bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitrogenase from Azotobacter chroococcum
Authors: YATES M. Geoffrey , PLANQUÉ Kees
Published: March 2005

A large‐scale purification of the nitrogenase components from Azotobacter chroococcum yielded two non‐haem iron proteins, both of which were necessary for nitrogenase activity and each had a specific activity of approximately 2000 ± 300 nmol of acetylene reduced/mg protein per min in the presence of saturating amounts of the other. This procedure freed the Mo‐Fe protein from a protein contaminant which had an electron paramagnetic resonance signal at g = 1.94.

Both proteins were purified to homogeneity as determined by disc gel electrophoresis and ultracentrifugal analysis. Both proteins were oxygen‐sensitive but not cold‐labile. Ultracentrifugal analysis indicated that both proteins dissociated to a slight degree at concentrations below 2 mg/ml.

The larger of the two proteins had a molecular weight of 227 000 and contained 1.9 ± 0.3 atoms of Mo, 23 ± 2 atoms of Fe, 20 ± 2 acid‐labile sulphide and 47 tryptophan residues/mol. The protein consists of 4 subunits of mol. wt 60000 (approx.). The reduced protein showed electron paramagnetic resonance signals at g = 4.29, 3.65 and 2.013 but not in the area of g = 5 to 6. Upon oxidation absorbance increased throughout the visible region of the ultraviolet visible spectrum, with a maximum difference between oxidised and reduced protein occurring at 430 nm.

The smaller protein had a molecular weight of 64000 and contained 4 g‐atoms of Fe and 4 acid‐labile sulphide groups/mol but no tryptophan. It had two subunits of mol. wt 30800. The reduced protein showed electron paramagnetic resonance signals in the g = 1.94 region which integrated to 0.17 electrons/mol. The protein retained almost full activity after oxidation with phenazine methosulphate. The ultraviolet visible spectrum of oxidised protein was clearly different from that of the oxygen‐inactivated protein: it had a sharp peak at 269 nm and a broad absorbance between 340 and 470 nm with a maximum difference between oxidised and reduced forms at 430 nm. Oxygen‐inactivated protein showed a sharp peak at 277.5.nm and broad peaks from 305 to 360, 400 to 425 and 435 to 475 nm.

Amino acid analyses of both proteins showed that most common amino acids were present with a preponderance of acidic residues. Analyses of compositional relatedness showed that the nitrogenase proteins from A. chroococcum were most closely related to those from A. vinelandii and least so to those from Clostridium pasteurianum.
The Effect of 8‐Hydroxyquinoline on Enzyme Synthesis in the Fission Yeast Schizosaccharomyces pombe
Authors: CREANOR James , MAY John W. , MITCHISON J. Murdoch
Published: March 2005

The effect of 8‐hydroxyquinoline, a rapid inhibitor of RNA synthesis, was followed on the activity of a number of enzymes in cultures of the fission yeast Schizosaccharomyces pombe. Two types of effect were found. In the first the activity continued to rise for a period and then remained constant. This occurred with alkaline phosphatase, basal and derepressed acid phosphatase, hexokinase, and derepressed sucrase and maltase at low cell density. It is consistent with control being exercised by an unstable mRNA or by an unstable stimulator of translation. In the second the activity increásed above the control values for several hours. This occurred with basal sucrase and maltase, and suggests a stable mRNA and an unstable inhibitor of translation. The extent of ‘superproduction’ of sucrase varied with cell density and with growth medium and this may be due to differences in the degree of translational inhibition. The possibility of a stable mRNA has interesting implications for the control of enzyme synthesis through the cell cycle.
The Reduction of Plastocyanin by Plastoquinol‐1 in the Presence of Chloroplasts
Authors: WOOD P. Muir , BENDALL Derek S.
Published: March 2005

The reduction of plastocyanin by plastoquinol‐1 was efficiently catalysed by disrupted chioroplasts or etioplasts in the dark. The reaction was inhibited by 2,5‐dibromomethylisopropyl‐p‐benzoquinone which inhibits photosynthetic electron transport between plastoquinone and cytochrome f. Evidence is presented that the reduction took place via cytochrome f, and that plastoquinone‐9 was not involved. Triton X‐100 and organic solvents were inhibitory, but partial fractionation was achieved without loss of activity by density gradient centrifugation in the presence of high digitonin concentrations. All active material contained cytochromes b‐559LP and b‐563 in addition to cytochrome f, but these b‐type cytochromes were not directly involved. Other 1‐electron acceptors could be used in place of plastocyanin, for instance ferricyanide and Pseudomonas cytochrome c‐551. The reaction can be applied to give a sensitive dark assay for active cytochrome f It is suggested that cytochrome f possesses two sites for interaction with redox reagents: a hydrophilic site with which plastocyanin reacts by electron transfer and a hydrophobic site with which plastoquinol reacts by hydrogen atom transfer.
Interactions of KB‐Cell Glycoproteins with an Adenovirus Capsid Protein
Authors: MEAGER Anthony , BUTTERS Terry D. , MAUTNER Vivien , HUGHES R. Colin
Published: March 2005

