bluuebunny/crossref_metadata_2025 huggingface dataset:
A Conformational Study of Glucagon
Authors: Gratzer W. B. , Beven G. H. , Rrattle H. W. E. , Bradbury E. M.
Published: March 2005

glucagon is a polypetide of 29 residues, which can be crystallised. Optical rotatory dispersion in dilute neutral, acid and alkaline solution suggests that the polypeptide chain is largely random, but contains about one turn of α‐helix, which is eliminated in 6 M guanidine hydrochloride. Using thermal difference spectra, nuclear magnetic resonance, and the temperature‐dependence of the optical retatory dispersion as criteria, it appears that glucagon possesses no difined tertiary structure in solution, but may be regarded as being in a state of α‐helix ⇌ random coik equilibrium, near the high‐temperature end of the transition range. That there is no steric objection to the formation of a highly α‐helical conformation is demonstrated by optical rotatory dispersion measurements in 2‐chloroethanol solution, in which the protein becomes largely helical. A rough calculation indicates that in aqueous solution at pH 2 the transition mid‐point is at about –40%%. From changes in the proton magnetic resonance spectrum when glucagon is transferred from an aqueous environment into 6 M guanidine hydrochloride, it is deduced that the short helical segment in the chain in the aqueous state contains most of the long aliphatic side chains, and since the sequence is known it has been possible to identify such as segment uniquely near the C‐terminal end. On standing in acid solution the vicosity increases and a birefringent gel is formed. Sedimentation studies indicate the formation of large aggregates. On further standingor warming, a precipitate appears which has the appearance of long fibrils in the electron microscope. Infrared spectra of the gel, of solid films and of the precipitated material show that in all these states the glucagon is in the form of antiparallel β‐chains.
Regulation of the Enzymes of the β‐Ketoadipate Pathway in Moraxella calcoacetica
Authors: Cánovas J. L. , Johnson B. F.
Published: March 2005

Mutants of Moraxella calcoacetica which synthesize constitutively β‐ketoadipate succinyl‐CoA transferase, but in which all other enzymes of the β‐ketoadipate pathway are still normally inducible, have been isolated by selection for the ability to grow with β‐ketoadipate. The constitutively synthesized transferase was identified as transferase III. This enzyme is induced in the wild type by adipate and its physiological function is probably adipate activation. The properties of these constitutive mutants confirm that the synthesis of transferase III is regulated independently of the synthesis of enzymes that mediate reactions of the β‐ketoadipate pathway.

Two phenotypically different classes of mutants which synthesize transferase II constitutively have also been isolated by the same selective procedure. Both classes of mutants synthesize constitutively the five enzymes that convert catechol to β‐ketoadipyl‐CoA. Strict coordinacy between four of these enzymes (muconate lactonizing enzyme, muconolactone isomerase, β‐ketoadipate enol‐lactone hydrolase II and β‐ketoadipate succinyl‐CoA transferase II) is maintained in one class of mutants. However in the other class this coordinacy is destroyed and muconate lactonizing enzyme is synthesized in succinate‐grown cells at a relative level much lower than those of the other three enzymes. These findings are discussed and evaluated in terms of earlier evidence concerning the regulation of the synthesis of enzymes of the catechol branch by M. calcoacetica.
The Synthesis of One‐Carbon Units from CO2 in Clostridium kluyveri
Authors: Jungermann K. , Thauer R. K. , Decker K.
Published: March 2005

Clostridium kluyveri was grown (a) on standard media, in which alternately [14C]bicarbonate, [U‐14C], [1‐14C], and 2‐14C]acetate plus ethanol were the labeled substrates, and (b) on standard media supplemented with either formate, glycine or serine, in which bicarbonate or formate were 14C‐labeled. Adenine, guanine and methionine were isolated from the cells and degraded.

(a) Carbon atoms 2 and 8 of the purines were found to be synthesized from CO2 (about 70%) and from the methyl group (about 20%) and the carboxyl group (about 10%) of acetate. The S‐methyl group of methionine was shown to be derived almost equally from CO2 (about 50%) and from the two‐carbon substrates, the methyl group of acetate accounting for about 35% and the carboxyl group for about 15%.

(b) The participation of CO2 in the synthesis of the S‐methyl group of methionine remained unaffected (about 50%) in the presence of serine, while it was decreased to below 5% in the presence of formate and increased to over 80% in the presence of glycine. The participation of formate was in the order of 55%.

These data demonstrate that (1) two major competitive pathways for the synthesis of onecarbon units exist in Clostridium kluyveri: C1‐units are formed predominantly by a reduction of CO2 and to a minor degree by a cleavage of serine, the usual pathway (C3‐serine = C2‐acetate) and (2) the relative rates of the two pathways are controlled by the glycine requirement of the growing organism.
Reactions of Enolic Sugar Derivatives
Authors: Herrmann K. , Lehmann J.
Published: March 2005

Chemically synthesized TDP‐d‐[U‐14C,5‐3H]glucose was converted to TDP‐4‐keto‐6‐deoxy‐d‐[U‐14C,6‐3H]glucose by an enzyme system system obtained from Escherichia coli B. The product contained only a minor amount of the tritium originally present in the starting material. Elimination of tritium from the starting material paralleled the formation of the keto‐product. This result is interpreted as indicating that a 5,6‐unsaturated hexose nucleotide is an intermediate in the overall conversion. Part of the tritium eliminated from the 5‐position of d‐glucose was reincorporated into the 6‐position of the end product. The mechanistic implications of these results are discussed.
Interactions between Neutral and Dibasic Amino Acids for Uptake by the Rat Intestine
Authors: Robinson J. W. L.
Published: March 2005

Kinetic studies on the interaction between neutral and dibasic amino acids for absorption by the rat intestine have been performed. l‐Arginine uptake is stimulated by l‐citrulline, the kinetics formally revealing “uncompetitive” stimulation. l‐Isoleucine inhibits l‐arginine uptake “uncompetitively”. In the case of l‐methionine and l‐leucine, stimulation occurs at low concentrations of the neutral amino acid, but inhibition occurs at higher concentrations. The double reciprocal plots bisect in the first quadrant. Arginine inhibits the uptake of both methionine and citrulline, the kinetics being consistent with partially competitive inhibition. These kinetic situations were observed by means of steady‐state observations, but appear to be paralleled when initial velocities are determined.

