bluuebunny/crossref_metadata_2025 huggingface dataset:
Receptor‐Mediated Gonadotropin Action in Ovary
Authors: AZHAR Salman , MENON K. M. Jairam
Published: March 2005

The effect of gangliosides and cholera enterotoxin on 125I choriogonadotropin binding, cAMP formation, protein kinase activation and progesterone production have been studied in isolated rat ovarian interstitial cells. A close correlation was observed between the amount of adenosine 3′:5′‐monophosphate (cAMP) produced in the cells and the extent of the activation of protein kinase in response to cholera enterotoxin and choriogonadotropin. Similarly cholera enterotoxin, like choriogonadotropin, stimulated steroidogenesis in a dose‐dependent and time‐dependent manner.

Addition of highly purified ganglioside mixtures abolished the stimulatory effect of cholera enterotoxin on cAMP and progesterone responses, while these agents had no effect on choriogonadotropin‐stimulated responses. Similar results were also obtained when different lutropin preparations were tested. Pretreatment of ovarian cells with gangliosides did not show any enhancement in the extent of steroidgenesis stimulated by toxin or trophic hormones compared to that seen with untreated cells.

Both gangliosides and cholera enterotoxin failed to block 125I‐labelled choriogonadotropin binding to ovarian cells. Individual gangliosides GD1a, GD1b, GT1 and GM1 [designated according to Svennerholm (1963) J. Neurochem. 10, 613–623] did not modify gonadotropin binding to ovarian cells. Similarly gangliosides isolated from rat ovary, bovine corpus luteum and rat brain also lacked the ability to modify 125I‐labelled choriogonadotropin binding to cells. Analysis of ovarian cells for gangliosides indicated that major ganglioside was GM3 with trace amounts of other gangliosides including GM1 and more complex gangliosides.

The results presented in this communication suggest that gonadotropin binding and subsequent cAMP and progesterone production are not influenced directly by gangliosides. In contrast, choleragen‐stimulated cellular events in the ovarian cells were completely inhibited by gangliosides. This suggests that these two agents exert their effects on the responsive system by interacting through separate components on the cell surface.
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.
The Enzyme‐Bound Copper of Dopamine β‐Monooxygenase
Authors: SKOTLAND Tore , LJONES Torbjørn
Published: March 2005

The enzyme‐bound copper of dopamine β‐monooxygenase reacted rapidly with the chelator bathocuproine disulfonate; the reaction in the presence of ascorbate was completed in 2 min at 25°C with 1 mM chelator. This reaction and also the reaction with EDTA could be used to prepare the apoenzyme, which in both cases was completely reactivated in less than 10 s. The reactivation data gave apparent Michaelis constants for copper of 0.03–0.2 μM. Trace amounts of copper in buffers and assay mixtures gave significant reactivation without added copper, unless they had been treated with a chelating resin. Titrations using the different chelation rates of free and enzyme‐bound copper indicated that four copper atoms are bound per enzyme molecule of four subunits. The native enzyme was more stable against thermal inactivation than the apoenzyme, but this stability was only partially restored by addition of copper to the apoenzyme.
Distribution of Inverted Repeat Sequences in Nuclear DNA from Physarum polycephalum
Authors: HARDMAN Norman , JACK Peter L. , BROWN J. P. , MCLACHLAN Alan
Published: March 2005

