bluuebunny/crossref_metadata_2025 huggingface dataset:
Accumulation of tRNAMetf on 80‐S Ribosomes in vitro under the Influence of a Met‐tRNA Deacylase from Rat‐Liver Microsomes
Authors: NYGÅRD Odd , HULTIN Tore
Published: March 2005

A Met‐tRNA deacylase has been partially purified from the 0.5 M KCI wash of rat liver microsomes. In preparative sucrose gradients, the active component sediments as a single band at about 6 S, corresponding to an estimated molecular weight of 1.7 × 105. The deacylase is specific for Met‐tRNA, without discriminating between Met‐tRNAf and Met‐tRNAm. Met‐tRNAf bound to the initiation factor IF‐MP in the ternary complex, IF‐MP. GTP. Met‐tRNAf, or to initiation‐factor‐dependent, complexes with 40‐S subunits or 80‐S ribosomes, is protected against deacylation. However, in the course of the initiation‐factor‐dependent joining of the 40‐S subunit complex to 60‐S ribosomal subunits, the bound Met‐tRNAf is exposed to added deacylase. Under these conditions, deacylation is inhibited by GTP. The tRNAMetf remains bound and accumulates on the 80‐S ribosomes.
The Functional and Fluorescence Properties of Escherichia coli RNA Polymerase Reacted with Fluorescamine
Authors: DAMJANOVICH Sándor , BÄHR Wolfgang , JOVIN Thomas M.
Published: March 2005

Fluorescamine (4‐phenylspiro[furan‐2,(3)1′‐phthalan]‐3,3′‐dione) reacts rapidly with Escherichia coli RNA polymerase and produces a fluorescent derivative which is inactivated to an extent dependent upon reagent concentration. Excess fluorescamine is rapidly hydrolysed. Reaction is with ɛ‐amino groups of lysine residues in all subunits as revealed by gel electrophoresis and fluorescence scanning.

The extent of inactivation and fluorescence yield are diminished in the presence of added template, a finding which provides evidence for the existence of reactive and essential amino groups which can be at least partially shielded by DNA in the binary complexes. The relative decrease of fluorescence is greatest in the ββ′ subunits. Holoenzyme and core enzyme show essentially the same behavior.

The inactivation of activity by fluorescamine is primarily at the level of initiation. Template binding and chain propagation are less affected.

The enzyme derivatized by fluorescamine shows an intense fluorescence with a peak at 490 nm and an excitation maximum at 390 nm. The fluorescence lifetime is in the range of 3–8 ns and the emission is highly polarized. In reactions carried out at high ionic strength the fluorescence yield is approximately double that at low ionic strength and insensitive to the presence of template.

Energy transfer is observed between the derivatized enzyme as donor and ethidium bromide as acceptor in the presence of template to which both the enzyme and intercalating dye are bound. The transfer efficiency is a function of the relative concentrations and of the conditions of reaction with fluorescamine. An average transfer distance of approx. 4–5 nm has been calculated suggesting a close proximity between bound polymerase and helical regions of the template.
Characterization of the pH 4.0 Endonuclease from Adenovirus‐Type‐2‐Infected KB Cells
Authors: REIF Ulrike , WINTERHOFF Ute , DOERFLER Walter
Published: March 2005

The properties of the pH 4.0 endonuclease from adenovirus‐type‐2‐infected KB cells were determined. The enzyme has a molecular weight of approximately 40 000. Its pH optimum is at pH 4.0, it is not inhibited by ethylenediaminetetraacetate (EDTA), and it is active at temperatures up to 60 °C. The enzyme cleaves adenovirus DNA in a stepwise manner. The limit digestion product has a molecular weight of 120 000–200 000. There is evidence that the cleavage reaction proceeds via an initial single‐strand nick. Under the conditions tested the endonuclease did not seem to reveal a high degree of specificity as to the recognition of cleavage sites, or else the sites recognized occurred very frequently.
Affinity Labeling of Rat‐Kidney γ‐Glutamyl Transpeptidase
Authors: INOUE Masayasu , HORIUCHI Seikoh , MORINO Yoshimasa
Published: March 2005

The reaction of γ‐glutamyl transpeptidase from rat kidney with a glutamine analog, 6‐diazo‐5‐oxo‐L‐norleucine, resulted in irreversible inactivation of the enzyme. The concentration of this reagent giving a half‐maximum rate of inactivation was 6 mM at pH 7.5. The inactivation was prevented by the presence of reduced glutathione in a competitive fashion, which indicates the active‐site‐directed nature of this reagent. The rate of inactivation was greatly accelerated in the presence of maleate, which is known to enhance the glutaminase activity of this enzyme. The presence of maleate increased the maximum velocity of the inactivation, but did not affect the affinity of the enzyme for 6‐diazo‐5‐oxo‐L‐norleucine.

Inactivation of the enzyme with 6‐diazo‐5‐oxo‐L‐[6‐14C]norleucine as well as with 6‐diazo‐5‐oxo‐L‐[1,2,3,4,5‐14C]norleucine resulted in a stoichiometric incorporation of radioactivity into the enzyme protein via covalent linkage. The amount of radioactivity incorporated was 1 mol 14C label 248 000 g enzyme protein.

A native enzyme preparation showing a single protein band on polyacrylamide gel electrophoresis gave four distinct bands upon sodium dodecylsulfate/polyacrylamide gel electrophoresis.

Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis of the 14C‐labeled enzyme, only the band moving the fastest towards the anode was found to contain radioactivity. This finding indicates that this protein band represents the catalytic component of the enzyme.
Repetitive Sequences in Protein A from Staphylococcus aureus
Authors: SJÖDAHL Jörgen
Published: March 2005

After tryptic digestion of intact Staphylococcus aureus the residual portion of protein A that was still bound to the cell wall was cleaved off with lysostaphin. From the two digests Fc‐binding fragments were isolated and the following characteristics observed. (a) There are four Fc‐binding, highly homologous regions, each consisting of 58–62 amino acid residues. (b) These regions are consecutively arranged from the N‐terminal part of the protein. (c) The residual C‐terminal part, approximately 150‐residues long, differs to a great extent with respect to primary and secondary structures from the four active regions. Furthermore, it is suggested that the protein is bound to the bacterial cell wall structure through this C‐terminal part.
Amino‐Acid Sequences of Heme‐Linked, Histidine‐Containing Peptides of Five Peroxidases from Horseradish and Turnip
Authors: WELINDER Karen Gjesing , MAZZA Gilbert
Published: March 2005

In a previous paper we have characterized five plant peroxidases, P1, P2, P3 and P7 of turnip and horseradish isoperoxidase C by peptide mapping studies, and only found two highly homologous sequences present in all. Both contained histidine. The findings supported previous suggestions of two histidine sequences near the peroxidase heme prostetic group. In the present paper we present the amino acid sequences around the histidine residues of all four turnip peroxidases, i. e. of 25 residues around the histidine proximal to heme, and 34 residues around the probably distally located histidine, and compare them with the histidine‐containing sequences of the complete amino acid sequence of horseradish isoperoxidase C. Substitutions of residues are rare close to these histidines, but more abundant with greater distances. The probably distal sequences of P1, P2, P3 and horseradish peroxidase C all contain two histidine residues, at positions 40 and 42. In P7, however, residue 40 is phenylalanine, a substitution presumably important to its abnormal physico‐chemical and enzymic properties.

Gel filtration profiles of tryptic digests of the turnip isoperoxidases confirm their previous classification into a P1, P2 and P3 group and a distinct P7 enzyme, but further prove the presence of several sites of carbohydrate attachment in P1, P2 and P3 peroxidases, like in horeseradish peroxidase C which has eight sites. P7 has one such site.
A Mutant ATP Synthetase of Escherichia coli with an Altered Sensitivity to N,N′‐Dicyclohexylcarbodiimide: Characterization in Native Membranes and Reconstituted Proteoliposomes
Authors: FRIEDL Peter , SCHMID Brigitte I. , SCHAIRER Hans U.
Published: March 2005

Dicyclohexylcarbodiimide‐resistant mutants of Escherichia coli were isolated and characterized. In one mutant the mutation is closely linked to the unc genes and affects the membrane‐integrated part of the ATP synthetase. The sensitivity of ATP synthetase functions to N,N′‐dicyclohexylcarbodiimide was compared in wild‐type and mutant membranes.

The membrane‐integrated part of the wild‐type ATP synthetase is highly sensitive to 50 μM dicyclohexylcarbodiimide, as measured by inhibition of ATPase activity, ATP‐dependent membrane energization and restoration of lactate‐dependent energization of ATPase‐depleted membranes. In mutant membranes this concentration has only a slight effect on these activities whereas a severe inhibition is obtained at 200 μM. Using the highly water‐soluble 1‐ethyl‐3(3‐dimethylaminopropyl)‐carbodiimide the activities of wild‐type and mutant membranes are inhibited to the same extent.

The ATP synthetase of wild‐type and mutant was partially purified and incorporated into liposomes. These showed an uncoupler‐sensitive ATP‐32Pi exchange and ATP‐dependent quenching of acridine‐dye fluorescence. The activities of mutant and wild‐type proteoliposomes exhibit the same pattern of sensitivity to dicyclohexylcarbodiimide as the corresponding membranes.
Ca‐Linked Phosphorylation of a Light Chain of Vertebrate Smooth‐Muscle Myosin
Authors: SOBIESZEK Apolinary
Published: March 2005

In vertebrate smooth muscle actomyosin and myofibrils a myosin light chain of molecular weight about 20000 becomes phosphorylated at the same Ca2+ concentration as required to stimulate the actin‐activated ATPase activity of myosin. Further, the degree of phosphorylation in the preparations as well as in various reconstituted actomyosins is proportional to their measured Ca2+ sensitivity. The phosphorylation process is very rapid and is essentially completed before the rise in ATPase activity. The enzyme responsible for the observed myosin phosphorylation is a specific myosin light chain kinase which is routinely co‐purified with myosin. This kinase is normally present in actomyosin and its removal together with tropomyosin leads to a complete loss of the actin‐activated ATPase activity. It is suggested that the Ca‐dependent phosphorylation of the light chain via the light chain kinase represents the initial step in the activation of myosin that leads to contraction. Relaxation is probably effected by an as yet uncharacterised light chain phosphatase.
Studies on a Carboxypeptidase Y Mutant of Yeast and Evidence for a Second Carboxypeptidase Activity
Authors: WOLF Dieter H. , WEISER Ulrike
Published: March 2005

