bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Forms and Hydrodynamic Properties of Acetylcholine Receptor from Electric Tissue
Authors: RÜCHEL Reinhard , WATTERS Diane , MAELICKE Alfred 
Published: March 2005

We have studied purified acetylcholine receptor proteins from Electrophorus electricus and Torpedo marmorata which function in both binding and reconstitution experiments. The molecular properties of these receptor‐channel complexes were analyzed under non‐denaturing conditions by polyacrylamide gradient gel electrophoresis, ultracentrifugation sedimentation studies and laser light scattering.The purified receptor proteins exist in different interconvertible forms depending on both the type and concentration of detergent present, and the presence or absence of an intersubunit disulfide bridge. Receptor purified in the absence of sulfhydryl‐blocking agents exists in two monomeric and two dimeric forms at very low detergent concentrations (0.01‐0.05% Tween 80). At intermediate detergent concentrations (0.4% Triton X‐100) one monomeric and one dimeric form are present. Only the monomeric form remains at high levels of detergent (2% Triton X‐100). This form has a sedimentation coefficient of 9.29 S, as measured by ultra‐centrifugation using Schlieren optics. If receptor is purified in the presence of sulfhydryl‐blocked agents, conververt dimmers into monomers by high concentrations of detergent does not occur. Disulfide‐reducing agents convert dimmers into monomers independent of whether the receptor's free sulfhydryl groups are blocked or not. These findings imply that dimmer formation is primarily due to hydrophobic interaction between monomers. When these interactions are reduced by high levels of detergent the sulfhydryl group(s) have remained unblocked.Monomers and dimmers bind α‐cobratoxin with the same affinity and kinetics. Dimers of disulfide‐linked monomers are not required for the reconstitution of a functional ion‐translocation system. The presence of EDTA during the purification of receptor proteins (as recommended by many groups to inhibit proteclysis) adversely influences the activity of the receptor in channel gating. These observations are discussed in terms of the requirements for a purification procedure yielding receptor preparations unaffected by proteolysis and functioning in both binding and ion translocation.The molecular weight of the receptor monomer was determined by several independent techniques and yielded values in the range of 250‐300×103. With a translational diffusion constant D20, w=2.95%10−7 cm2s−1 and a sedimentation coefficient s20, w=9.29 S, the frictional coefficient ratio f/fo=1.15 was calculated for the receptor monomer from Torpedo marmorata. This indicates a considerable asymmetry in the shape of the detergent‐solubilized receptor.
Reversible Cleavage of the Coalt‐Carbon Bond of Coenzyme B12 Catalysed by Methylmalonyl‐CoA Mutase from Propioniacterium shermanii
Authors: GAUDEMER Alain , ZYLER Jean , ZYLER Nicole , BARAN‐MARSZAC Maurice , HULL Willian E. , FOUNTOULAKIS Michael , KÖNIG Alfred , WÖLFLE Klaus , RÉTEY János 
Published: March 2005