Efflux of arginine from a preloaded tissue is stimulated by external neutral amino acids, though not to such an extent as by arginine itself. Similarly, the efflux of neutral amino acids is stimulated to a lesser extent by external arginine than by the same or different external neutral amino acids.

Intracellular methionine inhibits arginine efflux, and the efflux coefficient for arginine, determined from isotopic exchange experiments, is depressed by methionine. These results suggest that the stimulation of net arginine uptake by methionine may be explained in terms of an inhibition of arginine efflux. Intracellular citrulline and isoleucine have no effect on arginine efflux, however.

Preloading with neutral amino acids produces the same effect on arginine uptake as the presence of these amino acids in the incubation medium. Although preloading with unlabelled arginine has no influence on the subsequent uptake of labelled arginine, it does tend to neutralise the isoleucine‐inhibition or the citrulline‐stimulation of arginine uptake. Methionine stimulation is, on the other hand, not affected by this preloading. This and other results suggest an intracellular locus of action of citrulline and isoleucine. Indeed these two amino acids seem to act in a similar manner but with an opposite effect on arginine uptake.
Guanosine Triphosphate Dependent Enzymic Binding of Aminoacyl Transfer Ribonucleic Acid to Yeast Ribosomes
Authors: Ayuso M. S. , Heredia C. F.
Published: March 2005

The binding of phenylalanyl‐tRNA to purified yeast ribosomes at low magnesium concentrations is dependent upon one of the yeast supernatant transfer factors (factor A). This reaction requires GTP. The GTP analogue guanylyl methylene diphosphonate cannot be substituted for GTP but it acts as a good inhibitor. After incubation of the ribosomes with phenylalanyl‐tRNA, factor A and GTP practically no compound other than phenylalanyl‐tRNA itself is found linked to the ribosomes.

In addition to this enzymic type of binding, phenylalanyl‐tRNA is also bound to yeast ribosomes in the absence of factor A and GTP provided that the magnesium concentration is increased to about 20 mM. This non‐enzymic binding is effectively inhibited by deacylated tRNA and this inhibition is prevented if factor A and GTP are included in the incubation mixtures.

The binding of N‐acetylphenylalanyl‐tRNA to yeast ribosomes also requires GTP, and is dependent upon factor(s) other than the amino acid transfer factors, which are bound to the ribosomes.
Zusammensetzung der Phospholipide und deren Fettsäuremuster in Kaninchenerythrocyten und ghosts
Authors: Gercken G. , Brockmann U.
Published: March 2005

The phospholipids of rabbit erythrocytes were separated by thin‐layer chromatography on silica gel HR and determined quantitatively by the molybdenum blue method. Prior to thinlayer chromatography phosphatidic acid and phosphatidyl inositol were concentrated by column chromatography.

The total phospholipid contents were 313 ± 27 amoles1 per erythrocyte and 289 ± 13 amoles per ghost. The proportions of the individual phospholipids of erythrocytes in moles % were 36.3 of phosphatidyl choline, 32.6 of phosphatidyl ethanolamine, 15.7 of sphingomyelin, 12.4 of phosphatidyl serine, 1.6 of phosphatidic acid, 1.3 of phosphatidyl inositol, and < 0.1 of lysophosphatidyl choline. The same distribution was obtained in ghosts with the exception of a significantly higher concentration of sphingomyelin.

The fatty acid composition of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, ceramide trisaccharide, and sphingomyelin was analysed by gas‐liquid chromatography after the lipids had been fractionated by preparative thin‐layer chromatography.

For identification of the fatty acids, their methylesters were isolated on silica gel G‐silver nitrate plates according to degree of saturation.

Typical of sphingolipids were the low concentration of linoleic acid, the absence of arachidonic acid, the presence of lignoceric acid and the very high concentration of nervonic acid.

Phosphatidyl choline and phosphatidyl ethanolamine were hydrolysed by phospholipase A (ophiophagus hannah) in order to analyse the fatty acid distribution of the two positions. In position 1 of phosphatidyl choline and phosphatidyl ethanolamine the saturated fatty acids amounted to 83.6% and 65.6%, respectively. Unsaturated fatty acids, especially oleic acid and linoleic acid, also occurred in position 1. These acids were the main components in position 2.
Structure‐Function Relationships for some Metalloalkaline Phosphatases of E. coli
Authors: Lazdunski C. , Petitclerc C. , Lazdunski M.
Published: March 2005

The alkaline phosphatase of Escherichia coli is a zinc metalloprotein with a metal to enzyme ratio of 4. Inorganic phosphate protects the four zinc atoms against removal by EDTA. Cadmium and manganese can replace zinc on its four binding sites. Samples of phosphatase with different proportions of zinc and cadmium or of zinc and manganese have been prepared and tested for their enzymatic activity. These results together with others suggest a model which involves two functional and two structural zinc atoms. The zinc atoms necessary for the activity would have different apparent binding constants for the apoenzyme. This conclusion is in agreement with Cohen and Wilson's scheme postulated in 1966.

