bluuebunny/crossref_metadata_2025_split huggingface dataset:
Subunit composition of oxaloacetate decarboxylase and characterization of the α chain as carboxyltransferase
Authors: DIMROTH Peter , THOMER Anna 
Published: March 2005

Oxaloacetate decarboxylase from Klebsiella aerogenes was shown to be composed of three different subunits α, β, γ with Mr 65000, 34000 and 12000, respectively. On dodecylsulfate polyacrylamide gels the smallest of these subunits was heavily stained with silver but poorly with Coomassie brilliant blue. All three subunits were resolved and clearly detectable by high‐performance liquid chromatography in a dodecylsulfate‐containing buffer. Biotin was localized exclusively in the α chain.Freezing and thawing of the isolated membranes in the presence of 1 M LiCl released the α chain which was subsequently purified to near homogeniety by affinity chromatography on monomeric avidin‐Sepharose. No β or γ chain were detectable in this α chain preparation and no oxaloacetate decarboxylation was catalyzed. The isolated α chain, however, was a catalytically active carboxyltransferase as evidenced from the isotopic exchange between [1‐14C]pyruvate and oxaloacetate. The rate of this exchange reaction was about 9 U/mg protein and was completely independent of the presence of Na+ ions. The ease with which the α chain was released from the membrane characterize this subunit as a peripheral membrane protein. The β and γ chain, on the other hand, stick so firmly in the membrane that they are only released by detergents, thus indicating that these are integral membrane proteins.Limited tryptic digestion of oxaloacetate decarboxylase led to a rapid cleavage of the α chain, yielding a polypeptide of Mr 51000 which was devoid of biotin. Degradation of the β chain required prolonged incubation periods and was markedly influenced by Na+ ions which had a protective effect against proteolysis.A proton is required in the decarboxylation of oxaloacetate and CO2 arises as primary product. The other alternative, i. e. generation of HCO−3 with H2O as substrate, has been excluded.
NADH oxidation in submitochondrial particles protects respiratory chain activity against damage by adriamycin‐Fe3+
Authors: DEMANT Erland J. F. 
Published: March 2005

Oxidative decomposition of polyunsaturated fatty acid moieties of membrane lipid in pig heart submitochondrial particles, as initiated by ferric ion complexes of the antineoplastic drug adriamycin and concomitant inactivation of oxidase activities, is counteracted by EDTA, low oxygen pressure, a phenolic antioxidant and NADH oxidation through the respiratory chain but not by scavengers of reactive oxygen species. Protection by NADH is strengthened by removal of cytochrome c from the submitochondrial particles and by antimycin A but abolished by rotenone.Inhibition of cytochrome c oxidase activity by the adriamycin‐Fe3+ complex is reversible and activity is recovered upon cholate solubilization of the particles. ADP inhibits binding of the complex to the submitochondrial particles and protects both cytochrome c oxidase activity and membrane lipid.The results are discussed in relation to the possible role of mitochondrial function in protection against free‐radical‐mediated effects of adriamycin.
On the cytotoxicity of vitamin C and metal ions
Authors: SAMUNI Amram , ARONOVITCH Jacob , GODINGER Dina , CHEVION Mordechai , CZAPSKI Gidon 
Published: March 2005

The toxicity of ascorbate towards phage lambda and the phages T2–T7 has been investigated. At room temperature the T‐odd and lambda bacteriophages are highly susceptible to ascorbate‐induced damage, whereas the T‐even phages are practically resistant. The toxicity of ascorbate is dependent on the presence of copper (or iron) and oxygen, although oxygen is not required in the presence of H2O2. Hydrogen peroxide is essential for the ascorbate‐induced phage inactivation and the damage is prevented by catalase. At the concentrations used, most of the copper ions are bound to the phage particles. Chelating agents such as EDTA or histidine fully protect the phages, whereas salicylate only reduces the rate of phage inactivation. OH scavengers such as sucrose, formate, mannitol, tert‐butyl alcohol or poly(ethylene glycol) have no protective effect.Experiments with DNA labeled phages indicate that both phage adsorption and DNA injection are impaired as a result of the exposure to ascorbate and copper. The failure to express the viral genetic information as a result of single and double‐strand breaks in the DNA, probably also contribute to the loss of the plaque‐forming ability of the phages.The results are interpreted in terms of a ‘site‐specific’ Fenton mechanism according to which the binding of the transition metal ions to the biological target is a prerequisite for the production of damage. The bound metal ion is reduced either by O−2, ascorbate or other reductants and is subsequently reoxidized by H2O2 yielding OH˙ radicals. This cyclic redox reaction of the metal generates OH˙ radicals which react with vital macromolecules with a high probability of causing ‘multi‐hit’ damage. This ‘site‐specific’ formation of OH˙ radicals, which takes place near the target molecules, accounts both for the high damaging efficiency and for the failure of OH˙ scavengers to protect against it.
β‐Agarases I and II from Pseudomonas atlantica
Authors: MORRICE Lora M. , McLEAN Maitland W. , LONG William F. , WILLIAMSON Frank B. 
Published: March 2005

