bluuebunny/crossref_metadata_2025 huggingface dataset:
The Coagulation of Differently Sized Casein Micelles by Rennet
Authors: DALGLEISH Douglas G. , BRINKHUIS Jan , PAYENS Theodoor A.J.
Published: March 2005

Fractions of bovine casein micelles of different sizes were prepared by sucessive centrifugation steps, and dilute suspensions of the different fraction were reacted with rennet. The molecular weight increas with time after addition of rennet was measured by light scattering. After an initial lag stage, where the increase was zero, and a short intermediate stage, the molecular weight became linearly proportional to time, indicating complete conversion of the ϰ‐casein substrate by the enzyme. The slope of the final portion of the growth profile can be used to define values of the Smoluchowski rate constant governing the aggregation. No significant differences in this constant were found with mielles of different sizes, and the value of the rate constant suggested that the aggregation reaction of completely renneted micelles is only moderately retarded relative to the diffusion controlled process, even at room temperature.
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.
Catalytic and Molecular Modifications of Succinate Dehydrogenase by Monovalent Inoganic Anions
Authors: BONOMI Franco , PAGANI Silvia , CERLETTI Paolo
Published: March 2005

The enzyme activity and the oxidation state of soluble, activated, substrate‐reduced succinate dehydrogenase are modified by the presence of bromide. The anion inhibits the enzyme by two different mechanisms which depend on the ratio of bromide to sucinate. At high ratios binding of two bromide ions is required and a competitive inhibition is observed: removal of succinate from the substrate binding site (Kd= 0.1mM) leads to oxidation of the flavin. At lower ratios but with sufficient succinate to saturate a site with Kd= 1.52 mM, unompetitive inhibition by a single bromide ion is observed. Mechanisms, as well as the possible physiological signifinance of the novel type of regulation of succinate dehydrogenase, are discussed.
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.
Large‐Scale Purification and Phosphorylation of a Detergent‐Treated Adenosine Triphosphatase Complex from Plasma Membrane
Authors: FOURY Françoise , AMORY Antoine , GOFFEAU André
Published: March 2005

A new procedure for large‐scale preparation of plasma‐membrane‐bound ATPase from Saccharomyces cerevisiae is described. The crude membrane fraction is purified by selective extraction with three successive detergents: deoxycholate (0.025mg/mg protein), Triton X‐100 (0.25%) and lysophosphatidyclcholine (1 mg/mg protein). These treatments extract the mitochondria and strip the plasma membrane. From 1 kg commercial baker's yeast, 200 mg of plasma membrane proteins are isolated in 2‐3 days. Plasma‐membrane‐bound ATPase of specific activity of 10‐13 μmol Pi× min −1× mg protein −1is obtained with a yield estimated to 60%. Dodecylsulfate/ polyacrylamide gel electrophoresis shows three predominant polypeptides of Mr= 95000, 70000 and 56000 in the purified membrane fraction. The major polypeptide of Mr= 95000 identified as the ATPase submit is phosphorylated by millimolar concentrations of ATP. The phosphorylated intermediate reaches the steady‐state level in less than 100 ms and turns over very rapidly. It is hydrolyzed by hydroxylamine. Its formation is prevented by the ATPase inhibitors vanadate and Dio‐9, a plasma‐membrane ATPase inhibitor of unknown structure. At least four other membrane proteins are phosphorylated with much slower kinetics, presumably through the action of protein‐kinase(s).
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.
Activation by Polyamines of the Acetylation of endogenous Histones in Isolated Chromatin and Nuclei from Artemia
Authors: ESTEPA Inmaculada , PESTAÑA Angel
Published: March 2005

Polyamines have been shown to increase the rate of transfer of acetyl groups from acetyl‐CoA to endogenous hisstones in chromatin and nuclei from 50‐h‐old Artemia nauplii, with little changes in the rate of deacetylation. Polyamines showed a double specificity for both the effector, spermine, and the substrate, the histone H3. The same specificity was found for the activation of isolated histone acetyltransferase when assayed at subsaturating substrate concentration. The concentration of spermine required for half‐maximal activation was found to be 50‐60 μM.
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 FMN

A 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 solutions

Binding 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.
Formation of Leukotrienes E3, E4,E5 in Rat Basophilic Leukemia Cells
Authors: ÖRNING Lars , BERNSTRÖM Kerstin , HAMMARSTRÖM Sven
Published: March 2005

