bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydrosoluble Complexes of Sterols, Sterol Esters and Their Precursors from Zea mays L.
Authors: Rohmer Michel , Ourisson Guy , Brandt Roger D.
Published: March 2005

The study undertaken shows the existence of water‐soluble complexes of sterols, sterol esters and their precursors (triterpenes and 4α‐methylsterols) in a higher plant, Zea mays L. The leaves, roots and culture medium were treated separately in order to investigate the role and nature of these complexes. The sterols found in the water‐soluble form represent 0.1–1% by weight of the total sterols in the plant. The composition of the hydrosoluble fraction of the sterols and their precursors is different from that of the lipid‐soluble fraction, cholesterol, sitosterol and isofucosterol being preferentially complexed compared to campesterol and stigmasterol.

This complex was found in the microsomal supernatant after homogenisation and also in the culture medium. The sterols are released from the complex by a treatment with pyrogallol under basic conditions or after repeated extraction with petroleum ether. The complex is precipitated with ethanol but not with trichloroacetic acid. The behaviour of several sterols on treatment with a solution of starch was investigated. Starch was able to complex with a large number of phytosterols unspecifically. The implication of this complex for the transport of sterols, for the biosynthesis of steroids and the ecological impact is discussed.
The Effect of Structural Modifications of ATP on the Yeast‐Hexokinase Reaction
Authors: Hohnadel David C. , Cooper Cecil
Published: March 2005

The specificity of yeast hexokinase for ATP was examined with the aid of a number of synthetic and natural analogs. The main features observed are:

The presence of a 2‐amino group either alone or in combination with a 6‐amino or 6‐hydroxyl group inhibits catalysis.

The replacement of one or both hydrogen atoms of the 6‐amino group with methyl groups does not have a large effect on either binding or catalysis.

The replacement of the nitrogen at position 7 with a carbon atom has virtually no effect.

The movement of the glycosidic bond from the N9 to the N3 position has only a moderate effect on both Km and V.

The presence of both of the 2′‐ and 3′‐cis hydroxyls in the ribose is important for catalysis.

The replacement of ribose with arabinose or glucose produces a large decrease in catalysis. Inhibitor experiments were carried out with the ATP analog containing glucose instead of ribose. It was found to be competitive for ATP and uncompetitive for glucose under the conditions used. These results are consistent with an ordered reaction mechanism.
Composition du suc pancréatique humain
Authors: Clemente François , de Caro Alain , Figarella Catherine
Published: March 2005

The enzyme composition of human pancreatic juice has been studied by immunological techniques, using non‐activated and activated juice.

Antisera were prepared with activated and non‐activated pancreatic juice. Same samples of antisera were adsorbed with human serum. Immunoelectrophoresis was carried out at pH 8.6 and disc immunoelectrophoresis at pH 8.9 and 5.0. Gel‐diffusion media contained benzamidine, to prevent zymogen activation. Precipitation lines were characterized by activating the zymogens on the plates with trypsin, and identifying the enzymes produced using specific synthetic substrates.

Immunoelectrophoresis showed 15–20 antigenic constituents. Although disc immunoelectrophoresis gave slightly different results, those were consistent with the results of immunoelectrophoresis.

The following active enzymes were identified and localized: α‐amylase, lipase and carboxylester hydrolase, which is a lipolytic enzyme structurally and catalytically different from lipase.

Several zymogens were identified: two immunologically distinct trypsinogens, one procarboxypeptidase B, two forms of procarboxypeptidase A, which are immunologically identical, and two proelastases with some common antigenic determinants. After activation, both proelastases showed a weak but different degree of chymotryptic activity. Chymotrypsinogen was localized in two major anionic lines, and one, minor, cationic line. The possibility that one anionic immune precipitate represents a polymer is discussed. No chymotryptic activity was associated with procarboxypeptidase A.

Immunoelectrophoresis of activated pancreatic juice demonstrates electrophoretic and antigenic changes. Activation of one anionic trypsinogen gave rise to cationic trypsin, whereas after activation chymotrypsinogens and procarboxypeptidases A showed no important change.
Incorporation of 3‐Methyl‐2‐benzothiazolone Hydrazone into the Anticodon Loop of tRNAPhe from Yeast
Authors: Freist Wolfgang , Maelicke Alfred , Sprinzl Mathias , Cramer Friedrich
Published: March 2005

The incorporation of 3‐methyl‐2‐benzothiazolone hydrazone into the anticodon loop of tRNAPhe from yeast after excision of the base Y has been studied with respect to concentration, pH and temperature. Model reactions were performed at the monomer level with ribose and ribose 5‐phosphate and at the oligomer level with a tRNAPhe anticodon‐arm dodecanucleotide from which the base Y had been excised. The reaction is easily followed by ultraviolet spectroscopy. The reaction product exhibits weak fluorescence. The covalent attachment of 3‐methyl‐2‐benzothiazolone hydrazone to the nucleotide chain is stable. As shown by nucleoside analysis no side reactions occur and the incorporation of 3‐methyl‐2‐benzothiazolone hydrazone into tRNA is quantitative. After modification tRNAPhe is fully chargeable.
Biological Activities of Lipid A Complexed with Bovine‐Serum Albumin
Authors: Galanos Chris , Rietschel Ernst T. , Lüderitz Otto , Westphal Otto , Kim Yoon B. , Watson Dennis W.
Published: March 2005

Lipid A preparations from the lipopolysaccharides of four Salmonella minnesota R mutants and one Escherichia coli 0100 R mutant were assayed as soluble complexes with bovine serum albumin for lethality in mice, pyrogenicity in rabbits and complement inactivation. Although generally less active than endotoxic lipopolysaccharides, these lipid · albumin complexes nevertheless exhibited strong biological activity. These results indicate that lipid A contains the endotoxic principle of bacterial lipopolysaccharides.
A 13C Nuclear‐Magnetic‐Resonance Study of the Enzyme Cofactor Flavin‐Adenine Dinucleotide
Authors: Breitmaier Eberhard , Voelter Wolfgang
Published: March 2005

