bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of caldesmon on dynamic properties of F‐actin alone and bound to heavy meromyosin and/or tropomyosin
Authors: GAŁAZKIEWICZ Barbara , BELÁGYI Joseph , DABROWSKA Renata
Published: March 2005

The effect of caldesmon on the rotational dynamics of actin filaments alone or conjugated with heavy meromyosin and/or tropomyosin has been measured by the electron paramagnetic resonance (EPR) technique using a maleimide spin label rigidly bound to Cys374 of actin. The rotation of actin protomers in filaments and the angular distribution of spin probes on actin were determined by conventional EPR spectroscopy, while torsional motions within actin filaments were detected by saturation transfer EPR measurements.

Binding of caldesmon to F‐actin resulted in the reduction of torsional mobility of actin filaments. The maximum effect was produced at a ratio of about one molecule of caldesmon/seven actin protomers. Smooth muscle tropomyosin enhanced the effect of caldesmon, i.e. caused further slowing down of internal motions within actin filaments.

Caldesmon increased the degree of order of spin labels on F‐actin in macroscopically oriented pellets in the presence of tropomyosin but not in its absence. Computer analysis of the spectra revealed that caldesmon alone slightly changed the orientation of spin probes relative to the long axis of the filament. In the presence of tropomyosin this effect of caldesmon was potentiated and then approximately every twentieth protomer along the actin filament was affected.

Caldesmon weakened the effect of heavy meromyosin both on the polarity of environment of the spin label attached to F‐actin and on the degree of order of labels on actin in macroscopically oriented pellets. Whereas the former effect of caldesmon was independent of tropomyosin, the latter one was observed only in the absence of tropomyosin.
Evolutionary relationships among aminotransferases
Authors: MEHTA Perdeep K. , HALE Terence I. , CHRISTEN Philipp
Published: March 2005

A data base was compiled containing the amino acid sequences of 12 aspartate aminotransferases and 11 other aminotransferases. A comparison of these sequences by a standard alignment method confirmed the previously reported homology of all aspartate aminotransferases and Escherichia coli tyrosine aminotransferase. However, no significant similarity between these proteins and any of the other aminotransferases was detected. A more rigorous analysis, focusing on short sequence segments rather than the total polypeptide chain, revealed that rat tyrosine aminotransferase and Saccharomyces cerevisiae and Escherichia coli histidinol‐phosphate aminotransferase share several homologous sequence segments with aspartate aminotransferases. For comparison of the complete sequences, a multiple sequence editor was developed to display the whole set of amino acid sequences in parallel on a single work‐sheet. The editor allows gaps in individual sequences or a set of sequences to be introduced and thus facilitates their parallel analysis and alignment. Several clusters of invariant residues at corresponding positions in the amino acid sequences became evident, clearly establishing that the cytosolic and the mitochondrial isoenzyme of vertebrate aspartate aminotransferase, E. coli aspartate aminotransferase, rat and E. coli tyrosine aminotransferase, and S. cerevisiae and E. coli histidinol‐phosphate aminotransferase are homologous proteins. Only 12 amino acid residues out of a total of about 400 proved to be invariant in all sequences compared; they are either involved in the binding of pyridoxal 5′‐phosphate and the substrate, or appear to be essential for the conformation of the enzymes.
Supramolecular structure of lipopolysaccharide and free lipid A under physiological conditions as determined by synchrotron small‐angle X‐ray diffraction
Authors: SEYDEL Ulrich , BRANDENBURG Klaus , KOCH Michel H. J. , RIETSCHEL Ernst Th.
Published: March 2005

Lipopolysaccharides, the major amphiphilic components of the outer leaflet of the outer membrane of Gramnegative bacteria, may assume various three‐dimensional supramolecular structures depending on molecular properties (e.g. chemical structure) and on ambient conditions (e.g. temperature, concentration of divalent cations). We applied synchrotron small‐angle X‐ray diffraction to investigate the supramolecular structures of natural and synthetic Escherichia‐coli‐type lipid A, of lipid A from Salmonella minnesota, and of rough mutant lipopolysaccharides of E. coli and S. minnesota under physiological water content (> 90%) at different temperatures (20, 37, and 55°C) and at different lipid/divalent cation molar ratios (20:1 to 1:1).

We found that in the absence of divalent cations rough mutant lipopolysaccharide and free lipid A form unilamellar structures with the main reflections centered around 4.50 nm for free lipid A, 4.80 nm for Re lipopolysaccharide, and 5.90 nm for Rd1 lipopolysaccharide at 20°C, i.e. below the β↔α acyl‐chain‐melting transition temperature. Above this temperature, the reflections are shifted to 4.30 nm for free lipid A (at 55°C), 4.60 nm for Re lipopolysaccharide (at 37°C), and to 5.50 nm for Rd1 lipopolysaccharide (at 37°C). The addition of divalent cations leads (at lower concentrations, i.e. lipid/cation molar ratios 20:1 to 5:1) to sharper reflections expressing a higher state of order and to a shift of the center of the main reflections lying now at 5.10 nm for free lipid A, 6.40 nm for Re and 7.20 nm for Rd1 lipopolysaccharide at 20°C. At higher concentrations of divalent cations (e.g. lipid/cation molar ratio 1:1), an increasing tendency to form nonlamellar, inverted cubic structures is observed which is indicated by the occurrence of another main periodicity and/or of reflections with spacing ratios 1:√2, 1:√3 of the main periodicity. The tendency to assume inverted cubic structures is only weakly pronounced for rough mutant lipopolysaccharides but dominant for free lipid A even at physiological temperature and divalent cation concentration.
Control‐pattern analysis of metabolic pathways
Authors: HOFMEYR Jan‐Hendrik S.
Published: March 2005

