bluuebunny/crossref_metadata_2025 huggingface dataset:
Intranuclear Concentration of Lysosomal Hydrolases in Steroid Target Cells
Authors: SZEGO Clara M. , STEADMAN Rosemarie A. , SEELER Barbara J.
Published: March 2005

The present investigation was designed to determine whether the rapid intracellular redistribution of lysosomes to a perinuclear position in reproductive target cells after administration of specific gonadal hormones in vivo was accompanied by evidence of penetration of the nucleoplasm by lysosomal marker enzymes. Nuclei were prepared by conventional isolation and purification procedures from preputial glands or uteri that were excised from gonadectomized rats within 2–15 min after intravenous injection of physiological doses of estradiol‐17β, testosterone or saline control solutions. Control and experimental preparations were essentially “pure”, as judged by routine phase‐contrast microscopic criteria in general use. However, nuclei from target cells of animals given hormone contained substantial activities of representative lysosomal hydrolases, including acid phosphatase, β‐glucuronidase and acid ribonuclease II. In contrast, the samples originating from the target tissue of saline‐treated controls or from non‐target tissues contained minimal concentrations of these enzyme activities. After further purification of the nuclei by removal of the outer nuclear membrane through brief exposure to very low concentrations of Triton X‐100 in the cold, the resultant ‘ultrapurified’ nuclei retained significant concentrations of structurally latent lysosomal marker enzymes after hormonal pretreatment without evidence of appreciable contamination by enzymes of mitochondrial origin. Similar results were obtained with nuclei isolated by non‐aqueous procedures. These combined observations appear to exclude adsorption artifacts. The total activities of lysosomal enzymes in the unfractionated homogenates and the Mg2+‐dependent ribonuclease activity indigenous to the nuclear compartment were unaffected by prior hormone treatment. These results are consistent with the hypothesis that invasion of the nucleoplasm of specific target cells by lysosomal products may serve as a mechanism for triggering genic derepression in steroid hormone‐induced growth.
Cytochrome c: A Thermodynamic Study of the Relationship among Oxidation State, Ion‐Binding and Structural Parameters Cation Binding to Horse‐Heart Ferrocytochrome c
Authors: MARGALIT Rimona , SCHEJTER Abel
Published: March 2005

The specific binding of cations to horse heart ferrocytochrome c has been studied, using the gel filtration method. The cations investigated were: Mg2+, Co2+, cinchonine and proflavine. The stability constants are in the range of 5–8 mM−1, and the number of binding sites per protein molecule are 1 to 2. The temperature dependence of the stability constant for the Mg2+‐ferrocytochrome system was measured. The thermodynamic parameters were found to be: ΔH0obs=+ 12 kcal/mol, ΔG0obs, (25°C) =−5.6 kcal/mol and ΔS0obs=+ 57 cal × mob1× K−1.
The Determination of the Primary Structure of Histone F3 from Chicken Erythrocytes by Automatic Edman Degradation
Authors: BRANDT Wolf F. , VON HOLT Claus
Published: March 2005

Histone F3 has been fragmented into 10 non‐overlapping fragments.

With the aid of the automatic Edman degradation procedure for proteins using 3‐(N,N‐dimethylamino)propyne buffer the first 48 amino‐acid residues in the uncleaved protein have been positioned.

The remainder of the amino‐acid sequence of this protein was elucidated by subjecting the aligned peptides to the automatic degradation. The amino acids obtained during the degradation were quantitated and identified by gas chromatography and amino‐acid analysis.

The primary structure of this histone is compared to the corresponding fraction from calf which had been independently determined by another laboratory.
UDP‐glucuronyltransferase in Perfused Rat Liver and in Microsomes: Influence of Phenobarbital and 3‐Methylcholanthrene
Authors: BOCK Karl Walter , WHITE Ian N. H.
Published: March 2005

Liver microsomal UDP‐glucuronyltransferase (1‐naphthol as substrate) is activated about 10‐fold by treatment of native microsomes in vitro with Triton X‐100. Following pretreatment of rats with 3‐methylcholanthrene or phenobarbital the specific activities of detergent‐activated microsomes were increased by about 300% and 40%, respectively, while the corresponding increases in native microsomes were only about 70% and 10%, respectively. In isolated liver perfused with 0.5 mM 1‐naphthol, the rate of glururonidation of naphthol, as determined by the appearance of naphthol glucuronide in the perfusate, bile and liver tissue, was 0.1 μmol × min−1× g liver−1. This rate of glucuronidation was increased by 70% in rats pretreated with 3‐methylcholanthrene and by 140% following phenobarbital treatment, in contrast to the changes observed in microsomes.

A UDP‐glucuronic acid level of 0.3 μmol/g liver was determined in perfused liver from control rats. This level remained unchanged during perfusion with 0.5 mM 1‐naphthol. In the perfused liver from phenobarbital or 3‐methylcholanthrene‐treated rats, the UDP‐glucuronate level increased to 0.5 μmol/g liver.

In control, 3‐methylcholanthrene or phenobarbital‐treated rats, rates of glucuronidation of 1‐naphthol in the perfused liver system correspond best with the UDP‐glucuronyltransferase activities determined in native microsomes when the different UDP‐glucuronate levels, microsomal protein contents and liver weights are taken into account.

The results suggest that the enzyme is mostly latent in the microsomal membrane and that this latency is increased after pretreatment of rats with 3‐methylcholanthrene despite the marked enzyme induction.
Heme‐Linked Properties of Euglena Cytochrome f
Authors: BEN‐HAYYIM Gozal , SCHEJTER Abel
Published: March 2005

Oxidized Euglena cytochrome f has an absorbance band at 695 nm which disappears upon reduction. Various treatments that cause known changes of conformation of cytochrome c appear to have similar effects on cytochromef. Heating cytochrome f results in the gradual disappearance of the 695‐nm band; the thermodynamic parameters for this change of the tertiary structure at 26 °C and ionic strength 0.01 are: ΔF=+2.67 kcal/mole, ΔH=+ 33 kcal/mol and ΔS=+102cal × K−1× mol−1.

