bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of Animal Neurotoxins
Authors: Miranda François , Kupeyan Charles , Rochat Hervé , Rochat Catherine , Lissitzky Serge
Published: March 2005

Using the same method of purification of small molecular weight proteins worked out to obtain pure scorpion neurotoxins, four neurotoxins have been isolated from the venom of the cobra Naja haje.

N. haje venom from the Miami Serpentarium contains two neurotoxins (I and II) whereas the venom of the same species obtained from the Institut Pasteur contains three neurotoxins (I, II′ and III). Toxins I, II and II′ consist of 61 amino acid residues and toxin III of 71. All are basic proteins containing four or five disulphide bridges. It is likely that the difference observed in neurotoxin composition of N. haje venoms from two different sources has a taxonomical counterpart, each venom originating from a different sub‐species.
Phylogeny of the Neurohypophysial Hormones
Authors: Acher Roger , Chauvet Jacqueline , Chauvet Marie‐Thérèse
Published: March 2005

The neurohypophysial hormones of three species of birds belonging to three different families: the chicken Gallus gallus (Phasianidae), the goose Anser anser (Anatidae) and the turkey Meleagris gallopavo (Meleagridae) have been isolated and characterized by amino acid composition, chromatographic and electrophoretic migrations and pharmacological properties. Mesotocin ([Ile8]‐oxytocin) and arginine vasotocin ([Arg8]oxytocin) have been identified in each case. Birds seem therefore to have the same neurohypophysial hormones as amphibians and reptiles but to differ on the one hand from fishes which have not mesotocin except the lungfishes akin to amphibians, and on the other hand from mammals which have oxytocin and arginine vasopressin or lysine vasopressin. The neurohypophysial hormones have displayed a peculiar stability during the evolution of the tetrapodes since three successive classes, Amphibia, Reptilia and Aves, have the same active principles. A double shift mesotocin‐oxytocin and arginine vasotocin‐arginine vasopressin has occurred between mammalian reptiles or primitive mammals and modern mammals. Each change involves a single amino acid substitution, in position 8 for the first and 3 for the second. In each case the substitution occurs between two hydrophobic residues: leucine for isoleucine in the transition mesotocin‐oxytocin and phenylalanine for isoleucine in the transition arginine vasotocin‐arginine vasopressin. There is a contrast between the relative similarity of the known biological properties of mesotocin and oxytocin and the great stability of each hormone in a given zoological group, stability which seems hardly explained by selection pressure.
Chromatographic Separation of Membrane Proteins on Lipophilic Ion Exchange Resins
Authors: Weiss Hanns , Bücher Theodor
Published: March 2005

Aliphatic amines of increasing chainlength (butyl, capryl, lauryl, and palmityl) and increasing degree of unsaturation (stearyl, oleyl, and linoleyl) were bound by amide bonds to polyacrylic acid resin. Mitochondrial membrane proteins solubilized with cholate and deoxycholate were chromatographically separated on such resins.

The separation efficiency as measured by the number of protein fractions and their specific cytochrome content was highest with capryl or linoleyl resins while the free polyacrylic acid resin or the stearyl resin proved to be the most ineffective ones.

These effects are discussed in terms of free thermal mobility of the aliphatic chains necessary for hydrophobic interaction with the membrane proteins.
Influence of Bile Acids on the Activity of Rat Liver 3‐Hydroxy‐3‐methylglutaryl Coenzyme A Reductase
Authors: Hamprecht Bernd , Nüssler Christa , Waltinger Gudrun , Lynen Feodor
Published: March 2005

The rate limiting enzyme of cholesterol biosynthesis, 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, is found in the microsomal fraction of a liver homogenate. It is markedly inhibited in vitro by the cholesterol metabolites cholate, deoxycholate, chenodeoxycholate and their taurine conjugates only at unphysiologically high concentrations.

The inhibition by taurochenodoxycholate is non‐competitive and only partially reversible. Therefore, the effect of bile salts in vitro is interpreted as a non‐specific detergent effect.

Feeding of cholic acid in the diet prevents the diurnal rhythm of the activity of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase. The activity decreases with increasing time of feeding of cholic acid.

The specific activity of the enzyme decreases with increasing age of the animal.

It is concluded that the effect of bile salts in vitro on this enzyme cannot account for their effect in vivo. Rather, it is suggested that in vivo bile salts interfere with the de novo‐synthesis and/or the degradation of the enzyme.
Influence of Bile Acids on the Activity of Rat Liver 3‐Hydroxy‐3‐methylglutaryl Coenzyme A Reductase
Authors: Hamprecht Bernd , Roscher Rudolph , Waltinger Gudrun , Nüssler Christa
Published: March 2005

Cholic acid feeding strongly depresses the activity of the rate limiting enzyme of cholesterol synthesis in liver 3‐hydroxy‐3‐methylglutaryl CoA reductase. Experiments were undertaken to decide between (a) a direct influence of the bile acid itself in liver or (b) an indirect effect of the bile acid by improving the intestinal resorption of cholesterol, which itself then would depress the activity of hepatic 3‐hydroxy‐3‐methylglutaryl CoA reductase.

For this purpose lymph fistula rats were fed with cholic acid. Compared with the control animals, which did not obtain cholic acid, the experimental groups showed strong depression of 3‐hydroxy‐3‐methylglutaryl CoA reductase activity. This was only observed with animals killed during the dark period, i.e. when the rhythmically changing activity of the enzyme was high.

