bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fluorescence energy transfer between the myosin subfragment‐1 isoenzymes and F‐actin in the absence and presence of nucleotides
Authors: TRAYER Hylary R. , TRAYER Ian P. 
Published: March 2005

The unique fast‐reacting cysteine residue (SH1) of myosin subfragment 1 (S1), prepared by chymotryptic digestion, and cysteine 373 of actin have been labelled selectively with the fluorescent probes, N‐(bromoacetyl)‐N′‐(1‐sulpho‐5‐naphthyl)ethylenediamine (1, 5‐BrAEDANS) and 5‐(iodoacetamido)fluorescein (5‐IAF), whose spectral properties render them a particularly effective donor‐acceptor pair in fluorescence energy transfer studies. The transfer efficiency of 40–45% represented a spatial separation of the chromophores of about 5 nm, which is in reasonable agreement with the value of 6 nm reported earlier for similarly labelled S1, prepared by papain digestion, and actin [Takashi, R. (1979) Biochemistry, 18, 5164–5169]. This transfer efficiency did not change when the doubly‐labelled binary complex was formed: (1) with acto‐S1(A1) or acto‐S1(A2) at 10–200 mM KC1, pH 7–8 and different buffer conditions; (2) with either S1 isoenzyme and regulated actin (i. e. actin with tropomyosin and troponin) both in the presence and absence of Ca2+ or when the donor and acceptor attachment sites were reversed. Analysis of donor and acceptor polarized fluorescence showed that the chromophores are not randomly orientated (i.e. χ2≠ 2/3), but they do have some motion relative to either protein. From a knowledge of the limiting values for χ2, the intersite distance for donor and acceptor chromophores was calculated to be in the range 3.9–6.7 nm.Addition of MgATP to the doubly‐labelled acto‐S1 complex eliminated energy transfer but this was recovered when ATP hydrolysis was completed. By utilizing the known binding constants between S1, actin and either MgADP or MgAdoPP[NH]P (magnesium adenosine 5′‐[βγ‐imido]triphosphate) [Konrad, M. and Goody, K. (1982) Eur. J. Biochem. 128, 547–555; Greene, L. E. and Eisenberg, E. (1980) J. Biol. Chem. 255, 543–548], the concentrations of all species present at equilibrium were determined. Experimental conditions were chosen to maximise the amount of ternary acto‐S1‐nucleotide complex (∼ 50%) and minimise the amount of binary complex (≤ 2%). The spatial separation of the chromophore interaction sites in the ternary complex was found to be the same with both nucleotides and indistinguishable from that found with the binary complex. A similar strategy was employed to compare the conformations of the binary and ternary complexes by 1H‐NMR spectro‐scopy. In these experiments about 90% of the S1 was in the form of the ternary complex. There was no noticeable change in the acto‐S1 spectra upon addition of either MgAdo PP[NH]P or MgADP. These observations support the conclusion that there is no large change in structure in the ‘rigor’ binary acto‐S1 complex when it binds either ADP or Ado PP[NH]P.
Distribution of enzymes of fatty acid and ketone body metabolism in periportal and perivenous rat‐liver tissue
Authors: KATZ Norbert R. , FISCHER Wilhelm , GIFFHORN Susanne 
Published: March 2005

The zonal distribution of cytosolic acetyl‐CoA carboxylase, mitochondrial 3‐hydroxyacyl‐CoA dehydrogenase and 3‐hydroxybutyrate dehydrogenase was studied in microdissected liver tissue.

In fed male and female rats the activity of the lipogenic enzyme acetyl‐CoA carboxylase was 1.6‐times higher in the perivenous than in the periportal zone of the liver acinus.
3‐Hydroxybutyrate dehydrogenase, which is involved in the formation of the ketone body, 3‐hydroxybutyrate, exhibited a similar distribution pattern with a 1.5–1.8‐times higher activity in the perivenous than in the periportal zone.
In contrast, the activity of 3‐hydroxyacyl‐CoA dehydrogenase, the third enzyme in ß‐oxidation, was about equally distributed between the periportal and the perivenous zone of the liver acinus.