(5′R)‐2H1)Adenosine [(5′R):(5′S) = 85:15] was prepared by a procedure which involved inter alia the reduction of 6‐N‐benzoyl‐2′,3′‐O‐isopropylidene 5′‐oxoadenosine with a reagent obtained from LiAl2H4 and (‐)‐ isoborneol.
(5′S)‐(5′‐2H1)AdoCbl= 5′S):(5′R) = 74:26] (AdoCbl = 5′ deoxyadenosylcoalamin) was synthesised by reacting cobal(I)amin with (5′R)‐2′‐3′‐O‐isopropylidene‐5′‐tosyl‐(5′‐2H1) adenosine followed by acid hydrolysis to remove the isopropylidene protective group.
(5′R)‐2H1)AdpCbl [(5′S):(5′R) = 77:23] was prepared by reacting cobal (I) amin with (5′S)‐5′‐chloro 5′‐(5′‐2H1) deoxyadenosine [(5′S):(5′R= 80:20] obtained in turn from (5′R)‐(5′‐2H1)adenosine. The reaction sequence involved two consecutive inversions at the C‐5′ atom of adenosine.
Comparison of the 500‐MHz 1H‐NMR spectra of unlaelled, (5′S)‐ and (5′R)‐(5′2H1)AdCbl allowed assignment of the triplet at 0.58 ppm and the doublet at 1.525 ppm to the diastereotopic 5′‐HRe and 5′‐HSt atoms, respectively. On acidification, these two protons gave rise to two triplets at 0.11 ppm and 1.78 ppm indicating that torsion had occurred around the C‐4′‐C‐5′ bond.
Samples of (5′R)‐ and (5′S)‐(5′2H1)AdoCbl were incubated with metylmalonyl0‐CoA mutase from Propioniacterium shermanii Examination by 1H‐NMR spectroscopy at 500 MHz revealed partial los and stereochemical scrambling of the deuterium at the 5′ position. This indicates transient conversion of the C‐5′ atom into a torsiosymmetric group and hence cleavage of the cobalt‐carbon bond during interaction with the enzyme. The mechanism by which deuterium is lost remains to be elucidated.
The Interaction of the High‐Density Lipoprotein with Cultured Cells of Bovine Vascular Endothelium
Authors: TAUBER Jean‐Pierre , GOLDMINZ David , VLODAVSKY Israel , GOSPODAROWICZ Denis 
Published: March 2005

Previous studies have shown that high‐density lipoprotein (HDL) is mitogenic for low‐density vascular endo thelial cell cultures. In contrast, low‐density lipoprotein(LDL) at physiological concentration is cytotoxic for this cell type [Tauber et al. (1980) J.Clin.Invest. 66, 696‐709]. In order to relate these observations to the physico‐chemical events occurring after the interaction of these lipoproteins with the cells, we have studied the binding, internalization, and degradation of HDL as a function of cell density and organization and have compared the results to those obtained with LDL.Cultured vascular endothelial cells bind HDL to an extent which, on a molar basis, is similar to that of LDL. Since an 80% loss of the LDL receptor sites in cells that were preexposed to LDL was not associated with a detectable decrease in HDL binding, it is likely that HDL and LDL binding sites exist on the cell surface as distinct entities A 5‐10 fold molar excess of LDL over 125 I‐HDL was almost as effective as a similar excess of unlabeled HDL in reducing the binding of 125, indicationg that LDL can comete with HDL for HDL binding sites. In contrast, 125 I‐LDL binding and uptake were reduced by less than 20% in the presence of a 50‐fold molar excess of unlabeled HDL. Both uptake and degradation of HDL were 10‐20 fold lower than those of LDL. HDL degradation was only slightly or not inhibited by chloroquine at a concentration which almost fully inhibited the degradation of LDL. Confluent and highly orgainized endothelial cell cultures bind HDL and LDL particles to an extent which is half of the amount bound to sparse or subconfluent culture However. The formation of a resting endothelial cell monlayer was associated with only a twofold decrease in the uptake of HDL, as compard to a 10‐20‐fold decrease in the uptake and degradation of LDL. In both sparse and confluent endothelial cultures, uptake via fluid pinocytosis non‐specific adscorptive endocytosis could account for most of the HDL uptake. The different pathways of internalization of HDL when added at high concentration to sparse and actively growing cultures.
Subunit Stoichiometry of Cytochrome c Oxidase of Bovine Heart
Authors: VERHEUL Frans E.A.M. , DRAIJER J. Wim , DENTENER Iris K. , MUIJSERS Anton O. 
Published: March 2005