A partial depression of the enzyme by fasting can be excluded, because animals fed with or without cholic acid, respectively, ingest the same amount of food.
Steroids in Germfree and Conventional Rats
Authors: Eriksson Håkan
Published: March 2005

Gas chromatography‐mass spectrometry and radio‐gas chromatography were used to identify metabolites of [14C]corticosterone and [14C]pregnenolone in faeces and urine from germfree and conventional male rats. The metabolites were generally more polar than those in female rats and the main part of those which could be identified were found in the free steroid fraction. In the conventional male rats a number of pregnane‐3,11,16,20,21‐pentol isomers constituted the major corticosterone metabolites identified both in urine and in faeces. In addition 3α(and 3β),20β‐dihydroxy‐5α‐pregnan‐11‐one and 5α‐pregnane‐3α(and 3β),11β,20β,21‐tetrol were excreted in faeces. In the germfree male rats, minor amounts of some C19O3 and C21O3 steroids were excreted as monosulphates in faeces. However, 5α‐pregnane‐3α(and 3β),11β,21‐tetrols were the predominant steroids in this fraction. These compounds also constituted the major part of steroids identified in the disulphate fraction, which also contained 3α(and 3β),11β,21‐trihydroxy‐5α‐pregnan‐20‐one. All radioactive steroids excreted in the urine of germfree male rats were unconjugated. Although none of these compounds could be fully identified evidence was obtained for the presence of several C19O5, C21O4, C21O5, C21O6 and C21O7 steroids.

The findings are discussed in relation to previous information on the steroid metabolizing activities of the intestinal flora and sex differences in the metabolism of steroids in rats.
The Reduction of Diacetyl and Acetoin in Aerobacter aerogenes
Authors: Bryn Klaus , Hetland Øyvind , Størmer Fredrick C.
Published: March 2005

The reduction of diacetyl and acetoin has been shown to be catalyzed by one enzyme in Aerobacter aerogenes. The ratio of the two activities remains constant during purification into homogeneity, and the two activities appear in the same amount whne the growing cells ae exposed to acetate.
The Kinetics of Light Induced Carotenoid Changes in Rhodopseudomonas spheroides and their Relation to Electrical Field Generation across the Chromatophore Membrane
Authors: Jackson J. Barry , Crofts Antony R.
Published: March 2005

The shift of the carotenoid absorption spectrum in chromatophores prepared from Rhodo pseudomonas spheroides induced by different types of actinic illumination has been resolved into several distinct, kinetic phases. A saturating 20 ns pulse from a Q‐switched ruby laser elicited a spectral change more rapid than the response time of the mesuring apparatus (1 μs). A rapid change 10–20% greater than this was produced during a saturating 200 μs xenon flash. Additional slower phases (half‐resetimes approximately 300 μs and 5 ms) could be distingushed after the xenon flash. Continuous illumination produced a rapid change of similar extent to that produced by xenon flash but with a risetime limited by the light intensity, and an equally extensive slow change of half‐risetime approximately 83 ms. During prolonged illumination the carotenoid shift decayed by about 50% with an approximate half‐decay time of 30 s.

The effects of certain electron‐transport inhibitors, uncoupling agents, ion‐transporting antibotics and permeant anions on these phases and upon the decay after illumination have been investigated. None of these agents markedly affected the rapid phase of the shift induced by flash illumination. The relation of this phase to membrane potential was indicated by the fact that its decay was stimulated during electrophoretic ion flux. These observations are consistent with the suggestion tht the rapid phase of the carotenoid shift is in response to separation of charge across the chromatophore membrane, between reaction cente bacteriochlorophyll and the primary acceptor. The effects of these specific chemical modifies on the slower pahses of the carotenoid shift were complex. They are discussed in terms of a model in which thes phases reflect the generation of a membrane potential as a result of electrogenic oxidation‐reduction processees. Antimycin A partly inhibited electron flow through such an electrogenic site(s). Valinomycin delayed the formation of the membrane potential by catalysing an initial, electrophoretic K+ efflux. Uncoupling agents causeed a partial but permanent collapse of the electrical field. Permeant anions produced a slight lowering of the steady‐state electrical field by raising the membrane conductivity.

The contribution of the membrane potential to the high‐energy state leading to ATP synthesis is discussed.
Evidence of an Enzymatic Degradation of Insulin in Blood in vitro
Authors: Brodal Björn P.
Published: March 2005

Haemolyzed red cells from human and rat blood possess the ability to split enzymastically the insulin molecule into its A and B chain without further degradation.

The splitting of insulin incubated with different blood preparations, has been studied as a function of the degree of haemolysis, by the use of a trichloroacetic acid precipitation technique using 125I‐labelled insulin. Kinetic data are obtained using haemolysate as crude enzyme.
On the Conversion of Cholestanol into Allocholic Acid in Rat Liver
Authors: Björkhem Ingemar , Gustafsson Jan
Published: March 2005

The early steps in the conversion of cholestanol into allocholic acid were studied in homogenates of rat liver. The 20000 ×g supernatant fluid was found to catalyze 7α‐hydroxylation of cholestanol. Under the same conditions 7α‐hydroxylation of 5α‐cholestan‐3α‐ol and 5α‐cholestan‐3‐one was insignificant. The microsomal fraction fortified with NAD catalyzed oxidation of 5α‐cholestande‐3β,7α‐diol into 7α‐hydroxy‐5aL‐cholestan‐3‐one. The 100 000 ×g supernatant fluid catalyzed reduction of 7α‐hydroxy‐5α‐cholestan‐3‐one into 5α‐cholestane‐3α,7α‐diol. The same reaction was effected by a partially purified 3α‐hydroxysteroid dehydrogenase from rat liver in the presence of NADPH. The microsomal fraction fortified with NADPH catalyzed 12α‐hydroxylation of 5α‐cholestane‐3α, 7α‐diol at a rate about six times faster than that with either 5α‐cholestane‐3β,7α‐diol or 7α‐hydroxy‐5α‐cholestan‐3‐one. In rats with a biliary fistula 5α‐cholestane‐3β,7α‐diol and 5α‐cholestane‐3α,7α‐diol were converted into allocholic acid. The results indicate that the main pathway for the conversion of cholestanol into allocholic acid involves the following steps: cholestanl → 5α‐cholestane‐3β,7α‐diol → 7α hydroxy‐5α‐cholestan‐3‐one → 5α‐cholestane‐3α,7α‐diol → 5α‐cholestane‐3α,7α,12α‐triol → allocholic acid.
Formation of the Parasporal Inclusion of Bacillus thuringiensis
Authors: Somerville Hugh J.
Published: March 2005