An assignment of the 13C‐NMR spectrum of the enzyme cofactor flavin‐adenine dinucleotide (FAD) is given by comparing it with the 13C chemical shifts of the constituents of FAD, i.e. adenosine 5′‐diphosphate, riboflavin 5′‐monophosphate, riboflavin, adenosine and adenine. The 13C‐NMR spectrum of FAD shows three groups of signals:

a) At highest field the resonances of the methyl groups (‐18 to ‐21 ppm) of the isoalloxazine moiety.

b) At lowest field the signals of the carbon atoms of the isoalloxazine ring (‐117 to ‐161 ppm).

c) In the range of ‐47 to ‐88 ppm the resonances of the carbon atoms of the ribose and ribitol residue.
Metal‐Combining Properties of Human Lactoferrin
Authors: Teuwissen Bruno , Masson Pierre L. , Osinski Pierre , Heremans Joseph F.
Published: March 2005

By means of a Spectrophotometric titration method it was found that six of the tyrosyl residues of the metal‐binding proteins lactoferrin and transferrin were titratable by alkali in the metal‐free proteins but not in their iron protein complexes, suggesting the involvement of three tyrosyl groups in each of the two binding sites present in these proteins.

Seemingly discordant estimates of the number of binding tyrosyls were obtained using the difference spectra of the lactoferrin · Tb3+vs lactoferrin, lactoferrin · Fe3+vs lactoferrin, transferrin · Tb3+vs transferrin or transferrin · Fe3+vs transferrin. However, such discrepancies as were found could be explained in the light of the ligand‐field theory and need not invalidate the conclusions based on alkali‐titration data.
Isolation of mRNA from KB‐Cells by Affinity Chromatography on Polyuridylic Acid Covalently Linked to Sepharose
Authors: Lindberg Uno , Persson Torgny
Published: March 2005

Messenger RNA from polysomes of KB‐cells was isolated by affinity chromatography on columns of polyuridylic acid covalently linked to Sepharose. The mRNA molecules were retained by the resin apparently via their poly(A) segments by base pairing to the poly(U). Ribosomal RNA and transfer RNA were not retained by the columns and were thus removed from the mRNA. The mRNA was recovered to an extent of 90% and apparently in intact form.

This method allows studies of mRNA resulting from unabated synthesis and was used here in studies of the size distribution of different classes of cytoplasmic poly(A)‐containing RNA. The presence of poly(A)‐containing RNA in the non‐polysomal fractions of the cytoplasm was demonstrated. This putative mRNA was shown to constitute about 30% of the total cytoplasmic poly(A)‐containing RNA. Also by using the poly (U)‐Sepharose technique it was demonstrated that actinomycin D at low concentration, 0.04 μg/ml, supresses the appearance of mRNA on polysomes to an extent of 50%. This low concentration of the drug was previously though to effect only ribosomal RNA synthesis.
La glycéraldehyde‐3‐phosphate déshydrogénase du latex d'Hevea brasiliensis
Authors: Jacob Jean‐Louis , d'Auzac Jean
Published: March 2005

The cytoplasmic serum of Hevea brasiliensis latex contains two glyceraldehyde‐3‐phosphate dehydrogenases. These enzymes have been isolated and purified. One of them has an absolute specificity for NAD (EC 1.2.1.12); the other utilizes mainly NADP and reacts only slowly with NAD; it is similar to the non‐phosphorylating enzyme discovered by Arnon (EC 1.2.1.9). The NAD‐dependent enzyme has a molecular weight which is analogous to that of the other species of glyceraldehyde‐3‐phosphate dehydrogenases. Its optimum is around 8 and its Km‐values for the substrate, NAD and Pi respectively are: 0.1 mM, 0.12 mM and 2.6 mM.

The NADP‐dependent enzyme is probably composed of 4 subunits and its molecular weight is estimated to be about 200000. Its optimum pH is around 8.6 and it catalyses a reaction that is irreversible. Its Km‐values for the substrate, NADP and NAD respectively are: 1.2 mM, 10 μM and 12 mM.

Influence of some effectors like NADH, NADPH and adenosine phosphates on the enzymatic reaction has been studied. Only NADH has an influence. It inhibits competitively the reaction and the Ki value of the order 13 μM. It appears on the other hand, taking into account the influence of cysteine, that the NADP‐dependent enzyme is more resistant to xodative agents than the NAD‐dependent enzyme. NADP‐dependent enzyme is also inhibited by PO42‐, Mg2+, Ca2+, 3‐phosphoglycerate, glyceraldehyde iodoacetamide and citrate.
Comparison of the Conformation of Mammalian Globins in Solution
Authors: Vodrážka Zdenék , Hrkal Zbyněk , Kodíček Milan B. , Jandová Danuše
Published: March 2005

By studying the oligomeric state, chiroptical properties and the reactivity of sulfhydryl and phenolic groups, we compared the properties of human, horse, bovine, canine, rabbit and rat globins in solution.

The results indicate that, in spite of the differences in the amino acid sequence, all the mammalian globins studied exhibit analogical properties suggesting similar conformation.
Synthesis and Properties of Poly(2‐Methyladenylic Acid)
Authors: Ikehara Morio , Hattori Masao , Fukui Toshikazu
Published: March 2005

Chemically synthesized 2‐methyladenosine 5′‐diphosphate (m2ADP) was polymerized to yield poly(2‐methyladenylic acid), poly(m2A), using Escherichia coli polynucleotide phosphorylase. In neutral solution poly(m2A) exists with a significant stacking of bases as studied by ultraviolet, circular dichroism and thermal melting methods. In a solution of pH 5.0 poly(m2A) formed a double‐stranded protonated structure, which had melting temperatures, Tm of 90°C at 0.15 M salt concentration. Upon mixing with poly(U), poly(m2A) formed a 1:1 complex with Tmof 21°C at 0.15 M salt concentration.

