bluuebunny/crossref_metadata_2025 huggingface dataset:
ESR‐ und CD‐Spektren von pH‐abhängigen Konformationsisomeren des Hämoglobin(III)
Authors: Gersonde Klaus , Wollmer Axel
Published: March 2005

The native horse haemoglobin(III) forms two different pH‐dependent detergent associates. These two associates were related to the existence of two conformation isomers of the protein [11]. By aid of the direct verification of the conformation isomers on the native haemoglobin(III) the possibility should be excluded that the observed associates both originate from only one haemoglobin conformation, which undergoes a pH‐depending transformation of the protein structure in the course of the detergent association.

ESR und CD techniques confirm the existence of two isomers of the native haemoglobin. According to these findings haemoglobin(III) is a heterotropic allosteric protein, in which one (at room temperature) or two (at 77° K) proton binding sites interact with the binding site of the 6th ligand mediated by conformation. This interaction can directly be demonstrated in the ESR spectrum of the OH‐derivative by the appearance of distinct signals for both isomers (g′1= 2.68, g′2= 2.16, g′3= 1.82; g″1= 2.68, g″2= 2.14, g″3= 1.77).

The allosteric and protolytic reactions are described as a circuit process. By aid of computer simulation model curves are calculated, which allow determination of the actual allosteric and protolytic pK‐values. A simulation of the pH‐dependency of the ellipticity at 340 and 290 nm fits the experimental data, if allosteric pK‐values of 7.2 and 7.0 and protolytic pK‐values of 8.3 and 8.5 are presumed.

The α‐helix content remaining constant in the pH‐range concerned, the change of conformation affects the tertiary structure only.

Because of the common pH‐dependency the further three low spin signals (g1= 2.91, g2= 2.23, g3= 1.95) have to be attributed to the H2O‐Hb(III) in addition to the high spin signals (g′= 5.9, g′= 2.0), which characterize the low spin part in the physical high spin‐low spin equilibrium of this complex.

Further the origin of the conformational active CD bands is discussed.
Isotope Derivative Method for Determination of Microquantities of Inorganic Pyrophosphate in Biological Material
Authors: Flodgaard Hans
Published: March 2005

An isotope derivative method for determination of inorganic pyrophosphate (PPi) in biological material in amounts down to 10 pmoles is described. The method is based upon measurements of radioactivity in [5‐3H]UTP formed in UDP‐glucose (UDPG) pyrophosphorylase reaction using a highly purified preparation of UDPG pyrophosphorylase from calf liver and [5‐3H]UDPG. The optimal conditions for reaction between [5‐3H]UDPG and PPi was brought about by addition of phosphoglucomutase, NADP+ and glucose‐6‐phosphate dehydrogenase. The method was specific and no influence from endogenous metabolites could be demonstrated. By this method the total concentration of PPi in rat liver and guinea pig liver was found to be in the range from 0.5 mM to 1.4 mM.
Peptide Chain Initiation in Homologous and Heterologous Systems From Mitochondria and Bacteria.
Authors: Sala Francesco , KÜNTZEL Hans
Published: March 2005

Native ribosomal subunits from mitochondria of Neurospora crassa bind fMet‐tRNA and synthesize fMet‐puromycin at 5 mM Mg 2+. The reactions are stimulated by the trinucleotide ApUpG and by GTP. After washing the subunits with 1 M NH4CI the binding capacity is lost but can be restored by adding initiation factors from Escherichia coli

Unwashed mitochondrial 73 S ribosomes require E. coli initiation factors for binding fMet‐tRNA and forming fMet‐puromycin. Cytoplasmic 77 S ribosomes from Neurospora are inactive in these chain initiation reactions, even in the presence of bacterial initiation factors.

The requirement of the initiation reactions for AUG, GTP and initiation factors is similar in systems from E. coli, Bacillus subtilis and Neurospora mitochondria.

Ribosomes and initiation factors from E. coli and B. subtilis are completely interchangeable.

The results suggest that mitochondria from Neurospora use a bacteria‐like chain initiation mechanism.
Carbohydrate Components of Extraneuronal Gangliosides from Bovine and Human Spleen, and Bovine Kidney
Authors: Wiegandt Herbert , BÜCKING Hans Walter
Published: March 2005

The gangliosides from bovine spleen and kidney as well as human spleen, were extracted from the tissues. The carbohydrate‐portion of the gangliosides was characterized. Bovine spleen gangliosides and those isolated from bovine kidney contained the same sialo‐oligosaccharide moieties. Eleven different ganglosides from the bovine tissues and five gangliosides from human spleen were identified. They are derived from lactose, ganglio‐N‐triose, ganglio‐N‐tetraose, lacto‐N‐neotetraose or lacto‐N‐tetraose and differ in their content of sialic acid. Gangliosides from human tissue contain N‐acetyl‐neuraminic acid only. The sialic acid of the gangliosides from bovine spleen consist of either N‐acetyl‐neuraminic acid, N‐glycolyl‐neuraminic acid or both.
On the Antigenic Determinants of Glutamic‐Aspartic Transaminase
Authors: Patramani Iphigenia M. , Katsiri Katherine D. , Dimitropoulos Constantin G. , Kalogerakos Theodoros G. , Pavlatos Mary P. , Evangelopoulos Athanasios E.
Published: March 2005

Tryptic hydrolysates of denaturated glutamic‐aspartic transminase fractionated on a Sephadex G‐25 column, gave an elution diagram containing six main fractions. When peptide maps were developed from the transaminase digest 37 spots were detected on the paper sheet.