Inverted repeat sequences, capable of forming stable intra‐chain foldback duplexes, are shown using electron microscopy to be located in over 90% of fragments of nuclear DNA from Physarum polycephalum. A statistical treatment of the data indicates that, on average, foldback sequence foci are spaced every 7000 nucleotides and that they are distributed uniformly amongst the DNA chains. The majority of inverted repeat sequences give rise to the simple types of foldback structure observed in DNA from other eukaryotic species, but a significant proportion of the DNA fragments also contain novel foldback structures with a more complex appearance, referred to as ‘bubbld’ hairpins. The latter structures appear to be formed by the annealing of several distinct segments of homologous inverted repeat sequence, each separated by interspersed non‐foldback sequences of variable sizes up to 15000 nucleotides in length. The size, both of the foldback duplexes and of the intervening single‐chain segments of DNA, are not random. Instead, they appear to forma regular, arithmetic series of lengths. These obsevations suggest that the different segments of Physarum DNA from which foldback structures are derived contain nucleotide sequences that share a highly ordered and uniform pattern of structural organisation. These regular units of organisation in Physarum DNA in some cases cases extend over distances up to 50000 nucleotides in length.
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.
The Kinetics of Schiff‐Base Formation during Reconstitution of D‐Serine Apodehydratase from Escherichia coli with Pyridoxal 5′‐Phosphate
Authors: REED Thomas A. , SCHNACKERZ Klaus D.
Published: March 2005

Schiff base formation during reconstitution of D‐serine dehydratase (Escherichia coli) from its apoenzyme and pyridoxal 5′‐phosphate (pyridoxal‐P) has been studied by rapid kinetic techniques using absorbance changes at 436 nm. Three distinct reaction phases have been observed. The first is a very rapid change during which pyridoxal‐P is initially bound to the apoenzyme. This step has an equilibrium constant of 1500 M−1 and a forward reaction rate of the order of 2.6×106 M−1 s−1. The second phase shows a first‐order rate constant with a value dependent on pyridoxal‐P and corresponds to a first‐order step with a forward rate constant of 3.04 s−1 interacting with the initial equilibrium. The final phase is a slow first‐order reaction, the rate constant of which is approximately 0.01 s−1 and is independent of pyridoxal‐P concentration. The active pyridoxal species has been shown to be the free pyridoxal‐P as opposed to hemiacetal or hemimercaptal forms.
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.
Ribosomal Translocation Assayed by the Matrix‐Bound Poly(uridylic acid) Column Technique
Authors: BELITSINA Nadezhda V. , SPIRIN Alexander S.
Published: March 2005

The system of translation of cellulose‐bound poly(uridylic acid) by Escherichia coli ribosomes has been used for preparation of pre‐translocation state ribosomes in columns. Translocation has been induced by passing the elongation factor G (EF‐G) with GTP or its non‐cleavable analog (guanosine 5′‐[β,γ‐methylene]triphosphate) through the column. A method for quantitative comparison of translocation rates, and thus of effectiveness of translocation‐inducing factors, has been proposed. The method is based on an analysis of the profile of deacylated tRNA elution resulting from translocation in the column. The determination of the rate and amount of translocation has been done under different ionic conditions. It has been found that the Mg2+ concentration is a decisive factor of translocation in vitro: at high Mg2+ (30 mM) EF‐G cannot induce translocation, and lowering the Mg2+ concentration (to 10 mM) is required for EF‐G to become effective. Sufficiently low Mg2+ (3 mM) itself has proved to induce fast and complete translocation, without EF‐G.
Isolation and Characterization of Phosphorylated Oligosaccharides from α‐N‐Acetylglucosaminidase that Are Recognized by Cell‐Surface Receptors
Authors: VON FIGURA Kurt , KLEIN Udo
Published: March 2005

Adsorptive endocytosis of lysosomal enzymes by fibroblasts and hepatocytes involves binding to cell surface receptors that recognize on lysosomal enzymes a phosphorylated carbohydrate, most likely a mannose 6‐phosphate residue [Kaplan et al. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 2026–2030; Ullrich et al. (1978) Hoppe‐Seyler's Z. Physiol. Chem. 359, 1591–1598]. Loss of α‐N‐acetylglucosaminidase endocytosis after treatment with endoglucosaminidase H indicated that the recognition site of α‐N‐acetylglucosaminidase is located on N‐glycosidically linked oligosaccharides of the high mannose type. Acidic oligosacchrides with an average molecular weight of 2200 were liberated from α‐N‐acetylglucosaminidase by endoglucosaminidase H. These oligosaccharides were susceptible to degradation by alkaline phosphatase, α‐mannosidase and β‐N‐acetylglucosaminidase. At the non‐reducing terminal these oligosaccharides bear phosphorylated mannose and/or N‐acetylglucosamine residues.
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.
Translation of Biologically Active Messenger RNA from Human Placenta in Xenopus Oocytes
Authors: MOUS Jan , PEETERS Ben , VAN BELLEGEM Hilde , ROMBAUTS Wilfried
Published: March 2005