From this and previous findings, i.e. a bulky group at position 2 of the purine distorted the C═O group of the pyrimidine residues at position 2 and inhibited the Watson‐Crick‐type hydrogen bonding, a double‐stranded complex of Hoogsteen‐type for poly(m2A) · poly(U) was predicted.
Temperature‐Sensitive Mutants Affected in DNA Synthesis in Phage ϕ29 of Bacillus subtilis
Authors: Talavera Antonio , Salas Margarita , Viñuela Eladio
Published: March 2005

The DNA synthesis in Bacillus amyloliquefaciens infected with wild‐type phage ϕ29 and with temperature‐sensitive mutants has been studied in the presence of the drug 6‐(p‐hydroxy‐phenylazo)‐uracil. The drug inhibits DNA synthesis in uninfected bacteria while it does not affect DNA synthesis and phage development in ϕ29‐infected bacteria. DNA synthesis was blocked by infection at the restrictive temperature with the mutants F99 and K132, representatives of the two early functions identified so far, as well as with mutant C17, which affects a late function. The remaining mutants, belonging to eight complementation groups, synthesized DNA at the restrictive temperature. The proportion of the DNA resistant to DNAase was different, depending on the mutant.
Lipogenesis in Chicken Uropygial Glands
Authors: Tang Bernard Y. , Hansen Ian A.
Published: March 2005

The chicken uropygial gland secretes diester waxes. The component fatty acids are synthesized by gland tissue, not derived from plasma lipids or microbial sources. Injection of [14C]acetate into the gland in vivo results in labelling of the wax fatty acids and uropygiols. Fatty acid synthesis from acetate in the cytosol requires NADPH and ATP. The pattern of fatty acids produced differs from that synthesized by chicken liver cytosol, especially if propionate is also added. ATP citrate lyase (EC 4.1.3.8) and malic enzyme (EC 1.1.1.40) are very active but are much less affected by fasting and re‐feeding than the liver enzymes.
Interaction of RNA Polymerase from Escherichia coli with DNA
Authors: Dausse Jean‐Pierre , Sentenac André , Fromageot Pierre
Published: March 2005

The influence of phosphodiester bond interruptions on DNA transcription by core and holoenzyme was investigated. Different types of scissions were introduced in T7 DNA using pancreatic, micrococcal and spleen DNAases and sonication. It is shown that only pancreatic DNAase is able to make breaks competent for chain initiation by core enzyme. The competent sites are single‐strand scissions and not unpaired DNA gaps or frayed ends; the presence of a phosphate group at the 5′ or 3′ position is neither required nor inhibitory. Factor sigma restricts the utilisation of DNA scissions as initiation sites and thus exerts a negative control on RNA chain initiation. Evidence is presented that holoenzyme binds to most single‐strand breaks and very tightly to unpaired DNA ends produced by double‐strand breakage. On the other hand RNA polymerase has a lower affinity for natural DNA ends and for new DNA termini produced by sonication. These studies indicate that the utilisation of phosphodiester bond interruptions as initiation sites is modulated by the nature of the DNA interruption, possibly its base sequence environment, and by σ factor. This suggests a possible control of DNA transcription through specific cleavage of the template.
Hybridisation of Aldolase from Drosophila, Blocked at the Active Site, with Native C Aldolase from Calf Brain
Authors: Brenner‐Holzach Olga , Leuthardt Franz
Published: March 2005

Fructose‐1,6‐bisphosphate aldolase from Drosophila melanogaster, inactivated by reducing the Schiff base between the active‐site lysyl residue and the substrate, formed enzymatically active hybrids with native C aldolase from calf brain. The fact that hybridisation takes place indicates that blockage of the active site of Drosophila aldolase with reduced substrate does not significantly change the quaternary structure of the enzyme. Furthermore our results show that each subunit in the tetrameric structure of aldolase makes an independent contribution to enzymatic activity.
Formation and Reduction of Intermediate Acyladenylate by Aryl‐Aldehyde
Authors: Gross Georg G.
Published: March 2005

The enzyme aryl‐aldehyde: NADP oxidoreductase catalyzes a rapid exchange of PPi and ATP. The reaction requires the presence of an aromatic acid and Mg2+ ions. The exchange reaction has a pH‐optimum of approx. 8.5 and is inhibited by NADPH. The inhibition of the exchange reaction by hydroxylamine as well as the enzymic formation of benzoylhydroxamate confirm that an activated intermediate is involved in the reduction of aromatic acids. This compound was identified as acyladenylate which, however, is assumed not to exist in the free state under normal conditions. In a subsequent step the acyladenylate is reduced to aldehyde. This reaction depends on the presence of sulfhydryls and does not require Mg2+ ions. Both the activating and the reductive step are catalyzed by one enzyme.
Fatty‐Acid Elongation in a Mutant of Saccharomyces cerevisiae Deficient in Fatty‐Acid Synthetase
Authors: Orme Thomas W. , Lynen Feodor , Mcintyre Jean , Kühn Lothar , Schweizer Eckhart
Published: March 2005