The pK values obtained by equilibrium titrations of the heme‐linked acid and alkaline ionizations are roughly 2.1 and 9.4. Equilibrium studies of the alkaline pK reveal that more than one hemelinked proton is dissociated in this pH range.

The carboxymethylated product of the cyanide complex of oxidized cytochrome f has the same properties as cytochrome c carboxymethylated under the same conditions. The results suggest that a methionine sulfur is involved in iron coordination in oxidized Euglena cytochrome f.
The Dynamics of the Allomorphic Forms of Mouse Mitochondrial DNA
Authors: PORCHER Harald H. , KOCH Jürgen
Published: March 2005

Mouse mitochondrial DNA can be fractionated by means of band sedimentation into the allomorphic forms supercoiled circle (34 S), open circle (24 S) and linear rod (21 S). The separation of the ring‐open form based on its hydrodynamic properties is not necessarily an indication of the chemical properties of this form, i.e. a nick in the phosphodiester backbone of at least one strand of the duplex DNA. Circular duplex DNA molecules may show an ambiguous behaviour. During sedimentation the high frictional coefficient of open circles is observed. However, as judged by other criteria the molecules behave like covalently closed circular DNA with invariant topological winding (restricted uptake of an intercalating dye).

A large part of mouse mitochondrial DNA occurs as D‐loop mitochondrial DNA molecules which contain a displaced single‐stranded region in the duplex DNA molecule. The insertion of the new displacing strand reduces the rotation of the parental strands around each other and therefore reduces the number of right‐handed superhelical turns in the “covalently closed” circular DNA. For this reason, the bulk of the mouse D‐loop mitochondrial DNA molecules display a sedimentation behaviour (28 S) similar to that of ring‐open (nicked) molecules although these molecules contain a “covalently closed” phosphodiester backbone in each strand. Pulse‐chase‐pulse experiments, labeling firstly with [2‐14C]thymidine and secondly with [Me‐3H]thymidine, indicate that the different allomorphic forms of the mitochondrial DNA turn over at a different rate. The lowest turnover rate is observed in alkali‐stable supercoiled mitochondrial DNA and in D‐loop mitochondrial DNA. It is concluded that the accumulation of D‐loop mitochondrial DNA molecules in mouse cells is due to abortive replication events.
Generation in vivo of “Covalently Closed” Circular Mitochondrial DNA Free of Superhelical Turns
Authors: RASTOGI Anil K. , KOCH Jürgen
Published: March 2005

The non‐intercalating tryptanocidal drug berenil inhibits completely the synthesis of the mitochondrial DNA in cultured human cells. This interference appears to be fairly selective, as the effect on the synthesis of the nuclear DNA is relatively small.

The exposure of the cells to the drug results in a conversion in vivo of existing supercoiled mitochondrial DNA into DNA molecules with the hydrodynamic properties of ring‐open mitochondrial DNA. Dye‐buoyant‐density separation and titration with ethidium bromide indicate that this ring‐open mitochondrial DNA contains “covalently closed” circular DNA molecules which are free of superhelical turns.
Fluorescence Studies of Saccharide Binding to Wheat‐Germ Agglutinin (Lectin)
Authors: PRIVAT Jean‐Paul , DELMOTTE Francis , MIALONIER Gérard , BOUCHARD Philippe , MONSIGNY Michel
Published: March 2005

Wheat germ agglutinin was isolated in pure form by a method adapted from D. LeVine, M. J. Kaplan and P. J. Greenaway, Biochem. J. 129, 847–856 (1972). Amino‐acid composition calculated for a molecular weight of 23 500 showed that the protein contained four tryptophan residues and was rich in half‐cystine (38 residues) and glycine (51 residues).

Analysis of the fluorescence spectrum showed a characteristic tryptophan emission with a maximum centered at 348 nm. The spectrum is almost the same between pH 3.5 and 9.0. By adding N‐acetylglucosaminides (β‐d‐GlcNAc 1 → 4)n‐d‐GlcNAc, an enhancement of the fluorescence intensity was detected. Moreover, binding of the dimer and higher GlcNAc oligomers induced a 10‐nm shift of the emission maximum towards shorter wavelengths indicating that the environment of one or several tryptophan residues was altered by addition of ligands. The pH dependence of the fluorescence quantum yields shows that the tryptophan fluorescence is not altered by any ionisable group which possesses a pK in the range 4.0–9.0 in both the free and the complexed protein.

Association constants were determined by measuring the change in fluorescence intensity produced by addition of different glucosaminides. These constants increased with chain length up to the trimer. No cooperative effect could be found with any ligand. It was shown that the carbohydrate binding is abolished below pH 2.8 and that a protein carboxyl group of pKa= 3.9 is involved.

Our results are discussed and compared with the model proposed by A. K. Allen, A. Neuberger and N. Sharon, Biochem. J. 131, 155–162 (1973).
Accessible DNA in Chromatin
Authors: ITZHAKI Ruth F.
Published: March 2005

A study has been made of the action of DNAase I and of micrococcal nuclease on chromatin, on the complex of chromatin with polylysine and of DNAase I on DNA · protamine; a comparison of different assay methods of DNAase I action on chromatin is included here. Also, the nature of the polylysine‐binding zones in chromatin has been investigated.