Glycoprotein material extracted from human KB cells with a flurocarbon, trichlorotrifluoroethane (Arklone P), into a water‐soluble fraction binds to fibre, a structural protein of the adenovirus type‐5 capsid. The fibre‐binding glycoprotein(s) were purified by ion‐exchange chromatography on DEAE‐Sephadex and affinity chromatography on a fibre‐Sepharose support. The purification procedure also includes a trypsinization step which eliminates the bulk of contaminating KB cell proteins present in the aqueous fraction without appreciably affecting the activity of the fibrebinding glycoprotein(s). Some comparison is made of the membrane‐bound receptors for adenovirus and the water‐soluble fibre‐binding glycoprotein(s).
Purification and Sequence Analysis of the mRNA Coding for an Immunoglobulin Heavy Chain
Authors: COWAN Nicholas J. , SECHER David S. , MILSTEIN César
Published: March 2005

A mutant cell line (IF2) derived from the mouse myeloma MOPC 21 has been used for the isolation and sequence analysis of H‐chain mRNA. The IF2 cells synthesise an H‐chain of reduced size in which the CH1 homology region is missing. Sizing of the IF2 H‐chain mRNA and wild‐type H‐chain mRNA revealed that the deletion is expressed at the mRNA level. The mutant H‐chain mRNA sedimented at 16‐S, enabling effective resolution from 18‐S ribosomal RNA. In experiments using IF2 cells labelled with [32P]phosphate, the 16‐S mRNA was purified by oligo(T)‐cellulose chromatography. Polyacrylamide gel analysis of the poly(A)‐containing fraction showed the presence of a single radioactive band. Comparison of the mobility of this band relative to markers of known molecular weight revealed that the molecule contained about 1600 nucleotides. Digestion of the 32P‐labelled mRNA with T1 ribonuclease and two‐dimensional fractionation of the resulting oligonucleotides yielded a ‘fingerprint’ suitable for a preliminary sequence analysis. By using the established amino acid sequence of the IF2 H‐chain and a knowledge of the genetic code, 14 oligonucleotides were assigned within the constant region and four within the variable region of the IF2 H‐chain. This sequence data accounts for 19.5% of the coding region. Several other oligonucleotides, which could not be assigned within the coding region but which occurred in approximately molar yield, have also been partially characterised. These oligonucleotides are presumably derived from the untranslated regions of the mRNA.
Purine‐Nucleoside Phosphorylase from Salmonella typhimurium and Escherichia coli
Authors: JENSEN Kaj Frank
Published: March 2005

Purine nucleoside phosphorylase from Salmonella typhimurium has been subjected to kinetic analysis, i.e. determination of initial velocity patterns and product inhibition studies.

The kinetic results suggest that the enzyme works by a sequential reaction mechanism, where the nucleoside, phosphate, and pentose 1‐phosphate are all able to bind to the free enzyme, whereas it appears that the purine base binds after addition of the pentose 1‐phosphate.

The proposed mechanism is confirmed by substrate binding studies. In addition to the enzymesubstrate complexes suggested by the kinetics, the binding studies revealed a ‘dead end’ complex, consisting of enzyme, phosphate, and purine base. Similar binding experiments were carried out using the enzyme from Escherichia coli. The results suggest that this enzyme works by an identical reaction mechanism. The binding data are in agreement with the presence of six binding sites per native enzyme molecule, one binding site per subunit, for each ligand.

Both enzymes show normal Michaelis‐Menten kinetics for their substrates with the exception of phosphate, for which the double‐reciprocal plots are concave down. This behaviour is seen in both binding and velocity curves, and most likely is a result of negative cooperativity in the binding of phosphate to the enzyme.
31P Nuclear‐Magnetic‐Resonance Studies on the Developing Embryos of Xenopus laevis
Authors: COLMAN Alan , GADIAN David G.
Published: March 2005

The concentrations of nucleoside triphosphate, inorganic phosphate and the yolk proteins, phosvitin and lipovitellin, have been monitored in living embryos of Xenopus laevis by 31P nuclear magnetic resonance (NMR) spectroscopy. The nucleoside triphosphate levels remain relatively constant at about 3.5–4.5 nmol/embryo at least until the ‘spontaneous movement’ stage of development. By the swimming tadpole stage an inorganic phosphate resonance representing about 30 nmol/embryo becomes evident in the NMR spectrum. Computer manipulation also shows such a resonance, although smaller, to be present at a somewhat earlier developmental stage; these findings are confirmed biochemically. The major contribution to the NMR spectrum of oocytes, unfertilized eggs and early embryos is the yolk phosphoprotein resonance. On isolation of the yolk from the embryos it is possible to quantify the contribution to the NMR spectrum from the lipid‐phosphate and protein‐phosphate moieties of the yolk proteins. During development, as the yolk is used up, it is found that the protein‐phosphate resonance disappears at a greater rate than the lipid‐phosphate peak. The total phosphorus content of the embryo (∼ 200 nmol/embryo) is shown biochemically to remain constant during development; however, the total amount of phosphorus observed by NMR decreases by about 40% during development.

From the resonance positions of their α, β and γ phosphate groups it is deduced that the nucleoside triphosphate molecules are liganded in vivo to a divalent cation which is not manganese, but could be either magnesium or calcium. From the position of the inorganic phosphate resonance it is deduced that the internal pH of embryos where this resonance is evident is 6.8 ± 0.2.
Subcellular Localization of a Lesion in Protein Synthesis in Rabbit Reticulocytes Incubated at Elevated Temperatures
Authors: BONANOU‐TZEDAKI Sophia A. , ARNSTEIN Henry R. V.
Published: March 2005