The results indicate a predominance of lipogenesis in the perivenous, mainly glycolytic zone of the liver acinus. Furthermore, these data support the hypothesis that ß‐oxidation supplies energy for basic and anabolic processes like gluconeogenesis in the periportal zone, while it provides acetyl‐CoA for ketogenesis besides energy for basic needs in the perivenous zone.
Primary structure of histone H2A from nucleated erythrocyte of the marine worm Sipunculus nudus Presence of two forms of H2A in the sipunculid chromatin
Authors: KMIECIK Daniel , COUPPEZ Maurice , BELAICHE Denise , SAUTIERE Pierre 
Published: March 2005

The complete amino acid sequence (123 residues) of histone H2A from erythrocytes of the marine worm Sipunculus nudus, has been established from data provided by automated sequence analysis of large fragments generated by V8 staphylococcal protease digestion of histone H2A and by limited hydrolysis of the protein with α‐chymotrypsin and from structural studies of tryptic peptides of the protein.By comparison with calf homologous histone, the sipunculid histone H2A shows 6 deletions and 13 substitutions. Six of the substitutions are non‐conservative.Most of the evolutionary changes are mainly observed in the basic amino‐terminal and carboxy‐terminal regions of the molecule, which are the primary DNA‐binding sites. Few conservative point changes are observed in the central region (residues 18–118) which interacts strongly with histone H2B to form the dimer H2A‐H2B. 60% of the H2A molecules were found phosphorylated on the amino‐terminal residue, N‐acetyl‐serine. The high content of phosphorylated histone H2A in the sipunculid erythrocyte chromatin could probably be related to smaller repeat length (177 ± 5 base pairs) of nucleosomal DNA and to nuclear inactivation and chromatin condensation.
Topography of the total protein population from cultured cells upon fractionation by chemical extractions
Authors: LENSTRA Johannes A. , BLOEMENDAL Hans 
Published: March 2005

Chemical extractions are proposed as a major tool for a fractionation of cellular proteins. As a model system, proteins from cultured hamster lens cells have been divided by independent extractions into seven subcellular fractions, corresponding to water‐soluble proteins and the proteins from membranes, microfilaments (and other deoxycholate‐soluble proteins), intermediate filaments, microtubules, polysomes and nuclei respectively. The latter two fractions have been subfractionated yielding ribosomal proteins, the elongation and initiation factors of the protein‐synthesis machinery, chromatin proteins and non‐chromatin proteins.
The protein compositions of the fractions have been analyzed by one‐dimensional and two‐dimensional gel electrophoresis. This resulted in an almost complete topography of the proteins detected on two‐dimensional gels of total‐cell lysates.
Comparison of two‐dimensional patterns of proteins from the total‐cell lysate and proteins from hamster erythrocytes or from liver, muscle or brain tissue showed that the different cell types have only few proteins in common. Two proteins are common to all of these cell types, namely actin and a 68‐kDa protein. The latter protein was, like actin, vimentin and the tubulin subunits, also present in most cell fractions. Evidence is presented that this protein is identical to a 68‐kDa heat‐shock protein.
Dimeric glutamyl‐tRNA synthetases from wheat Kinetic properties and functional structures
Authors: THOMES Jean Claude , RATINAUD Marie Hélène , JULIEN Raymond 
Published: March 2005

The Michaelis constants in the tRNA aminoacylation reaction have been studied for the three dimeric glutamyl‐tRNA synthetases C, P and E. The values were found to be: for tRNA, 0.20 μM, 0.20 μM, 0.20 μM and 0.44 μM; for glutamic acid, 10 μM, 83 μM and 83 μM; for MgATP, 0.46 mM, 0.38 mM and 0.26 mM. MgATP concentrations higher than 2 mM induce pronounced inhibition.
The presence of the cognate tRNA is required for [32P]PPi‐ATP isotopic exchange. In the absence of tRNA no hyperbolic saturation of the enzymes by glutamic acid occurs in our experimental conditions.
Analysis of the enzymic activity as a function of enzyme concentration leads to the conclusion that the active forms are dimers which are in equilibrium with inactive monomers. The values of the dissociation constants Kd were found to be 43 nM, 53 nM and 87 nM for glutamyl‐tRNA synthetases C, P and E respectively.
Dogfish insulin
Authors: BAJAJ Mona , BLUNDELL Thomas L. , PITTS James E. , WOOD Stephen P. , TATNELL Michele A. , FALKMER Sture , EMDIN Stefan O. , GOWAN Linda K. , CROW Hal , SCHWABE Christian , WOLLMER Axel , STRASSBURGER Wolfgang 
Published: March 2005

