bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and DNA‐RNA Hybridization Properties of Small‐Molecular‐Weight Nuclear RNA Components from Baby‐Hamster‐Kidney Cells
Authors: Engberg Jan , Hellung‐Larsen Per , Frederiksen Sune
Published: March 2005

Several small‐molecular‐weight nuclear RNA components (snRNAs) have been isolated and purified from nuclei of baby hamster kidney cells. The proportion of the cell genome complementary to the different snRNA components was investigated by DNA‐RNA hybridization. The values for saturation range between 0.4 × 10−4% and 0.8 × 10−4% which correspond to about 2000 genes for each of the snRNA components per cell. For comparison the saturation value for rRNA is 2.2 × 10−4% which corresponds to about 200 genes per cell. Saturation experiments with mixtures of the snRNA components indicate that there are separate genes for the different snRNA components.

Hybridization competition experiments show that no base sequence homologies exist between the snRNAs and other RNAs such as rRNA, 5‐S RNA and tRNA.

The DNA complementary to the snRNAs is distributed throughout the band of the bulk DNA of buoyant‐density fractionated DNA. This is in contrast to the DNA complementary to rRNA which bands as a heavy satellite. Hence the snRNA components can not be fragments of the non‐conserved parts of the rRNA precursor.
Affinity Chromatography on Immobilised Adenosine 5′‐monophosphate
Authors: Craven David B. , Harvey Michael J. , Dean Peter D. G. , Lowe Christopher R.
Published: March 2005

N
6‐(6‐Aminohexyl)‐5′‐AMP has been prepared by a single step reaction involving the nucleophylic displacement of the thiol group from 6‐mercaptopurine riboside 5′‐phosphate by 1, 6‐hexanediamine. The structure of the product has been confirmed by physical, chemical and enzymic analyses. The 5′‐AMP derivative was coupled to Sepharose 4‐B and used as an affinity adsorbent in the separation of dehydrogenases and kinases.
Affinity Chromatography on Immobilised Adenosine 5′‐monophosphate
Authors: Harvey Michael J. , Dean Peter D. G. , Lowe Christopher R.
Published: March 2005

Icreased temperatures decrease the affinity of glycerokinase and yeast alcohol dehydrogenase for N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose. Energies of adsorption can be calculated from Arrhenius plots but need to be corrected for the effects of temperature on the ionic strength of the eluant. The energy of adsorption of lactate dehydrogenase using NADH as a specific eluant was shown to have a minimum value of 54.6 kJ/mol (13.0 kcal/mol). Linear gradients of temperature can be successfully employed for the elution of enzymes from affinity adsorbents.
Localization and Quantification of Circular DNA in Yeast
Authors: Clark‐Walker G. Desmond , Miklos George L. Gabor
Published: March 2005

Saccharomyces cerevisiae contains 62 molecules per cell of a class of covalently closed circular DNA termed omicron (oDNA) of buoyant density 1.701 g/cm3 with a major size class of 1.9 μm. The localization of oDNA has been investigated in a respiratory deficient mutant 2eρ−1 containing altered mitochondrial DNA (ρDNA) in the form of covalently closed circles. There are 81 ρDNA molecules per cell and they range in size from 0.05 μm to 1.6 μm and have a buoyant density of 1.676 g/cm3. When this mini‐circular ρDNA is used as a marker of cytoplasmic contamination, we find that purified nuclei do not contain oDNA. We conclude from the cytoplasmic location of oDNA that it does not represent a gene amplification product nor is it related to “informosomal” DNA.
Animal DNA‐Dependent RNA Polymerases
Authors: Mandel Jean‐Louis , Chambon Pierre
Published: March 2005

We have analyzed the parameters which influence the transcription in vitro of the allomorphic forms of Simian virus 40 (SV 40) DNA by mammalian RNA polymerases AI and B. Comparative studies with Escherichia coli core and holoenzyme RNA polymerase are also reported. Our studies demonstrate that the nature of the divalent cation present in the reaction, the ionic strength, and the physical configuration of SV40 DNA have marked effects on the template efficiency of this DNA for mammalian RNA polymerases. It was found that the transcription of superhelical SV40 DNA form I was almost completely Mn2+‐dependent, irrespective of the enzyme type. Under all incubation conditions, the open circular form II and particularly the linear form III were poorly transcribed by AI and B RNA polymerases, suggesting that both enzyme have great difficulty in initiating RNA synthesis on well base‐paired intact double‐stranded DNAs. The effects of rifamycin AF/013 and of spermine on RNA synthesis catalysed by the mammalian enzymes on SV40 DNA were also analyzed. Most of our results can be explained in the light of the recent discovery of the existence, in superhelical DNAs, of an alteration of the double helix which could be magnified by the presence of Mn2+.
The Effects of Bases and Nucleosides on the Intracellular Contents of Nucleotides and 5‐Phosphoribosyl 1‐pyrophosphate in Escherichia coli
Authors: Bagnara Aldo S. , Finch Lloyd R.
Published: March 2005

It has been reported that supplying preformed purine and pyrimidine bases and nucleosides to growing cells of Escherichia coli and other organisms results in the preferential utilization of these precursors for nucleotide biosynthesis, but the factors responsible for inhibition of the de novo pathways in vivo are not known. We have surveyed the effects of addition of purine and pyrimidine bases and nucleosides on the intracellular contents of nucleotides and 5‐phosphoribosyl 1‐pyrophosphate (P‐Rib‐PP) of Escherichia coli in an attempt to determine what some of these factors might be. The results show that initially adenine is converted mainly to ATP, guanine mainly to GTP and uracil mainly to UTP, while hypoxanthine is converted to both ATP and GTP. The corresponding nucleosides have similar effects except for adenosine, whose effects are similar to those of hypoxanthine and inosine. All purine precursors, except for guanosine, deplete the intracellular content of P‐Rib‐PP to 10% or less of its normal value. This finding indicates that the limitation in the concentration of P‐Rib‐PP for the first reaction of the de novo pathway is an important factor in the inhibition of that pathway when a preformed purine is available to support a salvage pathway of purine nucleotide biosynthesis. Our results also suggest that the decreased availability of P‐Rib‐PP may also cause immediate transient decreases observed in the levels of CTP and UTP, after the addition of a preformed purine precursor.

