bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleophilic Competition in Some β‐Galactosidase‐Catalyzed Reactions
Authors: Viratelle Odile M. , Yon Jeannine M.
Published: March 2005

A study of nucleophilic competition in β‐galactosidase‐catalyzed reactions has been performed with different substrates: o, m and p‐nitrophenyl β‐d‐galactosides, phenyl β‐d‐galactoside, o‐aminophenyl, β‐d‐galactoside, p‐aminophenyl, β‐d‐galactoside, o‐nitrophenyl α‐l‐arabinoside. For each substrate the limiting step and the individual rate constants have been determined. The rate of formation of the galactosyl‐enzyme intermediate, k2, does not correlate with the Hammett values, suggesting that this step remains more complex than a single chemical reaction.

A preliminary investigation of the effect of pH and Mg2+ concentration on each individual rate constant is presented. Both k2 and k3 seem to exhibit the same pH profile.

The action of various nucleophilic compounds on the galactosyl‐enzyme has been tested. A peculiar effect of 2‐mercaptoethanol has to be emphasized; the behaviour of this nucleophile has been analyzed and discussed.
The Effects of Ionic Strength on Termination of Transcription of DNAs from Bacteriophages T4, T5 and T7 by DNA‐Dependent RNA Polymerase from Escherichia coli, and the Nature of Termination by Factor ρ
Authors: Schäfer Rolf , Zillig Wolfram
Published: March 2005

Ionic strength influences (a) the equilibrium between the monomeric and the dimeric form of DNA‐dependent RNA polymerase from Escherichia coli, in the free state, (b) the primary binding of the enzyme to sites on the template, (c) the velocity of a state transition involved in initiation and (d) termination (or block) of transcription.

On T7‐ and T5‐DNA transcription is terminated, with release of the transcript, at both high and low ionic strength at the same sites but the enzyme is released only at high ionic strength allowing reinitiation whereas at low salt concentration the complex of enzyme with terminator and σ does not dissociate.

On T4‐DNA transcription at low ionic strength is arrested at a class of low‐ionic‐strength blocking sites without release of the transcript.

In the presence of “termination factor”ϱ and ϰ at low ionic strength transcription of T4, T5 and T7‐DNA is arrested at another class of blocking sites again without release of the transcript. The average relative positions of the three different types of blocking sites between the initiation site and the terminator have been determined for T4‐DNA.

Implications of these results are discussed.
Dissociation of Single Ribosomes as a Preliminary Step for Their Participation in Protein Synthesis
Authors: Ottolenghi Sergio , Comi Paola , Giglioni Barbara , Gianni Alessandro M. , Guidotti Guido G.
Published: March 2005

32P‐labelled derived 40‐S ribosomal subunits and 80‐S monoribosomes entered the polyribosome fraction of a rabbit‐reticulocyte lysate active in protein synthesis by a process that was dependent on time, energy and temperature. Both 40‐S and 60‐S subparticles from the labelled monoribosome entered the polyribosomes at the same rate. The addition of excess unlabelled 40‐S subparticles to the cell‐free system containing labelled monoribosomes increased the ratio of 60‐S to 40‐S‐subparticle radioactivity in the polyribosome fraction. This indicated a competition between added unlabelled and monoribosome‐derived 40‐S subparticles for entry into polyribosomes. From the evaluation of the translation time of mRNA, of the rate of monoribosome‐polyribosome exchange, and of the equilibrium distribution of the cycling labelled monoribosomes and unlabelled subparticles with polyribosomes it is concluded that the dissociation of single ribosomes into subunits is an obligatory step for their entry into the polyribosoma1 fraction and that this process is coupled with initiation of mRNA translation.
Some Reactions of Insulin at Non‐Polar Surfaces
Authors: Browne Marjorie , Cecil Rupert , Miller Janet C.
Published: March 2005

The behaviour of insulin at decane/water interfaces has been studied under a variety of conditions. A state of equilibrium has been shown to exist between insulin in solution and on the interface which appears to be independent of concentration. It is changed when a charge is introduced at the interface.

Dansyl chloride was added to the decane so as to label that part of the insulin molecule in contact with the interface. Reaction was confined to the B1 phenylalanine and evidence is given which shows that insulin adsorbed on the interface is dimeric. Two products were found with one and two dansyl groups respectively per dimer. The former is 50% active and the latter inactive by the mouse convulsion method. This is explained by assuming that the monomer is the active species.

Surface tension experiments were carried out to determine the area of the insulin dimer at the interface. The results obtained are consistent with the X‐ray data.
Messenger Ribonucleoprotein and Initiation Factors in Rabbit‐Reticulocyte Polyribosomes
Authors: Nudel Uri , Lebleu Bernard , Revel Michel , Zehavi‐Willner Tova
Published: March 2005

EDTA dissociation of reticulocyte polyribosomes liberates globin mRNA as a nucleoprotein. An attempt to compare the proteins forming the mRNA · protein complexes with the initiation factors of protein synthesis is presented here.

Two conditions are described under which initiation factors are removed from hemoglobin‐synthesizing ribosomes. One is after treating the polyribosomes with a high salt concentration; the other is by isolating ribosomes from reticulocytes at an advanced state of maturation which have lost their initiation factors. In both cases it is shown that the mRNA remains associated with the ribosomes and with the proteins forming the mRNA · protein.

Moreover both mRNA and mRNA · protein are translatable in a heterologous cell‐free system and their translation is stimulated to the same extent by the addition of reticulocyte initiation factors.