Rat basophilic leukemia (RBL‐1) cells incubated with ionophore A23187 and 5,8,11‐eicosatrienoic acid produced three slow‐reacting substances identified as leukotrienes C3,D3 and E3 by spectroscopic, chromatographic and enzymatic methods 5,8,11,14,17‐ Eicosapentaenoic acid was similarly converted by RBL‐1 cells to leukotrienes C5, D5 and E5Leukotrienes C4,D4 and E4 were also formed in these experiments from endogenous arachildonic acid Time‐course studies, incubations with 3H‐labeled leukotriene C3 and effects of acivicin [L‐(αS,5S)α amino‐3‐chloro 4,5‐dihydro‐5‐isoxazoleacetic acid; a γ‐glutamyl transpeptidase inhibitor] indicated that leukotrienes C and D are intermediates in the formation of leukotrienes E. L‐Cysteine enhanced the conversion of leukotriene C3 to leukotriene D3 and inhibited further degradation of leukotriene D3 to leukotriene E3.
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.
Protein Kinases Associated with the Adenovirus Single‐Stranded DNA‐Binding Protein
Authors: CAJEAN‐FEROLDI Chantal , LOEB Jacques , MEGUENNI Said , GIRARD Mare
Published: March 2005

Protein kinase activities copurifying with the 7200‐MrDNA‐binding protein of adenovirus on DNA‐cellulose chromatography and gel filtration in acrylamide/agarose have been partially characterized and purified. One of these kinases was found to phosphorylate efficiently the viral DNA‐binding protein in vitro and to be stimulated severalfold by the addition of histones, protamine, or polyamines. The kinase does not, however, phosphorylate histones, protamine, casein, or phosvitin. A second protein kinase was also recovered from single‐stranded DNA‐cellulose which is able to phosphorylate the 72000‐Mr DNA‐binding protein, but which is inhibited by the addition of histones. Phosphorylation in vitro of the 72000‐Mr DNA‐binding protein from the ts125 mutants of adenovirus by the histone‐stimulated protein kinase was found to be thermosensitive.
The Protochlorophyllide Holochrome of Barley (Hordeum vulgare L.)
Authors: SANTEL Hans‐Joachim , APEL Klaus
Published: March 2005

During the illumination of etiolated barley plants a rapid decline of the NADPH: protochlorophyllide oxidoreductase is observed. Within the first 5 min of continuous light approximately 90% of the enzyme activity present in dark‐grown barley plants disappears and, at the same time, the amount of enzyme protein is diminished by more than 60%. No stable polypeptide fragments have been found which might be formed during the light‐induced degradation of the enzyme protein. The rate of enzyme protein synthesis is not drastically affected at the beginning of the illumination period. During the subsequent light‐dependent chloroplast development a phytochrome‐induced decline in the rate of protein synthesis, concomittant with a continuous light‐dependent degradation of the enzyme protein, leads to a progressive decrease of the concentration of the enzyme. After 6 h of continuous light, when the rate of chlorophyll accumulation is at its greatest, only traces of the enzyme protein are visible and the enzyme activity is no longer detectable within the plants. In contrast to previous concepts of chlorophyll biosynthesis in higher plants, our results present evidence that the NADPH: protochlorophyllide oxidoreductase functions only for a short time period after the onset of light.
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.
Six Polymorphic Crystal Forms of α‐Cobratoxin
Authors: WALKINSHAW Malcolm D. , SAENGER Wolfram
Published: March 2005

Six different crystal forms of the long neurotoxin α‐cobratoxin isolated from the venom of Naja naja Siamensis have been obtained and the conditions for their crystallization are described. Heavy atom derivatives for two of these forms have been prepared and have proven suitable for high‐resolution X‐ray studies and, with one of them, a 0.28‐nm resolution structure has been determined. All but one of the crystal forms were grown between pH 2 and pH 3. Two novel suggestions are made which may be applicable to other protein crystallization studies. One involves cocrystallisation with mércurous iodide and the other describes a two‐step microdialysis procedure, first against salt solution, followed by dialysis against poly(ethylene glycol).
Proton‐Nuclear‐Magnetic‐Resonance Study on Molecular Conformations of Long Neurotoxins
Authors: ENDO Toshiya , INAGAKI Fuyuhiko , HAYASHI Kyozo , MIYAZAWA Tatsuo
Published: March 2005

