bluuebunny/crossref_metadata_2025 huggingface dataset:
Ribonucleoside 5′‐thiodiphosphates as Substrates for Escherichia coli Ribonucleotide Reductase
Authors: von Döbeln Ulrika , Eckstein Fritz
Published: March 2005

In an attempt to learn something about the substrate requirements of ribonucleotide reductase from Escherichia coli the following substrate analogues were examined: adenosine 5′‐O‐(1‐thiodiphosphate), adenosine 5′‐O‐(2‐thiodiphosphate) and cytidine 5′‐O‐(1‐thiodiphosphate). The thioanalogues were reduced by the enzyme and their reduction was regulated by nucleotide triphosphate effectors in the same way as the reduction of the normal ribonucleotide substrates. The rate of reduction, however, was markedly reduced, i.e. adenosine 5′‐O‐(1‐thiodiphosphate) about 2% the rate of ADP, cytidine 5′‐O‐(1‐thiodiphosphate) 11% the rate of CDP and adenosine 5′‐O‐(2‐thiodiphosphate) 25 the rate of ADP. The analogues competed with the ribonucleotide substrates for the substrate binding site and their Ki values were in the same concentration range as the Km, values for the corresponding unsubstituted ribonucleotides. Thus, the binding to the enzyme is presumably equally strong and the reduced rate of reduction is due to a slow‐down in the catalytic reaction.
Intracellular Hydroxylation of Non‐Helical Protocollagen to Form Triple‐Helical Procollagen and Subsequent Secretion of the Molecule
Authors: Uitto Jouni , Prockop Darwin J.
Published: March 2005

Cells liberated from the tendons of chick embryos by controlled enzymic digestion were used to examine the synthesis of the polypeptide chains of collagen separately from the secretion of collagen in the precursor form known as procollagen. When the cells were incubated under anaerobic conditions, the synthesis of collagen hydroxyproline was completely inhibited but the rate of protein synthesis remained about the same as the control rate for about 30 min. Under these conditions the cells synthesized the non‐hydroxylated precursor known as protocollagen and the protocollagen was retained in the cells. By incubating the cells with [14C]proline under anaerobic conditions and then exposing the system to air, it was possible to follow the intracellular hydroxylation of [14C]protocollagen to [14C]procollagen. The conformation of the intracellular [14C]protocollagen and [14C]procollagen was examined by proteolytic digestion under conditions in which the triple‐helical portion of collagen is resistant to digestion. When digestion with either pepsin or α‐chymotrypsin was carried out at either 30 ° C or 37 °C, all of the [14C]protocollagen was digested. After the same protein was hydroxylated to [14C]procollagen by exposing the cells to O2 a large fraction of the protein became resistant to proteolysis and was secreted. The results suggested that the intracellular hydroxylation of collagen polypeptides converts them from a non‐helical to a helical form and that the helical conformation probably is required for the protein to be secreted at an optimal rate.
Conformational Transitions of Polynucleotides in Polymer Media
Authors: Laurent Torvard C. , Preston Barry N. , Carlsson Birgit
Published: March 2005

The melting temperature of three polynucleotides, poly(deoxyadenylic‐thymidylicf‐acid (double helix), calf‐thymus DNA and Staphylococcus aureus phage 80α DNA, increases in the presence of two neutral polymers, dextran and poly(ethyleneglycol). The results have been interpreted as a steric exclusion phenomenon. Two strands of polynucleotides in random coil conformation occupy a larger volume than a helix structure and the equilibrium between the two conformational forms is therefore moved towards the formation of the more compact helix when the available volume in the solution is restricted. The experiments indicate that steric exclusion in polymer media can influence the conformation of other macromolecules.
An Approach for the Determination of Equilibrium Constant of Structural Motility
Authors: Vas Mária , Boross László
Published: March 2005

A kinetic approach is presented for the determination of equilibrium constant of a local conformational fluctuation of a protein molecule. As a result of this fluctuation a distinct side chain becomes temporarily accessible to a specific reagent. Determination of fluctuation constant, Kf, is based on the kinetic analysis of chemical modification of the side chain with a selective reagent which reacts faster than the conformational changes occur.

The use of p‐mercuribenzoate‐mercaptide of 2‐nitro‐5‐mercaptobenzoate as an–SH reagent is described to determine Kf of d‐glyceraldehyde‐3‐phosphate dehydrogenase around residue Cys‐153, when the hyper‐reactive side‐chain Cys‐149 was previously carboxymethylated (CM‐enzyme). The values of Kf are 0.034 ± 0.005 and 0.014 ± 0.003 in the case of CM‐apoenzyme and holoenzyme, respectively, in Tris‐HCl buffer, I= 0.05, pH 7.5, at 5 ° C. The free enthalpy change (ΔG) accompanying the conformational change which makes Cys‐153 accessible to the reagent, is 1.86 ± 0.08 and 2.34 ± 0.11 kcal/mol for CM‐apoenzyme and holoenzyme, respectively. The enthalpy change (ΔH) is + 3.9 ± 0.7 kcal/mol both in the absence and presence of coenzyme. The entropy change (ΔS) is + 7.6 ± 1.3 and + 6.3 ± 1.1 cal × degree−1× mol−1 for CM‐apoenzyme and holoenzyme, respectively.

