bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation from Porcine‐Intestinal Wall of a Vasoactive Octacosapeptide Related to Secretin and to Glucagon
Authors: Said Sami I. , Mutt Viktor
Published: March 2005

A polypeptide composed of 28 amino acid residues has been isolated from porcine upper intestinal wall. In addition to exhibiting several biological activities which may be attributed to a relaxant effect on vascular and systemic smooth muscle it stimulates exocrine pancreatic secretion in a secretin‐like fashion, with an apparent efficiency, on a molar basis, of 5–10% of that of secretin and also gives rise to hyperglycaemia with about 30% of the efficiency of glucagon. Structurally it resembles secretin and glucagon in having a histidyl‐seryl sequence N‐terminally. Of the amino acid residues commonly occurring in mammalian proteins the residues of cysteine/cystine, glycine, proline and tryptophan are absent from it.
Identification of Prostaglandins in the Gorgonian, Plexaura homomalla
Authors: Light Robley J. , Samublsson Bengt
Published: March 2005

Hexane extracts of the air‐dried cortex of the gorgonian Plexaura homomalla (Esper) were found to contain (15R)‐hydroxy‐9‐oxo‐prost‐5,10,13‐trienoic acid [i.e. (15.R)‐prostaglandin A2] and its 15‐acetate methyl ester in 0.2% and 1.3% yield from the cortex by Weinheimer and Spraggins in 1969. Chloroform‐methanol extracts of this organism have now been shown to contain these two compounds as major prostaglandin constituents, but also a more complex mixture of prostaglandin derivatives including 11α, (15R)‐dihydroxy‐9‐oxo‐prost‐5,13‐dienoic acid [i.e. (15R)‐prostaglandin E2] and the methyl esters of (15R)‐prostaglandin E2 and (15R)‐prostaglandin A2. (15R) Prostaglandin E2 was identified by its chromatographic behavior, by the chromatographic behavior and mass spectrum of its O‐methyloxime, methyl ester, tri‐methylsilyl ether derivative, and by oxidative ozonolysis to fragments of established structure. The configuration of the 15‐hydroxyl group was determined in the cleavage fragment 2‐hydroxy‐heptanoic acid by gas‐liquid chromatography of its methyl ester (–)‐menthyloxycarbonyl derivative. Similar degradations of material with chromatographic properties equivalent to prostaglondin A2 and prostaglandin E2 yielded 2‐hydroxyheptanoate with the S configuration, demonstrating the presence of small amounts of (15S)‐prostaglandins in the lipid extract.
Purification and Subunit Interactions of Yeast Hexokinase
Authors: Easterby John S. , Rosemeyer Michael A.
Published: March 2005

A method is described for the purification of two native isoenzymes of yeast hexokinase (A and B) in the presence of phenylmethyl sulphonylfluoride, an inhibitor of yeast proteases. In the absence of this inhibitor two proteolytically modified forms (A′ and B′) were obtained.

Investigation of the effects of solvent ionic strength and pH on the dissociation of the native form A and the proteolytically modified form A′ suggests that these enzymes have a common molecular weight and quaternary structure. Both forms exist as dimers of molecular weight 108000 ± 3000 in reversible dissociation equilibrium with monomers of molecular weight 54000 ± 1500.

Hexokinase A dissociates to monomer both with increasing ionic strength and in the presence of hexose substrates. Dissociation to constituent polypeptide chains of 54000 molecular weight is effected by maleylation or succinylation of the native enzyme, confirming its dimeric structure. Hexokinase A′ exhibits a pH‐dependent dissociation from dimer to monomer. This dissociation is attributable to two ionising groups on the monomer which are predominantly unionised in the dimer.
Animal DNA‐Dependent RNA Polymerases
Authors: Kedinger Claude , Chambon Pierre
Published: March 2005

This paper describes a method for the purification of calf thymus DNA‐dependent RNA polymerase B activity and its subsequent resolution into two enzymes, BI and BII. The method of purification of the combined B activities involved chromatography on phosphocellulose and hydroxyapatite and centrifugation through a glycerol density gradient. Enzymes BI and BII were separated by two successive chromatographic steps on DEAE‐cellulose. Addition of 30% glycerol to all buffers increased the stability of the enzymes throughout the purification. The polymerases were over 95% pure and free of contaminating nucleases. It seems unlikely that enzyme BII could derive from enzyme BI by a specific proteolytic process occurring during purification of the B activity.
A Glycopeptide from the Posterior Lobe of Pig Pituitaries
Authors: Holwerda Dirk A.
Published: March 2005

The primary structure of the glycopeptide from the posterior lobe of the pig pituitary is: Ala‐Ser‐Asp‐ Arg‐Ser‐Asn‐Ala‐Thr‐Leu‐Leu‐Asp‐Gly‐Pro‐Ser‐Gly‐Ala‐Leu.

The, carbohydrate is linked to the peptide chain via the asparagine residue.
The Amino‐Acid Sequence of Neurotoxin II of Androctonus australis Hector
Authors: Rochat Hervé , Rochat Catherine , Sampieri François , Miranda François , Lissitzky Serge
Published: March 2005

The complete amino acid sequence of neurotoxin II of the North African scorpion Androctonus australis Hector has been determined.