Immunological studies on the carboxypeptidase Y mutant prc1‐1 of Saccharomyces cerevisiae revealed the origin of mutation in the structural gene of carboxypeptidase Y. The absence of carboxypeptidase Y has no effect on growth, even after drastic changes of growth conditions. A double mutant (prc1−leu2−) lacking carboxypeptidase Y and auxotrophic for leucine is able to grow on the peptide benzyloxycarbonylglycylleucine (Cbz‐Gly‐Leu) as sole nitrogen source, indicating the existence of a second carboxypeptidase. Using a new peptidase test, the existence of this second enzyme, called carboxypeptidase S, was confirmed biochemically.
Purification and Properties of Coliphage 21 Repressor
Authors: BALLIVET Marc , REICHARDT Louis F. , EISEN Harvey
Published: March 2005

Repressor from the lambdoid phage 21 has been purified. It specifically binds to λimm21 and P22 DNA. Kinetics of the binding reaction to λimm21 DNA are second‐order, suggesting that the rate‐limiting step is the formation of a dimer.
Intramolecularly‐Quenched Fluorescent Peptides as Fluorogenic Substrates of Leucine Aminopeptidase and Inhibitors of Clostridial Aminopeptidase
Authors: CARMEL Amos , KESSLER Efrat , YARON Arieh
Published: March 2005

Fluorogenic oligopeptide derivatives of the type Lys(ABz)‐X‐ONBzl, where ABz is o‐amino‐benzoyl (anthraniloyl), X stands for Ala, Phe, or Ala‐Ala, and ONBzl is p‐nitrobenzyloxy, were synthesized and shown to be hydrolyzed by leucine aminopeptidase. The hydrolysis is accompanied by an increase in fluorescence due to disruption of the intramolecular quenching of the fluorescent anthraniloyl moiety by the nitrobenzyl ester group. The spectral characteristics of the compounds are not consistent with an energy transfer mechanism according to Förster, therefore the quenching is assumed to be caused by a direct encounter between the quenching and the fluorescent groups. The change in fluorescence that accompanies the enzymic hydrolysis of the first peptide bond was used for quantitative measurement of the activity of leucine aminopeptidase and for the determination of some of its kinetic parameters. A bacterial aminopeptidase from Clostridium histolyticum that is very similar to leucine aminopeptidase in its substrate specificity did not hydrolyze the above peptide derivatives. The hydrolysis of leucine p‐nitroanilide by this enzyme was found to be inhibited by the three peptides and the corresponding inhibition constants were determined.
The Purification, Characterization and Partial Sequence Determination of a Trout Testis Non‐histone Protein, HMG‐T
Authors: WATSON David C. , PETERS Erwin H. , DIXON Gordon H.
Published: March 2005

A specific non‐histone chromatin‐associated protein, having a high content of both acidic and basic amino acids has been isolated from the chromatin of rainbow trout (Salmo gairdneril) testis cells. The protein has been prepared by the extraction of chromatin with 0.35 M sodium chloride and purified by chromatography on carboxymethyl‐cellulose with a gradient of lithium chloride at pH 9.0. The amino acid sequence of the first 29 residues of the amino‐terminal region has been determined using an automatic protein sequencer. The primary structure of this protein differs from that of any of the histones yet sequenced and, therefore, cannot be a degradation product of any of them. Moreover, the N‐terminal amino acid sequence shows considerable similarity to the HMG‐1 and HMG‐2 chromosomal proteins described by Goodwin et al. [Eur. J. Biochem. 38. 14–19 (1973)] whose N‐terminal sequences were also determined in this laboratory.
Changes in the Sequence Diversity of Polyadenylated Cytoplasmic RNA during Testis Differentiation in Rainbow Trout (Salmo gairdnerii)
Authors: LEVY W. Beatriz , DIXON Gordon H.
Published: March 2005

We have compared the sequence diversity of polyadenylated cytoplasmic RNA derived from naturally maturing trout testis at three different stages of spermatogenesis, by performing hybridization experiments between cDNA synthesized on a template of polyadenylated RNA and a vast excess of polyadenylated RNA. Polyadenylated RNA from early testis has a base sequence complexity of 7.2 × 109 daltons. As testis maturation proceeds, there is a decrease in the complexity of polyadenylated mRNA sequences in testis cells and the relative abundance of individual mRNAs varies over a more narrow range. Heterologous hybridization reactions demonstrate that a substantial fraction of polyadenylated RNA sequences present in early testis cytoplasm is absent from late testis cytoplasm. Nevertheless, all the sequences of late testis mRNA are represented in the population of early testis mRNA molecules.
Phospholipid Binding and Self‐Association of the Major Apoprotein of Human and Baboon High‐Density Lipoproteins
Authors: ROSSENEU Maryvonne , BLATON Victor , VERCAEMST Rafaël , SOETEWEY Frederik , PEETERS Hubert
Published: March 2005

The purpose of this study was to establish a relationship between self‐association and phospholipid binding of the human and the baboon apoA‐I protein.

The enthalpy changes on binding dimyristoyl lecithin and lysolecithin to either the human or the baboon native apoA‐I protein were measured in a microcalorimeter. An endothermal process, most pronounced for the human apoprotein, was observed at low phospholipid levels. At higher phospholipid to protein ratios the binding was exothermal. Gel filtration experiments on Sephadex G‐200 showed that the native apoprotein of both species consists of dimers and tetramers. The baboon native apoA‐I protein contained a higher amount of dimers. After preincubation of the apoA‐I protein with lysolecithin, the enthalpy changes measured on subsequent binding of dimyristoyl lecithin were shifted towards more exothermal values compared to the curve for the native apoprotein. The amplitude of this shift corresponds to that of the endothermal process observed on binding dimyristoyl lecithin to the native apoprotein. This process was attributed to a phospholipid‐induced disaggregation of the apoA‐I protein.