The 270‐MHz proton NMR spectra were analyzed of the long neurotoxins α‐bungarotoxin fromBungarus multicinctus and Toxin B fromNaja naja.The aromatic proton resonances were completely assigned to individual nuclei for α‐bungarotoxin and in part for toxin B. The pH dependences of proton chemical shifts were analyzed by the nonlinear least‐square method, for obtaining PKavalues and protonation shifts. The PKavalues of Tyr‐25, an invariant residue of neurotoxins, are 12.1 for α‐bungarotoxin and 11.3 for toxin B, suggesting the presence of a strong hydrogen bond involving Tyr‐25 in α‐bungarotoxin. The Trp‐29 residues of both toxins show a common titration shift due to the carboxylate group of Asp‐31 and a similar structural arrangement of functionally invariant pair of Trp‐29 and Asp‐31 is implied. From the temperature dependences of the chemical shifts of His‐68 and a methyl group of α‐bungarotoxin, the local structure around His‐68 near the tail part is shown to be more flexible than the other part. The six main‐chain amide protons of α‐bungarotoxin exchange most slowly with solvent deuterons and are found by interproton nuclear Overhauser effects to be in the β‐sheet near the aromatic ring of Tyr‐25 residue. Hydrogen → deuterium exchange rates in 2H2O solution at 37 °C were measured of slowly exchanging amide protons of α‐bungarotoxin, toxin B, and two short neurotoxins, namely cobrotoxin and erabutoxin b. The two long neurotoxins have amide protons with half‐times longer than 3 h. The distributions of the half‐times of amide proton exchange indicate the structural rigidity of neurotoxins in the order, α‐bungarotoxin > toxin B > cobrotoxin ∼ erabutoxin b, in agreement with the order of neurotoxicity as reported previously by Chicheportiche et al. and by Lee and Chen.
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.
Partial Purification of the Messenger RNA for Eukaryotic Elongation Factor Tu from Artemia salina
Authors: VAN HEMERT Formijn J. , ROOBOL Kees , MÖLLER Wim
Published: March 2005

Polyadenylated RNA from developing Artemia salina cysts fractionated by centrifugation through a sucrose gradient containing methylmercuric hydroxide (CH3HgOH). Aliquots of each fraction were directly added to a rabbit reticulocyte lysate to program protein synthesis in vitro. The translation produts were assayed for eukaryotic elongation factor Ta (eFF‐Tu) by immunoprecipitation with an antibody raised in rabbits and purified by affinity chromatography. The immunoprecipitated radioactivity was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Sequences coding for eFF‐Tu sediment in the 20‐S region of the gradeint and form a major component of the poly(A)‐containing RNA. The mRNA of the 20‐S region, comprising about 10% of the poly(A)‐containing RNA fractionated on the gradient, has been translated in vitro and 30% of the translation products represent immunoprecipitable eFF‐Tu protein chains with an Mr of 50000.
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.
Conformation of Chromatin Oligomers
Authors: MARION Christian , BEZOT Pierre , HESSE‐BEZOT Christine , ROUX Bernard , BERNENGO Jean‐Claude
Published: March 2005

Quasielastic laser light scattering measurements have been made on chromatin oligomers to obtain information on the transition in their electrooptical properties, previously observed for the hexaneric structures [Marion, C. and Roux, B. (1978) Nucleic Acids Res. 5, 4431‐4449]. Translational diffusion coefficients were determined for mononucleosomes to octanucleosomes containing histone H1 over a range of ionic strength. At hign ionic strength, oligomers show a linear dependence of the diffusion coefficient upon the logarithm of number of nucleosomes. At low ionic strength a change occurs between hexamer and heptamer. Our results agree well with the recent sedimentation data of Osipova et al. [Eur. J. Biochem. (1980) 113, 183 –188] and of butler and Thomas [J. Mol. Biol.(1980) 140, 505–529] showing a change in stability with hexamer. Various models for the arrangements of nucleosomes in the superstructure of chromatin are discussed. All calculations clearly indicate a conformational change with the hexanucleosome and the results suggest that, at low ionic strength, the chromatin adopts a loosely helical structure of 28‐nm diameter and 22‐nm pitch. These results are also consitent with a discontinuity every sixth necleosome, corresponding to a turn of the helix. This discontinuity may explain the recent electric dichroism data of Lee et al. [Biochemistry (1981) 20, 1438–1445]. The hexanucleosome structure which we have previously suggested, with the faces of nucleosomes arranged radially to the helical axis has been recently confirmed by Mc Ghee et al. [Cell (1980) 22,87–96].With an increase of ionic strength, the helix becomes more regular and compact with a slightly reduced outer diameter and a decreased pitch, the dimensions resembling those proposed for solenoid models.
Synthesis of Human Interferon β1 in Escherichia coliInfected by a Lambda Phage Recombinant containing a Human Genomic Fragment
Authors: MORY Yves , CHERNAJOVSKY Yuti , FEINSTEIN Sheldon I. , CHEN Lousia , NIR Uri , WEISSENBACH Jean , MALPIECE Yves , TIOLLAIS Pierre , MARKS David , LADNER Martha , COLBY Clarence , REVEL Michel
Published: March 2005