It was demonstrated that the transaminase fractions (groups of peptides) as well as well as some of the individual peptides eluted from the map spots, possessed immunological activity. This was proved by their capacity to interact with rabbit pig heart antienzyme and rabbit antiserum against the pig‐heart whole tryptic transaminase hydrolysate as well as to inhibit the homologous transaminase‐antitransaminase reaction. Higher immunological reactivity was foud among peptides moving faster in electrophoresis and chromatography.

The catalytic activity of rabbit transaminases (heart, liver, skeletal muscles), was inhibited to a substantial degree by the rabbit anti‐pig heart transaminase. The above enzymes however, as was otherwise expected, gave negative precipitin reactions with antitransaminase. A comparative study of the reactivity between transaminases from pig, ox and rabbit and rabbit anti transaminases against pig and ox heart enzymes as well as against pig‐heart whole tryptic transaminase hydrolysate is presented here.
Studies on Extrachromosomal DNA Elements
Authors: Goebel Werner
Published: March 2005

The replication of the colicinogenic factor Col E1 has been studied in two mutants of Escherichia coli with different thermosensitive lesions in DNA replication. One of these mutants E. coli ts 120/6 (Col E1) presumably synthesizes a thermolabile initiator for DNA replication. This mutant is not capable of supporting the synthesis of the Col E1 DNA at the restrictive temperature. The other mutant E. coli ts CR 34/43 (Col E1 DNA at the restrictive temperature at a reduced rate. The Col E1 DNA forms synthesized under the non‐permissive conditions are abnormal. The banding pattern of the DNA in ethidium bromide‐cesium chloride gradients and the sedimentation profile in sucrose gradients suggest that multiple circular and possibly catenated forms of Col E1 DNA are generated under these non‐permissive conditions.

Mutant E. coli ts CR 34/43 (Col E1) shows a high colicin production upon induction with mitomycin C at 43°, while mutant E. coli ts 120/6 (Col E1) fails to synthesize colicin at the restrictive temperature.
Conformation du DNA dans la nucléoprotéoprotéine
Authors: Wilhelm F. Xavier , Champagne Madeleine H. , Daune Michel P.
Published: March 2005

The nucleoprotein of chicken erythrocytes was dissociated by NaCl. The free histone and the residual complex were separated by centrifugation or gel filtration in order to obtain a series of complexes with variable protein content. For each complex the viscosity, circular dichroïsm spectrum and thermal profile were determined and discussed in terms of the configuration and conformation of DNA inside the nucleoprotein.

A very small amount of protein is able to provoke a folding of the DNA which then behaves as a rigid rod. Subsequent binding of histones (up to 20—30%) is accompanied by an increased flexibility and a coiled configuration.

In the case of a protein/DNA ratio of 0.9, the intrinsic viscosity of the nucleoprotein is four times smaller than the viscosity of DNA in the same condition. Such a decrease could indicate an organization of the DNA in a supercoiled structure.

The circular dichroïsm spectrum of the DNA inside the nucleoprotein is very peculiar and bears no resemblance to any known state of DNA (A form or denatured). It is only the positive band between 250 nm and 300 nm which is strongly reduced, the negative one being practically unaffected. Similar results were obtained with solutions of DNA of high salt content.

During the progressive stripping of histones the intensity of the positive band remains unchanged with the release of F1 and begins to increase when F2 and F3 are simultaneously liberated.

In contrast to this absence of any structural change of DNA with the binding of F1, it is remarkable that the same binding (or release) affects the stability of roughly 50% of the DNA molecule. Although F1 represents about 20% of the total histone, and owing to the cooperative nature of the melting, it seems clear that histones are unequally distributed along the DNA and that F1‐type histones predominantly cover large sequences of nucleotides.
Miscoding by Escherichia coli tRNAs for Methionine, Cysteine and Valine in the Synthesis of Rabbit Globin
Authors: Hunter Anthony R. , Jackson Richard J.
Published: March 2005

Methionine, valine and cysteine tRNAs from E. coli have been used in the reticulocyte transfer system and the products of transfer investigated. Fractionated methionine tRNAF and methionine tRNAM both transfer methionine into the two methionine containing peptides in rabbit globin, and also transfer it into other peptides not corresponding to globin peptides. Little difference was observed in the patterns of transfer of methionine tRNAF and methionine tRNAM. The occurrence of the extra peptides is thought to be due to miscoding by the methionine tRNAs for valine positions coded by GUG. Valine tRNA transfers mainly into globin peptides, but some valine is transferred in non‐globin peptides. αT1 and βT1 both receive valine from E. coli valine tRNA, so that no special valine tRNA can be required for the initiation of globin chains. Cysteine tRNA transferred no detectable cysteine into the two cysteine containing peptides, but it did transfer cysteine into some arginine positions. From a consideration of the patterns of miscoding it has been possible to make tentative codon assignments for certain valine and arginine positions of rabbit globin. Possible causes of miscoding by E. coli tRNA in the reticulocyte transfer system are discussed.
5′‐P‐Forming Exonuclease from Actinomyces sp. of Coelicolor Group, Strain No. 5 (Exonuclease A5) Purification and Some Properties
Authors: Tatarskaya Rachel I. , Lvova Tatyana N. , Abrossimova‐Amelyanchik Nina M. , Korenyako Alexandra I. , Bayev Alexander A.
Published: March 2005

A new exonuclease named exonuclease A5 has been isolated from the culture medium of Actinomyces sp. of the coelicolor group, strain No. 5 (classification after Krassilnikov). The enzyme exists in the culture broth, as a complex with some other component(s), from which it can be obtained in a free state by non‐ionogenic detergent OP‐7 treatment. Its purification procedure consists of the gel‐filtration of the salted out protein on Sephadex G‐75 and the double chromatography on DEAE‐Sephadex A‐50 at two different pH values. The specific activity of the purified enzyme reaches as a rule 2000 units per mg of protein. The enzyme is free of nucleosidase and deaminase. The nuclease/phosphatase ratio of the best preparations corresponds to 2000—4000. The exonuclease A5 splits RNA and denatured DNA into 5′‐mononucleotides and does not hydrolyze bis‐p‐nitrophenylphosphate. The cleavage begins from the 3′‐end of the polynucleotide chain. The enzyme does not discriminate many modified nucleotides. It hydrolyses T‐even phage DNA as well as tRNA, native or chemically modified. Polynucleotide chains containing no modified bases are split more rapidly however.