Metabolic control analysis [Kacser and Burns (1973) Symp. Soc. Exp. Biol. 27, 65–104; Heinrich and Rapoport (1974) Eur. J. Biochem. 42, 89–95] leads to a description of the systemic properties of a metabolic system (expressed as control coefficients) in terms of the local kinetic properties of the individual enzyme‐catalyzed reactions (expressed as elasticity coefficients). This paper describes a non‐algebraic diagrammatic method which generates the mathematical expressions for flux or concentration‐control coefficients in terms of elasticity coefficients. According to a set of simple rules, ‘flux‐control patterns’ or ‘concentration‐control patterns’ are drawn on a metabolic diagram. Each control pattern represents a product of elasticity coefficients that occurs as a term in the expression for a control coefficient. The rules also generate the correct sign that precedes each term. The control patterns are then used to build the expressions for control coefficients. The procedure was developed in such a way that each control pattern can be understood in terms of a ‘chain of local effects’ which shows how a perturbation in the activity of an enzyme is propagated through the metabolic pathway.
Lactoferrin‐binding sites at the surface of HT29‐D4 cells
Authors: ROIRON Danielle , AMOURIC Michèle , MARVALDI Jacques , FIGARELLA Catherine
Published: March 2005

The binding of 125I‐lactoferrin to HT29‐D4 cells, a clone of HT29 cells, was studied and compared to the binding of 125I‐transferrin to the same cells. The binding of the two iron‐transport proteins is saturable and reversible suggesting the presence of specific receptors for each protein. Scatchard analysis suggests the existence of binding sites for lactoferrin with the relatively high equilibrium dissociation constant, Kd1 of 408 nM. Additionally, the cell is capable of binding large amounts of lactoferrin with very low affinity, probably in a non‐receptor intermediate fashion. The dissociation constant of transferrin and its receptor was calculated 9.29 nM which corresponds well to values found in the literature. In contrast to lactoferrin, the cell was capable of binding only low amounts of transferrin in a non‐receptor intermediate fashion. After chemical crosslinking of lactoferrin to the cell surface, the radiolabeled lactoferrin was found in a complex of molecular mass 300 kDa. Crosslinking of transferrin resulted in a complex of much higher molecular mass.

These data clearly show a binding site for lactoferrin different from the transferrin receptor. Only if competition experiments were performed with a high molar excess of both ligand proteins did a small percentage of either of the two ligands crossreact with the receptor for the other, possibly due to a structural similarity of the two glycoproteins.
A spin‐label electron spin resonance study of the binding of mitochondrial creatine kinase to cardiolipin
Authors: CHENEVAL Dominique , CARAFOLI Ernesto , POWELL Gary L. , MARSH Derek
Published: March 2005

The binding of the mitochondrial creatine kinase to aqueous dispersions of beef heart cardiolipin has been studied via the perturbation of the mobility of spin‐labelled cardiolipin, using electron spin resonance (ESR) spectroscopy. In the presence of creatine kinase (1:1 protein/lipid ratio, by mass), the ESR spectra of cardiolipin labelled in a single acyl chain [n‐(4,4‐dimethyl‐oxazolidinyl‐N‐oxy)stearoylcardiolipin] indicate a restriction of motion both at the C‐5 and C‐14 positions (n= 5, 14) of the lipid chains. The restriction in mobility was reversed by addition of phosphate or adriamycin, which are thought to inhibit the binding of creatine kinase to the mitochondrial membrane or to displace it from its binding site on the membrane. The effect of the protein on the chain mobility is consistent with surface binding of the protein; no positive evidence was obtained for penetration of the protein into the hydrophobic region of the membrane.
Synthetic fragments of β‐casein as model substrates for liver and mammary gland casein kinases
Authors: MEGGIO Flavio , PERICH John W. , MEYER Helmut E. , HOFFMANN‐POSORSKE Edeltraut , LENNON David P. W. , JOHNS R. Basil , PINNA Lorenzo A.
Published: March 2005

