bluuebunny/crossref_metadata_2025_split huggingface dataset:
Separation of the Two Non‐Identical Subunits of Lombricine Kinase from Lumbricus terrestris Muscle by Chromatography on Sepharose‐Mercurial
Authors: Der terrossian Elisabeth , Pradel Louise‐Anne , Kassab Ridha , Desvages Gisèble 
Published: March 2005

Of the dimeric phosphagen kinases (Mr= 80000) studied, lombricine kinase is the only one which contains only one essential thiol group and one binding site for ADP‐Mg. These facts suggest a non‐identity between the two subunits of this enzyme.The reversible labeling of the essential thiol group of this protein with 2,4‐dinitrofluorobenzene, followed by alkylation of the remaining thiol groups, allowed the separation of the two subunits by means of a Sepharose‐mercurial column. The Sepharose‐mercurial was prepared by treatment of Sepharose 4B with p‐aminophenyl mercuric acetate.Amino‐acid analyses, N‐ and C‐terminal determinations and fingerprintings were performed to detect the differences in the primary structure between the two subunits.Furthermore, the Sepharose‐mercurial column was used to purify the tryptic peptide containing the essential thiol group of lombricine kinase. By this process, it was possible to obtain, in one step, the peptide in a pure state.It is interesting to point out the applicability of this method for the purification of peptides or proteins containing essential thiol groups which can be specifically labelled by reversible inhibitors.
Conformational Properties of Some Viral DNAs
Authors: Champell P. , Brahms J. 
Published: March 2005

Oriented films of bacteriophage DNAs of low and intermediate (G + C) content and of different chemical composition were investigated by infrared linear dichroism: PBS1, DNA (28% G + C, uracil replaces thymine), T4, DNA (34%, G + C, glucosylated), T4* DNA (34% G + C, unglucosylated), T5, DNA (39% G + C). None of these phage DNAs exhibit a transition to a predominantly pure A form when relative humidity is lowered. At lower relative humidity, the infrared dichroism of the 1230‐cm‐1 band changes to mainly parallel orientation with respect to the DNA axis. The pattern of the tansition as a function of relative humidity indicates the presence at low relative humidity of a well‐defined form for which the O2–O3 line of the phosphate group was found to be directed approximately at 52°C with respect to the helix axis. This allows one to assign the lower‐humidity form of these phage DNAs as a predominant C‐like form.The comparison of T4 with T4* DNA indicates that glucosylation has no effect on conformational properties of phage DNAs, and particularly on the absence of the B to A structural change. The replacement of the thymine by uracil in DNA of PBS1, has not affected the structural properties of the B form at high humidity nor the absence of the main A form at lower humidity; the transition from the B form to a predominant C‐like form was observed.
The Role of Dolicholmonophosphate in Glycoprotein Biosynthesis in Saccharomyces cerevisiae
Authors: SHARMA Chandra Bhan , BABCZINSKI Peter , LEHLE Ludwig , TANNER Widmar 
Published: March 2005