Initiation factors are therefore different physically and functionally from the proteins of the mRNA · protein.
Fractionation of Membrane‐Bound Polysomes, Free Polysomes, and Nuclei from Tissue‐Cultured Cells
Authors: Birckbichler Paul J. , Pryme Ian F.
Published: March 2005

Free polysomes from many kinds of eukaryotic cells are liberated by cell lysis using the non‐ionic detergent Kyro EOB leaving a residue of nuclei with strands of rough endoplasmic reticulum attached. Subsequent treatment with a combination of Kyro EOB plus deoxycholate solubilizes the membrane‐bound polysomes. Polysomes prepared in this manner function well in amino‐acid‐incorporating systems in vitro. The nuclei remaining incorporate [14C]ATP into RNA. More active nuclei, however, are obtained when cells are disrupted by nitrogen cavitation.
A Study of the Interactions between Monomeric Tropocollagen and Glycosaminoglycans
Authors: ÖBrink Björn
Published: March 2005

Monomeric, lathyritic tropocollagen and a variety of well‐characterized glycosaminoglycans have been used in a light‐scattering study of the interaction that occurs between these macro‐molecules under physiological ionic conditions. Additional information has been obtained by turbidimetric and precipitation studies. A binding to collagen of chondroitin 4‐sulphates, derma‐tan sulphates, heparan sulphates, heparin and proteoglycans of chondroitin sulphate and dermatan sulphate was observed. The complex formation was electrostatic in nature and was abolished with increasing ionic strength.

Keratan sulphate did not bind to collagen, indicating either that uronic acid was a prerequisite for the interaction or that a charge density of more than one negative charge per disaccharide was required. Polysaccharides which contained l‐iduronic acid showed a stronger interaction than polysaccharides containing only d‐glucuronic acid. As a general rule the interaction seemed to increase with increasing chain length of the polysaccharide and increasing charge density.

The results of studies on mixtures of tropocollagen and proteoglycans indicated that the properties of the polysaccharide chains per se were of importance for the mode of reaction between the proteoglycans and collagen.

Hyaluronic acid was not to any significant extent bound to collagen. This polysaccharide and tropocollagen affected each other by a mutual steric exclusion with a concomitant increase in the activity of the tropocollagen molecules.
Differences in Nucleotide Sequences of Ribosomal RNA between the Liver and a Hepatoma of C3H/He Mice
Authors: Hashimoto Shuichi , Muramatsu Masami
Published: March 2005

Highly purified ribosomal 18‐S and 28S RNA have been prepared from the liver and the hepatoma (MH 134) of C3H/He mice labeled with 32P and subjected to pancreatic RNAase digestion. Analysis of oligonucleotide patterns with two‐dimensional electrophoresis indicated that there were slight but significant differences between the liver and the hepatoma both for 18‐S and 28‐S RNA. Remarkable was the fact that a tetranucleotide sequence, Y‐(G, G*)‐Cp where G* represents a modified G and Y, an unspecified pyrimidine nucleotide, and a hexanucleotide sequence, Y‐A‐A‐A‐A‐Up that were found in the 18‐S RNA of normal liver were always missing in the 18‐S RNA of the hepatoma.

Evidence was also obtained that both 18‐S and 28‐S RNA of these cells are not entirely homogeneous but consist of molecules with some heterogeneity with respect to nucleotide sequences.

These results were discussed under the concept of differential transcription of redundant and heterogeneous ribosomal genes in mammalian cells.
The Binding of Ethidium Bromide to Different Conformations of tRNA
Authors: Urbanke Claus , Römer Roland , Maass Günter
Published: March 2005

Ethidium bromide destabilizes the low‐temperature transitions of tRNAPhe (yeast) and tRNAVal (E. coli), in contrast to its effect upon the double‐helix to coil transitions in all other known cases. This fact leads to the interpretation of the low‐temperature transition as the unfolding of tertiary structure. Fluorescence temperature‐jump experiments show the binding of ethidium bromide to increase as a result of this unfolding. The number of binding sites and the binding constants are n= 3 and K= 2.3 μM−1 in the folded state (8°C) and n= 6 and K= 1.1 μM−1 in the unfolded state (32°C).
The Effects of Thioketo Substitution upon Uracil‐Adenine Interactions in Polyribonucleotides
Authors: Bähr Wolfgang , Scheit Karl‐Heinz , Faeber Peter
Published: March 2005

The physical and biological properties of two new poly(U) analogs, poly(2‐thiouridylic acid) or poly(s2U) and poly(2,4‐dithiouridylic acid) or poly(s2s4U) have been investigated. Ultraviolet absorption and circular dichroism spectra of poly(s2U) and poly(s2s4U) as well as those of the corresponding monomers were measured. Absorption‐temperature and absorption‐pH profiles of both poly(s2U) and poly(s2s4U) displayed sharp cooperative transitions. The phase transition diagrams of poly(s2U) and poly(s2s4U) show the high stability of their secondary structures. Neither poly(s2U) nor poly(s2s4U) formed helical complexes with poly(A). It was concluded from the corresponding phase transition diagrams that the reason for this behaviour is the larger thermodynamical stability of the poly(s2U) or poly(s2s4U) helices over the hypothetical complexes poly(s2U) · poly(A) or poly(s2s4U) – poly(A). Sedimentation velocity analysis of poly(s2U) and poly(s2s4U) revealed sedimentation coefficients S20,w of 28 S and 12 S, respectively. A more detailed analysis of poly(s2U) lead to the conclusion that poly(s2U) exists as an intra‐molecular associate of two anti‐parallel poly(s2U) strands in the form of a large hairpin. Consistent with their physical properties, poly(s2U) and poly(s2s4U) are not able to replace poly(U) as messengers in the cell‐free synthesis of poly(phenylalanine). Poly(s2U) is only poorly, poly(s2s4U) not at all phosphorolyzed by polynucleotide phosphorylase.
The Interactions of 2‐Thiopyrimidine Bases with Hydroxymercurybenzene Sulfonate
Authors: Scheit Karl‐Heinz , Faerber Peter
Published: March 2005

