bluuebunny/crossref_metadata_2025_split huggingface dataset:
ATP Synthesis and Hydrolysis in Chloroplast Membranes
Authors: SHOSHAN Varda , SHAVIT Noun 
Published: March 2005

Divalent antibodies against chloroplast coupling factor 1 inhibited the factor ATPase, ATP synthesis, hydrolysis and Pi‐ATP exchange in chloroplasts. These antibodies also inhibited coupled electron flow rates but not the basal or uncoupled rates.
Several types of non‐precipitating, modified antibodies prepared from the original antibody preparation strongly inhibited the ATPase and Pi‐ATP exchange reaction but had little effect on ATP formation.
It is suggested that the inhibition of ATP synthesis by the divalent antibodies is probably due to an indirect blocking of the active site, while the inhibition of ATP‐utilizing reactions by the modified antibodies is related to their effect on the transfer of ATP from a non‐catalytic to a catalytic site on coupling factor 1, via an energy‐dependent conformational change.
Specificity of Diacylglycerol Acyltransferase from Bovine Mammary Gland, Liver and Adipose Tissue towards Acyl‐CoA Esters
Authors: MARSHALL Michael O. , KNUDSEN Jens 
Published: March 2005

Microsomal diacylglycerol acyltransferase from bovine lactating mammary gland, liver and adipose tissue was capable of acylating microsomal‐bound 1,2‐dipalmitoylglycerol with acyl‐CoA of chain length C4–C18.
The activity of the liver and adipose enzymes towards butyryl‐CoA and hexanoyl‐CoA relative to longer‐chain acyl‐CoA was similar to that of the mammary enzyme. The Km and V values of the three enzymes with butyryl‐CoA and hexanoyl‐CoA were similar, except for the V values of the adipose enzyme which were higher.
Microsomal diacylglycerol acyltransferase from mammary gland and liver of non‐ruminants was also capable of utilizing butyryl‐CoA.
These results indicate that the unique presence of short‐chain acids in ruminant milk triacylglycerols is not caused by differences in specificity between the diacylglycerol acyltransferase from ruminant mammary and other tissues.
Surface Charge of Biological Membranes as a Possible Regulator of Membrane‐Bound Enzymes
Authors: WOJTCZAK Lech , NAŁĘCZ Maciej J. 
Published: March 2005

Glycerol‐3‐phosphate dehydrogenase of insect thoracic muscle mitochondria and arylsulphatase C of rat liver microsomes were inhibited by anionic surface‐active agents oleate, palmitoyl‐CoA and sodium dodecylsulphate, and activated by cationic surfactants cetyltrimethylammonium bromide and cetylpyridinium chloride. Dimethylaniline oxidase of rat liver microsomes were inhibited by cationic surfactants and activated by anionic surfactants. The inhibition of NADH dehydrogenase of rat liver submitochondrial particles by NAD+ was potentiated by the anionic surfactants and partly released by cetyltrimethylammonium bromide.
These surface‐active activators and inhibitors altered apparent Km values of the enzymes but did not change the activities at infinite substrate concentration (V values).
This effect of surfactants disappeared after solubilization of the membranes and re‐appeared after incorporating the solubilized enzyme into phospholipid vesicles.
Cationic surfactants decreased whereas anionic surfactants increased the negative surface charge and surface potential of mitochondria, submitochondrial particles and microsomes, as measured by free electrophoresis of the particles and binding of 8‐anilino‐1‐naphthalene sulphonate.
Calculations based on Km change and the change of surface potential agree well with the assumption that a change of the surface charge density alters local concentrations of the substrates and/or the products in the vicinity of the membrane.
Possible role of factors affecting the surface charge of biological membranes in controlling the activity of membrane‐bound enzymes is discussed.
Studies on the Multi‐Enzyme Complex of Yeast Fatty‐Acid Synthetase
Authors: WIELAND Felix , RENNER Leni , VERFÜRTH Cornelia , LYNEN Feodor 
Published: March 2005