A peculiarity of the enzyme is its small sensitivity to the 3′‐terminal P. An oligonucleotide with 3′‐terminal P is split with the enzyme into mononucleosidediphosphate (3′‐end), dinucleosidemonophosphate (5′‐end) and 5′‐mononucleotides. Thus the nucleotide sequence of tri‐ and tetranucleotides can be readily established. Dinucleosidemonophosphates are split only with large enzyme amounts. Dinucleotides with 3′‐terminal P are resistant to its attack. The oligonucleotide sequence bearing 5′‐terminal P can be completely split into 5′‐mononucleotides, the last internucleotide bond at the 5′‐end being easily attacked.

The above mentioned properties of exonuclease A5 will probably make it useful for determining the nucleotide composition of polynucleotides containing unusual bases, as well as for end analysis of nucleic acid fragments.
Sequence Studies on Bence Jones Proteins
Authors: Alescio‐Zonta Lucilla , Baglioni Corrado
Published: March 2005

One Bence Jones protein and two Bence Jones fragments (fragmentary chains) of K type, and one Bence Jones protein and two fragments of L type have been analyzed. These proteins have been aminoethylated and digested with proteolytic enzymes. The resulting peptides were separated by different procedures.

The peptides obtained were analyzed and could be located in most cases in the amino acid sequence of the corresponding protein by homology with published sequences. These tentative sequences were particularly satisfactory in the case of the three K type proteins. These belonged to subtype KI and showed considerable homologies with other KI proteins. It was more difficult to position peptides of the three L type proteins on the basis of homology with published sequences. Several peptides of one protein (BJ 98) which were completely sequenced by Edman degradation combined with proteolytic digestion could not be positioned.

Amino acid analysis of peptides obtained from Bence Jones fragments confirmed that these correspond to the N‐terminal varible region of Bence Jones proteins for K type fragments, whereas they extend for three residues over the C‐terminal constant region for some L type fragments. In any case, no peptide from the constant region of light chains has been isolated from tryptic digests of Bence Jones fragments.
Fatty Acyl Transferase
Authors: Schweizer Eckhart , Lerch Irmgard , Kroeplin‐Rueff Luistraut , Lynen Feodor
Published: March 2005

A method is described for the isolation of radioactively labeled coenzyme A from yeast which was grown on [1‐14C]pantothenic acid. From about 800 g of wet cells 38 μmoles of chromatographically pure S‐benzoyl‐[14C]coenzyme A were obtained.

It was shown that purified yeast fatty acid synthetase catalyzes a fatty acyl transfer reaction between labeled and unlabeled coenzyme A. All saturated fatty acids with chain lenghts between 6 and 18 C‐atoms were transferred at about equal rates. Malonate was transferred about three times faster, acetate, propionate and butyrate about ten times slower than palmitate. Almost no transfer of crotonate and β‐hydroxybutyrate was observed. The transferase was resistant to N‐ethylmaleimide and iodoacetamide inhibition. It is suggested that the binding sites involved in the acyl transfer reactions are the non‐thiol substrate binding sites of the fatty acid synthetase complex, and not the “central” or “peripheral” SH‐groups.

Both transferase and total fatty acid synthetase are inhibited by long chain fatty acyl CoA compounds. This inhibition increases with concentration and chain length of the acyl CoA derivative. It is assumed that there is a barrier for the transfer of fatty acids other than acetate from the outer transferase binding site to the active center of the fatty acid synthetase complex, and vice versa. By a local conformational change induced by the end products palmitate and stearate, bound to the “central” SH‐group, this block is released to allow the acids to be transferred from the enzyme to external coenzyme A. Simultaneously, this conformational change is assumed to block further chain elongation.
Structure of Porcine Secretin
Authors: Mutt Viktor , Jorpes J. Erik , Magnusson Staffan
Published: March 2005

Porcine secretin is a heptacosapeptide with the amino acid sequence His‐Ser‐Asp‐Gly‐Thr‐Phe‐Thr‐Ser‐Glu‐Leu‐Ser‐Arg‐Leu‐Arg‐Asp‐Ser‐Ala‐Arg‐Leu‐Gln‐Arg‐Leu‐Leu‐Gln‐Gly‐Leu‐ValNH2. Fourteen of its twenty‐seven amino acids occur in the same position as they do in porcine glucagon.
Studies on the Metabolism of C21 Steroids in Rat Liver
Authors: Gustafsson Jan‐Åke , Lisboa Belisário P.
Published: March 2005

Progesterone was incubated with 105000 ×g liver microsome preparations from adult male rats and the hydroxylated 3‐oxo‐δ4‐steroids formed were isolated and identified by thin‐layer chromatography and gas chromatography‐mass spectrometry. The following compounds were characterized: 2α‐, 6β‐, 6α‐, 7α‐, 15α‐, 15β‐ and 16α‐hydroxyprogesterone.
Mass Spectrometric Characterization of Ceramides
Authors: Hammarström Sven
Published: March 2005

Bovine brain cerebrosides were degraded to ceramides on a milligram scale, using a procedure by Carter et al. These were separated into the 2‐hydroxy acid and non‐hydroxy acid containing ceramides and subsequently according to degree of unsaturation of the constituent acids. Four groups of ceramides were obtained. Each group was separated into molecular species by gasliquid chromatography as the trimethylsilyl ether derivative. Structures of the molecular species were determined by mass spectrometry.
Localization of Two Species Specific Antigenic Determinants on the Peptide Chains of Calf Skin Collagen
Authors: Pontz Bertram , Meigel Wilhelm , Rauterberg Jürgen , Kühn Klaus
Published: March 2005

Antibodies against two types of species specific determinants, designated P and S, can be differentiated in rabbit antisera against acid soluble calf skin collagen. Using the passive hemagglutination inhibition technique, they were localized with collagen α‐chains, with peptides obtained by treatment of the α‐chains with cyanogen bromide and with fragments isolated from collagenolytic digests of native collagen.