The octapeptide Glu‐Ser‐Leu‐Ser‐Ser‐Ser‐Glu‐Glu, corresponding to the 14–21 sequence of bovine β‐casein A2 and 11 shorter and/or modified derivatives were synthesized and used as model substrates for three casein kinases: rat liver casein kinases 2 and 1 and a casein kinase isolated from the golgi‐enriched fraction of lactating mammary gland (GEF‐casein kinase). Casein kinase‐2 readily phosphorylates the octapeptide at its Ser‐4 residue with a Vmax value comparable to those obtained with protein substrates and Km values of 85 μM and 11 μM in the absence and presence of polylysine, respectively. These are the most favourable kinetic parameters reported so far with peptide substrates of casein kinase‐2. Stepwise shortening of the octapeptide from its N terminus promotes both a gradual decrease of Vmax and an increase of Km, this being especially dramatic in passing from the hexapeptide Leu‐Ser‐Ser‐Ser‐Glu‐Glu (Km 210 μM) to the pentapeptide Ser‐Ser‐Ser‐Glu‐Glu (Km 2630 μM). The tetrapeptide Ser‐Ser‐Glu‐Glu is the shortest derivative still phosphorylated by casein kinase‐2, albeit very slowly, and the tripeptides Ser‐Glu‐Glu and Glu‐Leu‐Ser were not substrates at all. Furthermore, the pentapeptide Ser‐Ser‐Ser‐Glu‐Glu was found to be a better substrate than Ser‐Ser‐Ala‐Glu‐Glu, Ser‐Ala‐Ser‐Glu‐Glu and Ser‐Ala‐Ala‐Glu‐Glu by virtue of its lower Km value. These data, while confirming that the motif Ser‐Xaa‐Xaa‐Glu is specifically recognized by casein kinase‐2, strongly suggest that additional local structural features can improve the phosphorylation efficiency of serine‐containing peptides which are devoid of the large acidic clusters recurrent in many phosphorylation sites of casein kinase 2. In particular, predictive structural analysis as well as NMR and C18 reverse‐phase HPLC elution profile data support the hypothesis that a β‐turn conformation is responsible for the remarkable suitability of the octapeptide Glu‐Ser‐Leu‐Ser‐Ser‐Ser‐Glu‐Glu and some of its shorter derivatives to phosphorylation mediated by casein kinase‐2.

While neither the peptide Glu‐Ser‐Leu‐Ser‐Ser‐Ser‐Glu‐Glu nor any of its derivatives were affected by casein kinase‐1, a rapid phosphorylation of the octapeptide by GEF‐casein kinase at Ser‐5 (not Ser‐4) was obtained. This phosphorylation site corroborates the concept that, with this protein kinase, the critical acidic residue in a peptide must be located at position +2, instead of +3 as in the case of casein kinase‐2. Consequently the tripeptide Ser‐Glu‐Glu, which is not affected by casein kinase‐2, is still slowly phosphorylated by GEF‐casein kinase.
Purification and isolation of choline acetyltransferase from the electric organ of Torpedo marmorata by affinity chromatography
Authors: RAEBER Alexander J. , RIGGIO Gaetano , WASER Peter G.
Published: March 2005

Choline acetyltransferase (EC 2.3.1.6) catalyzes the synthesis of the neurotransmitter acetylcholine from acetyl‐coenzyme A and choline. It has been purified from the electric organ of Torpedo marmorata by a new double‐affinity chromatography. Our rapid and specific purification procedure includes affinity chromatography on CoA‐Sepharose and then a second affinity chromatography on the enzyme's inhibitor {2‐[3‐(2‐ammonioethoxy)‐benzoyl]ethyl}trimethylammonium bromide coupled to Sepharose via a six‐carbon spacer arm. The final enzyme preparation has been purified 7300‐fold to a specific activity of 73 μmol acetylcholine formed min−1 mg protein−1. The isolated enzyme gave a single band on disc polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The relative molecular mass was determined to be 68300 ± 2100.
Metabolism of free sphingoid bases in murine tissues and in cultured human fibroblasts
Authors: KOBAYASHI Takuro , SHINNOH Nobue , GOTO Ikuo
Published: March 2005

Metabolism of free sphingoid bases present in normal tissues [Kobayashi, T., Mitsuo, K. & Goto, I. (1988) Eur. J. Biochem. 172, 747–752] was examined in mouse tissues and in human cultured fibroblasts. Subcellular fractionation studies of the mouse liver revealed most of free sphingoid bases to be in the membrane fractions. Fibroblasts from patients with Farber's disease contained concentrations of free sphingoid bases similar to those seen in the normal fibroblasts. When l‐cycloserine, a potent inhibitor of the first reaction of sphingoid base synthesis, was added to the culture medium, the concentration of free sphingoid bases in fibroblasts decreased, dose‐dependently. Thus, most of the free sphingoid bases in the tissue probably do not derive from the degradation of sphingolipids but are newly synthesized. Free sphingoid bases in microsomes from the brainstem and spinal cord were acylated or glycosylated when incubated with acyl‐CoA or UDP‐glycoside. However, the reaction for the synthesis of ceramide was much greater and more rapid than that of glycosylsphingosine synthesis. In liver microsomes, ceramide synthesis from endogenous free sphingoid bases was observed but synthesis of glycosylsphingosine was not evident. Therefore, the main pathway of metabolism of free sphingoid bases is presumably acylation (to ceramide) but not glycosylation (to lysoglycosphingolipid).
Growth control strength and active site of yeast plasma membrane ATPase studied by site‐directed mutagenesis
Authors: PORTILLO Francisco , SERRANO Ramon
Published: March 2005