Based on the value of Kt and our earlier kinetic data for mercaptide formation of Cys‐153 one can characterize the structural motility: the rate constants of exposure and masking of residue Cys‐153 of the apoenzyme are k+1, = 2.2 ± 0.2 min−1 and k−1= 65 ± 16 min−1. respectively, under the experimental conditions.
Energy‐Dependent Reverse Electron Flow in Chloroplasts
Authors: Rienits Keith G. , Hardt Haim , Avron Mordhay
Published: March 2005

An ATP‐driven rise in fluorescence yield (Q reduction) and cytochrome f oxidation in light‐triggered chloroplasts are demonstrated. Both effects are shown to be dependent on the creation and maintenance of the light‐triggered state. ATP‐induced reduction of Q depends upon the presence of an electron donor in the dark with a redox potential up to, but not exceeding, 300 mV. The effects are abolished by uncouplers or energy transfer inhibitors. 3‐(3,4‐Dichlorophenyl)‐1,1‐dimethylurea inhibits the ATP‐driven reduction of Q but not that of cytochrome oxidation. A location for a coupling site and the site of action of electron‐transport inhibitors is suggested on the basis of these observations.
Control of Lipid Metabolism in Adipose‐Tissue Hornogenates of Fasted Refed Rats
Authors: Winand Jacques , Furnelle Jacques , Hebbelinck Marcel , Christophe Jean
Published: March 2005

The 700 ×g fat‐poor internatant from epididymal adipose tissue homogenate of fasted refed rats was incubated at pH 6.5 with 0.5 mM [1‐14C]acetate in the presence of cofactors. After incubation, six lipid classes were separated by thin‐layer chromatography and their fatty acids analyzed by radio gas‐liquid chromatography. The incorporation of [1‐14C]acetate into glycerides was limited by the activity of acetyl‐CoA carboxylase, and was stimulated two to three‐fold by the addition of 0.1 mM unlabelled malonyl‐CoA.

The chain‐length of fatty acids labelled in vitro was controlled by the relative concentrations of malonyl‐CoA and acetyl‐CoA. Addition of unlabelled malonyl‐CoA influenced the operation of fatty acid synthetase in such a way that the ratio of radioactive palmitate to radioactive myristate increased markedly.

sn‐Glycerol 3‐phosphate stimulated the esterification of fatty acids into triglycerides. Under these conditions whereas the specific activity of free fatty acids was much higher than that observed in triglycerides, the specific activity of diglycerides was low, when compared to the same triglycerides. Newly synthesized monounsaturated fatty acids were preferentially esterified into triglycerides.

0.5 mM [1‐14C]palmitate was efficiently incorporated into triglycerides but not into diglycerides.
Inhibition of 3‐sn‐Phosphatidylcholine Biosynthesis in Baby‐Hamster Kidney‐21 Cells Infected with Semliki Forest Virus
Authors: Vance Dennis E. , Burke Derek C.
Published: March 2005

The infection of baby hamster kidney cells with Semliki Forest virus results in the inhibition of [Me‐3H]choline incorporation into 3‐sn‐phosphatidylcholine of the cytoplasmic membranes of intact cells. This inhibition was also observed in purified plasma membrane and endoplasmic reticulum. The major decrease of incorporation of label occurred between 4 and 6 h after infection.

Microsomes from baby hamster kidney cells infected with Semliki Forest virus exhibit approximately a 50% decrease in total activity and a 40% decrease in specific activity of the enzyme CDP‐choline: 1,2‐diglyceride cholinephosphotransferase. This decrease in enzymatic activity occurred between 4 and 6 h post‐infection. When compared to mock‐infected cells, the enzymatic activity from infected cells had a similar apparent Km for CDP‐choline and a similar half‐maximal stimulation value for 1,2‐diacyl‐sn‐glycerol. Experiments with cycloheximide and actinomycin D suggest that viral inhibition of host protein and RNA synthesis would not account for the depressed cholinephosphotransferase activity. However, since cycloheximide stimulates the choline‐phosphotransferase activity of mock‐infected cells, the results of these experiments must be interpreted with caution. A freely diffusable inhibitor was not detected in the microsomes from infected cells.

Infection of baby hamster kidney cells with a picornavirus (bovine enterovirus) did not inhibit or stimulate [Me‐3H]choline incorporation into 3‐sn‐phosphatidylcholine nor did it affect the activity of CDP‐choline : 1,2‐diglyceride cholinephosphotransferase.
Amphipathic Changes in Etioplast Membranes following Illumination
Authors: Trefry Timothy
Published: March 2005

Membrane preparations from dark‐grown protochlorophyllide‐containing pea leaves and from dark‐grown leaves illuminated so as to contain predominantly chlorophyllide or chlorophyll, have been compared by counter‐current distribution in a dextran‐poly(ethyleneglycol) two‐phase system. The major shift in the polarity of etioplast membranes which follows illumination appears to be correlated not with the photoreduction of protochlorophyllide but with its subsequent phytolation. It is suggested that phytolation is associated with a change in membrane components which alters the amphipathic balance of the developing chloroplast membrane.
Structure and Antigenicity of Hen Egg‐White Lysozyme Fragments
Authors: Matthyssens Gaston E. , Giebens Gonda , Kanarek Louis
Published: March 2005

Under controlled conditions of pepsin digestion of hen egg‐white lysozyme, large fragments with composition identical to sequence 57—107 and sequence (57—83) + (91—107), have been obtained.