Measurements of acid‐solubility and salt‐solubility during digestion show that DNAases I and II degrade almost all of the DNA in chromatin (from various tissues), unimpeded by the chromatin proteins which are released concurrently (and which eventually aggregate on to the degraded DNA). With micrococcal nuclease concurrent release of DNA and of protein occurs similarly; however, full degradation by this nuclease is impeded apparently by aggregation of released protein on to acid‐insoluble DNA. In the case of chromatin · polylysine and also DNA · protamine, about half of the DNA is degradable. The results indicate that discrepancies between the findings of previous workers are due to differences in assay of degradation, in conditions of digestion, in enzyme used and probably in methods of preparing chromatin resulting in products varying widely in composition. Also, the results support the previous suggestion that virtually all the DNA in chromatin is associated with protein yet is highly accessible [1], unlike DNA complexed with protamine or polylysine. The polylysine‐binding zones in chromatin are discrete, double‐stranded, protein‐associated regions of DNA which are probably heterogenous from one molecule to another.
Cell Envelope and Shape of Escherichia coli K12
Authors: GARTEN Wolfgang , HENNING Ulf
Published: March 2005

The protein from Escherichia coli ghosts can be separated electrophoretically into three major bands (I, II, and IV) and one minor band (III). The proteins corresponding to bands I, II and IV have been isolated. Protein IV is the lipoprotein described previously by Braun et al. and is thus a homogeneous polypeptide chain. Proteins I and II do not contain phosphorus, palmitic acid, or lipopolysaccharide but small amounts of carbohydrate, probably glucosamine (or a derivative).

Proteins I and II have been characterized by amino‐acid analyses, tryptic fingerprints, carboxy‐peptidase digestion, and cyanogen bromide cleavage. Based on cysteic acid the minimum chemical molecular weights of both proteins are the same as those found by electrophoreses: about 40000 (±∼ 10%) for I and about 28000 (±∼ 10%) for II. The numbers of tryptic peptides found do not argue against the assumption that each of the two proteins consists of identical polypeptide chains. However, the sum of the apparent molecular weights of the cyanogen bromide fragments of protein I is almost 80000. The amino acids liberated by carboxypeptidase A at near integral values per 40000 g of protein I correspond to 1 mol each of serine, glycine, alanine, isoleucine, tyrosine, phenylalanine, and to 2 mol valine per 40000 g of protein; however, only 0.5 mol leucine is released. Apart from revealing a rather hydrophobic C‐terminal region, the data thus include the possibility that protein I consists of two different but presumably rather similar polypeptide chains. Protein II is a tryptic fragment of a protein II* with a molecular weight near 40000. Protein II* can give rise to two other fragments by the action of other proteases. The apparent molecular weights of the cyanogen bromide fragments of protein II add up to that of protein II. However, although carboxypeptidase A liberates nearly 1 mol each of serine, alanine, valine, and leucine per 28000 g of protein II, it releases only about 0.5 mol each of glutamic acid, glycine and isoleucine. As for protein I, it cannot yet be excluded that protein II consists of two polypeptide chains. If so, the two must be very similar. Regardless as to whether proteins I or II consist of one or two polypeptide chains these must be arranged as repeating subunits in the outer membrane of the E. coli cell envelope.

The properties of proteins I and II allow them to be classified as intrinsic or integral membrane proteins.
Enzymes Involved in Ketone Utilization in Different Types of Muscle: Adaptation to Exercise
Authors: WINDER William W. , HOLLOSZY John O. , BALDWIN Kenneth M.
Published: March 2005

Activity levels of the enzymes involved in ketone utilization were compared in heart, fast‐twitch red, slow‐twitch red, and fast‐twitch white types of muscles in rats. 3‐Hydroxybutyrate dehydrogenase, 3‐ketoacid CoA‐transferase, and acetoacetyl‐CoA thiolase activities were lowest in white muscle, higher in slow red than in fast red muscle, and highest in the heart. The large differences between the four muscle types in the levels of these enzymes reflects differences in both mitochondrial content and composition. Differences in composition were evidenced by large dissimilarities between the different muscle types in the ratios of the activities of the enzymes of ketone oxidation to each other and to citrate synthase and cytochrome c. Of particular interest is the finding that, despite the fact that in the rat slow red muscle has a lower content of mitochondria than fast red muscle, 3‐hydroxybutyrate dehydrogenase activity is 140% higher and 3‐ketoacid CoA‐transferase is 70% higher in slow red than in fast red muscle.

A 14‐week‐long program of treadmill running induced increases in the levels of ketone utilization enzymes in all three types of skeletal muscle but not in heart; 3‐hydroxybutyrate dehydrogenase became measurable, though at very low levels, in white muscle; increased 2.6‐fold in slow red muscle, and 6‐fold in fast red muscle. 3‐Ketoacid CoA‐transferase increased 2‐fold in both fast red and white types of muscle, but only 26% in slow red muscle. Acetoacetyl‐CoA thiolase activity increased 40–45% in all three types of skeletal muscle. In contrast, citrate synthase and cytochrome c increased approximately 2‐fold in all three types of skeletal muscle. These adaptive changes tend to make skeletal muscles more like heart muscle in their enzyme patterns and may help to explain why physically trained, as compared to untrained, individuals do not develop post‐exercise ketosis.
Acetate, a Major End Product of Fatty‐Acid Oxidation in Hamster Brown‐Adipose‐Tissue Mitochondria
Authors: BERNSON Vibeke S. M. , NICHOLLS David G.
Published: March 2005

1. Brown‐adipose‐tissue mitochondria prepared from the cold‐adapted rat and guinea‐pig require malate for significant palmitoyl‐l‐carnitine oxidation. In contrast the mitochondria prepared from the cold‐adapted hamster (a hibernator) rapidly oxidize palmitoyl‐l‐carnitine in the absence of added malate.

2. The major end product of malate‐independent fatty acid oxidation in the hamster is identified as acetate. In the presence of malate or l‐carnitine, citric acid cycle intermediates or acetyl‐l‐carnitine are also produced.