β‐Agarase I and II were characterised by their action on agar‐type polysaccharides and oligosaccharides.β‐Agarase I, an endo‐enzyme, was specific for regions containing a minimum of one unsubstituted neoagarobiose unit [3,6‐anhydro‐α‐l‐galactopyranosyl‐(1 → 3)‐d‐galactose], hydrolysing at the reducing side of this moiety. Yaphe demonstrated that agar was degraded by this enzyme to neoagaro‐oligosaccharides limited by the disaccharide but with a predominance of the tetramer [Yaphe, W. (1957) Can. J. Microbiol. 3, 987–993]. β‐Agarase I slowly degraded neoagarohexaose but not the homologous tetrasaccharide. [1‐3H] Neoagarohexaitol was cleaved to neoagarotetraose and [1‐3H]neoagarobiitol. The highly substituted agar, porphyran was degraded to methylated, sulphated and unsubstituted neoagaro‐oligosaccharides which were invariably terminated at the reducing end by unsubstituted neoagarobiose.The novel enzyme, β‐agarase II, was shown to be an endo‐enzyme. Preliminary evidence indicated this enzyme was specific for sequences containing neoagarobiose and/or 61‐O‐methyl‐neoagarobiose. It degraded agar to neoagaro‐oligosaccharides of which the disaccharide was limiting and predominant. β‐Agarase II rapidly degraded isolated neoagarotetraose and neoagarohexaose to the disaccharide. With [1‐3H]neoagarohexaitol, exo‐action was observed, the alditol being cleaved to neoagarobiose and [1‐3H]neoagarotetraitol. Neoagarotetraitol was hydrolysed at 4% of the rate observed for the hexaitol. Porphyran was degraded to oligosaccharides, the neutral fraction comprising 24% of the starting carbohydrate. This fraction was almost exclusively disaccharides (22.4%) containing neoagarobiose (7.4%) and 61‐O‐methyl‐neoagarobiose (15%). β‐Agarase II is probably the ‘β‐neoagarotetraose hydrolase’ reported by Groleau and Yaphe as an exoenzyme against neoagaro‐oligosaccharides [Groleau, D. and Yaphe, W. (1977) Can. J. Microbiol. 23, 672–679].
Freeze‐fracture analysis of membrane appression and protein segregation in model membranes containing the chlorophyll‐protein complexes from chloroplasts
Authors: RYRIE Ivan J. 
Published: March 2005

Cation‐induced membrane appression and lateral segregation of chlorophyll‐protein complexes have been investigated by freeze‐fracture analysis of model membranes containing photosystem 1 and the light‐harvesting complex of photosystem 2. In light‐harvesting complex proteoliposomes, cations caused extensive membrane adhesion and a segregation of protein into appressed regions. A marked flattening of the appressed membranes, sometimes together with a co‐alignment of the particles on the opposing membrane faces, strongly suggests a direct transmembrane attraction between the protein particles. Photosystem 1 membranes were not appressed by cations but some clustering of the particles occurred, together with their exclusion from some regions of the lipid. By incorporating dipalmitoylglycerophosphocholine into the membranes, it is shown that a similar exclusion of the particles can occur due to liquid‐crystalline to gel state transitions of the lipids.Proteoliposomes containing both the light‐harvesting complex and photosystem 1 displayed cation‐induced membrane appression, but only between regions containing the light‐harvesting complex. Photosystem 1 was largely confined to unappressed membranes. Destacking occurred at low salt concentrations irrespective of whether photosystem 1 was present, showing that in proteoliposomes, and probably in thylakoids, this process does not require the presence of highly charged complexes from unappressed regions.
The identity of conformational states of reconstituted and native histone octamers
Authors: GREYLING H. Johann , SCHWAGER Sylva , SEWELL B. Trevor , von HOLT Claus 
Published: March 2005

