors: GRAZI Enrico , MAGRI Ermes , BALBONI Giancarlo
Published: March 2005

Arginases have been found to be located on the external side of the inner mitochondrial membrane of chicken kidney and liver.

Transamidinase has been detected within the liver mitochondrial matrix space.

Arginases and transamidinase act upon two different intracellular arginine pools.

Penetration of arginine into the matrix space occurs only in respiring mitochondria and in the presence of anions such as acetate and phosphate; d‐arginine, l‐ornithine, d‐ornithine and l‐lysine penetrate with the same modalities. l‐Histidine penetrates only kidney mitochondria.

Because of transamidinase compartmentation, the rate of creatine synthesis is influenced by the rate of penetration of arginine into the mitochondria.
The Binding of Maltose to ‘Virgin’ Maltose‐Binding Protein is Biphasic
Authors: HAZELBAUER Gerald L.
Published: March 2005

The biphasic binding properties of the galactose‐binding and maltose‐binding proteins of Escherichia coli may be important in the functioning of these proteins as recognition components of chemoreceptors. However, Richarme and Kepes [Eur. J. Biochem. 45, 127‐133 (1974)] have suggested that the biphasic binding curve of the galactose‐binding protein may be the result of isotopic dilution, during equilibrium dialysis, by unlabeled ligand retained by the binding throughout purification. Here the binding of maltose to maltose‐binding protein which has never previously been exposed to the sugar (‘virgin’ binding protein) is shown to be biphasic. This implies that the unusual binding properties are attributable to the maltose‐binding protein itself.
Properties of the Ribose‐Ring‐Opened Adenine Nucleotide, 2,2′[1‐(9‐adenyl)‐1′‐(tri‐,diphosphoryl‐oxymethyl)]‐dihydroxydiethylether in Mitochondrial Adenine‐Nucleotide Translocation
Authors: BOOS Karl Siegfried , SCHLIMME Eckhard , BOJANOVSKI Dubo , LAMPRECHT Walther
Published: March 2005

(Adenine‐14C) or (γ‐32P)‐labelled 2,2′(1‐(9‐adenyl)‐1′‐(tri‐, diphosphoryl‐oxymethyl)]‐dihydroxydiethylether (rroANP) was obtained from ANP by cleavage of the C‐2′‐C‐3′ bond by sodium periodate oxidation and subsequent borohydride reduction.

Binding of rroANP to rat liver mitochondria revealed carrier‐linked (atractyloside‐sensitive) and unspecific (atractyloside‐insensitive) binding but no transfer across the inner mitochondrial membrane. Kinetic data indicate rroANP as a competitive inhibitor for ANP uptake with Ki=9.3 × 10−5M. Experimental rroANP confirmed that an intact adenine base and three anionic charges of the phosphate chain are essential for the recognition between ANP‐carrier and nucleotide but insufficient for the induction of a transmembrane ANP exchange. In addition mobilisation of the carrier‐nucleotide complex requires an intact ribofuranoside ring system.
DNA Polymerases of Rat Liver
Authors: ZUNINO Franco , GAMBETTA Romolo , COLOMBO Ambrogio , LUONI Giuseppe , ZACCARA Adriano
Published: March 2005

Three distinct DNA‐dependent DNA polymerase activitives have been partially purified from normal rat liver. Soluble activities are separable into two distinct fractions (P1 and P2) by phosphocellulose chromatography. A low‐molecular‐weight DNA polymerase was isolated from purified nuclei. The enzymes were characterized according to chromatographic and sedimentation behavior, enzymological properties, and response to various inhibitors. The results indicate that fraction P1 corresponds to the high‐molecular‐weight enzyme and suggest that polymerase P2 may be derived from partial dissociation of the high‐molecular‐weight enzyme. The molecular weight of polymerase P1 was estimated to be about 250000 by Sephadex column chromatography. Both fraction P2 and nuclear DNA polymerase appeared to be low‐molecular‐weight enzymes. However, the molecular size of these activities was apparently different. The estimated molecular weights of nuclear and P2 enzyme are about 40000 and 25000, respectively.