Polysomal RNA was extracted from human term placenta and total poly(A)‐containing RNA purified by affinity chromatography on oligo(dT)‐cellulose. Poly(A)‐containing RNA constituted approximately 1.2% of the total polysomal RNA and 8% of this purified preparation was able to anneal with [3H]poly(U). When injected into Xenopus oocytes, this poly(A)‐rich RNA directed the synthesis of a polypeptide which is immunoprecipitable with a specific antiserum to human placental lactogen. The identity of authentic human placental lactogen and the immunoreactive polypeptide synthesized in the oocyted is suggested by their identical behaviour in dodecylsulfate gel electrophoresis and by the formation of identical cyanogen bromide peptides. No presursor of human placental lactogen can be detected in the oocytes. The messenger RNA for human placental lactogen is very stable in oocytes; it is translated efficiently for a period of at least 7 days.
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).
Translational Fidelity and Specificity of Ribosomes Cleaved by Cloacin DF13
Authors: TWILT Johan C. , OVERBEEK Gerrit P. , VAN DUIN Jan
Published: March 2005

The effect of cloacin DF13 cleavage on several functional properties of the ribosome has been studied in a translational system in vitro. Cleaved ribosomes synthesize relatively shorter polypeptide chains on synthetic and natural templates. Their translational specificity is, however, unchanged as judged by the read‐out of MS2 RNA. Here, cleaved as well as control ribosomes start translation only on the coat cistron of the phage RNA. Cloacin cleavage of ribosomes increases their fidelity of translation. Differential inhibition of translation of synthetic and natural template was not observed.
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.
Cyclic Nucleotide‐Independent Protein Kinases from Rabbit Reticulocytes
Authors: TUAZON Polygena T. , BINGHAM Elizabeth W. , TRAUGH Jolinda A.
Published: March 2005

Purified variants of αs1 casein and β casein have been used to examine the substrate‐specificity of two adenosine‐3′:5′‐monophosphate‐independent protein kinase activities purified from rabbit reticulocytes. These enzymes, identified as casein kinase I and II were stimulated by monovalent cations. Casein kinase I had a pH optimum between 6.8 and 8.0, used ATP as the phosphate donor and preferentially phosphorylated serine. Casein kinase II had optimal activity at pH 8.5 to 10, utilized both ATP and GTP in the phosphotransferase reaction and modified predominantly threonine residues. The chymotryptic peptides of the phosphorylated casein variants were analyzed by thin‐layer electrophoresis and ascending chromatography and the phosphorylated peptides were identified by autoradiography. In the β caseins, the phosphorylated peptides observed with casein kinase I were different from those obtained with casein kinase II. With the αs1 caseins, the same phosphorylated peptide was observed with both protein kinases. The phosphorylation sites in the αs1 and β caseins have been assigned on the basis of the amino acid analyses of the phosphorylated chymotryptic peptides and the phosphoserine and phosphothreonine determinations in conjunction with the known primary sequences. Casein kinase I phosphorylated Ser‐22 in β‐A2 casein and Ser‐41 in αs1‐A casein, suggesting that this protein kinase recognized the primary sequence Glu‐X‐Ser. Thr‐41 in β‐A2 casein and Thr‐49 in αs1‐A casein were the primary sites phosphorylated by casein kinase II. The recognition determinants for casein kinase II appeared to be Thr‐Gou‐Asp. When endogenous phosphate was removed from the caseins, the rate of phosphorylation of β casein was diminished by greater than six‐fold indicating that negative charge near the phosphorylation site facilitated phosphorylation.
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.
The Use of Biochemical Solid‐Phase Techniques in the Study of Alcohol Dehydrogenase
Authors: ANDERSSON Lars , MOSBACH Klaus
Published: March 2005