Dissociation constants for Me2+‐apophosphatase complexes were determined (Me2+= Zn2+, Ni2+, Cd2+, Co2+, Mn2+). These constants follow the order Ni2+ > Co2+ > Zn2+ > Cd2+= Mn2+. The optical rotatory dispersion spectra of these different metallophosphatases are very similar between 25° and 55° suggesting little change in protein structure. However, only Co2+ and Zn2+ alkaline phosphatases are active enzymes.
Microsomal Marker Enzymes and their Limitations in Distinguishing the Outer Membrane of Rat Liver Mitochondria from the Microsomes
Authors: Brunner G. , Bygrave F. L.
Published: March 2005

Repeated washing of rat liver mitochondria with sucrose medium removes all of the cyto‐chrome P‐450 while low but significant amounts of glucose 6‐phosphatase and NADPH cyto‐chrome c reductase are still retained. At the same time monoamine oxidase and succinate cyto‐chrome c reductase activities increase slightly. Cytochrome P‐450 and glucose 6‐phosphatase are completely retained by the microsomes even after five washings with sucrose medium.

The specific activities of glucose 6‐phosphatase and of NADPH cytochrome c reductase in the outer membrane of rat liver mitochondria subfractionated by “swelling‐shrinking” are about 4‐ to 8‐fold greater than in the intact mitochondria and can range from about 20–60% of the value found for microsomes. The cytochrome P‐450 content of the outer membrane fraction is less than 3 % of that of the microsomes.

From a consideration of these data it is concluded that NADPH cytochrome c reductase and glucose 6‐phosphatase are intimately associated with the outer membrane of rat liver mitochondria whereas cytochrome P‐450 is not. The limitations of these enzymes as microsomal “markers” is discussed.
Pyruvate Dehydrogenase, Substrate Specificity and Product Inhibition
Authors: Bremmer J.
Published: March 2005

Studies with intact mitochondria and with soluble pyruvate dehydrogenase indicate that pyruvate and α‐ketobutyrate are oxidized by the same enzyme (pyruvate dehydrogenase), while α‐ketovalerate is oxidized by a different enzyme.

Pyruvate and α‐ketobutyrate have about the same affinity for the enzyme, but pyruvate is oxidized at a much higher rate.

Acetyl‐CoA and propionyl‐CoA both behave as competitive inhibitors to CoA. The Ki for both is slightly higher than the Km for CoA. Accordingly the enzyme is only moderately inhibited by a high acetyl‐CoA/CoA ratio.

NADH behaves mainly as a competitive inhibitor to NAD. The Ki is significantly lower than the Km for NAD. Accordingly the enzyme is strongly inhibited by a high NADH/NAD ratio.

The significance of these properties of the enzyme for the regulation of the activity of pyruvate dehydrogenase in vivo is discussed.
The Effect of Antibiotics on the Coded Binding of Peptidyl‐tRNA to the Ribosome and on the Transfer of the Peptidyl Residue to Puromycin
Authors: ČErná J. , Rychlík I. , Pulkrábek P.
Published: March 2005

The effect of antibiotics on the coded binding of peptidyl‐tRNA to the ribosome and the transfer of the peptidyl residue from peptidyl‐tRNA to puromycin has been studied. The coded binding of polylysyl‐tRNA was inhibited only by tetracyclines, the binding of acetylphenyl‐alanyl‐tRNA was inhibited by tetracyclines and streptomycin and stimulated by spiramycin and carbomycin. The most potent inhibitors of the puromycin reaction with polylysyl‐tRNA were the macrolides erythromycin, spiramycin, carbomycin and oleandomycin, inhibiting by 50% at a concentration of 0.1 μM; neomycin (1 μM), chloramphenicol (100 μM), tetracyclines (100 μM), streptomycin (50 μM) and streptovitacin A (1 mM) were somewhat less effective. The puromycin reaction with acetylphenylalanyl‐tRNA was inhibited by antibiotics in a similar way, only erythromycin stimulated this reaction at concentrations between 0.1 and 500 μM.
An Analysis of the Polyoma Virus DNA‐RNA Hybridization Reaction in vitro
Authors: Hudson J. B.
Published: March 2005

A study was made of the parameters involved in the hybridization reaction between Polyoma virus DNA and its complementary RNA, with a view to establishing conditions suitable for routine measurement of the viral specific RNA synthesized in vivo. In general the reaction obeyed the same principles as those determined for the Escherichia coli DNA‐RNA system. An interesting result was the finding that the neutralized “random cyclic‐coil” form of Polyoma DNA had as good a hybridization capacity as the heat‐denatured naturally occurring double‐stranded circular form, under certain conditions; an observation which has important relevance to the structure of the cyclic‐coil DNA. An analysis was also made of the “competition hybridization” reaction, leading to the development of conditions minimizing the non‐specific effect displayed by various types of RNA.

Under the conditions derived for optimal assay, the level of viral RNA synthesis was found to be several times greater than previously reported values.
Fractionation in a Density Gradient of some Glycoproteins from Human Ovarian Cyst Fluids
Authors: Dunstone J. R.
Published: March 2005

Equilibrium density gradient sedimentation in caesium chloride has been used as an effective method of separating the components of sulphite‐reduced sparingly soluble glycoproteins from two human ovarian cyst fluids. An almost quantitative recovery of the carbohydrate and peptide constituents has been obtained. Some fractions had overall chemical compositions similar to those isolated previously by Dunstone and Morgan in 1965, while others contained much greater proportions of amino acids, especially of aspartic acid, glutamic acid and cystine.

The results suggest that the sparingly soluble glycoproteins are large macromolecules containing serologically active carbohydrate chains, bound to specific peptide fragments which form an integral part of larger macromolecules, the insolubility of which is due to the presence of –S‐S‐bonds.