From the N‐terminal sequence (43 residues) obtained by automatic phenylisothiocyanate degradation in the protein sequenator, and from the sequence of tryptic peptides obtained by digestion of the reduced and methylated toxin of which amino groups had been previously masked by reaction with ethylthioltrifluoroacetate the complete amino acid sequence of the protein has been inferred. It is composed of a single polypeptide chain of 64 ammo acid residues, including 8 half‐cystines, ended by an histidinamide residue at the C‐terminus.
The Effect of Ether Anaesthesia Stress on Cholesterol‐Side‐Chain Cleavage and Cytochrome P450 in Rat‐Adrenal Mitochondria
Authors: Simpson Evan R. , Jefcoate Colin R. , Boyd George S. , Brownie Alexander C.
Published: March 2005

Cholesterol side‐chain cleavage has been studied in intact adrenal mitochondria from rats subjected to stress by ether anaesthesia as a means of raising the plasma adrenocorticotrophin levels. Control rats were either injected with cycloheximide or kept in a quiescent state. After initiation of cholesterol side‐chain cleavage by addition of isocitrate, pregnenolone formation from endogenous cholesterol in intact rat‐adrenal mitochondria follows a biphasic time‐course of formation with an initial rapid phase lasting 3–5 min followed by a much slower rate of formation.

Pregnenolone formation from [4‐14C]cholesterol is linear if the tracer substrate is added to the mitochondria together with isocitrate. Preincubation of the mitochondria with [4‐14C]cholesterol prior to addition of isocitrate results in a biphasic [4‐14C]pregnenolone formation; the rate of the initial rapid phase depending on the duration of preincubation.

The effect of stress is to increase 2–3 times the rate of pregnenolone formation in the initial phase compared to the rates observed in mitochondria from quiescent or cycloheximide‐treated rats.

Depletion of cholesterol from adrenal mitochondria follows a similar pattern to pregnenolone formation, but the initial cholesterol content of the mitochondria is not affected by the pretreatment.

On the basis of these results it is suggested that only a fraction of the total mitochondrial cholesterol is readily available for cholesterol side‐chain cleavage and that this pool is increased by stress.

The cytochrome P450 type II spectral changes induced by addition of pregnenolone and isocitrate to the intact rat adrenal mitochondria have also been studied. The effect of stress was to increase pregnenolone‐induced difference spectrum in adrenal mitochondrial two‐ to three‐fold compared with cycloheximide‐treated animals. Similar results were found for the isocitrate‐induced type II difference spectrum.

The spectral changes are interpreted to mean that stress causes an increase in the cholesterol complex of side‐chain cleavage cytochrome P450. Metabolism of the remainder of the cholesterol in the mitochondria is limited by the rate of transport, or binding, to this reactive centre. It is proposed that the acute effect of stress, mediated by adrenocorticotrophin, is to increase the proportion of mitochondrial cholesterol in the readily available form perhaps by an increase in the rate of transport or binding of cholesterol to sites from which it can be readily metabolised.
Chemical and Immunological Properties of Reduced and Alkylated Polypeptide Chains of Bovine Fibrinogen
Authors: Gollwitzer Rotraut , Timpl Rupert , Becker Udo , Furthmayr Heinz
Published: March 2005

The α, β and γ chains were isolated from reduced and carboxymethylated bovine fibrinogen by chromatography on CM‐cellulose. Electrophoretically pure polypeptide chains could be obtained as judged by three different methods. The chains were soluble in buffers at or above pH 8 but exhibited non‐covalent aggregation. The molecular weights of the α, β and γ chains were estimated in dodecylsulfate‐polyacrylamide gel electrophoresis as 62000, 58000 and 48000, respectively. Each chain differed considerably from the other in amino acid composition as well as in the fragment pattern obtained after CNBr treatment. The occurrence of unresolved α/β material suggested microheterogeneity of the individual chains. Sulfitolysis yielded, at least for the α chain, less pure products.

Between 20% and 54% of the antibodies in rabbit antisera to native fibrinogen precipitated with the α and/or β chain. Absorption studies, gel precipitation and hemagglutination‐inhibition suggested a considerable antigenic homology of both chains. They could be clearly distinguished from the γ chain which reacted poorly in precipitation test but considerably better in passive hemagglutination. Cyanogen bromide cleavage did not destroy the serologic activity of the chains. These findings indicated that a considerable number of the antigenic determinants on fibrinogen are not dependent on intact conformation. However, antibodies specifically reacting with the native molecule occur as well.
Intermediates in the Reaction Catalyzed by Glutamate Dehydrogenase
Authors: Antonini Eraldo , Chiancone Emilia , Yielding K. Lemone , Shafer Jules A.
Published: March 2005

By studying the glutamate dehydrogenase catalyzed oxidation of glutamic acid under conditions where the concentration of enzyme exceeded that of NAD, it was possible to evaluate directly the first‐order rate constant (0.73 sec−1) for the decay of an intermediate associated with the burst in absorbance, which is seen at 340 nm in the pre‐steady state phase when the NAD concentration exceeded that of enzyme. A study of the wavelength dependence of the absorbance vs time plots when [enzyme] > [NAD] indicated that the intermediate associated with the burst is isosbestic with the ternary abortive complex E · Glu · NADH. This information made it possible to estimate the total amount of NADH (π) formed in the initial rapid phase when [NAD] > [enzyme].

A study of the dependence of the pre‐steady‐state kinetics on [NAD] revealed that between 27 and 1000 μM NAD, π was invariant at 0.4 μmol NADH formed in the burst phase per polypeptide chain. However, the velocity of the phase immediately following the burst phase increased between 27 and 1000 μM NAD, and the pseudo‐first‐order rate constant for the burst phase increased between 30 and 300 μM NAD and decreased between 300 and 1000 μM NAD. These results were taken to mean that all the NAD sites on glutamate dehydrogenase are not catalytically equivalent and that binding of NAD to sites which are not catalytically active causes a decrease in the rate of the burst phase and an increase in the rate of the phase following the burst.

