bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Some Properties of Chromatin Non‐Histone Proteins and Nuclear Sap Proteins
Authors: UMANSKY Samuil R. , ZOTOVA Rimma N. , KOVALEV Yurij I.
Published: March 2005

The properties of rat liver and thymus non‐histone and nuclear sap proteins were compared. The distribution of total, labile‐bound and 0.35 M NaCl extractable non‐histone proteins from one organ on polvacrylamide‐gel electrophoresis in the presence of sodium dodecylsulphate is quite similar. On electrophoresis non‐labelled and 32P‐labelled non‐histone and nuclear sap proteins from one organ differ from one another both qualitatively and quantitatively. We did not find an appreciable difference between non‐labelled non‐histone proteins isolated from liver and thymus. The distribution of 32Plabelled non‐histone proteins from various organs differs quantitatively rather than qualitatively. Non‐labelled and 32P‐labelled nuclear sap proteins from liver and thymus differ significantly. ‘Free’ nuclear sap proteins and the proteins of ribonucleoprotein particles from thymus nuclei contain a great quantity of identical polypeptides, whereas other polypeptides are specific to each of these protein fractions. Upon incubation of nuclei with [γ‐32P]ATP the label is incorporated into all the fractions of nuclear protein. The nuclear proteins are phosphorylated at decreasing rates in the order: labile‐hound non‐histone proteins > firmly bound non‐histone proteins > ‘free’ nuclear sap proteins = proteins of ribonucleoprotein particles > histones.

Nuclear sap and non‐histone proteins contain protein kinases capable of phosphorylating both these proteins and histones. Histone phosphorylation is sharply inhibited after addition of DNA, the protein kinases of nuclear sap phosphorylating less effectively the histones complexed with DNA than the non‐histone proteins. Both non‐histone and nuclear sap proteins contain fractions interacting in vitro with DNA. Denatured DNA hinds twice as much 32P‐labelled nuclear sap proteins and a little more 32P‐labelled non‐histone proteins than native DNA. Denatured DNA binds nonhistoric and nuclear sap proteins much more effectively than native DNA. It was shown by the membrane filter technique that the major part of the nuclear sap and non‐histone proteins interacting with native DNA binds to it non‐specifically. A certain portion of non‐histone and nuclear sap proteins interacts specifically with homologous denatured DNA. The possible role of non‐histone and nuclear sap proteins in the regulation of transcription is discussed.
Studies on the Pyridoxal Phosphate Site in Glycogen Phosphorylase b
Authors: CORTIJO Manuel , LLOR Juan , JIMENEZ Juan S. , GARCIA‐BLANCO Francisco
Published: March 2005

It is usually accepted that the adduct formed by reaction of pyridoxal phosphate with amines in aprotic solvents is a good model to simulate some properties of the pyridoxal phosphate site in glycogen phosphorylase. The chemical structure of this adduct was not very well established. An aldimine structure is supported by the infrared, electronic absorption and nuclear magnetic resonance spectra given in this work. Therefore, we conclude that, at neutral pH, the pyridoxal phosphate is bound to the glycogen phosphorylase through a Schiff base structure and embedded in a hydrophobic environment. The polarographic measurements reported in this paper could explain the fact that, at neutral pH, the pyridoxal phosphate can not be reduced onto phosphorylase by NaBH4.
Enzymic Synthesis of Lignin Precursors
Authors: WENGENMAYER Herta , EBEL Jürgen , GRISEBACH Hans
Published: March 2005

A cinnamoyl‐coenzyme A reductase catalyzing the NADPH‐dependent reduction of substituted cinnamoyl‐CoA thiol esters to the corresponding cinnamaldehydes was isolated from cell suspension cultures of soybean (Glycine max L. var. Mandarin). A 1660‐fold purification of the enzyme was achieved by (NH4)2SO4 fractionation, chromatography on DEAE‐cellulose, hydroxyapatite and Sephadex G‐100 and affinity chromatography on 5′‐AMP‐Sepharose. The apparent molecular weight of the reductase was found to be about 38000 on the basis of the elution volume from a Sephadex G‐100 column. Maximum rate of reaction was observed between pH 6.0 and 6.2 in 0.1—0.2 M citrate buffer at 30 °C. The enzyme was markedly inhibited by thiol reagents.

The reductase showed a high degree of specificity for cinnamoyl‐CoA esters. Feruloyl‐CoA was the substrate with the lowest Km value (73 μM) and highest V (230 nkat/mg) followed by 5‐hydroxyferuloyl‐CoA, sinapoyl‐CoA, p‐coumaroyl‐CoA, caffeoyl‐CoA and cinnamoyl‐CoA. No reaction took place with acetyl‐CoA. The Km value for NADPH varied with the type of substrate. Km values of 28, 120, and 290 pM were found with feruloyl‐CoA, sinapoyl‐CoA, and p‐coumaroyl‐CoA, respectively. The rate of reaction observed with NADH was only about 5 % of that found with NADPH.

The reaction products CoASH and NADP+ inhibited the reaction. The Ki values were in the range of 0.5—1 mM and the inhibition was of a noncompetitive (mixed) type. The role of the reductase in the biosynthesis of lignin precursors is discussed.
Two Forms of RNA Polymerase B in Yeast
Authors: DEZÉLÉE Sybille , WYERS Françoise , SENTENAC André , FROMAGEOT Pierre
Published: March 2005

Special care to prevent proteolysis during yeast RNA polymerase B purification leads to the appearance of two forms of enzymes, BI and BII, with different molecular weight (465000 and 435000, respectively). The two forms of enzyme can be separated by ion‐exchange chromatography or polyacrylamide gel electrophoresis. Their subunit structures were compared by sodium dodecylsulfate gel electrophoresis, the only observed difference between the two enzymes is in the molecular weight of the heaviest subunit which is 220000 for enzyme BI and 180000 for enzyme BII. Otherwise, the two enzymes have seven common subunits of molecular weights 150000, 45000, 26000, 22500, 14500, 12500 and 9000. Two additional polypeptide chains of 32000 and 16500 Mr are dissociated from the enzyme upon polyacrylamide gel electrophoresis or DEAF Sephadex chromatography.