Inhibition of lysozyme‐anti‐lysozyme precipitation by these fragments, showed that section 84—90 is important for the antigenic specificity of fragment 57—107. The position of one lysozyme antigenic region was suggested, namely section 85—90 combined with a region close to methionine 105, reacting with about 10% of total anti‐lysozyme antibodies. Structure evaluation of the purified peptides through circular dichroism, revealed that only 30% of the “native” alpha‐helicity amount (ie. the amount of helicity this fragment possesses when integrated in the lysozyme molecule) remained. We concluded that in the case of lysozyme, mainly long‐range interactions stabilize the native configuration. Secondly, that differences in antigenic specificity among related peptides are not due to differences in structure. Fragment‐antibody interaction could be explained by assuming the existence of a simple equilibrium Fr⇌ Fn between the completely random and the native fragment, or, through an “induced fint” mechaism.
Structure and Antigenicity of Hen Egg‐White Lysozyme Fragments
Authors: Matthyssens Gaston E. , Kanarek Louis
Published: March 2005

Purification of a lysozyme‐digest resulted in the isolation of two main regions of the molecule, namely sequence (57—107) denoted loop fragment (see the accompanying paper), and sequence (1—27) linked to sequence (122—129) through disulfide bridge I—VIII (Jollès et al., 1963), denoted NC‐peptide. Inhibition of lysozyme‐anti‐lysozyme precipitation showed that NC‐peptide reacted with about 10% of all goat anti‐lysozyme antibodies. It was suggested that these antibodies react with a conformation‐dependent determinant including tyrosine 20 and 30. In water or in aqueous salt solution, NC‐fragment retained about 20% of its “native” alpha‐helical amount (i.e. the amount of helicity this fragment possesses when integrated in the lysozyme‐molecule). Reduction and alkylation of the I—VIII disulfide bridge abolished most of the spatial conformation, as was obvious from the circular dichroism curve isolated N‐peptide. Long‐range interactions between the N and C terminal parts of the molecule, therefore, seem to stabilize the native lysozyme molecule.
Purification of Neurophysins by Affinity Chromatography
Authors: Frénoy Jean‐Pierre , Ménard Joël , Pesquiès Pierre , Corvol Pierre
Published: March 2005

(8‐Lysine)‐vasopressin was coupled to activated Sepharose by cyanogen bromide with 65%, yield. Lysine‐vasopressin antibodies were specifically retained on a lysine‐vasopressin Sepharose column and purified in a single step. They kept their ability to bind lysine‐vasopressin.

Pig neurophysins prepared from fresh porcine posterior pituitaries by 0.1 M HCOOH extraction and by Sephadex gel filtration, were applied to the lysine‐vasopressin‐Sepharose column. A major protein peak was retained and eluted by 0.1 M HCOOH. Polyacrylamide gel electrophoresis of this peak revealed two major protein bands and six minor bands. These proteins have been called neurophysins for several reasons: (1) they bind lysine‐vasopressin coupled to Sepharose, (2) they show no defined absorption at 275—300 nm, (3) they have a molecular weight on dodecylsulfate‐polyacrylamide gel electrophoresis of 11 000 and 15500 which is close to those already reported for neurophysins I and II, (4) the whole amino‐acid composition is similar to that already given for the purified neurophysins. This study demonstrates the usefulness of affinity chromatography for the purification of lysine‐vasopressin‐binding proteins. It shows the heterogeneity of porcine neurophysins.
The Intracellular Location of Pyruvate Carboxylase in Rat Liver
Authors: Weiss Gerhard , Lamartiniere Coral A. , Müller‐ohly Brigitte , Seubert Werner
Published: March 2005

Because of reported extramitochondrial location of pyruvate carboxylase in rat liver concluded from extraction studies and the importance of these findings for the concept of the regulation of gluconeogenesis, the intracellular location of this enzyme in rat liver was reinvestigated. The following results were obtained.

Reported differences in extraction properties between pyruvate carboxylase and glutamate dehydrogenase in rat liver are due to secondary binding of the latter enzyme to nuclei, cell debris, mitochondria and microsomes.

Rat‐liver pyruvate carboxylase is located exclusively in mitochondria, a location in another subcellular component is excluded.

Rat liver mitochondria are heterogeneous with respect to their enzyme content. Rate‐zonal centrifugation reveals asymmetric distribution of mitochondrial matrix enzymes: Ornithine aminotransferase is shifted towards slower‐sedimenting mitochondria, pyruvate carboxylase, malate dehydrogenase and aspartate aminotransferase in faster‐sedimenting mitochondria. Glutamate dehydrogenase as well as monoamine oxidase (outer membrane) and adenylate kinase (inter‐membrane space) are located in an intermediate position.

Electronmicrographs show that the mitochondria retain their intact outer membrane and most of them are in condensed conformation.

The biological significance of the observed heterogeneities with respect to the regulation of gluconeogenesis is discussed.
Sterol Transmethylation during Aerobic Adaptation of Yeast
Authors: Parks Leo W. , Anding Claude , Ourisson Guy
Published: March 2005

An analysis of the total sterol content has been made of a yeast strain during aerobic adaptation. Transmethylation occurs principally at the 4‐demethylsterol level, but conditions were obtained wherein a small fraction of the transmethylations were with 4α‐methyl precursors. Δ7‐Zymostenol has been tentatively identified as a product previously unreported in yeast. Esterification was observed in all sterol intermediates and products. Cell‐free cyclization of 2,3‐epoxy‐22‐methylene squalene to 24‐methylene lanosterol occurs with yeast.
Adenovirus Assembly
Authors: Boulanger Pierre A. , Puvion Francine
Published: March 2005

Cross‐linking of adenovirus hexon capsomers by glutaraldehyde in vitro has been studied as a chemical probe for quaternary structure of hexon protein and virus capsid assembly. Glutaraldehyde cross‐linking of hexons gave rise to three main oligomeric species, trimer, hexamer and nonamer, as analyzed on dodecylsulfate‐acrylamide gels, which corresponded in terms of complete hexon capsomers, to single hexon capsomer, capsomer dimer and trimer. The major species was found to be the trimer of the hexon polypeptide unit, viz. the intramolecularly cross‐linked hexon capsomer. This result indicated that intraoligomeric cross‐linking took place more readily than the interoligomeric reactions. As shown by electron microscopy the spatial arrangement of the cross‐linked hexon capsomer trimer was reminiscent of the structure of groups of three hexons clustered with 3‐fold symmetry to form the groups of nine hexons isolated from disrupted adenovirus capsid.
Modulation of Mitochondrial Adenylate Kinase by Citric‐Acid‐Cycle Intermediates
Authors: Pradhan Tapas K. , Criss Wayne E.
Published: March 2005

Modulation of rat liver mitochondrial adenylate kinase by citric acid cycle intermediates is reported.