The multi‐enzyme complex of fatty acid synthetase, Mr 2300000, was dissociated by acylation with dimethyl maleic anhydride under conditions which lead to an acylation of about 30% of the ɛ amino groups of lysine. The complete dissociation into the subunits α and β is demonstrated by analytical ultracentrifugation as well as disc gel electrophoresis.
This dissociation is reversible. Hydrolysis of the resulting protein dicarboxylic acid monoamides under mildly acidic conditions leads to the unmodified subunits, which can be reconstituted to form a complex displaying about 60% of the original activity.
The subunits were isolated by sucrose‐density‐gradient centrifugation and studied for the different partial enzyme activities involved in long‐chain fatty acid synthesis: malonyl, palmitoyl and acetyl transferase, enoyl reductase and dehydratase were shown to be exclusive functions of the β chains of the complex, confirming a pentafunctional role of this subunit.
The Binding of 1‐Anilino‐8‐naphthalene Sulfonate to the Hemocyanin of Octopus vulgaris
Authors: RICCHELLI Fernanda , SALVATO Benedetto 
Published: March 2005

The binding of 1‐anilino‐8‐naphthalene sulfonate (ansyl) to native and copper‐free hemocyanin of Octopus vulgaris has been studied in different conditions by measuring the fluorescence properties of the probe in the presence of hemocyanin.Native hemocyanin, either in the oxygenated or in the deoxygenated state, does not bind ansyl.The binding of ansyl with apohemocyanin induces a strong increase (from 0.004 to 0.6–0.7) of the quantum yield and a blue shift from 520 nm to 460 nm of the emission maximum indicating the presence of ansyl binding sites in the protein. Experimental evidence is reported that the binding occurs at the copper‐binding site of the protein.The dissociation constants of the ansyl‐hemocyanin complexes are equal to about 10−4 M, i.e. they are of the same order of those obtained with other proteins.The number of binding sites (n) of apohemocyanin for ansyl depends on the conformational state of the protein and ranges from 0.15–0.80 mol/mol protein (Mr 500000), depending on pH. ionic strength, and urea concentration. A negative interaction between the ansyl binding sites has been suggested.
Studies on the Allosteric Properties of Glycogen Synthase I
Authors: SØLLING Henrik 
Published: March 2005

The reaction mechanism of glycogen synthase I from human polymorphonuclear leukocytes is shown to be either a rapid equilibrium random bi‐bi mechanism or an ordered sequential mechanism with uridinediphosphoglucose (UDP‐Glc) as the first substrate and UDP as the second product. The rate equations are identical at saturating glycogen concentrations. A multisite enzyme model without subunit interaction is proposed. Three sites are distinguishable on the enzyme: the catalytic site, a site for the attachment of glycogen and the allosteric binding site for glucose 6‐phosphate (glucose‐6‐P). It is proposed that the enzyme can undergo allosteric transition between two states, α and β. The α state is induced by glucose‐6‐P (activation constant 14 μM) and has a low Km for UDP‐Glc (21 μM) and a dissociation constant for the product UDP of 12 μM. For the β state the corresponding values are 5–800 μM and 4 μM. The influence of modifiers on the kinetic constants of the rate equation is on K, not V. ATP, ADP and AMP were found to favour the β state by competing with glucose‐6‐P for the allosteric site and forming a dead‐end complex. Pi, PPi, SO42−, and glycerol 2‐phosphate also competed with glucose‐6‐P, but are in themselves activators, which, however, are not able to induce a complete transformation from the β to the α state. UTP, UDP, and UMP are competitive inhibitors of the substrate UDP‐Glc. In high concentrations small‐molecular‐weight anions also interfere with the catalytic site. The influence of divalent cations is indirect, depending on affinity to modifiers. Alone, Mg2+ has no effect, except that it is toxic to the enzyme.Under ‘physiological’ conditions a Michaelis constant for UDP‐Glc of 81 μM and an activation constant for glucose‐6‐P of 230 μM were estimated. It is concluded that glycogen synthase I is subject to allosteric control and under physiological conditions is not always fully active.The optimal condition for assay of glycogen synthase I activity requires that product inhibition is avoided, which may be achieved by including Mg2+ in the assay mixture. Also, the presence of Na2SO4 is advocated, however, at 2 mM.
Messenger RNA Competition in Living Xenopus Oocytes
Authors: ASSELBERGS Fred A. M. , VAN VENROOIJ Walther J. , BLOEMENDAL Hans 
Published: March 2005