The largest subunit of enzyme BI (Mr 220000) can be specifically cleaved in vitro by a yeast protease extract, generating a polypeptide chain indistinguishable from the largest subunit of enzyme BII. This proteolytic cleavage of enzyme BI in vitro is inhibited by phenylmethylsulfonyl fluoride and does not significantly change the activity of the enzyme with single‐stranded or double‐stranded DNA as template. The precursor‐product relationship of the different forms of class B RNA polymerises in eukaryotic cells is discussed.
Influence of DNA Acidification on DNA Premelting and Template Properties
Authors: SAROCCHI Marie‐Thérèse , DAUSSE Jean‐Pierre , GUSCHLBAUER Wilhelm
Published: March 2005

Acidification of a T7 DNA sample was found to be partly irreversible as ultraviolet difference spectra measured at various sub‐melting temperatures were different from those observed for a ‘normal’ DNA sample. This implies some subtle conformational change which is not reversed by return to neutral pH. In the same conditions, only poly(purine)· poly(pyrimidine) polymers behaved in a different manner, during premelting. according to whether they were previously acidified or not.

The properties of acidified and reneutralized T7 DNA were also investigated for Escherichia coli RNA polymerase binding and transcription. An inhibition of RNA synthesis and chain initiation was observed. The results suggest that the binding of the enzyme is affected. RNA synthesized is specific but there is a decrease in the number and in the stability of the RNA‐polymerase · DNA complexes.
Cell‐Cycle Dependence of Two Nuclear Histone Kinase Enzyme Activities
Authors: HARDIE D. Graham , MATTHEWS Harry R. , BRADBURY E. Morton
Published: March 2005

Growth‐associated histone kinases have been extracted from Physarum polycephalum nuclei and resolved into two components by ion‐exchange chromatography. The two component activities have different substrate specificities and different times of appearance in the cell cycle. It is proposed that the enzyme(s) phosphorylate H1 histone in vivo in G2 phase, possibly sequentially in time at different sites in the H1 amino acid sequence.
Proton Exchange of the Pro‐S Hydrogen at C‐1 in Dihydroxyacetone Phosphate D‐Fructose 1, 6‐Bisphosphate and d‐Fructosehate, d‐Fructose 1,6‐Bisphosphate and Catalysed by Rabbit‐Muscle Aldolase
Authors: LOWE Gordon , PRATT Rex F.
Published: March 2005

Rabbit muscle aldolase catalyses the stereospecific exchange of the pro‐S C‐1 hydrogen of dihydroxyacetone phosphate, d‐fructose 1,6‐bisphosphate and d‐fructose 1‐phosphate. The rate of exchange, however, is at least 400 times slower than the proton exchange of the pro‐S hydrogen at C‐3 in dihydroxyacetone phosphate.
Circulardichroismus von heterodetischen cyclischen Cystinyl‐Tripeptiden
Authors: OTTNAD Michael , HARTTER Peter , JUNG Günther
Published: March 2005

Circular Dichroism of Heterodetic Cyclic Cystinyl Tripeptides

The circular dichroism of two series of heterodetic cyclic tripeptides, t‐butoxycarbonyl‐l‐cysteinyl‐X‐cysteine disulfide methyl esters [X = Ala, Val, Leu, Phe, Glu(Obut), Lys(Z)] and l‐cysteinyl‐X‐cysteine disulfide methyl ester formates, and a corresponding series of t‐butoxycarbonyl‐l‐cysteinyl‐(glycyl)n‐cysteine disulfide methyl esters (n= 1–5) was investigated. The interaction between the disulfide group and the amide bonds results in a long‐wavelength Cotton effect at 300 nm. which exhibits a strong solvent dependence. In contrast to the long‐wavelength disulfide band and to the n‐π* amide Cotton effect a bathochromic shift is observed from less polar to strongly polar solvents. Within the series of N‐terminal protected tripeptide esters the influence of the central chiral amino acid X is small, but it increases in the non‐protected peptide esters. The circular dichroism is dominated by the influence of the conformation of the disulfide ring, which is essentially the same in both series. Due to interactions of the disulfide group with the amide chromophores, the sign of the long‐wavelength disulfide Cotton effect cannot be used for the determination of the helicity of the CSSC group. Infrared spectra of the protected compounds indicate trans‐peptide bonds in the solid state, which may also be true for the solutions.

A pronounced time‐dependence is observed for the circular dichroism of t‐butoxycarbonyl‐l‐cysteinyl‐glycyl‐cysteine disulfide methyl ester after dissolving in ethanol. This is due to a uniform conformational change of the ring structure as shown by spectroscopic analysis using extinction and ellipticity diagrams.

In the series of the cyclic cystinyl peptides containing glycyl residues a strong electronic disulfide‐amide interaction is observed for the tripeptide, being still observable for the tetra‐peptide. The comparison of the glycyl tripeptide with the tripeptides containing chiral central amino acids reveals a negative contribution to the circular dichroism at 260 nm due to these l‐residues.
Preparation and Specificity of Endonuclease IV Induced by Bacteriophage T4
Authors: BERNARDI Alberto , MAAT Jan , DE WAARD Adrian , BERNARDI Giorgio
Published: March 2005

Bacteriophage‐T4‐induced endonuclease IV suitable for DNA sequence analysis has been prepared by a modified and easily reproducible method.

The specificity of T4‐induced endonuclease IV has been investigated in order to verify whether this enzyme exhibits a single nucleotide recognition or a short sequence recognition.