Uracil as a pyrimidine precursor causes only a slight depletion of P‐Rib‐PP while uridine causes an initial increase in the P‐Rib‐PP content. It is concluded, therefore, that an increase in feedback inhibition, caused by elevated concentrations of uridine nucleotides, may be a major factor causing the inhibition of de novo biosynthesis of pyrimidine nucleotides when preformed pyrimidines are available, or that the inhibition may not be fully effective until repression and growth have diluted out the enzymes for the de novo biosynthesis.
Subunit Interactions in Enzyme Catalysis
Authors: Nari Joannès , Mouttet Christian , Fouchier Francis , Ricard Jacques
Published: March 2005

The effects of subunit interactions on the steady‐state rate of the reaction catalyzed by the enzymel‐phenylalanine ammonia‐lyase, have been investigated using the concepts of structural kinetics developed in a previous paper. Both theoretical and experimental methods have been proposed, which allow a choice among the possible kinetic models. These methods rest on the mathematical analysis of rate data obtained in the presence of substrate analogues, used as inhibitors.

In the absence of inhibitor, the reciprocal plots exhibit a significant downward curvature. If a substrate analogue is present in the reaction medium, the plots can be straight lines, or can even exhibit an upward curvature. If vs and vI are the steady‐state rate in the absence and in the presence of inhibitor, respectively, the plots (vs/vI)‐1 versus inhibitor concentration are either straight lines if the inhibitor is d‐phenylalanine, or parabolas if the inhibitor is benzoic acid.

The detailed analysis of these data as well as previous information obtained on the number of polypeptide chains, suggest that the enzyme is made up of two protomers (Mr 160X103), each protomer consisting of two. polypeptide chains: an α chain (Mr 75X103) and a β chain (Mr 85X103). The kinetic data indicate the existence of a hybrid conformation of the enzyme during substrate binding. Thus, phenylalanine ammonia‐lyase appears to follow neither the classical allosteric model of Monod, nor the simple sequential model of Koshland, but a partially‐concerted mechanism of subunit interactions. The binding of the substrate induces a conformation change in the corresponding subunit as well as the appearance of a new conformation of the unliganded protomer.

When d‐phenylalanine or benzoic acid are used as inhibitors, no hybrid enzyme · substrate · inhibitor complex is formed. It thus appears that the new unliganded conformation, called A′, is still able to bind the substrate, l‐phenylalanine, but cannot form a complex with d‐phenyl‐alanine or benzoid acid.

The behaviour of the free enzyme appears to be different with regard to D‐phenylalanine and benzoic acid. The unliganded phenylalanine ammonia‐lyasc can bind one molecule of d‐phenylalanine only, whereas it can bind two molecules of benzoic acid. It thus appears that the binding of d‐phenylalanine on a subunit, induces a conformation change of the unliganded subunit, the new conformation (A″) being unable to form a complex with any molecule of inhibitor. In the case of benzoic acid, on the other hand, the new conformation of the unliganded protomer is still able to bind an inhibitor molecule.
Effects of Potassium Iodide on Aspartate Aminotransferase
Authors: Burridge Noranna , Churchich Jorge E.
Published: March 2005

The absorption spectrum of the phosphopyridoxal form of the enzyme, aspartate aminotransferase, can be perturbed by the anion iodide over a wide range of pH values.

The binding of this anion to the catalytic site of the enzyme brings about a decrease in the absorption at 360 nm and a concomitant increase in the absorption band centered at 430 nm corresponding to the positively charged nitrogen of the aldimine linkage of the cofactor.

The absorption spectral changes are accompanied by variations in the fluorescence intensity emitted by the cofactor. The fluorescence studies reveal an efficient collisional interaction between the excited state of pyridoxal‐5‐P and the perturbing anion iodide. It is proposed that the active site of the enzyme aspartate aminotransferase is very accessible to the anion iodide.
A Spectroscopic Study of the Haemin–Human‐Serum‐Albumin System
Authors: Beaven Gilbert H. , Chen Shi‐Hua , D'albis Anne , Gratzer Walter B.
Published: March 2005

The interaction of haemin with human serum albumin has been reexamined. The absorption spectrum of the bound haem is identical with that of uncomplexed monomeric haemin in solution, and it is suggested, on the basis of an interaction of albumin with iron‐free protoporphyrin IX, that the iron is not implicated in the interaction with the protein. A ferric cyanide derivative, and a ferrous haem derivative of methaemalbumin can be recognised, but not azide or fluoride derivatives. The bound haemin gives rise to extrinsic Cotton effects, which are different in detail in the derivatives, and in the complex with protoporphyrin IX Spectrophotometric titrations show that there is one strong binding site for haemin and several weaker sites. The latter are associated with optical activity opposite in sign to that of the strong complex. The binding profiles are little affected by pH over a wide range, by ionic strength or by the presence of 40% (v/v) dimethylsulphoxide/water, in which the free haemin is monomeric. The binding of haemin to albumin has been followed by spectrophotometry, circular dichroism and fluorescence. The binding of haemin quenches the protein fluorescence, which progressively changes in character from tryptophan to tyrosine type. The haem at the primary binding site thus strongly quenches the tryptophan specifically. From fluorescence titrations at a range of protein concentrations, binding isotherms were constructed, and gave an association constant for the strong site of 50 μM−1. From binding isotherms based on absorption measurements we can infer the existence of at least four sites with much lower binding constants. A long‐chain fatty acid anion was found to compete with haemin only for the weaker binding sites, so that binding beyond one mole per mole of protein can be essentially eliminated. The open‐chain tetrapyrrole, bilirubin, was found, in agreement with earlier work, not to compete with haemin, as regards the strongest binding sites of either ligand. Between the weaker sites, however, evidence of competition was obtained.
Peptide‐Bond Formation on the Ribosome
Authors: Eckermann David J. , Greenwell Philip , Symons Robert H.
Published: March 2005

The acceptor substrate activity of peptidyl transferase of both Escherichia coli and rat liver ribosomes has been investigated using 3′‐N‐aminoacyl analogues of puromycin and 5′‐O‐phosphoryl and 5′‐O‐nucleotidyl derivatives of 3′‐N‐glycylpuromycin aminonucleoside. Two simple assay systems were used with washed ribosomes; the fragment reaction with CpApCpCpA‐(Ac‐L‐[3H]Leu) as donor substrate and the reaction with N‐acetyl‐L‐[3H]phenylalanyl‐tRNA as donor substrate in the presence of poly(U). The results can be summarized as follows.