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein‐synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs thus become endogenous mRNAs.These results, together with previous estimates of the translational efficiency of injected heterologous mRNA species, are compatible with the assumption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in a temporarily inactivated form.
The Influence of Electrostatic Interaction on the Proton‐Binding Behaviour of myo‐Inositol Hexakisphospahte
Authors: ZUIDERWEG Erik R. P. , VAN BEEK Gerard G. M. , DE BRUIN Simon H. 
Published: March 2005

The 31P nuclear magnetic resonance (NMR) spectrum of myo‐inositol hexakisphosphate (P6‐inositol) in solution consists of four resonances which shift and broaden as a function of pH. From the pH dependence of the resonance positions we calculated an overall hydrogen ion titration curve which corresponds very well with the potentiometric titration data. This implies that the same quantity, namely the degree of dissociation of protons from the phosphate groups, is determined in both experiments.The titration curve covers a pH range of 6.5 and exhibits a pseudo‐equivalence point. It is shown that electrostatic interaction between the six phosphate groups on P6‐inositol, becoming doubly charged at high pH, accounts for the observed extended titration range. A thermodynamic model from which the overall titration curve and the pH dependence of the phosphorous magnetic resonances can be calculated is presented. These calculations demonstrate that the pseudo‐equivalence points observed in the NMR and potentiometric titration data are a result of differences in electrostatic interaction between opposing and adjacent phosphate groups in the P6‐inositol molecule.The pH‐dependent line broadening of the phosphorous resonances is analysed in terms of chemical exchange. It is shown that the proton diffusion rate is enhanced and the hydroxyl‐ion diffusion rate is reduced due to the high charge present on the P6‐inositol molecule.The pH dependence of the 31P NMR spectrum of myo‐inositol pentakisphosphate isolated from chicken blood, is discussed in relation to that of P6‐inositol.
Bee Venom Neurotoxin (Apamin): Iodine Labeling and Characterization of Binding Sites
Authors: HABERMANN Ernst , FISCHER Kurt 
Published: March 2005

Apamin, the octadecapeptide from bee venom acting on the central nervous system, has been labeled with 125I at its single histidine residue. Apamin, monoiodoapamin, diiodoapamin, and iodide can be separated on thin‐layer chromatography. Diiodoapamin retains about half the toxicity of the original peptide. Partial iodination with 125I, leading mainly to the monoiodo derivative, did not measurably decrease the toxicity.
Labeled apamin binds to the insoluble, but not to the soluble part of homogenates made from rat cerebral cortex. The order of binding to homogenates is rat forebrain > rostral part of the brain stem > cerebellum > caudal part of the brain stem > spinal cord. Grey substance of rabbit forebrain binds much better than white matter. The binding site is enriched within fractions of rat cerebral cortex which sediment between 10000 and 100000 × g.
Apamin also binds to peripheral organs. In rats, the sequence of specific apamin binding by different organs is cerebral cortex ≫ (20 ×) liver > kidney or ileum whereas binding by skeletal muscle, salivary glands, and testes is hardly measurable. In rabbits, the sequence is cerebral cortex ∼ liver > adrenals > cerebellum > kidney > medulla oblongata, whereas skeletal muscle and ileal mucosa do not bind to a measurable degree. Among all organs tested, bovine adrenal cortex binds the most.
Binding data with rat cerebral cortex were obtained under a variety of conditions. A single type of binding sites occurred with a density of about 1–3 pmol/g rat forebrain (wet weight). A very high affinity (Kd in the range of 10–25 pM) was found in both equilibrium and velocity measurements.
With increasing temperature apamin binding is diminished. Labeled apamin is slowly degraded by the supernatant of rat brain homogenate at 37°C and 25°C, but not at 5°C.