Histone octamers were reconstituted from the following preparations: (a) natural histone H3‐H4 tetramer and histone H2A‐H2B dimer, either selectively extracted from chromatin with solutions of sodium chloride or prepared by dissociation of the natural octamer; (b) acid‐denatured core histones, either an unfractionated mixture or individually purified proteins.Complexes assembled from these histones elute from exclusion chromatography columns with octamer size as verified by cross‐linking with dimethylsuberimidate. The reconstituted octamers all crystallize in the same form of helical tubes as the natural octamer.
Cloning and characterization of the ribosomal genes of the sea‐urchin Paracentrotus lividus
Authors: PASSANANTI Claudio , FELSANI Armando , GIORDANO Roberto , METAFORA Salvatore , SPADAFORA Corrado 
Published: March 2005

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the PstI site of pBR 322 by the dG · dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping.The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot‐hybridization of the restriction fragments, using as probes nick‐translated 32P‐labelled cloned ribosomal DNA fragments or 18S and 26S sea‐urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 × 103 bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.
Structural studies on the phosphate‐free lipid A of Rhodomicrobium vannielii ATCC 17100
Authors: HOLST Otto , BOROWIAK Dietmar , WECKESSER Jürgen , MAYER Hubert 
Published: March 2005

The structure of the free lipid A from Rhodomicrobium vannielii ATCC 17100 was elucidated. It consists of a central β‐1′,6‐linked glucosamine disaccharide which is not substituted by phosphate. About 30% of the disaccharide molecules are substituted with mannopyranose in β‐1,4′‐linkage to the non‐reducing glucosamine. The reducing glucosamine can be directly reduced with NaBH4, indicating either that this glucosamine is not substituted at C1 or its substituent has been removed during the preparation of free lipid A or is removed during reduction with NaBH4. The following formula shows the ‘backbone’ structure of the free lipid A from Rm. vannielii ATCC 17100: β‐Manp(1→4)‐β‐GlcpN(1→6)GlcpN.3‐(R)‐Hydroxyhexadecanoic acid is linked to the amino group of the reducing glucosamine. The residue at the amino group of the non‐reducing glucosamine has not been identified. The hydroxyl groups of the central disaccharide are acylated with 3‐(tetradecanoyloxy)‐tetradecanoic acid, 3‐hydroxytetradecanoic acid, Δ14‐ docosenoic acid (Δ14‐C22:1) and acetyl groups. The hydroxyl groups of the mannose are not substituted.
Distance measurement by energy transfer
Authors: STEINHÄUSER Klaus G. , WOOLLEY Paul , DIJK Jan , EPE Bernd 
Published: March 2005

Ribosomal proteins L6, L11 and the complex [(L12)4· L10] were labelled specifically at their respective single thiol groups, either with the acetylaminoethyl‐dansyl or with the acetamidofluorescein fluorophore. The labelled proteins were then reconstituted, singly or in pairs, into ribosomal 50 S subunits; the presence of the label had no observable effect on the composition, shape or activity of the reconstituted subunits.The distances between the labelled thiol groups were measured by a fluorescence energy transfer method detailed elsewhere [Epe, B. et al. (1983) Proc. Natl Acad. Sci. USA, 80, 2579–2583] and were found to be: for L6–L10, 60 Å (6.0 nm); for L6–L11, 46 Å (4.6 nm); for L10–L11, 56 Å (5.6 nm). Reversal of the direction of energy transfer by exchanging labels gave duplicate distances which differed, on average, by about 4%. The distance between the fluorescent labels on L10 and L11 in the [23 S‐RNA · L10 · L11 · (L12)4] ribonucleoprotein complex was the same as in the 50S subunit, but all three distances were greater in 50S subunits which had been reconstituted without the final activation step (incubation at 50°C). This suggests a tightening of the L6/L10/L11 domain of the 50S subunit during the activation step.
Modification of ribosomal proteins in sea urchin eggs following fertilization
Authors: TAKESHIMA Kazuhito , NAKANO Eizo 
Published: March 2005