The conditions of the fragment reaction impose severe restrictions on the structural requirements for acceptor substrate activity, especially for E. coli ribosomes. Of the 21 puromycin analogues tested, only 3′‐N‐L‐phenylalanyl‐puromycin aminonucleoside and its 5′‐O‐cytidylyl derivative gave high (>50% of that of puromycin) activity with E. coli ribosomes while all other analogues gave essentially negligible (<5% of that of puromycin) activity. However, with rat liver ribosomes, 3′‐N‐L‐phenylalanyl‐puromycin aminonucleoside gave high activity while all other analogues containing a single benzene ring gave low (5‐24% of that of puromycin) to moderate (25–50% of that of puromycin) activity. These observations plus the low activity of 5′‐O‐cytidylyl (3′‐N‐L‐phenylalanyl)‐puromycin aminonucleoside with rat liver ribosomes represent the most marked differences found between the two types of ribosomes.

In the N‐acetyl‐L‐[3H]phenylalanyl‐tRNA assay system with E. coli ribosomes, there was low to moderate activity of a number of the more hydrophobic aminoacyl analogues of puromycin which had negligible activity in the fragment reaction. With rat liver ribosomes, there was a general enhancement of activity of aminoacyl analogues above that seen in the fragment reaction. With the phosphoryl and nucleotidyl analogues, however, only 5′‐O‐cytidylyl(3′‐N‐L‐phenylalanyl)‐puromycin aminonucleoside showed high activity with both types of ribosomes and the results were essentially similar to those of the fragment reaction. In general, therefore, the structural requirements for activity of aminoacyl analogues, but not of nucleotidyl analogues, tended to be more stringent in the fragment reaction as compared with the N‐acetyl‐L‐[3H]phenyl‐alanyl‐tRNA assay. It is suggested that the methanol present in the fragment reaction may cause this by disruption of hydrophobic binding at the puromycin binding site.
Covalent Structure of Calf‐Thymus ALK‐Histone
Authors: Sautielère Pierre , Tyrou Danièle , Laine Bernard , Mizon Jacques , Ruffin Pierre , Biserte Gérard
Published: March 2005

Highly purified calf thymus ALK‐histone (histone rich in alanine, lecine and lysine) was isolated from F2a2 histone fraction by ion‐exchange chromatography on Biorex 70 followed by gel filtration chromatography on Sephadex G‐100. Peptides obtained by enzymatic hydrolyses (trypsin, chymotrypsin, thermolysin) of native or maleylated protein were fractionated by ion‐exchange chromatography on Chromobeads P.

Thermolysin peptides which account for 127 of the 129 residues of the protein provide overlaps of tryptic and chymotryptic peptides and led us to establish the complete amino‐acid sequence of ALK‐histone as follows: Ser1Gly‐Arg‐Gly‐Lys‐Gln‐Gly‐Gly‐Lys‐Ala‐10Arg‐Ala‐Lys‐Ala‐ Lys‐Thr‐Arg‐Ser‐Ser‐Arg20‐Ala‐Gly‐Leu‐Gln‐Phe‐Pro‐Val‐Gly‐Arg‐Val30‐His‐Arg‐Leu‐Leu‐Arg‐Lys‐Gly‐Asn‐Tyr‐Ala40‐Glu‐Arg‐Val‐Gly‐Ala‐Gly‐Ala‐Pro‐Val‐Tyr50‐Leu‐Ala‐Ala‐Val‐Leu‐Glu‐Tyr‐Leu‐Thr‐Al60‐Glu‐Ile‐Leu‐Glu‐Leu‐Ala‐Gly‐Asn‐Ala‐Ala70‐Arg‐Asp‐Asn‐Lys‐Lys‐Thr‐Arg‐Ile‐Ile‐Pro80‐Arg‐His‐Leu‐Gln‐Leu‐Ala‐Ile‐Arg‐Asn‐Asp90‐Glu‐Glu‐Leu‐Asn‐Lys‐Leu‐Leu‐Gly‐Lys‐Val100‐Thr‐Ile‐Ala‐Gln‐Gly‐Gly‐Val‐Leu‐Pro‐Asn110‐Ile‐Gln‐Ala‐Val‐Leu‐Leu‐Pro‐Lys‐Lys‐Thr120Glu‐Ser‐His‐His‐Lys‐Ala‐Lys‐Gly‐Lys129.

The amino end is acetylated. The sequence of ALK‐histone is characterized by two hydro‐phobic regions, the first from Val‐43 to Ala‐70 and the second from Val‐100 to Pro‐117 and presents many repetitive or analogous sequences.

The NH2‐terminal sequence of ALK‐histone is highly basic and shows a remarkable analogy with that of GRK‐histone.