Under given experimental conditions, a reproducible sequence of biochemical events can be measured during sporulation in Bacillus thuringiensis. Electron microscope studies reveal that, during its formation, the crystalline inclusion of Bacillus thuringiensis is closely associated with the exosporium. This association persists through several serial sections and can also be demonstrated in a mutant strain which is biochemically blocked soon after crystal formation.

Two asporogenic crystalliferous strains produce a toxin active on larvae of Plutella maculipennis whereas two sporogenic, acrystalliferous strains are not toxic.

Data from immunological assays and from pulse‐chase and label‐chase experiments with [14C]leucine indicate that the crystal protein is synthesized at the time of appearance of crystal antigens. It is suggested that the crystal is synthesized and assembled on the exosporium.
Characterization of the Complex Containing Rapidly Labelled RNA in EDTA‐Treated Polyribosomes from Rat Liver
Authors: Olsnes Sjur
Published: March 2005

Polysomes were isolated from livers of rats sacrificed 30 min after an intraperitonal injection of [3H]orotic acid. The labelled material liberated from the polysomes upon treatment wth EDTA sedimented considerably more rapidly than did purified RNA from the same preparation.

CsCl‐gradient centrifugation of the labelled material and sedimentation studies of the purified labelled RNA indicated that the sedimentation rate of the rapidly labelled material was correlated neither to the protein content nor to the length of the ribonucleoprotein strand.

The rapidly labelled material liberated from unfragmented polysomes banded in a CsCl‐gradient as a single peak. When the polysomes were fragmented by mild RNase treatment prior to the addition of EDTA, two peaks appeared, one with buoyant density of 1.43 and another one with the same buoyant density as that of the 30 S ribosomal subunit (1.54), indicating that it represented material bound to the subunit. After extensive fragmentation most of the radioactive material banded at 1.43 and this was considered to represent ribonucleoprotein fragments with no particle attached.

The labelled material with buoyant density 1.43 sedimented slowly, whereas the materila that banded with the 30 S ribosomal subunits sedimented together with the ribosomal subunits.

The rapidly labelled material from polysomes treated with deoxycholate had a buoyant density which was slightly higher than that of the small ribosomal subunit, but lower than the buoyant density of the big subunit. After RNase‐treatment of the deoxycholate‐treated polysomes the liberated ribonucleoprotein fragments has a higher bouyant density than that of either ribsomal subunit, indicating that protein had been removed from the ribonucleoprotein strand.

The data confirm that the rapidly labelled RNA is present in polysomes as a ribonucleoprotein strand of high protein content. The results indicate that after treatment of the polysomes with EDTA this strand remains bound to one or a few particles (presumably 30 S ribosomal subunits) which can be detached by mild RNase treatment.
Structure primaire de la caséine β bovine
Authors: Ribadeau Dumas Bruno , Grosclaude Francçois , Mercier Jean‐Claude
Published: March 2005

In a previous communication [1] we have reported the isolation and composition of the tryptic and ‘CNBr’ peptides that account for the complete peptide chain of the bovine β‐casein A2. These peptides had been respectively numbered T1 to T14 and CN1 to CN7.

The positioning of peptidde T1 at the amino terminus of the protein was elucidated by comparing the results of Edman degradations carried out on this peptide with those obtained by Kalan et al. [2] for the whole protein molecule. Peptide CN1, which gave peptide T1 by tryptic hydrolysis, accounts for the 92 amino‐terminal residues of β‐casein A2.

Similarly peptide T4 is carboxyl terminal since it does not contain any lysyl or arginyl residue. Furthermore the amino acids liberated from this peptide by carboxypeptidase A are those obtained from the whole protein by Peterson et al. [3]. Peptide CN7, which has the same carboxyl terminal sequences, accounts for the 24 carboxyl terminal residues of the protein.

The comparison of the fragments obtained by tryptic hydrolysis from the intact β‐casein A2 moelcule and from the ‘CNBr’ eptides have allowed to locate all these ‘CNBr’ peptides, and to assign the correct position of the tryptic fragments from CN2, CN4, CN5, CN6 and CN7, which contain two, one or none arginyl or lysyl residues.

The alignment of the tryptic fragments from CN1, which contains six arginyl or lysyl residues, was elucidated by taking use of some previously obtained pptides resulting from incomplete tryptic clavages of the whole β‐casein A2 molecule.

Finally the tryptic peptides from CN3, which contains three Lys or Arg residues, were positionned after analysis of fragments liberated from this ‘CNBr’ peptide by hydrolysis with thermolysin.

The order of the tryptic and ‘CNBr’ peptides in the β‐casein A2 can then be written: H‐T1‐T9‐T11‐T3‐T2‐T10‐T6‐T12‐T7‐T14‐T5‐T8‐T13‐T4‐OH, and H‐CN1‐CN5‐CN6‐CN2‐CN4‐CN3‐CN7‐OH.
Studies of the Energy‐Transfer System of Submitochondrial Particles
Authors: Nordenbrand Kerstin , Ernster Lars
Published: March 2005

Submitochondrial particles elicit a fluorescence of 8‐anilino‐naphthalene‐1‐sulfonate which is increased when the particles are brought into the energized state.