Gel filtration data showed a decreased extent of aggregation in the apoA‐I protein preincubated with lysolecithin. This sample consisted exclusively of dimers. Ultracentrifugal flotation of the complexes formed between the apoA‐I protein, and respectively dimyristoyl lecithin and sphingomyelin indicated that preincubation with lysolecithin increased the extent of complex formation.

These results suggest that the dimeric form of the apoA‐I protein possesses the highest affinity for phospholipids. Any dissociation of higher polymers enhances the phospholipid‐binding capacity of the human and the baboon apoA‐I protein.
Purification and Properties of Pyrazon Dioxygenase from Pyrazon‐Degrading Bacteria
Authors: SAUBER Klaus , FRÖHNER Christoph , ROSENBERG Gideon , EBERSPÄCHER Jürgen , LINGENS Franz
Published: March 2005

Chromatography on DEAE‐cellulose and gel filtration on Sephadex revealed that pyrazon dioxygenase from pyrazon‐degrading bacteria consists of three different enzyme components. No component alone oxidizes the phenyl moiety of pyrazon, only when the three components are combined can oxidation be detected. Following electron paramagnetic resonance and ultraviolet measurements the protein nature of the three components was determined: component A1 (molecular weight about 180000, red‐brown in colour) is an iron‐sulphur protein. The existence of approximately two moles of iron and two moles of inorganic sulphur per mole of protein was demonstrated. This enzyme component was purified to homogeneity in disc electrophoresis. Component A2 is a yellow protein of a molecular weight of about 67000. FAD was shown to be the prosthetic group of this protein. Component B (molecular weight about 12000, brown in colour) is a protein of the ferredoxin type, which was purified to homogeneity, as demonstrated by disc electrophoresis.

A hypothetical scheme for the cooperation of the three components is proposed: component A2 accepts as cosubstrate NADH and functions as a ferredoxin reductase. The ferredoxin, component B, has the function of an electron carrier. The conversion of the substrates is effected by component A1, the terminal dioxygenase.
Conformations of Purine Ribosyl 5′‐Nucleotides Bound to Glycogen Phosphorylase b
Authors: MORANGE Michel , KOLB Annie , BUC Henri , CHACHATY Claude , LANGLET Gérard
Published: March 2005

The conformation of 5′‐nucleotides in the active site of glycogen phosphorylase b has been deduced from linewidth measurements of protons H‐1′, H‐8 and H‐2. It is shown by selective deuteration of the purine ring in position 8 that the orientation of the base is anti in the case of strong activators like AMP and syn in that of weak activators like IMP. The orientation correlation time of the nucleotides in the active site is nearly that of the enzyme, i.e. 160 ns at 21 °C.
The “b5‐like” Domain from Chicken‐Liver Sulfite Oxidase: A New Case of Common Ancestral Origin with Liver Cytochrome b5 and Bakers' Yeast Cytochrome b2 Core
Authors: GUIARD Bernard , LEDERER Florence
Published: March 2005

Limited chymotryptic digestion of chicken‐liver sulfite oxidase destroys its ability to oxidize sulfite. From the digest can be isolated a heme‐binding fragment of molecular weight about 11000. Its purification is described, as well as its characterization by a number of methods (absorption spectroscopy, circular dichroism, electrophoretic mobility, immunochemical reactivity, amino acid analysis). The heme spectrum shows no detectable difference with that of the native enzyme.

The N‐terminal sequence of this sulfite oxidase core is reported (34 residues). It shows a strong similarity to that of liver microsomal cytochrome b5 and bakers' yeast cytochrome b2 core. The sequence comparison is discussed in terms of structural similarity to cytochrome b5. Our data suggest a common evolutionary origin for the three b‐type cytochromes.
Ribonuclease H Levels during the Response of Bovine Lymphocytes to Concanavalin A
Authors: BÜSEN Werner , PETERS J. Hinrich , HAUSEN Peter
Published: March 2005

Several enzymes with ribonuclease H specificity have been identified in bovine lymphoid tissue. Their nomenclature and some of their properties have been reported previously. In this study the time course of the induction of the different ribonuclease H activities during the stimulation of resting bovine lymph‐node cells with concanavalin A was investigated. The activity of one of these enzymes (ribonuclease H IIb) increases in parallel with the induction of uridine incorporation and is well separated from the induction of ribonuclease H I, which increases together with DNA synthesis. These data indicate that the different ribonuclease H activities serve different physiological functions. They suggest that ribonuclease H I belongs to the set of enzymes which are involved in DNA synthesis.
Fourth Expansion and Glucose Perturbation of the Dictyostelium Kinetic Model
Authors: WRIGHT Barbara E. , TAI Anna , KILLICK Kathleen A.
Published: March 2005

The kinetic model of carbohydrate metabolism has been expanded to include: (a) the accumulation of α and β‐cellulose, insoluble cell‐wall glycogen and mucopolysaccharide; (b) the role of RNA turnover as a source of carbon for end‐product synthesis and as a buffer regulating the level of uridine nucleotides in this metabolic network; and (c) the role of purine‐nucleoside phosphorylase, 5′‐AMP nucleotidase, nucleosidediphosphate kinase and polynucleotide phosphorylase. One of many predictions based on this model is that cells differentiating in the presence of glucose will produce sorocarps with an abnormally high trehalose to cellulose ratio. External perturbation of either the model or of developing cells by glucose increases the levels of sorocarp trehalose and glycogen, 5‐fold and 6‐fold respectively. Evaluation of the experimental data and the simulation analyses have allowed several predictions to be made concerning the compartmentation of metabolites and the permeability of cells to glucose during differentiation.
Association Kinetics and Binding Constants of Nucleoside Triphosphates with G‐Actin
Authors: WAECHTER Felix , ENGEL Jürgen
Published: March 2005