A cluster of five basic residues His‐His‐Lys‐Ala‐Lys‐Gly‐Lys is present at the COOH‐terminal end.
Inhibition of Rat‐Liver RNA Polymerase in vitro by Aflatoxin B1 in the Presence of a Microsomal Fraction
Authors: Akinrimisi E. Olabisi , Benecke Bernd J. , Seifart Klaus H.
Published: March 2005

Aflatoxin B1 inhibits rat liver nucleoplasmic RNA polymerase B (40–50%) and cytoplasmic RNA polymerase C activities (25–35%) if applied in vivo. Nucleolar RNA polymerase A activity is not inhibited under the same conditions. Aflatoxin B1 has no effect on the activities of purified RNA polymerase enzymes A, B and C or on [3H]UTP incorporation of isolated rat liver nuclei in vitro. Aflatoxin B1, upon preincubation with a rat liver microsomal fraction, however, is apparently converted to a compound which then inhibits the activities of purified nucleoplasmic RNA polymerase (20–40%), cytoplasmic RNA polymerase (10–20%) and the incorporation of [3H]‐UTP into isolated nuclei (38%). Nucleolar RNA polymerase activity is not affected under these conditions. The microsomal fraction, which most effectively converts aflatoxin B1 to an inhibitor of RNA polymerase is also the most effective cellular fraction catalysing the metabolism of aflatoxin B1.
Thyrotropin‐Binding Properties of Isolated Thyroid Cells and Their Purified Plasma Membranes
Authors: Verrier Bernard , Fayet Guy , Lissitzky Serge
Published: March 2005

Plasma membranes have been prepared from porcine thyroid cells stimulated by dibutyryl adenosine 3′:5′‐monophosphate in culture. These cells show the histiotypic organization in follicles and the metabolic properties of gland follicular cells. The plasma‐membrane fractions have a density in sucrose of 1.18 g/ml (about 80%) and 1.16 g/ml (about 20%). The yield is 2 mg per 6 × 108 cells. Electron microscopy showed vesicular structures and sheets of membranes often joined by membrane junctions. Both showed the triple layer characteristic of the unit membrane ultrastructure. Less than 3% of contamination by microsomes was derived from the assay of NADPH‐cytochrome c reductase and NADH oxidase. No contamination by mitochondria was indicated from succinate dehydrogenase activity measurements. NaF‐and thyrotropin‐stimulated adenylate cyclase, 5′‐nucleotidase and alkaline phosphatase are concentrated in the plasma‐membrane preparations.

Specific binding of 125I‐labeled thyrotropin of high specific radioactivity (2.5 Ci/μmol) and preserved biological activity was compared to that in intact cells and to the plasma membranes from which they derived. The rate constants of the thyrotropin association with cell or membrane (0.56 and 0.11 nM−1 min−1, respectively) and dissociation (0.36 and 0.25 min−1, respectively) were measured independently at 35°C (cells) and 27°C (membranes). The association constants derived from these rate constants (1.5 and 0.4 nM−1 for cells and membranes) are very similar to that (1.9 and 0.6 nM−1) determined independently from equilibrium data. Therefore plasma membranes purified from cultured thyroid cells represent an excellent material to study the molecular events linked to receptor activation.

The relation between thyrotropin concentration and adenylate cyclase activation of membranes was studied in time conditions of equilibrium binding of thyrotropin. The semilogarithmic plot of thyrotropin concentration correlates sigmoidally with the percentage of adenylate cyclase activation. Half‐maximum stimulation is obtained for a thyrotropin concentration of 1.8 nM which correlates remarkably with the dissociation constant of 1.78 nM determined for the binding of the hormone to membranes.
The Denaturation of Lactoferrin and Transferrin by Urea
Authors: Teuwissen Bruno , Schanck Karin , Masson Pierre L. , Osinski Pierre A. , Heremans Joseph F.
Published: March 2005

Lactoferrin and transferrin were submitted in their metal‐free forms as well as in their ironsaturated forms to various concentrations of urea, in the presence or absence of 2‐mercaptoethanol. The progressive unfolding of the proteins was followed by their optical activity in the visible region.

The change in the three‐dimensional structure of lactoferrin when binding iron was found to be more profound than in transferrin whose stability is more dependent on disulphide bridges. In the absence of 2‐mercaptoethanol, the concentrations of urea which caused 50% denaturation at pH 7.4 were found to be respectively 4 M for apolactoferrin, 4.9 M for apotransferrin, 5.5 M for Fe·transferrin and 8 M for Fe·lactoferrin. In the presence of 2‐mercaptoethanol, these values became respectively 2 M for apotransferrin, 2.5 M for apolactoferrin, 3.9 M for Fe·transferrin and 6.7 M for Fe·lactoferrin.

The loss of iron‐binding ability, estimated from absorbance at 460 nm, was found to follow strictly the evolution of the [m']546 parameter.
The Reaction of the Iron‐Sulfur Protein Hydrogenase with Carbon Monoxide
Authors: Thauer Rudolf K. , Käfer Barbara , Jungermann Kurt , Zähringer Marianne
Published: March 2005

The iron‐sulfur protein hydroenase from Clostridium pasteurianum was shown to be specifically inhibited by carbon monoxide. Cyanide (10 mM), azide (10 mM), and fluoride (10 mM) were without effect.

The inhibition of hydrogenase by carbon monoxide was studied under pre‐steady‐state and under steady‐state conditions. The data obtained from both studies indicate a 1:1 stoichiometry of the reversible reaction between an independent active site of hydrogenase and carbon monoxide. The second‐order rate constant of the formation (k+1) and the first‐order rate constant of the dissociation (k−1) of the hydrogenase · carbon‐monoxide complex at pH values between 6.5 and 8.5 and at 25°C were found to be 5.7 s−1mM−1 and 0.013 s−1, respectively. The equilibrium constant K was calculated from pre‐steady‐state data to be 440 mM−1 and from steady‐state data to be 420 mM−1.