The P‐determinant is labile to proteolytic attack (pronase, chymotrypsin, pepsin). It was detected in a 300 Å region at the C‐terminus of both types of α‐chains. The S‐determinant is resistant to proteases and was found to occur in an interior region of the α2‐chain. The serological activity is contained in one of the collagenolytic peptides.
The Oxidation of Malate by Mitochondria Isolated from Cauliflower Buds
Authors: Macrae Alasdair R. , Moorhouse Ralph
Published: March 2005

Pyruvate is the major product of malate oxidation by isolated cauliflower bud mitochondria when pyruvate dehydrogenase is inhibited. Pyruvate formation, which requires Mg++, Mn++ or Co++, is stimulated by added NAD.

The effects of ADP, 2,4‐dinitrophenol and respiratory inhibitors on pyruvate formation indicate that the reaction is linked to mitochondrial electron transport.

No enzymic decarboxylation of oxaloacetate to pyruvate by the mitochondria can be observed. With added malate oxaloacetate breaks down in the mitochondria, probably to malate using NADH generated by a mitochondrial malic enzyme.

Acetyl‐CoA and glutamate, which compete for oxaloacetate in the mitochondria, do not inhibit pyruvate formation from malate.

A solubilised NAD requiring malic enzyme is present in sonicates of the mitochondria.

It is concluded that oxaloacetate is not an intermediate in pyruvate formation during malate oxidation by cauliflower bud mitochondria, and that a direct conversion of malate to pyruvate by a mitochondrial NAD requiring malic enzyme occurs.
Préparation de deux fractions d'histones par extraction saline
Authors: Wilhelm F. Xavier , Wilhelm Marcelle L. , Champagne Madeleine H.
Published: March 2005

A large‐scale procedure for the preparation of two histone fractions by salt dissociation of the chicken erythrocytes nucleoprotein is proposed. The very lysine‐rich histone is selectively removed from the nucleoprotein by NaCl in the range of 0.30 M to 0.55 M and the erythrocytes specific fraction in the range of 0.55 M to 0.80 M. Further purification of the two fractions is achieved by chromatography on a CM‐cellulose column at pH 7. This method enables us to prepare large quantities of these two histone fractions in a relatively short time (3 days). These two proteins are fairly pure as shown by amino‐acid analysis, N‐terminal amino‐acid determination and electrophoresis on polyacrylamide gel.

The α‐helical conformation of the two fractions was determined from their optical rotatory dispersion in aqueous and saline solutions. In saline solutions the histones have the highest percentage of α‐helix conformation (20% to 30%).

If a comparison is made between the optical rotatory dispersion spectrum of these histones prepared by salt extraction and the optical rotatory dispersion spectrum of the same histones prepared more drastically by acid extraction and precipitation, no difference can be detected in the limit of experimental error. If a two‐term Drude equation is used to analyse optical rotatory dispersion measurements, one can give evidence, especially in the case of the very lysine‐rich fraction, of a structure different from the α‐helix.
Metabolic Differentiation of Rabbit Skeletal Muscle as Induced by Specific Innervation
Authors: Golisch Günter , Pette Dirk , Pichlmaier Heinz
Published: March 2005

Cross‐innervations and re‐innervations were performed on the slow (“red”) m. soleus and the fast (“white”) m. extensor digitorum of the rabbit. Innervation of the slow soleus muscle with “fast” motor neurons causes a significant change of the metabolic type of the muscle. This transformation consists in a parallel increase of the activity levels of glycogen phosphorylase, of all the investigated glycolytic enzymes (phosphofructokinase, triosephosphate isomerase, triosephosphate dehydrogenase, phosphoglycerate kinase, glycerate phosphomutase, phosphopyruvate hydratase, lactate dehydrogenase), of adenylate kinase and mitochondrial glycerolphosphate dehydrogenase. A concomitant and parallel decrease is found in the activity levels of extramitochondrial hexokinase, and alanine aminotransferase, of all the investigated enzymes of the citric acid cycle (intramitochondrial citrate synthase, and succinate dehydrogenase, extra‐ and intramitochondrial malate dehydrogenase, and NADP isocitrate dehydrogenase), of intramitochondrial 3‐hydroxyacyl‐CoA dehydrogenase and glutamate dehydrogenase, and of extra‐ and intramitrochondrial aspartate aminotransferase. In spite of the marked changes in absolute activities, no alterations occur with regard to the ratios of enzymes and enzyme groups which by comparison of functionally different muscles have been classified as constant. Changes in the activity ratios concern the same enzymes which in previous studies have been shown to be characteristic of the metabolic type (phosphorylase/hexokinase, triosephosphate dehydrogenase/citrate synthase, lactate dehydrogenase/citrate synthase, triosephosphate dehydrogenase/3‐hydroxyacyl‐CoA dehydrogenase, adenylate kinase/creatine kinase). The alterations show that a conversion of the metabolic type from “slow” to “fast” takes place and indicate a specific influence of the innervation on metabolic differentiation in muscle, that is the control of a specific pattern of enzyme synthesis. Due to the obvious failure of innervation of the fast extensor muscle with “slow” motor neurons from the tibial nerve, no significant changes were observed in this muscle.
Presence of One Polypeptide Chain in Valyl and Isoleucyl tRNA Synthetases from Escherichia coli
Authors: Berthelot Francis , Yaniv Moshe
Published: March 2005