The dissociation of the complex between 1:N6‐ethenoadenosine 5′‐triphosphate (ɛP) and G‐actin was initiated by dilution to concentrations between 1 μM and 5 nM and monitored by the fluorescence change of ɛP. The results were quantitatively explained by a two‐step mechanism: a reversible dissociation of the actin · nucleotide complex followed by a fast irreversible inactivation of nucleotide‐free G‐actin. Under normal conditions (0.8 mM CaCl2, pH 8.2, 21 °C), the rate‐limiting step was the dissociation of the nucleotide· G‐actin complex. The half‐time of the dissociation of ɛP from G‐actin was 290 s as compared to only 13 s for the following denaturation step of nucleotide‐free actin. 1 mM EDTA highly accelerated the dissociation step and, regardless of its concentration, the complex dissociated quantitatively within 1 min. Addition of Ca2+ within 20 s after EDTA addition induced a re‐association of ɛP with nucleotide‐free but still native G‐actin. This reversal was kinetically resolved by means of a multimixing stopped‐flow apparatus. The association rate constant was 6 × 106 M−1 s−1. From the association and dissociation rate constant, a value of 2.5 × 109 M−1 was calculated for the binding constant of ɛP to G‐actin. The binding constant of ATP (1.4 × 1010 M−1) was derived from the relative binding constant of ɛP and ATP as determined by fluorescence titration of ɛP · G‐actin with ATP. These binding constants are 103–104 times higher than values reported earlier on the basis of more indirect data.
Antibodies and Autoantibodies of Glycogen Phosphorylase b: Inactivation of Pig and Rabbit Enzymes
Authors: TZARTOS Socrates J. , EVANGELOPOULOS Athanasios E.
Published: March 2005

Pig skeletal muscle glycogen phosphorylase b was purified using ammonium sulfate fractionation, DEAE‐Sephadex A‐50 and Sephadex G‐200 column chromatography. The purified enzyme was used to immunize rabbits in the presence or in the absence of complete Freund adjuvant.

Antibodies against pig phosphorylase in pure form were isolated from rabbit antisera using insoluble immunoadsorbents of pig phosphorylase. Autoantibodies against the rabbit enzyme were obtained from the same antisera using insoluble immunoadsorbents of rabbit phosphorylase. Complete inactivation of pig phosphorylase was accomplished by an antibody/enzyme molar ratio equal to 4 and autoantibody/enzyme molar ratio equal to 130. Complete inactivation of rabbit phosphorylase was accomplished by an antibody enzyme molar ratio equal to 250 and auto‐antibody/enzyme molar ratio equal to 160. Passive haemagglutination technique gave positive results with minimum amounts of 0.02 μg/ml and 0.8 μg/ml for pig and rabbit phosphorylase respectively.

Kinetic experiments have shown that antibodies and autoantibodies act as noncompetitive inhibitors of both enzymes with respect to AMP and glucose 1‐phosphate but exhibit a mixed type of inhibition with respect to glycogen. When glycogen hydrolysates were used as substrate in place of intact glycogen molecules a pronounced decrease in the inhibitory capacity of antienzyme on the enzyme was demonstrated.
Thyrotropin Binding to and Adenylate Cyclase Activity of Porcine Thyroid Plasma Membranes
Authors: VERRIER Bernard , PLANELLS Richard , LISSITZKY Serge
Published: March 2005

Plasma membranes have been purified from porcine thyroid gland homogenate by discontinuous sucrose gradient centrifugation. The preparations contained specific binding sites for thyrotropin but not for luteinizing hormone or the β subunits of thyrotropin and luteinizing hormone. Optimum conditions of 125I‐labeled thyrotropin binding were pH 6.0–6.5 and 37°C. Thyrotropin binding was reduced by divalent (Ca2+, Mg2+) and monovalent cations (Na+, K+, Li+), 50% inhibition being obtained at 10 mM and 50 mM respectively. Displacement curves of 125I‐labeled bovine or porcine thyrotropin by the unlabeled hormone from three species was in the order of increasing concentrations (bovine > porcine > human) which is the order of decreasing biological activity of these hormone preparations in the assay in vivo in the mouse. The validity of the results was established by controlling that porcine membranes bound the native and the 125I‐labeled hormones with equal affinity. A single type of high‐affinity (Kd= 0.28 nM) binding sites was detected for bovine and porcine thyrotropins. In contrast, porcine plasma membranes bound human thyrotropin with a lower affinity (Kd= 70 nM). A good correlation was found at equilibrium and in the conditions of the cyclase assay, between receptor occupancy and adenylate cyclase activation for the three hormones.
A Nuclear Elongation Factor of Transcription from Physarum polycephalum in vitro
Authors: ERNST Gerold Hans , SAUER Helmut Wilhelm
Published: March 2005

Homogenates of Physarum plasmodia contain a factor which stimulates UMP incorporation on native DNA by solubilized homologous RNA polymerases in vitro. The factor is a heat‐sensitive protein and has been located in nuclei. It does not alter the template activity of DNA nor the initiation frequency of transcription. The factor interacts with free or bound RNA polymerase molecules (only at 37 °C and at low ionic strength) and yields larger transcripts in vitro.