The incorporation and metabolic alteration of a variety of dietary fatty acids has been studied in wild‐type Saccharomyces cerevisiae and a fatty‐acid auxotroph which carries a genetic defect in its fatty‐acid synthetase complex. This mutant grows when supplemented with a singleven or odd chain‐length fatty acid ranging from 13–17 carbon atoms. Although incapable of fattyacid synthesis from acetate de novo, the mutant yeast still elongates the dietary fatty acids by one or more C2 units. The extent of elongation is dependent on the chain length of the fatty acid included in the growth medium. It is most evident with tridecanoic acid and insignificant with palmitic acid. All saturated fatty acids are desaturated to an appreciable but varying degree to the corresponding monounsaturated acids. No degradation to lower homologues is observed in the wild‐type, nor in the mutant cells. No evidence was obtained to indicate participation of the mutant fatty‐acid synthetase complex in the elongation process described. In wild‐type yeast, intracellular fatty acid biosynthesis is unimpaired by the addition of free fatty acids to the growth medium and apparently proceeds by a much faster rate than the uptake and elongation of fatty acids from the growth medium. A membrane‐bound enzyme analogous to acid : CoA ligase has been isolated from bakers' yeast and its chain‐length specificity studied. The optimal chain length with saturated fatty acids is 15 carbon atoms. Unsaturated fatty acids are generally better substrates. The role of fatty acid activation in determining the rate of fatty‐acid‐dependent yeastmutant growth is discussed.
The Mechanism of Reaction of Methionyl‐tRNA Synthetase from Escherichia coli
Authors: Blanquet Sylvain , Fayat Guy , Waller Jean‐Pierre , Iwatsubo Motohiro
Published: March 2005

The interactions of methionyl‐tRNA synthetase, a tetrameric enzyme, with various ligands involved in the methionine‐activation reaction (methionine and analogues, ATP, AMP, PPi, methioninyl adenylate and methionyl adenylate) were examined by equilibrium dialysis, absorbance difference spectroscopy and fluorescence measurements at equilibrium and in a stoppedflow apparatus.

Two binding sites for methionine and four for ATP were found by equilibrium dialysis. The number of binding sites and the association constants of these ligands for the enzyme were not affected by the presence of Mg2+ or EDTA. From ligand‐induced fluorescence and absorbance variations, two binding sites were measured for methioninyl adenylate, a structural analogue of methionyl adenylate.

The interaction of methionyl‐tRNA synthetase with most of the ligands investigated gave rise to characteristic fluorescence variations of large amplitude. Thus, saturating concentrations of methioninyl adenylate and methionine enhanced the fluorescence emission by 40% and 26%, respectively, while interaction with chemically synthesized methionyl adenylate resulted in 27% quenching. ATP, AMP or PPi, alone or in combinations, did not affect the emission spectrum. However, when ATP was added to enzyme saturated with methionine, the fluorescence level shifted from the initial value of 126% to 82%. Similary, additon of PPi to enzyme saturated with ATP and methionine raised the level of fluorescence from 82% to 112%. In none of the cases investigated did addition of Mg2+ or EDTA affect the final level of ligand‐induced fluorescence modifications. Titration experiments were conducted to measure the association constants of the ligands for the enzyme.

The rates of formation of methionyl adenylate and of reversal of this reaction by PPi were determined by stopped‐flow measurements. Comparison of reaction rates in the presence of Mg2+ or EDTA reveals a dramatic enhancement of the rates of both reactions in the presence of Mg2+.
Inhibition of Q β Replicase by Excess Template
Authors: Kondo Masatoshi , Weissmann Charles
Published: March 2005

Initiation of RNA synthesis by Qβ replicase is inhibited by increasing the molar ratio of Qβ RNA to enzyme beyond about 1.5. In long‐term incubations the inhibition preferentially affects synthesis of plus strands and leads to an accumulation of single‐stranded minus strands. It is postulated that plus strands compete with minus strands for the same template site.
A Myoglobin‐Type Haemoglobin of Chironomus thummi thummi
Authors: Wollmer Axel , Buse Gerhard , Sick Hinrich , Gersonde Klaus
Published: March 2005

A monomeric haemoglobin of the insect Chironomus thummi thummi is described which does not exhibit a Bohr effect. The thermodynamic parameters ΔG, ΔH, and ΔS are given for the oxygen association. There is a difference of 4 cal per degree in the ΔS values for the acid and alkaline pH. The absence of a Bohr effect at physiological temperatures is explained by the fact that there is a point of intersection at 25 °C in the −ΔG versus T plot for acid and alkaline pH. This point is designated the “isoaffinity temperature”. In the case of other monomeric Chironomus haemoglobins a Bohr effect is observable because their isoaffinity temperatures are remote from the physiological temperature range (haemoglobin III = 184 °C, haemoglobin IV = 377 °C).

No conformation isomers can be distinguished by circular dichroism and absorption measurements at 25 °C; isosbestic triangles are not encountered in this case.

Electron spin resonance spectroscopy, however, carried out at 77 K, reveals the existence of at least two conformation isomers distinguished by specific high‐ and low‐spin signals. In case of high‐spin H2O‐Hb(III) the acid conformation is characterized by a rhombic distortion of the intramolecular electric field and strong interaction of the d‐electrons of the iron with the two nuclei of the bound imidazole. Transition to the alkaline conformation is followed by a change to axial symmetry and weak interaction of the d‐electrons with the imidazole.

In the case of OH‐Hb(III) two sets of low‐spin signals describe two conformations with different rhombic symmetry.

By comparing the positions of histidine residues in haemoglobins I, III and IV possible Bohr proton‐binding sites are discussed.
The Membrane Structure Studied with Cationic Dyes
Authors: Dell' Antone Paolo , Colonna Raffaele , Azzone Giovanni Felice
Published: March 2005

Three effects concerning the organic cations, the aggregation, the metachromasy and the pKα shift, have been studied in comparison at high dye concentrations, with natural and synthetic polyanions and with energized submitochondrial particles.

The metachromatic effect has been obtained by increase of the free cationic dye concentration or by interaction of the dye with natural, synthetic polyanions and energized submitochondrial particles. The metachromatic effect is dependent on the pH of the medium, the presence of ionized acidic groups, and the ionic strength of the medium.