Several amino acids which are conserved in cation‐pumping ATPases with phosphorylated intermediate have been mutagenized in the yeast plasma membrane H+ ‐ATPase. The mutant genes have been selectively expressed in a yeast strain where the wild‐type ATPase is only expressed in galactose medium. A series of mutants with decreasing levels of activity demonstrates that the ATPase is rate‐limiting for growth and that decreased ATPase activity correlates with decreased intracellular pH. Enzymatic and transport studies of mutant ATPases indicate that (a) Lys474 is the target for the inhibitor fluorescein 5′‐isothiocyanate and this residue can be replaced by either arginine or histidine with partial retention of activity; (b) the sensitivity to inhibition by vanadate is affected by the mutations Thr231 → Gly, Cys376 → Leu, Lys379 → Gln and Asp634 → Asn; (c) the mutation Ser234 → Ala causes uncoupling between ATP hydrolysis and proton transport and reduces the ATP content of the cells; (d) the mutation Asp730 → Asn, which affects a polar residue conserved in hydrophobic stretches of H+ ‐ATPases, abolishes ATPase activity and proton transport but not the formation of a phosphorylated intermediate.
Frameshifting in the synthesis of Escherichia coli polypeptide chain release factor two on eukaryotic ribosomes
Authors: WILLIAMS Joanna M. , DONLY B. Cameron , BROWN Chris M. , ADAMSKI Fran M. , TROTMAN Clive N. A. , TATE Warren P.
Published: March 2005

A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF‐2) to bypass an in‐frame termination codon within the coding sequence. The nucleotide sequence preceding the in‐phase stop codon within RF‐2 mRNA is complementary to the 3′ anti‐(Shine‐Dalgarno sequence) region found in prokaryotic 16S rRNA and Weiss et al. (1988) have concluded that this pairing triggers the frameshift event. In vitro production of RNA coding for RF‐2, suitable for translation on eukaryotic ribosomes, has enabled testing of whether eukaryotic ribosomes can frameshift at this sequence. The 18S rRNA of eukaryotic ribosomes does not contain the 3′ anti‐(Shine‐Dalgarno sequence) region. The prokaryotic RF‐2 gene and the gene for the other release factor, RF‐1, which does not contain an in‐frame stop codon, were subcloned into transcription vectors such that the RNA transcripts produced in vitro would resemble a typical eukaryotic mRNA. These RF‐1 and RF‐2 RNAs both synthesized a major product of Mr approximately 45000 when translated in vitro within reticulocyte lysate; the size expected for full length RF‐1 and RF‐2 molecules. The RF‐2 product was immunoprecipitated by RF‐2‐specific antibodies, including those to regions of the protein encoded in the mRNA downstream from the frameshift site. The putative premature termination product, an oligopeptide of 25 amino acids, was not detected, but a chemically synthesized derivative was shown to be very unstable within the translation system. Although it was not possible therefore to calculate an absolute efficiency of frameshifting, the relative efficiency of the translation of RF‐2 RNA was estimated to be 10–20% of that of RF‐1 RNA in the reticulocyte system. This was similar to the relative synthesis of the two proteins in a plasmid‐DNA‐directed prokaryotic transcription/translation system. These results show that in vitro on eukaryotic ribosomes where the Shine‐Dalgarno‐type interaction is not possible, high efficiency frameshifting around the in‐phase stop codon in the RF‐2 mRNA can still occur.
Glucose‐6‐phosphate dehydrogenase
Authors: JEFFERY Jonathan , BARROS‐SÖDERLING Jane , MURRAY Lynda , WOOD Irene , HANSEN Robert , SZEPESI Bela , JÖRNVALL Hans
Published: March 2005

The primary structure of glucose‐6‐phosphate dehydrogenase from rat liver has been determined, showing the mature polypeptide to consist of 513 amino acid residues, with an acyl‐blocked N‐terminus. This structure is homologous to those of both other eutherian and marsupial mammals (human and opossum), thus characterizing a mammalian type enzyme to which the human form, notwithstanding its large number of genetic variants, conforms. The mammalian type differs from the fruit fly enzyme by about 50%. Known mutant forms exhibit further differences, widely distributed along the polypeptide chain. Structural patterns show glucose‐6‐phosphate dehydrogenases to consist of a few variable regions intermixed with relatively constant segments.
Restriction of poliovirus RNA translation in a human monocytic cell line
Authors: LÖPEZ‐GUERRERO José Antonio , CARRASCO Luis , MARTÍNEZ‐ABARCA Francisco , FRESNO Manuel , ALONSO Miguel Angel
Published: March 2005