3. Factors controlling the formation of these products are investigated. Acetate production is highly dependent on the rate of fatty acid oxidation, and on the acetyl‐carnitine/carnitine ratio.

4. The rate of acetate production in the presence of malate is independent of the incubation temperature over the range from 37°C to 10°C, while the entry into the citric acid cycle is decreased 10‐fold.

5. Acetate production is most pronounced under conditions where a high acetyl‐CoA/CoA ratio would be predicted to occur.

6. It is suggested that the acetate pathway is of particular significance in brown adipose tissue from hibernators as a means of allowing fatty acid oxidation, and hence heat production, to occur at temperatures where the citric acid cycle is apparently unable to function.
A Dynamic Correlation between Ribose Conformation and Glycosyl Torsion Angle of Dissolved Xanthosine Studied by Continuous‐Wave‐Mode and Pulsed Nuclear‐Magnetic‐Resonance Methods
Authors: LÜDEMANN Hans‐Dietrich , WESTHOF Eric , RÖDER Oskar
Published: March 2005

The solution conformation of xanthosine in liquid N2H3 has been determined by nuclear magnetic resonance. The correlation times for rotational diffusion were derived from 13C relaxation measurements. A Karplus analysis of the high‐resolution spectra of the ribose moiety yields the conformation of the sugar from the H–H coupling constants. The mole fraction of the sugar in the N conformation is 0.4 at room temperature and increases slightly as the temperature decreases. From nuclear Overhauser enhancement studies, and T1 measurements of the various protons it is deduced that the N ribose is correlated with an anti conformation of the base (Y∼ 210°) while the S form of the sugar is coupled to a glycosyl torsion angle in the syn range (Y ∼ 50°–90°).
Utilization by Yeast of d‐Lactate and l‐Lactate as Sources of Energy in the Presence of Antimycin A
Authors: PAJOT Patrick , CLAISSE Maurice L.
Published: March 2005

Saccharomyces cerevisiae cells contain d and l‐lactate: cytochrome c oxidoreductases. The physiological role of these enzymes is investigated by inhibiting the respiratory chain with antimycin A and observing how the vital functions (respiration, protein biosynthesis, growth) are altered. When d or l‐lactate are provided as the source of energy, stoichiometric amounts of the antibiotic decrease the growth rate about five‐fold, the oxygen uptake two‐fold and the energetic efficiency eleven‐fold.

In the presence of antimycin A, it was often proposed that only a very short chain consisting of one of the lactate dehydrogenases, cytochrome c and cytochrome oxidase could be involved in the oxidation of lactate. Consistent with this scheme are the measurements of lactate and oxygen uptake and the observation that keto acids are released into the external medium, which also allow one to rationalize the growth yield and the amino acid incorporation results.
Hamster Brown‐Adipose‐Tissue Mitochondria
Authors: NICHOLLS David G.
Published: March 2005

Hamster brown‐adipose‐tissue mitochondria, swollen in the chloride, bromide or nitrate salts of sodium, or potassium in the presence of nigericin, do not contract on the addition of oxidizable substrate. If albumin is added to the swollen mitochondria a rapid and complete contraction occurs on substrate addition; however no contraction was observed of mitochondria swollen in KCl or potassium phosphate plus valinomycin or in sodium phosphate. Contraction is interpreted as being a consequence of ion expulsion linked to respiration‐dependent proton translocation. GDP inhibits respiration‐independent chloride permeation but does not prevent chloride efflux from the matrix during respiration in the presence of albumin; respiratory control is retained in this condition after completion of contraction. The stoichiometry of chloride extrusion is approximately 2Cl−/O with succinate as substrate in the presence of albumin, and is unchanged if GDP is additionally present. In the presence of albumin the chloride conductance of the inner membrane is approximately 8 nequiv. Cl−× min−× mg−× mV−. The further addition of GDP decreases the conductance, and no chloride efflux is observed if the membrane potential is below 40 mV. The effectiveness of purine nucleotides in inhibiting chloride permeation in non‐respiring systems is attributed to the low membrane potentials generated by diffusion. The possibility that the sites of electrical anion permeation and purine‐nucleotide‐sensitive proton (or hydroxyl ion) permeation are identical is discussed.
On the Structure of Phenylalanine tRNA from Yeast
Authors: SPRINZL Mathias , KRÄMER Erich , STEHLIK Dietmar
Published: March 2005

2‐Thiocytidine incorporated into tRNAPhe from yeast by tRNA nucleotidyl transferase can be alkylated by iodoacetamide and its derivatives. Using this method a spin label was introduced specifically into tRNAPhe and the product, tRNAPhe‐C‐(s.l.)s2C‐A, can still be aminoacylated by phenylalanyl‐tRNA synthetase. Electron paramagnetic resonance spectra obtained between 10° and 70°C for tRNAPhe‐C‐(s.l.)s2C‐A and the pentanucleotide C‐A‐C‐(s.l.)s2C‐A (a model compound) were evaluated for the correlation time, τc, of the reorientation of the spin label in its environment and for the motional anisotropy parameter, ɛ. Spin‐labeled pentanucleotide is characterized by a spin label motion with an activation energy, EA, of 1.67 × 10−3 J × mol−1× K−1 (7.0 kcal × mol−l×°C−1) and a strongly anisotropic motion with a long axis of the rotational ellipsoid along the N‐Obond of the radical. This motion is temperature independent. In comparison spin label bound to the tRNA molecule is more immobilised and its motion is more isotropic. In addition, the slope of log τc(T) and the nature of the motion changes at a critical temperature Tcr which, in the absence of magnesium, varies with ionic strength. In the presence of magnesium Tcr is 47.5°C, whereas the midpoint temperature of the optical melting curve, Tm, is 75°C. The melting transition monitored by the spin label is a cooperative process associated with the melting of a higher‐ordered structure of the tRNAPhe molecule.
Absence of a Low‐Molecular‐Weight DNA Polymerase from Nuclei of the Yeast, Saccharomyces cerevisiae
Authors: WINTERSBERGER Ulrike
Published: March 2005