The carbon monoxide inhibition of hydrogenase could be reversed bylight. The light sensitivity indicates that in the active site iron is being attacked by carbon monoxide.
Determination of Binding Parameters from Scatchard Plots
Authors: Weder Hans Georg , Schildknecht Jürg , Lutz Rudolf A. , Kesselring Paul
Published: March 2005

Procedures for graphic and numerical parameter fitting from Scatchard plots for the case of two different independent sets of equivalent binding sites are described. These methods are compared with other procedures and their advantages and disadvatages are discussed. Estimates of how much one parameter can be changed without changing the Scatchard curve significantly are made. This variability of the parameters is discussed. It is shown that low saturation points are of considerable importance in evaluating the binding parameters of the high affinity binding site. An example of the parameter fitting procedures is given and the utility and limitations of the procedures are discussed.
Structural Aspects of Sheep‐Heart Phosphofructokinase
Authors: Brennan Stephen O. , Davis Paul F. , Midwinter Graeme G.
Published: March 2005

The sedimentation pattern of the purified enzyme has been ivestigated over a protein concentration range of 0.7 to 14.5 mg cm−3. Two distict 7‐S and 30‐S boundaries were observed at all concentrations. A minor amount of 19‐S material was also present. The 30‐S boundary was asymmetric, and its concentration dependence was characteristic of a polymerising system in rapid reversible equilibrium.

The dissolved crystallin enzyme, under similar conditions, sedimented as single asymmetric 30‐S peak. X‐ray crystallography indicated unit cell dimensions of 60×25×22 nm, implying a protein weight per asymmetric unit of about 106 daltons.

Dodecylsulphate‐gel electrophoresis indicated a protomer molecular weight of 80000 to 85000, which was consistent with a corrected sedimentation coefficient of 3.8 S and a molecular weight of 90000 for maleyl‐phosphofructokinase, and with a corrected sedimentation coefficient of 3.9 S for the urea‐dissociated enzyme.

When maleylation was carried out on carboxymethylphosphofructokinase in 7.5 M urea, the enzyme was further dissociated to a subunit with molecular weight 40000. Peptide mapping of tryptic peptides, in which peptides containing arginine, histidine, tryptophan and tyrosine were located, was consistent with an 85000‐Mr form composed of two identical subunits.
The Neutral Proteases of Human Granulocytes
Authors: Ohlsson Kjell , Odsson Inge
Published: March 2005

Elastolytic enzymes were isolated from the extract of the lysosome‐like granules of human leukaemic myeloid cells by chromatography on Sephadex G‐75, ɛAhx‐Sepharose 4 B, elastin‐Sepharose 4 B and preparative agarose‐gel electrophoresis. The elastase were cationic proteins but appeared as three proteins with a slight difference in electrophoretic mobility. The three elastases degraded native, insoluble elastin with a pH optimum of about 8.5. The granulocyte elastases were inactivated by α1‐antitrypsin and α2‐macroglobin. The amino‐acid composition of the three elastases was the same except for a variation in arginine content. Dodecylsulfate‐electrophoresis suggested molecular weights in the range 33000–36000. The s20, w0 value determined for one of the elastases was 2.6 S. The three elastases gave the reaction of identity on immunodiffusion according to Ouchterlony and crossed immunoelectrophoresis. They showed the same type of reaction with elastase partly purified from normal human granulocytes, which also contained three separate elastolytic enzymes.
Metabolism of Isolated Kidney Tubules
Authors: Guder Walter G. , Wieland Otto H.
Published: March 2005

Pyruvate dehydrogenase activity was measured in extracts of isolated kidney cortex tubules, prepared by collagenase treatment. The measured activities were comparable to those described previously for whole kidney homogenates.

Incubation of tubules in vitro in the absence of exogenous substrates led to a steady increase in enzyme activity over 30 min. This increase was not significantly changed by the addition of several gluconeogenic substrates including lactate, glutamine, glutamate, glycerol, fructose and malate. 1 mM oleate, 5 mM acetate and 20 mM 2‐oxoglutarate however, prevented this increase completely. In contrast, glucose and dihydroxyacetone accelerated the activation of pyruvate dehydrogenase during incubation in vitro. 20 mM pyruvate very rapidly activated pyruvate dehydrogenase with a following decrease below the activity of control experiments without substrate. 1 mM oleate and 20 mM 2‐oxoglutarate counteracted the increase in pyruvate dehydrogenase activity caused by glucose and dihydroxyacetone and had no effect in the presence of 20 mM pyruvate.

Determination of the inactive part of pyruvate dehydrogenase after addition of purified pyruvate dehydrogenase phosphatase revealed that all changes observed were probably caused by enzymatic interconversion of pyruvate dehydrogenase.

The results are compared with previously published observations on the regulation of pyruvate dehydrogenase in vivo and confirm the role of fatty acids in the regulation of kidney cortex pyruvate metabolism.
Translational Control of Antibody Synthesis
Authors: Stevens Ronald H.
Published: March 2005

Mouse plasma cytoma cells (5563) contain 2.5 times more L‐chain mRNA than H‐chain mRNA. The ratio of L‐ to H‐chain protein produced however appears related to the growth rate of the cells. During exponential growth, up to 2.5 times more L‐chain than H‐chain protein is produced, but as cells approach stationary growth phase, L‐ and H‐chain protein approach equal synthesis. The apparent decrease in L‐chain synthesis upon shifting from exponential to stationary growth appears not to be due to a relative reduction in peptide elongation but rather reflects differences in ribosome loading. During exponential growth, L‐chain polyribosomes contain 6–8 ribosomes/L‐chain mRNA whereas in stationary growth the figure drops to less than 3–4 ribosomes/L‐chain mRNA. A similar shift of polyribosome size is noted with total cellular polyribosome.

Polyribosomes engaged in H‐chain synthesis remain fully loaded with ribosomes during conditions of L‐chain polyribosome size reduction. The size of polyribosomes synthesizing H‐chain protein can be reduced however under conditions of an increase in the intracellular level of H2L2.
Δ4‐3β‐Hydroxysteroid Dehydrogenase Activity in Rat Liver
Authors: Lax E. Rodney , Schriefers Herbert
Published: March 2005

A simple optical method for the assay of Δ4‐3β‐hydroxysteroid dehydrogenase activity in cell‐free preparations of rat liver was designed. This test is based on the fact that under the chosen conditions no reduction of the Δ4‐bond occurs. Thus the Δ4‐3β‐hydroxysteroid dehydrogenase activity can be directly measured by the production of Δ4‐3β‐ketosteroids, the sum of which in turn may be quantitated by their isonicotnic acid hydrazone formation.