Cytochrome c oxidase from bovine ehart was dissociated into its protein subunits by sodium dodecylesupphaste, the subnuntis were separated on a preparative scale by sodium dodecylsuplhate gel permeation chromatography. The subunits elute upon gel chromatography in order of decreasing apparent relative molecular mass (I, 40000; II, 26000; III, 21000; IV, 17000; V and VI, 12000; VII, 10000 and VIII, 6000), The very hydrophobic subunits I and III tend to form small aggregates both in the presence and absence of sodium dodecylsulphate. The molar ratio of the subunits was determined by two methods: firstly by quantitataive amino acid analysis of each subunit peak, and secondly from the absorbance of each subunit at 280 nm caused by tryptophan and tyrosine. We conclude that the subunits I to VI are present in 1:1 ratio; our fraction VII contains two stoichiometric polypeptides which may or may not be identical. Fraction VIII contains enough protein for four stoichiometric chains which may belong to three different types.The 12 stoichiometric chains add up to a Mr of about 170000 if the sizes of I and III are 40000 and 21000, respectively. After correction for the presence of aggregates, subunits I and III appear to be present in more than 1:1 stoichiometric amounts will respect to other subunits, which probably means that subunits I and III aare considerably larger than hitherto assumed. This is in line with recently published mtDNA sequence work [Anderson, S. et al. (1981) Nature, 290, 457‐465].
Characterization of Isolated Rat‐Liver Cells Made Permeable with Filipin
Authors: GANKEMA Henk S. , LAANEN Elly , GROEN Albert K. , TAGER Joseph M. 
Published: March 2005

When isolated rat‐liver cells were incubated for 1 min at 37°C with filipin at a concentration of 50μM, the plasma membrane became permeable to sucrose, inulin, glycerol 3‐phosphate and other low‐molecular‐weight compounds. Upon removal olf the filipin and subsequent incubation of the cells at 37°C there was a gradual leakage of lactate dehydrogenase from the cells. However, the leakage of lactate dehydrogenase could be prevented for about 10 min by including glutathione and ATP in the incubation medium.The filipin‐treated cells were able to metabolize phosphorylated sugars. The conversion of fructose 1,6‐bisphosphate to fructose 6‐phosphate, glucose 6‐phosphatae and glucose was inhibited by AMP but not by high concentrations of fructose 1,6‐bisphosphate.The results indicate that filipin‐treataed cells can be used to study the kinetic parameters of enzymes in their macromolecular environment in situ.
Effects of Chromatic Illumination on Cyanobacterial Phycobilisomes
Authors: BRYANT Donald A. , COHEN‐BAZIRE Germaine 
Published: March 2005

Pseudanabaena 7409 is a chromatically adapting cyanobacterium which photocontrols the synthesis of both phycoerythrin and phycocyanin [Tandeau de Marsac (1977) J. Bacteriol. 130, 82‐91]. Phycobilisomes, isolated from cells grown in either green or red light, have been dissociated and the component biliproteins purified and characterized.Phycobilisomes isolated from cells grown in green light were composed of allophycocyanin B, allophycocyanin, two phycocyanin subunits (one α‐type and one β‐type subunit), phycoerythrin and eight uncolored polypeptides. When dissociated phycobilisomes were chromatographed on DEAE‐cellulose at pH 5.5, most of the phycocyanin was recovered as part of a large (17.3 S) multiprotein complex with phycoerythrin (molar ratio 1:1). This complex also contained five of the uncolorerd polypeptides found in innntact phycobilisomes isolated from cells grown in green light.Phycobilisomes isolated from cells grown in red light were composed of allophycocyanin B, allophycocyanin, four phycocyanin subunits (two α‐type and two β‐type subunits), and six uncolored polypeptides. When these phycobilisomes were dissociated, the phycocyanin was recovered as a large (21.0 S) multiprotein comples which was composed of the four phycocyanin subunits types and four uncolored polypeptides. Thia complex was morphologically identical to the rod‐like stacks of discs about 6x12nm which form the peripheral rods of intact phycobilisomes.Each of the four phycocyanin subunits found in the complex isolated from the phycobilisomes of cells grown in red light was purified to homogeneity and characterized. Amino acid compositions of the four subunits indicated that each subunit was a unique gene product. Two of the subunits of the complex were apparently identical to those of the phycocyanin purified from phycobilisomes isolated from cells grown in green light. These studies suggest that one pair of phycocyanin subunits was synthesized constitutively (i.e. irrespective of the light wavelength to which the cells were exposed during growth) while the synthesis of the second pair of phycocyanin subunits was specifically induced during growth in red light.
The Photoregulated Expression of Multiple Phycocyanin Species
Authors: BRYANT Donald A. 
Published: March 2005