1. In a crude particulate fraction from yeast mannose is transferred from GDP‐mannose to an endogenous “lipid” fraction, the monophosphates of dolichol‐14 to dolichol‐18. The same membrane fraction also catalyzes the mannosylation of glycoproteins. More than 80% of the total radioactivity in the glycoprotein fraction obtained from GDP‐[14C]mannose is released by β‐elimination. The small‐sized radioactive products of β‐elimination are mannose, mannobiose and mannotriose.2. Ageing of the particulate fraction leads to a drastic loss of mannosyl transfer activity from GDP‐[14C]mannose to dolichol monophosphate. To the same extent the amount of [14C]mannose obtained after β‐elimination of the glycoproteins decreases. The amount of radioactive mannobiose and mannotriose is, however, much less affected.3. With decreasing GDP‐mannose concentrations the amount of [14C]mannose obtained after β‐elimination increases as compared to the amount of radioactive mannobiose and mannotriose. The Km‐value for the incorporation of mannosyl groups directly linked to serine and/or threonine, therefore, is lower than the Km‐value for the transfer of subsequent mannosyl residues.4. The mannosylation of dolichol monophosphate from GDP‐[14C]mannose proceeds in the presence of Mg2+ and Mn2+ ions as well. When solely Mg2+ is present in the incubation mixture, β‐elimination of the glycoprotein produced yields almost exclusively mannose, and it is only in the presence of Mn2+ that the normal β‐elimination pattern is observed.5. Incubation of the particulate fraction with dolichol‐monophosphate‐[14C]mannose results in the formation of radioactive glycoprotein; again most of the radioactivity is released by β‐elimination but only [14C]mannose is obtained as product. When dolichol‐monophosphate [14C]mannose is incubated together with non‐radioactive GDP‐mannose, β‐elimination yields radioactive mannose, mannobiose and mannotriose. Mannobiose is found to be labelled in the reducing end only.6. The results are consistent with the assumption that dolichol monophosphate is involved in the mannosylation of a specific position in yeast glycoproteins, i.e. in the formation of mannosyl linkages to serine and/or threonine. The subsequent mannosylation proceeds directly from GDP‐mannose in a reaction obligatorily requiring Mn2+.7. After β‐elimination of the methanol‐insoluble material mannosylated in the presence of dolichol‐monophosphate [14C] mannose the residual insoluble radioactivity is to a large extent transformed into dialyzable material by pronase. Mannose, therefore, is transferred from dolichol‐monophosphate mannose also to glycoprotein positions not involving OH‐groups of serine or threonine.
On the 3α‐Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni
Authors: SKÅLHEGG Bjømn A. 
Published: March 2005

The steroid‐induced 3α‐hydroxysteroid:NAD+‐oxidoreductase from Pseudomonas testosteroni (ATCC 11996) exhibits reversible, concentration‐dependent monomer‐dimer transitions. The dimerization is demonstrable by gel chromatography and by precipitations with ammonium sulphate in concentrated solutions of the enzyme. The dimer formation leads to a considerable decrease in the specific activity, especially when androsterone is used as substrate. In this case the formation of dimers results in a 90 % reduction of the specific activity found with the monomers. The described effect is demonstrable, but less pronounced, when tetrahydrocortisone or deoxycholate are the substrates.Higly purified preparations of the 3α‐hydroxysteroid dehydrogenase prepared by repeated isoelectric focusings at different pH gradients have shown the enzyme to be composed of at least two isoenzymes. One major part focusing at pH 6.2 and a minor part focusing at pH 5.8.
Properties of the Assimilatory Nitrate Reductase from Aspergillus nidulans
Authors: McDONALD Donald W. , CODDINGTON Alan 
Published: March 2005

1. Nitrate reductase has been partially purified from Aspergillus nidulans. It has a sedimentation coefficient of 7.6 (± 0.1) S and a molecular weight of 190000 (± 10000) from sucrose density gradient centrifugation and gel‐filtration studies. There is spectroscopic evidence for the presence of a haemoprotein component.2. It possesses two associated activities, cytochrome c reductase and reduced viologen dye: nitrate reductase. A study of the effects of heat and inhibitors on the three activities suggests that the molecule consists of two functional parts. One of these is heat labile, contains FAD and a haemoprotein and catalyses the transfer of electrons from NADPH to cytochrome c. The other part is heat stable, contains molybdenum and catalyses the transfer of electrons from reduced viologen dyes to nitrate.3. The Km values for nitrate, NADPH and cytochrome c are reported. In each case the Km for one substrate is not affected by varying the concentration of the other, suggesting a lack of interaction between their respective binding sites. Product inhibition by NADP+ and nitrate under saturating and non‐saturating concentrations of NADPH and nitrate suggests a random order rapid‐equilibrium mechanism for the enzyme.
The Lipid Composition and Na+‐K+‐Dependent Adenosine‐Triphosphatase Activity of the Salt (Nasal) Gland of Eider Duck and Herring Gull
Authors: Karlsson Karl‐Anders , Samuelsson Bo E. , Steen Göran O. 
Published: March 2005