The polyanion‐induced metachromasy requires the ionization of the acidic groups of the polyelectrolyte. The metachromasy with neutral red requires an acidic pH if induced by increase of the dye concentration or by chondroitin sulphuric acid, whereas it takes place also at alkaline pH if induced by polystyren sulphonic acid or by energized particles. It is inhibited by increase of ionic strength in the case of agar and chondroitin sulphuric acid, but not in the case of the energized particles or polystyrene sulphonic acid.

Interaction of neutral red with polystyrene sulphonic acid or energized particles results also in a large apparent pKα shift, which represents the mechanism for obtaining the metachromatic effects at alkaline pH. The apparent pKα, as measured from the extinction of the alkaline band is: 6.0 at 400 μM neutral red, 6.7 at 20 μM neutral red, 7.0 with chondroitin sulphuric acid, 7.1 with the deenergized particles, 8.0 with polystyrene sulphonic acid and 8.1 with energized particles. From thermodynamic considerations it is suggested that the pKα shift requires a decrease of the activity coefficient of the dye following the formation of ion pairs with anionic groups of the membrane. The pKα shift may be taken as a tool for discriminating between a dyeinduced and a membrane‐induced metachromatic effect.

A model is proposed for the energized membrane, based upon electrostatic and hydrophobic interactions of the cationic dyes with a layer of oriented nucleophilic sites in an environment of intermediate polarity.
The Iodination of Chymotrypsinogen and Chymotrypsin in Crystal and Solution
Authors: Wright H. Tonie , Weber Bruce H.
Published: March 2005

Single crystals of active and phenylmethanesulfonyl‐treated γ‐chymotrypsin and γ‐chymotrypsin were iodinated. The sites of iodination and their degree of occupancy were determined crystallographically. It was found that the pattern of iodination depends upon the form of chymotrypsin being iodinated, the pH, and whether the enzyme has been irreversibly inhibited. The effect upon γ‐chymotrypsin catalytic activity of the iodination of specific residues was observed toward acetyl tyrosine ethyl ester.

Chymotrypsinogen was iodinated in solution under slightly varying conditions; it was impossible to iodinate chymotrypsinogen crystals in either native or glutaraldehyde crosslinked form. The iodochymotrypsinogens were activated by trypsin under slow and rapid conditions and the catalytic properties of the resulting chymotrypsins examined. The activity of the iodochymotrypsins toward acetyl tyrosine ethyl ester depends upon both the conditions of iodination and those of activation.

The crystallographic results are compared with those obtained previously for γ‐ and α‐chymotrypsins and possible explanations for observed differences are advanced. We propose from the kinetic data that the slow interconversion of α‐ and γ‐chymotrypsins is controlled by the ionization of the tyrosine‐146 sidechain.
Complexes of Aminoacyl‐tRNA Synthetases with tRNAs as Studied by Partial Nuclease Digestion
Authors: Hörz Wolfram , Zachau Hans G.
Published: March 2005

Complexes of tRNAPhe and tRNASer with their cognate aminoacyl tRNA synthetases have been exposed to a number of nucleases under conditions of partial digestion in order to gain information on the topography of the complexes. tRNAPhe was found to be strongly protected by yeast phenylalanyl tRNA synthetase at both nuclease‐sensitive sites, the dihydrouridine loop and the anticodon. tRNASer, on the other hand, was selectively shielded by yeast seryl tRNA synthetase at one of the two nuclease targets, the G‐C‐C‐A terminus, whereas splitting at the other target, the anticodon, was not affected at all by the synthetase.

There was complete specificity of shielding: tRNAs were only protected by their respective synthetases. The complexes were sufficiently stable to allow an analysis of protection not only at pH 5.5 but also at pH 7.0. Phenylalanyl tRNA synthetase protected one mole of tRNAPhe, seryl tRNA synthetase two moles of tRNASer against nuclease attack. Denatured tRNAPhe did not bind to phenylalanyl tRNA synthetase.

Two effects of ligands on complex stability have been observed. The analog phenylalaninol‐AMP increased the protection by phenylalanyl tRNA synthetase of tRNAPhe but not of Phe‐tRNAPhe; ATP abolished the shielding of the G‐C‐C‐A terminus of tRNASer by seryl tRNA synthetase.

A number of tRNAPhe and tRNASer fragments was studied. It was concluded that the terminal 7 nucleotides in tRNAPhe are not required for complex formation. As expected, combinations of homologous tRNA half molecules resembled the intact tRNAs in the degree of protection. When the half molecules were tested separately or in heterologous combinations, phenylalanyl tRNA synthetase afforded some protection for the pG‐half molecule of tRNAPhe and seryl tRNA synthetase for the C‐C‐A‐half molecule of tRNASer.
A Reinvestigation of Shigella flexneri O‐Antigens by Methylation Analysis
Authors: Lindberg Bengt , Lönngren Jörgen , Rudén Ulla , Simmons D. Alastair R.
Published: March 2005

Methylation studies of Shigella flexneri lipopolysaccharides indicate that the primary side chains of the O‐specific regions of these polymers have saccharide repeating units with the structure N‐acetylglucosaminyl‐(1 → 2)‐rhamnosyl‐(1 → 2)‐rhamnosyl‐(1 → 3)‐rhamnose or N‐acetyl‐glucosaminyl‐(1 → 2)‐rhamnosyl‐(1 → 3)‐rhamnosyl‐(1 → 2)‐rhamnose. The repeating units are bound together by (1 → 3)‐linkages.
Isolation of a Glucose‐Binding Lipoprotein from Yeast Plasma Membrane
Authors: Hoeák Jaroslav , KOTYK Arnost
Published: March 2005

Baker's yeast membranes were solubilized with Tween 80, the material was chromatographed on Sephadex G‐150 and hydroxyapatite and finally treated with ammonium sulphate and in an isopycnic gradient of sucrose, to yield a lipoprotein complex which appears homogeneous in disc electrophoresis. The complex binds d‐glucose, d‐xylose, d‐arabinose, l‐sorbose, but not d‐galactose and l‐arabinose, showing thus the specificity of the typical glucose carrier from baker's yeast. The binding is suppressed by dodecylsulphate and by treatment at 90°C, but not by 2,4‐dinitrophenol or N‐ethylmaleimide.