The interaction of hydroxymercurybenzene sulfonate (HOHgBzS) with 2‐thiouridine (s2Urd) and 2‐thiocytidine (s2Cyd) by means of absorption‐difference spectroscopy and spectrophotometric titration was investigated. The absorption spectra of the reaction mixtures displayed isosbestic points indicating uniform reactions. Scatchard plots of the titration data revealed the formation of 1:1 complexes with a dissociation constant for s2Urd · HOHgBzS and s2Cyd · HOHgBzS of Kd= 0.55 μM and Kd= 0.54 μM, respectively. The interactions of poly(s2U) and poly(s2c) with hydroxymercurybenzene sulfonate were established by their corresponding absorption difference spectra. The binding of hydroxymercurybenzoate (HOHgBz) to poly(s2C) and poly(s2U) was investigated by means of equilibrium dialysis. The association of HOHgBz to 2‐thiocytosine bases in poly(s2C) follows a simple hyperbolic binding isotherm. The Scatchard plot of the binding data disclosed that only a fraction (n= 0.32) of the s2CMP residues in poly(s2C) was accessible to the interaction with HOHgBz. The dissociation constant of this interaction was found to be Kd= 4.3 μM. The binding of HOHgBz to the helical poly(s2U) is a cooperative process as indicated by the sigmoidal binding cuve. Under saturating conditions all s2UMP residues in poly(s2U) interact with the ligand.
The Subunit Structure of Rabbit‐Skeletal‐Muscle Phosphorylase Kinase, and the Molecular Basis of Its Activation Reactions
Authors: Cohen Philip
Published: March 2005

Phosphorylase kinase was isolated from rabbit skeletal muscle in a state approaching homogeneity as judged by the criteria of ultracentrifugal analysis, ion‐exchange chromatography, and antigen‐antibody precipitation in agar. The purified enzyme showed four protein‐staining components upon acrylamide gel electrophoresis in the presence of sodium dodecylsulphate, termed α, α′, β and γ. A variety of precautions designed to eliminate the possible action of proteases and ensure complete reduction and denaturation of the protein had no influence on the gel pattern. The molecular weights of the four components determined by gel electrophoresis, and supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride, following partial separation of the chains by gel filtration on Sephadex G‐200 in the presence of sodium dodecylsulphate, were:–α= 145000, α′= 140000, β= 128000 and γ= 45000. The α' component was present only in trace amounts and the evidence suggests it may be derived from the α component. The three subunits α, β, and γ, were found to exist in equimolar quantities by densitometric analysis of acrylamide gels, by gel filtration on Sephadex G‐200 in the presence of sodium dodecylsulphate, and by carboxymethylation with iodo[14C]acetate, suggesting a minimal binding molecular weight, αβγ, of 318000. The molecular weight of the native enzyme was determined as 1.28 × 106, demonstrating that phosphorylase kinase is composed of 4.0 αβγ units.

Activation of the enzyme by incubation with adenosine‐3′:5′‐phosphate‐dependent protein kinase, adenosine 3′:5′‐phosphate and Mg‐ATP, was accompanied by the phosphorylation of one site on the β subunit, although a second site on the α subunit was phosphorylated at a slower rate. Activation of the enzyme by proteolysis resulted from a limited cleavage of the α subunit. The products of proteolytic attack suggest that the α and β subunits may be structurally related. The γ subunit was not phosphorylated, was resistant to proteolysis and distinct from the (α+β) subunits in its amino acid composition. The possible functions of the chains, and the implications of the activation reactions to the nervous and hormonal control of glycogenolysis in vivo are discussed.
Labeling of Cytoplasmic Liver RNA by [6‐14C]Orotic and 5‐Fluoro[2‐14C]orotic Acids
Authors: ČIhák Alois , Garret Charleton , Pitot Henry C.
Published: March 2005

Orotic and 5‐fluoroorotic acid are utilized differentially for the synthesis of the known species of cytoplasmic hepatic RNA. 5‐Fluoroorotic acid is incorporated preferentially into a fraction of non‐ribosomal RNA which has several properties in common with messenger RNA. The incorporation of both orotate and 5‐fluoroorotate into cytoplasmic RNA is influenced by the administration in vivo of several inhibitors but to different degrees. Actinomycin D blocks the incorporation of orotic acid into ribosomal RNA with relatively little effect on the incorporation of 5‐fluoroorotate into non‐ribosomal RNA. 5‐Azacytidine administration leads to an alteration in the incorporation of orotic as well as 5‐fluoroorotic acid into cytoplasmic RNA, these effects being dependent on the length of pretreatment with the analogue. 5‐Azacytidine given 24 h earlier causes an enhancement of orotic acid incorporation into liver RNA, while no such effect on the labeling of total liver RNA by 5‐fluoroorotic acid is seen. Short‐term treatment (2 h) with 5‐azacytidine leads to the depressed utilization of orotic as well as 5‐fluoroorotic acid for RNA synthesis. 5‐Azaorotate administration results in an inhibition of the incorporation of orotic and 5‐fluoroorotic acid into total liver RNA without affecting their utilization during later phases of the treatment. The observed differences in the incorporation of labeled orotic and 5‐fluoroorotic acid into individual fractions of hepatic cytoplasmic RNA are discussed in relation to their known labeling characteristics.
Effect of an Additional Peptide Extension of the N‐Terminus of Collagen from Dermatosparactic Calves on the Cross‐Linking of the Collagen Fibres
Authors: Bailey Allen J. , Lapière Charles M.
Published: March 2005