Below 1000 μM glutamate, a pronounced lag in the onset of the burst phase is seen. This lag suggests that at low glutamate concentration binding of NAD and/or glutamate to the enzyme becomes slower than reduction of E · Glu · NAD.
Studies on the Biosynthesis of the Branched‐Chain Sugars from the Quinocycline Complex
Authors: Matern Ulrich , Grisebach Hans
Published: March 2005

The incorporation of [2‐14C]pyruvate and [1‐14C]acetate into the 2,6‐dideoxy‐4‐C‐[1′‐hydroxy‐ethyl]‐l‐xylo‐hexopyranose (compound I) moiety of quinocycline A and into the 2,6‐dideoxy‐4‐C‐[1′‐oxoethyl]‐l‐xylo‐hexopyranose (compound II) portion of quinocycline B produced by Streptomyces aureofaciens was investigated. The methylglycosides of compounds I and II were subjected to periodate oxidation to yield, respectively, acetaldehyde and acetic acid from the branch carbon atoms. Acetaldehyde was oxidized to acetic acid, which was then degraded by Schmidt‐degradation. With [2‐14C]pyruvate 87 to 90% of the radioactivity incorporated into compound I or II was located in the C2 branch without randomization of the label. In contrast, with [1‐14C]acetate only very low activity was incorporated into the sugars and no 14C was localized in the C2 branch. These results prove that the C2 branch in compounds I and II originates from C‐2 and C‐3 of pyruvic acid.

When 14C‐labelled quinocycline B was incubated for 17 h with S. aureofaciens, 13% was converted to quinocycline A. Under the same conditions only 2% of [14C]quinocycline A was converted to quinocycline B. It can therefore be concluded that quinocycline B is the precursor of quinocycline A. This result is in agreement with the assumption that hydroxyethylthiamine pyrophosphate is the immediate precursor for the oxoethyl group of the branched sugar II, which after transfer to the aglycone is reduced to compound I. Experiments with the thiamine antagonist neopyrithiamine did not show inhibition of incorporation of radioactivity from [2‐14C]‐pyruvate into the hydroxyethyl branch of compound I. However, this result does not exclude the possibility that thiamine pyrophosphate participates in the reaction.
Regulation of the Catabolic Qrnithine Carbamoyltransferase of Pseudomonas fluorescens
Authors: Stalon Victor , Ramos Fernando , Piérard André , Wiame Jean‐Marie
Published: March 2005

The properties of the catabolic ornithine carbamoyltransferase of Pseudomonas fluorescens are compared to those of the anabolic transferase of the same organism. The comparison is also extended to a mutationally modified enzyme which, in contrast to the catabolic enzyme, is able to synthesize citrulline in vivo. All three activities are measured in the direction of citrulline synthesis.

The catabolic ornithine carbamoyltransferase and the mutationally modified enzyme have an approximate molecular weight of 400000 while that of the anabolic enzyme is 100000.

The catabolic transferase exhibits a number of regulatory properties which are not observed with the anabolic enzyme:

Its saturation by carbamyolphosphate is intensively cooperative. Its half saturation concentration is more than one hundred times higher than the Km of the anabolic enzymes; this explains its inability to perform an anabolic function in vivo.

Phosphate, a substrate in the physiological direction of the reaction and a low‐energy‐level indicator, acts as an activator of the enzyme; it abolishes the cooperativity of the carbamoylphosphate and increases the capacity of the enzymes.

ATP, a product of the catabolic pathway and a high‐energy‐level signal, reduces the capacity of the enzyme.

Putrescine, another product of the catabolic pathway, and its analogs, inhibit the enzyme by decreasing its apparent affinity for carbamoylphosphate.

The catabolic transferase of the mutant IRC204M1 shares all those properties with the wild‐type enzyme, but, owing to a reduced cooperativity of carbamoylphosphate, it is able to perform an anabolic function at reduced rate.

Thus, the comparison of the regulatory properties of the catabolic ornithine carbamoyltransferase and of the modified enzyme provides a kinetic explanation for the inability of the catabolic enzyme to play an anabolic role and this without the need of referring to compartmentation of channeling of the anabolic and catabolic pathways.
Regulation of the Catabolic Ornithine Carbamoyltransferase of Pseudomonas fluorescens
Authors: Stalon Victor
Published: March 2005

The catabolic ornithine carbamoyltransferase from Pseudomonas fluorescens catalyzes the phosphorolysis of citrulline leading to the formation of ornithine and carbamoylphosphate. The initial rates have been measured in the experimentally most accessible direction of the reaction, the carbamoylation of ornithine. The plots of reaction rates against carbamoylphosphate (substrate), phosphate (activator), and putrescine (inhibitor) are sigmoid shaped. The homotropic interactions between carbamoylphosphate molecules are decreased in the presence of phosphate and increased in the presence of putrescine.

The allosteric properties of the enzyme are accounted for by the concerted transition theory proposed by Monod, Wyman and Changeux. The treatment of the kinetic data according to the method of Blangy, Buc and Monod suggests that the enzyme is an octamer made up of eight identical protomers which can exist in equilibrium in two conformational states which differ in their dissociation constant for the substrate, activator and inhibitor. The values of these constants for the binding of carbamoylphosphate, phosphate and putrescine, and the allosteric transition constant have been determined for the wild‐type enzyme and for the enzyme of a mutant which can use the catabolic enzyme for anabolic purpose.
Purification and Some Properties of Citrate Synthase from Penicillium spiculisporum
Authors: Måhléan Anders
Published: March 2005