When rabbit reticulocytes are incubated at 43–45 °C their rate of protein synthesis rapidly decreases, compared to a control 37 °C incubation. Lysates prepared from cells incubated at this supra‐optimal temperature have an equally decreased capacity for endogenous, but not poly(uridylic acid)‐directed, protein synthesis. Subcellular fractionation traced the lesion to the crude ribosomal pellet, 0.5 M KCl ribosomal wash and postribosomal supernatant of the temperature‐shocked cells. Preparation of purified ribosomal subparticles showed, however, that they were as active as the control in protein synthesis. In this paper we present evidence that the decreased activity of the heated lysate, 0.5 mM KCl wash and postribosomal supernatant is due to an inhibitor and can be overcome by the addition of 0.5 M KCl or supernatant from control cells. The results are discussed in terms of the inactivation of a component, essential for initiation of endogenous protein synthesis, which is probably partitioned between ribosomes and supernatant. We also suggest that the decreased protein synthetic activity of the heated cells may be related to their decreased synthesis of haem.
The Reaction Kinetics of Fluoride and Ammonia with Acid and Alkaline Ferrimyoglobin in a Crystalline State
Authors: CHANCE Britton , RUMEN Nevenka M.
Published: March 2005

Differences between the reactivity of amorphous and crystalline myoglobin have been studied by the rapid‐flow method combined with dual‐wavelength spectrophotometry. The binding of ammonia to the hydroxide compound has a half‐time of 55 ms. The reverse reaction has a half‐time of 70 ms. At pH 7.0 the relative half‐times of combination and dissociation with fluoride are 10 min for crystalline and 1.8 min for amorphous materials. Reactivity of the crystals to fluoride at pH 6.0 greatly increased as compared with pH 8.7. Half‐time at pH 8.7 is 10 min, while at pH 6.0 the half‐time is 2.5 s for the ‐crystalline material and 1.4 s for the amorphous material. The exchange of fluoride by azide at pH 6.0 is 3.1‐fold faster in amorphous material than in crystalline material.
Processing of the 17‐S Escherichia coli Precursor RNA in the 27‐S Pre‐Ribosomal Particle
Authors: HAYES Françoise , VASSEUR Marc
Published: March 2005

An RNase activity probably involved in the maturation of 16‐S pre‐ribosomal RNA in Escherichia coli has been partially purified from crude cell extracts. When 27‐S ribosome precursor particles are incubated with this enzyme preparation in vitro, their 17‐S RNA is converted to a product with the same electrophoretic mobility as mature 16‐S rRNA. Fingerprint analysis of this product shows that it contains the 3′‐OH but not the 5′‐P terminus of mature 16‐S rRNA. Generation of the normal 5′‐P terminus seems to require a factor present in cell extracts since incubation of the 27‐S precursor particle in an extract obtained after centrifugation at 30000 g causes conversion of the 17‐S RNA to a 16‐S species containing both termini of mature 16‐S rRNA.

Preliminary experiments suggest that correct maturation of the 5′ end of the 17‐S precursor RNA requires a system in which protein synthesis can take place.
Replacement of Lysine by Arginine, Phenylalanine and Tryptophan in the Reactive Site of the Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) and Change of the Inhibitory Properties
Authors: JERING Helmut , TSCHESCHE Harald
Published: March 2005

The reactive‐site sequence of a proteinase inhibitor can be written as…‐P3‐P2‐P1‐P1′‐P2′‐P3′‐…, where ‐P1‐P1′‐ denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin‐kallikrein® inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive‐site residue P1, lysine‐15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon.

Starting from native (virgin) inhibitor, reactive‐site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des‐Lys15‐inhibitor by reaction with carboxypeptidase B. Inactive des‐Lys15 ‐inhibitor was reactivated by enzymic replacement of the P1 residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme inhibitor complexes were subjected to kinetic‐control dissociation, and the semisynthetic virgin inhibitors were isolated.

The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikreina and plasmin. The homologues with either lysine or arginine in the P1 position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg15‐homologue is a slightly more effective kallikrein inhibitor than the Lys15‐inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys15‐inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P1 residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz).
Characterization of the Oxygenase Activity in a Mutant of Chlamydomonas reinhardi Exhibiting Altered Ribulosebisphosphate Carboxylase
Authors: NELSON Patricia E. , SURZYCKI Stefan J.
Published: March 2005

A previously described Mendelian mutant of Chlamydomonas reinhardi, ac i72, exhibiting altered ribulosebisphosphate carboxylase activity and unable to grow on minimal medium is examined for changes in ribulosebisphosphate oxygenase activity. The ribulosebisphosphate oxygenase activity of the enzyme purified from both wild type and ac i72 is compared over a pH range from 7.0 to 9.5. Both enzymes exhibit maximum activity at pH 9.0. However, the ac i72 enzyme is twice as active as the wild type enzyme at a physiological pH of 7.0.

The studies in vivo of the products of CO2 fixation of ac i72 and wild type cells in the presence of high and low O2 concentration shows that due to a lower level of carboxylation, the ac i72 cells fix CO2 at half the rate of wild type cells. In ac i72, 24% of the photosynthetically fixed 14C is channelled into the water‐soluble fraction as opposed to 6% in wild type. Thin‐layer chromatography of the watersoluble fraction showed extensive accumulation of components of the glycolate pathway in ac i72 as compared to wild type. This indicates that the oxygenase activity of the enzyme prevails in ac i72 in vivo. Since a high concentration of glycolate is toxic to cells of C. reinhardi, the high oxygenase activity of ac i72 provides an explanation for the inability of ac i72 to grow phototrophically even though its rate of CO2 fixation is half that of wild type. This toxicity to glycolate is overcome by growth under amber illumination or low O2 concentration.
New Assay for Pregnenolone and Progesterone 17α‐Hydroxylase Based on the Specific Substitution of a Tritium Situated on Carbon 17
Authors: KREMERS Pierre
Published: March 2005

A new assay for the measurement of steroid 17α‐hydroxylase activity in beef adrenals is described. This method is based on the biochemical mechanism of the enzymic reaction, i. e. the direct and stereospecific substitution of the proton located on the hydroxylated position. Progesterone or pregnenolone specifically labelled on the 17 position are solubilized in the incubation mixture with the help of Tween 80 and incubated under optimal conditions. The tritium enzymically released from the substrate is found in the medium as a molecule of water which is then distilled under reduced pressure and counted by liquid scintillation.