DNA from a human adult was fragmented by partial digestion with restriction endonuclease EcoRI and cloned in λ Charon 4A. Clone C15, with a human DNA insert of 17 × 103 bases, was identified as containg a gene for the fibroblast, interferon, interferon β1. Restriction mapping shows that this gene, located on a 1840‐base EcoRI fragment, is not interrupted by introns. Moreover, we show that this human genomic DNA fragment is able to direct the synthesis of active human interferon β1 in Escherichia coli. Interferon activity of up to 7 × 106 U/1 was recovered from phage lysates by chromatography on CiBacron blue–Sepharose, and had the same immunological properties and species specificity as interferon produced by human fibroblasts.
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.
Activation and Partial Proteolysis of Variant Glucocorticoid Receptors, Studied by Two‐Phase Partitioning
Authors: ANDREASEN Peter A. , GEHRING Ulrich
Published: March 2005

In cultured lines of mouse lymphoma cells, resistance to glucocorticoids is frequently associated with the occurrence of glucocorticoid receptors with an abnormally low affinity (nt−) C an abnormally high affinity (nti) for nucleiand DNA. We have investigated whether the abnormal affinities for DNA are correlated with alterations in charge and surface properties of the receptors, that would be revealed through the partition coefficeint in aqueous dextran/poly(ethylene glycol) two‐phase systems.

We have found that none of teh receptor variants is defective in the activation step per se, and that only the nti receptors are abnormal in partition properties. Partial proteolysis of wild‐type and nt− receptors with α‐chymotrypsin products forms which are indistinguishable from the nti receptors with respect to partition coefficeints, Upon α‐chymotrypsin treatment the wild‐type receptors attain DNA‐binding properties identical to those of the nti receptors, while the nt− receptors, in spite of some increase in DNA affinity, still bind less firmly to DNA than the α‐chymotrypsin‐treated wild‐type receptors, α‐Chymotrypsin treatment of the various receptor types also produces and increase in the inding to dextran sulphate, but the dextran sulphate affinity is higher and varies less between different receptor types than the DNA affinity. Trypsin‐treated receptors were found to be devoid of affinity for DNA and dextran sulphate.

We conclude that the increased DNA affinity of the nti receptors is due to a gross change in teh structure of the receptors, while the decreased DNA affinity of the nt− receptors is caused by more subtle alterations in the polyanion binding domain of the receptors.
The Imidazole‐Promoted Inactivation of Horse‐Liver Alcohol Dehydrogenase
Authors: DAHL Knut H. , McKINLEY‐McKEE John S.
Published: March 2005

The effect of imidazole on the inactivation of liver alohol dehydrogenase by alkylation of Cys‐46 with iodoacetate, bromoacetate, 2‐bromopropionate, 3‐bromopropionate, 2‐bromobutyrate and iodoacetamide has been studied at pH 7.0. Imidazole promoted inactivation with all the compounds but 2‐bromobutyrate.

Enzyme inativation with haloacids was faster in a ternary enzyme‐imidazole‐haloacid complex compared to a binary enzyme‐haloacid complex. Inactivation with iodoacetamide, which is a direct bimolecular reaction, was faster with the binary enzyme‐imidazole complex as compared to the free enzyme. Results of haloacid and iodoaetamide inactivation in the presence of imidazole were fitted, by nonlinear regression analysis, to the rate expressions for the proposed mechanisms and the kinetic parameters resulted. Imidazole was also found to promote inactivation of the obalt‐substituted and cadmium‐substituted liver alcohol dehydrogenases.