Various parameters of the anilino‐naphthalene sulfonate response were investigated in “EDTA particles” from beef‐heart mitochondria in the energized and non‐energized states. The results reveal striking differences between teh two types of anilino‐naphthalene sulfonate response in several respects, including the effects of pH and ionic strength, the number of binding sites, the dissociation constant, and the quantum yield.

Conditions are established which result in a maximal ratio of energy‐dependent to non‐energy‐dependent anilino‐naphthalene sulfonate response and a virtually linear relationship of both types of response to particle concentration. It is shown that, under such conditions, there is a linear inverse relationship between the steady state of energy‐dependnet anilino‐naphthalene sulfonate response and the respiratory rate of the particles as measured with succinate as substrate in the presence of oligomycin and increasing concentrations of an uncoupler. Extrapolation of this straight line to respiratory rate = 0 allows an estimation of the extent of anilinonaphthalene sulfonate response at complete respiratory control, i.e., in the maximally energized state.

Experiments with different substrates and electron‐transport inhibitors reveal that the linear relationship between the extent of energy‐dependent anilino‐naphthalene sulfonate response and the respiratory rate in the presence of increasing concentrations of uncoupler holds only when the cytochrome b‐c1 step of the respiratory chain is rate‐limiting. The available evidence indicates that the energy‐dependent anilino‐naphthalene sulfonate response originates mainly or exclusively from a restricted region or locus of the membrane, which is closely related to energy‐coupling site II of the respiratory chain.

With ATP as the source of energy, the anilino‐naphthalene sulfonate response increases with low, and decreases with high, concentrations of oligomycin, in a fashion similar to that earlier found for oxidative phosphorylation and its reversal.
Effect of Glucuronides on Glucuronide Biosnthesis
Authors: Hänninen Osmo , Marniemi Jukka
Published: March 2005

1‐Naphthyl and phenolphthalein glucuronides were found to be potent inhibitors of p‐nitro‐phenyl glucuronide synthesis catalyzed by rat liver microsomal fraction. They did not compete with theaglycone, p‐nitrophenol, but with glucuronyl donor, UDPglucuronic acid. The free aglycones, 1‐naphthol and phenolphthalein, were, however, more powerful inhibitors. They competed with p‐nitrophenol andnot with UDPglucuronic acid.Also the o‐aminophenyl glucuronide synthesis in rat liver slices was inhibited by 1‐naphthyl and phenolphthalein glucuronides and free 1‐naphthol.

The results indicate that the glucuronyl residue transfer to the aglycone totally block the affinity of the enzyme for the compound, although it is reduced. Glucuronides and their aglycones are apparently bound by different sites in the enzyme and glucuronides attach to UDPglucuronic acid binding site.
Two types of Pyruvate Kinase in Mucor rouxii
Authors: Terenzi Héctor F. , Roselino Eduardo , Passeron Susana
Published: March 2005

Extracts of mycelium of Mucor rouxii have been shown to contain two forms of pyruvate kinase named Type I and II. They can be separated by DEAE‐cellulose chromatography and by electrophoresis in polyacrylamide gel.

Extracts from ungerminated sporangiospores or yeast‐like cells contained only the Type I form. Pyruvate kinase Type II appears during aerobic germination of spores or during conversion of yeast‐like cells to mycelium.

Both pyruvate kinases are similar in their molecular weight, pH range of activity, Km with respect to substrates and their response to activators (Mn++ and fructose 1,6‐diphosphate).
Chemical Fixation of Enzymes to Cyanogen Halide Activated Polysaccharide Carriers
Authors: Axéan Rolf , Ernback Sverker
Published: March 2005

Water‐insolubel chymotrypsin, trypsin and papain have been synthesized by covalent fixation of the enzymes to beads of cross‐linked dextran (Sephadex®), beads of agarose, and cellulose powder. The fixation procedure involves two simple steps: (a) activation of the polysaccharide polymer by a cyanogen halide; (b) coupling of the protein to the activated polymer in slighly alkaline medium. The activation process can be controlled so as to retain certain useful or essential properties of the carrier material, such as particle shape and swelling capacity. The enzyme conjugates of Sephadex and agarose are therefore especialy suitable for use in the column format. The coupling process is so mild that a large portion of the inherent enzymic activity is conserved. However, the steric properties and other features of the carrier framework often prevents full manifestatuion of the fixed activity. Catalysts that are highly efficient toward small substrates are easily obtainable. Teh agarose‐bound enzymes can even exhibit high activities toward large proteins. Conjugates of the latter type should therefore be particularly useful for the digestion of prtoeins.
Structure of Pig Skin Dermatan Sulfate
Authors: Malmström Anders , Fransson Lars‐Åke
Published: March 2005

The presence of sulfated uronic acid residues in dermatan sulfate has been known for some time. In view of the copolymeric nature of pig skin dermatan sulfate, it was of interest to see whether the extra sulfate groups were attached to iduronic acid or glucuronic acid residues.