The experiments emphasize the value of equilibrium density gradient sedimentation in the study of complex proteinpolysaccharide systems.
20β‐Hydroxysteroid : NAD Oxidoreductase Activity towards 3β‐Hydroxypregn‐5‐en‐20‐one Sulphate and Related Free Steroids
Authors: Pocklington T. , Jeffery J.
Published: March 2005

The sulphate of 3β‐hydroxypregn‐5‐en‐20‐one is now reported to be a substrate for crystalline 20β‐hydroxysteroid: NAD oxidoreductase prepared from Streptomyces hydrogenans. Utilization of this steroid sulphate was not independent of activity towards pregn‐4‐ene‐3,20‐dione or 3β‐acetoxypregn‐5‐en‐20‐one, but the total rate of reaction for equimolar mixtures of 3β‐hy‐droxypregn‐5‐en‐20‐one sulphate and either pregn‐4‐ene‐3,20‐dione or 3β‐acetoxypregn‐5‐en‐20‐one was less than that calculated for the simplest type of mutual competitive inhibition, particularly at low substrate concentrations.

The Michaelis constant for NADH was similar (about 5 μM) with each of these compounds as substrate.

3β‐Hydroxypregn‐5‐en‐20‐one, and its 17α‐hydroxy derivative behaved anomalously, and their behaviour is discussed. 3β,17α,21‐Trihydroxypregn‐5‐en‐20‐one was a good substrate and had kinetic constants similar to those of 17α,21‐dihydroxypregn‐4‐ene‐3,20‐dione.
Action de la canavanine sur la synthèse du RNA chez E. coli
Authors: Simonnet G. M. , Chapeville F.
Published: March 2005

Incorporation of uracil or UDP into RNA does not take place in Escherichia coli arg− RCstr in absence of arginine. Canavanine, a structural analogue of arginine, partially reestablishes RNA synthesis.

In the strain E. coli arg− RCrel where RNA synthesis takes place in absence of arginine, canavanine shows inhibition. RNA synthesis will start again in this strain, if canavanine is removed from the culture medium. The presence of chloramphenicol together with canavanine suppresses the inhibitory effect of the latter upon RNA synthesis.

In both arg− strains RNA synthesis will proceed, if nucleoside triphosphates are furnished to the bacteria. Canavanine has no effect on the utilisation of nucleoside triphosphates.

The experiments reported suggest that canavanine acts upon the triphosphonucleoside kinases which appear to be inhibited by this arginine analogue in the relaxed strain.
Interactions of Ribosomal RNA with Synthetic and Natural Polybases
Authors: Krèumers P. , Fredericq E.
Published: March 2005

The formation of complexes between RNA and calf thymus histone or polysine has been studied at molar ratios (basic groups to phosphoric groups) in the range 0.1 to 0.2.

The flexible polyelectrolyte behavior displayed by RNA in its reduced viscosity curves at low ionic strength disappears in the presence of the polybases.

The thermal profiles of RNA are shifted to slightly higher temperatures by the polybases.

Measurements of optical rotatory dispersion indicate an increase of ordered structure in the presence of the polybases, mostly polylysine.

Ultraviolet spectra of RNA have been measured from 186 to 300 nm, in native condition, at low ionic strength and at pH 3.2. Strong hyperchromic effects appear in the 188 nm absorption band under denaturing conditions. In the presence of histone or polylysine, the hyperchromic effects are markedly lowered.

It is concluded that both polybases combine to RNA, strengthening and extending the ordered areas and stiffening the molecular structure.
Étude par dichroïsme circulaire des interactions hème‐flavine‐protéine dans le cytochrome b2 (l‐lactate oxydoréductase) de la levure
Authors: Iwatsubo M. , Risler J. L.
Published: March 2005

The circular dichroism spectra of bakers' yeast l‐lactate dehydrogenase and some of its derivatives (without flavin and/or heme) have been investigated in order to obtain some information about the interactions between the prosthetic chromophores or between these chromophores and the protein moiety.

The following derivatives were studied: (a) the cytochrome b2 (flavohemoprotein), (b) the apocytochrome b2 (following FMN removal), (c) the noyau cytochromique b2 (low molecular weight core isolated following proteolytic degradation), (d) the apo‐noyau cytochromique b2 (obtained by removal of the heme from the latter).

It is shown that the circular dichroism spectra of the protein moieties in the ultraviolet region (200–250 nm) are not affected, either by the removal of the flavin from the flavocytochrome b2, or by the removal of the heme from the noyau cytochromique b2. The significance of the values calculated for the α‐helical content is discussed. The oxido‐reduction state of the ferric ion does not affect the circular dichroism spectra in this region.

The circular dichroism bands associated with the heme chromophore are essentially the same in the apocytochrome b2 and in the noyau cytochromique b2, i. e. both without the flavin moiety, but are markedly changed in the flavocytochrome b2, in particular with an inversion of the strong γ band.

Noticeable differences are observed with a homologous enzyme extracted from the yeast Hansenula anomala, especially in the γ band region.

In conclusion there appears to be no prosthetic group‐protein interaction detectable by circular dichroism measurements, i. e. no important modification of the peptide bond geometry occurs when the flavin is removed from the enzyme or when the oxido‐reduction state of the heme is varied. However, a strong effect is exerted by the flavin on the symmetry of the heme environment, this effect being probably an indirect one, via the protein moiety, as discussed in this paper.
Étude en culture continue du phénomène de répression transitoire
Authors: Gaillard R.
Published: March 2005

A chemostat is used to study the kinetics of establishment of β‐galactosidase catabolite repression in unrepressed cultures of Escherichia coli. A state of catabolite repression near to zero is obtained in a continuous culture in which the growth is limited by the carbon source. An excess of carbon source is abruptly added into the culture vessel at the beginning of the kinetics. The transient repression phenomenona can be obtained both with a constitutive strain and with inducible strains in the presence of an inducer. They can be obtained with one substrate, the concentration of this substrate alone being changed during the kinetics.