The 5′‐terminal dinucleotides and 3′‐terminal nucleotides of oligonucleotides released by T4 induced endonuclease IV from three single‐stranded DNAs (from bacteriophages ØX174, fd, M 13) have been analysed. In different DNAs, 74–82% of the 5′‐terminal dinucleotides end in 5′‐deoxycytidylic acid; small but significant levels of several dinucleotides ending in 5′‐deoxyadenylic acid, 5′‐thymidylic acid and 5′‐deoxyguanylic acid are also found. As far as 3′‐terminal nucleotides are concerned all nucleotides are present with a large predominance of thymidylic acid.
Solubilization and Purification of Rat, Liver Microsomal 1,2‐Diacylglycerol: CDP‐choline Cholinephosphotransferase and 1,2‐Diacylglycerol CDP‐ethanolamine Ethanolaminephosphotransferase
Authors: KANOH Hideo , OHNO Kimiyoshi
Published: March 2005

The procedure, which involved 2‐step sonication of microsomes at pH 7.4 and then at pH 8.5 in the presence of sodium deoxycholate and subsequent dialysis, resulted in 4–5‐fold purification of choline‐phosphotransferase and ethanolaminephosphotransferase with the yield of 40–50%.

Ethanolaminephosphotransferase was further purified 8.5‐fold over microsomes by sucrose density gradient centrifugation of the partially purified preparation, while cholinephosphotransferase activity was considerably lost during this procedure. No separation of the two transferases from each other was achieved at this step.

Cholinephosphotransferase required Mg2+ as cofactor, and microsomal phospholipids for its maximal activity. On the other hand, Mn2+ was more effective than Mg2+ as cofactor for ethanolaminephosphotransferase, and this enzyme was inhibited by microsomal phospholipids.

Both transferases were stimulated several‐fold by sodium deoxycholate and also showed similar optimal pH ranging from pH 8.0 to 8.5.

Km values for 1,2‐diacylglycerol emulsion were 81.0 μM for cholinephosphotransferase and 63.0‐μM for ethanolaminephosphotransferase, respectively. CDP‐choline and CDP‐ethanolamine competitively inhibited, with the‐ same Ki value (both 350 μM), ethanolaminephosphotransferase and cholinephosphotransferase, respectively. The Ki values obtained were much greater than the corresponding Km values for the cytidine substrates (36.4 μM for CDP‐choline and 22.0 μM for CDP‐ethanolamine).

The partially purified enzymes were further treated with Triton X‐100. When enzyme activities were assayed with Mg2+, cholinephosphotransferase, although considerably inactivated, was partially separated from ethanolaminephosphotransferase by sucrose density gradient centrifugation of Triton‐treated preparations. Furthermore, cholinephosphotransferase (but not ethanolaminephosphotransferase) itself was partially separated into Mg2+‐requiring and Mn2+‐requiring components. In contrast, ethanolaminephosphotransferase assayed with either Mg2+ or Mn2+ formed a single peak together with Mn2+‐requiring cholinephosphotransferase.
Spectroscopic Investigation of Binary and Ternary Coenzyme Complexes of Yeast Alcohol Dehydrogenase
Authors: KARLOVIĆ Damir , AMIGUET Pierre , BONNER Francis J. , LUISI Pier Luigi
Published: March 2005

Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100‐fold. The formation of the ternary enzyme NAD pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290‐nm region. Significant optical changes also accompany the formation of the ternary enzyme NADH acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme‐induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.
Immunochemical Studies on Lipopolysaccharides from Wild‐Type and Mutants of Escherichia coli K‐12
Authors: MAYER Hubert , RAPIN Anette M. C. , SCHMIDT Günter , BOMAN Hans G.
Published: March 2005

Lipopolysaccharides from a number of mutants of Escherichia coli K‐12 were investigated by means of chemical and serological methods. Inhibition of passive hemagglutination and inhibition of precipitation show that l‐rhamnose is the immunodominant sugar in the lipopolysaccharide from wild‐type E. coli K‐12, The disaccharide rhamnosyl‐KDO (where KDO is 3‐deoxy‐d‐manno‐octulosonic acid) was isolated and characterized after mild acid hydrolysis of the lipopolysaccharide. It is concluded that rhamnose is present in the innermost part of the core as a side‐chain substituent on KDO.

From crosses between an E. coli K‐12 donor and E. coli O8, hybrids were obtained which contained either one or both of the donor rna and rfb clusters. Serum absorption studies with lipopolysaccharides from these hybrids indicated that the histidine‐linked rfb cluster is responsible for the presence of rhamnose in the K‐12 core oligosaccharide.

Using paper chromatography of 32P‐labelled lipopolysaccharides we have found heterogeneous lipopolysaccharide in two strains as well as some differences between two wild‐type strains. The latter difference is believed to be due to varying contents of KDO‐linked ethanolamine phosphate. The overall results presented together with those described in the companion paper clearly show that the core oligosaccharide in E. coli K‐12 has a structure different from the types previously described for other strains of E. coli (designed coli R 1 to coli R4).
Cell‐Wall Lipopolysaccharides of Ampicillin‐Resistant Mutants of Escherichia coli K‐12
Authors: PREHM Peter , STIRM Stephan , JANN Barbara , JANN Klaus , BOMAN Hans G.
Published: March 2005

The lipopolysaccharides of ampicillin‐resistant cell‐wall‐defective mutants of Escherichia coli K‐12 were analyzed. From their lipopolysaccharides the respective core oligosaccharides were obtained. Following dephosphorylation, the core oligosaccharides were methylated and analyzed by gas chromatography/mass spectrometry. From core‐defective mutants substructures of the K‐12 core were obtained. Analysis of the lipopolysaccharide preparations from wild‐type K‐12 indicated the presence of several core structures with different degrees of completion. The lipopolysaccharide preparation was degraded and the oligosaccharide mixture was partially resolved by gel filtration chromatography. Methylation, gas chromatography and mass spectrometry of the oligosaccharides permitted the tentative formulation of the K‐12 core structure. Alternative interpretations for this heterogeneity are discussed.
Functional Relationships between a Reticulocyte Polypeptide‐Chain‐Initiation Factor (IF‐MP) and the Translational Inhibitor Involved in Regulation of Protein Synthesis by Haemin
Authors: CLEMENS Michael J.
Published: March 2005