Hyaluronidase‐degraded dermatan sulfate was fractionated on Dowex‐1 to yield fractions of varying sulfate content. It was observed that the degree of sulfation was invesely proportional to the glucuronic acid content. Oversulfated dermatan sulfate was subsequently treated with chondroitinase‐AC to yield a completely glucuronic acid‐free polymeric product still bearing all the extra sulfate groups. More direct evidence for the presence of sulfated induronic acid moieties was obtained by Smith degradation of oversulfated, chondroitinse‐AC treated dermatan sulfate. A fast‐moving component isolated by preparative electrophoresis had the molar ratio sulfate; hexosamine 1.8:1 and contained exclusively L‐iduronic acid.
Influence of Conterions on the Radius of Gyration of Phenylalanine Specific Transfer RNA as Determined by Small Angle X‐Ray Studies
Authors: Pilz Ingrid , Kratky Otto , von der Haar Friedrich , Cramer Freidrich
Published: March 2005

The influence of the ion and water could shell on the structure of polyelectrolytes is very important especially for small angle studies. In the present work the change in the radius of gyration of phenylalanine specific tRNA under the influence of various counterions was investigated. tRNAphe(yeast) was studied in diffierent solutions at pH 7.5, each containing only one certain kind of cation. As cations lithium, cesium, barium and spermidin were used. The influence of the ions on the radius of gyration of tRNAphe(yeast) was considerable. The value of R was 23.6 Å with Li+ ions, 26.2 Å with Cs+ ions and 26.3 Å with Ba++ions; these results are discussed.
Les nitrate‐réductases bactériennes
Authors: Forget Pierre
Published: March 2005

Solubilization and Purification. Crude extract is perpared from cells of Micrococcus denitrificans grown anaerobically in the presence of NO3−. Nitrate reductase A is solubilized by an alkaliacetone treatment of particles. The purification consists of four steps: ammonium sulfate precipitation at 35% saturation; Sephadex gel filtration; discontinuous elution from two successive phosphate gel columns, the first eluted by acid phophate bffer, the second by alakline. The enzyme is purified 50‐fold with a yield of 17%. Polyacrylamide gel electrophoresis and ultracentrifugation show the prepartion to be nearly homogeneous (95% purity).

Properties of the Purified Enzyme. Isoelectric point: pH 4.2. Molecular weight: (filtration on Sephadex and ultracentrifugation) 160000. Presence of flavins: no FMN and FAD. Absorption spectra: purified preparations have a brown color. Absorption increases continuously from 550 to 280 nm but does not exhibit any peaks in the visible or near‐ultraviolet. In the visible, the higher the specific absorption the higher the iron content of the protein. Heavy metals: the enzyme contains iron but the content varies from one preparation to another, 4 to 8 atoms per mole (Mw= 160000). Some of the iron is lost during purification and 8 atoms sees the value closer to reality. Traces of molybdenum are revealed by analysis, but may be acontamination although we cannot discard the hypothesis that this metal is normally present in the enzyme and lost during the purification. In ths case, it must be assumed that molybdenum is not necessary for the activity of nirate reductase with the artificaial electrons donors used. Labile sulfide: approximately one labile sulfide is found per iron atom. Amino acid composition: high content in aspartic and glutamic acids; low content in cysteine residues. Electron donors: the enzyme uses the reduced forms of viologen indicators, FMNH2, FADH2 but not NADH and NADPH. Substrates: NO3−, C1O3− and BrO3− are substrates. Inhibitors: CN− and N3− are inhibitors. The observed kinetics with 33.3 and 3.33 μM N3− are those of competitive inhibition. The nitrate reductase affinity for azide is one thousand times greater than for nitrate or chlorate. Versene and 1, 10‐phenanthroline are not inhibitors. Influence of pH: activity is maximal at pH 5.6‐5.8 with NO3− and at 6.2–6.4 with C1O3−. Iron function: the following observations suggest this metal plays an essential role in nitrate reductase A catalysis: (a) the higher the iron content of the purified fraction the greater its activity; (b) the 2 inhibitors, CN− and N3−, complex heavy metals; (c) dithionite causes a lowering of the absorption between 300 and 500 nm and this effect is reversed by the addition of nitrate; (d) the competitive inhibition by azide indicates that the iron may be at the active center and may play an important role in the formation of the enzyme‐substrate complex.

Conclusion. Nitrate reductase A of M. denitrificans is a non‐heme iron protein.
Binding of Aminoacyl‐tRNA to Ribosomes Programmed with Bacteriophage MS2‐RNA
Authors: Voorma Harry O. , Benne Rob , Den Hertog Tyronne J. A.
Published: March 2005

The secondary and tertiary structure of bacteriophage RNA is possibly concerned in the regulation of the synthesis of the three proteins encoded by this RNA. The accessibility of the beginning of the three cistrons for ribosomes should therefore be different.

We decided to use the aminocyl‐tRNA binding assay to obtain proof of difference in accessibility. Binding studies with aminoacyl‐tRNA to ribosomes, programmed with bacteriophage MS2‐RNA, revelaed that, besides formylmethionyl‐tRNA, alanyl‐tRNA is also extremely well bound to the initiation complex. This result was taken to indicate that the beginning of the coatgene is the major ribosomal binding site with intact RNA. In experiments with other aminoacyl‐tRNAs it was shown that seryl‐ and lysyl‐tRNA rection. In most experiments no significant binding of arginyl‐tRNA could be recorded. Similar experiments were performed with framented MS2‐RNA of which it is known that the secondary and tertiary structure content is much lower. Binding results with fragmented MS2‐RNA revealed that the accessibility of the beginning of the different cistrons reached similar levels as indicated by the amount of binding of the following aminoacyl‐tRNAs: alanyl‐, seryl‐, lysyl‐ and arginyl‐tRNA. The synthesis of the formylmethionyl‐peptides : F‐Met‐Ala, F‐Met‐Ser, F‐Met‐Arg, F‐Met‐Ala‐Ser and F‐Met‐Ser‐Lys does occur in this binding reaction, although the efficiency of the dipeptide reaction is different for the various dipeptides involved.
Study of Heme‐protein Linkage in Cytochrome b2
Authors: Groudinsky Olga
Published: March 2005