The level of the factor in vitro fluctuates: it is gradually reduced during spherulation and reaches its maximum in mid S phase of the cell cycle of Physarum.
The Molecular Defect in a Case of (Cystathionine β‐Synthase)‐Deficient Homocystinuria
Authors: GRIFFITHS Roger , TUDBALL Norman
Published: March 2005

Cystathionine β‐synthase activity isolated from fibroblast cultures obtained from the skin of a normal and a homocystinuric individual were both cross‐reactive with normal human liver cystathionine β‐synthase antibody.

Isoelectric focusing revealed a substantial difference in the isoelectric points of the normal and abnormal fibroblast enzymes.

Treatment of purified samples of normal and abnormal fibroblast enzymes with sodium dodecylsulphate followed by polyacrylamide gel electrophoresis indicated that both normal and abnormal enzymes were composed of two sub‐units of molecular weights 53000 and 70000.

A combination of urea and sodium dodecylsulphate treatment revealed that the respective 53000 molecular weight sub‐units were different.

It has been concluded that the molecular defect in the case of pyridoxine non‐responsive homocystinuria examined in the present investigation arises as a result of an alteration in the structural gene which codes for the lower molecular weight sub‐unit of cystathionine β‐synthase.
The Kinetics of Reoxidation of Reduced Benzylamine Oxidase
Authors: OLSSON Bengt , OLSSON Jadwiga , PETTERSSON Gösta
Published: March 2005

The mechanism of reoxidation of reduced benzylamine oxidase has been investigated at different pH between 6 and 10 by steady‐state and transient‐state kinetic methods.

The reoxidation process involves minimally a second‐order interaction between reduced enzyme and oxygen leading to the formation of a spectrally modified enzyme intermediate, and a subsequent first‐order step converting this intermediate into free enzyme. The variation with pH of rate constants according to such a reaction scheme is reported.

Under aerobic conditions the oxygen‐independent reaction represents the main rate‐limiting step in the catalytic process at alkaline pH. At neutral or acid pH the interaction between reduced enzyme and oxygen becomes mainly rate‐limiting, indicating that the concentration of oxygen may be a critical factor controlling enzyme activity under physiological conditions.

The spectrally modified intermediate formed during the reoxidation process exhibits a dif‐ference‐absorption band centered around 290 nm in comparison to free enzyme, and an additional difference‐absorption band at 470 nm in comparison to reduced enzyme. These data indicate that formation of the intermediate, besides leading to a reappearance of the 470‐nm absorption band dis‐appearing on reduction of the enzyme, results in a spectral perturbation of one or several aromatic amino‐acid residues in the protein. This perturbation could possibly reflect a conformational change of the enzyme.
Determination of the Solution Conformation of Adenosine 2′:3′‐Monophosphate by Nuclear Magnetic Resonance with Lanthanide Probes
Authors: FAZAKERLEY G. Victor , WOLFE Melanie A.
Published: March 2005

The aqueous solution conformation of adenosine 2′:3′‐monophosphate at pH 2.5 has been determined by a nuclear magnetic resonance method utilizing lanthanide ions as shift and relaxation probes. The ribose conformation is best described as a rapid equilibrium of 2′‐endo(3′‐exo) and 3′‐endo(2′‐exo) conformations in a ratio of approximately 2 to 1. The orientation of the base relative to to ribose is restricted to a narrow range about χcn=−70°.
The Immunochemistry of Peptidoglycan
Authors: SEIDL Peter H. , SCHLEIFER Karl H.
Published: March 2005

An albumin‐peptide conjugate was synthesized, which carries pentaglycine peptides with C‐terminal glycine residues as found in the interpeptide bridges of the peptidoglycan of many staphylococci. Immunization of rabbits with this synthetic immunogen yielded antisera containing predominantly antibodies against the peptide moiety of the conjugate. The quantitative precipitin and the Ouchterlony agar gel reaction with several synthetic protein‐peptide‐conjugates, immunoaffinity chromatography of the antisera on Sepharose‐(Gly‐Gly‐Gly‐Gly‐Gly)n and hapten inhibition studies with several synthetic peptides and peptide derivatives demonstrated that the antibodies were highly specific for oligoglycine peptides with C‐terminal glycine. These antibodies also reacted strongly with staphylococcal peptidoglycans with an interpeptide bridge composed of pentaglycine peptides or of pentaglycine peptides in which part of the glycine residues were replaced by L‐serine. In contrast, all the peptidoglycans lacking interpeptide bridges composed of glycine residues gave no precipitin reaction at all. The final proof for identical determinant groups of albumin‐(CH2CO‐Gly‐Gly‐Gly‐Gly‐Gly)31 and the staphylococcal peptidoglycans applied in the precipitin reaction was furnished by double gel diffusion studies and by hapten inhibition of the precipitin reaction between antisera to albumin‐(CH2CO‐Gly‐Gly‐Gly‐Gly‐Gly)31 and the corresponding peptidoglycans.