Δ4‐3β‐Hydroxysteroid dehydrogenases are localized in both cytoplasmic and microsomal fractions, exhibiting higher activity with NAD than with NADP. Microsomal enzyme activities show a significant sex difference which is more pronounced with NADP as coenzyme than with NAD (male: female activity ratios 5.8 with NADP, 1.9 with NAD). No sex differences were observed in the cytoplasmic enzyme activity. These results indicate the existence of two NAD‐dependent enzyme activities of which the microsomal activity shows sexual differences. On the basis of the male:female activity ratios a further separate NADP‐dependent microsomal enzyme activity can also be distinguished.
Template Specificity of Eucaryotic DNA‐Dependent RNA Polymerases
Authors: Flint Sarah Jane , Chesterton C. James , DE Pomerai David I. , Butterworth Peter H. W.
Published: March 2005

The routine procedures used to prepare DNA from eucaryotic cells and nuclei give rise to material which is considerably degraded. This degradation takes the form of haplotomic (single‐stranded) breaks arising from deoxyribonuclease action action and diplotomic cleavage (double‐stranded scission) of the DNA duplex due to mechanical shearing during the isolation procedure. Attempts to study the template specificities of the eucaryotic DNA‐dependent RNA polymerases must take into account how these modifications to the structure of the DNA affect its template properties for these enzymes. Techniques are described which permit an accurate assessment of the state of integrity of the DNA. Treatment of the DNA with pancreatic deoxyribonuclease or sonication is shown to give rise to single‐stranded interruptions in the duplex which are competent sites for the initiation of RNA synthesis by both forms AI and B rat liver RNA polymerases. However both these treatments also give rise to diplotomic cleavage of the DNA. This results in an inhibition of RNA synthesis which may be correlated with the formation of non‐productive complexes by the RNA polymerases at DNA ends. Therefore the degree of template activation by pancreatic deoxyribonuclease and sonication for RNA synthesis as seen in the assay in vitro is a delicate balance between two opposing effects and is highly dependent upon the polymerase : DNA ratio. This data serves to emphasise that experiments designed to study the template specificity of eucaryotic RNA polymerases must make use of new, sophisticated techniques for the preparation of a template which is both intact and of high molecular weight.
Structure of the Porcine Vasoactive Intestinal Octacosapeptide
Authors: Mutt Viktor , Said Sami I.
Published: March 2005

The amino acid sequence of the porcine vasoactive intestinal octacosapeptide is His‐Ser‐Asp‐Ala‐Val‐Phe‐Thr‐Asp‐Asn‐Tyr‐Thr‐Arg‐Leu‐Arg‐Lys‐Gln‐Met‐Ala‐Val‐L ys‐Lys‐Tyr‐Leu‐Asn‐Ser‐Ile‐Leu‐Asn‐NH2. Its amino acid residues 1, 2, 6 and 7, counted from the N‐terminus, are identical to those in the corresponding positions in both porcine glucagon and secretin. The residues in positions 3, 12, 13, 14 and 23 are identical to those in secretin, but not in glucagon, and position 10 is occupied by a tyrosyl and position 28 by an asparaginyl residue, like in glucagon but not in secretin. If in addition to identical amino acid residues also chemically similar residues, such as isoleucine in position 26 of the octacosapeptide, as compared to leucine in secretin and glucagon, are taken into consideration then the similarity between these three polypeptides is still more evident.

At a more remote level there is some structural resemblance also between these peptides and the other four peptides from the intestinal wall, cholecystokinin‐pancreozymin, motilin, the gastric inhibitory peptide and substance P, the structures of which are known.

The elucidation of the structure of the octacosapeptide was facilitated by the finding that pancreatic kallikrein preferentially cleaved only one of the three bonds in its N‐terminal cyanogen bromide heptadecapeptide that are susceptible to cleavage with trypsin.
Undialyzable Growth Factors for Lactobacillus bifidus var. pennsylvanicus
Authors: GyÖRGY Paul , Jeanloz Roger W. , von Nicolai Hubertus , Zilliken Fritz
Published: March 2005

Lactobacillus bifidus var. pennsylvanicus requires human milk constituents for growth. All growth factors so far known represent low‐molecular derivatives of d‐glucosamine (2‐amino‐2‐deoxy‐d‐glucose). Some higher‐molecular undialyzable sialic acid‐containing oligosaccharides from human milk and some sialo‐glycoproteins from different body fluids have been tested for growth promoting activity. Isolation, purification and some structural properties of the oligosaccharides are described. Without pretreatment by neuraminidase, none of the substances exhibit any growth promoting activity. After incubation with neuraminidase from Vibrio cholerae, the activity of the oligosaccharides increased in close correlation to the content of N‐acetyl‐d‐glucosamine to the same extend as ethyl‐2‐acetamido‐2‐deoxy‐β‐d‐glucopyranoside, the most active derivative of N‐acetyl‐d‐glucosamine known. The activity of α1‐acid glycoprotein from human serum, urinary glycoprotein and submaxillary mucin was considerably lower, possibly due to the different linkages of sialic acid and their more complex structures. The possible protective effect of sialic acid bound to oligosaccharides and glycoproteins against degradation by bacterial enzymes is discussed.
Isolation and Characterisation of a Mitochondrially Synthesized Precursor Protein of Cytochrome Oxidase
Authors: Werner Sigurd
Published: March 2005

Cytochrome oxidase was prepared from Neurospora crassa. An individual subunit of the enzyme (subunit 3, apparent molecular weight 21000) was isolated by recycling gel chromatography.

Specific antibodies to cytochrome oxidase and to subunit 3 were produced.