After a phase of severe repression of short duration, it is possible to observe another phase of resumption of the β‐galactosidase synthesis which does not follow the normal law of enzyme synthesis. During this phase the amount of β‐galactosidase is growing linearly as a fonction of time, while the cells are growing exponentially. The continuation of this linear synthesis is not affected by an abrupt slowing down of the cell growth. This independence between the β‐galactosidase synthesis and the growth can be explained by uncoupling between the transcription of the last messenger RNAs and their translation into enzyme. It must be assumed that during the linear synthesis the existing messenger RNA is stabilised by the physiological conditions of catabolite repression.
Factors in the Iodination of Histidine in Proteins
Authors: Wolff J. , Covelli I.
Published: March 2005

Factors that determine the reactivity toward iodine of the histidyl residues of various proteins are compared. The proteins studied include lysozyme, ribonuclease, insulin, lactic, malic and glutamic dehydrogenases, phosphoglucomutase, thyroglobulin, trypsin, chymotrypsin and β‐lactoglobulin. Relatively fewer histidyl residues than tyrosyl residues are iodianated at pH 8.5 both in water and in guanidine. The fraction of iodine present in histidyl residues increases with increasing pH. Diiodohistidine is formed primarily in proteins that do not contain tryptophane.

Thyroglobulin can be iodinated to contain 8% histidine iodine. However, mono‐ and diiodohistidine formation in equilibrium‐labelled thyroids accounts for < 1% of the total iodine.

Histidine iodination requires the dissociable proton of the imidazole ring as shown by lack of iodination of N‐methylated model compounds. The assumption that iodination proceeds by an attack on the imidazole anion (rather than via an N‐I intermediate) is supported by the finding that the sterically hindered 2‐t‐butyl imidazole is iodinated.

It is concluded that, although conformational factors such as electrostatic facilitation and ligand binding influence the reactivity of certain histidyl residues, the major factors in histidyl iodination are the instrinsic properties of the residue.
Biosynthesis of 18‐Hydroxytestosterone in the Human Foetal Liver
Authors: Lisboa B. P. , Gustafsson J.‐Å.
Published: March 2005

After incubation of testosterone with 105 000 X g microsomes of human foetal liver, a C19 steroid with a 3‐oxo‐Δ4‐structure was isolated by ascending one‐dimensional thin‐layer chromatography. Gas chromatography‐mass spectrometry revealed that the compound was identical with 18‐hydroxytestosterone. The significance of this finding in relation to earlier reports of 18‐hydroxylated steroids in human urine is discussed.
The Acylation of Diacylglycerols in Pig Liver
Authors: ÅKesson B.
Published: March 2005

A pig liver microsomal fraction catalyzed the formation of radioactive triacylglycerols and 1,2‐diacyl‐sn‐glycerols when incubated with different X‐1,2‐diacylglycerols, ATP, CoA, MgCl2 and radioactive fatty acids.

Analysis of the radioactive diacylglycerols suggested that they were formed by acylation at position 1 of 2‐monoacylglycerols, which were produced by hydrolysis of the added X‐1,2‐diacylglycerols.

No difference between 1,2‐diacyl‐sn‐glycerols of different degree of unsaturation and various rac‐1,2‐diacylglycerols as substrates for triacylglycerol and diacylglycerol formation could be detected.

1,3‐Dioleoyl‐sn‐glycerol was about half as effective as rac‐1,2‐dioleoylglycerol as substrate for triacylglycerol synthesis and caused no formation of radioactive diacylglycerols.
Preparation of Crystalline Nucleoside Diphosphate Kinase from Baker's Yeast and Identification of 1‐[32P]Phosphohistidine as the Main Phosphorylated Product of an Alkaline Hydrolysate of Enzyme Incubated with Adenosine [32P]Triphosphate
Authors: Edlund B. , Rask L. , Olsson P. , Wålinder O. , Zetterqvist Ö. , Engström L.
Published: March 2005

A nucleoside diphosphate kinase has been highly purified from baker's yeast, and has been obtained in a crystalline form from an ethanol‐containing medium. During short‐time incubation with [32P]ATP, the enzyme was phosphorylated to an extent of about 3–4 phosphoryl groups per mole of enzyme assuming a molecular weight of 105. This indicates an intermediate phosphorylation of the enzyme. 1‐[32P]Phosphohistidine was shown to be the dominating radioactive degradation product in an alkaline hydrolysate of the 32P‐labelled enzyme. In addition, 3‐[32P]‐phosphohistidine and Ne‐[32P]phospholysine were obtained in small amounts.

The results indicate that the amino acid sequence around the reactive 1‐phosphohistidine of the yeast enzyme is different from that of human erythrocytic and bovine liver nucleoside diphosphate kinase.
Composition of Rat Liver Triacylglycerols and Diacylglycerols
Authors: ÅKesson B.
Published: March 2005

Different fractions of rat liver triacylglycerols and X‐1,2‐diacylglycerols were prepared by thin‐layer chromatography on silver nitrate‐containing silica gel.

The positional distribution of fatty acids in these fractions and unfractionated triacylglycerols and diacylglycerols was analyzed by a scaled‐down version of Brockerhoff's method.

Both in tri‐ and diacylglycerols position 1 contains mainly saturated fatty acids, while most fatty acids in position 2 are unsaturated. Position 3 in triacylglycerols contains mainly unsaturated fatty acids and has a large proportion of highly unsaturated fatty acids.