Citrate synthase has been purified 300 to 400‐fold to a maximum specific activity of 50—60 U/mg protein. It has similar properties to the majority of the eucaryotic citrate synthases which have been studied, although it is inhibited less by ATP. The Km for acetyl‐CoA is 30 μM and for oxaloacetate 10 μM. The apparent Km for acetyl‐CoA in the presence of 2.5 mM ATP is 50 μM and, in the presence of 5 mM ATP, 80 μM. The Ki for ATP is 3—4 mM. The ATP inhibition is reduced by Mg2+. At 20 μM acetyl‐CoA 12% inhibition results from the transition from 5 mM AMP to 5 mM ATP in presence of a slight excess of Mg2+. The size of citrate synthase as estimated from gel‐filtration is about that of serum albumin. No close relationship between citrate synthase and 2‐decylcitrate synthase from Penicillium spiculisporum is apparent. If citrate is considered to be the ultimate precursor of decylcitrate, the extracted amount of citrate synthase is hardly sufficient to account for the observed production unless the enzyme operates at or near conditions of maximal velocity, which, in presence of adenine nucleotides, requires considerable effective substrate concentrations.
Ein „Kurzschluß”‐Weg der Nicotinsäuresynthese bei Rhodotorula glutinis
Authors: Böttcher Fritz , Birnbaum Dieter , Samsonova Ida A.
Published: March 2005

Nicotinic acid in Rhodotorula glutinis is synthesized from tryptophan via N'‐formylkynurenine, kynurenine, 3‐hydroxykynurenine and 3‐hydroxyanthranilic acid. Auxotrophic mutants, with blocked synthesis at various steps between tryptophan and 3‐hydroxyanthranilic acid, can grow with 100 μg/ml anthranilic acid (concentration in the agar‐agar). The usually required concentration of substances such as nicotinic acid or other substances after the blocked step is about 2 μg/ml. Auxotrophs with a block between 3‐hydroxyanthranilic acid and nicotinic acid are not able to grow with anthranilic acid. Therefore mutants requiring nicotinic acid with a blocked step between tryptophan and 3‐hydroxyanthranilic acid can synthesize nicotinic acid from anthranilic acid, a substance formed in the first step of tryptophan synthesis from chorismic acid, by eluding the blocked pathway.

The same ability has been found in prototroph revertants with the original block of the auxotrophs but with a second mutation in the gene of anthranilate synthetase. As a result of this mutation the feedback inhibition by tryptophan is disturbed and anthranilic acid is produced in excess. When the culture medium of the prototrophs is analysed by paper chromatography after adding anthranilic acid to the growing culture, 3‐hydroxyanthranilic acid as one of the formed products can be found.

Summarizing these facts it is evident, that there exists a “short‐cut”‐pathway of nicotinic acid synthesis in Rh. glutinis, which is realized by direct hydroxylation of anthranilic acid to 3‐hydroxyanthranilic acid. This “short‐cut”‐pathway however is only possible, when anthranilic acid in the cell is present in excess.
Fatty‐Acid Biosynthesis
Authors: Landriscina Clemente , Gnoni Gabriele V. , Quagliariello Ernesto
Published: March 2005

The relationship between the two elongation syntheses of fatty acids, one present in microsomes and the other in mitochondria, and the system for synthesizing palmitate de novo has been studied in rat liver. The results indicate that microsomes are much more active than mitochondria in elongating and/or desaturating fatty acids synthesized by cytoplasmic system.

Fatty acids synthesized by cell sap in the presence of added microsomes are found principally esterified into phospholipids, this indicates that these compounds in the microsomal fraction are actively deacylated and reacylated.

ATP is required both for chain elongation and for the esterification of newly synthesized fatty acids.

Evidence has been obtained that acyl groups are elongated and/or esterified into complex lipids as acyl‐CoA and not as acyl‐ACP.

The high activity of palmityl thioesterase in the cell sap with respect to the other subcellular fractions points out the importance of this enzyme in the fatty acid synthesis de novo.
Thermodynamic and Kinetic Studies on the Cooperative Binding of Proflavine to Linear Polyanions
Authors: Schwarz Gerhard , Klose Sigmar
Published: March 2005

The cooperative binding of proflavine with poly(acrylic acid), poly(α‐l‐glutamic acid) and polyphosphate has been investigated. In all cases the equilibrium experiments could be described by a recently proposed theory based on a linear Ising lattice of equivalent binding sites. Thermodynamic and spectroscopic parameters are evaluated as a function of ionic strength and discussed in terms of a simplified structural model of dye aggregation on the polyion. Application of the temperature‐jump technique generally resulted in a fast initial process which is in most cases followed by a substantially slower further relaxation effect of more or less amplitude. The first process can be quantitatively interpreted in terms of a diffusion controlled reaction mechanism according to the basic model theory. The slow effect depends very much on the nature of the polymer. It seems to be caused by structural changes of the dye aggregates upon polymer complex formation.
Fluorescence Studies on Substrate Binding to Seryl‐tRNA Synthetase from Yeast
Authors: Engel Günter , Heider Henning , Maelicke Alfred , von der Haar Friedrich , Cramer Friedrich
Published: March 2005

The interactions of seryl‐tRNA synthetase with its ligands have been investigated by fluorescence measurements. Using a microscale fluorescence technique and an excitation wavelength of 303 nm the inner filter effect was suppressed.

The fluorescence intensity of the seryl‐tRNA synthetase at 342 nm (maximum emission) was quenched by 33%, by adding tRNASer, whereas no effect was produced by the tRNAs which are specific for phenylalanine and tyrosine. For tRNASer an association constant of 2.1 × 106 M−1 per enzyme subunit was calculated from the fluorescence titration. The emission maximum of the synthetase did not shift when tRNA was added.

Under similar conditions ATP caused only 6%, quenching of the enzyme fluorescence from which an intrinsic association constant of 105 M−1 for about 10 moles ATP per mole subunit could be derived. On the other hand, when the enzyme was saturated with tRNA, addition of ATP increased the fluorescence intensity. Performing the titration in the reverse order, the same fluorescence end point was achieved. The addition of CTP to the preformed complex between tRNASer and the synthetase did not induce any change in the fluorescence intensity.