1. The lipid composition of the salt (nasal) gland of eider duck and herring gull, an organ with a high transport capacity for sodium ions, was characterized in detail.2. The three major lipids of both animals were cholesterol, ethanolamine phosphoglycerides and choline phosphoglycerides. The major fatty acids of ethanolamine phosphoglycerides were stearic and arachidonic acid, and the major fatty acids of choline phosphoglycerides were palmitic, oleic and arachidonic acid. Small amounts of plasmalogens were found.3. Other phospholipids identified were sphingomyelins, diphosphatidylglycerols, inositol phosphoglycerides and serine phosphoglycerides.4. The major glycosphingolipids were sulphatides, with similar structure for the two animals. A sulphate group was identified in position 3 of galactose, bound to ceramide mainly containing the C20 homologue of sphingosine and saturated 2‐d‐hydroxy C22, C23 and C24 fatty acids. No branched‐chain or trihydroxy bases were present.5. An earlier unknown glycolipid, xylopyranosylceramide, was found in small amounts in herring gull but not in eider duck. Glucosyl‐and galactosylceramides were present in both animals.6. The ratio of Na+‐K+‐dependent ATPase and sulphatides was similar to ratios found for other tissues with increased capacity for the transport of sodium ions. No other lipid resembled sulphatides in this respect.
The Determination of the Primary Structure of Histone F3 from Chicken Erythrocytes by Automatic Edman Degradation
Authors: BRANDT Wolf F. , VON HOLT Claus 
Published: March 2005

Histone F3 has been fragmented into 10 non‐overlapping fragments.With the aid of the automatic Edman degradation procedure for proteins using 3‐(N,N‐dimethylamino)propyne buffer the first 48 amino‐acid residues in the uncleaved protein have been positioned.The remainder of the amino‐acid sequence of this protein was elucidated by subjecting the aligned peptides to the automatic degradation. The amino acids obtained during the degradation were quantitated and identified by gas chromatography and amino‐acid analysis.The primary structure of this histone is compared to the corresponding fraction from calf which had been independently determined by another laboratory.
UDP‐glucuronyltransferase in Perfused Rat Liver and in Microsomes: Influence of Phenobarbital and 3‐Methylcholanthrene
Authors: BOCK Karl Walter , WHITE Ian N. H. 
Published: March 2005

Liver microsomal UDP‐glucuronyltransferase (1‐naphthol as substrate) is activated about 10‐fold by treatment of native microsomes in vitro with Triton X‐100. Following pretreatment of rats with 3‐methylcholanthrene or phenobarbital the specific activities of detergent‐activated microsomes were increased by about 300% and 40%, respectively, while the corresponding increases in native microsomes were only about 70% and 10%, respectively. In isolated liver perfused with 0.5 mM 1‐naphthol, the rate of glururonidation of naphthol, as determined by the appearance of naphthol glucuronide in the perfusate, bile and liver tissue, was 0.1 μmol × min−1× g liver−1. This rate of glucuronidation was increased by 70% in rats pretreated with 3‐methylcholanthrene and by 140% following phenobarbital treatment, in contrast to the changes observed in microsomes.A UDP‐glucuronic acid level of 0.3 μmol/g liver was determined in perfused liver from control rats. This level remained unchanged during perfusion with 0.5 mM 1‐naphthol. In the perfused liver from phenobarbital or 3‐methylcholanthrene‐treated rats, the UDP‐glucuronate level increased to 0.5 μmol/g liver.In control, 3‐methylcholanthrene or phenobarbital‐treated rats, rates of glucuronidation of 1‐naphthol in the perfused liver system correspond best with the UDP‐glucuronyltransferase activities determined in native microsomes when the different UDP‐glucuronate levels, microsomal protein contents and liver weights are taken into account.The results suggest that the enzyme is mostly latent in the microsomal membrane and that this latency is increased after pretreatment of rats with 3‐methylcholanthrene despite the marked enzyme induction.
Heme‐Linked Properties of Euglena Cytochrome f
Authors: BEN‐HAYYIM Gozal , SCHEJTER Abel 
Published: March 2005

Oxidized Euglena cytochrome f has an absorbance band at 695 nm which disappears upon reduction. Various treatments that cause known changes of conformation of cytochrome c appear to have similar effects on cytochromef. Heating cytochrome f results in the gradual disappearance of the 695‐nm band; the thermodynamic parameters for this change of the tertiary structure at 26 °C and ionic strength 0.01 are: ΔF=+2.67 kcal/mole, ΔH=+ 33 kcal/mol and ΔS=+102cal × K−1× mol−1.The pK values obtained by equilibrium titrations of the heme‐linked acid and alkaline ionizations are roughly 2.1 and 9.4. Equilibrium studies of the alkaline pK reveal that more than one hemelinked proton is dissociated in this pH range.The carboxymethylated product of the cyanide complex of oxidized cytochrome f has the same properties as cytochrome c carboxymethylated under the same conditions. The results suggest that a methionine sulfur is involved in iron coordination in oxidized Euglena cytochrome f.
The Dynamics of the Allomorphic Forms of Mouse Mitochondrial DNA
Authors: PORCHER Harald H. , KOCH Jürgen 
Published: March 2005