The rate of initiation of protein synthesis in rabbit reticulocyte lysates is regulated by a translational inhibitor protein which is activated in the absence of added haemin. The effects of this inhibitor on amino acid incorporation are overcome by the protein synthesis initiation factor IF‐MP which binds Met‐tRNAf in a ternary complex with GTP and which can transfer this complex to small ribosomal subunits. Addition of this factor to haemin‐deficient lysates prevents loss of polysomes and regenerates polysomes from 80‐S single ribosomes, thus confirming an effect at the level of polypeptide initiation. The ability of the initiation factor to overcome the effects of various concentrations of the translational inhibitor suggests that the inhibitor inactivates the factor catalytically rather than stoichiometrically.

In a system in vitro consisting of salt‐washed 40‐S ribosomal subunits, initiator Met‐tRNAf and GTP, the initiation factor IF‐MP transfers Met‐tRNAf to the subunits in the absence of any other factor or mRNA. Equilibrium buoyant density gradient analysis in CsCl shows that formaldehyde‐fixed subunits carrying Met‐tRNAf bound under these conditions have a buoyant density approximately 0.02 g/cm3 lower than the bulk of salt‐washed subunits, suggesting that approximately 100000 daltons of additional, protein are associated with these subunits. This is in marked contrast to the amounts of protein bound to subunits incubated with Met‐tRNAf and GTP in the presence of a crude ribosomal salt‐wash fraction. The translational inhibitor has no effect on formation of the ternary complex IF‐MP. Met‐tRNAf. GTP but does impair the factor‐catalysed transfer of Met‐tRNAf to washed subunits. The possible mechanisms of action of the inhibitor on polypeptide chain initiation are reviewed in the light of these results.
Intracellular Degradation and Deamidation of α‐Crystallin Subunits
Authors: VAN KLEEF Frans S. M. , WILLEMS‐THIJSSEN Willy , HOENDERS Herman J.
Published: March 2005

The subunits AA2 and N2, present in α‐crystallin from the nucleus of young and old bovine lenses, were isolated and characterized. It was found that AA2 is identical to a shorter A‐chain and hence it was designated as A1–101. The subunit N2 turned out to be identical with a shorter B‐chain and‐was designated as B1/2−170. Characterization of the subunit N1, present only in α‐crystallin from the cortex of old bovine lenses, suggested that this subunit is a modified B‐chain, probably a deamidation product; it was designated as Bo.

Comparison of the appearance of degraded and deamidated chains in relation to the age of Uhc fiber cells in old bovine lenses with that in calf lenses revealed that the observed specific limited degradation of the subunits of α‐crystallin increased with older age of the tissue. The deamidation process was found not to be related to the aging of the tissue. Eventually, a clear picture concerning the heterogeneity and fickleness of theα‐crystallin subunit structure was obtained.
Sulfated Bile Acids in Germ‐Free and Conventional Mice
Authors: EYSSEN Hendrik J. , PARMENTIER Guido G. , MERTENS Jan A.
Published: March 2005

Sulfated and non‐sulfated bile acids were determined in the intestines and in the feces of 7‐month‐old germ‐free and conventional male mice.

The bile acid pools in the gall bladder and small intestine were 21.13 mg/100 g body weight in germ‐free and 11.50 mg in conventional mice. The bile acid pools in the cecum and large intestine of germ‐free mice were 3.03 mg/100 g body weight as compared to 1.24 mg in conventional mice. Fecal bile acid excretion was 2.93 mg and 4.12 mg/100 g body weight in 24 h in germ‐free and conventional mice respectively.

The major bile acids from germ‐free mice were cholic acid, α‐muricholic acid and β‐muricholic acid. Small amounts of chenodeoxycholic and allocholic acid were also present. In addition to these primary bile acids the following secondary bile acids were identified in conventional mice: lithocholic, deoxycholic and ω‐muricholic acid.

In both germ‐free and conventional animals significant amounts of chenodeoxycholic and cholic acid were present as the 7‐monosulfate esters. The sulfate esters of these bile acids did not exceed 2% of the total bile acids in the small intestine, but accounted for approximately 50 of the bile acids in the cecum and the large intestine. In contrast, the muricholic acids were nearly exclusively found in the non‐sulfate fraction.

Alkaline hydrolysis without prior solvolysis of the sulfate esters resulted in loss of bile acids and production of artifacts. Hence, the bile acids of the mouse cannot be analysed by methods involving alkaline deconjugation unless a solvolysis step is included in the procedure.
The Use of Mercurated Nucleoside Triphosphate as a Probe in Transcription Studies in vitro
Authors: BEEBEE Trevor J. C. , BUTTERWORTH Peter H. W.
Published: March 2005

Purified form A RNA polymerase and the endogenous, nuclear form A RNA polymerase are shown to incorporate 5‐mercuri‐uridine 5′‐triphosphate (Hg‐UTP) into RNA in vitro. The Km for both Hg‐UTP and UTP are in the region of 10 μM for the purified enzyme.

The RNA products formed in nucleoli by endogenous RNA polymerase A have similar base compositions (G + C‐rich) whether UTP or Hg‐UTP is provided as the substrate in vitro.

Sulphydryl‐Sepharose chromatography of RNA synthesised in vitro by nucleoli allows separation of this material from the endogenous RNA, when the former is synthesised in the presence of Hg‐UTP.