The antibody to cytochrome oxidase precipitates the enzyme from detergent‐treated mitochondria. The precipitated cytochrome oxidase contains seven polypeptide subunits. The growth labelling kinetics of subunit 3 within the enzyme complex was studied.

The antibody to subunit 3 recognizes an antigen in solubilized mitochondria, which is not identical with cytochrome oxidase. This component was isolated from mitochondria by immunological techniques. It was identified as a precursor protein, which has the following characteristics, (a) The protein is translated on mitochondrial ribosomes. (b) The precursor has a half‐life of about 2 min. (c) Precursor protein and subunit 3 display identical apparent molecular weights, (d) It is tightly associated with mitochondrial membranes. (e) The precursor represents about 0.5‰ of the mitochondrial membrane protein.

It is concluded that the isolated precursor is a genuine precursor protein of subunit 3 of cytochrome oxidase.
Polysaccharide Biosynthesis in Acetobacter xylinum
Authors: GarcÍA Rodolfo C. , Recondo Eduardo , Dankert Marcelo
Published: March 2005

The incorporation of radioactivity in vitro from UDP‐[14C]glucose into endogenous acceptors in a crude enzymatic preparation from Acetobacter xylinum is described.

A fraction of the total incorporated radioactivity could be extracted with organic solvents, like butanol. The remainder was mostly solubilized by hot alkali, leaving an insoluble fraction.

No incorporation was observed in the absence of magnesium ions. Mn2+ could only partially replace Mg2+, and had an inhibitory effect when both ions were present.

Analysis of the butanol‐soluble fraction permitted the isolation of at least three components characterized as: lipid‐diphosphate α‐glucose, lipid‐diphosphate α‐cellobiose and lipid‐monophosphate β‐galactose.

In the presence of [β‐32P]UDP‐glucose the system incorporated radioactivity only into the diphosphate derivatives, indicating the transfer of a α‐glucose 1‐phosphate group from UDP‐glucose. Experiments with doubly labelled substrate confirmed these results.

The role these compounds might play in polysaccharide biosynthesis is discussed.
Fluorescent Labels on Ribosomes The Interactions of rRNA and Proteins
Authors: Pochon François , Ekert Bernard , Perrin Martine
Published: March 2005

Both rRNA and protein components of Escherichia coli ribosomes have been specifically labelled without any important loss of activity for polyphenylalanine synthesis in the cell‐free system. Three adenylic residues per 70‐S ribosome were labelled by an anthracenyl derivative and the ribosomal proteins by dansylation of four amino acid residues per 70‐S particle. The association between the two complementary subparticles, the formation of the binary and ternary complexes in the presence of mRNA and tRNA were investigated using the concomitant measuring of fluorescence intensity and polarisation of the two kinds of labels. The formation of these complexes involves the rRNA and the ribosomal protein components of the 30‐S and 50‐S sub‐particles directly and/or indirectly. The mobility of the messenger bound to the 70‐S ribosome in the presence of tRNA is indicated by use of a polyadenylic acid labelled with the same marker as that for rRNA. Reversible conformational changes are observed during acidic‐alkaline titrations and melting curves of fluorescent ribosomal particles. The irreversible denaturation occurs at the same pH and melting temperature for the rRNA and the ribosomal components: The results are interpreted in terms of highly specific interactions between the rRNA and the proteins, slight modification in the strength and/or nature of these interactions leading to a conformational change of the whole ribosomal particle.
Isotope Effects and Hydrogen Transfer during Simultaneous Metabolism of Ethanol and Cyclohexanone in Rats
Authors: Cronholm Tomas
Published: March 2005

The metabolism of ethanol in vivo was studied by measurements of tritium isotope effects and extent of hydrogen transfer from ethanol to a reducible substrate of liver alcohol dehydrogenase. The specific radioactivity of ethanol in bile increased by a factor of two in 6–8 h following administration of [1‐3H]ethanol, 11 mmol/kg. This indicates a tritium isotope effect of about 1.6 on the association of [1‐3H]ethanol to the alcohol dehydrogenase‐NAD complex or on the rate of conversion of the ternary complex.

Cyclohexanone was used as the reducible substrate of liver alcohol dehydrogenase. The major metabolite in bile was cyclohexyl glucuronide. During the simultaneous metabolism of [1,1 ‐2H2]‐ethanol with a deuterium excess of 95 atoms % the cyclohexanol formed had a deuterium excess of about 65 atoms %. This dilution cannot be explained by the isotope effect and is most likely due to the reversible dissociation of the enzyme‐NADH complex resulting in exchange with a less‐labelled free‐NADH pool.

Ethanol metabolism increased the rate of cyclohexanone elimination, probably by increasing the concentration of alcohol dehydrogenase‐NADH complex. A large dose of cyclohexanone decreased the ethanol‐elimination rate, possible due to competitive inhibition.
Anion‐Exclusion and Anion‐Exchange Chromatography of Nucleotides
Authors: Singhal Ram P.
Published: March 2005

Anion‐exclusion chromatography, previously shown to resolve nucleosides, has been applied to separate nucleotides on “cation‐exchange” columns at acid pH values. The study of pH and ionic effects indicates that these separations occur by anion repulsion, not by cation exchange. Cation exchange, previously held reponsible for these separations, is inoperative since nucleotides at acid pH values bear net negative charges, as do the fixed anions of the resin. The separations can be explained by anion‐exclusion chromatography) which involves two factors: repulsion between identically charged solute and resin ions, and partition between aqueous eluant and organic resin matrix. The results indicate that separations by anion exclusion are as good as or better than those by anion‐exchange chromatography.

Anion‐exchange chromatography has also been reinvestigated to develop systems that can be used to separate the four major nucleotides in single eluants and to separate most nucleosides, nucleotides and other RNA hydrolysis products in one analysis.