Analysis of the highly disorganized fibres of dermatosparactic bovine skin revealed a marked decrease in the inter molecular cross‐linking of the aldimine bond type. However, after removal of the additional peptides from the procollagen in solution with a crude procollagen peptidase preparation, normal fibres were formed on reprecipitation, and analysis of these fibres indicated the formation of normal reducible cross‐links. These studies clearly demonstrate that one effect of the additional peptide extension is to inhibit the formation of both uniform fibres and fibres capable of producing the normal cross‐links.
Binding of Radioactive Homopolynucleotides to RNA
Authors: Fraser Ronald S. S. , Loening Ulrich E.
Published: March 2005

A simple method is described for the characterisation and assay of messenger RNAs which contain polyadenylic acid sequences. Total RNA is mixed with radioactive polyuridylic acid or polythymidylic acid, which bind to polyadenylic acid sequences. When the mixture is fractionated by gel electrophoresis or sedimentation, the radioactivity profile then shows the distribution of those RNAs which contain polyadenylic acid sequences. The amount of homopolynucleotide bound is measure of the amount of messenger RNA present. The detection and assay of 10‐S haemoglobin mRNA in mouse‐reticulocyte total RNA is shown as an example of the technique.
Effect of Erythropoietin on the Peroxidase and Tyrosine‐Iodinase Activity of Mouse Thyroid and Submaxillary Gland
Authors: Banerjee Ranajit K. , Chowdhury Rajyasree Ray , Datta Asoke G.
Published: March 2005

During an attempt to explore the mechanism of cobalt and vitamin B12 effect on the peroxidase and tyrosine iodinase activity, it was observed that cobalt and not vitamin B12in vivo elicits a high plasma titer of a humoral factor, erythropoietin, which caused increased 59Fe incorporation into red blood corpuscles. This erythropoietin was extracted from plasma or kidney and partially purified. This partially purified erythropoietin preparation when injected into starved rat caused increased 59Fe incorporation into red blood corpuscles. The potency of the partially purified erythropoietin preparation from plasma and kidney was compared. This partially purified erythropoietin, as well as sheep step‐I erythropoietin, when administered to mice gave rise to a response almost similar to cobalt administration. It appears that the effect of cobalt on the peroxidase or tyrosine iodinase activity might be mediated through the elevation of the erythropoietin titer of plasma. Moreover, this erythropoietin was found to stimulate both peroxidase and tyrosine iodinase activity of the enzyme preparation of submaxillary gland in experiments in vitro. Erythropoietin, purified further in our laboratory, also produced the same effect. Neuraminidase‐treated erythropoietin, in contrast, failed to execute any such stimulatory effect on the iodination of tyrosine.
The Activity of Individual Molecules of Hybrid β‐Galactosidase Reconstituted from the Wild‐Type and an Inactive‐Mutant Enzyme
Authors: Melchers Fritz , Messer Walter
Published: March 2005

A technique capable of detecting individual molecules of β‐galactosidase has been used to measure the enzyme activity of single hybrid β‐galactosidase molecules reconstituted from mixtures of varying proportions of wild‐type and lac−aba mutant enzyme. Reconstituted molecules exhibit activities of 0, 1/4, 2/4, 3/4, 4/4 of the wild‐type enzyme, depending on the ratio of wild‐type to mutant subunits. These results are expected if each of the four active sites of the Escherichia coliβ‐galactosidase is independently active.
Inhibition of Protein Biosynthesis in Escherichia coli B by Tri‐l‐ornithine
Authors: Barak Zeev , Sarid Sara , Katchalski Ephraim
Published: March 2005

Triornithine completely inhibited growth of Escherichia coli B in the minimal liquid medium M63. The di‐, tetra‐ and pentapeptides showed no effect on the growth of the bacterium.

Triornithine, when added to a bacterial culture, inhibited the incorporation of 14C‐labeled amino acids into the protein fraction, but did not suppress the incorporation of [3H]uridine and [3H]thymidine.

The tripeptide inhibited translation of the induced β‐galactosidase mRNA and also inhibited the synthesis of alkaline phosphatase initiated by derepression.

Triornithine had no effect on the reaction in vitro of T4‐DNA‐directed RNA synthesis.

Incubation of E. coli B cells with triornithine impaired the protein biosynthetic system of the treated cells in vitro. By this treatment the biological activity of the ribosomal fraction is impaired within the bacterial cell.

Triornithine, even at a high concentration, did not inhibit in vitro the polypeptide synthesis in a cell‐free system.
Inhibition of T4 Maturation by Tri‐l‐ornithine
Authors: Barak Zeev , Sarid Sara , Katchalski Ephraim
Published: March 2005

Triornithine inhibited the development of new T4D phages within the bacteria. The plaque‐forming capability of free phages was not affected.

Triornithine inhibits the synthesis of early, as well as late protein in T4‐infected Escherichia coli B cells.

Triornithine was found to enhance [3H]uridine incorporation into viral mRNA. [3H] Thymidine incorporation into T4 DNA was not affected by the tripeptide when added after the time interval during which all of the enzymes concerned with viral DNA synthesis have already been formed.
Two Forms of Microsomal Palmitoyl‐Coenzyme A Synthetase
Authors: Brandes Ruth , Zohar Yizhak , Arad Rivka , Shapiro Benyamin
Published: March 2005

Two palmitoyl‐CoA synthetase activities have been demonstrated in rat liver microsomes. One, catalyzing palmitoyl‐CoA synthesis from ATP, palmitate and CoASH as well as from palmitoyl‐AMP and CoASH. A second enzyme reacts with the first constituents only and is inactive with palmitoyl‐AMP. The first enzyme, but not the second catalyzes the reaction between palmitoyl‐AMP and PPi to yield ATP and palmitic acid.