In‐vitro‐synthesised nucleolar RNA hybridises with cot profiles similar to 28‐S ribosomal RNA, when made with either Hg‐UTP or UTP.

Hybridisation studies using DNA excess suggest that little competition occurs between the in vitro transcripts and the endogenous nucleolar RNA.

Size analysis of in vitro transcripts show that although some degradation occurs during isolation, purification and hybridisation of the RNA species, most of the RNA remains larger than 5 S throughout.
Studies on the Formation of Acetoin from Acetaldehyde by the Pyruvate Dehydrogenase Complex and Its Regulation
Authors: ALKONYI Istvái , BOLYGÓ Elek , GYÓCSI László , SZABÓ Dénes
Published: March 2005

Pyruvate dehydrogenase complex was isolated from pigeon breast muscle involving steps of isoelectric precipitation, poly(ethyleneglycol) fractionation and separation on a glycerine gradient in the ultracentrifuge.

Arsenite, a potent inhibitor of the dihydrolipoyl transacetylase, did not affect the formation of acetoin from acetaldehyde, indicating that the pyruvate dehydrogenase component was operative in this reaction.

Production of acetoin by the pyruvate dehydrogenase complex is subject to regulation by phosphorylation and dephosphorylation, the dephosphorylated form only being active.

The inhibition by acetaldehyde of the pyruvate dehydrogenase complex could be partly explained by the formation of acetoin as an alternative reaction.
Synthesis, Separation, Identification and Interconversion of Riboflavin Phosphates and Their Acetyl Derivatives: A Reinvestigation
Authors: SCOLA‐NAGELSCHNEIDER Gisela , HEMMERICH Peter
Published: March 2005

A reinvestigation of flavin phosphate synthesis, separation, identification, and interconversion was made in view of contradictory results in the literature. It has been confirmed that monochlorophosphoric acid is the best agent for selective 5′‐monophosphorylation of riboflavin and derivatives. This reaction yields, however, invariably up to 20% of an isomer, which has been separated by preparative thick‐layer chromatography and shown to be the 4′‐monophosphate. All the earlier authors failed to detect this isomer which does not bind to flavodoxin. It equilibrates in dilute mineral acid to yield an 8:2 mixture of 5′‐phosphate to 4′‐phosphate by phosphate migration.

The formation of 2′,3′,4′‐triacetyl‐flavin mononucleotide, according to Christie, S. M. H., Kenner, G. W. & Todd, A. R. (1954) J. Chem. Soc., 46–52, upon acid‐catalysed acetylation of pure FMN, was confirmed. The same reaction under base catalysis, however, does not yield 2′,3′,4′‐triacetyl‐flavin mononucleotide as claimed by Khomutova, E. D., Shapiro, T. A., Mezentseva, M. V. & Berezovskii, V. M. (1965) Otd. Ohshch. i Tekhn. Khim., 241–244, Chem. Absir. 65, 5516a, but in fact up to 80% 2′,3′‐diacetyl‐flavin 4′: 5′‐cyclophosphate as the main product, which is stable under neutral and weak acidic conditions and does not hydrolyse to 2′,3′‐diacetyl‐flavin 5′‐monophosphate as claimed by McCormick, D. B. (1974) J. Heterocycl. Chem. 11, 969–974.

The various flavin phosphates and their acetyl derivatives have been identified by pH‐titration, electrophoresis, and proton magnetic resonance spectrometry, which permit direct analyses of crude reaction products as well as a rapid purity check of commercial FMN.
Purification and Properties of 3‐Hexulosephosphate Synthase from Methylomonas M 15
Authors: SAHM Hermann , SCHÜTTE Horst , KULA Maria‐Regina
Published: March 2005

3‐Hexulosephosphate synthase, the first enzyme of the ribulose monophosphate cycle, was purified 15‐fold from methanol‐grown Methylomonas M 15. The purification procedure involved chromatography on DEAE‐cellulose, Sephadex G‐75, and DEAE‐Sephadex A‐50. The purified enzyme was more than 95% pure as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was calculated to be 43000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels gave a single band corresponding to a molecular weight of 22000.

The enzyme catalyzes specifically the condensation of formaldehyde with ribulose 5‐phosphate to yield d‐arabino‐3‐hexulose 6‐phosphate. The Km values were found to be 1.1 mM for formaldehyde and 1.6 mM for ribulose 5‐phosphate. A bivalent cation is essential for activity and stability of the enzyme, Mg2+ and Mn2+ serve best for this purpose. The optimum of pH for enzyme activity is 7.5–8.0.
Biosynthesis of Gramicidin S with the Aid of Dipeptides by Gramicidin S Synthetase
Authors: VON DUNGEN Adolf , VATER Joachim , KLEINKAUF Horst
Published: March 2005

Dipeptides l‐phenylalanyl‐proline, d‐phenylalanyl‐proline, prolyl‐valine, valyl‐lysine, lysylleucine and leucyl‐phenylalanine, derived from the sequence of gramicidin S, are substrates of the gramicidin S synthetase. When any of these dipeptides are used to replace the two corresponding amino acids in the reaction assay, cyclodecapeptide antibiotic synthesis occurs, and requires the whole multienzyme system. Active esters, like the thiophenyl and p‐nitrophenyl esters of d‐phenylalanylproline are unable to promote gramicidin S biosynthesis with the gramicidin S synthetase system or with the heavy enzyme alone.
Photolysis of Desmosine and Isodesmosine by Ultraviolet Light
Authors: BAURAIN Roger , LAROCHELLE Jean‐François , LAMY François
Published: March 2005

Desmosine and isodesmosine were separated by ion‐exchange and paper chromatography, after acid hydrolysis of purified elastin from beef ligamentum nuchae. The fractions obtained by ion‐exchange chromatography were clearly mixtures of related compounds. The desmosine fraction could be resolved into seven compounds and the isodesmosine into four by paper chromatography.