The regulation of phycocyanin synthesis in response to growth in chromatic illumination was studied in 69 strains of cyanobacteria. Cyanobacteria (24 of 31 strains examined), which chromatically adapt by modulating the synthesis of both phycocyanin and phycoerythrin, controlled phycocyanin synthesis through the differential, photoregulated expression of two phycocyanin species (two α‐type and two β‐type subunits). For these strains the expression of one pair of phycocyanin subunits was constitutive (i.e. irrespective of the light wavelength in which the cells were grown); the expression of the second pair of phycocyanin subunits occurred specifically during growth in red light. Two facultatively heterotrophic cyanobacteria, Calothrix strains 7101 and 7601, synthesized both the constitutive and the inducible pairs of phycocyanin subunits when grown heterotrophically in the dark after transfer from either red or green light.No evidence for the existence of multiple and/or photoregulated phycocyanin species was found for cyanobacteria (25 strains) incapable of chromatic adaptation, nor for cyanobacteia (13 strains) which chromatically adapt by modulating the synthesis of phycoerythrin alone.
A 13C Nuclear‐Magnetic‐Resonance Study on Free Flavins and Megasphaera elsdenii and Azotobacter vinelandii Flavodoxin
Authors: VAN SCHAGEN Cees G. , MÜLLER Franz 
Published: March 2005

Selectively 13C‐enriched free and protein‐bound flavins in the oxidized and two‐electron reduced state were investigated by the 13 C nuclear magnetic resonance technique. FMN in aqueous solution and N‐(3)‐menthyl‐2′,3′4,'5′‐tetraacetylriboflavin in an apolar solvent were used as reference compounds for the protein‐bound FMNA comparison of th chemical shifts of FMN in aqueous solution with those of N(3)‐methyltetraacetyl‐ riboglavin in CHCL3 reveals that FMN is strongly polarized yielding a pseudo‐ionic molecule stabilized by hydrogen bonding of H2O with C‐2α of the isoalloxazine molecule. The interpretation is fully supported by the one‐bond)1J) 13C coupling constants. The chemical shifts combined with the coupling constants indicate the change of charge distribution within the molecule when going from apolar to polar solutionsBinding of FMN to Megasphaera elsdenii and to Azotobacter vinelandii apoflavodoxins affects the resonances due to C‐2 and C‐10 a as compared to free FMN.These shifts together with the coupling constants indicate the formation of a hydrogen bound between C‐2α of FMN and an amino acid residue of the apoprotein. In M. Elsdenii flavodoxin such a hydrogen bond also exists with C‐4α which is not observed in A vinelandii flavodoxin.The chemical shifts to the two‐electron reduced derivatives of FMN and N(3)menthyl‐2′,3′,4′,5′,‐tetraacetyl‐ riboflavin clearly indicate that the FMN derivative possesses a more planar conformation than its analog; it shows pH‐independent, From the pH‐dependence of the chemical shifts of the atoms C‐10a, C‐2 and C‐4a. The chemical shift of C‐4 is pH‐independent. From the pH‐ dependence of the chemical shifts an ionization constant of 6.7 is calculated.The chemical shifts of protein‐bound FMN in the reduced state are very similar to those of the anionic, reduced FMN. The data show that in both flavodoxins studied the prosthetic group when reduced is bound in the anionic form an possesses approximately a coplanar confornation.The results are discussed with respect to the possible biological implications. It is suggested that specific interaction of the prosthetic group with the apoprotein, the ionization state and the degree of planarity of the reduced prosthetic group are factors determining the biological functions of flavodoxins and possibly the functions of flavoproteins in general.
The Catabolism of Phosphatidylinositol by an EDTA‐Insensitive Phospholipase A1and Calcium‐Dependent Phosphatidylinositol Phosphodiesterase in Rat Brain
Authors: HIRASAWA Keisuke , IRVINE Robin F. , DAWSON Rex M. C. 
Published: March 2005