These results are discussed with respect to the binding process and binding sites at which tRNA and ATP are attached to the synthetase and the possible involvement of the tryptophan residues.
Partial Purification and Characterization of Glyoxalase I from Porcine Erythrocytes
Authors: Mannervik Bengt , Lindström Lars , Bártfai Tamás
Published: March 2005

Glyoxalase I from porcine erythrocytes has been purified about 2000‐fold by ion‐exchange chromatography on SE‐Sephadex, CM‐cellulose and DEAE‐cellulose columns. The isoelectric point was estimated to be at about pH 5, and the molecular weight was 52000 as determined by gel filtration. The kinetics of the enzymatic reaction was studied and the Km value was determined to be 0.19 mM, which differs significantly from the Km value of the yeast glyoxalase I. The enzymatic activity is completely inhibited by the chelating agents EDTA and o‐phenanthroline, and activity is regained by addition of Mg2+, Mn2+, and Ca2+. The reactivation is time‐dependent, which indicates that a metal · enzyme complex is the catalytically active species. An apparent dissociation constant of 0.6 mM for the Mg2+· enzyme complex was determined.

Notable differences between glyoxalase I from yeast and from porcine erythrocytes appear in molecular weight, kinetics, and reactivation of chelator‐inhibited enzyme.
The Binding of Steroid Hormones to Degranulated Microsomes from Rat‐Liver Endoplasmic Reticulum
Authors: Blyth Carol A. , Cooper Michael B. , Roobol Anne , Rabin Brian R.
Published: March 2005

Smooth reticular membranes from male rat liver contain high affinity binding sites for oestradiol. Similar membranes from females contain analogous sites for testosterone.

Rough membranes from which polysomes have been removed by EDTA (degranulated membranes), contain no high affinity binding sites for either hormone.

Ethyl acetate extracts of degranulated membranes contain materials which block the high affinity binding sites on smooth membranes of the same but not the opposite sex.

These extracts promote the attachment of ribosomes to smooth membranes of the same but not the opposite sex.

The ethyl acetate extracts from male and female degranulated membranes contain substances which behave like oestradiol and testosterone, respectively.
Hemoglobin St Luke's or α95Arg2 (G2) β2
Authors: Bannister William H. , Grech Joseph L. , Plese Charles F. , Smith Linda L. , Barton Betty P. , Wilson Jerry B. , Reynolds Cecelia A. , Huisman Titus H. J.
Published: March 2005

A new hemoglobin variant, designated hemoglobin‐St Luke's, was detected by routine starch‐gel electrophoresis in four members of a Maltese family. The carriers of this abnormality are clinically and hematologically normal. Structural analyses showed the replacement of prolyl residue in position 95 (G2) of the alpha chain by an arginyl residue. The relative amount of hemoglobin‐St Luke's present in the red cell hemolysates of its carrier is approximately 10%, compared to about 23% for hemoglobin‐G Georgia and 15% for hemoglobin‐Rampa (the other two variants in which prolyl residue α95 (G2) is replaced). Sedimentation velocity studies of hemoglobin‐St Luke's in NaCl solutions of increasing concentration up to 2 M indicate that the oxy derivative is extensively dissociated into dimers in 0.1 M NaCl at neutral pH; whereas under the same conditions, the deoxygenated molecule retains a tetrameric structure. In the deoxy state, appreciable tetramer formation occurs even in 2 M NaCl. In addition, association‐dissociation in oxy‐or cyanferrihemoglobin St Luke's is both pH and temperature dependent.
Structural Studies on the Lipopolysaccharide of a Rough Strain of Escherichia coli
Authors: Morton James J. , Stewart John C.
Published: March 2005

The determination of the 2‐keto‐3‐deoxyoctonic acid (KDO) content of a lipopolysaccharide obtained from Escherichia coli ATCC 12408 using the thiobarbituric acid and semicarbazide methods for its estimation points to the presence of a KDO trisaccharide unit in the structure. On partial hydrolysis of the lipopolysaccharide by the method of Painter, using polystyrene sulphonic acid, up to 60% of the KDO was lost. A core oligosaccharide, with a molecular weight in the region of 3500 to 5000 and containing heptose, glucose, galactose, KDO and phosphorus in the molar ratio of 2:1.44:0.6:0.3:1.3, was isolated on fractionation of the dialysate on Sephadex G‐15. Sodium borohydride reduction of the oligosaccharide results in the loss of both reducing power and KDO, indicating a linear‐type structure with KDO only at the reducing end. Removal of phosphate from the oligosaccharide had no significant effect on its molecular weight. Periodate oxidation, followed by borohydride reduction, and methylation studies showed the presence of certain structural features similar to those already found for the core region of lipopolysaccharides from other gram‐negative bacteria.
Deficiency in Initiation Factors of Protein Synthesis in Stationary‐Phase Escherichia coli
Authors: Scheps Ruth , Revel Michel
Published: March 2005

Initiation factor activities for natural messenger RNA translation are markedly reduced in extracts from stationary phase Escherichia coli, as compared to growing cells. Immunological techniques demonstrate that the amount of initiation protein IF3 is actually decreased in non‐growing cells. These changes result in a modified mRNA specificity in the stationary cell extracts.
Studies on Seed‐Oil Triglycerides
Authors: Gurr Michael I. , Blades Jon , Appleby Robert S.
Published: March 2005

Until 10–12 days after flowering the lipid of Crumbé seeds accounts for less than 1% of the total seen weight. Most of this lipid is phospholipid and glycolipid, the fatty acid composition of which resembles that of green tissue.