Destnosine was maximally degraded by irradiation at 274 nm and isodesmosine at 285 nm. These wavelengths did not correspond to the absorption maxima of the cross links, but to shoulders of the main absorption peaks.

When irradiated at their optimum wavelengths, but at various pH, both desmosine and isodesmosine seemed quite stable at pH greater than 8.5. Between pH 8 and 5, the photolytic rate was maximum and decreased slightly at more acidic pH. Below pH 4.0, one of the products of photolysis was free lysine.

In analogy to the mechanism of the photolytic degradation of N‐methyl pyridinium chloride, it appears that the (iso)desmosines were degraded via the formation of an open amino aldehyde, which was hydrolysed at acid pH to give free lysine and a substituted glutaconic aldehyde.
Human‐Polymorphonuclear‐Leucocyte Neutral Protease and Its Inhibitor
Authors: STEVEN Frank S. , MILSOM David W. , HUNTER John A. A.
Published: March 2005

Human polymorphonuclear leucocytes were obtained from the synovial fluids of patients with inflamed knee joints suffering either from Reiter's syndrome or from rheumatoid arthritis. The polymorphonuclear leucocytes were collected by gentle centrifugation followed by disruption and their subcellular fractionation by centrifugation in 0.34 M sucrose to provide a granule fraction and a post granule supernatant fraction. 0.5M KC1 extraction of the granule fraction yielded neutral protease activity, similar to trypsin, when assayed against fluorescein‐labelled polymeric collagen fibrils. The post‐granule supernatant fraction contained an inhibitor towards the neutral protease and trypsin. The inhibition of the neutral protease was found to be time‐dependent, this inhibition being released after 1.5–2 h. In contrast, the inhibition of trypsin was irreversible and this property was used to devise an assay procedure for the inhibitor.
Photosynthetic Activities of Membrane Preparations of the Blue‐Green Alga Phormidium luridum
Authors: BINDER Andres , TEL‐OR Elisha , AVRON Mordhay
Published: March 2005

Techniques have been developed for the isolation of whole spheroplaste and membrane preparations from the blue‐green alga Phormidium luridum which are active in photosystem‐II‐sensitized and photosystem‐I‐sensitized electron‐transport reactions and in photophosphorylation. Attention is called to the necessity of supplying magnesium and phosphate throughout the preparative and assay procedures for obtaining active membranes.

Selective washing of a proper membrane preparation is shown to remove a new protein factor, termed protein factor 1, which restores the ability of the preparation to catalyze photosystem‐I‐sensitized reactions.
8‐Azidoadenine Analogs of NAD+ and FAD
Authors: KOBERSTEIN Rudolf
Published: March 2005

The synthesis and purification of the 8‐azidoadenine analogs of NAD+ (azido‐NAD+) and FAD (azido‐FAD) from 8‐azidoadenosine 5 ‐phosphate and NMN+ or FMN, respectively, is described. The coenzyme analogs are characterized by absorption. nuclear magnetic resonance and circular dichroism spectra. The two latter methods indicate a folded structure of azido‐NAD+ and azido‐FAD. Upon irradiation at 300 mn in aqueous solution, a change of the ultraviolet absorption spectra of the coenzyme analogs indicates photolysis of the azido group. The coenzyme properties of azido‐NAD+ are demonstrated with lactate, glutamate and alcohol dehydrogenase yielding 14, 154 and 60% respectively, of the I observed with NAD+. Concomitantly the Km values of the coenzyme analogs are 1.7, 3.5 and 3‐fold higher than those of NAD+. Azido‐FAD is shown to be coenzyme of apo‐glucose oxidase. The recovery of activity, however, is much slower in the presence of azido‐FAD than with FAD. A final value of 66% of the activity with FAD is obtained. With apo‐d‐amino acid oxidase, azido‐FAD is completely inactive, although it is specifically bound to the enzyme.
Formation of Dolichol Monophosphate 2‐Deoxy‐d‐glucose and Its Interference with the Glycosylation of Mannoproteins in Yeast
Authors: LEHLF Ludwig , SCHWARZ Ralph T.
Published: March 2005

A crude membrane fraction from Saccharomyes cerevisiae has been found to catalyze 2‐deoxy‐d‐glucose transfer from GDP‐2‐deoxy‐d‐glucose to endogenous lipid and glycoprotein acceptor. Evidence will be represented that the glycolipid formed has properties characteristic of doliehol monophosphate 2‐deoxy‐d‐glucose. The 2‐deoxy‐d‐glucosyl group can further be transferred fron the glycolipid into a membrane‐bound polymer fraction. More than 95% of the radioactivity incorporated can be released by β‐elimination, indicating an O‐glycosidic linkage to serine or threonine. The only radioactive product obtained is 2‐dcoxv‐d‐glucose. When dolichol monophosphate 2deoxy‐[14C]glucose is incubated together with non‐radioactive GDP‐mannose subsequent β‐elimination yields no higher oligosaccharides in contrast to an experiment where dolichol monophosphate [14C]mannose and GDP‐mannose are used as donors. The results are consistent with the assumption that the non‐physiological nucleotide sugar interferes with GDP‐mannose for mannosylation and terminates further elongation of the serine threonine‐bound oligomannose side chains. UDP‐2deoxy‐D‐glucose, used as donor, results also in the formation of a glycolipid. In this case, however, no polvprenol derivative is formed. Instead, the glycolipid displays properties characteristic of a sphingolipid or a sterol glucoside.
A Novel Pathway for the Synthesis of Solanidine in the Isolated Chloroplast from Greening Potatoes
Authors: RAMASWAMY N. Krishnan , BEHERE Arun G. , NAIR P. Madhusudanan
Published: March 2005