The results obtained with this new method are comparable with those obtained with a conventional method using a 14C‐labelled substrate.
An Intracellular Endonuclease of Bacillus subtilis Specific for Single‐Stranded DNA
Authors: CIARROCCHI Giovanni , FORTUNATO Adriana , COBIANCHI Fabio , FALASCHI Arturo
Published: March 2005

We have fractionated from extracts of Bacillus subtilis the DNase activity specific for singlestranded DNA; the activity separates in two main fractions on Sephadex G‐200, a larger one (Mr < 400000) and a smaller one (Mr∼ 30000). We have purified the smaller, more abundant fraction nearly 3000‐fold. The purified enzyme has a pH optimum close to 8, is activated by Ca2+, and is inhibited by EDTA; the enzyme hydrolyses single‐stranded DNA at a rate approximately 40 times greater than double‐stranded DNA. The mode of action is endonucleolytic on both substrates. but the possibility that the two activities may reside on different molecules is not ruled out. The products have 5′‐P and 3′‐OH ends.

The enzyme is different from those purified from the culture media of the same organism in several respects; the latter are all extracellular enzymes, they are not specific for single‐stranded DNA (except one) and have all an exonucleolytic mode of action.
Guanylate Cyclase : Assay and Properties of the Particulate and Supernatant Enzymes in Mouse Parotid
Authors: DURHAM John P.
Published: March 2005

A new, very sensitive, rapid and reliable assay for guanylate cyclase has been established based on conversion of [32P]GTP to [32P]guanosine 3′: 5′‐monophosphate and its separation on Dowex 50 and aluminium oxide columns. The optimum conditions for the assay of mouse parotid guanylate cyclase have been established and using this procedure the properties of the enzyme have been investigated. The enzyme was found in both the particulate and supernatant fractions. The particulate enzyme was activated 12‐fold by Triton X‐100 and the supernatant enzyme activity increased 2‐fold. In the presence of detergent guanylate cyclase activity was distributed 85% in the particulate and 15% in the supernatant fractions, respectively. The particulate activity was localised in a plasma membrane fraction.

Guanylate cyclase activity was also assayed in a wide variety of other tissues. In all cases enzymatic activity was found in both the particulate and supernatant fractions. The distribution varied with the tissue but only the intestinal mucosa had a greater proportion of total guanylate cyclase activity in the particulate fraction than the parotid.

The two enzymes showed some similar properties. Their pH optima were pH 7.4, both enzymes were inhibited by ATP, dATP, dGTP and ITP, required Mn2+ for activity and plots of activity versus Mn2+ concentration were sigmoidal. However, in many properties the enzymes were dissimilar. The ratios of Mn2+ to GTP for optimum activity were 4 and 1.5 for the supernatant and plasma‐bound enzymes, respectively. The slope of Hill plots for the supernatant enzyme with varying Mn2+ was 2. The particulate enzyme plots also had a slope of 2 at low Mn2+ concentration but at higher concentrations (above 0.7 mM) the Hill coefficient shifted abruptly to 4. Calcium ions reduced sigmoidicity of the kinetics lowering the Hill coefficient, activated the enzyme at all Mn2+ concentrations but had no effect on the Mn2+: GTP ratio with the supernatant enzyme while with the plasma membrane enzyme Ca2+ had no effect on the sigmoid form of the kinetics at low Mn2+ but prevented the shift to a greater Hill coefficient at higher Mn2+, inhibited the activity at low Mn2+ and shifted the Mn2+ : GTP optimum ratio to 4.

For the particulate enzyme plots of activity versus GTP concentration were sigmoid (n = 1.3), while the supernatant enzyme exhibited hyperbolic kinetics.
Action of Nucleases on Double‐Stranded RNA
Authors: EDY Victor Gordon , SZEKELY Maria , LOVINY Therese , DREYER Christine
Published: March 2005

Double‐stranded RNAs from Penicillium chrysogenum virus have been treated with RNAse III, pancreatic RNAse A and RNAse T1 and the degradation of the RNAs has been studied under different conditions. It was found that only the two former enzymes cut across both strands, RNase T1 cannot cleave double strands.

RNase III was shown to digest double‐stranded RNA by a two step process: an initial phase of specific cleavage is followed by random degradation. In the first phase the enzyme exhibited a definite preference for some specific base pattern.

Partial or complete degradation with pancreatic RNase A could also be achieved in media with high salt concentration provided that the enzyme : substrate ratio was increased together with the salt concentration. By combining different assay techniques, the process of degradation was followed from the early stages to complete digestion and the breakdown products were characterised. It is suggested that a structural change in the enzyme molecules enables them to act on double‐stranded RNA.