After 10–12 days the oil content of the seed rises rapidly, due mainly to the synthesis of new triglyceride. Oil synthesis levels off at about 30 days when the oil comprises 35% of the weight of the seed.

The fatty acids characteristic of green tissue, C16:0, C18:2 and C18:3 decrease in proportion to the total fatty acids as the seeds mature. Erucic acid, which is not present before 6 days rises rapidly from 8 days to becomes the major seed oil fatty acid in the mature seeds (53%). Oleic acid and C22:1 increase in proportion reaching a maximum at 12 days

The triglycerides of the mature seed oil are composed of 5 major molecular species, having 2, 3, 3, 4 and 5 double bonds per molecule, respectively.

Position two is occupied entirely by C18 fatty acids with Δ9 unsaturation, namely C18:1, C18:2 and C18:3. No erucic acid occurs at position 2. The acids at position two are cleaved preferentially by the lipase from Geotrichum candidum.

Position 1 is occupied preferentially by saturated acids or erucic acid. Position 3 is occupied almost entirely by erucic acid.
Modification of Protein Properties by Change in Charge
Authors: Shiao Daniel D. F. , Lumry Rufus , Rajender Shyamala
Published: March 2005

Succinylated derivatives of bovine chymotrypsinogen A and α‐chymotrypsin were prepared by treatment of the native proteins with succinic anhydride; 60% of the 6‐amino groups of the 13 lysine residues form stable succinylated products. The large negative charge at neutral pH is reflected in electrophoretic and titration behavior but absorption spectra, rotatory‐dispersion spectra and reversible unfolding behavior indicate that the succinylated species are conformationally similar to the parent proteins. Succinylated chymotrypsin is active as an esterase with N‐acetyl‐l‐tryptophan ethyl ester with a small decrease in the maximum‐velocity parameter. The pH dependence suggests that three ionizing groups influence catalytic behavior. The apparent pKa values analyzed in terms of three groups are shifted relative to α‐chymotrypsin. Thermal‐unfolding studies of succinylated chymotrypsinogen demonstrate the presence of sub‐states near pH 7 but is consistent with two‐state behavior at other pH values.
Investigations of the Structure of Glutamine Synthetase from Pig Brain
Authors: Stahl Jürgen , Jaenicke Lothar
Published: March 2005

Glutamine synthetase from pig brain was purified to complete homogeneity. The sedimentation behavior, direct electron optical evidence and tracing of the difference spectra during disaggregation and denaturation by urea and detergents at different pH values and different ionic strengths lead to the conclusion that the enzyme (s= 15 S, ƒ/ƒ0= 1.26, Mr= 370000) is a dihedral, cubic octamer which consists of two enzymatically inactive half molecules (s= 8.6 S, ƒ/ƒ0= 1.36, Mr= 185000). These tetrads are formed in 2 M urea and, by dialysis against buffer, reaggregate to the native enzyme. In the tetramer, the monomers (s= 2.8 S, Mr= 46000) are in one plane, narrowly and relatively strongly associated. The dissociation of the binding forces, which is irreversible, is only possible at high concentrations of denaturants. With low concentrations of dodecylsulfate, the enzyme forms a catalytically active complex which, below pH 6.5, stacks together and precipitates in the form of helical aggregates. On addition of more dodecylsulfate, inactive dissociates arise which bind a maximum of 130 molecules of detergent per subunit. Although pig brain and sheep brain glutamine synthetases are protein‐chemically, biophysically and immunologically strikingly similar, hybridization of their tetrameric half molecules did not succeed.
Circular Dichroism of Avian‐Erythrocyte Chromatin and Ethidium Bromide Bound to Chromatin
Authors: Williams Ross E. , Lurquin Paul F. , Seligy Verner L.
Published: March 2005

The circular dichroism of mature gander erythrocyte chromatin was compared with that of partially dehistonized chromatin and deproteinized DNA. The ellipticity at 275 nm for native chromatin was found to be three‐times less than that for DNA. The 275‐nm transition was gradually increased in magnitude upon removal of histones. Selective removal of the very lysine‐rich histone I and the erythrocyte‐specific histone V, but not histone I alone, resulted in a significant increase in transcription in vitro and ethidium bromide primary binding properties of chromatin. Concomitant with these changes a “blue shift” of the positive DNA band from 282 nm for native chromatin to 275 nm for purified DNA was observed.

Optimal conditions for circular dichroism studies in ethidium bromide bound to DNA and native chromatin were determined.

The circular dichroic spectra of ethidium bromide bound to native chromatin and partially deproteinized chromatin were found to be essentially the same as that exhibited by ethidium‐bromide · DNA complexes. However, the ellipticity of the dye bound to native chromatin was smaller than that when ethidium bromide was bound to DNA. The 310‐nm dye transition was increased after the removal of chromosomal proteins. Selective removal of histone I and V was found to generate the most notable increment in ellipticity.