This duality has been shown by: (a) partial separation of the two types of activity by fractional solution in Triton X‐100, (b) kinetic behaviour testing competition between both reaction mixtures at saturating concentrations and (c) the varying distribution of the two activities in microsomes obtained from rats under different dietary regimes or when diabetes was induced by alloxan.
Parallel Partial Purification of Cytoplasmic and Mitochondrial Aconitate Hydratases from Rat Liver
Authors: Guarriero‐Bobyleva Valentina , Volpi‐Becchi Maria Angela , Masini Alberto
Published: March 2005

A rapid method allowing parallel purification of cytoplasmic and mitochondrial aconitate hydratases of rat liver is described. The two enzymes were purified 25 to 35‐fold. The isoelectric points and Km values in the production of cis‐aconitate with citrate or d‐isocitrate as substrates, were measured for both enzymes. Substrate activation at high concentrations of citrate and d‐isocitrate has been observed.
Activation of Rat‐Skeletal‐Muscle 5′‐Adenylate Deaminase by Non‐Substrate Analogues of Adenosine 5′‐Phosphate
Authors: Maguire M. Helen , Atkinson Maurice R. , Tonkes Peter G.
Published: March 2005

Analogues of adenosine 5′‐phosphate (AMP), i.e. the 2‐chloro, 2‐methylthio, 2‐methoxy, 2‐ethylamino and 2‐chloro‐N6‐methyl derivatives of AMP were not deaminated by 5′‐adenylate deaminase from rat skeletal muscle, but instead stimulated the deamination of AMP. The effects of the 2‐methylthio, 2‐chloro and 2‐chloro‐N6‐methyl derivatives and K+ on the sigmoidal variation of 5′‐adenylate deaminase activity with AMP concentration were studied. At non‐saturating AMP concentrations and low K+ levels the 2‐chloro, 2‐methylthio and 2‐chloro‐N6‐methyl derivatives stimulated the deamination of AMP; the 2‐chloro and 2‐methylthio derivatives activated also at high K+ levels. The three analogues reduced the slopes of Hill plots of the velocity of AMP deamination in the absence of K+ or at low K+ levels, and reduced the AMP concentration for half‐maximal activity. Kavalues for the 2‐methylthio derivative at 1.0 mM K+ were 30 μM at 0.25 mM AMP and 100 μM at 0.125 mM AMP. ATP stimulated the deamination of AMP at low K+ levels, and synergistically increased the activation caused by the 2‐methylthio derivative. The 2‐chloro, 2‐methylthio and 2‐chloro‐N6‐methyl derivatives were not inhibitors of 5′‐adenylate deaminase. It is proposed that these 2‐substituted AMP analogues do not bind to the catalytic site of 5′‐adenylate deaminase, and modify enzyme activity by acting at a regulatory site.
Structure of Protocollagen Proline Hydroxylase from Chick Embryos
Authors: Olsen Bjørn R. , Berg Richard A. , Kivirikko Kari I. , Prockop Darwin J.
Published: March 2005

Protocollagen proline hydroxylase was purified from chick embryos with an affinity‐column procedure which yielded pure enzyme in a tetramer form. Electron microscopy of the enzyme after it was largely dissociated into monomers indicated that the monomers were rod‐shaped with a diameter of 3.31 ± 0.07 nm (S.E.M.) and a length of 6.95 ± 0.07 nm. In preparations of the enzyme in which the protein was partially dissociated, dumb‐bell and V‐shaped structures were seen which morphologically consisted of two sub‐structures each with dimensions similar to those of the isolated monomers. The results suggested that these structures were dimers in which the monomers were joined at one end with an angle between their longitudinal axes.

The largest regular protein structures seen in preparations of the enzyme were about 8 × 8 nm and in some of these structures four sub‐structures were seen, indicating that they were tetramers. On the basis of the data presented here we propose a model of the tetramer in which two V‐shaped dimers were interlocked.

Further studies demonstrated that the single‐ring and four‐ring structures seen by electron microscopy of enzyme preparations obtained by a previous purification procedure were not the enzyme.
Characteristics of Nuclear‐Ribosomal and DNA‐Like Ribonucleic Acids Differentially Extracted by Hot‐Phenol Fractionation
Authors: Markov George G. , Arion Vitali J.
Published: March 2005

The purpose of the present study was to define more exactly the conditions of the method of hot‐phenol fractionation of nuclear RNA (Georgiev and associates). The thermal RNA fractions obtained were characterized by agar‐gel electrophoresis, determination of the specific radioactivity and base composition analysis, both of the total and newly synthesized RNA.

It was found that the fraction extracted at 0–4 °C (considered as cytoplasmic RNA in the literature) contained some nuclear ribosomal precursor RNAs, expecially 32‐S and 23‐S RNAs.

The composition of the different thermal RNA fractions strongly depended on the extraction conditions (time, pH, volume ratiosetc.). Conditions were well‐defined (15‐min extraction at each temperature, pH 6.2) for obtaining, as separate fractions, the nuclear RNA species of ribosomal type (40‐°C RNA,i.e.that obtained at 40 °C) and two classes of DNA‐like RNA (63‐°C and 85‐°C RNA). The 55‐°C RNA fraction was found to be of mixed character (rRNA + DNA‐like RNA) regardless of the extraction conditions.