An investigation of heme ligands in cytochrome b2 has been attemptd by means of rose bengal sensitized photooxidation of “apo” and “holo” cytochrome b2 core. When “apo” cytochrome b2 core is photooxidizxed heme binding sites are progressively lost and histidine residues selectively destroyed. In “holo” cytochrome b2 core the presence of the heme prosthetic group prevents the photooxidation of histidines. It is concluded tat at least one histidine, or possibly two, is involved as an iron ligand. Evidence is given that cysteine residues are not implicated in the heme binding. A methionine‐iron bond does not seem to exist.
Distinct Antignic Specificities of Alanine Peptide Determinants Attanched to Protein Carriers via Terminal Amino or Carboxyl Groups
Authors: Schechter Israel , Clerrici Enrico , Zazepitzki Etti
Published: March 2005

L‐Alanine chains, serving as haptenic groups were coupled to proteins in opposite directions. There was no cross‐precipitation reaction between “anti‐amino‐alanyl” antibodies (obtained by immunization with conjugates containing alanine chains with free α‐amino groups) and “anti‐alanyl‐carboxyl” antibodies (obtained by immunization with conjugates containing alanine chains with free α‐carboxyl groups). The properties of the combining sites of these antibodies were evaluated by means of the hapten‐inihibtion method. For the inhibition of the precipitin reaction, series of free peptides and peptide derivatives (blocked in th amino or carboxyl function) composed of L‐ and D‐alanine residues were employed. The results led to the conclusion that the overall properties of the combining sites in both systems, but not their specificity, were quite similar. The size of the combining site of “anti‐amino‐alanyl” antibodies is such as to accommodate a tripeptide, whereas that of “anti‐analy‐carboxyl” antibodies is such as to accommodate tri‐to a tetrapeptide. In both systems the most exposed portion of the antigeic determinant is immunodominant. Thus, “anti‐amino‐alanyl” antibodies bind very poorly peptides in which the amino terminal end was altered (e.g., by replacing the N‐terminal L‐alanine residue by a D‐residue, or by N‐acetylation), but will combine very effectively with amides of L‐alanine peptides. Correspondingly, “anti‐alanyl‐carboxyl” antibodies react very poorly with alanine peptides in which the aminio terminal end was altered, e.g., they do not bind amides of L‐alanine peptides. Both types of antibodies can react with free L‐alanine peptides, but in their combining sites these peptides are probably bond in oppoiste direction. Thus, antibodies with distinctly different specificities can be elicited against L‐alanine peptide determinants, depending on their mode of attachment in the immunogen.

It is also shown that more precise information on the combining sites is obtained by comparing pairs of haptens of equal size which differ from one another in a particular feature, rather than from studying the effect of elongation in a homologous series of haptens.
Transfer Ribonucleic Acid from Mycoplams laidlawii A
Authors: Feldmann Horst , Falter Hermann
Published: March 2005

tRNA and an enzyme fraction which contain aminoacyl‐tRNA‐synthetases and tanformylase, were prepared from Mycoplasma laidlawii A. After chromatography of tRNA on benzoylated‐DEAE‐cellulose, amino acid acceptor activites were determined. One or more species of tRNA were found for the following amino acid (number of isoaccepting species in brackets): alanine (1), arginine (2), cysteine (1), glycine (1), isoleucine (2), leucine (3), methionine (2), phenylalanine (1), serine (3), tyrosine (1), and valine (3). The mthionine specific tRNA was separated into a homologously formylatable and a non‐formylatable species.

Cross‐reactions in heterologous systems from M. laidlawii A, Escherichia coli, and yeast were studied in some detail for methionine and phenylalanine. Aminoacylation was possible in all combinations of methionine specific tRNAs and enzymes. Transformylase of mycoplasma formylates tRNA from E. coli, but none of the methionine specific tRNAs from yeast.

The content of minor nucleotides in tRNA from M. laidlawii A was determined mainly from [32P]tRNA by two‐dimensional thin layer chromatography.

The findings are compared with those reported for other mycoplasma species and discussed in view of the genetic relationship of mycoplasma other microorganisms.
The Control of Liver Glycogen Synthetase Phosphatase by Phosphorylase
Authors: Stalmans Willy , De Wulf Henri , Hers Henri‐Géry
Published: March 2005

The activation of glycogen synthetase in a liver gel filtrate and the latency by which it is normally preceded have been studied in conditions where the level of phosphorylase a was greatly modified by the addition of either the pure enzyme or of antibodies directed against it. In the presence of liver or muscle phorphorylase a, glycogen synthetase phosphatase was nearly inactive; phosphorylase b had only a slight effect. Antibodies, obtained in the rabbit against dog liver phosphorylase, strongly depressed the activity of phosphorylase a in a mouse liver preparation; they simultaneously allowed an immediated activation of glycogen synthetase.

These results indicate that the lag period that precedes that activation of glycogen synthetase in a liver filtrate is the time required for the conversion of phosphorylase a into b by phosphorylase phosphatase. The previously reported shortening of the lag period by glucose, caffeine and nicotinamide or by a tratment with glucocorticoids is entirely explained by the faster inactivation of phosphorylase in the same conditions. The elongation of the lag period by glycogen and its supporession by AMP and magnesium remained however to be explained. It has been shown that the dose of glycogen that prolongs the lag period also inhibits phosphorylase posphatase, wherease AMP and magnesium counteract the inhibition of synthetase phosphatase by phosphorylase a.
Antigenic Properties of Bacillus licheniformis Cell Wall Components
Authors: Hughes R. Colin , Thurman Paul F. , Salaman Myer R.
Published: March 2005

Antibodies directed against mucopeptide were obtained by immunization of rabbits with Bacillus licheniformis N. C. T. C. 6346 cell walls extracted chemically to remove the teichoic acids. The teichuronic acid component present in whole walls or alkali extracted walls of the organism was not significantly immunogenic in rabbits. The mucopeptide antisera reacted strongly with sonicates of Bacillus licheniformis and Bacillus subtilis mucopeptides and the homologous reaction was inhibited by soluble fragments of low molecular weight derived from mucopeptide. The best inhibition of the homologous reaction was obtained with fragments containing peptide and hexosamine material isolated from the soluble products of lysozyme treatment of Bacillus licheniformis mucopeptide.