A rat brain supernatant and microsomal fraction contained a phospholipase A1enzyme which hydrolysed phosphatidylinositol at pH 8 in the absence of calcium. Triolein and phosphatidylcholine were not attacked under the same incubation conditions.
No evidence could be obtained for a phospholipase A2in the microsomal preparation, and in the presence of Ca2+the release of fatty acid observed was due to phosphatidylinositol phosphodiesterase followed by diacylglycerol lipase action.
Brain phosphatidylinositol phosphodiesterase showed extensive activity in the alkaline range (7–8.5) as well as at pH 5–5.5. The activity at higher pH values required higher calcium concentrations and disappeared on purification of the soluble enzyme by ammonium sulphate fractionation.
In general the ratio between inositol 1,2‐(cyclic)phosphate and inositol 1‐phosphate produced by phosphodiesterase action decreased with increasing pH.
Properties of Three Different Molecular Forms of the α2Plasmin Inhibitor
Authors: CLEMMENSEN Inge , THORSEN Sixtus , MÜLLERTZ Sten , PETERSEN Lars C. 
Published: March 2005

The α2plasmin inhibitor prepared by affinity chromatography on plasminogen‐Sepharose 4B appears mainly as a molecular form with Mr67000 but also as forms with Mr65000 and 60000. The 67000‐Mr and 60000. The 67000‐Mrform spontaneously converts to the lower‐molecular‐mass forms. The rate of conversion is temperature‐dependent. All three molecules react with specific immuno globulin against α2plasmin inhibitor. The plasminogen‐binding capacity of the 67000‐Mr form is lost upon its conversion into the 65000‐Mrform, but it has retained its capacity to inhibit plasmin. Upon conversion to the 60000‐Mr form the capacity to inhibit plasmin is also lost. The presumed catalytic effect of a copurified serine proteinase on these reactions could not be substantiated since the addition of a number of known serine proteinases and serine proteinase inhibitors did not influence the rate of conversion of the 67000‐Mrform to the modified forms. The functional properties of the 65000‐Mrnon‐plasminogen‐binding α2plasmin inhibitor form were compared to those of the 67000‐Mrplasminogen‐binding form. In a clot lysis assay the plasminogen‐binding inhibitor was a more efficient inhibitor of fibrinolysis than the non‐plasminogen‐binding inhibitor. Plasminogen was displaced from fibrin more readily by the plasminogen‐binding inhibitor than by the non‐plasminogen‐binding form, and activation of Glu‐plasminogen by urokinase and tissue activator was more rapid with the plasminogen‐binding inhibitor than with the non‐plasminogen‐binding form.Kinetic studies showed that the plasminogen‐binding α2plasmin inhibitor reacted more readily with plasmin than the non‐plasminogen‐binding form and that the inactivation of plasmin by the former was a biphasic process in the absence of 6‐aminohexanoic acid, but monophasic in its presence. Inactivation of plasmin by the non‐plasminogen‐binding α2plasmin inhibitor was a monophasic reaction both in the absence and the presence of 6‐aminohexanoic acid and identical to the reaction with the plasminogen‐binding form in the presence of 6‐aminohexanoic acid.
The Essential Carboxyl Group in Restriction Endonuclease EcoRI
Authors: WOODHEAD J. Lesley , MALCOLM Alan D. B. 
Published: March 2005