The represent results are consistent with recent interpretations on how ethidium bromide interacts with chromatin; native chromatin has fewer ethidium bromide primary binding sites than deproteinized DNA; the mode of this dye interaction in these two systems is nearly the same, if not identical.
The Molecular Structure of 6,7‐Dimethyllumazine‐N‐1‐β‐d‐ribofuranoside, a Uridine Derivative in the syn‐Conformation
Authors: Saenger Wolfram , Ritzmann Götz , Pfleiderer Wolfgang
Published: March 2005

The molecular structure of the uridine analog 6,7‐dimethyllumazine‐N‐1‐β‐d‐ribofuranoside has been determined from 4114 three‐dimensional X‐ray data. The substance crystallized in the monoclinic space group P21 with two crystallographically independent molecules per asymmetric unit. The intensity data were measured diffractometrically, and the positions of the 46 non‐hydrogen atoms were located by direct methods. The atomic parameters were refined by full‐matrix least‐squares methods to a discrepancy index of 5.9%. Both molecules are in the unusual syn conformation with C(2′)‐endo sugar puckering and a gauche, gauche conformation about the C(4′)‐C(5′) bond; thus an intramolecular O(5′)‐H … O(2) hydrogen bond can be formed. The molecules are packed such that the “hydrophobic” pteridine heterocycles are stacked in endless ribbons which are separated by regions containing the sugar residues. The lumazine substituents at O(2), O(4) and N(3)‐H but not N(5) and N(8) are involved in hydrogen bonding. A discussion of the possible biochemical consequences are given.
Protein A from Staphylococcus aureus
Authors: Sjöholm Ingvar , Ekenäs Anna‐Karin , Sjöquist John
Published: March 2005

Protein A, a cell wall protein from Staphylococcus aureus, has been chemically modified by acetylation with acetylimidazole. All the four tyrosines present in the molecule as well as 40 out of 52 lysines were acetylated. The conformation of protein A was not changed by the acetylation.

The ability of protein A to react with the Fc fragment of immunoglobulins was decreased to 40%, but was restored to 60%, when acetylated protein A was O‐deacetylated with hydroxylamine. The reactivity of protein A against specific rabbit antiserum was not decreased by acetylation. The significance of unmodified tyrosines and lysines for the protein A reactivity is discussed.
Nucleotide Sequences of Fragments of R17 Bacteriophage RNA from the Region Immediately Preceding the Coat‐Protein Cistron
Authors: Adams Jerry M. , Cory Suzanne , Spahr Pierre F.
Published: March 2005

The known nucleotide sequence preceding the coat protein cistron in R17 bacteriophage RNA has been extended by 23 residues towards the 5′‐terminus of the RNA. 32P‐labelled R17 RNA was partially digested with Escherichia coli ribonuclease IV and the resulting fragments were resolved by electrophoresis on polyacrylamide gels. Fingerprinting techniques were then used to identify fragments with sequences overlapping the known sequence for the ribosomal‐binding site at the beginning of the coat protein cistron. Three such fragments (59, 31 and 26 nucleotides long) were found routinely and a slightly larger fragment was found in one experiment using a different batch of enzyme. The nucleotide sequences of the fragments were studied by established radiochemical techniques and all four fragments were found to have the same 3′‐terminus. The nucleotide sequence determined for the largest fragment is A‐A‐U‐C‐A‐G‐G‐C‐A‐A‐C‐G‐G‐C‐U‐C‐U‐C‐U‐A‐A‐A‐U‐A‐G‐A‐G‐C‐C‐C‐U‐C‐A‐A‐C‐C‐G‐G‐A‐G‐U‐U‐U‐G‐A‐A‐G‐C‐A‐U‐G‐G‐C‐U‐U‐C‐U‐A‐A‐C‐U‐U‐U where AUG is the initiation codon for the coat protein cistron. The sequence preceding this AUG contains three possible nonsense codon but whether any of them is actually used to terminate synthesis of the R17‐A protein is not known; most likely the untranslated sequence between the A protein and coat protein cistrons is at least 26 residues long. The sequence reported here shows little homology with the known sequences preceding the R17‐A protein and RNA synthetase cistrons nor with any of the sequences preceding the cistrons in Qβ phage RNA; however, it is almost identical to the sequence preceding the coat protein cistron of the closely related phage f2. The nucleotide sequence which we have determined can be folded into the form of two hydrogen‐bonded loops.
Étude de la dissociation, par le désoxycholate de sodium, des polyparticules ribonucléoprotéiques nucléaires du cerveau de rat
Authors: Stévenin James , Jacob Monique
Published: March 2005

The action of sodium deoxycholate (10g/l) on nuclear particles containing DNA‐like RNA from rat brain was studied. Under our experimental conditions, the size of the RNA remained unchanged when the particles were treated with deoxycholate. Therefore, modifications of the properties of the particles cannot be attributed to the release of latent RNAases by the detergent.

The density, in CsCl, of the particles increased from 1.40 to 1.46–1.64 g/ml and their sedimentation coefficient decreased 30 to 50%, depending on the conditions of treatment with deoxycholate.

It was calculated that an average 65% loss of proteins (as determined by density measurements) should cause a 32% reduction in the sedimentation coefficient of 150 S particles; the observed reduction was 39%. The agreement indicates that deoxycholate did not reveal hidden breaks in RNA, but only released proteins.

Native particles were converted to 40‐S monoparticles upon mild ribonuclease hydrolysis, as is already known. Their density decreased slightly (from 1.41 to 1.39 g/ml) corresponding to a 10% loss of RNA. Complexes of a density between that of particles and released proteins were never observed. Such complexes would be expected if the RNA was progressively hydrolysed without modification of the protein moiety. In contrast, when the enzymatic action was allowed to proceed, particles disappeared progressively without further change in density.

The behaviour of deoxycholate‐treated particles upon ribonuclease hydrolysis was different. The enzyme transformed the treated particles of high density (1.48 g/ml) into 5–20 S ribonucleoproteins of density 1.40 g/ml. Complexes of lower density were not detected.