The infection of the human monocytic cell line U‐937 by poliovirus was characterized by a low level of virus production and a slow progression of the cytopathic effect. Infection took place in > 99% of the cells as revealed by a limiting dilution assay. No viral protein synthesis was evident in the infected U‐937 cells when analyzed by polyacrylamide gel electrophoresis. However, a low level of poliovirus RNA translation was detected by immunofluorescence analysis using a mixture of polyclonal antisera against non‐structural proteins. Although there was only a low level of viral protein synthesis, a gradual accumulation of viral mRNA took place in U‐937 cells as revealed by RNA blot analysis. Similar results were obtained when the erythroleukemic cell line K‐562 was used as a host cell for poliovirus. RNA extracted from infected U‐937 cells was efficiently translated in rabbit reticulocyte extracts giving rise to a pattern of viral polypeptides similar to that detected when virion‐purified RNA was the template used for the in vitro translation assay, suggesting that the poliovirus RNA present in infected U‐937 cells is functional. The existence in U‐937 cells of a discriminatory mechanism which differentially interferes with poliovirus RNA translation is discussed.
Characterisation by mass spectrometry and 500‐MHz proton nuclear magnetic resonance spectroscopy of penta‐ and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins)
Authors: HOUNSELL Elizabeth F. , LAWSON Alexander M. , STOLL Mark S. , KANE David P. , CASHMORE Geoffrey C. , CARRUTHERS Robert A. , FEENEY James , FEIZI Ten
Published: March 2005

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500‐MHz 1H NMR are described of the major penta‐ and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied after depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio‐Gel P4 chromatography and HPLC. Eleven penta‐ and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J. 256, 397–401] and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features:

Sequences in these oligosaccharides not commonly found in mucins so far studied are chain‐terminating GlcNAα1–4Gal. repeating‐type‐I (Galβ1–3GleNAc) backbones, the backbone branch GlcNAcβ1–6(GlcNAcβ1–3)Gal and the backbone sequence GlcNAcβ1–6Galβ1‐ in the absence of a substitutent at C3 of galactose.
Structure dynamics of the hemoglobin mutants Hb Hôtel Dieu, HbG Philadelphia, HbJ Mexico, Hb St. Mandé and Hb San Diego, studied by nanosecond‐laser‐flash photolysis
Authors: FONTAINE Marie‐Pierre , LINDQVIST Lars , BLOUQUIT Yves , ROSA Jean
Published: March 2005

The kinetics of the change from the carboxy to the deoxy conformation of the mutated hemoglobins mentioned in the title and of normal human adult hemoglobin were determined from measurements of light absorption changes occurring up to 50 μs after nanosecond‐laser photodissociation of the corresponding CO complexes.

The spectral evolution of the mutated hemoglobins was found to be similar in its main features to that of normal hemoglobin. The kinetics could be decomposed into two phases with rates 1.1–1.8 × 106s−1 and 0.17–0.34 × 106s−1 (except Hb St. Mandé which displayed only the faster phase). Study of the mutated subunits of HbJ Mexico (α subunit) and Hb Hôtel Dieu (β subunit) showed that they convert exponentially to the stable deoxy state after photodeligation at the same rates as the corresponding subunits of normal Hb: 1.1 × 106s−1 (α) and 0.3 × 106s−1 (β). The results indicate that there is no direct correlation between the kinetics of spectral relaxation in the time range studied and the oxygenation properties for these hemoglobins. However, there is some indication that the kinetics are dependent upon the region of mutation.
Non‐identical behavior of the Ca2+ ‐ATPase in the terminal cisternae and the longitudinal tubules fractions of sarcoplasmic reticulum
Authors: MÉSZÁROS László G. , IKEMOTO Noriaki
Published: March 2005

The kinetics of Ca2+ dissociation from and binding to the Ca2+‐ATPase and the coupled tryptophan fluorescence changes were compared in the heavy and the light sarcoplasmic reticulum (SR) fractions. It was found that in the light SR both the dissociation of Ca2+ from the Ca2+ ‐ATPase and the coupled tryptophan fluorescence change took place in a biphasic fashion, but they were monophasic in the heavy SR. On the other hand, the time courses of both the Ca2+ binding and the coupled tryptophan fluorescence increase were biphasic, and virtually indistinguishable between the heavy and the light SR fractions. Submicromolar ruthenium red altered the kinetics of both the Ca2+ dissociation and the coupled fluorescence change in heavy, but not in light SR; the monophasic time course characteristic of the heavy fraction became biphasic, similar to that found for light SR in the absence of ruthenium red. Extraction of non‐ATPase proteins from the heavy SR vesicles also changed the kinetics of the Ca2+ dissociation‐coupled fluorescence decrease in the heavy SR from monophasic to biphasic. These results suggest that the difference between the heavy and light SR Ca2+‐ATPase in the Ca2+ dissociation kinetics is most probably produced by a ruthenium‐red‐sensitive interaction of the Ca2+‐ATPase with additional protein(s), which is are present exclusively in the heavy SR, rather than being due to a difference inherent to the Ca2+‐ATPase polypeptide itself.
Dependence of the Calvin cycle activity on kinetic parameters for the interaction of non‐equilibrium cycle enzymes with their substrates
Authors: PETTERSSON Gösta , RYDE‐PETTERSSON Ulf
Published: March 2005