The relative abundance of different 1,2‐diacyl‐sn‐glycerol units in diacylglycerols and triacylglycerols was similar except for highly unsaturated units. This suggests a random utilization of 1,2‐diacyl‐sn‐glycerols with 1–3 double bonds for triacylglycerol biosynthesis. The highly unsaturated diacylglycerols probably originate from phospholipids.

From the positional distribution of fatty acids in total triacylglycerols the proportions of different triacylglycerol fractions were predicted from the assumption that each fatty acid in any position is combined with fatty acids in proportion to their relative amounts in the other positions. The observed distribution agreed well with the predicted one‐.
Dihydropteridine Reductase
Authors: Nielsen K. H. , Simonsen V. , Lind K. E.
Published: March 2005

The activity of the enzyme dihydropteridine reductase, which catalyses the reduction of quinoid dihydropteridine cofactor in the presence of NADH or NADPH, was measured in a system containing H2O2 and peroxidase. Peroxidase and H2O2 continually reoxidize the reduced tetrahydropteridine cofactor to the quinoid dihydropteridine cofactor, and thus maintains the concentration of the latter virtually constant. A method for calculation of the actual concentration of dihydropteridine cofactor and for correction of the dihydropteridine reductase activity to allow for variations in the dihydropteridine cofactor concentration have been derived. Even though dihydropteridine reductase can utilize either NADH or NADPH as the reductant, the former is 15 to 45‐fold more active than the latter. This specificity for the nucleotide cofactor has been found for dihydropteridine reductases prepared from five differnt mammalian species.
Purification and Properties of a Trypsin Inhibitor from Barley
Authors: Mikola J. , Suolinna E.‐M.
Published: March 2005

A trypsin inhibitor present in ungerminated barley in relatively high concentration (0.45 mg/g) has been obtained in apparently pure form. The inhibitor is a heat‐stable protein. Molecular weights of 14400 and 14055 were calculated from the sedimentation equilibrium and amino acid composition respectively. 1 μg of purified inhibitor inhibits 1.6–1.9 μg of pure trypsin in hydrolyses of casein, haemoglobin, and benzoyl‐dl‐arginine‐p‐nitroanilide. The inhibitor preparations are totally inactive against chymotrypsin and pepsin, proteolytic enzymes of germinating barley, and a number of microbial proteinases.
Ribonucleoside Diphosphate Reductase. Purification of the two Subunits, Proteins B1 and B2
Authors: Brown N. C. , Canellakis Z. N. , Lundin B. , Reichard P. , Thelander L.
Published: March 2005

Ribonucleoside diphosphate reductase from Escherichia coli B consists of two non‐identical subunits, called proteins B1 and B2. After purification from extracts of a partially derepressed thymine‐requiring mutant, both subunits were obtained in essentially pure form.

Criteria of purity included the demonstration of constant specific enzyme activity in peaks from final chromatographic or electrophoretic steps and the behaviour of the proteins during ultracentrifugation and analytical gel electrophoresis.

Determinations of molecular weights by sedimentation equilibrium centrifugation gave values between 160000 and 200000 for protein B1 and 78000 for protein B2. Evidence was obtained for some dissociation of protein B1 during centrifugation.

Separately, each subunit was completely inactive; together, under the proper conditions, they catalyzed the reduction of the 5′‐diphosphates of cytidine, uridine, adenosine and guanosine to the corresponding deoxyribonucleotides.

With the exception of deoxyadenosine 5′‐triphosphate, the regulatory effects of nucleoside triphosphates were identical to those observed earlier with less pure preparations of enzyme. The general inhibitory effect of deoxyadenosine 5′‐triphosphate at concentrations higher than 1 μM was obtained as before. In addition, lower concentrations of this nucleotide had a novel stimulatory effect on the reduction of cytidine diphosphate.
14CO2‐Bildung aus [3‐14C]Serin in roten Blutzellen
Authors: Rapoport S. , Müller M. , Knöfel R.
Published: March 2005

Substrate‐free incubated erythrocytes and reticulocytes of rabbits form 14CO2 from [3‐14C]‐serine.

The 14CO2 formation from [3‐14C]serine exhibits a maturation‐dependent maximum, which is about ten times higher than that of erythrocytes.

The production of 14CO2 from [3‐14C]serine requires the splitting off of the hydroxymethyl group with formation of glycine. Serine undergoes neither desamination nor transamination. This is indicated by the fact that neither pyruvate, nor lactate or alanine are labelled. The β‐carbonatom of [3‐14C]serine is found in the porphyrines and the 14CO2 formed from serine is diluted by inactive formate.

In erythrocytes the 14CO2 is formed exclusively from formate by the peroxidative action of catalase. If catalase is irreversibly inhibited with 3‐amino‐1,2,4‐triazole the 14CO2 formation from [3‐14C]serine, is completely suppressed.

In reticulocytes there exists a second catalase‐independent pathway of 14CO2 formation from [3‐14C]serine, which is proposed to be as follows: condensation of formyl‐tetrahydrofolate with succinyl‐CoA with formation of α‐ketoglutar‐semialdehyde, its oxidation to α‐ketoglutarate and oxidative decarboxylation to succinate. This pathway is indicated by the dilution of 14CO2 from [3‐14C]serine by glycine and particularly by the incorporation of the β‐carbon‐atom of serine in α‐ketoglutarate.
Circular Dichroism of Bovine Plasma Albumin—Dye complexes
Authors: Shechter E.
Published: March 2005