The DNA polymerase activity associated with purified nuclei of the yeast, Saccharomyces cerevisiae, was analyzed by sucrose gradient centrifugation. Using different salt concentrations there was no polymerase activity found sedimenting in the 3–4‐S region. On the contrary, the nuclear enzyme shows a main peak around 8 S, similar to the DNA polymerases from yeast cytoplasm. Other properties tested (e.g. behaviour on ion‐exchange columns, dependence of activity on salt concentration, inhibition by p‐chloromercuribenzoate etc.) were also found identical with those of the cytoplasmic DNA polymerases.
Synthesis of Apamin, a Neurotoxic Peptide from Bee Venom
Authors: VAN RIETSCHOTEN Jurphaas , GRANIER Claude , ROCHAT Hervé , MIRANDA Francois , LISSITZKY Serge
Published: March 2005

The apamin sequence has been synthesized by the solid‐phase procedure. The synthetic peptide showed the same physicochemical and chemical properties as natural apamin in the following tests: paper electrophoresis, amino acid analyses after acid and enzymatic hydrolyses, sequence analysis, electrophoreses after tryptic and chymotryptic digestions. Synthetic apamin was as active as natural apamin in the neurotoxic assay in mice (LD50, after subcutaneous injection, for the 20‐g mouse: 58 μg).
Flavocytochrome b2: Simulation Studies of the Electron‐Transfer Reactions among the Prosthetic Groups
Authors: CAPEILLÈRE‐BLANDIN Chantal
Published: March 2005

Simulation studies by digital computer were undertaken in order to test and clarify the interpretations deduced from experimental data concerning the electron transfer mechanism from l‐lactate to flavocytochrome b2, which were presented in a preceding paper in this journal.

The reaction scheme proposed as the “best” one is composed of 7 steps. It allows the best fitting of the time courses established for the oxidized flavin (Flox), the flavin semiquinone (Flsq), the fully reduced flavin (Flred), and the reduced haem (Hred); it can be extended to 1 s. This scheme also allows a good simulation of the general shape of preequilibrium titration curves obtained at a 200‐ms reaction time for Hred and Flsq, and a valuable simulation of the reduction electron paramagnetic resonance time course established for Hred and Flsq at low lactate concentration. The agreement between experimental and simulated curves led to an estimation of some rate constants experimentally unknown, relative (in particular) to the electron exchange between flavin and haem and between couples of flavins. Another interest of these stimulation studies was to point out the obligatory involvement of a slow final step to perform the flavocytochrome b2 full reduction; this step could be controlled by some conformational change of the protein.
Stimulation of Proteinase K Action by Denaturing Agents: Application to the Isolation of Nucleic Acids and the Degradation of ‘Masked’ Proteins
Authors: HILZ Helmuth , WIEGERS Ulrich , ADAMIETZ Peter
Published: March 2005

Hydrolysis of serum albumin by proteinase K was strongly (> 7‐fold) stimulated by urea and dodecylsulfate in a dose‐dependent manner. With an oligopeptide as substrate, however, proteinase K was inactivated by dodecylsulfate. This indicates that the apparent activation of proteinase K by urea and dodecylsulfate is caused primarily by denaturation of the protein substrates.

Although dodecylsulfate inhibited ribonuclease activity in the test‐tube completely, it could not prevent RNA degradation during isolation of polysomal RNA, to which ribonuclease had been added, because of the reversible nature of the dodecylsulfate inhibition. Complete protection of RNA, however, was achieved by a combination of dodecylsulfate and proteinase K.

The combined action of the detergent and proteinase K was also effective in degrading “masked” proteins in a poly(adenosine diphosphoribose) preparation which could not be attacked by the proteinase alone.
Antibodies to Adenosine 5′‐Monophosphate: Purification and Specificity
Authors: DROCOURT Jean‐Louis , LENG Marc
Published: March 2005

Antibodies to adenosine‐5′‐monophosphate were produced in rabbits by injecting a conjugate of the nucleotide (oxidized with periodate) with bovine serum albumin. Nucleotide‐specific antibodies were isolated by affinity chromatography on oligoadenylic acids/agarose column. Pure immunoglobulin G antibodies were obtained by gel filtration on Sephadex G‐200. These antibodies, as analyzed by double diffusion react with adenosine 5′‐monophosphate‐bovine serum albumin, slightly with inosine‐5′‐monophosphate conjugate and not at all with the other nucleotide conjugates. The association constants for adenosine‐5′‐monophosphate‐antibody complex formation obtained by dialysis equilibrium and fluorescence measurements, are in good agreement. This latter technique was used to study on one hand the influence of temperature and salt on complex formation, on the other hand the interaction of the antibodies with AMP derivatives. The phosphate group, the ribose and the base are recognized by the antibody, but the C‐8 atom of adenine residues is not directly involved in the binding.
Two‐Dimensional Polyacrylamide‐Gel Electrophoresis of the Proteins and Glycoproteins of the Human Erythrocyte Membrane
Authors: ANSELSTETTER Volker , HORSTMANN Hans‐Joachim
Published: March 2005

A two‐dimensional polyacrylamide gel electrophoresis technique has been developed, improving the analytical separation of some proteins and glycoproteins of the human erythrocyte membrane.

Freshly prepared membranes are totally solubilized, subjected to dodecylsulfate – polyacrylamide gel electrophoresis in the first dimension, followed by electrophoresis in the second dimension, using a detergent‐free polyacrylamide gradient gel.

By this method the proteins of the human erythrocyte membrane could be resolved into a two‐dimensional pattern, which has been shown to be highly reproducible with respect to various blood‐groups and within one blood‐group from specimen to specimen.