The appearance of fragments of density similar to that of native particles during RNAase action on treated particles, as well as the absence of complexes of density lower than 1.39 g/ml during RNAase action on native particles, are in favour of the type “folded ribonucleoprotein strand” structure for the monoparticle. The experimental results are in contradiction with the informofer structure proposed by Georgiev's group. The reasons for the discrepancy are not clear.
Integrity of the DNA Template, a Prerequisite for the Faithful Transcription of Xenopus rDNA in vitro
Authors: Hecht Ralph M. , Birnstiel Max L.
Published: March 2005

RNA polymerase from Micrococcus luteus transcribes in vitro the cistrons specifying ribosomal RNA in Xenopus laevis DNA. The rDNA template is transcribed asymmetrically in the same sense as in vivo; furthermore the rDNA spacer which is not transcribed in vivo is also poorly transcribed by the bacterial RNA polymerase. The essential conditions for such near fidelity to rDNA transcription in vivo is the use of native rDNA which exhibits a high, single‐strand molecular weight of 8–10 × 106. Regardless of the cation present during the reaction it is shown by critical hybridization and hybridization‐competition studies that 90% of the RNA synthesized on this template (cRNA) is complementary to the ribosomal cistrons. It is also shown that in short incubation the M. luteus RNA polymerase initiates RNA synthesis preferentially near the 18‐S cistron and that some of the transcribed spacer DNA coding for the RNA excess of the ribosomal 40–S precursor is located adjacent to the 18‐S cistron. In longer incubations the “intact”, large molecular weight primer allows the polymerase to read through to the 28‐S cistron while “nicked” rDNA inhibits this polymerization.

Both “nicked” or “intact” native rDNA produce an asymmetric cRNA but the latter exhibits a higher degree of asymmetry and loci specificity. Denatured rDNA produces a cRNA which shows no strand specificity and no loci specificity, i.e. 50% of the cRNA is complementary to each strand of rDNA and about half its sequences are complementary to the spacer DNA. In support, the cRNA is capable of competing out all labelled 28‐S and 18‐S RNA hybridization. It is also shown that the RNA sequences complementary to spacer rDNA are unique to rDNA and are not located elsewhere in the genome of Xenopus laevis.
Magnetic Circular Dichroism of Tryptophanyl Residues in Proteins
Authors: Mcfarland Thomas M. , Coleman Joseph E.
Published: March 2005

The magnetic circular dichroism (MCD) induced in the wavelength region of absorption by the aromatic chromophores of a number of proteins has been examined. The major contribution to the MCD is from the tryptophanyl chromophores and to a much lesser extent the tyrosyl chromophores. The magnetically induced CD of tryptophanyl residues is independent of the environment of the residue as indicated by the insensitivity of the MCD to protein denaturation. These characteristics make MCD a valuable analytical tool for the determination of tryptophan in proteins. Determinations of tryptophan content by this method on 9 proteins containing 0–6 tryptophanyl residues per molecule are reported. Certain features of MCD and natural CD are compared with particular attention to azurin and several mammalian carbonic anhydrases which have large natural ellipticity bands associated with the aromatic chromophores.
Optical Properties of the Hexon of Adenovirus
Authors: Day Loren A. , Franklin Richard M. , Pettersson Ulf , Philipson Lennart
Published: March 2005

The ultraviolet absorbance, circular dichroism (CD) and optical rotatory dispersion of the hexons of adenovirus type 2 have been measured and analyzed in terms of secondary structural features. A curve‐fitting method for the CD spectra from 190 nm to 240 nm indicates 170%‐α‐helical, 26%β, and 56%, “random” conformations; the classical parameter b0, indicates from 15 to 20%α‐helical conformation. The far ultraviolet absorbance indicates considerable hypochromicity in comparison to the absorbance expected from the chromophore composition of the protein, which indicates very high α‐helical content. We have applied corrections for absorbance flattening in the analysis of all the spectra and demonstrate the relatively large magnitude of such corrections in the regions of intense absorbance for this protein complex of molecular weight around 400000. In spite of such corrections, the discrepancy in apparent α‐helical contents indicated by ultraviolet hypochromism and by optical rotatory methods persists.
Some Physicochemical Properties of Protein A from Staphylococcus aureus
Authors: Björk Ingemar , Petersson Bengt‐Åke , Sjöquist John
Published: March 2005

The molecular weight of protein A, isolated from Staphylococcus aureus by lysostaphin digestion, was found to be 42000 by sedimentation equilibrium analyses and by gel chromatography on Sepharose 6B in 6M guanidine hydrochloride. Hydrodynamic studies revealed a frictional ratio of 2.1—2.2 and an intrinsic viscosity of 29 ml/g. Both these parameters suggest that protein A is not a typical globular protein but rather has a markedly extended shape.
Isolation of Active Ribosomal Subunits from Yeast
Authors: van der Zeijst Ben A. M. , Kool Addy J. , Bloemers Henri P. J.
Published: March 2005

We have designed a method for the large scale isolation of pure and highly active subunits from yeast cytoplasmic ribosomes. The method consists of the incubation of yeast protoplasts in the presence of 1 mM NaN3. This causes the disappearence of polysomes and the formation of 80‐S ribosomes that can be dissociated in 0.5 M KCl. In this way over 90% of the ribosomes can be dissociated into subunits. The subunits are isolated by centrifugation in a B‐XV zonal rotor using an equivolumetric sucrose gradient. When this separation is performed at 5°C, dimerization of 40‐S particles to 65‐S particles occurs and no pure subunits can be obtained. At 10°C, there is no dimerization and the 60‐S + 40‐S subunits obtained are less than 1% cross‐contaminated. In poly(U)‐directed protein synthesis one subunit pair polymerizes phenylalanine residues at a rate of 1.7 residue per min. About 70% of the subunits were estimated to participate in this synthesis. Subunits prepared in the absence of dithiothreitol still bound phenylalanyl‐tRNA, but were inactive in phenylalanine polymerization. The 60‐S subunits prepared without dithiothreitol were as active as those prepared in the presence of dithiothreitol when their peptidyltransferase activity was tested.