Insulin from an elasmobranch, the spiny dogfish (Squalus acanthias) has been purified to near homogeneity by means of acid‐ethanol extraction and salt precipitation. The amino acid sequences of the performic‐acid‐oxidised A and B chains have been determined and exhibit some unusual features. The A chain contains a total of 22 amino acids; only the insulin from coypu (a member of the Rodentia suborder, Hystricomorpha), has previously been reported to contain an extension past the A21 asparagine. The B10 histidine, which is involved in the formation of the insulin hexamers in higher vertebrates through the co‐ordination of zinc, is present in this elasmobranch insulin. Several substitutions relative to bovine insulin occur in the proposed receptor binding region (A5Gln→His, B21Glu→Pro, B22Arg→Lys, B25Phe→Tyr). In spite of these substitutions, the maximal response in the rat epididymal fat cell assay is the same for bovine and dogfish insulins; the concentration required to produce the half‐maximal response is, however, approximately threefold greater for dogfish insulin than that of bovine insulin.The use of interactive computer graphics model‐building predicts that the dogfish insulin can attain a threedimensional structure very similar to that of bovine insulin; circular dichroic spectra are presented which support the model‐building studies.
β‐Agarases I and II from Pseudomonas atlantica. Purifications and some properties
Authors: MORRICE Lora M. , McLEAN Maltland W. , WILLIAMSON Frank B. , LONG William F. 
Published: March 2005

The agarose‐degrading system of Pseudomonas atlantica has been re‐examined. In addition to the previously reported extracellular endo‐β‐agarase [Yaphe, W. (1966) in Proceedings 5th International Seaweed Symposium, pp. 333–335] a second, membrane‐bound endo‐enzyme activity, β‐agarase II has been discovered. These two enzymes act in concert to degrade agarose to neoagarobiose [3,6‐anhydro‐α‐l‐galactopyranosyl‐(1→3)‐d‐galactose] and also to degrade partially 6‐O‐methylated agarose to neoagarobiose and 61‐O‐methyl‐neoagarobiose. Novel assays were devised for β‐agarase II and the associated disaccharidase, neoagarobiose hydrolase. These allowed the critical purification of β‐agarase I and II.β‐Agarase I was purified 670‐fold from the bacterial medium by a new method using ammonium sulphate precipitation and gel filtration on Sephadex G‐100. The enzyme was resolved from the small amount of extracellular β‐agarase II. Dodecylsulphate/polyacrylamide gel electrophoresis indicated a homogeneous protein and a molecular weight of 32 000. Activity was observed against agar over the pH range 3.0–9.0 and optimally at pH 7.0. The enzyme could be used indefinitely at 30°C but only for up to 2 h at 40°C.β‐Agarase II was partially purified (5‐fold) from the soluble fraction of disrupted cells by chromatogm raphy on Sephadex G‐100, hydroxyapatite and DEAE‐Sepharose CL‐6B. This preparation was free of β‐agarase I and disaccharidase. β‐Agarase II was stimulated by NaCl, optimally in the range 0.10–0.20 mol dm−3 (2.4‐fold the activity at 0.010 mol dm−3 NaCl). Alkali earth metal (0.002 mol dm−3 CaCl2 or 0.005 mol dm−3 MgCl2) gave 1.2‐fold the normal activity. Optimum activity was over pH 6.5–7.5.
Detection of conformational changes in chloroplast coupling factor 1 by 8‐anilino‐1‐naphthalene‐sulphonate fluorescence changes
Authors: PICK Uri , FINEL Moshe 
Published: March 2005

Chloroplast coupling factor 1 (CF1) contains a high‐affinity binding site for 8‐anilino‐1‐napthalene sulphonate (ANS, Kd= 5–6 μM). The binding of ANS to the enzyme is associated with a fluorescence enhancement and a blue‐shift in the emission spectrum. ANS only slightly inhibits ATP hydrolysis by CF1.
Adenine nucleotides and inorganic phosphate induce a fast ANS fluorescence quenching of about 50% which is due to a decrease in the affinity of the enzyme for ANS (Kd increases from 6 μM to 22 μM) and in the fluorescence quantum yield of the bound probe (by 33%) but not in the number of ANS sites (n=1). Conversely, Mg and Ca ions induce a fluorescence enhancement of bound ANS. Inactivation of the enzyme enhances ANS fluorescence, eliminates the response to adenine nucleotides and inorganic phosphate but increases the response to divalent metals.
The affinity of latent CF1 for ADP (Kd=12 μM) is considerably higher than for ATP (Kd=95 μM) in buffer containing EDTA. The Kd for inorganic phosphate is 140 μM. Mg increases the apparent affinity for ATP (Kd=28 μM) but not for ADP or Pi.
Binding of ATP to the tight‐sites does not inhibit the ADP or Pi‐induced fluorescence quenching but decreases the affinity for ADP (Kd=34 μM) and for inorganic phosphate (Kd=320 μM). These results suggest that the ADP and phosphate binding sites are different but not independent from the tight sites.
Activation of a Mg‐specific ATPase in CF1 by octyl glucoside decreases the affinity for ADP and inorganic phosphate by about threefold but increases the affinity for ATP. ATPase activation of CF1 also increases the Ki for ADP inhibition of ATP hydrolysis.
ATPase activation also influences the ANS responses to Ca and Mg. Ca‐ATPase activation increases the fluorescence enhancement and the apparent affinity for Ca whereas Mg‐ATPase activation specifically increases the Mg‐induced fluorescence enhancement.
The fluorescence of CF1‐bound ANS is enhanced by Dio‐9 and quenched by phloridzin, quercetin, Nbf‐Cl and FITC. Nbf‐Cl and FITC completely inhibit the ADP‐induced fluorescence quenching whereas Dio‐9 inhibits the Mg‐induced fluorescence enhancement. ANS does not relieve the quercetin or phloridzin inhibition of ATP hydrolysis indicating that these inhibitors do not compete with ANS for a common binding site.