The molecular weight of the protein (per binding site) appears to be about 5000, that of the lipoprotein (from analysis of P and N) about 35000. The lipid part contains phosphatidylcholine, phosphatidylethanolamine and probably lysophosphatidylcholine. There are a minimum of 3 × 106 glucose‐binding sites per cell.
The Presence of an Exonuclease in Highly Purified DNA Polymerase from Bakers' Yeast
Authors: Helfman William B.
Published: March 2005

Two DNA polymerase activities from bakers' yeast can be separated by DEAE‐cellulose chromatography, one of which can be dissociated from its DNAase activity by subsequent chromatography on phosphocellulose. Both of the DEAE‐cellulose polymerases prefer nicked double‐stranded DNA and utilize single‐stranded DNA efficiently only at high ratios of enzyme to template. DNA polymerase activity is completely inhibited by p‐hydroxymercuribenzoate, suggesting that cysteine is essential for activity.

The DNAase has been shown to be coincident with polymerase activity during chromatography. This enzyme hydrolyzes single‐stranded DNA preferentially, releasing deoxymononucleotides as the sole product. The rate of hydrolysis is unaffected by the presence of a 2′,3′‐dideoxy terminus on poly(dT).
Steroid‐Binding Properties of a Plasma‐Membrane Fraction from Rat Liver
Authors: Blyth Carol A. , CLARK Rosemary P. , Freedman Robert B. , HAMMOND Jane , James Derrick W. , RABIN Brian R. , Ridge David , VINTER Jill , Williams David
Published: March 2005

The binding curves of oestradiol and testosterone to plasma membranes from male or female rat livers show no specific sites of high affinity.

Plasma membranes from male and female rat livers show no disulphide interchange activity. It is concluded that no steroid‐promoted association of ribosome with plasma membranes occurs.

Microsomes prepared from a homogenate from which plasma membranes have already been isolated, show a slightly higher concentration of specific high‐affinity steroid‐binding sites than do standard preparations of microsomes. It is concluded that standard microsomal preparations are contaminated only to a slight extent by fragments derived from the plasma membrane.
The Primary Structure of the Major Parvalbumin from Hake Muscle
Authors: Capony Jean‐Paul , PECHÈRE Jean‐François , Rydèn Lars , DEMAILLE Jacques
Published: March 2005

Work on the amino acid sequence of the major parvalbumin from hake muscle has been pursued with the aim of obtaining large fragments and overlapping peptides permitting the tryptic peptides sequenced previously to be ordered and the structure of the N‐terminal portion of the molecule to be established.

Cyanogen‐bromide cleavage at the single methionine and tryptic digestion limited to the single arginine after maleylation were found useful in this respect and, with the help of the previous data, allowed the last 44 residues of the molecule to be placed unambiguously.

Additional information was obtained from a chymotryptic digest of the protein which resulted in almost complete elucidation of the sequence of the first 24 residues and valuable knowledge on two other fragments, 12 and 29‐residues long, respectively.

Application of both limited and extensive peptic‐digestion, the first one supplemented with the diagonal technique for cysteine peptides, permitted the remaining gaps to be filled, yielding an unique sequence of 108 residues now in full agreement with the amino acid composition of the protein.

The location of the three amide groups of this parvalbumin is described and completes the description of its primary structure.
Carbamoylation of Proteins Following Administration to Rats of Carbamoyl Phosphate and Cyanate and Effects on Memory
Authors: Crist Robert D. , Grisolia Santiago , Bettis Carol J. , Grisolia James
Published: March 2005

Both [14C]carbamoyl phosphate and sodium [14C]cyanate injected in rats were extensively incorporated into blood proteins, red cells and tissues, including brain. The rate of cyanate binding to blood protein plateaus 4 h after intraperitoneal administration of [14C]cyanate. The rapid disappearance of free [14C]cyanate was shown to be largely due to protein binding and the release of 14CO2 was found to decrease sharply at 4 h when cyanate binding is essentially complete.

Retention of carbamoylated proteins was measured after injection of [14C]cyanate, and at 110 days, when bound radioactivity in blood protein was negligible, brain protein retained about 50% of the radioactivity found at 6 h.

A dose‐related decrease in learning ability was observed when rats injected with 50, 100 and 150 μmol cyanate per day were tested in a Lashley maze No. III filled with water. This retardation persisted when the animals were retested nine weeks after termination of cyanate injections. When retested at eight months, no retardation could be demonstrated. At this time, only 10% modified proteins should remain in the brain according to the data presented on retention of carbamoylated proteins.

The application of this data to a biochemical model for learning based on synaptic facilitation by modified proteins is discussed, as well as the implied effect on humans treated for sickle‐cell disease by administration of cyanate or carbamoyl phosphate.
Studies on the Apparent Instability of Neurospora Tryptophan Synthase
Authors: Yu Peter‐Hao , KULA Maria‐Regina , Tsai Hsin
Published: March 2005

Neurospora tryptophan synthase prepared by a standard procedure was found to be contaminated with endogenous protease(s). Such a protease is responsible for the apparent instability of the tryptophan synthase. The rate of inactivation of tryptophan synthase was found to be directly proportional to the level of proteolytic activity. The protease is synthesized during all phases of cell growth. However, the proteolytic activity is most pronounced in the aged cultures.