Synthesis of chlorophyll always preceded the synthesis of solanine in cold‐stored potatoes in the presence of dim light (30–40 lux). Isolated chloroplasts from green peelings were able to fix C02. The evidence obtained showed a direct reduction of CO2 to formate, which, was the primary product of fixation. Apart from this the chloroplasts were capable of incorporating NaH14C03, [14C]formate, [2‐14C]glycine, [2‐14C]pyruvate, [2‐14C]acetate, [2‐14C]mevalonate and [U‐14C]serine into the sol1tnidine moiety of the alkaloid. The intermediates in the pathway between CO2 and acetate have been identified as formate, glycine, serine and pyruvate. The localization of the enzymes involved in the reaction sequence viz. serine hydroxymethyl‐transferase, and pyruvic dehydrogenase complex, in the isolated chloroplast has been established.
Coenzyme Properties of NAD+ Bound to Different Matrices through the Amino Group in the 6‐Position
Authors: SCHMIDT Hanns‐Ludwig , GRENNER Gerd
Published: March 2005

A method for the synthesis of N6‐(2‐aminoethyl)‐NAD+ is given. The binding of this NAD+ derivative to different soluble and insoluble supports and the direct coupling of NAD+ to epoxyactivated Sepharose are described. Proofs are given that NAD+ is bound through the amino group in 6‐position and the NAD+ derivative through the aliphatic amino group of the side chain.

Non‐enzymic reduction of the bound coenzyme to an almost quantitative extent is possible in all cases, but the enzymic reduction is largely influenced by the support. While N6‐(2‐aminoethyl)NAD+ coupled to soluble dextran is nearly completely reducible by different dehydrogenases with a velocity of about 40% of that for free NAD+, the coenzyme bound to different insoluble matrices is very slowly reduced. Only 5% of the coenzyme derivative bound to BrCN‐activated Sepharose are reducible, but 40% when it is bound through a spacer. From capacity determinations evidence is given that, even in this coenzyme gel, only those coenzyme molecules are useful in affinity chromatography which are on the surface of the gel grains; it is supposed that this may be due to the slow diffusion of an enzyme into the inner parts of an affinity gel.
Pyridoxal 5′‐Phosphate and the Regulation of Ornithine Decarboxylase Activity and Stability
Authors: CLARK Jeffrey L. , FULLER Joyce L.
Published: March 2005

There are two forms of ornithine decarboxylase with respect to pyridoxal 5′‐phosphate (pyridoxal‐P) affinity in exponentially‐growing Swiss 3T3 mouse fibroblasts: form I (Km∼ 10 μM) accounts for 30% of the total activity, and form II (Km∼ 0.4 μM) the remainder.

Each form of the enzyme is in rapid equilibrium with ornithine and pyridoxal‐P; neither form recognizes the Schiff base between ornithine and pyridoxal‐P as a substrtate.

Total pyridoxal‐P concentrations indicate that both forms may normally be at least partially active in vivo.

Upon stimulation of 3T3 cells by pituitary growth factors, form I becomes undetectable within 4 h. As total activity increases over 10‐fold during this time and continues to increase thereafter, a possible conversion of form I to form II could account for this increase only if the Km change reflects other changes in preexisting enzyme.

The rates of cofactor dissociation are apparently the same for each form and neither rate changes with the growth state. Since rapid equilibrium kinetics apply, the forms apparently differ in their rate of cofactor association.

The half‐lives of the two forms in vivo are the same in unstimulated cells when measured concurrently. Also, the half‐life of total activity decreases markedly upon stimulation as form II becomes dominant. These and other observations are not consistent with pyridoxal‐P serving a major protective function for the enzyme in vivo.
Reconstitution of Reduced Nicotinamide Adenine Dinucleotide Oxidase Activity with Menadione in Membrane Vesicles from the Menaquinone‐Deficient Bacillus subtilis AroD
Authors: BISSCHOP Arnold , KONINGS Wil N.
Published: March 2005

Membrane vesicles from the menaquinone‐deficient Bacillus subtilis aroD contain a low content of menaquinone and consequently oxidize reduced nicotinamide adenine dinucleotide (NADH) at low rate. Supplementation of the membrane vesicles suspension with the menaquinone‐analogue menadione, results in an incorporation of menadione in the membranes. The incorporated menadione increases with the external menadione concentration up to a maximum of 7 nmol of menadione bound per mg membrane protein.

The NADH oxidase activity of the membrane vesicles increases linearly with the menadione content and a 35‐fold stimulation is obtained in fully reconstituted membrane vesicles; this maximal NADH oxidase activity is about two‐fold higher than the NADH oxidase activity in membrane vesicles from wild‐type B.subtilis W23. Supplementation of membrane vesicles from B. subtilis W23 with menadione also results in a stimulation of the NADH oxidase activity but only a stimulation of 1.6‐fold is maximally obtained. The NADH oxidase activities in reconstituted B.subtilis aroD and B. subtilis W23 membrane vesicles are similarly affected by respiratory chain inhibitors, indicating that menadione occupies physiological sites of menaquinone.

NADH and the non‐physiological electron donor ascorbate + phenazine methosulphate are the best energy sources for active amino acid transport in membrane vesicles from B.subtilis W23. Membrane vesicles from B subtilis aroD accumulate amino acids in the presence of ascorbate phenazine methosulphate, but not with NADH. However, membrane vesicles from this mutant, reconstituted with menadione, demonstrate NADH‐driven transport activity. This activity increases linearly with the NADH oxidase activity, but maximal transport activities are reached under conditions where the NADH oxidase activity is not yet maximal. These results indicate that the rate of energy supply is the limiting factor for transport at low NADH oxidase activities and that the transport system itself becomes the limiting factor under conditions of high NADH oxidase activities. Under energy‐limiting conditions 135–235 molecules of NADH have to be oxidized in order to transport one molecule of amino acid. At all levels of energy supply a competition by the different amino acid transport systems for the available energy could not be observed. These observations indicate that only a fraction of the energy, generated by the respiratory chain, is used for the transport of an amino acid and that the bulk of the energy dissipates via other channels in the membrane vesicles.
The Structure of Artemia salina Haemoglobins
Authors: MOENS Luc , KONDO Masatoshi
Published: March 2005

Haemoglobins of Artemia salina were investigated during development and the results obtained are summarised as follows.

