bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
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.
UDP‐glucuronyltransferase in Perfused Rat Liver and in Microsomes
Authors: ABOU‐EL‐MAKAREM Mahmud M. , BOCK Karl Walter 
Published: March 2005

In perfused rat liver and in fistula rats the formation of bilirubin conjugates was studied after labeling with [14C]bilirubin,5‐amino [14C]levulinic acid and [14C]hemin. The latter two compounds were used to study heme degradation to bilirubin from intrahepatic and extrahepatic sources, respectively. Bilirubin glucuronides were the major conjugates in fistula bile. In liver perfusion bile the proportion of non‐glucuronide conjugates was increased. After a high dose of hemin (2.5 gmol) bilirubin glucuronides were decreased compared with other bilirubin conjugates both in fistula bile and in liver perfusion bile. In addition green pigments were formed. These alterations were reversed in chronically hemin‐treated rats in which heme oxygenase had been induced.The interference of UDP‐glucose and UDP‐glucuronic acid with bilirubin glucuronidation and glucosidation was studied in liver microsomes. UDP‐glucose did not affect bilirubin glucuronidation in native microsomes in which UDP‐glucuronyltransferase activity is constrained. When this constraint was released by various treatments altering membrane structure UDP‐glucose markedly inhibited bilirubin glucuronidation. However, under these conditions bilirubin glucosidation was unaffected by UDP‐glucuronic acid.The results suggest that the release of the constraint of UDP‐glucuronyltransferase in vivo may lead to a decrease of the proportion of bilirubin glucuronides to other bilirubin conjugates in bile.