The method enables especially the investigation of the hydrophobic and very likely integrated membrane proteins and glycoproteins. Thus, band III [Fairbanks, G., Steck, Th. & Wallach, D. F. H., Biochemistry, 10, 2606–2617 (1971)] could be shown to consist of five proteins, one of them being the major glycoprotein of the human erythrocyte membrane. The two spectrin bands differed considerably in their two‐dimensional patterns.

The value of the given method for the investigation of membrane defects, which may be linked with various diseases of human erythrocytes, could be demonstrated in the case of two patients suffering from congenital dyserythropoetic anaemia.
Initiation of Protein Synthesis in Ehrlich Ascites Tumour Cells
Authors: PAIN Virginia M. , HENSHAW Edgar C.
Published: March 2005

Binding of methionyl‐tRNAf to native 40‐S ribosomal subunits is thought to be an early stage in the process of polypeptide chain initiation, and [35S]Met‐tRNAf· 40‐S‐subunit complexes can be isolated from Ehrlich ascites tumour cells following a brief incubation with [35S]methionine. To determine whether this step is subject to modulation by physiological conditions, we have estimated the extent of binding of Met‐tRNAf to native 40‐S ribosomal subunits in Ehrlich cells under nutritional conditions known to affect the rate of protein synthesis in these cells. Deprivation of either an essential amino acid, lysine, or of glucose, results in a substantial reduction in the proportion of native 40‐S subunits which have Met‐tRNAf associated with them, and refeeding of lysine to cells deprived of this amino acid partially reverses this effect within 10 min. These effects on the concentration of Met‐tRNA · 40‐S‐subunit complexes are paralleled by changes of similar magnitude in the rate of protein synthesis and in polyribosome profiles.

Native 40‐S subunits can be separated by equilibrium density gradient analysis on caesium chloride into two species, with buoyant densities approximately 1.40 and 1.49 g × cm−3. In cells deprived of either lysine or glucose, the radioactivity from [35S]methionine is bound exclusively to the particle of buoyant density 1.40 g × cm−3. In well‐fed cells, or in starved cells shortly after refeeding, a significant proportion of the label is associated with a region of the CsCl gradient corresponding to a particle of higher density.

The results suggest that the binding of Met‐tRNAf to native 40‐S ribosomal subunits can be greatly affected by physiological conditions which alter the rate of protein synthesis. This is consistent with a regulatory role for this step in the sequence of reactions involved in initiation of translation.
Interaction between the Exocellular DD‐Carboxypeptidase‐Transpeptidase from Streptomyces R61, Substrate and β‐Lactam Antibiotics
Authors: FRÈRE Jean‐Marie , GHUYSEN Jean‐Marie , PERKINS Harold R.
Published: March 2005

The interaction between the exocellular DD‐carboxypeptidase‐transpeptidase of Streptomyces R61 and β‐lactam antibiotics is a multistep process during which a rather stable enzyme · antibiotic complex is formed. This mechanism of interaction is compatible with Lineweaver‐Burk plots that are typical of a competitive inhibition of the hydrolysis of the peptide donor by the antibiotic. In fact, however, the same Lineweaver‐Burk plots can be obtained on the basis of a non‐competitive type of inhibition. At present, a choice between the two models cannot be made.
Synthesis of S‐100 Protein in Monolayer Cultures of Rat‐Glial Cells
Authors: LABOURDETTE Gérard , MARKS Alexander
Published: March 2005

Synthesis and degradation of the brain‐specific S‐100 protein were measured in monolayer cultures of clonal rat glial cells C 6. The cells synthesize a small but definite amount of S‐100 protein in logarithmic growth. The increase in accumulation of S‐100 protein which occurs at the end of logarithmic growth is brought about by changes in synthesis.
Amino‐Acid Sequence of Toxin III of Naja haje
Authors: KOPEYAN Charles , MIRANDA François , ROCHAT Hervé
Published: March 2005

The complete amino acid sequence of toxin III of Naja haje (72 residues) has been established mainly by use of a protein sequenator (identification of 70 residues). The two C‐terminal residues have been determined by digestion with carboxypeptidases A and B.

Addition of succinylated protein or peptide greatly improved the performance of the sequenator for the Edman degradation of peptides: on one peptide (39 residues) degradation went to step 34 with a protein program and on two peptides (10 and 13 residues) degradation reached the last amino acid with a peptide program (use of dimethylbenzylamine).

Amino acid analysis of tryptic peptides obtained by digestion of the C‐terminal cyanogen bromide peptide are in full agreement with the sequence established by automatic degradation. The sequence of toxin III of Naja haje is unique and is very similar to that of Naja niveaα (although there are 9 differences), of Naja melanoleuca b (11 differences) and also to that of Naja naja A (18 differences).
Polyadenylation of RNA in vitro in Isolated Chromatin and Nuclei
Authors: DE POMERAI David I. , BUTTERWORTH Peter H. W.
Published: March 2005

Poly(A) is added post‐transcriptionally to RNA transcribed in vitro by endogenous form B DNA‐dependent RNA polymerase bound to the template in isolated nuclei or chromatin. It is also added to processed fragments of the products from α‐amanitin‐resistant RNA polymerases A and/or C. The poly(A) segments are of similar size to those found in nuclear RNA pulse‐labelled in vivo and are added onto the 3′ terminus of RNA chains (whether pre‐existing, completed during the incubation in vitro or created by fragmentation of larger RNA transcripts). That poly(A) addition is not directly mediated by any of the nuclear DNA‐dependent RNA polymerases is shown by the differential sensitivities of RNA and poly(A) syntheses to increasing ionic strength and transcriptional inhibitors such as the exotoxin from Bacillus thuringiensis.
Heart Hexokinase: Quaternary Structure Changes Accompanying the Binding of Regulatory Molecules
Authors: EASTERBY John S.
Published: March 2005

Heart hexokinase monomer has a molecular weight of 97000 and s020,w 5.2 S. It exists in equilibrium with dimer of 194000 molecular weight and s020,w 8.1 S. The proportions of monomer and dimer present in the absence of added ligands are 91% and 9% respectively. The existence of these forms may be demonstrated by separation on electrophoresis or chromatography.