Mouse mitochondrial DNA can be fractionated by means of band sedimentation into the allomorphic forms supercoiled circle (34 S), open circle (24 S) and linear rod (21 S). The separation of the ring‐open form based on its hydrodynamic properties is not necessarily an indication of the chemical properties of this form, i.e. a nick in the phosphodiester backbone of at least one strand of the duplex DNA. Circular duplex DNA molecules may show an ambiguous behaviour. During sedimentation the high frictional coefficient of open circles is observed. However, as judged by other criteria the molecules behave like covalently closed circular DNA with invariant topological winding (restricted uptake of an intercalating dye).A large part of mouse mitochondrial DNA occurs as D‐loop mitochondrial DNA molecules which contain a displaced single‐stranded region in the duplex DNA molecule. The insertion of the new displacing strand reduces the rotation of the parental strands around each other and therefore reduces the number of right‐handed superhelical turns in the “covalently closed” circular DNA. For this reason, the bulk of the mouse D‐loop mitochondrial DNA molecules display a sedimentation behaviour (28 S) similar to that of ring‐open (nicked) molecules although these molecules contain a “covalently closed” phosphodiester backbone in each strand. Pulse‐chase‐pulse experiments, labeling firstly with [2‐14C]thymidine and secondly with [Me‐3H]thymidine, indicate that the different allomorphic forms of the mitochondrial DNA turn over at a different rate. The lowest turnover rate is observed in alkali‐stable supercoiled mitochondrial DNA and in D‐loop mitochondrial DNA. It is concluded that the accumulation of D‐loop mitochondrial DNA molecules in mouse cells is due to abortive replication events.
Generation in vivo of “Covalently Closed” Circular Mitochondrial DNA Free of Superhelical Turns
Authors: RASTOGI Anil K. , KOCH Jürgen 
Published: March 2005

The non‐intercalating tryptanocidal drug berenil inhibits completely the synthesis of the mitochondrial DNA in cultured human cells. This interference appears to be fairly selective, as the effect on the synthesis of the nuclear DNA is relatively small.The exposure of the cells to the drug results in a conversion in vivo of existing supercoiled mitochondrial DNA into DNA molecules with the hydrodynamic properties of ring‐open mitochondrial DNA. Dye‐buoyant‐density separation and titration with ethidium bromide indicate that this ring‐open mitochondrial DNA contains “covalently closed” circular DNA molecules which are free of superhelical turns.
Fluorescence Studies of Saccharide Binding to Wheat‐Germ Agglutinin (Lectin)
Authors: PRIVAT Jean‐Paul , DELMOTTE Francis , MIALONIER Gérard , BOUCHARD Philippe , MONSIGNY Michel 
Published: March 2005

Wheat germ agglutinin was isolated in pure form by a method adapted from D. LeVine, M. J. Kaplan and P. J. Greenaway, Biochem. J. 129, 847–856 (1972). Amino‐acid composition calculated for a molecular weight of 23 500 showed that the protein contained four tryptophan residues and was rich in half‐cystine (38 residues) and glycine (51 residues).Analysis of the fluorescence spectrum showed a characteristic tryptophan emission with a maximum centered at 348 nm. The spectrum is almost the same between pH 3.5 and 9.0. By adding N‐acetylglucosaminides (β‐d‐GlcNAc 1 → 4)n‐d‐GlcNAc, an enhancement of the fluorescence intensity was detected. Moreover, binding of the dimer and higher GlcNAc oligomers induced a 10‐nm shift of the emission maximum towards shorter wavelengths indicating that the environment of one or several tryptophan residues was altered by addition of ligands. The pH dependence of the fluorescence quantum yields shows that the tryptophan fluorescence is not altered by any ionisable group which possesses a pK in the range 4.0–9.0 in both the free and the complexed protein.Association constants were determined by measuring the change in fluorescence intensity produced by addition of different glucosaminides. These constants increased with chain length up to the trimer. No cooperative effect could be found with any ligand. It was shown that the carbohydrate binding is abolished below pH 2.8 and that a protein carboxyl group of pKa= 3.9 is involved.Our results are discussed and compared with the model proposed by A. K. Allen, A. Neuberger and N. Sharon, Biochem. J. 131, 155–162 (1973).
Accessible DNA in Chromatin
Authors: ITZHAKI Ruth F. 
Published: March 2005