Determination of the molecular weight of valyl and isoleucyl tRNA synthetases under denaturing conditions leads to the conclusion that both enzymes are made of a single polypeptide chain of a molecular weight of 110000 approximately. The presence of one amino acid binding site per chain was determined by equilibrium dialysis.
Isolation and Characterization of Cyanogen Bromide‐Cleaved Peptides from a Collagenase Digest of α 1‐Chain of Calf Skin Collagen
Authors: Mark Klaus v. d. , Rexrodt Friedrich , Wendt Peter , Kühn Klaus
Published: March 2005

Molecular fragments of various lengths are formed by treating acid‐soluble collagen with collagenase from Clostridium histolyticum. Fragments 78, 105 and 125 nm long can be isolated by fractional ammonium sulphate precipitation. Their position in the collagen molecule has been determined by electron optical investigations. Denaturation of the individual fragments yielded peptides of the α1‐ and α2‐chains which could be separated by CM‐cellulose chromatography.

These peptides, α1 (78) and α1 (105), are in the region of two of those obtained by cyanogen bromide cleavage, namely, α1‐CB7 and CB6; α1 (125) is superimposed on α1‐CB3, CB7 and CB6. In order to obtain smaller fragments of these CB peptides of the α1‐chain, the three fragments from collagenase treatment were cleaved further with cyanogen bromide and the resultant peptides were isolated and characterized. Their position along the α1‐chain was determined electron microscopically after renaturation to the triple helical structure.

Each fragment of the α1‐chain yields two chromatographically and electrophoretically distinguishable peptides which have been designated α1 and α1*. The difference between the two was found to reside in the –COOH end of the molecule: the α1* peptide contains seven amino acids more than the α1.
The Identification of 3‐O‐Methyl‐l‐Rhamnose (l‐Acofriose) as Constituent of the Lipopolysaccharide of Rhodopseudomonas capsulata
Authors: Weckesser Jürgen , Mayer Hubert , Drews Gerhart
Published: March 2005

From the lipopolysaccharide of the gram‐negative bacterium Rhodopseudomonas capsulata (Athiorhodaceae) a lipophilic sugar was isolated by chromatographic procedures and characterized by mass spectrometry as belonging to the group of 3‐O‐methyl‐6‐deoxy‐hexoses. Demethylation showed that the parental sugar had the same properties in borate electrophoresis as rhamnose. Comparison with an authentic sample of 3‐O‐methyl‐rhamnose in gas liquid chromatography, paper chromatography and borate electrophoresis showed that the sugar under examination is 3‐O‐methylrhamnose (acofriose). Investigation of its optical rotation indicates that the sugar has the l‐configuration. l‐Acofriose (3‐O‐methyl‐l‐rhamnose) has not hitherto been found in lipopolysaccharides of gram‐negative bacteria.
A Soybean Proteinase Inhibitor
Authors: Harry John B. , Steiner Robert F.
Published: March 2005

Examination of the thermodynamic parameters and activation energies for the association of the Bowman‐Birk soybean proteinase inhibitor with trypsin and with chymotrypsin suggests that conformational changes may occur upon association. The data suggest that the nature of the two complexes may be somewhat different, and indicate that there is no pronounced interdependence of the kinetics, as well as the equilibria, for the sequential combination of the inhibitor with the two enzymes.
Studies on the Oxidative Fraction of Rabbit Muscle Glyceraldehyde‐3‐phosphate Dehydrogenase
Authors: Cantau Bernard N. , Jaureguiberry Ginette J. , Pudles Julio
Published: March 2005

In the work presented here, we have examined the most favorable conditions that lead to the isolation of the oxidative fraction of glyceraldehyde‐3‐phosphate dehydrogenase by chymotryptic hydrolysis.

NAD+ as well as glyceraldehyde‐3‐phosphate protect the apoenzyme against proteolysis and therefore do not favor the formation of the oxidative fraction. Nicotinamide mononucleotide, and d‐glyceraldehyde modify none of the properties related to the kinetics of proteolysis and do not change the yield in oxidative fraction.

Because it oxidizes the sulfhydril groups of the active center of the enzyme, o‐iodosobenzoic acid destroys the protective action of NAD+ with regards to proteolysis, but it also decreases the yield in oxidative fraction.

Only AMP has been found to render the apoenzyme more sensitive to chymotryptic digestion and to greatly increase the yield in the oxidative fraction.

The effects of glyceraldehyde‐3‐phosphate, NAD+ and AMP on the modification of the arsenolytic activity during chymotryptic hydrolysis have enabled us to determine the dissociation constants of the enzyme for these three effectors.

We have also examined the enzymatic properties of the purified oxidative fraction. Based on the two enzymatic activities retained in the purified oxidative fraction, the following values have been obtained:

Km for d‐glyceraldehyde = 61 μM, Km for NAD+= 120 μM, Km for p‐nitrophenylacetate = 6.3 μM.

The phosphorylating, phosphatase and NADH‐X activities are absent.

As a result of proteolysis, it is possible to dissociate three classes of catalytic functions: (1) the phosphorylating, phosphatase and NADH‐X activities, (2) the oxidative activity and (3) the esterase activity.
Cross‐Linkages in Collagen
Authors: Zimmermann Bodo K. , Pikkarainen Jarmo , Fietzek Peter P. , Kühn Klaus
Published: March 2005

Three different types of intermolecular bonds were demonstrated to be present in calf collagen.