In the presence of the regulatory molecule glucose 6‐phosphate, the dimer form of the enzyme is favoured. The glucose 6‐phosphate mediated dimerisation is abolished in the presence of phosphate or ATP · Mg and less effectively by free ATP. Glucose has no effect on the monomer‐dimer equilibrium.

On prolonged storage of hexokinase in glucose 6‐phosphate polymers are also formed and polymerisation is further enhanced by removal of the ligand.
Solution Conformation of Virginiamycins (Staphylomycins)
Authors: ANTEUNIS Marc J. O. , CALLENS Roland E. A. , TAVERNIER Dirk K.
Published: March 2005

The 1H (at 300 MHz) and 13C nuclear magnetic resonance spectra of virginiamycins S and S4 and vernamycin Bα have been unravelled and analyzed. Together with model building and theoretical considerations, this allows the detailed description of their solution conformations. The depside bond can rotate and gives to the backbone some conformational mobility. The orientation of the depsicarbonyl bond depends on the surrounding. Apparent discrepancies between the different methods that are applicable for the disclosure of the nature of peptide H‐bonding, have found a rational explanation.
The Use of Primed Synthesis by DNA Polymerase I to Study an Intercistronic Sequence of ΦX‐174 DNA
Authors: DONELSON John E. , BARRELL Bart G. , WEITH H. Lee , KÖSSEL Hans , SCHOTT Herbert
Published: March 2005

A decadeoxynucleotide complementary to ten nucleotides in the major ribosome‐protected fragment of ΦX‐174 plus‐strand DNA has been chemically ksynthesized and used as a primer for DNA Polymerase I on ΦX‐174 plus‐strand DNA as template. The sequence of the first 40 nucleotides incorporated onto the decadeoxynucleotide has been determined. This sequence extends further the sequence of the intercistronic region preceding gene G and shows the presence of another termination codon. The sequence was determined by using manganese as the activating cation for DNA polymerase I which allows ribonucleotides to be incorporated as well as deoxyribonucleotides. The ribo‐substituted product was then cleaved specifically at the ribonucleotide residues to generate a series of overlapping ribo‐terminated fragments whose sequences were sufficient to determine the complete sequence of the first 40 nucleotides. No evidence for misincorporation by DNA polymerase I in the presence of manganese was detected.
The Photochemical Inactivation of Peptidyl Transferase Activity
Authors: WAN Kwong K. , ZAHID Nasir D. , BAXTER Ross M.
Published: March 2005

The photochemical oxidation of the 50‐S ribosomal subunit results in a rapid irreversible loss of peptidyl transferase activity. The first‐order rate of inactivation occurring during the first forty minutes suggests that a single reactive group is being inactivated. The pH profile of inactivation exhibits a maximum at pH 7.5.

Erythromycin at a low concentration (0.04 μmol) affords significant protection. Puromycin also exerts a protective effect but at higher concentrations. Chloramphenicol, sparsomycin and lincomycin did not exert a protective effect.

The loss in catalytic activity was not accompanied by a loss in substrate binding affinity of the donor and acceptor substrates.
Properties of the High‐Molecular‐Weight Protein (Spectrin) from Human‐Erythrocyte Membranes
Authors: GRATZER Walter B. , BEAVEN Gilbert H.
Published: March 2005

The high‐molecular‐weight protein component from human erythrocytes has been isolated and its solubility properties studied. In physiological solvent conditions the spectrin is not aggregated and is unaffected, both in hydrodynamic properties and conformation, as judged by circular dichroism and intrinsic fluorescence, by the addition of calcium ions. When the pH is decreased an opalescence first sets in, which corresponds to an associated fibrous state of the protein, and when a critical pH is reached precipitation ensues. The precipitation profile is characterised by extreme sharpness, of the kind observed in the phase separation of polyacid–polybase mixtures or of polyampholytes. The addition of calcium ions displaces this precipitation edge towards higher pH. Sodium ions have a similar but smaller effect. The position of the profile is significantly displaced in aged spectrin preparations, or those from frozen erythrocyte ghosts. Fresh preparations of spectrin consist predominantly of a component sedimenting at 9.7 S, with a minor component at 4.4 S (and traces of higher aggregates). The pattern is independent of the ionic strength, or of the presence or absence of calcium ions. The proportion of the small component increases with time, and in spectrin preparations from frozen ghosts it invariably predominates. At low concentrations of guanidine hydrochloride the larger component gives place progressively to the smaller, and vanishes completely when the concentration of the denaturant reaches 1 M. The two components coexist at concentrations below this, and are not in rapid interconversion equilibrium. On recovery of the protein from the guanidine hydrochloride by dialysis, the original pattern of two components is regained. On the other hand the larger component is not found in the material recovered from guanidine hydrochloride solutions of preparations that contain only the small component at the outset. The recovered spectrin is similar to the starting material in its circular dichroism, in its pH‐precipitation profiles, and the manner in which the latter is modified by calcium ions. Molecular weight determination by sedimentation equilibrium shows that the 4.4‐S species has a molecular weight of some 230000, which is also the value derived from the extrapolated sedimentation coefficient in 6 M guanidine hydrochloride, and thus corresponds to single chains (of which two or more species are resolved in acrylamide gel electrophoresis in the presence of sodium dodecylsulphate); the 9.7‐S species, which characterises what is evidently the native state of the extracted spectrin, is found to be a dimer. The frictional coefficients of the monomer and dimer are appreciably different. That of the dimer is compatible with a somewhat asymmetric structure, but by no means to the extent expected for a myosin‐like or paramyosin‐like molecule.
Trypsin‐Kallikrein Isoinhibitor K (Type Kunitz) from Snails (Helix pomatia)
Authors: DIETL Thomas , TSCHESCHE Harald
Published: March 2005