The 85‐°C RNA contained the “giant” RNA molecules, had the highest specific radioactivity and a (G + C)/(A + U) ratio of about 0.80. These findings are in agreement with the view that 85‐°C RNA is the very first product of transcription. 63‐°C RNA was also DNA‐like but different from 85‐°C RNA with respect to electrophoretic profile, specific radioactivity and base composition. A high content of adenylic acid (above 30%) was found in the region 8 to 18 S of 63‐°C RNA. It is suggested that poly(A) segments are attached at the 18‐S stage of the processing of 63‐°C RNA as a precursor to the cytoplasmic mRNA.

The results presented demonstrate that under well‐defined conditions the method of the hot‐phenol fractionation of Georgiev may be used as a reproducible procedure for obtaining different types of nuclear RNA without preliminary isolation of nuclei and nucleoli.
The Time Course of Recombination of Human Chorionic Gonadotropin Subunits Studied with Immunological Methods, Circular‐Dichroic Measurements and Bioassay
Authors: Merz Wolfgang E. , Hilgenfeldt Ulrich , Brockerhoff Peter , Brossmer Reinhard
Published: March 2005

The restitution of immunological properties of native human chorionic gonadotropin by recombination of the subunits, α and β, was studied by immunological methods (qualitative and two‐dimensional immunoelectrophoresis and two immunodiffusion methods). Recombination of the subunits at different incubation times was carried out with three different mixture ratios. The bulk of the recombined hormone is formed during the first hour. The immunological behaviour of recombined hormone corresponds largely to that of the starting material used for the preparation of the subunits. In addition to the recombined hormone material a further recombination product appears which can be detected neither in the subunit preparations nor in the native hormone. It seems to be formed in high concentrations immediately after the beginning of incubation and to decrease rapidly thereafter. With an excess of β subunit in the recombination mixture, however, it does not disappear completely. The second product possesses antigenic determinants different from those of the recombined hormone. The circular dichroic spectra of the recombination mixtures display variations in dichroic absorption during the course of recombination, as well as a third maximum. With increasing incubation time, the third maximum decreases and is shifted to the far ultraviolet region, where it finally overlaps with the extremum at 195 nm.

The biological activity of the reconstituted material reaches as much as 75% of the starting material.
Evidence for a Catalytic‐Centre Heterogeneity of Molybdoferredoxin from Clostridium pasteurianum
Authors: Zumft Walter G. , Mortenson Leonard E.
Published: March 2005

Molybdoferredoxin, the high‐molecular‐weight component of clostridial nitrogenase has been separated into two components by gradient chromatography on DEAE‐cellulose. One component when combined with azoferredoxin, the low‐molecular‐weight component of nitrogenase, does not reduce acetylene, evolve hydrogen from dithionite or hydrolyse ATP; the other does. Compared with the active component, the inactive species lacks molybdenum and approximately two thirds of the iron. Both species can be identified by their electron paramagnetic resonance spectra. Active molybdoferredoxin has resonances at g= 4.27, 3.78 and 2.01, whereas the inactive species has signals at g= 2.05 and 1.92. Apart from the difference in their catalytic centre, the protein moiety of both species seems to be identical as based on subunit composition, sedimentation and electrophoretic behaviour, amino acid composition and immunological reaction with a molybdoferredoxin antiserum. The method currently employed for purification of nitrogenase proteins and the fractionation of molybdoferredoxin is described. The consequence of this finding influences previously reported physico‐chemical data of molybdoferredoxin.
Murex trunculus Haemocyanin
Authors: Wood Edward J. , Peacocke Arthur R.
Published: March 2005

The native or whole molecule of the haemocyanin from the whelk, Murex trunculus had a sedimentation coefficient of 102.7 S, and combination of this with the diffusion coefficient of 1.03 × 10−7 cm2 s−1 led to a molecular weight of 8.85 × 106. The parameter β of Scheraga and Mandelkern was 2.14 × 106, and the frictional ratio f/f0 was 1.5 indicating that the molecule did not deviate markedly from the spherical. Electron microscopy showed that the molecule was a short cylinder, about 35 nm in diameter.

When the pH was raised dissociation occurred into halves and then into smaller subunits which were referred to as “twentieths”, but which were heterogeneous. This dissociation took place at pH values greater than 7.6 in the absence of Mg2+, or at pH values near 10.0 in presence of 0.01 M MgCl2. Whole molecules were re‐formed from halves upon lowering the pH, but “twentieths”, once formed, did not re‐associate. The products of dissociation were separable by preparative ultracentrifugation and by zone electrophoresis. The proportion of wholes, halves, and twentieths formed was determined by the pH and Mg2+ concentration and was not influenced by rotor speed or by protein concentration. It was considered that there was not a dynamic equilibrium between wholes, halves and twentieths.

In 6 M guanidine‐HCl in the presence of 2‐mercaptoethanol a single species of molecular weight about 220000 was formed. In detergent‐gel electrophoresis there was rather poor evidence for a component of molecular weight about 50000, which whould correspond with one oxygen‐binding site containing 2 copper atoms.
Subcellular Distribution of Succinate‐Semialdehyde Dehydrogenase in Rat Brain
Authors: Sims Kenneth L. , Davis Gary A.
Published: March 2005

The report details the subcellular localization of succinate semialdehyde dehydrogenase in rat brain. The localization of this enzyme, which is important in the catabolism of 4‐aminobutyrate and brain intermediary energy metabolism, is compared with glutamic acid decarboxylase and lactate dehydrogenase. A unique significant over‐recovery of succinic semialdehyde dehydrogenase activity was observed in sucrose gradient subfractions, an over‐recovery which could be eliminated by repeated freeze‐thaws or Triton treatment of fractions. Triton treatment also revealed a significant amount of this activity in the synaptosomal fraction not apparent in the standard procedure, but the major enrichment of activity was found in the mitochondrial fraction. Our results indicate that a striking separation exists in the cellular localization of the synthetic reaction for 4‐aminobutyrate in brain and the catabolic system represented by succinate semialdehyde dehydrogenase.
Synthesis of Rabbit Globin by Reticulocyte Postribosomal Supernatant and Heterologous Ribosomes
Authors: Jacobs‐Lorena M. , Baglioni Corrado
Published: March 2005