A new haemoglobin (haemoglobin N) was induced de novo in the hatching nauplius, but not in the prenauplius nor in the embryo.

Haemoglobin N seemed not to be synthesised in the adult.

The adult contained three distinct haemoglobins (haemoglobins I, II and III) in a quantitative ratio of 1: 6: 3, respectively, and these haemoglobins were not detected in nauplius.

Isoelectric points determined were 5.7, 5.6, 5.7. and 5.9, for haemoglobin N, I, II and III, respectively.

All four haemoglobins contained one large polypeptide (Mr= 1.1–1.3 × 105) and possibly two minor polypeptides (Mr= 8–9.5 × 104 and Mr= 5.4 × 104, respectively).
Phospholipid and Lipopolysaccharide in Proteus mirabilis and Its Stable Protoplast l‐Form
Authors: GMEINFR Jobst , MARTIN Hans Herbert
Published: March 2005

Cells of the stable, protoplast L‐form of Proteus mirabilis contain 1.5 to 2 times more extractable lipid, mostly phospholipid, per dry weight than cells of the bacterial form.

Under identical conditions of cultivation the qualitative and quantitative composition of the phospholipid is very similar in both cell forms. The range of mole percentages of individual phospholipid species is 76–80 for phosphatidylethanolamine, 10–13 for phosphatidylglycerol, 3.9–5.5 for diphosphatidylglycerol and 1.0–2.1 for lysophospholipid. However, all phospholipid species in the L‐form differ from those of the bacterial form by a lower content of long‐chain fatty acids and a higher content of short‐chain fatty acids.

Growth of the L‐form in the presence of growth‐stimulating horse serum results in a change of phospholipid composition accompanied by the uptake of phosphotipid and fatty acids from the serum into L‐form phospholipid.

L‐form protoplasts synthesize the same two types of Lipopolysaccharide, I and II, that were previously identified in the bacterial form of Proteus mirabilis. However, only small amounts of the more hydrophilic lipopolysaccharide II are present in the L‐form. Lipopolysaccharides from both cell forms have virtually identical polysaccharide compositions but differ strikingly in the relative content of fatty acids in their lipid‐A moieties. Molar ratios of tetradecanoic acid, hexadecanoic acid and 3‐hydroxytetradecanoic acid are 5:1:6 in the bacterial form and 5:0.1:6 in the L‐form grown in serum‐free medium.

The observed differences between the bacterial form and the protoplast L‐form are interpreted as results of the adaptation of the L‐form to life in the state lacking an envelope by formation of a physically more stable but still sufficiently fluid protoplast membrane.

A rapid method based on fatty acid analysis for the simultaneous quantitative determination of phospholipid and lipopolysaccharide content of whole cells is reported.
The Non‐Covalent Binding of Small Molecules by Ligandin
Authors: TIPPING Edward , KETTERER Brian , CHRISTODOULIDES Lucia , ENDERBY Graham
Published: March 2005

Equilibrium dialysis studies have been made of the binding of a number of small molecules by rat ligandin. Direct measurements of binding together with competition experiments indicated that bromosulphophthalein, oestrone sulphate and dehydroepiandrosterone sulphate each bind at the same single primary binding site with association constants of 1.1 × 107. 6.6 × l05 and 2.6 × 105 l/mol respectively at pH 7.0, 10.16 M, 4 oC. As well as bromosulphophthalein and dehydroepiandrosterone sulphate, a number of strucurally similar organic anions including 2‐hydroxyoestradiol‐glutathione. oestrone glucuronide, N‐methyl‐4‐aininoazo benzene‐glutathione and several bile acids, were able to displace oestrone sulphate from ligandin in a manner consistent with competition at a single binding site. From these experiments association constants for the competing ligands were derived; these were in the range 1 × 104‐1 × 106 l/mol.

Ligandin was found to bind a number of compounds for which, because of their low aqueous solubilities relative to their binding affinities, complete binding isotherms could not be obtained. These included several steroids (but not cortisol), 20‐methylcholanthrene, diethylstilboestrol, oleate and palmitate. Oestrone sulphate was able to compete with these ligands for binding and the results of the competition experiments were interpretable in terms of 1 :1 competition at a single binding site.

In general the conjugation of non‐polar ligands with sulphate or glutathione resulted in increased affinities, but such increases were relatively small (∼ 15% in terms of free energy) implying that the main driving force for the binding of both the conjugated and unconjugated species was the hydrophobic effect. This conclusion is borne out by the oobservations that both oestrone and its sulphate showed slight increases in affinity with increase in ionic strength, as would be expected for hydrophobic interactions.

As well as non‐polar compounds and organic anions, ligandin was also found to bind sulphate and glucuronate to a measurable degree, and to interact quite strongly with glutathione. For the latter compound a single binding site was found with an associat.ion.constant of 1 × 105 l/mol. Glutathione was able to cause the dissociation of the ligandin‐oestrone sulphate complex, but this effect was not explicable in terms of simple 1 : 1 competition.

Both oestrone and oestrone sulphate were bound most strongly at pH 6‐ 7, the affinity of the protein for these ligands falling off quite sharply on either side of this maximum.

The affinities of ligandin for bromosulphophthalein, steroids and their conjugates, diethylstilboestrol and N,N‐dimethyl‐4‐aminoazohenzenc are similar in magnitude to those of serum albumin and aminoazodye‐binding protein A (B. Ketterer, E. Tipping, J. F. Hackney and D. Beale, 1976).