The protease which has been purified by Sephadex G‐200 column chromatography and poly‐acrylamide‐gel electrophoresis exhibits the properties of an endopeptidase. It is relatively insensitive to p‐mercuribenzoate and is insensitive to EDTA. It is very sensitive, however, to the proteolytic inhibitor phenylmethanesulfonyl fluoride.

During the course of this study, a potent protease inhibitor has also been isolated from Neurospora. It is concentrated in a fraction precipitated with ammonium sulfate at 30 to 45% saturation. The isolated inhibitor is heat‐resistant, non‐dialyzable and is excluded by Sephadex G‐25. It strongly inhibits the Neurospora protease but has no effect on trypsin.
Regulation of Yeast Inorganic‐Pyrophosphatase Activity by Divalent Cations
Authors: Baykov Alexander A. , AVAEVA Svetlana M.
Published: March 2005

The stability constants of the magnesium and calcium complexes of inorganic pyrophosphate and the manganese complex of ATP were measured with a specific ion electrode at pH 5.6 and 25°C. These data were used to calculate the concentrations of free metals, free substrates, and the metal · substrate complex in their mixtures. The effect of these variables on the activity of the inorganic pyrophosphatase from baker's yeast was investigated and it was concluded that the active enzyme · substrate complex has at least three sites specific to Mg2+ or Ca2+ and two sites specific to Mn2+ per protein molecule. Calcium inhibits the enzymatic hydrolysis of inorganic pyrophosphate due to displacement of three magnesium ions from the enzyme complex with the substrate.

A simple semi‐automatic analyzer is described which measures inorganic phosphate in solution at concentrations down to 2 μM.
Phoratoxin, a Toxic Protein from the Mistletoe Phoradendron tomentosum subsp. macrophyllum (Loranthaceae)
Authors: Mellstrand S. Tore , SAMUELSSON Gunnar
Published: March 2005

In a simplified procedure for isolation of phoratoxin, the dried plant material (stems and leaves) was extracted with 2% aqueous acetic acid. The extract was treated with polyamide to remove colored impurities and subjected to gel filtration on Sephadex G‐25. The high‐molecular‐weight fraction was chromatographed on SE‐Sephadex, using a gradient elution technique, whereby pure phoratoxin was obtained.

The material was homogeneous by standard criteria of protein purity. The molecular weight, determined in the ultracentrifuge by the sedimentation equilibrium method, is 5000. Phoratoxin has the following amino acid composition (residues/mole): alanine 2, arginine 3, aspartic acid 4, 1/2‐cystine 6, glycine 6, histidine 1, Isoleucine 3, leucine 1, lysine 4, phenylalanine 1, proline 2, serine 5, threonine 5, tryptophan 1, tyrosine 1, valine 1. The molecular weight, calculated from the amino acid composition, is 4884. The material contains no reducing sugars, amino sugars or sialic acids. The LD50, determined by intraperitoneal injection in mice, is 0.57 ± 0.05 mg/kg bodyweight.
The Occurrence of Repetitive Glycopeptide Sequences in Bovine Submaxillary Glycoprotein
Authors: Pigman Ward , MOSCHERA John , Weiss Michael , TETTAMANTI Guido
Published: March 2005

Bovine submaxillary mucin, after removal of its sialic acid component, is cleaved by trypsin into two principal glycopeptide fractions; their final yield corresponds to 70% by weight of the original mucin and 85% of the original amount of hexosamine in the mucin. These glycopeptides have amino acid and carbohydrate compositions that are very similar to those of the original and desialyzed mucin. The main glycopeptide contains about 28 amino acids; the second one seems to be a covalently linked trimer of the former and is resistant to further action by trypsin. The protein core of bovine submaxillary mucin is composed mainly of several hundred covalently bound sequences of these glycopeptides. These results are compatible with those reported by Downs and Pigman (1969) for the products obtained by chemical cleavage of this mucin.
Phosphate Transfer between 2,3‐Bisphosphoglycerate and Monophosphoglycerate Catalyzed by Bisphosphoglycerate Phosphatase
Authors: de Verdier Carl‐Henric , GROTH Torgny L.
Published: March 2005

2,3‐Bisphosphoglycerate phosphatase from human erythrocytes was extensively purified with the use of various chromatographic procedures and electrofocusing. All detectable phosphoglyceromutase activity was removed as tested with an optical assay at pH 7.6.

At the pH optimum of the phosphatase reaction (pH 6.5) the enzyme transfers phosphate groups from 2,3‐bisphosphoglycerate to monophosphoglycerates (3‐phosphoglycerate or 2‐phosphoglycerate). The transfer is more than hundred times faster than the hydrolyzing activity.

Chloride and 2‐phosphoglycolate stimulate the hydrolyzing but not the transferring activity.

Data from seven experiments with differently labelled phosphoglycerates were analyzed in terms of rate constants defined by a mathematical model for the different transfer reactions.

With the direction of phosphate transfer expressed from 2,3‐bisphosphoglycerate to mono‐phosphoglycerate the constants were estimated to have the following values (ml · min−1·μmol−1): C‐3 → C‐2 0.006 ± 0.001, C‐2 → C‐2 0.024 ± 0.003, C‐3 → C‐3 0.009 ± 0.006 and C‐2 → C‐3 0.034 ± 0.008.

The values indicate that the velocity of the transfer from C‐2 in 2,3‐bisphosphoglyerate to the vacant carbon atom in 2‐phosphoglycerate is about the same or possibly somewhat higher than the velocity of the transfer to 3‐phosphoglycerate.

