bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and Characterization of the Polypeptide Components from Light‐Harvesting Pigment‐Protein Complex B800–850 of Rhodopseudomonas capsulata
Authors: SHIOZAWA Judith A. , CUENDET Pierre A. , DREWS Gerhart , ZUBER Herbert 
Published: March 2005

The light‐harvesting bacteriochlorophyll‐carotenoid‐protein complex B800–850 was been isolated from membranes of the phototroph‐negative mutant strain Y5 of Rhodopseudomonas capsulata. The three polypeptides of the complex have been found to be soluble in chloroform‐methanol (1:1, v/v) in the presence of 0.1 M ammonium acetate. They were extracted from the complex and separated by gel filtration on Sephadex LH‐60 in the same solvent mixture.Minimum molecular weights based on amino acid composition are 12000, 9300, and 5100. Values previously determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis are 14000, 10000 and 8000. The two smaller polypeptides (polarities 31% and 39%) are completely soluble in chloroform/methanol/ammonium acetate while the largest and most polar (41%) polypeptide is only partially soluble. The largest polypeptide contains no tryptophan. The middle polypeptide contains no cysteine and arginine, while the small polypeptide lacks cysteine. Methionine is shown to be the amino terminus for the small and middle polypeptides by two independent methods (Edman degradation and dansylation). Both methods also indicated that the N terminus of the 14000 polypeptide seems to be blocked. Partial N‐terminal amino acid sequences were obtained for the two smaller polypeptides. No homology between the two proteins was observed.
Citrate Lyase Deacetylase of Rhodopseudomonas gelatinosa. Isolation of the Enzyme and Studies on the Inhibition by l‐Glutamate
Authors: GIFFHORN Friedrich , RODE Hergo , KUHN Anita , GOTTSCHALK Gerhard 
Published: March 2005

Citrate lyase deacetylase or acetyl‐S‐(acyl‐carrier protein) enzyme thioester hydrolase (acetate) (EC 3.1.2.‐), was purified 3100‐fold with a yield of 3.8% from cell extracts of Rhodopseudomonas gelatinosa. The final enzyme preparation gave a single protein band upon polyacrylamide‐gel electrophoresis in the absence or in the presence of sodium dodecylsulfate. The molecular weight of the native enzyme was estimated by gel filtration to be 14300 ± 1000. Sodium dodecylsulfate/polyacrylamide gel electrophoresis yielded a molecular weight of 7300 ± 600 indicating that the enzyme consisted of two subunits.Citrate lyase deacetylase acted as an S‐acetyl enzyme thioesterhydrolase because it catalyzed the conversion of citrate lyase (S‐acetyl form) into citrate lyase (Sulfhydryl form) and acetate.Citrate lyase deacetylase was strongly inhibited by l‐glutamate. The half‐maximal inhibitor concentration was 7.5 × 10−4 M, and a Ki‐value of 1.2 × 10−4 M was determined. The mode of inhibition appeared to be of the linear mixed type. l‐Glutamate was bound by citrate lyric deacetylase but not by citrate lyase.The pool concentrations of l‐glutamate inR. gelatinosa were 10 mM when citrate was present in substrate amounts and 2.7 mM after total consumption of citrate. Simulation of conditions in vivo using homogeneous enzyme preparations of citrate lyase and citrate lyase deacetylase, and glutamate concentrations of 10 mM and 2.7 mM respectively, revealed that the rate of citrate lyase inactivation increased from 8% within 15 min during growth on citrate to 50% within 15 min in the absence of citrate.
Properties and Characterization of a Spinach Chloroplast RNA Polymerase Isolated from a Transcriptionally Active DNA‐Protein Complex
Authors: BRIAT Jean‐François , MACHE Régis 
Published: March 2005