Activation of adenylate kinase in the presence of Mg2+ was dependent upon the following. (1) It was activated by citrate, cis‐aconitate, isocitrate, fumarate, and malate but not by 2‐oxo‐glutarate, succinate, or oxaloacetate. (2) Protonated forms of the intermediates were most effective. (3) Citrate caused a decrease from 6.9 to 4.1 in the Km for ATP. (4) The activation was not a simple citrate‐Mg2+ chelation phenomenon. (5) The Ka for citrate was 90 μM. (6) Activation was very rapid. (7) Citrate caused a decrease from 2.1 to 1.3 in the slopes of Hill plots with increasing ATP. (8) Measurements in differential absorbancies in the presence and absence of citrate and Mg2+ indicated structural changes in adenylate kinase upon modulator binding. (9) Non‐additive effects of saturating concentrations of several citric acid cycle intermediates could be interpreted as a common binding site for each of the intermediates. It is postulated that the citrate modulation of adenylate kinase involves an alteration in the structure of the enzyme.
Gluconeogenesis from Serine by the Serine‐Dehydratase‐Dependent Pathway in Rat Liver
Authors: Sandoval Ignacio V. , Sols Alberto
Published: March 2005

Isolated perfused livers from rats made deficient in pyruvate carboxylase by feeding an avidin‐rich, biotin‐deficient diet produced much less glucose than those from the corresponding normal animals (0.23 ± 0.08 and 0.83 ± 0.05 μmol/g liver per min, respectively). The ability to form glucose from alanine was also markedly impaired, while that from fructose was not similarly affected. Parenteral administration of biotin led within 3 h to full recovery of the activity of hepatic pyruvate carboxylase, as well as to restoration of the ability to form glucose from serine, both by the isolated liver and by the whole animal (measured in the latter case by the formation of [14C]glucose from [14C]serine).

In the perfused liver, gluconeogenesis from hydroxypyruvate and glycerate, the specific intermediates of the potential alternative pathway from serine to glucose mediated by serine pyruvate transaminase, was smaller than that from serine.

The serine dehydratase in the liver of fasted rats is in terms of V (2.3 ± 0.7 U/g) and Km (approx. 40 mM) quantitatively suited to account for the rate of gluconeogenesis from serine in the perfused liver, both when offered as the only precursor at high concentration and when offered as part of a mixture of precursors at physiological concentrations. In contrast, the efficiency of the so‐called serine pyruvate transaminase to act on serine in physiological conditions appears to be utterly insufficient to permit a major contribution to gluconeogenesis from serine by this means.

These results indicate that gluconeogenesis from serine in rat liver proceeds essentially by the serine‐dehydratase‐mediated pathway.
Structural Studies on Phenylalanine Transfer Ribonucleic Acid from Yeast with the Spectroscopic Label Formycin
Authors: Maelicke Alfred , Sprinzl Mathias , von der Haar Friedrich , Khwaja Tasneem A. , Cramer Friedrich
Published: March 2005

Formycinmonophosphate (FMP) has been incorporated in to the 3′‐terminus of phenylalanine tRNA from yeast by tRNA nucleotidyl transferase. tRNAPhepCpCpF can be amino‐acylated with the cognate synthetase exhibiting the same Km and an about 50‐times lowered V compared to tRNAPhepCpCpA. Quantum yield of FMP fluorescence in tRNAPhepCpCpF is about four times lower than in the monomer. Comparative studies of tRNAPhepCpCpF and the oligonucleotide CpApCpCpF show that the differences between the fluorescence properties of tRNAPhepCpCpF and the monomeric formycin‐nucleotide are due only to stacking interactions and do not arise from the tertiary structure of tRNA. After periodate oxidation of the 3′‐terminal ribose of tRNAPhepCpCpF the full fluorescence of formycin is reconstituted. This emphasises the important role of the intact ribose ring for stabilization of stacking units. Periodate oxidation of tRNAPhepCpCpF followed by borohydride reduction leads to a tRNA that acts as competitive inhibitor in the aminoacylation reaction. This, together with its high quantum yield makes it a valuable tool in fluorescence spectroscopic studies of the aminoacylation reaction.
The Vitamin A Transporting System in Porcine Plasma
Authors: Rask Lars
Published: March 2005

The retinol‐binding protein from porcine serum has been isolated and characterized. Its molecular weight and amino‐acid composition were determined and compared to these data from its human, monkey and rat counterpart. In anology to these proteins pig retinol‐binding protein is under physiological conditions bound to a larger plasma protein and has a carboxyl‐terminal residue of arginine suggesting a general mechanism for the delivery of vitamin A to the tissue cells.
Selective Carbethoxylation of the Histidine Residues of Actin by Diethylpyrocarbonate
Authors: Hegyi György , Premecz György , Sain Béla , Mühlrád András
Published: March 2005

Histidine residues of actin were carbethoxylated with diethylpyrocarbonate. Four and three histidine residues per actin monomer were rapidly carbethoxylated in the G and F form, respectively. After partial Carbethoxylation of G‐actin a non‐polymerizable fraction could be separated by ultracentrifugation. The analysis of these fractions showed that one of the four fast‐reacting histidines of G‐actin plays an essential role in polymerization.