A basic proteinase inhibitor, isoinhibitor K, was purified by SE‐Sephadex C‐25 column chromatography from the mixture of acid‐stable and heat‐stable isoinhibitors of the snail (Helix pomatia). Isoinhibitor K is homogeneous in polyacrylamide gel, cellulose acetate and polyacrylamide‐dodecylsulfate electrophoresis. From the electrophoretic mobility in dodecylsulfate‐polyacrylamide gel an apparent molecular weight of 6500 ± 200 was estimated. From the amino acid composition the inhibitor consists of 58 amino acid residues. It contains three disulfide bridges, a C‐terminal valine and a lysine residue at the reactive site. Isoinhibitor K inhibits the enzymes: bovine trypsin and chymotrypsin, porcine plasmin and pancreatic kallikrein®, the trypsin‐like component of Streptomyces griseus proteinase‐pronase E, and fungi proteinase K from Tritirachium album Limber, which is only inhibited very slightly in contrast to the effect of the mixture of isoinhibitors. The inhibitory effect of isoinhibitor K against these enzymes is compared to that of the mixture or of other isoinhibitors. The following enzymes are not inhibited by isoinhibitor K: Aspergillus proteinase P and alkaline bacillus proteinase 2231 (Röhm), which both are inhibited by the mixture of isoinhibitors. Porcine elastase, bacterial proteinase N (M) (Röhm), and a trypsin‐like proteinase from wheat are not inhibited, porcine acrosin and porcine serum kallikrein only to a very minor extent by the mixture of isoinhibitors. Reactive‐site peptide‐bond cleavage during inhibition could not be detected. Thus, the inhibitory behaviour is just as broad in specificity and as unusual as that of the trypsinkallikrein inhibitor (Kunitz) from bovine organs. The N‐terminus is blocked by pyroglutamic acid. Isoinhibitor K is the main component of the isoinhibitors secreted into the mucus and amounts to 35–40% of the mixture.
Regulation of Rat‐Liver Glycoprotein: N‐Acetylneuraminic Acid Transferase Activity by Pyrimidine Nucleotides
Authors: BERNACKI Ralph J.
Published: March 2005

CMP‐N‐acetylneuraminic acid : glycoprotein sialyltransferase activities were assayed in rat liver microsomal fractions using desialylated fetuin as the substrate acceptors for N‐acetylneuraminic acid. It was found that cytidine nucleotides specifically depressed enzyme activities. CMP was shown to act as a competitive inhibitor with an apparent Ki of 0.62 mM. N‐Acetylneuraminic acid at 1.15 mM had no effect on enzyme activities. Uridine nucleotides at 1.15 mM, especially UDP, increased enzyme activities. UDP may act as an allosteric activating agent increasing the apparent V. Other nucleotides, sugars and nucleotide‐sugars at similar concentrations affected sialyltransferase activities only slightly. A general mechanism is proposed for the regulation of glycosyltransferase activities by free nucleotides.
Enzymic Properties of Nitrated α‐Chymotrypsin and δ‐Chymotrypsin
Authors: SVENSSON K. Birte , IMHOFF Jean‐Marie , KEIL Borivoj
Published: March 2005

Chymotrypsinogen A and α‐chymotrypsin are both nitrated at tyrosines 146 and 171 by reaction with tetranitromethane. This substitution was essentially without influence on the overall rate constant for hydrolyses of N‐acetyl‐l‐tryptophan methyl ester and N‐acetyl‐l‐tyrosine ethyl ester catalyzed by α‐chymotrypsin and δ‐chymotrypsin, prepared by fast tryptic activation of nitrated chymotrypsinogen. With both ester substrates Km was doubled for nitrated α‐chymotrypsin.

Nitrated α‐chymotrypsin, nitrated δ‐chymotrypsin and δ‐chymotrypsin could all bind Nacetyl‐L‐tryptophan methyl ester at alkaline pH, in contrast to α‐chymotrypsin. The dissociation constant, Kd, of the complex of α‐chymotrypsin and basic pancreatic trypsin inhibitor was lowered ten‐fold relative to the constant obtained with unmodified α‐chymotrypsin. The nitrated δ‐chymotrypsin and δ‐chymotrypsin showed identical Kd values.

The nitrated α‐chymotrypsin is inactivated faster at pH 8.0 and 8.5 than α‐chymotrypsin and apparently by a different mechanism.
The Importance of Escherichia coli Ribosomal Proteins L1, L11 and L16 for the Association of Ribosomal Subunits and the Formation of the 70‐S Initiation Complex
Authors: KAZEMIE Mirabotalib
Published: March 2005

50‐S subunits were washed with LiCl solutions of different concentrations. After washing with 1 M LiCl solution the particles lost their ability to attach either to 30‐S subunits or to the AUG · 30‐S subunit · fMet‐tRNAfMet complex or to a poly(U) · 30‐S subunit · Phe‐tRNAPhe complex. Those proteins which were removed by LiCl were fractionated on a Sephadex G‐100 column. Of the fractionated proteins only the combinations L1 and L11 or L1 and L16 were essential for the association of 50‐S 1.0 cores (particles prepared by washing 50‐S subunits in 1.0 M LiCl) with 30‐S subunits. These three proteins were also required for the formation of a stable complex between 50‐S 1.0 cores, mRNA, 30‐S subunits and aminoacyl‐tRNA.