High specificity, high affinity and prevalence of binding sites within the nervous system indicate, together with the rank order of potency of apamin derivatives, a role of binding in poisoning. However, final proof will need a direct comparison between binding and the action of apamin on isolated systems.
The Primary Structure of the Coat Protein of the Broad‐Host‐Range RNA Bacteriophage PRR1
Authors: DHAESE Patrick , VANDEKERCKHOVE Joël S. , VAN MONTAGU Marc C. 
Published: March 2005

The complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N‐bromo‐succinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl‐Edman degradation procedure, which was slightly modified for application on insoluble peptides.PRR1 coat protein contains 131 amino acids, corresponding to molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Qβ, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1, Qβ and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage coat proteins.
Synthesis and Activation of Asparagine in Asparagine Auxotrophs of Saccharomyces cerevisiae
Authors: RAMOS Fernando , WIAME Jean‐Marie 
Published: March 2005

l‐Asparagine synthesis in Saccharomyces cerevisiae is performed by a glutamine‐dependent asparagine synthetase of the type found in higher organisms.Auxotrophy for asparagine has been obtained in two classes of mutants. In class I, asparagine synthetase activity is cancelled. These mutants combinetwo mutations, asnA− and asnB−. Neither asnA− nor asnB− mutation alone. leads to total auxotrophy. Partial auxotrophy as well as a strong decrease in enzyme activity result from asnA− mutation. No change is detectable in cells with the asnB− mutation alone. This, and Jones' report [J. Bacteriol. 134, 200–207 (1978)] of auxotrophy resulting from the combination of two mutations, are strong supports for asparagine synthesis being an unusual biosynthetic operation. In class II, auxotrophy results from a single mutation which leads to a modification of the efficiency of the asparaginyl‐tRNA synthetase (asnRS− mutation). This auxotrophy is cancelled if asparaginase I activity (the only one present in ∑ 1278b wild type) is cancelled by casnI− mutation. This latter mutation allows an increase in the asparagine pool which is able to compensate for the asparaginyl‐tRNA synthetase partial defect of the asnRS− mutant.
Association of the Prophage P1ban Protein with the dnaB Protein of Escherichia coli
Authors: EDELBLUTH Claus , LANKA Erich , VON DER HUDE Wilhelm , MIKOLAJCZYK Maria , SCHUSTER Heinz 
Published: March 2005

The constitutive synthesis of the dnaB analog (ban) protein by prophage P1bac permitted the construction of an otherwise non‐viable Escherichia coli strain bearing the unsuppressed (sup−) amber mutation dnaB266. Growth of this strain is cryosensitive and the ban protein furnished by prophage P1bac does not support bacteriophage λ replication [D'Ari et al., J. Mol. Biol. 94, 341–366 (1975)]. Recently a P1bac crr prophage mutant was isolated with the ability to confer cryoresistant (crr) growth to E. coli sup+dnaB266. The presence of prophage P1bac crr in E. coli sup−dnaB266 makes the strain permissive for λ growth [D. Touati‐Schwartz (1978) in DNA Synthesis‐Present and Future (Molineux, I. & Kohiyama, M., eds) pp. 683–692, Plenum Press, New York.P1ban protein is isolated by means of a dnaB‐complementation assay from P1bac crr lysogens of four E. coli mutants containing either thermo‐sensitive or thermo‐resistant dnaB protein. Monomers of the phage protein (ban) are found to be associated with monomers of the host dnaB protein in the form of heteromultimers as was reported earlier for P1bac lysogens [Lanka et al., J. Biol. Chem. 253, 4746–4753 (1978)]. The ban protein is, however, overproduced by P1bac crr when compared to the corresponding P1bac prophage. Overproduction of ban protein results in a thermoresistance of DNA synthesis in crude cell extracts and thermo‐resistant heteromultimers composed of ban and thermo‐sensitive dnaB subunits. The overproduction of ban protein is also demonstrated in the P1bac crr lysogen of E. coli sup+dnaB266 by precipitation with dnaB antibody.The results suggest that cryosensitive growth of E. coli sup+dnaB266 (P1bac) and its inability to support λ replication is due to a cellular deficiency in ban protein molecules rather than to an inefficacy of ban in the absence of an effective dnaB protein.
The Mitochondrial COB Region in Yeast Codes for Apocytochrome b and Is Mosaic
Authors: HAID Albert , SCHWEYEN Rudolf J. , BECHMANN Helga , KAUDEWITZ Fritz , SOLIOZ Marc , SCHATZ Gottfried 
Published: March 2005