We report on the circular dichroism induced in two dyes, 2,7‐dibromo‐4‐hydroxy mercurifluorescein and pyridoxal‐5‐phosphate, bound to bovine plasma albumin. We show that the optical activity induced in 2,7‐dibromo‐4‐hydroxy mercurifluorescein, covalently bound to the unique sulfhydryl residue of the protein, makes it possible to follow the pH dependent transitions of bovine plasma albumin occurring in the pH ranges 3 to 5 and 6 to 8. The optical activity induced in the dye is important only for the contracted state of the protein obtained near the isoionic point. On the other hand we show that the optical activity induced in pyridoxal‐5‐phosphate, a dye probably bound to the ɛ‐amino group of a particular lysine residue, is independent of pH between pH 5 and 8.
Optical Properties of Oriented Dye‐LiDNA Complexes Prepared with the Wet Spinning Method
Authors: Rupprecht A. , Rigler R. , Forslind B. , Swanbeck G.
Published: March 2005

For the study of the spatial arrangement of dyes bound to DNA a recently developed wet spinning method for DNA has been modified in order to prepare highly oriented dye‐DNA complexes. After bathing in alcoholic solutions containing LiCl and either acridine orange, proflavin or chrysazin, spun LiDNA deposits were dried and thereby coalesced into films of highly oriented dye‐LiDNA complexes. The method is reproducible and it is possible to obtain samples of practically any desired dimensions.

The maxima of absorption and emission spectra were characteristic for these dyes when bound to DNA. The films exhibited a strong negative dichroism. The dichroic ratios indicated that the absorption vectors of these dyes would be inclined more than 65° to the DNA helix axis. Preliminary polarized fluorescence measurements have been performed using the recently developed method of Nishijima and coworkers. To analyze the fluorescence intensity curves the recent theoretical study of Desper and Kimura has been extended for uniaxial orientation (The Appendix) to account for the occurrence of non‐coinciding absorption and emission vectors of the single chromophoric group. The application of this extended theory to simple models indicated that the angles of inclination to the DNA helix axis of the absorption and emission vectors of acridine orange and proflavin would be confined to the interval 65°–90°. The angles between the absorption and emission vectors in these dyes were estimated to lie within an interval of 20°–30°. It is concluded that improved experimental conditions and use of a computer to analyze the polarized fluorescence curves would give more precise information.

Wide‐angle X‐ray diffraction photographs of the specimens showed the undistorted crystalline B form pattern which might indicate that the dye molecules have not penetrated the DNA crystallites in the present modification of the wet spinning method. Other methods for introducing the complexing agents in the wet spinning method are discussed and the wide applicability of these samples of oriented DNA complexes for physico‐chemical studies is stressed.
The Conversion of N‐Formyl‐l‐Aspartate into Nicotinic Acid by Extracts of Clostridium butylicum
Authors: Scott T. A. , Bellion E. , Mattey M.
Published: March 2005

The rate of synthesis of nicotinic acid by partially purified cell extracts of Clostridium butylicum was increased when the substrates l‐aspartate and formate were replaced by N‐formyl‐l‐aspartate. Aminopterin (0.2 mM) inhibited the synthesis from l‐aspartate and formate, but not from N‐formyl‐l‐aspartate. N‐Formyl‐d‐aspartate did not support the synthesis of nicotinic acid.

In the presence of aminopterin, radioactivity from 14C‐labelled N‐formyl‐aspartate was incorporated into nicotinic acid by partially purified cell extracts with only low dilution of the molar specific radioactivity.The pattern of labeling in the synthesised nicotinic acid showed that C‐2, 3, 7, and 6 of the nicotinic acid were derived from C‐2, 3, 4 and the formyl carbon of N‐formyl‐aspartic acid.

Partially purified extracts of Cl. butylicum synthesised N‐formyl‐aspartic acid from aspartate and formate and this formylation was inhibited by 0.2 mM aminopterin.
Thrombosthenin M
Authors: Cohen I. , DE Vries A. , Bohak Z. , Katchalski E.
Published: March 2005

Thrombosthenin M was isolated from equine blood platelets by lysis of the platelets with n‐butanol at pH 7.0 at low ionic strength (I= 0.05), separation of the insoluble thrombosthenin by centrifugation, and extraction of thrombosthenin M from the sediment with 0.02 M pyrophosphate buffer pH 9.5. Contaminating ribonucleic acid was digested with ribonuclease and the protein finally purified by gel filtration on Sephadex G‐200. The purified thrombosthenin M was found to be free of thrombosthenin A and of fibrinogen. Thrombosthenin M, similarly to actin‐free muscle myosin, possesses a Ca++‐activated but not a Mg++‐activated ATPase activity, and shows neither a drop in viscosity nor superprecipitation on addition of ATP. Ultracentrifugal analysis of the thrombosthenin M preparation obtained (pH7.0, I= 0.6) revealed that about 90% of the protein sediments with a sedimentation coefficient of 7.2 S and about 10% with a sedimentation coefficient of 33 S. The thrombosthenin M preparation yielded a single band on disc electrophoresis; some of the protein failed, however, to penetrate the gel.

Incubation of thrombosthenin M with thrombin resulted in the appearance of one mole of carboxyl terminal arginine per 105 g of thrombosthenin M. The ATPase activity of the thrombosthenin M was not affected by thrombin. Incubation of equine platelets with thrombin resulted in the disappearance of the electrophoretic band corresponding to thrombosthenin M. Antiserum against thrombosthenin M was found to inhibit the clot retraction of thrombin‐treated platelet‐rich plasma.
Labeling of α‐P of Nucleoside Triphosphates by in vivo Incorporation of 32P in Rat Liver
Authors: Landin R. M. , Moulé Y. , Gaye P.
Published: March 2005

Rat liver RNAs labeled in vivo by 32P and hydrolyzed by snake venom phosphodiesterase into 5%‐nucleotides show a specific radioactivity always higher for AMP than that found for GMP, UMP and CMP. The difference of labeling is found on ribosomal and nuclear RNA as well as on the internal chain of tRNA.