Iodoacetate is known to inhibit horse liver alcohol dehydrogenase by selectively reacting with Cys‐46 in each of the two active sites of the dimeric enzyme. In this study, a method is described in which horse liver alcohol dehydrogenase is carboxymethylated while the enzyme is strongly bioaffinity‐bound to N6‐[N‐(6‐aminohexyl)carbamolymethyl]‐NADH‐substituted Sepharose 2B as a ternary complex in the presence of isobutyramide. A low degree of coenzyme‐ligand substitution (about 20 nmol/ml settled gel) was used in order to restrict enzyme‐coenzyme interaction on the gel to a single subunit. After the modification reaction by iodoacetate on the solid phase, the enzyme was eluted from the NADH‐substituted gel and its properties studied in free solution. Based on the incorporation of [14C]carboxymethyl groups, it is concluded that the reaction takes place at the active center of the ‘free’ (not bioaffinity‐bound) subunit; amino acid analysis showed that S‐carboxymethylcysteine had been formed. Also, the monoalkylated enzyme preparation was half as active as native enzyme when ethanol or benzaldehyde were used as substrates. Furthermore, after alkylation on the solid phase the enzyme loses about 50% of its original NADH‐binding capacity which was determined fluorometrically by titrating with NADH in the presence of isobutyramide. From these results it is suggested that both subunits in native horse liver alcohol dehydrogenase are kinetically equivalent.
Effect of Triamcinolone on Renal and Hepatic Phosphoenolpyruvate Carboxykinase in the Newborn Rat
Authors: MENCHER David , RESHEF Lea
Published: March 2005

The effects of triamcinolone on renal and hepatic phosphoenolpyruvate carboxykinase activity in the developing rat were investigated. The hormone induced increases in pre‐existing enzyme activity of both tissues in fetal and neonatal rats, yet did not cause the primary appearance of phosphoenolpyruvate carboxykinase activity in utero.

Neonatal hepatic phosphoenolpyruvate carboxykinase activity was increased 2–3‐fold by triamcinolone from the 3rd to the 15th postnatal day. This was shown to be additive to the effect of Bt2cAMP on enzyme activity. The increases in phosphoenolpyruvate carboxykinase activity were demonstrated to be due to increased synthesis of the enzyme, which was accompanied by a proportionate increase in the amount of functional phosphoenolpyruvate carboxykinase mRNA, as measured by the polyribosomal and poly(A)‐containing RNA directed cell‐free synthesis of the enzyme.

The demonstration of a triamcinolone effect on kidney and liver phosphoenolpyruvate carboxykinase activity in fetal and neonatal rats provides support for a possible role of glucocorticoids in the regulation of phosphoenolpyruvate carboxykinase activity during development.
Purification and Reverse Transcription of the Messenger RNA Coding for the Insect Protein, Calliphorin, Isolated from Larvae of the Blowfly, Calliphora vicina R.‐D.
Authors: ALONSO Angel , FLYTZANIS Constantin N. , SCHÄTZLE Ursula , SCHELLER Klaus , SEKERIS Constantin E.
Published: March 2005

Starting from poly(A)‐containing RNA prepared from the fat body of larvae of the blowfly Calliphora vicina, we have purified an mRNA coding for the protein calliphorin, which is a major blowfly protein accounting for approximately 9% of the total poly(A)‐containing mRNA activity in the fat body of 5‐days‐old larvae, as demonstrated by translation in vitro. The mRNA for this protein was purified by sucrose gradient centrifugation under denaturing conditions and identified by cell‐free translation. The peak fraction of the gradient shows three bands of about 20 S after formamide/acrylamide gel electrophoresis. Using reverse transcriptase and Calliphora mRNA isolated directly from the acrylamide gel electrophoresis or from sucrose gradients, we synthesized a cDNA probe. This cDNA hybridizes with the template, showing a major kinetic component with a molecular weight of 2.8 × 106, fitting well with the three bands observed in the electrophoresis. Hybridization of the cDNA with total sonicated Calliphora DNA shows annealing only at very high cot values, indicating that Calliphora mRNA is transcribed from the unique portion of the fly genome.
Amino Acid Sequence of Neurotoxin III of the Scorpion Androctonus australis Hector
Authors: KOPEYAN Charles , MARTINEZ Gérard , ROCHAT Hervé
Published: March 2005