The transfer velocity from 2,3‐[3‐32P]bisphosphoglycerate to 3‐phosphoglycerate is much lower. Due to the experimental conditions the results do not allow determination of the velocity of the transfer from C‐3 to 2‐phosphoglycerate with any high degree of precision. The C‐3 to C‐2 transfer however, is slower than the transfer from C‐2 in 2,3‐bisphosphoglycerate.
Hormonal Interactions at the Molecular Level
Authors: Camier Maryse , ALAZARD Robert , Cohen Paul , PRADELLES Philippe , Morgat Jean‐Louis , FROMAGEOT Pierre
Published: March 2005

Bovine neurophysins I and II have been obtained in a highly purified state using isoelectric focusing. The interactions of oxytocin and [8‐lysine]vasopressin with these proteins have been investigated by thin‐film equilibrium dialysis using highly radioactive hormones retaining their full biological activities.

Binding of both oxytocin and [8‐lysine]vasopressin to neurophysins I and II as a function of pH leads to typical “bell‐shaped” curves. The patterns obtained indicate functional differences in the binding of oxytocin to neurophysin I compared with neurophysin II. No significant change was detected in the case of [8‐lysine]vasopressin.

Scatchard plots indicate that both hormones bind neurophysins I and II with similar affinities. They demonstrate that at pH 5.7 one oxytocin molecule and two [8‐lysine]vasopressin molecules can be bound per neurophysin I or II monomer.

Ca2+ and Mg2+ had no effect on the binding process of either [8‐lysine]vasopressin or oxytocin. Iodination on the tyrosyl ring of [8‐lysine]vasopressin and oxytocin resulted in the suppression of their binding ability.

Competition experiments between oxytocin and vasopressin show that each compound inhibits the binding of the other peptide in a non‐competitive fashion.
Aggregation of Chlorophylls in vitro
Authors: Dijkmans Huguette
Published: March 2005

In water‐methanol solutions, chlorophyll a forms aggregates absorbing at 745 ± 10 nm or at 710 ± 10 nm. In all cases, chlorophyll also gives a form with no absorption at wavelengths between 350 and 1000 nm, which is considered as an important aggregate of chlorophyll molecules (sieve effect). It is assumed that, in the immediate environment of the chlorophyll a molecules, the water/methanol ratio is the determining factor of the type of aggregate obtained and that water and chlorophyll compete for the “aggregation sites” of chlorophyll.
Aggregation of Chlorophylls in vitro
Authors: Dijkmans Huguette , AGHION Jacques
Published: March 2005

Aqueous methanol promotes chlorophyll a aggregation and also that of chlorophyllide a, pheophytin a and pheophorbide a. Comparing the aggregation features of all these molecules leads to the assumption that the phytyl chain of chlorophyll a and its Mg atom play antagonist roles in the association of chlorophyll molecules in polar solvents.
Analysis of Bacteriophage‐M 13‐DNA Replication in an Escherichia coli Mutant Thermosensitive in DNA Polymerase III
Authors: Staudenbauer Walter L. , OLSEN William L. , Hofschneider Peter Hans
Published: March 2005

The various stages of M 13 DNA replication were investigated in an Escherichia coli dnaE mutant thermosensitive in DNA polymerase III after a shift to the restrictive temperature (45°C). The following results were obtained. (1) DNA polymerase III is not needed for the conversion of the infecting single‐stranded phage DNA into the parental replicative form. (2) Semi‐conservative replicative form replication is completely blocked in the absence of a functional DNA polymerase III. (3) Synthesis of phage‐specific DNA continues after a temperature shift late in the infection. However, due to an inherent thermosensitivity of M 13 single‐stranded circular DNA production no free single strands are detected.
Studies on Energy‐Linked Reactions
Authors: Avner Philip R. , GRIFFITHS David E.
Published: March 2005

The genetics of the two major phenotypic classes of oligomycin‐resistant yeast mutants have been investigated. All the class II mutants (oligomycin specific) tested show typical cytoplasmic inheritance and the determinants appear located on mtDNA. Evidence is presented that this type of resistance can be conferred by at least two distinct groups of nonallelic cytoplasmic determinants. The class I mutants which show cross resistance to a variety of inhibitors and uncouplers of energy conservation reactions exhibit behaviour on genetic analysis which is not fully understood. Tetrad analysis indicates that a nuclear gene is concerned but many anomalous observations, discussed here, suggestive of the involvement of a cytoplasmic element remain to be explained.
Identification of Polysomes Synthesizing Thyroglobulin
Authors: Assart Gilbert V , DUMONT Jacques E.
Published: March 2005

In preliminary experiments, we have shown that membrane‐bound thyroid ribosomes specifically synthesize thyroglobulin. This article presents more precise characterization of the thyroglobulin‐synthesizing polysomes.

A population of heavy membrane‐bound polysomes has been isolated from horse thyroid in the detergent‐treated pellet of centrifugation at 27000 ×g.

Immunoprecipitation experiments performed with labelled nascent peptides have revealed that this class of polysomes is specifically engaged inthyroglobulin synthesis.

The pattern of [3H]uridine incorporation into these polysomes suggest that the thyroglobulin template has a very slow turnover. This is further supported by the unique resistance of these polysomes during long‐time incubations of thyroid slices with actinomycin D.
Demonstration of Two Types of Proteins Synthesized in Isolated Rat‐Liver Mitochondria
Authors: Kadenbach Bernhard , HADVÁRY Paul
Published: March 2005

Isolated rat‐liver mitochondria incorporate various labeled amino acids into two types of proteins. About one third of the incorporated radioactivity is soluble in chloroform—methanol (2:1, v/v), the other part is insoluble.

The incorporation into the chloroform‐methanol‐soluble proteins is sensitive to chloramphenicol and ethidium bromide, but insensitive to cycloheximide.

The rate of incorporation into the chloroform‐methanol‐soluble proteins is different from that into the insoluble proteins, depending on the time of incubation, the intactness of the mitochondria and the sucrose concentration of the incubation medium.

The possibility of the existence of two different types of protein synthetic systems in mitochondria is discussed.