The high labeling of AMP of [32P]RNA compared to the other 5′‐phosphates is not due to contaminants but to the very high radioactivity of the α‐P of the ATP precursor in comparison to that of other nucleoside triphosphates, especially after a short incorporation time. The inequality of labeling decreases with time.
A Method for the Large Scale Preparation of the Avian Erythrocyte Specific Histone F2C
Authors: Johns E. W. , Diggle J. H.
Published: March 2005

A new method is described for the large scale preparation of the avian erythrocyte specific histone, F2C. The method is based on the selective extraction of F2C, using perchloric acid, subsequent to the removal of the very lysine‐rich histone, F1, with 0.5 M NaCl.
Metabolism and Excretion of C19‐ and C18‐ Steroids by Human Skin
Authors: Oertel G. W. , Treiber L.
Published: March 2005

After intravenous administration of [7α‐3H]dehydroepiandrosterone [35S]sulfate (3H/35S = 2.88) to a normal male subject various 3H‐labeled C19‐ and C18‐steroid metabolites could be isolated from the fractions of free steroids and double‐labeled sulfoconjugates (3H/35S = 3.16) in skin extracts, containing a total of 1.36% of the injected 3H‐activity. The specific activity of 3H‐labeled steroids in skin extracts was found to be significantly lower than that of corresponding compounds in the 24‐hour urine.

Whereas in skin extracts up to 95% of steroids present were isolated from the fractions of sulfoconjugates, more than 90% of steroids in sweat extracts could be detected in the fraction of free steroids.

From such data, as well as from the qualitative and quantitative composition of steroid fractions in skin and sweat it was concluded, that under in vivo conditions endogenous sulfoconjugated dehydroepiandrosterone undergoes a direct metabolism in human skin tissue. Subsequently, sulfoconjugated metabolites probably suffer hydrolysis prior to excretion in sweat.
Zn2+ and Co2+‐Alkaline Phosphatases of E. coli
Authors: Lazdunski C. , Lazdunski M.
Published: March 2005

For the Zn2+‐phosphatase, the ratio k3/k2 (pH 8.4) of the dephosphorylation (k3) and phosphorylation (k2) rates is very similar, about 1.4–1.7, for substrates which are very different chemically such as p‐nitrophenyl phosphate, β‐glycerophosphate, 5′‐AMP and ATP. This means that k2 is nearly the same for all these substrates. On the other hand with o‐carboxyphenyl phosphate, phosphorylation is the determining step.

The Co2+‐enzyme has a much lower specific activity than the Zn2+‐phosphatase. The substitution of Zn2+ by Co2+ decreased the rate of phosphorylation by a factor of 6.4 for o‐carboxyphenyl phosphate and 16 for the above mentioned substrates; no such decrease is observed on k3. The phosphorylation is inhibited by nucleophilic agents which otherwise activate the Zn2+‐enzyme in the same conditions. The Co2+‐phosphatase offers a unique opportunity to compare the free energy of binding of the different substrates to form the Michaelis complex; those free energies are strikingly similar, they vary between – 6.1 and – 7.0 kcal. This is further evidence that specificity of the enzyme is not directed against the R part of RO PO3 even in such a peculiar case as ATP. Neither the Zn2+ nor the Co2+‐enzyme are able to catalyse the hydrolysis of a P‐N bond. Organic phosphates (P‐O bond) bind better to the phosphatase than p‐chloro‐anilido‐phosphonic acid (P‐N bond). The exchange of Zn2+ by Co2+ decrease only slightly the affinity for the active center of organic substrates or inhibitors; a factor of 1.78 for o‐carboxyphenyl phosphate and 2.65 for p‐chloroanilido‐phosphonic acid. The difference is more pronounced for inorganic phosphate; a factor of 23 (pH 9.16) and 27 (pH 8.5).

Two ionisable groups are important for the binding of the substrate to the active center. One must be in the basic form, it has a pK of 7.1 and 7.4 in the Zn2+ and Co2+‐phosphatase, respectively. The other must be acidic, the corresponding pK values are 8.6 and 8.9, respectively. With both metallo‐enzymes the phosphorylation step is pH‐independent between pH 6 and 9. The dephosphorylation step is dependent upon the ionization of a group which must be in the basic form and has a pK of 7.5 for the Zn2+‐phosphatase.

The enzyme remains dimeric in the course of the catalysis and it seems that there is only one active site phosphorylated per dimer.

k
2 and k3 are decreased in 2H2O indicating possibly rate‐determining proton transfers. The enthalpy and entropy of activation corresponding to both steps have been calculated.
Purification of the Staphylococcal 6‐Phospho‐β‐d‐Galactosidase
Authors: Hengstenberg Wolfgang , Penberthy William K. , Morse Melvin L.
Published: March 2005

The 6‐phospho‐β‐d‐galactosidase of Staphylococcus aureus has been purified and partially characterized. The native protein, and the sodium dodecyl sulfate treated protein, have a molecular weight of about 50 000. The amino terminal amino acid was found to be threonine. Some kinetic data such as Km, Vmax, pH optimum, heat inactivation, and inactivation by certain metal ions are reported.
Acetolactate Decarboxylase from Aerobacter aerogenes
Authors: Løken Jan P. , Størmer Fredrik C.
Published: March 2005

Acetolactate decarboxylase has been highly purified from A. aerogenes.

The purified enzyme does not need manganese for its activity as earlier described, and it is able to decarboxylate acetohydroxybutyrate, the second intermediate in the biosynthesis of isoleucine. The pH optimum in phosphate is 6.2–6.4.