For rapid screening of the different peptidoglycans, a latex agglutination test was elaborated. Purified antibodies were adsorbed to latex particles, and the titers with the particular peptidoglycans were then determined. The test was highly sensitive, in that 10 nanograms of peptidoglycan could still be detected.
Studies of Glutamate Dehydrogenase
Authors: DAVID Michael , RASCHED Ihab R. , SUND Horst
Published: March 2005

In phosphate buffer at pH 7.0, 5,5′‐dithio‐bis(2‐nitrobenzoic acid), N‐ethylmaleimide or iodo‐acetamide do not alter the activity of beef liver glutamate dehydrogenase. Iodoacetate, however, inactivates the enzyme irreversibly by alkylation. Combined addition of the coenzyme NADH and the substrate 2‐oxoglutarate or the effector GTP protects against this inactivation.

The alkylation reaction is independent of pH between pH 6–9 indicating that amino, imidazole or phenolic groups are probably not involved in this reaction. Titration of the thiol groups, after denaturation of the enzyme, revealed the loss of approximately one group per polypeptide chain. However, this is not due to the exclusive alkylation of a cysteine residue, since alkylation with iodo‐[2‐14C]acetic acid also labels a methionine residue. 50% of the label is incorporated into methionine‐169 and only 7% into cysteine‐115, the remaining radioactivity is distributed in minor quantities (4%) in several unidentified residues. A probable cause of the erroneous thiol groups titration is discussed.
A Mathematical Model for the Influence of Anionic Effectors on the Phosphofructokinase from Rat Erythrocytes
Authors: OTTO Matthias , HEINRICH Reinhart , JACOBASCH Gisela , RAPOPORT Samuel
Published: March 2005

The influence of the positive effectors AMP, sulphate, glucose 1,6‐bisphosphate and the negative effector 2,3‐bisphoglycerate on rat erythrocyte phosphofructokinase has been investigated. The kinetic data have been fitted to the Monod‐Wyman‐Changeux model as well as to a model based on a closed association‐dissociation equilibrium. The application of the fitting procedure yields for both models a good correspondence between theoretical and experimental data and equal results with respect to the action of the effectors on the enzyme. The corresponding dissociation constants for the binding of the positive effectors to the active state are: AMP 35 μM, sulphate 0.43 mM and glucose 1,6‐bisphosphate 15 μM. 2,3‐Bisphosphoglycerate as an inhibitor stabilizes the inactive state (dissociation constant: 1.4 mM).

A preliminary discrimination between the Monod‐Wyman‐Changeux model and the association‐dissociation model has been attempted.
Aminopeptidase N from Escherichia coli
Authors: MURGIER Maryse , PELISSIER Corinne , LAZDUNSKI Andrée , BERNADAC Alain , LAZDUNSKI Claude
Published: March 2005

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30–40%) by osmotic shock or by converting Escherichia coli K 10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75–85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N‐ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I‐labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and β‐galactosidase to fluorescamine in intact cells. Only 16% of the total β‐galactosidase was labeled with this fluorescent reagent whereas 44–45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places.

Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.
Mitochondrial Heredity of Resistance to 3‐(3,4‐Dichlorophenyl)‐1,1‐dimethylurea, an Inhibitor of Cytochrome b Oxidation, in Saccharomyces cerevisiae
Authors: COLSON Anne‐Marie , THE VAN Luu , CONVENT Bernard , BRIQUET Michel , GOFFEAU André
Published: March 2005

3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (diuron), an inhibitor of cytochrome b oxidation, has been used for the selection of three resistant mutants, (diur) of Saccharomyces cerevisiae. The mutant diur‐64 exhibits in vivo cross‐resistance to antimycin A while diur‐34 and diur‐1 are more sensitive to antimycin A than the parental strain. The three mutants exhibit mitochondrial inheritance according to the following criteria: mitotic segregation of diuron‐resistant and diuron‐sensitive diploids is obtained among the diploid progeny of a cross between diur and dius; non‐Mendelian segregation of diuron resistance (4:0) is observed in spores of tetrads issued from diuron‐resistant diploid; extensive ethidium bromide treatment leads to the formation of Q– mutants which no longer transmit diur and dius alleles.

Evidence for two distinct diuron‐resistant loci were obtained by allelism tests. Recombination analysis shows that diuron‐resistance is not located in the polar region of the mitochondrial genome. The diur loci are not linked to the erythromycin locus since the upper limit in recombinants frequency (26%) for a non‐polar region is obtained between diur and eryr. A low recombinants frequency (3%) is observed in crosses between diur‐34 mutation and the two mutants cob1 and cob2 suggesting that diur‐34 might be located between these two cytochrome‐b‐deficient loci.

The resistance to diuron is also expressed in vitro since the oxidation rates of succinate by sonicated submitochondrial particles from the mutants are clearly less sensitive to diuron than that of the wild type.
Synergistic Interaction of the Streptogramins with the Ribosome
Authors: CONTRERAS Antonio , VÁZQUEZ David
Published: March 2005

Quantitative binding studies of [G‐3H]streptogramin A and [G‐3H]streptogramin B with high‐salt‐washed ribosomes were carried out in the presence of a minimum of 10% (v/v) ethanol because of the antibiotic insolubility in water. It was observed that the presence of streptogramin A increases the affinity of [G‐3H]streptogramin B for the ribosome. Thus the dissociation constant for [G‐3H]‐streptogramin B interaction with the ribosome is Kd= 13.3 nM in the presence of streptogramin A and Kd= 59 nM in its absence. Furthermore the values for the dissociation constants for [G‐3H]‐streptogramin B interaction in the presence of 50% (v/v) ethanol, were Kd= 0.13 μM in the presence of streptogramin A and Kd= 0.70 μM in its absence. This increased affinity of [G‐3H]streptogramin B in the presence of streptogramin A can explain the synergistic effects of mixtures of streptogramins A and B at the ribosome level.
The Purification and Properties of NADP‐Dependent Isocitrate Dehydrogenase from Ox‐Heart Mitochondria
Authors: MACFARLANE Neil , MATHEWS Brian , DALZIEL Keith
Published: March 2005