Kinetic model studies and control analyses of the Calvin photosynthesis cycle have been performed to characterize the dependence of the cycle activity on maximum velocities and Kmvalues for the interaction of the non‐equilibrium cycle enzymes and ATP synthetase with their substrates under conditions of light and carbon dioxide saturation. The results show that Km values have no major influence on the cycle activity at optimal concentrations of external orthophosphate. The maximum cycle activity is controlled mainly by the catalytic capacities of ATP synthetase and sedoheptulose‐bisphosphatase, and is close to the maximum cycle flux that can be supported by these two enzymes.
Regulation of protein phosphatase‐1G from rabbit/skeletal muscle
Authors: HUBBARD Michael J. , COHEN Philip
Published: March 2005

The glycogen‐associated form of protein phosphatase‐1 (PP‐1G) is a heterodimer comprising a 37‐kDa catalytic (C) subunit and a 161‐kDa glycogen‐binding (G) subunit, the latter being phosphorylated by cAMP‐dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C‐terminal to site 1 in the primary structure. The sequence in this region is:

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At physiological ionic strength, phosphorylation of glycogen‐bound PP‐1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP‐1G, while the phospho‐G subunit remained bound to glycogen. Dissociation reflected a ≥ 4000‐fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate‐limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti‐site‐1 Fab fragments to glycogen‐bound PP‐1G.

At near physiological ionic strength, PP‐1G and glycogen concentration, site 2 was autodephosphorylated by PP‐1G with a t0.5 of 2.6 min at 30°C, ∼ 100‐fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type‐2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the α subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type‐2A and type‐2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10‐fold less effective than site 2 as a substrate for all four phosphatases.

In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP‐1 activity towards the glycogen‐metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen–protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin‐stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP‐1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.
Muscarinic acetylcholine receptor‐stimulated binding of guanosine 5′‐O‐(3‐thiotriphosphate) to guanine‐nucleotide‐binding proteins in cardiac membranes
Authors: HILF Gerhard , GIERSCHIK Peter , JAKOBS Karl H.
Published: March 2005

Receptor‐regulated binding of the labeled GTP analog, guanosine 5′‐O‐(3‐thiotriphosphate) ([35S]GTP[S]), to guanine‐nucleotide‐binding proteins (G‐proteins) was studied in porcine atrial membranes enriched in muscarinic acetylcholine (mACh) receptors. Binding of [35S]GTP[S] to the membranes was not or only slightly affected by the cholinergic agonist, carbachol, unless a second nucleotide was simultaneously present in the binding assay. This additional nucleotide requirement was best fulfilled by GDP, being maximally effective at 0.1–1 μM. In contrast, the GDP analog, guanosine 5′‐O‐(2‐thiodiphosphate), could not replace GDP in promoting carbachol‐induced increase in [35S]GTP[S] binding. In addition to GDP, agonist‐induced stimulation of [35S]GTP[S] binding to porcine atrial membranes required the presence of Mg2+, being half‐maximally and maximally effective at about 30 μM and 300 μM, respectively. Addition of NaCl, which decreased control binding measured in the presence of GDP alone, had no effect on the maximal extent of agonist‐stimulated binding, but reduced the potency of carbachol in stimulating [35S]GTP[S] binding. Under optimal conditions, carbachol increased the binding of [35S]GTP[S] without apparent lag phase up to about 2.5‐fold, with half‐maximal and maximal increase being observed at 5–10 μM and 100 μM, respectively. The agonist‐induced stimulation was competitively antagonized by the mACh receptor antagonist, atropine. The number of GTP[S] binding sites under receptor control was two–three‐fold higher than the number of mACh receptors in the porcine atrial membranes used. Pretreatment of the membranes with pertussis toxin under conditions leading to 95% ADP‐ribosylation of the toxin‐sensitive G‐protein α‐subunits markedly reduced agonist‐stimulated [35S]GTP[S] binding, with, however, about 30% stimulation still remaining. The data presented indicate that agonist‐stimulated binding of [35S]GTP[S] to G‐proteins can be a sensitive assay for measuring receptor‐regulated G‐protein activation in native membranes and, furthermore, suggest that one agonist‐activated mACh receptor can activate two or three cardiac G‐proteins, being mainly members of the pertussis‐toxin‐sensitive G‐proteins.
Direct photoaffinity labeling of leukotriene binding sites
Authors: FALK Eugen , MÜLLER Michael , HUBER Michael , KEPPLER Dietrich , KURZ Gerhart
Published: March 2005

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeiing taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock‐freezing of the samples, irradiation‐induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state.

After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time‐dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67000 was labeled. This polypeptide was identified as albumin.

Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25000, was identified as subunit 1 of glutathione transferases by immunoprecipitation.