Side‐to‐side bond: this fixes the quarter staggered array of the molecules within the fibrils and can casily be cleaved by pepsin.

Head‐to‐tail bond: this connects molecules by their dissimilar ends, is located in the region of the 30 nm overlap, and is relatively pepsin resistant.

End‐to‐end bond: this links adjacent molecules by their like ends, is located in terminal regions of the molecules, and can be easily cleaved by pepsin.

Polymeric molecules which were connected solely by end to‐end bonds were extracted from calf skin collagen. Brief treatment with pepsin cleaved side‐to‐side and end‐to‐end bonds in calf Achilles tendon collagen and yielded polymeric molecules which were linked exclusively by head‐to‐tail bonds. Polymeric chains (γ‐ and higher components) were isolated by gel chromatography subsequent to denaturation of end‐to‐end and head‐to‐tail polymers. Polymeric molecules containing the original cross linkages could be reformed by renaturation of these polymeric chains.
Specific Inactivation of Yeast Hexokinase Induced by Xylose in the Presence of a Phosphoryl Donor Substrate
Authors: DelaFuente Gertrudis
Published: March 2005

Yeast hexokinase can be inactivated by xylose in the presence of MgATP. The effect is highly specific for xylose. The nucleotide and divalent metal specificities are similar to those of the hexokinase reaction. The dissociation constants, as deduced from the effect of the concentration of the inactivating agents on the rate of inactivation, are about 0.2 mM for MgATP and about 10 mM for xylose. These values are similar to the Michaelis constant and Ki value for these compounds as substrate and competitive inhibitor, respectively, in the hexokinase reaction. Sugars capable of binding at the sugar substrate subsite protect against xylose‐induced inactivation with efficiencies closely related to their respective Ks or Ki values. The dissociation constant of glucose, as deduced from its protective effect, is about 0.04 mM.

When resting yeast is incubated aerobically with 0.1 M xylose and 1% ethanol, a marked loss of hexokinase activity takes place.

Among the possible mechanisms of this irreversible inactivation there is evidence against the involvement of phosphorylation or glycosylation of the protein as well as of a change in its degree of polymerization.
Chorismate Mutase from Euglena gracilis
Authors: Weber Hermann L. , Böck August
Published: March 2005

One of the key enzymes in the regulation of aromatic acid biosynthesis, chorismate mutase, has been purified about 3000‐fold from cell‐free extracts of Euglena gracilis. The enzyme possesses an apparent molecular weight of 160 000. Judged by our criteria Euglena possesses only one species of chorismate mutase.

The purified enzyme is sensitive to inhibition by tyrosine and phenylalanine and to activation by tryptophan; the extent of inhibition and activation is strictly pH‐dependent. The inhibition by phenylalanine and by tyrosine is partially competitive with chorismate. Substrate saturation data of the effector‐free enzyme analyzed by the Hill equation yield values of n of 2.0 at pH 8.4 and of close to 1.0 at pH 7.0, thus indicating strong cooperative interactions under the former and little or no interactions under the latter condition. At pH 7.0 the presence of the inhibitors results in an increase of the value of n suggesting cooperativity under this condition. Little or no cooperative interaction between the inhibitor sites was observed (n′= 1.2). In the presence of tryptophan chorismate mutase exhibits strict Michaelis kinetics (n= 1.0 at pH 7.0 and 8.4).
Steroids in Germfree and Conventional Rats
Authors: Eriksson Håkan , Gustafsson Jan‐Åke
Published: March 2005

Gas‐liquid chromatography, radio‐gas chromatography and gas chromatography‐mass spectrometry were used to identify steroid metabolites in the monosulphate and disulphate fractions of faeces from germfree female rats and in the monosulphate fraction of faeces from conventional female rats after intraperitoneal administration of [4‐14C]pregnenolone and [4‐14C]‐corticosterone. The germfree rats excreted the following steroids: monosulphate fraction: 3β, 17α‐dihydroxy‐5α‐pregnan‐20‐one, 5α‐pregnane‐3α,16α,20α‐triol, 2α,3β,16α‐trihydroxy‐5α‐pregnan‐20‐one and 3α,15β,16α‐trihydroxy‐5α‐pregnan‐20‐one; disulphate fraction: 3α‐ and 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one. The following steroids were identified in faeces from conventional rats: oestriol, 3α,15α(and 15β)‐dihydroxy‐5α,17α(and 17β)‐pregnan‐20‐one,3α,19‐dihydroxy‐5α,17α‐pregnan‐20‐one, 5α‐pregnane‐3α,16α,20‐triol, 3β,11α‐dihydroxy‐5α‐pregnan‐20‐one. 2α, 3α‐dihydroxy‐5α,17α‐pregnan‐20‐one (tentatively identified), and 3α,17α‐dihydroxy‐5α‐pregnan‐20‐one. The different steroid patterns in faeces from germfree and conventional rats are explained by the metabolic reactions carried out by the intestinal flora. Radio‐gas chromatography showed that 3α‐ and 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one and 3β,11α‐dihydroxy‐5α‐pregnan‐20‐one were metabolites of corticosterone. The formation of 3β,11α‐dihydroxy‐5α‐pregnan‐20‐one and of the 17α‐pregnane derivatives by intestinal microorganisms is discussed.
Study on a Family of Cystine‐Containing Fragments from the Variable Part of Pig Immunoglobulin k‐Chains
Authors: Novotný Jiří , Franěk František , ŠOrm František
Published: March 2005

From the tryptic hydrolyzate of pig immunoglobulin χ‐chains 6 cystine‐containing fragments were isolated. Amino acid analysis showed that they contained 37–70 amino acid residues and were of a variable nature. There exists a linear relationship between the specific electrical charge of the fragment and the ionic strength of the buffer by which the fragment is eluted from a column of QAE‐Sephadex.