Removal of the carbethoxy groups from the histidine residues by hydroxylamine treatment restored the ability to polymerize.

The specificity of the reaction was tested by the use of [14C]diethylpyrocarbonate.

After tryptic digestion of non‐polymerizable carbethoxylated actin a single histidyl peptide was isolated by gel filtration and diagonal electrophoresis. The histidine residue essential for polymerization was identified as histidine‐40.
Distribution and Some Properties of Acid Phosphatase in the 7,12‐Dimethylbenz[a]anthraccne‐Induced Rat‐Mammary Carcinoma
Authors: Nicholson Robert I. , Davies Malcolm
Published: March 2005

Differential centrifugation and gel‐chromatography techniques have been employed to study the distribution and molecular forms of acid phosphatase in rat mammary tumours induced by 7,12‐dimethylbenz[a]anthracene. With p‐nitrophenylphosphate as substrate three distinct forms of acid phosphatase activity were separated on gel chromatography.

Enzyme I (Mr > 200000) and enzyme II (Mr 120000) were found to have similar biochemical properties and differential centrifugation indicated that both enzymes are present in a sedimentable fraction. Disruption of a mitochondrial‐lysosomal fraction by freezing and thawing released 45% of the total acid phosphatase of the fraction into a soluble phase. Treatment with detergent solubilized the bound enzyme. The possibility of two forms of acid phosphatase, one bound and one freely soluble, in the lysosomes of the rat mammary tumour is discussed.

Enzyme III (Mr 30000) is localized in the soluble fraction of the tumour cell and is biochemically distinct from enzymes I and II.

The overall findings are examined in relation to the properties and distribution of acid phosphatase in other tissues.
Repeated Nucleotide Sequences in the Loach Genome
Authors: Kupeijanova Natalia S. , Timofeeva Margarita Ja.
Published: March 2005

Reassociation kinetics of loach DNA has been studied.

It has been shown that 50% of the loach genome consists of repeated nucleotide sequences. Most of them belong to an “intermediate” repetitive fraction of DNA, repetition frequency being equal to 104−102.

Close study of the intermediate repetitive fraction of DNA reassociation kinetics reveals the existence of discrete repetitive DNA groups in the loach genome. The DNA fractions corresponding to discrete repetitive groups were isolated and their reassociation kinetics, nucleotide composition and thermal stability were studied. The differences established allow us to postulate that DNA sequences of different repetitive groups perform distinct functions. Further investigations are to be undertaken to examine this assumption and clarify the role of each repetitive group in the genome.
Evidence for a Guanosine‐3′: 5′‐Monophosphate‐Binding Protein from Rat Cerebellum
Authors: Sold Guido , Hofmann Franz
Published: March 2005

Guo‐3′:5′‐P and Ado‐3′:5′‐P (guanosine and adenosine 3′: 5′‐monophosphate) are bound by a supernatant fraction of homogenates from rat cerebellum. Treatment of the supernatant fraction with trypsin destroyed the ability of the supernatant fraction to bind either of the cyclic nucleotides. For Ado‐3′:5′‐P a hyperbolic saturation curve was obtained and an apparent Kd of 8.5 nM was estimated. At a saturating concentration of 0.1 μM between 6 and 8 pmol per mg extracted protein was bound. For Guo‐3′: 5′‐P two different types of binding sites were observed; one nonspecific site from which Guo‐3′: 5′‐P could be displayed by low concentrations of Ado‐ 3′:5′‐P and one, which was specific for Guo‐3′: 5′‐P. The apparent Kd of the specific site for Guo‐3′:5′‐P was 12 nM and at a saturating concentration of 0.1 μM 1.5 pmol per mg extracted protein were bound. The Guo‐3′:5′‐P binding protein was partially separated from an Ado‐3′: 5′‐P‐dependent protein kinase by ammonium sulfate precipitation.
Acid Denaturation of Human Carbonic Anhydrase B
Authors: Flanagan Michael T. , Hesketh T. Robin
Published: March 2005

The acid denaturation of human carbonic anhydrase B has been followed using fluorescence spectroscopy. On lowering the pH of the protein solution through the acid unfolding transition region the quantum yield was increased and the wavelength of maximum fluorescence emission red shifted.

The rate of unfolding of the protein, as measured fluorimetrically, was shown to obey first‐order kinetics. Arrhenius plots at two ionic strengths were linear between 7 °C and 35 °C. In the pH range 3.0 to 3.8 the rate constant was second order with respect to hydrogen ion concentration. Approximately 30% of the free energy of activation for unfolding was shown to be an electrostatic contribution.

Circular diehroism spectra showed that the first, reversible unfolding step is followed by a slower irreversible step, the rate of which is reduced by the presence of Zn2+.

The irreversibly unfolded protein aggregated on increasing the pH, the onset of aggregation occurring sharply at pH 3.6.
Antigenicity of p‐Azobenzenearsonate‐Containing Oligopeptides in Rabbits
Authors: Levin Howard , Becker Martin , Sela Michael
Published: March 2005

The denned compounds containing p‐azobenzenearsonate (Aba), Aba‐Tyr, Aba‐Tyr‐Tyr, Aba‐Tyr‐(Tyr)2, Aba‐Tyr‐(Tyr)3, (Tyr)3‐Aba‐Tyr and Aba‐Tyr‐(Ala)3 (where Tyr is l‐tyrosine and Ala is l‐alanine) were synthesized and were investigated immunologically in New Zealand white rabbits. After immunization with the peptides in complete Freund's adjuvant the antibody responses were monitored by both a hapten‐modified bacteriophage inactivation assay and quantitative antibody precipitation. The anti‐Aba antibody response was shown to increase as tyrosine residues were added to the Aba‐Tyr moiety, with the most significant jump in titer occurring between Aba‐Tyr‐Tyr and Aba‐Tyr‐(Tyr)2. In contradistinction, no increase in immunogenicity was observed when three alanyl residues were used to lengthen the peptide chain.