RNAse T1, being unable to cleave double strands, provides a useful tool for studying the secondary structure of RNA molecules. Treatment with different nucleases yielded some new information on the structure of different RNA species in Penicillium stoloniferum virus.
The Purification and Properties of the Trimethoprim‐Resistant Dihydrofolate Reductase Mediated by the R‐Factor, R 388
Authors: AMYES Sebastian G. B. , SMITH John T.
Published: March 2005

The R‐factor R388 mediates the production of a trimethoprim‐resistant dihydrofolate reductase. This enzyme has a different‐ molecular weight and pH profile to the trimethoprim‐sensitive enzyme of the Escherichia coli host. The R‐factor mediated enzyme was separated completely from the host E. coli enzyme by DEAE‐cellulose ion‐exchange chromatography. The purified R‐factor enzyme was about 20000 times less susceptible to trimethoprim than the E. coli enzyme and although it was inhibited competitively by trimethoprim, its inhibitor constant (Ki) was 20000 times greater than that of the host enzyme. The R388 and E. coli enzymes also differed in their substrate specificity requirements. In addition, the R388 enzyme surprisingly conferred high level resistance to the broad spectrum dihydrofolate reductase inhibitor, amethopterin. The possible origins of the R388 enzyme are discussed.
Localization of a Prostaglandin F2a Receptor in Bovine Corpus luteum Plasma Membranes
Authors: POWELL William S. , HAMMERSTRöM Sven , SAMUELSSON Bengt
Published: March 2005

The distribution of a prostaglandin F2a receptor in various subcellular fractions from bovine corpora lutea obtained by differential and gradient centrifugation paralleled very closely the distribution in these fractions of 5′‐nucleotidase, a marker enzyme for plasma membranes. The fractions most enriched in the receptor and 5′‐nucleotidase were relatively free of mitochondria and lysosomes but were contaminated to some extent by elements of the endoplasmic reticulum. From these results it can be concluded that the prostaglandin F2a receptor is localized on the plasma membranes of the corpus luteum cells. A simple method is described for the purification of plasma membranes from bovine corpora lutea by differential centrifugation.
Glycosaminoglycan Biosynthesis in Arterial Wall
Authors: LEVY Peggy , PICARD Jacques
Published: March 2005

Hexosaminyltransferase and glucuronyltransferase are well known for their role in the biosynthesis of proteoglycans. These two enzymes are characterized in rough and smooth membranes, obtained following subcellular fractionation of aortic media‐intima. They require the presence of Mn2+ or Mg2+ for activity. The optimum concentration for these two cations is 5mM. The optimum pH for hexosaminyltransferase and glucuronyltransferase is approximately 6.8 in Tris buffer, and their optimum temperature is 30 °C. Hexosaminyltransferase has an apparent Km of 0.27 nM. Glucuronyltransferase has an apparent Km of 0.21 nM.

Uptake of labeled sugars by endogenous proteoglycans is inhibited by puromycin. Hexosaminyl‐transferase and glucuronyltransferase are present in both rough and smooth submicrosomal fractions. The different endogenous glycosaminoglycans are labeled during our incubation experiments. The greatest incorporation is noted for hyaluronic acid and heparan sulfate; the least is seen for chondroitin sulfate.

The results obtained in vitro for incorporation of labeled precursors into endogenous proteo‐glycans are consistent with those observed during the study in vivo of the turnover of these macromolecules.
Inhibition of Lobster‐Muscle Arginine Kinase by Homologous Antibodies and Study of an Antibody Population Directed against a Tyrosine‐Containing Antigenic Structure
Authors: BENYAMIN Yves , ROBIN Yvonne
Published: March 2005

The effect of highly inhibitory rabbit antibodies on the enzymic activity of lobster‐muscle arginine kinase has been studied. 100% inhibition was obtained at a 30‐fold molar excess of antibody. The guanidine substrate l‐arginine did not protect the enzyme from subsequent inhibition by its antibodies. On the other hand, the metal‐nucleotide substrate Mg‐ATP, or the substrate analog Mg‐AMP, protected about 50 % the enzyme activity, the extent of the protection being irrespective of the presence or of the absence of l‐arginine and of the value of the molar ratio Ab/Ag.

An antibody population directed against the antigenic determinant containing the essential tyrosine residue of the enzyme has been isolated by the affinity chromatography. No inhibitory activity was found in that fraction. Most of the inhibitory activity was found in the remaining antibody populations.

It is concluded that the combination of the tyrosine‐containing antigenic determinant with its specific antibodies is not responsible for the inactivation of the enzyme upon antibody binding and that the environment of the nucleotide substrate binding site could be implicated in the inhibitory action.
Physical Studies of Chromatin
Authors: BOSELEY Paul G. , BRADBURY E. Morton , BUTLER‐BROWNE Gillian S. , CARPENTER Brian G. , STEPHENS Ronald M.
Published: March 2005

Experiments have been carried out to define clearly which histone combinations can induce a higher order structure when combined with DNA. The criterion for a higher order structure being the series of low‐angle X‐ray diffraction maxima nominally at 5.5 nm, 3.7 nm, 2.7 nm and 2.2 nm. Such a pattern, with resolution similar to that of H1‐depleted chromatin, is readily attainable by recombining histones H2A + H2B + H3 + H4 with DNA using a salt‐gradient dialysis method. However, the use of urea in the recombination procedure is shown to be detrimental to the production of a higher order structure.

Low‐angle ring patterns are not obtained by recombining DNA with single pure histories or any combination of histone pairs except H3 + H4. The diffraction maxima from the latter are, however, weaker than those from chromatin and there are pronounced semi‐equatorial arcs. The presence of a third histone, either H2A or H2B in the H3 + H4 recombination mixture tends to distort the recognised low‐angle pattern.