The purification of NADP‐linked isocitrate dehydrogenase from ox heart mitochondria is described. The molecular weight from gel filtration, sedimentation equilibrium and gel electrophoresis is 90000 ± 4000, and there are two subunits in the molecule each of which binds NADPH with enhancement of the coenzyme fluorescence. The amino‐acid composition is reported, and the absorption coefficient, A2801% estimated from dry weight measurements is 11.8 cm−1.
Complexity of the Structure of Particles Containing Heterogeneous Nuclear RNA as Demonstrated by Ribonuclease Treatment
Authors: STEVENIN James , GALLINARO‐MATRINGE Héléne , GATTONI Renata , JACOB Monique
Published: March 2005

Brain ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNA) and a large number of polypeptides ranging from 23000 to 150000 molecular weight were treated with pancreatic and T1 ribonucleases at low (0.1 or 0.5 μg plus 5 or 25 units/ml), high (2 μg plus 100 units/ml) and very high (20 μg plus 1000 units/ml) concentrations.

At low enzyme concentration, a large fraction of the particle material accumulated at 35–45 S. The accumulation was more marked for proteins in the 30000–38000 molecular weight range. Heterogeneous complexes containing a large spectrum of proteins with a characteristic distribution were disclosed between 60 and 200 S. At high ribonuclease concentration the total quantity of proteins at 35–45 S decreased, but the proteins of 30000–38000 molecular weight predominated. Heterogeneous complexes were also present. Finally, at very high enzyme concentration, the particles were almost entirely hydrolyzed. Only a small amount of heterogeneous complexes subsisted at 30–190 S.

The RNA content of the remaining ribonucleoprotein complexes as well as its size decreased upon ribonuclease treatment as shown by CsCl density determination and electrophoretic analysis. The results suggested that the stability of the complexes depended upon protein‐protein as well as RNA‐protein interactions. They also showed that sequences up to 200–300 nucleotides were protected by proteins against ribonuclease.

The results were compatible with the existence of at least 3 constituents in the particles. The previously defined monoparticle population accumulating at 35–45 S was heterogeneous in respect to ribonuclease sensitivity and protein composition. The 30000–38000 molecular weight proteins accumulating at low ribonuclease and predominating afterwards were assumed to belong to monoparticles α. Monoparticles β contained a larger range of proteins more easily released by the enzymes. The heterogeneous complexes were a third constituent whose relationship with the monoparticles war not established yet. A large fraction of the particle phosphoproteins was associated to these complexes.

On the basis of our experiments in vitro it is assumed that monoparticle α can be preferentially isolated from the nuclei under conditions where endogeneous ribonuclease is high and (or) not inhibited. This might explain why, in certain cases, only one or a few proteins were described in nuclear particles instead of the complete set present in the native particles.
An Analysis of the Bovine Genome by Density Gradient Centrifugation: Fractionation in Cs2SO4/3,6‐Bis(acetatomercurimethyl)dioxane Density Gradient
Authors: CORTADAS Jordi , MACAYA Gabriel , BERNARDI Giorgio
Published: March 2005

The fractionation of calf thymus DNA by centrifugation in density gradients of Cs2SO4/BAMD, where BAMD = 3,6‐bis(acetatomercurimethyl)dioxane, is described. A large‐scale separation of (dG + dC)‐rich DNA fractions has been obtained, allowing the relative amounts of minor and satellite components in the bovine genome to be precisely assessed.
Nickel Cytochrome c. Effect of Protein Moiety on the Metal Ion Coordination
Authors: FINDLAY Meredith C. , CHIEN James C. W.
Published: March 2005

Nickel cytochrome c has been synthesized by the reaction of metal‐free porphyrin cytochrome c with Ni(II) ions in 0.6 M glycylglycine and 4 M KSCN. Electronic spectra and susceptibility measurement showed the nickel to be in a high‐spin octahedral configuration exemplifying the strong influence of the protein moiety as a macrocyclic ligand on the coordination chemistry of the metal ion. Nickel cytochrome c has the same electrophoretic mobility, helicity and pK values of conformational transitions as the native enzyme. At high pH, the partially denatured nickel cytochrome c becomes dimeric. Nitric oxide reacts with nickel cytochrome c to form the nitrosyl derivative with g¶= 2.187 and g⊥= 2.140. Reaction of NO with nickel protoporphyrin IX dimethyl ester in toluene, pyridine, or methylthioethanol produced no stable nitrosyl products, clearly demonstrating the effect of protein on metal ion ligation.
Snake Venom Toxins
Authors: STRYDOM Daniēl J.
Published: March 2005

The third most abundant component of black mamba venom, named FS2, was sequenced with the aid of sequenator studies and peptides derived by tryptic and chymotryptic digestion. Cyanogen bromide digests provided extra information to support the proposed structure. This protein is a homologue of the short neurotoxins of snake venom, but is much less toxic. Its structure is quite different from both neurotoxins and the other mamba proteins, called angusticeps types (neurotoxin homologues). Comparison of the known angusticeps‐type toxins from mamba venom with mamba neurotoxins and each other leads to proposals that these proteins of low toxicity are inventions of the group of mambas and that three different, as yet unknown, functions will be associated with the three subgroups that are discernable.