The amino acid sequence of neurotoxin III, purified from the venom of the North African scorpion Androctonus australis Hector, has been determined by Edman degradation using a liquid‐phase sequence. Carboxypeptidase A hydrolyses confirmed not only the sequence of the five last residues but also the presence of a free α‐carboxylic group at the C‐terminus. Edman degradation was conducted on one hand with the Quadrol [N,N,N′,N′‐tetrakis(2‐hydroxypropyl)ethylene diamine] program and S‐alkylated proteins before or after coupling with sulfophenylisothiocyanate (the first 34 residues were thus identified), on the other hand on tryptic and chymotryptic peptides with a dimethylbenzylamine program (residues 1–23 and 31–34 were confirmed, the positions of residues 35–64 were established). Neurotoxin III was found to belong to the same group of scorpion toxins active on mammals as neurotoxin I purified from the same venom (50 homologous positions exist in the two proteins).
Intrinsic Fluorescene of Cowpea‐Chlorotic‐Mottle‐Virus Protein
Authors: KRÜSE Jacob , VERDUIN Benedictus J. M. , VISSER Antonie J. W. G.
Published: March 2005

Changes in the environment of the aromatic amino acid residues in the protein subunits of cowpea chlorotic mottle virus were studied with fluorescence techniques. The fluorescence properties of the protein in nucleoprotein particles, empty protein shells and dimers of the coat protein subunit were determined.

According to fluorescence spectra (excitation at 295 nm, emission maxima at 342–346 nm depending on pH and ionic strength) the tryptophan residues are located in a polar environment. With excitation at 235 nm a strong shoulder at about 300 nm in the total emission spectrum can be observed. This emission must be attributed to the tyrosine residues in the protein and can also be observed as a fast component in the fluorescence decay curve. The lifetimes were compared with those of the other bromoviruses which are different in amino acid composition. From excitation spectra of cowpea chlorotic mottle virus it can be concluded that energy is transferred from tyrosine to tryptophan residues.

Increase in the quantum yield of the tryptophan emission of the nucleoprotein particles upon raising the ionic strength and pH are attributed to a diminishing interaction between the protein and the RNA. The fluorescence properties of the protein hardly change when the empty protein shells are dissociated into protein dimers.

Quenching studies with potassium iodide and acrylamide indicate that the tryptophan residues in the protein are attainable more easily at low ionic strength than at high ionic strength.
Synthesis of Carbonic Anhydrase in Rabbit and Chicken Reticulocyte Lysates
Authors: CRAMER Frans , GOULD Hannah , BARLOW Sandra , CARTER Nicholas
Published: March 2005

The synthesis of carbonic anhydrase, the second most abundant soluble protein of red blood cells, is shown to occur in rabbit and chicken reticulocyte lysates. This translation product was identified by chloroform/ethanol extraction, polyacrylamide gel electrophoresis is sodium dodecylsulphate and peptide mapping. In rabbit reticulocyte lysates, predominantly one of the two red cell isozymes, carbonic anhydrase I, is synthesised. The proportion of carbonic anhydrase synthesis (0.2–0.8% of total protein synthesis) in vitro is comparable to that (0.2–1.0%) in vivo for both rabbit and chicken reticulocytes.
A Method for Designing Peptide Substrates for Proteases
Authors: POZSGAY Marianne , ELÖDI Pál , GÁSPÁR Rezsö , BAJUSZ Sándor
Published: March 2005

The kinetic parameters of 25 peptidyl‐p‐nitroanilide substrates were investigated with subtilisin Carlsberg as model enzyme.