The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene‐metabolizing enzymes, and transport systems.
NMR studies of mobility within protein structure
Authors: WILLIAMS Robert J. P.
Published: March 2005

NMR studies of dynamics within structure have revealed that a quite new approach to protein structure and its relation to function is necessary. This approach requires the consideration in detail of the following:

Local movements of groups and small segments to allow fast recognition and fitting. The motion concerns on/off rates as well as binding. The observations affect surface/surface recognition, e.g. of antigen/antibody as well as of substrate and protein.

Somewhat larger interdomain or N‐ and C‐terminal segments which allow rearrangement. Cases in point are the movement of segments in blood‐clotting proteins or in histones.

Relative motion of helices in hinges. These actions are likely in such enzymes as kinases and P‐450 cytochromes.

Relative motion of helices within domains (relative to other helices or sheets) in mechanical devices (triggers) e.g. in calmodulin.

General motion in random proteins. Examples extend from rubber‐like proteins (entropy sensors), some glycoproteins, to proteins carrying peptide hormones to be generated only after hydrolysis.

Order → disorder transitions locally as in osteocalcin and metallothionine.

Swinging arm motions associated with special sequences such as (Ala‐Pro)n.

Of great interest is the power of NMR to look at proteins which are relatively large, up to 50 k Da proteins, and to isolate certain zones of interest. This needs careful temperature dependent studies and analysis of separated domains [72] as well as the use of a great variety of pulse sequences [15] and of nuclei other than protons.

In this article I have illustrated the different possibilities using work in my own group. This is done to lessen the burden of extensive review. I fully realise that the range of examples is now large. I would stress though that the production of the necessary technology was the endeavour of several of us within the Oxford Enzyme Group from 1970 to 1985, i.e. from 270–600 MHz Fourier‐transform NMR spectroscopy.

While all of these features have been demonstrated by NMR methods there are parallel developments both using X‐ray diffraction methods and theoretical approaches. All these procedures are changing the view of protein structure to one which incorporates dynamics all the way from conventional vibronic/rotational coupling to the disordered motions characteristic of random polymers. It is the understanding of dynamics that leads to an appreciation of function.
Identification and properties of a novel type of Na+‐permeable amiloride‐sensitive channel in thyroid cells
Authors: VERRIER Bernard , CHAMPIGNY Guy , BARBRY Pascal , GERARD Corinne , MAUCHAMP Jean , LAZDUNSKI Michel
Published: March 2005

Amiloride‐sensitive cationic channels are present in the apical membrane of porcine thyroid cells in primary culture. An amiloride‐sensitive (K0.5= 150 ± 28 nM where K0.5 is the concentration of unlabelled ligand which reduces the specific binding of the same labelled ligand by 50%) 22Na+‐flux component (Km for Na+ at 18 mM) has been identified which was also blocked by the potent amiloride derivative phenamil (K0.5= 47 ± 21 nM). The most potent inhibitor of Na+/H+ exchange, ethylisopropyl‐amiloride, hardly inhibited this 22Na+‐influx component at a concentration of 21 μM. Amiloride binding sites were characterized using [3H]phenamil. The tritiated ligand binds to a single family of binding sites in thyroid membranes with a Kd value of 50 ± 10 nM and a maximal binding capacity of 5 ± 1 pmol/mg protein. Patch‐clamp experiments have directly demonstrated the existence of a phenamil‐ and amiloride‐sensitive cationic channel, with a conductance of 2.6 pS, which is permeable to sodium, but not very selective (PNa‐/PK+= 1.2). This channel is an important element in the regulation of the resting membrane potential of thyroid cells.
Preparation and properties of a cross‐linked complex between ferredoxin–NADP+ reductase and flavodoxin
Authors: PUEYO José Javier , SANCHO Javier , EDMONDSON Dale E. , GÓMEZ‐MORENO Carlos
Published: March 2005

The electrostatically stabilized complex between Anabaena variabilis ferredoxin–NADP+ reductase and Azotobacter vinelandii flavodoxin has been covalently cross‐linked by treatment with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1:1 stoichiometry of the two flavoproteins. Immunochemical cross‐reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH–ferricyanide diaphorase activity although the Km for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH–cytochrome‐c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1–2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome‐c reductase activity is observed on adding ferredoxin to the cross‐linked complex. Stopped‐flow data show that covalent cross‐linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP+ show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin– NADP+ reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross‐linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of somiquinone and quinol forms.
NMR studies of the Escherichia coli trp aporepressor
Authors: HYDE Eva I. , RAMESH Vasudevan , ROBERTS Gordon C. K. , ARROWSMITH Cheryl H. , TREAT‐CLEMONS Lynda , KLAIC Branimir , JARDETZKY Oleg
Published: March 2005