A chloroplast RNA polymerase has been isolated from a transcriptionally active spinach plastid DNA‐protein complex.The properties of the complex and of the reconstituted system have been compared. The crude enzyme, is at least sevenfold less active when compared with the complex. RNA synthesis by the reconstituted system is sensitive to high ionic strength and heparin, contrarily to RNA synthesis by the chloroplast DNA‐protein complex. On the other hand, rifampicin has no inhibitory effect whatever on the transcriptional system used. The RNA polymerase isolated is more efficient with denatured DNA than with double‐stranded DNA and the best template is chloroplast DNA.The crude RNA polymerase isolated migrates in a peak of 11 S in glycerol gradient centrifugation and is located in a single band in non‐denaturing polyacrylamide gel electrophoresis.About 30 polypeptides (Mr 15000–180000) are part of the complex and only eight of them are found in the RNA polymerase preparation. Only five polypeptides are always present with the same yield. They are probably the subunits of the RNA polymerase. The molecular weight of these subunits ranged from 15000–69000, even if the isolation of the enzyme was performed in the presence of protease inhibitors.
Correlation of Enzymatic Activity and Anticoagulant Properties of Phospholipase A2
Authors: VERHEIJ Hubertus M. , BOFFA Marie‐Claire , ROTHEN Christophe , BRYCKAERT Marie‐Claude , VERGER Robert , de HAAS Gerard H. 
Published: March 2005

Some highly purified phospholipases A from the venom of viperidae, crotalidae and elapidae were found to have anticoagulant properties. All phospholipases which exhibited anticoagulant properties are characterized by a high isoelectric point, but not all strongly basic phospholipases are anticoagulant. Anticoagulant phospholipases hydrolyse highly packed monomolecular films of phospholipids without any lag time while non‐anticoagulant phospholipases present considerable induction times indicative of a low penetrating power.When the ester linkages in the procoagulant lipids were replaced by the non‐hydrolysable ether bonds, the mixture retained its clotting ability even in the presence of phospholipases, thus suggesting that anticoagulant phospholipases prevent clot formation by hydrolysis of phospholipids. This was confirmed by chemical modification of phospholipases, viz. alkylation of the activecentre histidine with 1‐bromo‐octan‐2‐one. This modification yielded proteins which had lost their anticoagulant properties but which retained a high affinity for phospholipids.
The Inhibition and Activation of Ca2+‐Dependent Phosphatidylinositol Phosphodiesterase by Phospholipids and Blood Plasma
Authors: DAWSON Rex M. C. , HEMINGTON Norma , IRVINE Robin F. 
Published: March 2005

Phosphatidylinositol‐phosphodiesterase (EC 3.1.4.10) was strongly inhibited when ovophosphatidylcholine and saturated phosphatidylcholines with acyl chain lengths of more than eight carbon atoms were mixed with its substrate. In contrast, dihexanoylglycerophosphocholine produces a marked activation attended by a breakdown of the bilayer structure of the substrate.
C12, C14 and C16 lysophosphatidylcholines gave a progressive inhibition of the enzyme with increasing chain length; C10 lysophosphatidylcholine activated the reaction.
The enzyme was strongly inhibited by blood plasma and serum and by blood lipoproteins.
Phosphatidic acid, and certain lysophosphatidic acids (oleoyl and palmitoylglycerophosphates) activated the hydrolysis at a physiological pH, whereas decanoylglycerophosphate had little effect.
Phosphatidic acid at a concentration of 1% molar total lipid P produced an enhancement of the hydrolysis of the phosphatidylinositol substrate contained in a lipid environment which approximated to the inner (cytoplasmic) lamella of the plasma membrane bilayer of the liver sinusoidal cell surface.
The possible role of phospholipid molecules adjacent to phosphatidylinositol in controlling the hydrolysis of the latter in the proximity of a cell surface receptor is discussed. It is suggested that phosphatidic acid formed by the action of diacylglycerol kinase could amplify any increased hydrolysis of phosphatidylinositol which results from stimulation of the cell by an agonist.
Purification and Characterization of Nucleoside Diphosphatase from Rat‐Liver Microsomes
Authors: OHKUBO Iwao , ISHIBASHI Teruo , TANIGUCHI Naoyuki , MAKITA Akira 
Published: March 2005