Studies of the specificity of the anti‐Aba antibodies produced against the peptides, using inhibition of the hapten‐modified phage inactivation, indicated that, while the specificity of antibodies is direct mainly to the Aba‐Tyr moiety in these compounds, the antibodies did demonstrate specificity for the homologous immunizing antigen.

The data suggests that the area adjacent to the Aba‐Tyr group on these small immunogens is important for its recognition as an immunogen and also for its ability to determine the specificity of the antibody induced.
Replication of the Minicircular DNA of Escherichia coli 15
Authors: Messing Joachim , Staudenbauer Walter L. , Hofschneider Peter Hans
Published: March 2005

During the replication of the minicircular DNA of Escherichia coli 15 an open circular form II has been identified as a precursor of superhelical form I. Pulse‐labeled form II DNA contains a newly synthesized linear strand which is non‐covalently associated with a circular template strand. Equilibrium centrifugation of isolated linear strands in alkaline CsCI shows that the incorporation of label occurs with equal frequency in either of the complementary strands indicating a symmetrical mechanism of replication. Labeling of replicative intermediates is observed in short pulses at 25 °C. Upon alkali denaturation these molecules yield small labeled DNA fragments (4–6 S) suggesting a discontinuous mode of strand synthesis.
Characterization of DNA from Differentiated Cells
Authors: de Jiménez Estela Sánchez , González José Luis , Domínguez José Luis , Saloma Enrique Sánchez
Published: March 2005

DNAs from different chicken tissues were purified. After denaturation, the reassociation reaction of two different molecular weight DNAs was measured by the hydroxyapatite method. The results of these experiments indicated that, under the generally accepted standard conditions, the chicken genome is comprised of 20%, repetitive DNA. Furthermore, it was found that a significant amount of the repetitive DNA was interdispersed among the unique DNA. It can be concluded from the kinetic data that there was not a significant difference either in the amount or in the distribution of the unique and repetitive sequences of the DNA samples tested. Isolation and further characterization of the repetitive sequences were carried out.
Blood‐Group Serology of Fractions Obtained from the Human Erythrocyte Membrane
Authors: Anstee David J. , Tanner Michael J. A.
Published: March 2005

The activity of a large number of human erythrocyte antigens was monitored through a procedure which fractionates the erythrocyte membrane proteins. A significant amount of the erythrocyte “ghost” protein could be removed by aqueous extractants without affecting the activity of any antigen. Subsequent extraction of the residual “ghosts” with pyridine solution resulted in a separation of blood group M, N and Pr activities from blood group A, P1, P and I activities. A number of antigens including c, C, D, k, Kpb, Jka and Fya were labile to this treatment. Gel filtration in the presence of detergent showed that the MN and Pr activity was associated with the erythrocyte sialoglycoprotein. In agreement with Whittemore et al. (1969) two types of blood‐group‐A antigen could be distinguished on the basis of their partition properties on butanol extraction. Type OS‐A antigen can be extracted into butanol, while type WS‐A antigen is retained in the aqueous phase. The characteristics of blood‐group‐P antigen were similar to that of type OS‐A antigen, while blood‐group‐I antigen and type WS‐A antigen showed similar behaviour.
Two Isoenzymes of Glucosephosphate Isomerase from Spinach Leaves and Their Intracellular Compartmentation
Authors: Schnarrenberger Claus , Oeser Angelika
Published: March 2005

From spinach leaves (Spinacea oleracea L.) two isoenzymes of glucosephosphate isomerase can be separated by DEAE‐cellulose ion‐exchange chromatography. Isoenzyme 1 eluting at high ionic strength shows a faster mobility during disc gel electrophoresis than isoenzyme 2 eluting at low ionic strength. For both isoenzymes the pH‐optima and Km‐values were very similar for either direction. However, the Km‐values with glucose‐6‐phosphate as substrate (Km= 8.0 mM and 5.9 mM for isoenzyme 1 and 2, respectively) were 20‐fold higher than with fructose‐6‐phosphate as substrate (Km= 300 μM for both isoenzymes). The molecular weight of both isoenzymes was estimated by sedimentation velocity centrifugation analysis to be 120000. The subcellular distribution reveals that isoenzyme 1 is located within the chloroplasts and isoenzyme 2 in the non‐particulate cell fraction (cytosol).

The results give further support to the view that in plants there exist two sets of isoenzymes for the oxidative pentose phosphate cycle, one set located in the chloroplasts, the other one in the non‐particulate cell fraction (cytosol).
On the Translational Control of Histone Synthesis
Authors: Breindl Michael , Gallwitz Dieter
Published: March 2005

Histone mRNA was isolated from polyribosomes and other cell fractions of synchronized HeLa cells and quantitated by its translational capacity in a rabbit reticulocyte lysate. Inhibition of DNA synthesis with hydroxyurea (5 mM) results within 30 to 60 min in a loss of 80–85% of biologically active histone mRNA from polyribosomes. In contrast, actinomycin D (5 μg/ml) during the first hour of its action, only stops further accumulation of histone mRNA on polyribosomes in early S‐phase cells. Under conditions of interruption of DNA replication biologically active histone mRNA does not appear in other cell fractions suggesting that it is immediately destroyed in the cytoplasm.