Immune sera obtained from rabbits immunized with intact Bacillus licheniformis N. C. T. C. 6346 walls containing mucopeptide, teichuronic acid and teichoic acids contained antibodies directed mainly against the teichoic acids. The immune response to the mucopeptide component was greatly reduced compared to that obtained when wall preparations lacking teichoic acids were used as immunizing antigens.
Studies on Hog Intestine Mucosa Peroxidase
Authors: StelmaszyŃSKA Teresa , ZgliczyŃSKI Jan M.
Published: March 2005

A method has been developed for isolating and purifying peroxidase from hog intestinal mucosa. The enzyme exhibits an absorbance ratio A417 nm/A280 nm of 0.906.

Studies on the homogeneity of intestinal peroxidase by electrophoresis on polyacrylamide gel and cellulose acetate strips showed its high degree of purification; the enzyme showed a single band at pH 8.45 with the cathodic mobility.

Intestinal peroxidase is a haemoprotein. Its absorption spectrum, as well as that of its derivatives (CN‐peroxidase, reduced peroxidase, CN‐reduced peroxidase and pyridine haemochromogen) is of the same type as the spectrum of lactoperoxidase and its corresponding derivatives.

Kinetic studies on the reaction of intestinal peroxidase with hydrogen peroxide and guaiacol showed that this haemoprotein has the properties of true peroxidase (donor: H2O2 oxidoreductase). The k1 value was determined as 5.9 × 106 M−1× sec−1, and Km (H2O2) was 260 μM. Optimum pH of the studied peroxidase was in the range pH 7.5–8.

Intestinal peroxidase, with respect to deamination and decarboxylation of amino acids, does not resemble the myeloperoxidase.
The Regulative Properties of UDPglucose: d‐Fructose‐6‐phosphate 2‐Glucosyltransferase (Sucrose Phosphate Synthetase) from Vicia faba Cotyledons
Authors: DE Fekete Maria A. R.
Published: March 2005

Sucrose phosphate synthetase of cotyledons of Vicia faba was purified about 130‐fold by means of ammonium sulfate and protamine fractionation. After freezing and thawing an activator could be separated by centrifugation from the purified enzyme. The regulatory properties of the activator‐bound and activator‐devoid enzyme were compared. Under the normal conditions of assay the activator‐devoid enzyme had scarcely any activity, but could be activated by citrate, a number of dicarboxylic acids and protamin. The saturation curves of these effectors were sigmoidal. Also the substrate saturation curves were sigmoid shaped in absence of activator but the presence of activator converted them to a hyperbolic form.

Quite different was the effect of citrate on the activity of the activator‐bound enzyme: at low concentration it inhibited the production of sucrose‐6‐phosphate. At 10–20 mM citrate a minimum of activity could be measured. At higher citrate concentrations a reactivation occurred. This activator‐bound preparation could be further inhibited, but not reactivated, by UDP, UTP, ATP, ADP and other phosphates which had scarcely any influence on the activity of the activator‐devoid enzyme. Experiences where the influence of citrate concentration was investigated on the UTP‐inhibited enzyme suggest that both effectors share binding sites.

The role of citrate in the control of the activities of sucrose phosphate synthetase (activation) and phosphofructokinase (inhibition), both enzymes competing for the branchpoint metabolite fructose‐6‐phosphate, is discussed.
Effects of Benzimidazole on the Oxygen‐Induced Biosynthesis of Hemoproteins in Yeast
Authors: Sels André A. , Verhulst Anne‐Marie
Published: March 2005

Benzimidazole almost completely inhibited respiratory adaptation of yeast under conditions where total protein synthesis was only partially inhibited.

The effects of benzimidazole on the synthesis of individual hemoproteins were examined. The production of enzymatically active cytochrome oxidase was almost totally blocked; this accounts for the interruption of respiratory adaptation. The biosynthesis of the NADH (and succinate): cytochrome c oxidoreductase segments, which include the cytochromes b and c1, was only partially inhibited. The induced synthesis of iso‐1‐cytochrome c displayed a singular insensitivity to the action of the drug. The mechanism of inhibition by benzimidazole appears to be different from that of cycloheximide and chloramphenicol.
A Study of Methylated Polyadenylic Acid
Authors: Thrierr Claude , Leng Marc
Published: March 2005

Copolymers with different contents of adenine and N1‐methyl adenine residues have been studied, polyadenylic acid being methylated by dimethylsulfate. Thermal stability, circular dichroism and light scattering measurements are in favor of a two‐strand structure at neutral pH. Thermal stability of these products is much less sensitive to ionic strength than that of most polynucleotides, and in some cases independent of ionic strength. These copolymers interact or not with poly(U), depending on the percentage of N1‐methyl adenine residues. Copolymers with high contents of methyladenine interact with poly(A) at neutral pH. These copolymers do not bind DNA‐dependent RNA polymerase. Some results at acid pH are also reported.
Interaction of Lipopolysaccharides and Lipid A with Complement
Authors: Galanos Chris , Rietschel Ernst Th. , LÜDERITZ Otto , Westphal Otto
Published: March 2005