It is concluded that the histone pair H3 + H4 is essential for the formation of a regular higher order structure in chromatin, although for a complete structural development the presence of H2A + H2B is also required.
The Formation of threo‐11‐Hydroxy‐trans‐12: 13‐epoxy‐9‐cis‐octadecenoic Acid by Enzymic Isomerisation of 13‐l‐Hydroperoxy‐9‐cis,11‐trans‐octadecadienoic Acid by Soybean Lipoxygenase‐1
Authors: GARSSEN Gerrit J. , VELDINK Gerrit A. , VLIEGENTHART Johannes F. G. , BOLDINGH Jan
Published: March 2005

The interaction of soybean lipoxygenase‐1 with 13‐l‐hydroperoxy‐9‐cis,11‐trans‐octadecadienoic acid (13‐hydroperoxy‐linoleate), the product of the enzymic dioxygenation of linoleic acid, yields either a yellow or a purple‐coloured enzyme species depending on the amount of product used. With an excess of 13‐hydroperoxy‐linoleate a labile purple‐coloured enzyme species is formed which reverts to a yellow‐coloured form with concomitant conversion of the hydroperoxy compound. In this reaction 13‐hydroperoxy‐linoleate isomerises into threo‐11‐hydroxy‐trans‐12: 13 epoxy‐9‐cis‐octadecenoic acid as could be concluded from nuclear magnetic resonance and mass spectral data.

Experiments with 13‐[18O2]hydroperoxy‐linoleate showed a high retention (70%) of the two hydroperoxy oxygen atoms in the end product.
Levansucrase of Bacillus subtilis: Kinetic and Thermodynamic Aspects of Transfructosylation Processes
Authors: CHAMBERT Régis , GONZY‐TRÉBOUL Genéviève
Published: March 2005

Simple kinetic considerations derived from the ping‐pong mechanism previously proposed for levansucrase of Bacillus subtilis allowed us to predict an easily operated method to approach the kinetic studies of exchange and hydrolytic activities of this enzyme. The experimental kinetic pattern obtained from the study of both activities is in close agreement with those predicted by theoretical approach. The combination of kinetic results enabled us to determine with a good accuracy the values of the apparent rate constant of the step of fructosylation of the enzyme from the sucrose‐enzyme Michaelis complex and the apparent rate constants of the steps of defructosylation of the fructosyl enzyme to water or to glucose. The standard free energy reaction coordinate diagram for the transfructosylation process from sucrose to water was constructed. We found that the high energy of the glycosidic linkage of sucrose is preserved in the fructosyl‐enzyme intermediate. The temperature dependence studies of the rate constants of fructosylation and defructosylation of the enzyme show that the entropy of activation for the two steps of defructosylation of the fructosyl enzyme are nearly the same. However the enthalpy of activation for the transfructosylation step to water is greater than that to glucose. We attempted to explain this discrepancy. Furthermore comparison of mechanism and efficiency of enzymatic and acid catalysis of sucrose hydrolysis was developed.
A Study of the Lysyl Residues in the Basic Pancreatic Trypsin Inhibitor using 1H Nuclear Magnetic Resonance at 360 MHz
Authors: BROWN Larry R. , DE MARCO Antonio , WAGNER Gerhard , WÜTHRICH Kurt
Published: March 2005

Fourier transform 1H nuclear magnetic resonance (NMR) experiments at 360 MHz using convolution difference techniques to improve the spectral resolution were employed to investigate the resonances of the lysyl residues in bovine pancreatic trypsin inhibitor. The observations in both native protein and in chemically modified protein containing Ne‐dimethyllysine show that three of the four lysines extend predominantly freely into the solvent, whereas lysine‐41 is involved in an intramolecular interaction with tyrosine‐10. Since in the single crystal structure tyrosine‐10 is involved in an intermolecular interaction with arginine‐42 of the neighboring protein molecule, the NMR data thus reveal a local conformation difference for bovine pancreatic trypsin inhibitor in solution and in the crystalline form which appears to result primarily from intermolecular interaction in the crystal lattice.
A DNA Polymerase from Ustilago maydis
Authors: BANKS Geoffrey R. , HOLLOMAN William K. , KAIRIS Michael V. , SPANOS Ad , YARRANTON Geoffrey T.
Published: March 2005

A DNA polymerase from Ustilago maydis has been purified to apparent homogeneity. The native enzyme possesses a subunit structure consisting of 50000 and 55000‐dalton monomers. The apparent sedimentation coefficient of the polymerase activity in the absence of salt is 8.4 S (Mr= 180000−200000), that in its presence (0.6 M NaCI or 0.12 M KCI) being 6.3 S (Mr= 80000–100000). Low concentrations of EDTA also converted the 8.4‐S to a 6.3‐S form, whereas magnesium ions catalysed the reverse association. The enzyme has an absolute requirement for both a DNA or RNA template and a DNA primer. For homopolymer templates the primer requirement was satisfied by a short complementary oligodeoxynucleotide, but oligoribonucleotides were extremely inefficient primers. With the template‐primer poly(dA) (dT)12, the enzyme added an average of 50 dTMP nucleotides on to each primer molecule, whereas with poly(rA) (dT)12, this figure was 300. The enzyme also possesses an associated deoxyribonuclease activity. No other DNA polymerase activity was detected in cell‐free extracts of U. maydis.
Characterization of Polysomes from Xenopus Liver Synthesizing Vitellogenin and Translation of Vitellogenin and Albumin Messenger RNA's in vitro
Authors: BERRIDGE Michael V. , FARMER Stephen R. , GREEN Christopher D. , HENSHAW Edgar C. , TATA Jamshed R.
Published: March 2005

Conditions are described for the isolation of polysomes from the liver of Xenopus laevis. The method involves homogenization of liver in 0.2 M Tris‐HC1 pH 8.5, treatment with 2 % Triton X‐100 and subsequent sucrose density gradient fractionation of polysomes from a 10000 × g supernatant.