The possible translational control of histone synthesis in HeLa cells was investigated with special consideration of the existence of messenger‐specific initiation factors. Cell‐free extracts from synchronized HeLa cells in which DNA and histone synthesis were inhibited with hydroxyurea, fully retain their capacity to translate added homologous histone mRNA as compared with extracts from synchronized S‐phase cells. Using a partially purified protein‐synthesizing system from rat liver supplemented with initiation factors from synchronized HeLa cells differing in their histone‐synthesizing capacity, it was shown that factors from hydroxyurea‐treated cells are as active in stimulating the translation of histone mRNA, globin mRNA and tobacco mosaic virus RNA as are initiation factors from S‐phase cells. The results suggest that messenger discriminating translation factors are not involved in the regulation of histone synthesis in HeLa cells or they are very labile and inactivated during preparation.
Mode of Degradation of Ribosomes in Regenerating Rat Liver in vivo
Authors: Tsurugi Kunio , Morita Tomi , Ogata Kikuo
Published: March 2005

The degradations of structural proteins and RNAs of ribosomes in regenerating rat liver were investigated with the following results.

Using a modification of the double isotope technique of Arias et al. (1969), it was found that almost all the structural proteins of both large and small ribosomal subunits, separated by dodecylsulfate or two‐dimensional polyacrylamide gel electrophoresis, were apparently degraded at the same rates.

Studies on the changes in the specific activities of the RNA and protein moieties of the two ribosomal subunits after administration of [Me‐3H]methionine as a common precursor showed that their specific activities decreased in parallel, with the same apparent half‐life of about 4.5 days.

These results suggest that structural components of rat liver ribosomal subunits are degraded as a unit.
On the Mechanism of Glucose‐6‐Phosphate Dehydrogenase Regulation in Mouse Liver
Authors: Hizi Amnon , Yagil Gad
Published: March 2005

The rates of synthesis and degradation of glucose‐6‐phosphate dehydrogenase in the liver of male C57BL mice are followed by a modified immunochemical method. Mice are given labeled amino acids for a predetermined period, and the labeled enzyme is isolated from liver homogenates with goat anti‐enzyme, followed by rabbit anti‐goat IgG serum. The precipitates are either counted directly or analyzed on acrylamide gels. The rate of glucose‐6‐phosphate dehydrogenase synthesis is found to be independent of the state of induction or repression of the animal. The synthesis of the enzyme constitutes 0.20–0.25% of the synthesis of all soluble liver proteins. This rate is maintained even during periods of most rapid formation or disappearance of enzyme activity. Quantitative immunoprecipitation shows that similar amounts of precipitable antigen are present in induced and non‐induced liver homogenates. The results indicate that the large reversible increase in activity of glucose‐6‐phosphate dehydrogenase, observed when animals are transferred from a high‐fat to a fatless diet, does not involve the synthesis of a new enzyme protein, and that mechanisms involving modulation of existing enzyme molecules ought to be considered for the adaptation of this enzyme in mouse liver.
Protocatechuate 3,4‐Dioxygenase
Authors: Fujisawa Hitoshi , Uyeda Motoji
Published: March 2005

Cobalt‐containing protocatechuate 3,4‐dioxygenase was purified and crystallized from Pseudomonas aeruginosa which was grown in a medium containing cobalt in place of iron. Although it was indistinguishable from the normal protocatechuate 3,4‐dioxygenase upon ultracentrifugation and acrylamide gel electrophoresis, it had very low enzyme activity compared with the normal enzyme and showed absorption spectra with a shoulder in a region of 420 nm quite different from the normal protocatechuate 3,4‐dioxygenase. It showed distinct electron‐spin resonance signals at g= 4.31 and at g= 2.004. Some other spectral and catalytic properties of the cobalt‐containing protocatechuate 3,4‐dioxygenase were studied and these results indicate the possibility that l‐cysteine residues participate in the ligand of the iron in the active site of the enzyme.
Ligand‐Specific Bohr Effect in Haemoglobins
Authors: Sick Hinrich , Gersonde Klaus
Published: March 2005

The CO binding properties of monomerie (Hb III and IV), dimeric, and tetrameric Chironomus haemoglobins as well as of the monomer‐analogue valence hybrid (αMmet2βmetβdeoxy) of Hb M Iwate were studied and compared with the respective O2 binding properties. All these haemoglobins show hyperbolic O2 binding curves and a Bohr effect. The amplitudes of the O2 Bohr effect curves range from Δlog (PO2(1/2), = 0.3 (Hb III) to 2.4 (tetrameric Chironomus haemoglobin).

The ligands CO and O2 differ not only in affinity but also with regard to the Bohr effect. The CO affinities of the various Chironomus haemoglobins are 60 to 490 times larger than the O2 affinities and the amplitudes of the CO Bohr effect curves are 1.25 to 8 times smaller than those of the respective O2 Bohr effect curves. In the case of Hb M Iwate, however, the CO Bohr effect is larger than the O2 Bohr effect by the factor 1.25 (2,3‐bisphosphoglycerate‐reacted Hb M) and 1.4 (stripped Hb M).