The resonances in the aromatic region of the 1H‐NMR spectrum of the Escherichia coli trp aporepressor have been assigned to amino acid type by two‐dimensional correlated spectroscopy (COSY), homonuclear Hartmann‐Hahn (HOHAHA) spectroscopy and nuclear Overhauser enhancement spectroscopy (NOESY) techniques and studies of the pH dependence of the chemical shifts, in combination with selective deuteration of the protein. Complete sequence‐specific assignments of the aromatic resonances have been made by comparing the observed inter‐residue NOEs with those expected on the basis of the crystal structure of the protein [Zhang, R.‐G., Joachimiak, A., Lawson, C. L., Shevitz, R. W., Otwinowski, Z. & Sigler, P. B. (1987) Nature 327, 591–597]. The latter experiments have also permitted the sequence‐specific assignment of some of the high‐field methyl resonances. The complete assignment of the aromatic region of the spectrum, in particular of resonances from residues at the dimer interface, opens the way to detailed studies of the conformational effects of corepressor and operator binding.
The core region of Proteus mirabilis R110/1959 lipopolysaccharide
Authors: RADZIEJEWSKA‐LEBRECHT Joanna , MAYER Hubert
Published: March 2005

The complete core structure present in the lipopolysaccharide of the R mutant R110/1959 from Proteus mirabilis (Proteus type II core) was investigated using methylation analysis and a number of degradation methods such as Smith degradation and β‐elimination. These studies combined with earlier work on a Rc‐type mutant of P. mirabilis O28 (R4/O28) which shares the same inner core region, allowed formulation of the complete core structure of the Proteus type II core as shown in Scheme 1.

image

[Structural proposal of the core region of P. mirabilis R110/1959 (Proteus type II core). Incomplete substitutions are indicated by broken arrows.]

A characteristic feature of the Proteus core of type II is the presence of two units of D‐galacturonic acid (DGalA); one in terminal, the other one in a chain‐linked position. In addition, the presence of the two isomers of glycero‐D‐manno‐heptose (LDHep and DDHep) and the lack of galactose are conspicuous. DDHep occupies a terminal position in the external core region, whereas the three units of LDHep in addition to dOclA form, as in other enterobacterial core types, the internal core region.

The taxonomic significance of the presence of DGalA in the Proteus type II core, but also in all R cores of other Proteeae investigated so far, will be discussed.
Sequential 1H‐NMR assignment and solution structure of bovine pancreatic ribonuclease A
Authors: RICO Manuel , BRUIX Marta , SANTORO Jorge , GONZALEZ Carlos , NEIRA José Luis , NIETO José Luis , HERRANZ José
Published: March 2005

Assignments for 1H‐NMR resonances of most of the residues of bovine pancreatic ribonuclease (RNase A) have been obtained by sequence‐specific methods. Identification and classification of spin systems have been carried out by two‐dimensional phase‐sensitive correlated spectroscopy (360 MHz) and single relayed coherence transfer spectroscopy. Sequence‐specific assignments have been achieved by phase‐sensitive two‐dimensional nuclear Overhauser effect spectroscopy. To overcome the problem of spectral overlap use has been made of (a) an exhaustive analysis of partly exchanged RNase A (spectra in D2O), (b) a comparison with the subtilisin‐modified enzyme (RNase S) and (c) small spectral perturbations caused by changes in pH and temperature. The secondary structure elements have been identified from the observed sequential, medium and long‐range nuclear Overhauser effects together with data from amide‐exchange rates. All information collected leads to the conclusion that the crystal and the solution structures are closely similar.
The Na+ ‐motive terminal oxidase activity in an alkalo‐ and halo‐tolerant Bacillus
Authors: SEMEYKINA Anna L. , SKULACHEV Vladimir P. , VERKHOVSKAYA Marina L. , BULYGINA Evgenia S. , CHUMAKOV Konstantin M.
Published: March 2005

An alkalo‐ and halo‐tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5S RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m‐chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na+ concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N′,N′‐tetramethyl‐p‐phenylenediamine (TMPD) in a cyanide‐sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine.

Operation of the Bacillus FTU terminal oxidase resulted in the generation of a ΔΨ which, in the Na+ medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K+ medium. CCCP discharged ΔΨ even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium as if Na+ ‐ and H+ ‐coupled oxidative phosphorylations were operative in the Na+ and K+ media, respectively.

Inside‐out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K+ increased the Na+ uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter. being inhibitory for respiration.

The data obtained indicate that in Bacillus FTU there is not only H+ ‐motive but also Na+ ‐motive terminal oxidase activity.
Purification and characterization of subforms of the guanine‐nucleotide‐binding proteins Gαi and Gαo
Authors: LANG Jochen
Published: March 2005

Five different pertussis‐toxin‐sensitive guanine‐nucleotide‐binding proteins (G proteins) were purified from bovine brain. Immunochemical characterization of α subunits identified two Gαo proteins (Gαo‐I and Gαo‐II), two 41‐kDa Gαi proteins (Gαi‐I and Gαi‐II) and the 40‐kDa Gαi2 protein. Site‐directed antisera specific for Gαo proteins did not differentiate between Gαo‐I and Gαo‐II. However, in situ peptide mapping using polyacrylamide gel electrophoresis revealed distinct cleavage products with different proteases for each of these proteins. Additionally comparison of Rf values demonstrated a slightly faster migration for Gαo‐II than for Gαo‐I, which is the only type of Gαo protein present in cell membranes of the neuroblastoma/glioma cell line NG 108–15. The importance of these structural differences and possible functional implications are discussed.