Nucleoside diphosphatase was purified from rat liver microsomes more than 3000‐fold with a 16% yield using a procedure including concanavalin‐A‐Sepharose and phenyl‐Sepharose column chromatography. The purified enzyme had a specific activity of 2500 units/mg protein and appeared homogeneous by gel electrophoresis. The enzyme had a sedimentation coefficient of 6.5 S by sucrose‐density gradient centrifugation, and a Stokes' radius of 4.8 nm was estimated by the gel filtration technique. Its molecular weight is 130000, but only one single band of Mr 65000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 9% carbohydrates. The enzyme had a pH optimum of 7. 5, an isoelectric point of 4.85 and a Km of 2. 5 mM for UDP.On the basis of direct measurement of metal content in the native enzyme, the rat liver nucleoside diphosphatase was found to be a metalloenzyme containing 0.9 mol zinc and 0.1 mol manganese/mol 65000‐Mr subunit. Metal‐free nucleoside diphosphatase has been prepared. The activity of the metal‐free enzyme was restored by the addition of several divalent cations, zinc being the most effective.
Purification and Primary Structure Determination of the N‐Terminal Blocked Protein, L11, from Escherichia coli Ribosomes
Authors: DOGNIN Mai J. , WITTMANN‐LIEBOLD Brigitte 
Published: March 2005

Protein L11 was isolated from the 50‐S subunit of Escherichia coli ribosomes, using two salt extractions and two chromatographic separations on CM‐cellulose. The unusual behavior of the protein when run on sodium dodecyl sulfate electrophoresis showed multiple bands.The complete primary structure of protein L11 is presented in detail. Its sequence was derived from peptides obtained by digesting the protein with trypsin, chymotrypsin, thermolysin, Staphylococcus aureus protease and, after modification, with trypsin. Chemical cleavage was performed with cyanogen bromide. Sequencing of the various peptides was achieved by manual micro‐dansyl‐Edman degradations and automatic methods. The N‐terminal residue of the protein is blocked and was not degradable in the liquid‐phase sequenator by the Edman method. It was identified by a combination of enzymatic cleavage and mass spectrometry.Protein L11 contains three methylated amino acid residues, a Nα‐trimethylalanine, and two residues of Nα‐trimethyllysine. Their behaviour and influence in the sequence elucidation are described. The protein contains 141 amino acid residues and has a molecular weight of 14874. Secondary structure predictions of the protein are given, and its sequence is compared with those of other E. coli ribosomal proteins.
A Critical Period of Ecdysterone Action on Sensitive Clones of Drosophila Cultured in vitro: the Maturation of the Cells
Authors: BEST‐BELPOMME Martin , COURGEON Anne‐Marie 
Published: March 2005

Ecdysterone‐sensitive clones cultured in vitro were isolated from established cell lines of Drosophila melanogaster. The clones FC and 89K are ecdysterone‐inducible for two enzymatic activities: acetylcholinesterase and β‐galactosidase. No activity could be detected in untreated cells, whereas after treatment with 50–250 nM ecdysterone, the activity appeared after one day and increased during 3–4 days.We wanted to modulate the response of the cells by varying the conditions of the hormonal stimulus. Mimicking the physiological situation of Drosophila (the ecdysterone peak corresponding to the molts is preceded by low levels) we pretreated the cells with a subthreshold concentration (1–5 nM) for 2 days and then we added the stimulating concentration of 50–250 nM ecdysterone. The enzymatic activities were then detectable within the following hours and the final level of induction was about twice the one of cells without pretreatment.Thus, the continuous presence of a subthreshold concentration of ecdysterone provokes the maturation of the cells which become able to respond to the hormonal stimulus by a quicker and higher enzymatic induction.The cellular maturation seems to be a critical period. It is altosid‐sensitive. Altosid (a juvenile hormone analog) abolishes the effects of the ecdysterone‐induced maturation.
Glucosyltransferase Activities in Liver Mitochondria
Authors: GATEAU Odile , MORELIS Renee , LOUISOT Pierre 
Published: March 2005