The ion‐exclusion separations are compared with those of anion‐exchange chromatography. The advantages and disadvantages of both methods are discussed. A typical example of the analysis of a nucleoside‐nucleotide mixture derived from the total hydrolysis of tRNAs for the assay of the base comparison and for the analysis of terminal end groups is described.
NADPH‐Dependent Formation of Thymidine diphosphodihydrostreptose from Thymidine diphospho‐d‐glucose in a Cell‐Free System from Streptomyces griseus and Its Correlation with Streptomycin Biosynthesis
Authors: Ortmann Rainer , Matern Ulrich , Grisebach Hans , Stadler Peter , Sinnnwell Volker , Paulsen Hans
Published: March 2005

When thymidine diphospho‐d‐[U‐14C]glucose was incubated with a cell‐free extract from a streptomycin‐producing strain of Streptomyces griseus in the presence of NADPH, Radioactive dTDP‐Dihydrosteptose Was formed in addition to radioactive dTDP‐4‐keto‐6‐deoxyglucose and dTDP‐rhamnose. Dihydrostreptose was identified by chromatographic, gas chromatographic and electrophoretic comparison with synthetic dihydrostreptose characterized by its nuclear magnetic resonance spectrum.

dTDP‐dihydrostreptose is very labile and decomposes to dihydrostreptose monophosphate and further to free dihydrostreptose.

dTDP‐dihydrostreptose and dTDP‐rhamnose were also obtained when dTDP‐4‐keto 6‐deoxyglucose was the substrate of the reaction. Dihydrostreptose synthesis could be inhibited completely by 1 mM p‐chloromercuribenzoate, whereas rhamnose formation was not inhibited.

In the course of fermentation the activities of arginine: x‐amidinotransferase, an enzyme involved in streptomycin biosynthesis, and of the enzymatic activity for dTDP‐dihydrostreptose formation ran parallel and reached a maximum before onset of streptomycin formation.

A good correlation between the activity of “dTDP‐dihydrostreptose synthase”, arginine : x‐amidinotransferase activity and streptomycin production was found in several strains of S. griseus. No dihydrostreptose formation could be detected in a hydroxystreptomycin‐producing strain of S. griseocarneus.

Streptomycin but not dihydrostreptomycin could be detected in the fermentation medium of the S. griseus strain used for preparation of the cell‐free extract. The role of dTDP‐dihydrostreptose in streptomycin biosynthesis is at present unknown.
Synthesis of a Protein‐Reactive ATP Analog and Its Application for the Affinity Labeling of Rabbit‐Muscle Actin
Authors: Faust Uwe , Fasold Hugo , Ortanderl Fhanz
Published: March 2005

The synthesis of a protein‐reactive ATP analog, S‐dinitrophenyl‐6‐mercaptopurine riboside 5′‐triphosphate, started from isopropylidated 6‐mercaptopurine. After dinitrophenylation, the thioester riboside was phosphorylated with POCI3 and the monophosphate was converted to the triphosphate by activation with the aid of phosphoric acid diphenylester chloride, and reaction with inorganic pyrophosphate.

The analog, labelled with 32P in the β and γ‐positions, was used for covalent binding of the nucleotide to rabbit muscle actin. In preparation for this affinity labelling, the one superficial—SH group of the protein had been alkylated with N‐ethylmaleimide. The nucleotide was able to depolymerize N‐ethylmaleimide‐F‐actin, and became attached to the G‐form at a ratio of 0.8 molecules per actin monomer. The product could be repolymerized to the F‐form with liberation of inorganic phosphate. The product could not be depolymerized after that by ATP solutions at low ionic strength.

A single radioactively labelled peptide was discerned in fingerprints of a tryptic digest.
Studies of Glutamate Dehydrogenase: Chemical Modification and Quantitative Determination of Tryptophan Residues
Authors: Witzemann Veit , Koberstein Rudolf , Sund Horst , Rasched Ihab , Jörnvall Hans , Noack Klaus
Published: March 2005

The effect of the modification of beef liver glutamate dehydrogenase with 2‐hydroxy‐5‐nitrobenzyl bromide was studied with respect to the association‐dissociation equilibrium as well as the enzymatic and regulatory properties. Upon incorporation of 2.1 molecules of the reagent per polypeptide chain, the association of the enzyme is strongly reduced, whereas the enzymatic activity is only slightly decreased (80% maximum velocity). The Km values and the regulation by ADP and GTP, respectively, remain unaltered. Evidence is presented that only tryptophan residues are modified. Magnetic circular dichroism measurements of the modified enzyme suggest partial disubstitntion of tryptophan residues. From the reduced incorporation of 2‐hydroxy‐5‐nitrobenzyl groups at high enzyme concentration it is concluded that tryptophan is located at the association areas of the enzyme.

Quantitative determination of the tryptophan content of glutamate dehydrogenase with 2‐hydroxy‐5‐nitrobenzyl bromide, magnetic circular dichroism and peptide analysis yields four tryptophan residues per polypeptide chain contrary to three residues previously suggested from structural analysis. The fourth tryptophan, not present in the reported sequence, is recovered in a chymotryptie peptide Glu‐Trp.
Studies on Two Initiation Factors of Protein Synthesis from Rat‐Liver Cytoplasm
Authors: Grummt Friedrich
Published: March 2005

Two initiation factors of protein synthesis designated here as IF‐S1 and IF‐S2 have been isolated from rat liver cytoplasm. The factors are separated by gel filtration of a supernatant obtained at 150000 ×g on Sephadex G‐200 from one another and are purified further by chromatography on hydroxyapatite and DEAE‐cellulose columns. Both factors stimulate poly‐(phenylalanine) synthesis at low Mg2+ concentrations in the presence of elongation factors EF‐1 and EF‐2. The factor EF‐S1 stimulates the binding of AcPhe‐tRNA and fMet‐tRNA to the small ribosomal subunit. This reaction is dependent on GTP; β,γ‐methylene‐guanosine triphosphate can replace GTP. IF‐S2 is active in the coupling of 40‐S and 60‐S subunit to active 80‐S initiation complexes. This activity depends on poly(U), on Phe‐tRNA and partially on GTP. Similarities and possible analogies between the initiation factors described here with those described by other authors arc discussed.