A number of lipopolysaccharides derived from Salmonella and Escherichia coli S and R mutant strains were tested for toxicity and anticomplementary activity in the absence of added antiserum. Although all preparations were toxic, only a few exhibited high anticomplementary activity, while others proved to be of low or negligible activity. It was found that isolated lipid A from both active and inactive lipopolysaccharides was strongly anticomplementary as well as toxic, when made water‐soluble with the aid of suitable carriers. Treatment of R form lipopoly‐saccharide with Mg2+ or Ca2+ led to complete precipitation of the lipopolysaccharide with consequent loss of toxicity and anticomplementary activity. This treatment had practically no effect on the anticomplementary activity and toxicity of S form lipopolysaccharides. When lipopolysaccharide, after reaction with complement, was reisolated and purified, the resulting preparation was found to be non‐toxic and of negligible anticomplementary activity. No detectable alterations in either the sugar or the fatty acid composition could be detected. The only significant change was the loss of solubility in water. Treatment of the reisolated lipopolysaccharide with EDTA completely restored solubility in water, toxicity, and anti‐complementary activity.
The Effect of Alkylation on Substrate‐ and Effector‐Binding Sites in Pigeon Liver NAD Kinase
Authors: Apps David K.
Published: March 2005

At pH 7.5, inactivation of NAD kinase by bromoacetyl pyridine occurs in three independent pseudo‐first‐order stages.

Below pH 7.5, only two stages are distinguishable.

The final stage alkylation leads to total inactivation, the earlier stages to the formation of an enzyme species with modified properties.

Effects of pH on the time courses of inactivation suggest that the first stage may involve a variable number of sulphydryl groups.

The final stage probably involves alkylation of a sulphydryl, with abolition of NAD binding.

No central role in catalysis can be postulated for any of the groups modified by bromoacetyl pyridine; two groups known to participate in catalysis and the binding of ATP · Mg are apparently not alkylated.

Inhibition by L‐malate and by free ATP may occur by attachment of these effectors at separate modifier sites.
The Origin and Nature of the Methionine Residue Initiating the Synthesis of Haemoglobin in vivo and in vitro
Authors: Hunter Anthony R. , Jackson Richard J.
Published: March 2005

The methionine residue which is transiently incorporated into the N‐terminal position during the initiation of both chains of rabbit globin, is provided solely by rabbit reticulocyte methionyl tRNAF and is not blocked or modified at any stage of the initiation process. This is true both for initiation observed in vitro and in vivo. All internal methionine residues of rabbit globin are provided by methionyl tRNAM.
Studies on the Protein Moiety of Endotoxin from Gram‐Negative Bacteria
Authors: Wober Wolfgang , AlaupoviĆ Petar
Published: March 2005

Conjugated protein isolated from the endotoxin complex of Serratia marcescens 08 by 1% acetic acid hydrolysis was characterized by analytical ultracentrifugation, electrophoretic and immunological properties and chemical analysis. The chemical composition of conjugated protein and other degraded fragments of endotoxin showed that all characteristic constituents of lipid A were present exclusively in conjugated protein. Successive treatment of conjugated protein with trypsin and pronase resulted in the isolation of corresponding tryptic and pronase cores which were characterized by an increased content of lipid A constituents. Lipid A was separated as an entity from the conjugated protein only after hydrolysis with 0.1 N hydrochloric acid. This result confirms our previous proposal that lipid A is covalently linked to the protein moiety in whole endotoxin of S. marcescens 08. “Conjugated” protein isolated by acetic acid hydrolysis and “simple” protein prepared by hot aqueous phenol treatment of whole endotoxin differ from one another not by the presence or absence of lipid A but rather by the presence of the entire lipid A moiety in the conjugated protein and only a fragment of lipid A in the simple protein.

Conjugated protein and its tryptic and pronase cores were immunogenic and possessed a common antigenic determinant with simple protein and its corresponding cores. Toxicity studies revealed that both conjugated and simple proteins retained to different degrees the toxic properties of whole endotoxin. Sodium dodecyl sulfate had an enhancing rather than diminishing effect on the toxicity of endotoxin and its various fragments. The importance of lipid A as the toxic factor of endotoxins was established not only indirectly by determining the toxicity of each fragment containing lipid A, but also directly by demonstrating that lipid A dispersed in a Tris‐dodecyl sulfate buffer displayed toxic properties similar to those of the lipopolysaccharide preparation.
Acid Soluble Calf Skin Collagen
Authors: Rauterberg Jürgen , Kühn Klaus
Published: March 2005

The α1‐chain of acid soluble calf skin collagen was cleaved with cyanogen bromide, and the resulting peptides were separated by ion exchange and molecular sieve chromatography. Eight different peptides could be isolated as expected from the seven methionyl residues of the chain. Their size was determined by gel filtration, dodecyl sulfate disc electrophoresis, measurement of electron micrographs of renatured peptides, and amino acid analysis. Peptides, differing in length between 19 and 270 residues were found. The C‐terminal peptide α1‐CB6 occurred in two variants (CB6a and CB6b), differing in size, chromatographic behaviour and amino acid composition. The results suggest that CB6a contains about 15 amino acid residues more than CB6b.

The amino acid compositions of the peptides were in good agreement with the composition of the α1‐chain. All CNBr‐peptides, including the sum of CB6a and CB6b, were isolated in approximately equimolar amounts, thus excluding the possibility of repeating sequences within the chain. The order of the CNBr‐peptides was established by electron microscopy and by comparative amino acid analysis of overlapping collagenase derived peptides and uncleaved CNBr‐peptides. It was found to be the same as in α1 from rat skin collagen.