Mitochondrial mutants of Saccharomyces cerevisiae defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome.The mutants mapped between OLI1 and OLI2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho− deletion mapping and recombination analysis.The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A–E, with A being closest to OLI2 and E being closest to OLI1. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence.

Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2→OLI1. The mutant closest to OLI1 accumulates an apocytochrome b which is slightly larger than that of wild type.
A mutant in cluster C exihibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red.
Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome aa3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown.
Zygotic complementation tests reveal at leas two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A+C+D+E).
Spin Transition of Camphor‐Bound Cytochrome P‐450
Authors: LANGE Reinhard , HUI BON HOA Gaston , DEBEY Pascale , GUNSALUS Irwin C. 
Published: March 2005

The kinetics of the spin transition in the heme iron of the cytochrome P‐450 substrate complex have been observed by stopped‐flow measurements between 4°C and –27°C. Large displacements in the spin equilibrium are induced by small changes in the concentration of hydrogen or potassium ions. The kinetic and thermodynamic data indicate that the spin transition is rate‐limited by conformational changes of the protein.The spin transition appears to be governed by the local paH, modulated in turn by external factors: a satisfying kinetic analysis is attained only by accounting for the difference between local paH (paH,in) and bulk paH (paH,out), as described in the preceding paper. Indeed, the low‐spin to high‐spin rate constant obeys a local paH titration curve with a pK= 5.4 ± 0.1 at − 17°C.
Solution Conformation of Glycosaminoglycans: Assignment of the 300‐MHz 1H‐Magnetic Resonance Spectra of Chondroitin 4‐Sulphate, Chondroitin 6‐Sulphate and Hyaluronate, and Investigation of an Alkali‐Induced Conformation Change
Authors: WELTI David , REES David A. , WELSH E. Jane 
Published: March 2005

Complete assignments are given for the 1H nuclear magnetic resonance (NMR) spectra at 300 MHz of chondroitin 4‐sulphate, chondroitin 6‐sulphate and hyaluronate in deuterium oxide solution, supported by spin decoupling and computer simulation. Coupling constants and chemical shifts are as expected from spectra of the model glycosides, methyl β‐d‐glucopyranosiduronate, methyl 2‐acetamido‐2‐deoxy‐β‐d‐glucopyranoside and methyl 2‐acetamido‐2‐deoxy‐β‐d‐galactopyranoside, when allowance is made for systematic influences on chemical shifts of interglycosidic linkages and sulphate substitution.As reported elsewhere, addition of alkali causes the hyaluronate spectrum to sharpen considerably. This is taken to indicate that segmental motion is enhanced by disruption of some system of inter‐residue bonding on ionisation of hydroxy groups. Concomitant changes in chemical shifts are seen mainly for H‐2 of the glucuronate residue, and the CH3 and H‐2 of the acetamidodeoxyglucose residue. Similar effects are not seen for chondroitin sulphates, either in line widths or chemical shifts.Comparison of the spectra of hyaluronate, chondroitin sulphates, and the model glycosides, indicates that proton chemical shifts are sensitive to the conformation differences between the polysaccharides in alkaline solution, but do not detect the differences in neutral solution that are known from NMR relaxation to be present. The altered configuration and/or substitution pattern of the acetamidodeoxyhexose residue in hyaluronate compared with chondroitin sulphates appears to have a critical influence on overall conformation in both alkaline and neutral solution.