Vitellogenin synthesis was induced in male Xenopus liver by oestradiol treatment. Polysomes were isolated and vitellogenin‐synthesizing polysomes characterized by their association with monospecific 125 I‐labelled rabbit anti‐vitellogenin antibody and by reaction with rabbit anti‐vitellogenin immunoglobulins followed by indirect immunoprecipitation with goat anti‐rabbit antibody.

Changes in liver polysome content following oestrogen treatment of male Xenopus are correlated with the appearance of vitellogenin synthesis using an Organ culture assay.

RNA extracted from livers of oestradiol‐treated male Xenopus and from purified polysomes is shown to code for the synthesis of vitellogenin‐specific immunoprecipitable polypeptides in a rabbit reticulocyte cell‐free protein‐synthesizing System, a major component having a molecular weight of 210000. Xenopus liver RNA is also shown to code for the synthesis of an albumin‐specific immunoprecipitable polypeptide of 74000 molecular‐weight which coelectrophoresed with Xenopus albumin.
New Coenzymically‐Active Soluble and Insoluble Macromolecular NAD + Derivatives
Authors: ZAPPELLI Piergiorgio , ROSSODIVITA Antonio , PROSPERI Giulio , PAPPA Rosario , Luciano RE
Published: March 2005

Reaction in dimethyl sulfoxide of nicotinamide 8‐bromoadenine dinucleotide with the disodium salt of 3‐mercaptopropionic acid afforded nicotinamide‐8‐(2‐carboxyethylthio)adenine dinucleotide. a new NAD+ analogue functionalized at the adenine C‐8 position by an ω‐carboxylic side chain. Carbodiimide coupling of the latter derivative to high‐molecular‐weight water‐soluble (polyethyleneimine, polylysine) and insoluble (aminohexyl‐Sepharose) polymers gave the corresponding macromolecular NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine analogue showed a substantial degree of efficiency relative to free NAD+ with yeast alcohol dehydrogenase (47%) but a considerably lower one with rabbit muscle lactate dehydrogenase (3%); the polylysine analogue showed a low degree of efficiency with both enzymes (5 – 6 %).
Hamster Brown‐Adipose‐Tissue Mitochondria
Authors: NICHOLLS David G.
Published: March 2005

The inner membrane of hamster brown adipose tissue mitochondria possesses a mechanism for the conductance of protons (or hydroxyl ions) and halide anions which may be specifically inhibited by exogenous purine nucleoside di‐ or triphosphates. The mechanism of the nucleotide interaction is examined. The added nucleotides can inhibit the ion conductances without equilibrating with the matrix pools of purine nucleotides. ADP translocation is completely sensitive to atractylate, and no mechanism for GDP translocation could be detected. The nucleotides act on the conductance mechanism without covalent modification. A purine nucleotide binding site is described which is distinct from the adenine nucleotide translocase, does not bind atractylate, has a capacity of 0.7 nmol · mg−1, and affinities, specificities and a pH dependency closely corresponding to the conditions required for the inhibition of the ion conductances. The binding site is not apparent in rat liver mitochondria. A causal relationship is suggested between the occupation of this site by added purine nucleotides, and the inhibition of the ion conductance pathway. The role of the pathway in the physiological control of non‐shivering thermogenesis by the tissue is discussed.
N‐Acetyl‐9‐O‐l‐lactylneuraminic Acid, a New Acylneuraminic Acid from Bovine Submandibular Gland
Authors: SCHAUER Roland , HAVERKAMP Johan , WEMBER Margret , VLIEGENTHART Johannes F. G. , KAMERLING Johannis P.
Published: March 2005

The acylneuraminic acid fraction, obtained on mild acid hydrolysis of glycoproteins from bovine submandibular glands, contains approximately 2 % N‐acetyl‐9‐O‐l‐lactylneuraminic acid. The compound has been isolated and purified by ion‐exchange and cellulose column chromatography. The structure has been elucidated using thin‐layer chromatography, colorimetry, gas‐liquid chromatography/mass spectrometry, periodate oxidation and specific lactate dehydrogenases. An evaluation of the different analytical methods is given.
Differentiation of the Nucleotide Pyrophosphatases of Rat‐Liver Plasma Membrane and Endoplasmic Reticulum by Enzymic Iodination
Authors: BISCHOFF Erwin , WILKENING Jörn , TRAN‐THI Thuy‐Anh , DECKER Karl
Published: March 2005

Enzymic iodination of isolated rat hepatocytes and of rat livers perfusedin situwas used to discriminate between the nucleotide pyrophosphatases of endoplasmic reticulum and of plasma membranes. The location of the latter on the cell surface could also be substantiated by this method. The activity of the microsomal enzyme increased after phenobarbital treatment of the animals. The nucleotide pyrophosphates from both subcellular fractions were solubilized, purified to electrophoretic homogeneity, and their125I content determined. The labelling of the enzyme obtained from plasma membranes was several‐fold higher than that of the nucleotide pyrophosphatase from endoplasmic reticulum. This indicates a bimodal distribution of nucleotide pyrophosphatase in rat liver and the accessibility of the plasma membrane enzyme from the extracellular space.
Histidyl Transfer Ribonucleic Acid Synthetase from Salmonella typhimurium
Authors: DI NATALE Paola , SCHECHTER Alan N. , CASTRONUOVO LEPORE Giuseppina , DE LORENZO Francesco
Published: March 2005