Cys‐46 is alkylated as a metal‐thiol complex. Imidazole, when binding to the active‐site metal, donates σ‐electrons to the metal atom, which distributes the inccreased electron density further to the other ligands. The increased nucleophilicity of the sulphur of Cys‐46 results in promoted alkylation. Proof that the imidazole promotion effect is caused by a displaced electron distribution in the active‐site coordination unit is provided by imidazole also promoting the alkylation of the model thiol, zinc‐meraptoethanol.
Purifiation and Characterisation of Naphthalene Oxygenase from Corynebacterium renale
Authors: DUA Ramji Das , MEERA Srinivasan
Published: March 2005

Naphthalene oxygenase has been purified 420‐fold from naphthalene‐adapted Corynebacterium renale The purified enzyme was obtained in 32% yield and gave an apparent single band on polyacrylamide gel electrophoresis. It has a molecular weight of approximately 99000 and contains two non‐identical polypeptide chanins of molecular weights 43000 and 56000. The enzyme requires molcular oxygen for its activity and gave a single product, cis‐1,2‐dihydroxy‐1,2‐dihydronaphthalene. The absorption spectrum of the enzyme protein shows a maximum at 278 nm and no absorption in the Soret region, indicating that it is non‐heme protein. Absence of cytochrome P‐450 was also confirmed when the protein, treated with CO, showed no absorption at 450 nm. The enzyme followed Michaelis‐Menten kinetics with Km values of 2.9 mM and 1.42 mM for naphthalene and NADH respectively. It exhibited maximal activity at 30°C and pH 6.5. Studeis on the stoichimetry of the reaction showed that one molecule of oxygen is consumed for each molecule of NADH oxidised or product formed.
Lack of Evidence for a Tetrahedral Intermediate in the Hydrolysis of Nitroanilide Substrates by Serine Proteinases
Authors: MARKLEY John L. , TRAVERS Franck , BALNY Claude
Published: March 2005

We have used a stopped‐flow apparatus to reinvestigate reports, based on the observation of ‘burst’ kinetics, of an intermediateprior to the acyl‐enzyme complex in hydrolyis reactions of anilides catalyzed by trypsin and elastase [M.W. Hunkapiller, M.D. Forgac and J.H. Richards (1976) Biochemistry 15, 5581–5588; D.D. Petkov (1978) Biochim. Biophys. Acta, 523, 538–541; A.L. Fink and P. Meehan (1979) Proc. Natl Acad. Sci. USA, 76, 1566–1569; P. Compton and A.L. Fink (1980) Biochem. Biophys. Res. Commun. 93, 427–431]. We studied the hydrolysis of seferal anilide substrates by bovien and porcine elastase between ‐30°C and 20°C. In no case did we record true ‘burst’ kinetics. We show that confusing spectral changes can arise from incomplete mixing, thermal gradients, or heterogeneity of the substrate. We conclude that there is no solid spectroscopic evidence at present for the existence of a tetrahedral intermediate in the hydrolysis of amides by serine proteinases.

The substrate N‐acetyl‐l‐alanyl‐l‐prolyl‐l‐alanine 4‐nitroanilide is a mixture of two isomers trans and cis about the l‐alanyl‐l‐propyl peptide bond. It appears that elastase hydrolyzes the cis isomer more rapidly than the trans isomer and this could lead to false ‘burst’ kinetics.

We describe the construction of the stopped‐flow apparatus designed for cryoenzymology used for this work that has novel features and is adaptable to a variety of spetrophotometers. Solutions can be handled under anaerobic conditions. A window allows teh drive syringes to be observed or exposed to light for photochemical experiments. The apparatus operates over the temperature range −35°C to +25°C. The dead time is under 5 ms. A recording system is described that permits one to follow reations over a wide time scale covering half‐times of the order of several milliseconds to hours.
A General Secondary‐Structure Model for Procaryotic and Eucaryotic RNAs of the Small Ribosomal Subunits
Authors: STIEGLER Patrick , CARBON Philippe , EBEL Jean‐Pierre , EHRESMANN Chantal
Published: March 2005