The ligand‐specific differences in the magnitude of the Bohr effect cannot be explained solely by the allosteric interaction connected with the trans‐effect of the complex. The differences are discussed in terms of steric influences on the binding geometry of the 6th ligand by protein side chains at the distal side of the haem group.
Phosphorylation of Proteins in Rat Liver
Authors: JERGIL Bengt , OHLSSON Rolf
Published: March 2005

Smooth and rough endoplasmic reticulum and free ribosomes from rat liver each show cyclic‐AMP‐stimulated protein kinase activity utilizing exogenous substrates. The protein kinases of smooth and rough endoplasmic reticulum can be divided into two classes, one which is extracted by ionic media, and one more firmly attached enzyme fraction which is solubilized by Triton X‐100. The protein kinase of free ribosomes is extracted by ionic media. All the three microsomal fractions support an endogenous phosphorylation of proteins which is only slightly stimulated by cyclic AMP. The endogenous phosphorylation shows two pH optima at pH 6.5 and 8.5. The phosphate is incorporated into seryl and threonyl residues of several protein species. Two major phosphoproteins are present in both smooth and rough endoplasmic reticulum, while a third major phosphoprotein is present only in the smooth fraction. There are also several minor phosphoproteins in the two fractions. The endogenous phosphorylation is initially rapid, especially in smooth and rough endoplasmic reticulum where it reaches a maximum after 10–15 min incubation. The endogenously phosphorylated microsomal fractions also support an endogenous dephosphorylation, which is rapid initially, but which leaves approximately 60% of the phosphoryl groups unhydrolyzed. Like the protein kinase the proteins of smooth and rough endoplasmic reticulum which can undergo endogenous phosphorylation can be divided into two classes, one which is extracted in ionic media and one more tightly bound which is solubilized by Triton X‐100. Protein kinase, cyclic‐AMP‐binding material, and protein substrates extracted from smooth and ruogh endoplasmic reticulum by salt or detergent all show recoveries substantially exceeding 100%, suggesting that these activities are partly masked while associated with membrane material.
Partition of Proteins in a Three‐Phase System
Authors: HARTMAN Annika , JOHANSSON Göte , ALBERTSSON Per‐Åke
Published: March 2005

A system consisting of three liquid phases has been used for partition of proteins. It is composed of water and three high‐molecular compounds: dextran, Ficoll and poly(ethyleneglycol) the latter partly converted into a derivative. All the three phases have a high water content, 71–79%, and dissolve proteins. Hemoglobin, serum albumin, lysozyme and hexokinase, as well as yeast lysate, have been partitioned in the system. The distribution of some glycolytic enzymes present in the lysate has been determined. At low pH, the proteins are found in the bottom phase. With increasing pH the affinity successively increases first for the middle phase and then for the top phase. The pH at which accumulation in the middle phase occurs is specific for each protein. Under certain conditions different isoenzymic forms can be separated by a single partition step. Practical applications of the three‐phase system are suggested.
A Film Detection Method for Tritium‐Labelled Proteins and Nucleic Acids in Polyacrylamide Gels
Authors: BONNER William M. , LASKEY Ronald A.
Published: March 2005

A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X‐ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5‐diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to RP Royal “X‐Omat” film at ‐70 °C. Optimal conditions for each step are described. β‐particles from 3H interact with the 2,5‐diphenyloxazole emitting light which causes local blackening of an X‐ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C. 3000 dis. 3H/min in a band in a gel can be detected in a 24‐h exposure. Similarly 500 dis./min can be detected in one week.

When applied to the detection of 35S and 14C in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 35S or 14C/min in a band in a gel can be detected in 24 h.
The Role of NADH in Uncoupled Microsomal Monoxygenations
Authors: STAUDT Heinrich , LICHTENBERGER Fritz , ULLRICH Volker
Published: March 2005

The stoichiometry of the liver microsomal monoxygenase system from phenobarbital‐pretreated rabbits has been investigated with cyclohexane, n‐hexane and perfluoro‐n‐hexane as substrates. With cyclohexane an almost 1:1:1 stoichiometry for cyclohexanol: NADPH: O2 was obtained. With the uncoupler perfluoro‐n‐hexane no product was formed and 2 moles of NADPH were oxidised per mole of oxygen. The stoichiometry of n‐hexane hydroxylation suggested that partial uncoupling with this substrate had occurred. NADH addition together with NADPH had no significant effect on the hydroxylation of cyclohexane but led to a more than additive hydroxylation in the case of n‐hexane. Oxygen uptake with perfluoro‐n‐hexane and NADPH was almost doubled in the presence of NADH. It was shown that during uncoupling an increased oxidation of NADH via cytochrome b5 occurs. It was concluded that active oxygen which is not used for monoxygenations is reduced to water by the NADH‐cytochrome b5 system and that this sparing effect could be the main mechanism of the well‐known synergistic action of NADH on microsomal hydroxylations.
Antibodies to Chick‐Tendon Procollagen
Authors: DEHM Peter , OLSEN Bjørn R. , PROCKOP Darwin J.
Published: March 2005

Antisera were prepared by immunizing a rabbit with procollagen synthesized and secreted by cells from chick embryo tendons. Specific antibodies were then purified from the antisera by using an immunoadsorbent which contained the isolated NH2‐terminal extensions of procollagen. The purified antibodies were shown to react specifically with intact procollagen as well as with procollagen in which the interchain disulfide bonds were ruptured by reduction under non‐denaturing conditions. The antibodies also reacted with the pro‐α1 chain but not the pro‐α2 chain isolated from the procollagen. There was no reaction after the intrachain bonds in the pro‐α chains of the procollagen were reduced under denaturing conditions and alkylated.

In the course of characterizing the antibodies it was shown that the NH2‐terminal extensions on the two pro‐α1 chains and the one pro‐α2 chain of type I procollagen are non‐identical. Also, it was shown that the serum of chick embryos contains an antigen which reacts with the specific antibodies to procollagen.