For a series of 12 substrates, the contribution of various side chains to the affinity constant was computed by regression analysis. From these contributions the sequence of a new and better substrate, N‐benzyloxycarbonyl‐arginyl‐norleucyl‐norleucyl‐p‐nitroanilide (Z‐Arg‐Nle‐Nle‐Nan) was predicted. The compound was synthesized and assayed. Its calculated 1/Km value, 43.5 mM−1, was in a good agreement with the value of 40.0 mM−1 that was determined experimentally.

On expanding the series to 19 substrates, it was found that the productivity of enzyme‐substrate binding is influenced primarily by those subsites which have a significantly greater contribution to the affinity constants than others.

The additivity principle applied reasonably well for the contribution of individual side chains to the kinetic parameters. This fact suggests that regression analysis can be used for the prediction of the amino acid sequence of better substrates than those already tested, probably not only for subtilisin but also for other proteolytic enzymes.
Identification and Characterization of a 19‐S Complex Containing a 27000‐Mr Protein in Artemia salina
Authors: DE HERDT Erik , SLEGERS Herman , KONDO Masatoshi
Published: March 2005

The cytoplasm of the cryptobiotic Artemia salina gastrula contains a large quantity of a unique 19‐S complex. This particle is a specific aggregated form of a 27000‐Mr protein, having a molecular weight of 106 and an apparent buoyant density of 1.25–1.26 g/cm3 in sucrose and 1.31 g cm3 in CsCI. The relative quantity of this 19‐S complex decreases significantly with respect to 80‐S monoribosomes during the postgastrula development. Biochemical and immunological studies indicate that the 27000‐Mr protein is one of the RNA‐binding proteins [Ovchinnikov et al., FEBS Lett. 88, 21–26 (1978)] but is absent in the protein components associated with poly(A)‐containing ribonucleoprotein complexes. The possibility is also suggested that the 27000‐Mr protein and Artemia elongation factor eEF‐Ts might be related to each other on the basis of amino acid composition and immunological cross‐reactivity.
Glutathione S‐Conjugate Formation from 1‐Chloro‐2,4‐dinitrobenzene and Biliary S‐Conjugate Excretion in the Perfused Rat Liver
Authors: WAHLLÄNDER Axel , SIES Helmut
Published: March 2005

The isolated hemoglobin‐free perfused rat liver was used as a model to study the formation of a glutathione S‐conjugate and its disposition into the biliary as well as other extracellular (caval perfusate) compartments. 1‐Chloro‐2,4‐dinitrobenzene was chosen as substrate because it reacts with the glutathione‐S‐transferases at high activity to give rise to an S‐conjugate, S‐(2,4‐di‐nitrophenyl)‐glutathione, a product readily discernible from the parent compound.

S‐(2,4‐Dinitrophenyl)‐glutathione formation rates were equal to the infusion rates of 1‐chloro‐2,4‐dinitrobenzene up to 0.3 μmol × min−1× g liver−1. Steady‐state rates of S‐conjugate release were maintained until the glutathione content of the liver had decreased to about 40% of the control value.

Biliary excretion of the S‐conjugate was practically quantitative at low rates of S‐conjugate formation, up to about 30 nmol × min−1× g liver−1. and release of S‐conjugate into the caval perfusate gradually increased with the rate of formation. Maximal biliary S‐conjugate concentration was 36 mM.

Choleresis associated with biliary S‐conjugate excretion amounted to 18.6 pi of extra bile per μmol of net S‐conjugate excreted. This would correspond to a limiting concentration of 54 mM S‐conjugate in the water compartment associated with biliary transport, e.g. exocytotic vesicle in the context of an exocytosis‐like mechanism of bile formation.

The system described here is suited for further investigation of properties of bile formation, as is illustrated by the sensitive detection of changes in the concentration of S‐conjugate in the perfusate upon Ca2+‐depletion.