Analysis of the ribosomal proteins in sea urchin eggs by two‐dimensional polyacrylamide gel electrophoresis revealed postfertilization changes in the proteins of both the small and the large subunits. Five egg‐ribosomal proteins (S7, S16, S19, L19, L31) appeared to undergo rapid changes to the corresponding embryo‐specific proteins. These changes were completed within 30 min after fertilization, and identical electrophoretic patterns were observed among the different developmental stages of embryos. One of the five proteins, S7, showed an increase in the phosphorylated form. The remainder showed qualitative shifts to the corresponding embryo‐specific proteins; however, peptide map analyses revealed the existence of common structural units between the corresponding proteins.These modifications were observed in the three species of sea urchin studied (Pseudocentrotus depressus, Hemicentrotus pulcherrimus and Anthocidaris crassispina), except in the case of one protein (L31). Purification of ribosomes by different procedures based on high‐salt treatment gave the same results with respect to the egg‐specific and embryo‐specific proteins.
Synergistic inhibition of [3H]muscimol binding to calf‐brain synaptic membranes in the presence of l‐homocysteine and pyridoxal 5′‐phosphate
Authors: GRIFFITHS Roger , WILLIAMS David C. , O'NEILL Cora , DEWHURST Ian C. , EKUWEM Clement E. , SINCLAIR Crawford D. 
Published: March 2005

1. l‐Homocysteine and pyridoxal 5′‐phosphate (PxyP) inhibited [3H]muscimol binding to freeze‐thawed, Triton‐treated calf brain membranes (containing high‐affinity muscimol‐binding sites: Kd 9.5 ± 0.6nM, Bmax 5.2 ± 0.2 pmol/mg protein). The homocysteine–pyridoxal‐5′‐phosphate (Hcy‐PxyP) thiazine complex had no effect.2. l‐Homocysteine was found to be a partially competitive inhibitor, thus demonstrating an allosteric inhibition with Ki value of 1.96 mM for free receptor and Ki of 13 mM for receptor‐muscimol complex.3. PxyP was shown to be a two‐site pure competitive inhibitor of [3H]muscimol binding with cooperativity of PxyP binding such that Ki values for PxyP of 20 mM and 2.1 mM were found.4. l‐Homocysteine and PxyP when added simultaneously to binding assays, caused a greater degree of inhibition than that observed at the same total specific concentration of either inhibitor alone. This synergistic inhibitory effect was shown to be due to a homocysteine‐induced increase in the affinity of PxyP‐binding sites.5. Three alternative models are suggested to explain the observed synergistic effects whereby it is proposed that PxyP and [3H]muscimol binding is mutually exclusive, while l‐homocysteine with PxyP and l‐homocysteine with muscimol, exhibit non‐exclusivity.6. Non‐linear regression analysis of binding data was undertaken in order to substantiate conclusions drawn from graphical procedures and in an attempt to ascertain which mathematical model best fitted the experimental data describing the synergistic inhibitory effects of l‐homocysteine and PxyP.7. This synergistic inhibitory effect of PxyP and l‐homocysteine on the post‐synaptic γ‐aminobutyric acid receptor may provide a basis for explanation of the mechanism of homocysteine‐induced seizures.
Classification of phospholipases A2 according to sequence. Evolutionary and pharmacological implications
Authors: DUFTON Mark J. , HIDER Robert C. 
Published: March 2005

The sequences of 32 phospholipases A2 (EC 3.1.1.4) were systematically compared on the basis of polypeptide chain length and similarity at selected amino acid positions around the active site. Two difference matrices were constructed and the various groupings present in the data were expressed in dendrogram form. The two methods of comparison yielded different results, and this is seen as a consequence of separate aspects of phospholipase evolution being highlighted in each case.It appears that, although Elapid snake venom phospholipases are very similar in terms of overall conformation, the area around their active sites distinguishes them into two major groups, namely the Asian Elapids and the marine/Australasian Elapids. Further, the Asian Elapids seem to have active‐site vicinities which are closer to those in the mammalian pancreatic phospholipases. The relevance of the classifications to structure/activity relationships (especially β‐neurotoxicity) and phospholipase evolution is discussed.
Characterization of β‐galactosidase–lactose‐permease chimaeras of Escherichia coli
Authors: GRIESSER Hans‐Werner , MÜLLER‐HILL Benno , OVERATH Peter 
Published: March 2005