Cell‐free protein synthesis has been studied in heterologous systems using rabbit reticulocyte postribosomal supernatant and ribosomes obtained from HeLa or mouse myeloma cells. These cell‐free systems are quite active and incorporate labelled amino acids linearly for more than 30 min. The proteins synthesized have been analyzed by acrylamide gel electrophoresis, column chromatography and paper electrophoresis of the tryptic peptides. More than 70% of the protein synthesized with either type of ribosomes is α chain of rabbit hemoglobin; a small amount of β chain is also synthesized. This result can only be explained by the presence in the postribosomal supernatant of reticulocytes of messenger RNA for hemoglobin. This mRNA is translated more efficiently than the endogenous mRNA present on HeLa or myeloma ribosomes; previous measurements of the mRNA of reticulocyte postribosomal supernatant indicate that it is less on a weight basis than 1/20 of the mRNA associated with the corresponding amount of ribosomes used in an incubation. The preferential translation of mRNA present in the supernatant, presumably in a ribonucleoprotein particle, is discussed.
ω‐Hydroxylation of Steroid Side‐Chain in Biosynthesis of Bile Acids
Authors: BjÖRKHEM Ingemar , Gustafsson Jan
Published: March 2005

ω‐Hydroxylation (26‐hydroxylation) of various C27‐steroids by the microsomal and mitochondrial fraction was studied. Assay conditions for the microsomal 26‐hydroxylation were determined with 5β‐cholestane‐3α,7α‐diol as substrate. NADPH was the required cofactor. NADH stimulated the hydroxylation at suboptimal concentrations of NADPH. The hydroxylation was inhibited markedly by carbon monoxide. Treatment with phenobarbital inhibited the reaction whereas starvation or biliary drainage had no significant effect. The rates of microsomal 26‐hydroxylation of different C27‐steroids expressed in nmol/mg protein per 20 min were cholesterol, <0.1; 5‐cholestene‐3β,7α‐diol, <0.1; 7α‐hydroxy‐4‐cholesten‐3‐one, 0.3; 7α,12α‐dihydroxy‐4‐cholesten‐3‐one, 0.9; 5β‐cholestane‐3α,7α‐diol, 1.8; 5β‐cholestane‐3α,7α, 12α‐triol, 2.7.

Assay conditions for the mitochondrial 26‐hydroxylation were determined with cholesterol and 5β‐cholestane‐3α,7α‐diol as substrates. The rate of reaction was faster with an NADPH‐generating system than with NADPH. The reaction was inhibited markedly with carbon monoxide. Biliary drainage inhibited the reaction whereas starvation or treatment with phenobarbital had no significant effect. The rates of mitochondrial 26‐hydroxylation of different C27‐steroids expressed in nmol/mg protein per 20 min were: cholesterol, 0.3; 5‐cholestene‐3β,7α‐diol, 0.9; 7α‐hydroxy‐4‐cholesten‐3‐one, 1.7; 7α,12α‐dihydroxy‐4‐cholesten‐3‐one, 0.9; 5β‐cholestane‐3α,7α‐diol, 0.9; 5β‐cholestane‐3α,7α,12α‐triol, 2.0.

The results are consistent with previously postulated pathways concerning biosynthesis of cholic acid and chenodeoxycholic acid in which 5β‐cholestane‐3α,7α,12α‐triol and 5β‐cholestane‐3α,7α‐diol, respectively are the main substrates for the 26‐hydroxylase. The possibility is discussed that pathways involving intermediary formation of 7α,26‐dihydroxy‐4‐cholesten‐3‐one and 7α,12α,26‐trihydroxy‐4‐cholesten‐3‐one also are of importance in the biosynthesis of bile acids.
A Transport System for 2‐Keto‐3‐deoxy‐d‐gluconate Uptake in Escherichia coli K12
Authors: Lagarde Alain E. , Pouysségur Jacques M. , Stoeber François R.
Published: March 2005

We have presented kinetic evidence for the occurrence in Escherichia coli K 12 of an active transport system responsible for 2‐keto‐3‐deoxy‐d‐gluconate uptake. It has been studied in constitutive strains PK4 and CAK 101 since it is completely cryptic in the wild‐type strain.

The accumulation is temperature‐dependent, coupled to electron and energy‐yielding reactions, and is inhibited by thiol reagents.

2‐Keto‐3‐deoxy‐d‐gluconate is not transported through the phospho‐enolpyruvate‐transferase system.

Entry reaction displays saturation kinetics obeying the Michaelis‐Menten laws. The Km value has been found to be 0.25 ± 0.05 mM (at 30 °C and pH 7.0), by measuring initial rates of entrance as well as steady‐state levels. Both the operator‐constitutive PK4 strain and the regulator‐constitutive CAK101 strain exhibit the same affinity but apparent V are 10 and 20 nmol × mg‐1× min‐1, respectively.

The 2‐keto‐3‐deoxy‐d‐gluconate permeation system is endogenously induced in a strain lacking 2‐keto‐3‐deoxy‐d‐gluconate kinase, when grown on any intermediary product of the hexuronide catabolic pathway, able to enter the cell and anterior to 2‐keto‐3‐deoxy‐d‐gluconate in the sequence. We presented further evidence making it very likely that 2‐keto‐3‐deoxy‐d‐gluconate itself is the true inducer.

The constitutive synthesis of the transport system is sensitive to the catabolite repression exerted by glucose and pyruvate.