Structural requirements for substrate binding to histidyl‐tRNA synthetase from Salmonella typhimurium have been investigated using ATP analogues. Ki values and the relative binding affinity of the enzyme for these analogues have been determined in the tRNA aminoacylation reaction. The enzyme is highly specific for ATP: no binding was found for GTP, CTP, TTP and UTP, dATP is a very poor substrate for acylation of tRNA, with a Km 40‐fold higher than that of ATP. Binding of adenosine 5′‐triphosphate requires interactions of the amino group of adenosine and the sugar moiety; the 2′ and the 5′ positions of the ribose appear to be essential for recognition; the phosphate groups enhance the binding. AMP is a noncompetitve inhibitor with ATP.

The interaction of histidyl‐tRNA synthetase, a dimeric enzyme, with histidine and ATP was examined by fluorescence measurements at equilibrium and by equilibrium dialysis. Binding with l‐histidine is significantly tighter at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 7.5 by equilibrium dialysis and is 1 mol ATP/mol enzyme and, variably, close to 2 or 1 mol histidine/mol enzyme.
Ion‐Binding to Phospholipids
Authors: HAUSER Helmut , DARKE Arthur , PHILLIPS Michael C.
Published: March 2005

The binding of Ca2+ to monolayers and bilayers of phosphatidylserine has been investigated as a function of pH, ionic strength (NaCl concentration) and Ca2+ concentration using surface and colloid chemical techniques. The molar ratio of lipid to bound calcium decreases to 2 as the Ca2+ concentration is increased to about 0.1 mM. At [Ca2+] > 0.1 mM a 1:1 complex is formed.

The apparent binding constant Ka ranges from about ∼ 106‐−104 l/mol depending on the Ca2+ concentration. After allowing for electrostatic effects and neighbour group interactions, the intrinsic binding constant Ki of the phosphorylserine polar group at pH 7 (I= 0.01 M), where it carries a net negative charge of one, is ∼ 104 l/mol; consistent values for Ki were obtained using several independent approaches. Ka for Ca2+ binding decreases with increasing NaCl concentration because the monovalent cations compete with Ca2++ for the same binding site. Na+ and K+ are equally effective in displacing 45Ca2+ adsorbed to monolayers of phosphatidylserine, both with respect to the kinetics and the equilibrium of the displacement. Ka for the reaction between phosphatidylserine and monovalent cations is about 103‐fold smaller than that of Ca2+. An investigation of the binding of Mn2+ to phosphatidylserine by both surface chemical and nuclear magnetic resonance methods shows that this cation has a similar binding constant to that of Ca2+. The Ca2+ ‐binding capabilities of monolayers containing only carboxyl groups (i.e. arachidic acid) and phosphodiester groups (i.e. dicetyl phosphate) have also been determined; the apparent pK for the –COOH group in monolayers is ⋧ 9 and that for the phosphodiester group is < 4. Since these groups do not retain the same pK values when they are in close proximity in the phosphorylserine group, the relative contributions of the two groups to the binding of Ca2+ to phosphatidylserine, is not obvious.
Transcription of Specific Genes in Isolated Nuclei from HeLa Cells in vitro
Authors: UDVARDY Andor , SEIFART Klaus H.
Published: March 2005

Isolated HeLa cell nuclei were employed to catalyze the synthesis of RNA in vitro. In the presence of low concentrations of α‐amanitin (1 μg/ml), used to suppress the formation heterogeneous nRNA, these nuclei synthesize RNA very efficiently for extended periods of time (at least 60 min) at an elongation rate of about seven nucleotides per second. The product, analyzed on sucrose density gradients and polyacrylamide gels was found to exist of two predominant size classes. Synthesis of the 45‐S ribosomal precursor was completely resistant even to high concentrations of α‐amanitin (150 μg/ ml) and hence was catalyzed by enzyme A (or I). A limited degree of processing of the 45‐S precursor occurred in vitro. In addition, a second RNA class of low molecular weight (4–8 S) was synthesized by HeLa cell nuclei in the presence of 1 μg/ml α‐amanitin in vitro. Analysis on 8% polyacrylamide gels resolved the RNA into four distinct components. Their synthesis was resistant to low (1 μg/ml) but clearly sensitive to high (150 μpg/ml) concentrations of α‐amanitin. Consequently the synthesis of all these small‐molecular‐weight RNA species is catalyzed by RNA polymerase C (or III). For the assessment of the initiation frequency of the individual classes of RNA, a new technique was developed independent of labelling the 5′ end of the RNA molecule with the γ‐phosphate of the initiating nucleotide. It employs the double labelling of an RNA molecule with two different isotopes added sequentially at different stages of completion of the chain. From the incorporation ratio of the two isotopes into a particular class of RNA, conclusions can be drawn concerning their initiation frequency. The results obtained have shown a high reinitiation frequency for the small‐molecularweight RNA species at all stages of the incubation reaction. In contrast, reinitiation of the 45‐S precursor RNA occurs only to a limited extent in isolated HeLa cell nuclei in vitro.