Mitochondria, and specially outer mitochondrial membranes, incorporate d‐[125C]glucose from UDP‐d‐[14C]glucose into products extracted with organic solvents and into a residual precipitate, with a pH optimum of about 6.5 in (2‐N‐morpholine‐ethane)‐sulfonic acid (MES) buffer.The chloroform/methanol (2:1, v/v) extract contains two products. The major [14C]glucolipid is stable to mild alkali, but releases [14C]glucose upon mild acid hydrolysis. It is retained on DEAE‐cellulose (acetate form) and is eluted with the same ionic strength as an hexosyldolichyl monophosphate diester. This [14C] glucolipid has the same chromatographic behaviour as dolichyl‐mannosylphosphate in neutral, acidic and basic solvent systems; and its biosynthesis is greatly increased by exogenous dolichylmonophosphate. the other [14C]glucolipid is stable upon mild acid hydrolysis and is not retained on DEAE‐cellulose. On silicic acid it is eluted with acetone. The biosynthesis of this compound is stimulated by exogenous ceramide. This glucolipid has the same chromatographic mobility in different solvent systems as glucosylceramide isolated from the liver of a patient with Gaucher's disease. Biosynthesis of these two glucolipids is inhibited by UDP, but only biosynthesis of dolichylglucosyl monophosphate is reversible with this nucleotide.The biosynthesis of these different glucosylated derivatives is stimulated by the addition of divalent cations (Mn2+, Mg2+). The effect of these two metal ions on dolichylglucosyl monophosphate and glucosylceramide formation is studied in different conditions.
A γ‐like DNA Polymerase in Spinach Chloroplasts
Authors: SALA Francesco , AMILENI Anna R. , PARISI Bruno , SPADARI Silvio 
Published: March 2005

A DNA polymerase has been extracted from spinach chloroplasts and purified by chromatography on DEAE‐cellulose and hydroxyapatite. A great similarity between the purified chioropiast polymerase and the mammalian mitochondrial DNA polymerase γ was found by several criteria: preference for the synthetic primer‐template (dT)12–18· poly(rA), optimal requirement for Mn2+ (0.1–1.0 mM), KCl (100 mM) and pH (8–9), high relative molecular mass (approximately 105000), resistance to aphidicolin and inhibition by N‐ethylmaleimide. Some peculiar features of the chloroplast DNA polymerase have, however, been noticed.The mammalian DNA polymerase y has been suggested to be responsible for the replication of mitochondrial DNA. Thus, both the presence of a γ‐like DNA polymerase in chloroplasts and the similarities between the chloroplast and the mitochondrial DNA (absence of a nucleosomal structure and presence of displacement loops) lead to the suggestion that chloroplast DNA is also replicated by a γ‐like DNA polymerase and that the γ polymerases present in eukaryotes are, therefore, involved in a strand‐displacement DNA synthesis.An α‐like DNA polymerase activity, present and predominant in crude leaf extracts, was practically absent from purified chloroplast preparations.
Biogenesis of Glyoxysomes
Authors: ZIMMERMANN Richard , NEUPERT Walter 
Published: March 2005

Biosynthesis of isocitrate lyase, a tetrameric enzyme of the glyoxysomal matrix, was studied in Neurospora crassa, in which the formation of glyoxysomes was induced by a substitution of sucrose medium by acetate medium.