Fragments, the disulfide bond of which was split by oxidative sulfitolysis, were split by thermolysin and the hydrolyzates were compared by the method of peptide maps. All the maps of fragments tested contained the peptide Ile‐ThrSer‐CysSSO3‐Arg (where CysSSO3=S‐sulfo‐cysteine) which represents the vicinity of the half‐cystine 24 of pig χ‐chains, as shown by us previously. The nature of the peptide maps indicates that the individual fragments originate in the same section of the primary structure of the N‐terminal half of the χ‐chains. Each of the isolated fragments was a mixture of variants with an identical specific charge but with some amino acid replacements.
Regulation of Gluconeogenesis in the Guinea Pig Liver
Authors: Söling Hans‐Dieter , Wilims Berend , Kleineke Jochen , Gehlhoff Maria
Published: March 2005

Because of differences in the pattern of enzyme activities involved in glycolysis and gluconeo‐genesis between rat and guinea pig liver, the regulation of gluconeogenesis in guinea pig liver was studied.

The following results were obtained:

Starvation increases the capacity for gluconeogenesis from pyruvate in vivo.

Gluconeogenesis from l‐lactate and from pyruvate in isolated perfused livers is greater in the guinea pig than in the rat, whereas gluconeogenesis from other precursors is similar in both species.

Long chain and medium chain fatty acids inhibit gluconeogenesis from l‐lactate in isolated perfused rat liver. In the guinea pig liver the difference does not result from a lower rate of fatty acid oxidation but from a greater reduction in the cytoplasmic NAD+/NADH system.

During enhanced fatty acid oxidation in guinea pig liver and in rat liver, similar changes in the concentrations of both acetyl‐S‐CoA and CoA‐SH are observed.

With high concentrations of pyruvate as the gluconeogenic precursor, both fatty acids and ethanol lead to a slight stimulation of glucose formation by supplying hydrogen equivalents to the cytosolic NAD+/NADH system.

Both glucagon and dibutyryl‐Ado‐3′:5′‐P stimulate ureogenesis and ketogenesis in isolated perfused guinea pig liver. However, the stimulation of gluconeogenesis by glucagon and Ado‐3′:5′‐P normally observed in isolated perfused rat livers was not found in isolated perfused guinea pig livers.

Partially purified pyruvate carboxylases from rat and guinea pig liver do not differ with respect to the apparent Km values for pyruvate and ATP, and also the apparent Ka values for acetyl‐S‐CoA at different pH values. At constant concentrations of MgATP2− or MnATP2− the pH‐optima for the guinea pig enzyme are significantly lower.

5‐Methoxyindole‐2‐carbonic acid inhibits gluconeogenesis in guinea pig liver in the same way as in rat liver. Quinolinate, on the other hand is far less inhibitory in guinea pig liver and does not inhibit at all in isolated perfused pigeon livers.

Major species differences are found when the activities of the rate limiting enzymes involved in gluconeogenesis are measured under Vmax conditions. Pyruvate carboxylase activity is in the same range in rat and guinea pig liver; pyruvate kinase activity is considerably lower, phospho‐enolpyruvate carboxykinase activity is considerably higher in guinea pig liver.

According to our results gluconeogenesis in guinea pig liver from lactate or pyruvate is mainly regulated by the concentration of pyruvate.

The difference in the regulation of gluconeogenesis between rat and guinea pig liver probably results from a difference in the compartmentalization of phosphoenopyruvate carboxykinase. A higher rate of futile cycling between phosphoenolpyruvate and pyruvate in rat liver, due to the higher activity of pyruvate kinase in relation to the activities of phosphoenolpyruvate carboxykinase and pyruvate carboxylase, may also contribute to the observed differences.
Konformationsinduzierter Circulardichroismus in Hämoglobinen von Chironomus th. thummi
Authors: Wollmer Axel , Sick Hinrich , Gersonde Klaus
Published: March 2005

The conformational isomers of Chironomus haemoglobin first postulated on the strength of the O2‐binding properties [24] and demonstrated by electron spin resonance spectroscopy [23] could now be confirmed by measurement of the circular dichroism.

All the circular dichroism bands of the oxidized haemoglobin from Chironomus in the visible and Soret region are induced or influenced conformationally. The pH‐dependency of the extinction and the ellipticity of the individual bands follows a circuit process with four reaction steps, two of which describe the allosteric transformation and two the protolytic reaction of water at the 6th coordination point of the haem iron. With the aid of fit calculations we get the protolytic constants pK′2= 8.1 and pK”2= 8.3 and the allosteric constants pKa1= 7.3 and pKa2= 7.1.

The existence of conformationally induced circular dichroism bands becomes obvious, if one compares the separated haemoglobins showing different degrees of allosterism. Thus for instance the individual haemoglobin IV, showing the smallest amplitude of the Bohr effect, is characterized by a minimal conformationally‐induced change of ellipticity. The individual haemoglobin VIII with the highest amplitude of Bohr effect shows the strongest conformationally‐induced circular dichroism bands.

The pH‐dependency of the absorption spectra in the visible range displays further characteristics of conformational isomerism. The 4‐component system to be expected from the circuit process reveals at sufficient distance between the allosteric and protolytic pK values a system of two 2‐component mixtures, which can be transformed into each other depending on pH and thus produce “isosbestic triangles”.

The helix content of the Chironomus haemoglobins is not influenced by the described change of conformation and amounts to 60 and 70% according to the individual haemoglobins.

The results are discussed under the aspect of isomerism of conformations and allosterism controlled by the trans‐effect of the 5th ligand of the haem iron.
A propos de I'actomyosine et de quelques unes de ses propriétés physico‐chimiques
Authors: Arnaud Yves , Leger Jean J. , Gruda Julian , Leroux Louis
Published: March 2005

The physicochemical properties of the actomyosin molecule were established by hydrodynamic and light‐scattering techniques. These results do not depend on the method of preparation.