As with the nuclear enzyme, polymerase P2 (but not P1) appeared to be free of detectable exonuclease activity. All of these polymerases showed a marked preference for initiated polydeoxyribonucleotide templates. The rat liver polymerases differed in their ability to use poly[d(A‐T)] primer‐template, as is shown by the ratios of their activity with this synthetic polymer to that with activated DNA: 0.5, 2.75, and 1.34 for P1, P2, and nuclear polymerase, respectively. Denatured DNA was a poor template for both enzymes P1 and P2, but it was inert as template for the nuclear enzyme.

Although each of these polymerases required all four deoxynucleoside triphosphates for maximal activity, they catalyzed a high rate of synthesis in the absence of one or more deoxynucleoside triphosphates. Such a ‘limited’ synthesis was much more extensive for polymerase P2 and nuclear enzyme than for P1. Polymerase P1 was the most sensitive of the three to sulphydryl reagents, ethidium bromide, heparin, and single‐stranded DNA. The responses of P2 and nuclear enzymes to various inhibitors were very similar. However, these two enzymes respond differently to heat and high ionic strength.
Independent Regulation of the Nuclear and Mitochondrial DNA Synthesis Induced by either Simian Virus 40 or Serum in Mouse Fibroblast 3T3 Cells
Authors: FISCHER‐FANTUZZI Lia , MARIN Guglielmo , VESLO Cesare
Published: March 2005

Resting cultures of 3T3 cells (an established line of mouse fibroblasts) were released from density inhibition by either infection with Simian virus 40 or addition of serum. The increased rate of thymidine incorporation into DNA, induced by these two agents, was measured in the presence and in the absence of three inhibitory conditions (cycloheximide or dibutyryladenosine 3′:5′‐monophosphate added to the medium, or lack of anchorage). The inhibition was found to be quite similar in cultures stimulated by virus or serum; under the same conditions, however, the incorporation into mitochondrial DNA was much less inhibited than that into nuclear DNA. The experiments also suggest that new protein synthesis may not be necessary, for either virus or serum, to start the inductive mechanism.
Luminescence Studies of Saccharide Binding to Wheat Germ Agglutinin (Lectin)
Authors: PRIVAT Jean‐Paul , MONSIGNY Michel
Published: March 2005

The fluorescence and phosphorescence emission of wheat germ agglutinin are reported.

Fluorescent tryptophan residues of wheat germ agglutinin are found highly exposed to solvent: fluorescence quenching induced by temperature fits with a single Arrhenius critical energy close to that of tryptophan in solution; the whole fluorescence emission is susceptible to iodide ion quenching and data reveal the homogeneity of fluorescence arising from only one type of tryptophan exposition.

Energy transfers are analyzed at singlet and triplet state level. Tyrosine fluorescence at 25 °C is very weak. Results obtained from the relative excitation fluorescence quantum yield and from intrinsic fluorescence polarization show that a large amount of energy absorbed by tyrosine at 280 nm is transferred to tryptophan residues. However, tyrosine fluorescence is highly increased at 70 °C although disulfide bridges are not reduced. The phosphorescence spectrum at 77 K in 50%, ethylene glycol is finely structured with several resolved vibrational bands at 405, 432 and 455 nm. Phos‐phorescence decay can be fitted with a single exponential. Lifetime is independent of excitation wave‐length. Its value is very close to that of free tryptophan.

Influence of tri‐N‐acetyl‐chitotriose binding on luminescence properties are investigated. Results are analyzed in terms of steric tryptophan‐ligand relationships. It is shown that all the fluorescent chromophores are concerned by the ligand binding but all fluorescence emission is still susceptible to iodide ion quenching. There is no change induced in energy transfer at the singlet state level and no modification in triplet state population.