Translation of Neurospora mRNA in reticulocyte lysates yields a product which has the same apparent molecular weight as the subunit of the functional enzyme. Using N‐formyl[35S]methionyl tRNAMetf as a label, the translation product shows the same apparent size which indicates that the amino terminus has no additional ‘signal’‐type sequence.
Read‐out systems employing free and membrane‐bound polysomes show that only free ribosomes are active in the synthesis of isocitrate lyase.
Isocitrate lyase synthesized in reticulocyte lysate is released into the supernatant and is soluble in a monomeric form. It interacts with Triton X‐100 to form mixed micells in contrast to the functional tetrameric form.
Transfer of isocitrate lyase synthesized in vitro into isolated glyoxysomes is suggested by results of experiments in which supernatants from reticulocyte lysates are incubated with a particle fraction isolated from acetate‐grown cells. No transfer occurs when particles from non‐induced cells are employed. Resistance to added proteinase is used as a criterion for transmembrane transfer.

The data support a post‐translational transfer mechanism for isocitrate lyase. They suggest that isocitrate lyase passes through a cytosolic precurscr pool as a monomer and is transferred into glyoxysomes.
Synthesis in vitro of Keratin Polypeptides Directed by mRNA Isolated from Newborn and Adult Mouse Epidermis
Authors: SCHWEIZER Jürgen , GOERTTLER Klaus 
Published: March 2005

A method is described which allows the isolation of undegraded total cellular RNA from living epidermis in a one‐step procedure. Epidermal tissue is homogenized in 4 M guanidinium thiocyanate/2% sarkosyl buffer and centrifuged through a 5.7 M CsCl cushion. Under appropriate density conditions in the homogenate, large amounts of RNA are sedimented free of DNA and protein to the bottom of the tubes. A prerequisite for the applicability of this method is the preparation of pure epidermis under conditions which efficiently inactivate endogenous RNases.Oligo(dT) affinity chromatography of total RNA yields 3–4% of polyadenylated RNA. Translation of poly(A)‐rich fractions from both newborn and adult mouse epidermis in a cell‐free reticulocyte system leads to the synthesis of proteins with electrophoretic mobilities identical to those of native keratin polypeptides of the corresponding tissue. Similar to native keratin polypeptides, these proteins are highly insoluble and can selectively be enriched by high‐salt extraction of the translation assays. The CNBr cleavage pattern of these purified proteins is identical to that of native keratins. There is no evidence for the synthesis of precursors of these proteins.
Further Electron Microscope Studies on Pyruvate Carboxylase
Authors: MAYER Frank , WALLACE John C. , KEECH D. Bruce 
Published: March 2005

Using negative staining and electron microscopic tilting techniques in conjunction with modelling experiments, the fine structure of chicken, sheep and rat pyruvate carboxylases has been studied. The overall configuration appears to be a tetrahedron‐like structure consisting of two pairs of subunits in two different planes orthogonal to each other with the opposing pairs of subunits interacting with each other on their convex surfaces.The predominant form of the enzyme particles mounted and stained in the presence of acetylcoenzyme A consisted of a compact, triangular outline enclosing three readily visible intensity maxima. When samples were mounted in the absence of acetyl‐coenzyme A the molecules were more ‘open’ predominantly rhomboid structures. From tilting experiments it is concluded that the rhomboid images found in the absence of acetyl‐coenzyme A represent partly or wholly flattened forms of the tetrahedron‐like molecule. A feature of the enzyme when mounted in the absence of acetyl‐coenzyme A was the existence of a ‘cleft’ along the longitudinal midline of each subunit, suggesting that the subunits may consist of two distinct domains.
Separation and Characterization of an Autolytic Endo‐β‐glucosaminidase from Bacillus cereus
Authors: KAWAGISHI Shigenori , ARAKI Yoshio , ITO Eiji 
Published: March 2005