A study has been made of the action of DNAase I and of micrococcal nuclease on chromatin, on the complex of chromatin with polylysine and of DNAase I on DNA · protamine; a comparison of different assay methods of DNAase I action on chromatin is included here. Also, the nature of the polylysine‐binding zones in chromatin has been investigated.Measurements of acid‐solubility and salt‐solubility during digestion show that DNAases I and II degrade almost all of the DNA in chromatin (from various tissues), unimpeded by the chromatin proteins which are released concurrently (and which eventually aggregate on to the degraded DNA). With micrococcal nuclease concurrent release of DNA and of protein occurs similarly; however, full degradation by this nuclease is impeded apparently by aggregation of released protein on to acid‐insoluble DNA. In the case of chromatin · polylysine and also DNA · protamine, about half of the DNA is degradable. The results indicate that discrepancies between the findings of previous workers are due to differences in assay of degradation, in conditions of digestion, in enzyme used and probably in methods of preparing chromatin resulting in products varying widely in composition. Also, the results support the previous suggestion that virtually all the DNA in chromatin is associated with protein yet is highly accessible [1], unlike DNA complexed with protamine or polylysine. The polylysine‐binding zones in chromatin are discrete, double‐stranded, protein‐associated regions of DNA which are probably heterogenous from one molecule to another.
Cell Envelope and Shape of Escherichia coli K12
Authors: GARTEN Wolfgang , HENNING Ulf 
Published: March 2005

The protein from Escherichia coli ghosts can be separated electrophoretically into three major bands (I, II, and IV) and one minor band (III). The proteins corresponding to bands I, II and IV have been isolated. Protein IV is the lipoprotein described previously by Braun et al. and is thus a homogeneous polypeptide chain. Proteins I and II do not contain phosphorus, palmitic acid, or lipopolysaccharide but small amounts of carbohydrate, probably glucosamine (or a derivative).Proteins I and II have been characterized by amino‐acid analyses, tryptic fingerprints, carboxy‐peptidase digestion, and cyanogen bromide cleavage. Based on cysteic acid the minimum chemical molecular weights of both proteins are the same as those found by electrophoreses: about 40000 (±∼ 10%) for I and about 28000 (±∼ 10%) for II. The numbers of tryptic peptides found do not argue against the assumption that each of the two proteins consists of identical polypeptide chains. However, the sum of the apparent molecular weights of the cyanogen bromide fragments of protein I is almost 80000. The amino acids liberated by carboxypeptidase A at near integral values per 40000 g of protein I correspond to 1 mol each of serine, glycine, alanine, isoleucine, tyrosine, phenylalanine, and to 2 mol valine per 40000 g of protein; however, only 0.5 mol leucine is released. Apart from revealing a rather hydrophobic C‐terminal region, the data thus include the possibility that protein I consists of two different but presumably rather similar polypeptide chains. Protein II is a tryptic fragment of a protein II* with a molecular weight near 40000. Protein II* can give rise to two other fragments by the action of other proteases. The apparent molecular weights of the cyanogen bromide fragments of protein II add up to that of protein II. However, although carboxypeptidase A liberates nearly 1 mol each of serine, alanine, valine, and leucine per 28000 g of protein II, it releases only about 0.5 mol each of glutamic acid, glycine and isoleucine. As for protein I, it cannot yet be excluded that protein II consists of two polypeptide chains. If so, the two must be very similar. Regardless as to whether proteins I or II consist of one or two polypeptide chains these must be arranged as repeating subunits in the outer membrane of the E. coli cell envelope.The properties of proteins I and II allow them to be classified as intrinsic or integral membrane proteins.