Taking advantage of aging of the solutions, and by taking into account the hydrodynamic pressure that acts during ultracentrifugation experiments, it is possible to obtain an homogenous analysis of the results of hydrodynamic and light‐scattering measurements. It seems likely that some discrepencies reported in the litterature may be explained by these observations.

In solution, actomyosin is a cylindrical molecule whose main axis is about 5800 Å long, and 1160 Å in diameter; the molecular weight is about 18 ± 4 × 106; the greater part of this cylinder is occupied by solvent.

Actomyosin is composed of F‐actin and A‐myosin which may be isolated by ultracentrifugation in the presence of ATP. In solution, actomyosin is a fibrillar molecule, whose axis is formed by F‐actin (4 × 106) and, on which 28 molecules of A‐myosin (5 × 105) are attached.

A model for the structure of actomyosin in solution is proposed.
Studies on the α‐Methylglucoside Permease of Escherichia coli
Authors: Gachelin Gabriel
Published: March 2005

The sequential events which occur in the membrane of Escherichia coli and lead to the antecedent appearance of α‐methylglucoside 6‐phosphate when cells are exposed to extracellular α‐methylglucoside have been analyzed.

It has been shown that phosphorylation is the rate limiting step in α‐methylglucoside phosphate synthesis in vivo. However, phosphorylation of α‐methylglucoside was not found to be the only step occurring in the thickness of membrane.

It was shown that a step of transport was necessarily taking place before phosphorylation. In the absence of phosphorylation (due to genetic inactivation of any part of the enzymatic complex devoted to phosphorylation, or to chemical inactivation of one among its components), this step was expressed as a facilitated diffusion of unaltered α‐methylglucoside. The demonstration of this facilitated passage rests on the following evidence: equalization of α‐methylglucoside concentrations in intracellular and extracellular compartments faster than when due to free passive diffusion alone; requirement of the interaction of α‐methylglucoside with a stereospecific site, distinct from EII, with a Km of 20 μM; finally, existence of counterflows suggesting that the passage was ensured by a mobile carrier, mediating both influx and efflux of α‐methylglucoside.

This step of stransport was shown to be required in intact cells for a normal efficiency of phosphorylation in vivo. However it was not expressed in vivo as a facilitated diffusion but very likely as a facilitated access to the site of phosphorylation of EII.

From these findings, and from an analysis of various mutants, it was concluded that a concerted change of conformation occurs when EII was phosphorylating, which renders the step of transport unlimiting. Some consequences of the dissociation of the α‐methylglucoside phosphate synthesis into two sequential steps are discussed. Mainly this model provides a simple explanation for the polarity exhibited by EII, the absence of phosphorylation of intracellular α‐methylglucoside and its continuous exchange.
Involvement of Intracellular Phospholipase C in Autolytic Fragmentation of Cytoplasmic Membranes of Bacillus cereus
Authors: Koga Yosuke , Kusaka Iwao
Published: March 2005

Stoichiometry of changes in lipid composition during autolysis indicated that phospholipase C was involved in autolysis of the membranes of Bacillus cereus. Phosphatidylethanolamine and phosphatidylglycerol, which were main constituents of the membrane lipid, were hydrolysed by phospholipase C.

The lipid‐rich material, which was released from the cell during autolysis, was found to be a kind of lipoprotein. Diglyceride, a product of phospholipase C, accounted for 69% of the total lipid in the released materials. Lipid/protein ratio of the material was calculated to be 1.56 (mg lipid/mg of protein). Electron microscopy of the material indicated that the material consisted mainly of membranous vesicles of about 0.2μm in diameter and had an “unit membrane” structure.

From these results, it was concluded that the cytoplasmic membranes of B. cereus might be broken down at the site where phospholipid was hydrolysed by phospholipase C and the fragment was released into the medium.
Structure primaire de la caséine αS1 bovine
Authors: Mercier Jean‐Claude , Grosclaude François , Ribadeau‐Dumas Bruno
Published: March 2005

Our previous studies [1, 2] on maleyl bovine αs1‐B casein were concerned with the isolation and amino acid composition of the tryptic peptides designated as Tm peptides. The sequence of the COOH‐terminal Tm peptide containing 48 residues was also reported.

In the present study, we report the overlaps of all the cyanogen bromide peptides as well as the Tm peptides from αs1‐B casein.

The αs1‐B casein which contains five methionyl residues was cleaved with cyanogen bromide. The six expected peptides obtained by this treatment and designated as CN peptides, were separated initially on Dowex‐50 X 2 and further purified by Sephadex column chromatography. The amino acid composition of these six purified CN peptides was determined. The results were in perfect agreement with the amino acid composition of αs1‐B casein deduced from the analysis of Tm peptides.

The three Tm peptides from αs1‐B casein which contained methionyl residues were also cleaved by cyanogen bromide. The fragments obtained were purified by either paper chromatography or Sephadex column chromatography and the amino acid composition of these purified peptides was determined. Of the eight peptides thus obtained, three were found identical with the CN peptides obtained directly by BrCN treatment of the αs1‐B casein. The remaining three CN peptides from αs1‐B casein for which identical fragments could not be located in the Tm peptides, were digested with trypsin (in two cases after maleylation) and the resulting fragments were purified by Sephadex column chromaography. These tryptic peptides provided the remaining gaps. This permitted us to locate the relative position of all the CN peptides in the αs1‐B casein molecule. These results also indicate the location of all the Tm peptides except 2 in the αs1‐B casein molecule. Further, the chymotryptic peptides from one of the CN fragments of αs1‐B casein provided the missing overlaps.