Escherichia coli strains have been isolated in which 3, 39 or 805 5′‐end codons of lacZ, the gene for the cytoplasmic enzyme β‐galactosidase are fused to codon 9 of lac Y, the gene for lactose permease. Lactose‐permease‐deficient cells, carrying the lacZ‐ Y fusions on F lac pro episomes, are phenotypically positive on eosin/methylene blue/lactose or on melibiose plates, demonstrating that the β‐galactosidase ‐lactose‐permease chimaeras transport lactose and melibiose in vivo.The apparent affinity for β‐d‐galactopypanosyl 1‐thio‐β‐d‐galactopyranoside (GalSGal) in cells is similar to that of the wild‐type gene product. The maximum velocity of active GalSGal transport is reduced in all three fusion strains. Both lactose and p‐nitrophenyl α‐d‐galactopyranoside inhibit GalSGal uptake.As demonstrated by immunoblot experiments the chimaeras cross‐react with polyclonal antibodies directed against native lactose permease and they are present in the cell envelope fraction of homogenates. Their apparent molecular weights upon electrophoresis in NaDodSO4/polyacrylamide gels correspond to those expected from their respective primary sequences, taking into account the migration properties of wild‐type lactose permease.−It is proposed that substitution of eight N‐terminal lactose permease residues by N‐terminal β‐galactosidase residues neither prevents membrane incorporation of permease nor completely impairs the ability to transport galactosides actively. Alternative interpretations of the experimental results are discussed.
Chemical modification of the lactose carrier of Escherichia coli by plumbagin, phenylarsinoxide or diethylpyrocarbonate affects the binding of galactoside
Authors: NEUHAUS Jean‐Marc , WRIGHT J. Keith 
Published: March 2005

The effects of chemical modification of the lactose carrier of Escherichia coli on galactoside binding (in overproducing strains) and on transport was examined. Both the modifying reagents diethylpyrocarbonate and rose bengal and the thiol reagents phenylarsinoxide and plumbagin can completely inhibit the binding of the substrate p‐nitrophenyl α‐d‐galactopyranoside to the carrier. If care is taken to inhibit galactoside binding only partially, the loss of transport is found to parallel the loss of binding sites. The modified carrier molecules are completely inactive, while the remaining active carrier molecules evince normal transport and binding parameters. The binding of galactoside protects the carrier partially against these forms of chemical modification.In view of these observations, the results of previous chemical modification studies [Padan, E., Patel, L. and Kaback, H. R. (1979) Proc. Natl Acad. Sci. USA, 76, 6221–6225; Konings, W. N. and Robillard, G. T. (1982) Proc. Natl Acad. Sci. USA, 79, 5480–5484] must be re‐interpreted. Our results stress the utility of studying substrate binding, the first step in the transport cycle.
Interferon‐inducing, pyrogenic and proclotting enzyme of horseshoe crab activation activities of chemically synthesized lipid A analogues
Authors: MATSUURA Motohiro , KOJIMA Yasuhiko , HOMMA J. Yuzuru , KUBOTA Yoshiyuki , SHIBUKAWA Nobuyuki , SHIBATA Masatoshi , INAGE Masaru , KUSUMOTO Shoichi , SHIBA Tetsuo 
Published: March 2005

Interferon‐inducing, pyrogenic and proclotting enzyme of horseshoe crab activation activities of chemically synthesized lipid A analogues were investigated and compared with the same activities of a natural lipid A. These analogues are nonphosphorylated, C‐1 or C‐4′ monophosphorylated and C‐1,4′ bisphosphorylated derivatives of β‐1,6‐linked d‐glucosamine disaccharide possessing both ester‐bound and amide‐bound fatty acid substituents. Fatty acid substituents of the analogues are tetradecanoyl (C14), (R)‐3‐hydroxytetradecanoyl (C14‐OH) or (R)‐3‐tetradecanoyloxytetradecanoyl [C14‐O‐(C14)] groups.The biological activities of the samples were assayed after solubilization with triethylamine and complexing with bovine serum albumin. Interferon‐inducing activity was exhibited by both the C‐1 monophosphorylated compounds examined. Ester‐bound and amide‐bound fatty acid substituents of these compounds are both C14 or C14 and C14‐OH, respectively. Nonphosphorylated, C‐4′ monophosphorylated and C‐1,4′ bisphosphorylated compounds possessing the same fatty acid substituents as those of the C‐1 monophosphorylated compounds showed no detectable interferon‐inducing activity. C‐4′ monophosphorylated compounds possessing C14‐OH as ester‐bound and C14‐OH or C14‐O‐(C14) as amide‐bound fatty acid substituents exhibited interferon‐inducing activity, but nonphosphorylated compounds possessing the same fatty acid substituents did not. None of the analogues exhibited significant pyrogenicity nor proclotting enzyme of horseshoe crab activation activity under the conditions employed in this study.