We have carried out studies on type II restriction endonucleaseEcoRI, which cleaves the DNA sequence , as indicated. The active form of the enzyme consists of two subunits, each 31063 molecular weight. A water‐soluble reagent, 1‐cyclohexyl‐3‐(2‐morpholinoethyl)carbodiimide metho‐p‐sulphonate, which reacts with carboxyl groups and also with tyrosine and cysteine residues, has been found to inactivate this enzyme.Results are presented which show the following. (1) This specific inactivation is not due to modification of lyrosine or cysteine residues. (2) There is one carboxyl group per subunit which, when modified with carbodiimide, inactivates the enzyme. (3) φX174 DNA (which does not contain EcoRI sites) partically protects the enzyme from the carbodiimide; protection is unaffected by the additional presence of Mg2+, but significantly greater with Co2+φX174DNA.
Evidence from Inhibitor Studies for Conformational Changes of Citrate Synthase
Authors: BAYER Ernst , BAUER Barbara , EGGERER Hermann 
Published: March 2005

Substrate analogue CoA derivatives were applied as inhibitors of citrate synthase. Substitution of the acyl‐CoA oxygen next to sulfur by hydrogen was without marked influence on the affinity.
Carboxymethyl‐CoA, a structural analogue of enolic acetyl‐CoA. Ks of the binary inhibitor‐enzyme comples was high (230 μM) but that of the ternary inhibitor‐oxaloacetate‐enzyme complex was 0.07 μM. Both enzyme sub‐units bond the inhibitor independently, also in the presence of oxaloacetate.
(3R,S)‐3,4‐Dicarboxy‐3hydroxybutyl‐CoA, an analogue of citryl‐CoA, inhibited the overall reaction noncompetitively against acetyl‐CoA and against oxaloacetate; it was a competitive inhibitor against the hydrolysis and cleavage reactions of (3S)‐citryl‐CoA. Kinetic data suggest that this inhibitor represents and intermediate analogue.
The results given above indicate conformational changes of the synthase during the catalytic cycle. In the proposed mechanism the free enzyme represents a hydrolase which in the presence of oxaloacetate, by a wellknown conformational change, is converted into a ligase. If both substrates are presents, the ligase is reconverted into the hydrolase upon formation of the intermediate, (3S)‐citryl‐CoA.
Selective Inhibition by Sodium Butyrate of Glucorticoid‐Induced Tyrosine Aminotransferase Synthesis in Hepatoma Tissue‐Cultured Cells
Authors: TICHONICKY Lydie , SANTANA‐CALDERON Maria Angelica , DEFER Nicole , GIESEN Eva Maria , BECK Gisèle , KRUH Jacques 
Published: March 2005

Sodium butyrate at a 5 mM concentration prevents the induction of tyrosine aminotransferase in hepatoma culture cells, without affecting the basal level of the enzyme. This effect is reversible immediately after the removal of butyrate, or after a lag, if butyrate was present for more than 2 h.Neither the amount of cellular RNA nor the rate of total RNA synthesis were affected by sodium butyrate. Furthermore butyrate does not inhibit protein synthesis: [35S] methionine incorporation into preteins, measured in a reticulocyte lysate system, shows to significant difference between the translation capacity of the RNAs from butyrate‐treated cells and from dexamethasone‐induced or uninduced cells. Nevertheless, when tyrosine aminotransferase was isolated from the amount of the enzyme synthesized in the presence of RNAs from dexamethasone/butyrate‐treated cells. These experiments indicate that the treatment of the cells with butyrate decreases the activity of the specific messenger RNA for tyrosine aminotransferase to a level close to the basal level.Butyrate does not prevent the penetration of the hormone and has a limited effect on the translocation of the glucocorticoid‐receptor complex to the nucleus.We therefore conclude that the effect of sodium butyrate might be inherent either to an impairment of an early step of the hormone‐chromatin interaction or to an alteration of the transcription process of some specific genes. The rapidity and the complete reversibility of the effects of butyrate suggest that it does not irreversibly alter the structure which are the targets of its action.