ANS may be used, therefore, as a sensitive probe to detect conformational changes in CF1 in response to activation or inactivation and to binding of substrates and of inhibitors.
On the formation of an oxygen‐tolerant three‐component nitrogenase complex from Azotobacter vinelandii
Authors: SCHERINGS Gerard , HAAKER Huub , WASSINK Hans , VEEGER Cees 
Published: March 2005

Conditions are defined in which the oxygen‐labile nitrogenase components from Azotobacter vinelandii can be protected against oxygen inactivation by the so‐called Fe/S protein II. It is demonstrated that oxygen protection can be achieved by complex formation of the three proteins. Complex formation was studied by gel chromatography. Only when the three proteins are in the oxidized state and MgCl2 is present, can an oxygen‐tolerant complex be isolated. Quantitative SDS/polyacrylamide gel electrophoresis of such complexes, yielded an average ratio of nitrogenase component 2/nitrogenase component 1 (Av2/Av1) of 2.4 ± 0.5. Protection by Fe/S protein II was correlated with the amount of [2 Fe‐2 S] clusters present in the protein and not by the amount of protein. Measurements of the amount of iron and sulfide of Fe/S protein II showed that the iron and sulfide content of the protein was variable. The maximum values found indicate that Fe/S protein II contains two [2Fe‐2S] clusters per dimer of 26 kDa. Full protection by Fe/S protein II was obtained with a ratio of Fe/S protein II/Av1 of 1.1 ± 0.2; the Fe/S protein II containing two [2Fe‐2S] clusters per dimer of 26 kDa. When Fe/S protein II contains less [2 Fe‐2S] clusters, more protein is necessary to obtain full protection. The three‐component nitrogenase complex is also oxygen stable in the presence of MgATP or MgADP.Analysis in the ultracentrifuge showed that the major fraction of the reconstituted complex has a sedimentation coefficient centered around 34S. A small fraction (<30%) sediments with values centered around 111 S. This suggests an average mass for the oxygen‐stable nitrogenase complex of 1.5 MDa. Taking into account the determined stoichiometry of the individual proteins, the molecular composition of the oxygen‐stable nitrogenase complex is presumably 4 molecules of Av1, 8–12 molecules of Av2 and 4–6 molecules of Fe/S protein II containing two [2Fe‐2S] clusters per dimer of 26 kDa.
Synthetic peptides reproducing the site phosphorylated by cAMP‐dependent protein kinase in protein phosphatase inhibitor‐1
Authors: CHESSA Gavino , BORIN Gianfranco , MARCHIORI Fernando , MEGGIO Flavio , BRUNATI Anna Maria , PINNA Lorenzo A. 
Published: March 2005

The hexapeptide (Arg)2‐Pro‐Thr‐Pro‐Ala (T1) and octapeptide (Arg)2‐Pro‐Thr‐Pro‐Ala (T5), reproducing the phosphorylatable site of protein phosphatase inhibitor‐1, a physiological target of cAMP‐dependent protein kinase, and five related peptides were synthesized by the method in solution.The phosphorylation rates of such peptides by cAMP‐dependent protein kinase and their kinetic parameters have been determined and compared with those of the hexapeptide (Arg)2‐Ala‐Ser‐Val‐Ala, reproducing the phosphorylatable site of rat liver pyruvate kinase. The results obtained show that both the presence of threonine instead of serine and the adjacent C‐terminal proline represent highly unfavourable factors seriously impairing the protein kinase reaction by both increasing Km and depressing V. On the other hand the N‐terminal proline is compatible with high phosphorylation rates and the row of four rather than two consecutive arginines improves the phosphorylation efficiency by lowering tenfold the Km, without affecting the V. The extension of the hexapeptide T 1 on its C‐terminal side to give the derivative (Arg) 2‐Pro‐Thr‐Pro‐Ala‐Thr‐Val‐Ala has no significant effect on the kinetic parameters. Moreover no relationship between the phosphorylation efficiency and the predicted secondary structures around the target residue could be evidenced.Therefore the local structural features of the phosphorylatable site of inhibitor‐1 cannot fully account for the fast phosphorylation of this regulatory protein. Other factors must optimize the protein kinase reaction.
A solution equivalent of the 2Zn→4Zn transformation of insulin in the crystal
Authors: RENSCHEIDT Heribert , STRASSBURGER Wolfgang , GLATTER Uwe , WOLLMER Axel , DODSON Guy G. , MERCOLA Daniel A. 
Published: March 2005