By testing a wide number of sugars and derivatives as possible substrates, only d‐glucuronate is a strictly competitive inhibitor (Ki= 1.5 mM). Since a specific hexuronate permease has been described elsewhere, we were led to demonstrate that d‐glucuronate could be taken up through both its own specific system with high affinity and through the 2‐keto‐3‐deoxy‐d‐gluconate specific system with lower affinity.

The exit reaction rate is first oder with respect to internal concentration, temperature‐dependent (exit rate constant kex is 0.0180 min‐1 at 30 °C) but is not carrier‐mediated.
The Flux of Pyruvate in Perfused Rat Heart
Authors: Mowbray John , Ottaway James H.
Published: March 2005

It was possible to find a steady‐state lactate concentration in a closed‐circuit heart perfusion containing added glucose. Insulin but not growth hormone (4 μg/ml) increased the equilibrium concentration in perfusate by 200%. Intracellular lactate also remained constant and was about twice that in perfusate: this distribution is difficult to explain whether ionic or undissociated lactate is the permeating species.

Pyruvate accumulated linearly in perfusate, reaching a peak after 30 min perfusion and then declined non‐linearly. Insulin increased the peak concentration and growth hormone slowed the decline after the peak. The intracellular pyruvate concentration stayed constant and, except in the first few minutes, was lower than the perfusate concentration.

Pyruvate addition at zero time could inhibit pyruvate accumulation but altered neither the non‐linear decline, the intracellular pyruvate level nor the lactate equilibrium.

The [NAD]/[NADH] ratio, after 10 min of pre‐perfusion on open circuit, was 23. During 2 h of perfusion the ratio rose slowly and then declined. If either insulin or growth hormone were present in the medium, the ratio fell to about 11 during the first few minutes of perfusion and remained low for 90 min. These values could not easily be correlated with either the varying extracellular, or the constant intracellular [lactate]/[pyruvate] ratios.

The glucose and oxygen consumption of the hearts during perfusion was measured, and the change in glycogen concentration in the tissue. It was then possible to draw up a balance sheet which indicated that the control hearts oxidised little other than carbohydrate for the 60 min of observation, but both insulin and growth hormone‐treated hearts oxidised significant quantities of fatty acid during the first 15 min. It appears that growth hormone, like insulin, has an immediate effect on heart metabolism. The addition of pyruvate at zero time appeared to stimulate carbohydrate oxidation in hearts perfused with insulin, even though the hormone had been present in the pre‐perfusion fluid.

Consideration of the variation in the extracellular [lactate]/[pyruvate] ratio with length of perfusion, and its lack of relationship to the intracellular [lactate]/[pyruvate] ratio, together with similar reports of other workers, leads us to conclude that neither lactate nor pyruvate establish a concentration gradient across the heart cell membrane which is based on passive diffusion. The discrepancy is particularly marked with respect to pyruvate. Our results strongly suggest that the extracellular [lactate]/[pyruvate] ratio cannot be used as an index of the cytoplasmic redox potential in perfused heart.

A tracer study of pyruvate metabolism in the reproducible non‐steady‐state conditions described can offer an explanation for the behaviour of the observed metabolic parameters: this study is reported in the following papers.
Identification of the Lipid Intermediate in Yeast Mannan Biosynthesis
Authors: Jung Peter , Tanner Widmar
Published: March 2005

The unsaponifiable lipid fraction of Saccharomyces cerevisiae contains a phosphorylated component which accepts a mannosyl residue from GDP‐mannose and transfers it to growing mannan chains. Both reactions are catalyzed by membrane‐bound enzymes. The lipid component has been purified more than 1000‐fold (based on lipid‐bound phosphate). After dephosphorylation with an orthophosphoric monoester phosphohydrolase this acceptor was shown by mass spectroscopy to be identical with authentic yeast dolichol. The “lipid” intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol. The “lipid” intermediate intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol monophosphate.

Yeast dolichol is a mixture of five homologous polyprenols with saturated α‐isoprene units, as shown by Dunphy et al. in 1967. All these homologues seem also to be present in the cells as monophosphates. As compared to free dolichols the phosphorylated forms only amount to about 10–20%.
Pyruvate Kinase
Authors: Carbonell Juan , Marco Roberto , Felíu Juan E. , Sols Alberto
Published: March 2005

Kinetic studies of the pyruvate kinases of rat and human tissues have led to the identification of three classes of isoenzymes with qualitative differences in regulatory properties.

Class L, the major component in liver extracts and a minor component of kidney extracts, shows markedly sigmoidal kinetics with respect to the concentration of phosphoenolpyruvate, allosteric inhibition by ATP and alanine, and activation by fructose 1,6‐bisphosphate. Class A, present in adipose tissue, and the major component in kidney and the minor one in liver, shows slightly sigmoidal substrate saturation curves and is allosterically inhibited by alanine and activated by fructose 1,6‐bisphosphate. Class M, present in muscle and brain, has none of the above regulatory properties.

The activities of the pyruvate kinases of classes L and A respond immediately to changes in the concentration of the effectors alanine and fructose 1,6‐bisphosphate.

The two regulatory isoenzymes are strongly inhibited by three amino acids: alanine, cysteine, and phenylalanine. Cysteine is the stronger inhibitor for class L, while phenylalanine is the stronger one for class A. The allosteric inhibition is stereospecific for the l‐amino acids in contrast with the weak isosteric inhibition of muscle pyruvate kinase by l‐ or d‐alanine as analogue of the product.

The allosteric inhibition of pyruvate kinase L by ATP is highly specific in contrast with a rather wide specificity of this enzyme for nucleoside diphosphates as acceptor substrates.