An autolytic endo‐β‐glucosaminidase, capable of cleaving the glycoside linkages of N‐unsubstituted glucosamine in the glycan moiety of cell wall peptidoglycan, was purified 470‐fold from a salt extract of the 20000 x g precipitate fraction obtained after sonication of a lysozyme‐resistant strain of Bacillus cereus. The properties of this enzyme were studied.
The purified enzyme preparation was also active towards the glycan chain of fully N‐acetylated cell wall peptidoglycan.
The endo‐β‐glucosaminidase was inactive towards the cell wall peptidoglycan unless the peptide portion of this polymer was removed either by the action of N‐acetylmuramyl‐l‐alanine amidase or by the treatment with alkali in aqueous dimethyl sulfoxide.
Studies on the action of this enzyme towards chemically modified glycans revealed that the carboxyl groups of muramic acid residues are indispensable to a substrate for this enzyme.
Complex Reaction Pathway of Aryl β‐Xyloside Degradation by β‐Xylanase of Cryptococcus albidus
Authors: BIELY Peter , VRŠANSKÁ Maria , KRÁTKÝA Zdeněk 
Published: March 2005

The extracellular endo‐1,4‐β‐xylanc se of the yeast Cryptococcus albidus catalyzes degradation of aryl β‐xylosides by other reactions than simple hydrolytic cleavage. Liberation of phenol or β‐nitrophenol from the corresponding β‐xylosides is accompanied by formation of xylose oligosaccharides and only small amounts of xylose. With the aid of phenyl β‐[U‐14C]xyloside synthesized from [U‐14C]xylose, it was established that the reaction followed a complex pattern with the rate of phenyl β‐xyloside digestion and appearance of various products varying markedly with time. The reaction involves multiple transglycosylic reaction leading first to phenyl glycosides of xylooligosaccharides, which are subsequently hydrolyzed mainly to xylobiose and xylotriose.At concentrations of phenyl β‐xyloside lower than 100 mM the reaction exhibited a significant lag phase, which was followed by period during which the rate of the degradation of the substrate could be determined. The rate showed a strong sigmoidal dependence on phcnyl‐β‐xyloside concentration. The lag phase could be eliminated and the initial rate accelerated by addition of xylose oligosaccharides, which are hydrolyzed by β‐xylanase. After disappearance of the added oligosaccharides, the reaction transitioNatly ceased and then resumed again at a rate comparable to the control without added oligosaccharides. It is proposed that β‐xylanase utilizes for degradation of phenyl β‐xyloside two reaction pathways differing in the nature of glycosyl donors.
p‐Toluenesulfonyl Chloride as an Activating Agent of Agarose for the Preparation of Immobilized Affinity Ligands and Proteins
Authors: NILSSON Kurt , MOSBACH Klaus 
Published: March 2005

A number of biomolecules were coupled covalently by nucleophilic displacement to agarose preparations substituted with tosyl groups. In one series of experiments N6‐(6‐aminohexyl)‐adenosine 5′‐monophosphate and N6‐(6‐aminohexyl)adenosine 2′,5′‐bisphosphatewere bound by their terminal amino groups to the polysaccharide support. It could be shown that from a mixture of lactate and 6‐phosphogluconate dehydrogenase the immobilized monophosphate showed bio‐affinity only for NAD+‐dependent lactate dehydrogenase, whereas the immobilized bisphosphate showed affinity only for the NADP+‐dependent 6‐phosphogluconate dehydrogenase. Furthermore, the immobilized monophosphate (5 μmol/g wet gel) was applied for the single‐step purification of lactate dehydrogenase from crude beef heart extract.To demonstrate the immobilization of proteins, soybean trypsin inhibitor (75 mg/g dry support) was immobilized to tosylated agarose, tested as affinity chromatography material and shown to bind 60 mg trypsin/g dry gel. Horseradish peroxidase and horse liver alcohol dehydrogenase were used as model enzymes. Although no optimization had been attempted, the former (approximately 70 mg/g dry support) had a coupling yield of approximately 18% with a specific activity (relative to soluble enzyme) of approximately 10%, whereas approximately 60% of alcohol dehydrogenase was coupled (approximately 100 mg/g dry support) with a specific activity of approximately 25%.