Circular dichroic spectroscopy clearly reveals a solvent‐induced conformational change of insulin in the presence of zinc ions. The spectral change corresponds to an increase in helix content. The transition observed in solution is an equivalent of the 2Zn → 4Zn insulin transformation in the crystal. This is inferred from a series of observations. (1) The spectral effects are compatible with the refolding of the B‐chain N‐terminus into a helix known from crystal studies. (2) The spectral effects are induced by the very same conditions which are known to induce the 2Zn → 4Zn insulin transformation in the crystal (i.e. threshold concentrations of NaCl, KSCN, NaI, for example). (3) They fail to be induced by the same conditions that fail to induce the crystal transformation (e.g. Ni2+ instead of Zn2+).It is concluded that the potential to undergo the transition resides in the hexamer since neither insulin dimers nor monomeric des‐pentapeptideB26–30‐insulin respond detectably to high halide concentration. Secondly the ability of zinc ions to accommodate tetrahedral coordination allows the transition which is not permitted by other divalent metal ions. Thirdly the transition is independent of the off‐axial tetrahedral zinc coordination sites since it occurs in [AlaB5]insulin which lacks the B5 histidine necessary for their formation. A symmetrically rearranged hexamer thus appears possible with two tetrahedrally coordinated zinc ions on the threefold axis; this is consistent with the observation that in native insulin two zinc ions per hexamer are sufficient to produce the full spectral effect. The amount of additional helix derived from the circular dichroic spectral change, however, cannot settle whether the transition comprises only three or all six of the subunits to yield a symmetrical hexamer. Finally the transformation in solution evidently still occurs in an intramolecularly A1‐B29‐cross‐linked insulin in spite of the partially reduced flexibility.
Changes in membrane ionic conductance, but not changes in slip, can account for the non‐linear dependence of the electrochemical proton gradient upon the electron‐transport rate in chromatophores
Authors: COTTON Nicholas P. J. , CLARK Adam J. , JACKSON J. Barry 
Published: March 2005

Decrease in the rate of cyclic electron transport (JE) measured from the absorbance changes associated with reaction centre bacteriochlorophyll led to a less than proportionate decrease in the membrane potential (ΔΨ) measured by electrochromism. In principle this result can be explained either by a ΔΨ‐dependent slip in the H+/e− coupling ratio (nE) or by a ΔΨ‐dependent change in the membrane ionic conductance. Simultaneous measurement of the membrane ionic current (JDIS) did not reveal any significant changes in the H+/e− ratio (JDIS/JE) and showed that conductance changes (JDIS/ΔΨ) account quantitatively for the curved dependence of ΔΨ on JE.Simultaneous recordings of JDIS and the extravesicular pH from cresol‐red absorbance changes, suggest that protons are the main current‐carrying species across the chromatophore membrane at high values of ΔΨ in the presence and absence of F0‐ATPase inhibitor. At reduced ΔΨ the flux of other ions outweighs the hydrogen ion current.
Histone H1 kinase from mouse plasmacytoma
Authors: QUIRIN‐STRICKER Christiane 
Published: March 2005

A cAMP‐independent protein kinase which phosphorylates histone H1 to a high level and which may correspond to the mitotic H1 kinase has been partially purified and characterized from mouse plasmacytoma microsomes [Quirin‐Stricker, C., and Schmitt, M. (1981) Eur. J. Biochem. 118, 165–172]. The present study compares the microsome‐associated and the chromatin‐associated histone H1 kinases isolated from mouse plasmacytoma cells. The results indicate that the two H1 kinases are indistinguishable by several criteria.The molecular structure of the microsome‐associated histone H1 kinase has been determined (a) by exclusion chromatography on Ultrogel, (b) by electrophoresis in non‐denaturing polyacrylamide gels of graded porosity and (c) by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the H1 kinase activity peak from an AcA‐34 Ultrogel column. All these techniques gave the same result: H1 kinase may exist in a native form as a monomeric enzyme with an apparent relative molecular mass of 90000 ± 8000.