A onsensus on the folding of the Escherihia coli 16‐S ribosomal RNA is emerging and several complete nucleotide sequences of small ribosomal subunit RNAs, covering diverse types of organisms and organelles, are now available. We therfore investigated the extent of both nucleotide sequence and secondary structure conservation that may exist between the E. coli16‐S RNA and other ribosomal RNAs. All the RNA molecules examined could be folded into secondary structure schemes that illustrated remarkable preservation of many structural motifs as well as striking nucleotide sequence conservation compared with the E. coli molecule. This study presents a unitary scheme for the structural organization of the small ribosomal subunit RNAs. The evolutionary constraints on both primary and secondary structures most likely reveal the basic role of some restricted RNA regions in the function of the rebosome.
Regulation of Protein Synthesis in the Plasmodial Phase of Physarum polycephalum
Authors: TURNOCK Geoffrey , CHAMBERS Jane , BIRCH Barbara
Published: March 2005

The rate of protein synthesis changes very little during the first 2–3 h (S phase) of the nuclear division cycle in plasmodia of Physarum polycephalum and then increases continuously during G2 phase, so that by the end of the cycle the rate has doubled relative to that in S phase. Protein synthesis appears to continue during mitosis.

Fractionation of extracts of plasmodia, labeled with [3H]lysine for 1h, by two‐dimensional electrophoresis indicated that most if not all proteins are synthesized throughout the nuclear division cycle. However, two metabolically stable polypeptides, the synthesis of which occurs predominantly in G2 phase, were detected.

Using a double‐labelling procedure, the differential rates of synthesis of 30 relatively abundant polypeptides were measured in relation to the nuclear division cycle. As a group, their differential rates of synthesis increase during the cycle so that their actual rates of synthesis increase 4–6‐fold. This implies that their synthesis is regulated over and above any simple change due to a doubling in the number of genes during S phase.
Analysis of Site Occupancies in [32P] Phosphorylated Pyruvate Dehydrogenase Complexes by Aspartyl‐Prolyl Cleavage of Tryptic Phosphopeptides
Authors: SALE Graham J. , RANDLE Philip J.
Published: March 2005

Activity of mammalian pyruvate dehydrogenase complexes from all sources so far tested is regulated by phosphorylation involving three sites. To facilitate understanding of the precise biological roles of the individual phosphorylation sites a method has now been developed, using pig heart [32P]phosphorylated complexes, which enables unambiguous measurement of their occupancies. Established methods of tryptic digestion give two peptides that contain the three phosphorylation sites: TA contains site 1 and 2: TB contains site 3. Thus, while occupancy of site 3 may be determined unequivocally by tryptic digestion, occupancies of site 1 and 2 cannot. The present paper shows that peptide TA may be specifically and quantitatively cleaved by formic acid at an Asp‐Pro bond located between the two phosphorylation sites. Equal amounts of two new peptides each containing a different phosphorylation site (site 1 or site 2) are produced. The 32P‐labelled peptides may be completely separated and quantified by high‐voltage paper electrophoresis at pH 2. A combination of tryptic digestion (determination of 32P in site 3) and formic acid cleavage of peptide TA (determination of 32P in sites 1 and 2) thus enables unequivocal assignment of occupancies of individual phosphorylation sites to > 95% accuracy. This method has been used to show that during phosphorylation and inactivation of pig heart complexes (inactivated to between 1.5% and 90%) > 98% of the observed inactivation was primarily attributable to phosphorylation of site 1; the contribution of site 2 was < 2% if at all. Relative initial rates of phosphorylation, site 1: site 2: site 3 were approximately 90:3:1.
An Investigation of the Active Site of Lactate Dehydrogenase with NAD+ Analogues
Authors: SAMAMA Jean‐Pierre , MARCHAL‐ROSENHEIMER Nicole , BEIELLMANN Jean‐Francois , ROSSMANN Michael G.
Published: March 2005

The kinetic properties of 18 NAD+ analogues, with alterations to the nicotinamide moiety, have been studied with respect to dogfish M4, rabbit M4 and beef H4 lactate dehydrogenases.

The size of the groups present at C‐3 of the pyridinium can be increased quite extensively without loss of coenzyme activity. Groups tested were thioamide, methyl, ethyl, diazoketone and chloroacetyl.

Substitutions at positions C‐4 and C‐5 prevent proper positioning for hydride transfer and can hinder binding to the enzyme. The kinetic properties of pyridine‐adenine dinucleotide and its 3‐iodo derivative reveal the bidning role of the amide at C‐3 whereas 3‐cyanopyridine‐adenine dinculeotide is a strong inhibitor.