bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel approach to the study of the regulation of hormone‐sensitive lipase in rat adipocytes
Authors: MOONEY Robert A. , EBERSOHL Richard D. , McDONALD Jay M.
Published: March 2005

Use of a digitonin‐permeabilized rat adipocyte preparation overcomes inherent problems which occur when currently used broken cell systems are utilized for studying the regulation of hormone‐sensitive lipase. The effect of digitonin on plasma membrane permeability was concentration‐dependent being nearly maximum at 20 μg/ml as assessed by (a) leakage of 85% cellular lactate dehydrogenase after 30 min, (b) the efflux of 72% preloaded cellular (86Rb) rubidium within 10 min and (c) immediate inhibition of glucose oxidation.

Hormone‐modulated rates of lipolysis were preserved in this preparation. Following maximal activation of lipolysis in adipocytes with catecholamines, the rate of lipolysis in intact cells and digitonin‐treated cells was elevated 26‐fold and 20‐fold respectively, while the rate in homogenates from these cells was elevated only 2.8‐fold. Insulin suppressed catecholamine‐dependent activation of lipolysis by at least 90% when subsequently measured in intact cells and digitonin‐treated cells. Insulin suppression was only 56% when measured in homogenates.

The hormone‐sensitive lipase in permeabilized cells, as opposed to intact cells and homogenates, was activated by cyclic AMP to a degree that approached activation by catecholamines. In homogenates, cyclic AMP (1.0 mM) plus ATP (0.25 mM) activated the lipase only 36%, while neither alone had any effect. In digitonin‐permeabilized cells, however, exogenous cyclic AMP alone activated lipolysis in a concentration‐dependent manner with 1 μM, 30 μM and 1.0 mM cyclic AMP activating lipolysis by 41%, 250% and 1300% respectively. In contrast, lipolysis in intact cells was activated by 0%, 25% and 250% by 1 μM, 30 μM and 1.0 mM cyclic AMP. Also in digition‐treated preparations, ATP alone activated lipolysis 40%, but ATP plus cyclic AMP activated lipolysis to only 74% of the level due to cyclic AMP alone.

These studies indicate that the permeabilized adipocyte preparation is an excellent system for investigating the mechanism of regulation of the hormone‐sensitive lipase by permitting manipulation of the intracellular environment while preserving the physiological response of the lipase.
The vanadate complex of the calcium‐transport ATPase of the sarcoplasmic reticulum, its formation and dissociation
Authors: MEDDA Pankaj , HASSELBACH Wilhelm
Published: March 2005

Vanadate binding to different sarcoplasmic reticulum membrane preparations was determined by measuring bound vanadate colorimetrically and by phosphorylating the vanadate‐free enzyme fraction with [γ‐32P] ATP. Colorimetry allowed the study of the dependence of equilibrium vanadate binding on ionized magnesium and the displacing effect of ionized calcium at vanadate concentrations > 0.1 mM only. At saturating magnesium concentration the enzyme binds 6–8 nmol vanadate/mg protein and half‐maximum saturation is reached at 40 μM. Vanadate is displaced from the enzyme when its high‐affinity calcium‐binding sites are saturated and conversely calcium is solely displaced from its high‐affinity binding sites by vanadate. The phosphorylation procedure allowed the measurement of equilibrium binding as well as the kinetics of vanadate binding and release at vanadate concentrations below 0.1 mM. Half‐times of 30 s and 3 s were observed for vanadate release induced by 0.1 mM and 1 mM calcium respectively. Millimolar concentrations of ATP are required for vanadate displacement. Under equilibrium conditions the enzyme displays an affinity for vanadate of 1.6. 106 M−1. The dependence on the concentration of vanadate of the rate of vanadate binding yielded an affinity of only 1–104 M−1. Closed vesicles bind vanadate much more slowly than calcium‐permeable preparations. The initial rate of calcium‐induced vanadate dissociation is accelerated considerably when the vesicles are made calcium permeable. The rate of vanadate dissociation from calcium‐permeable vesicles reaches half‐maximum values at 1–2 mM calcium indicating that the internal low‐affinity calcium‐binding sites must first be occupied in order to release bound vanadate. The results suggest that vanadate binding leads to a transition of the external high to internal low‐affinity calcium‐binding sites.
NAD+ analogue binding to glyceraldehyde‐3‐phosphate dehydrogenase
Authors: WALLÉN Leif , BRANLANT Guy
Published: March 2005

A series of NAD+ analogues, modified on the pyridinium ring, have been tested for their enzymic properties in reactions with d‐glyceraldehyde‐3‐phosphate dehydrogenase form sturgeon muscle, rabbit muscle and Bacillus stearothermophilus. The observed activity, inhibition and binding data are correlated to the structure of the enzyme and coenzyme analogue by model building on a Vector General interactive graphic display system using coordinates from the B. stearothermophilus holoenzyme structure.

Most of the analogues with substituents in the pyridinium‐3 position could be bound to glyceraldehyde‐3‐phosphate dehydrogenase, either in manner similar to NAD+ or in a completely different way with the substituted pyridinium ring rotated 110° or more around the glycosidic bond. This indicates different possible modes of binding of NAD+ analogues within the pyridinium binding subsite.

Analogues with substituents in the pyridinium‐4 position are shown to be weakly bound to glyceraldehyde‐3‐phosphate dehydrogenase. This is explained by a strong interaction of the substituent in the 4 position with the residues Asn‐313 and Cys‐149.
Calcium/calmodulin‐regulated guanylate cyclase and calcium‐permeability in the ciliary membrane from Tetrahymena
Authors: SCHULTZ Joachim E. , SCHÖNEFELD Ulrich , KLUMPP Susanne
Published: March 2005

A guanylate cyclase of high specific activity was localized in the ciliary membrane from Tetrahymena pyriformis. Purity of cilia was checked by electron microscopy and purity of membrane fractions isolated by a sucrose density gradient by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Enzyme activity was due to the presence of endogenous calmodulin as evidenced by the inhibition of guanylate cyclase by addition of antiserum against calmodulin from Tetrahymena or soybean. Removal of endogenous calmodulin by La3+ ‐treatment of ciliary membranes resulted in loss of guanylate cyclase activity. In addition to protozoan calmodulins, the original activity could also be restored by the nonhomologous calmodulins from soybean and pig brain but not by calcium‐binding proteins like Dictyostelium calmodulin, parvalbumin, and troponin C, lacking the trimethyllysine characteristic for mammalian calmodulins. However, only calmodulins from the protozoans Tetrahymena and Paramecium stimulated guanylate cyclase activity in excess of the initial activity. This indicates that the guanylate cyclase either contains two binding sites for calmodulin with different specificities or that a single, but only partially occupied binding site is modified possibly by hydrolytic exo‐proteases during membrane preparation. The ciliary membrane from Tetrahymena contains a discrete calcium‐permeability as demonstrated by calcium‐flux measurements using the calcium indicator dye arsenazo III. In analogy to the excitable ciliary membrane of the larger relative Paramecium, the ciliary membrane of Tetrahymena may thus carry the voltage‐sensitive calcium‐channels known from electrophysiological studies.
Subunit composition of oxaloacetate decarboxylase and characterization of the α chain as carboxyltransferase
Authors: DIMROTH Peter , THOMER Anna
Published: March 2005

Oxaloacetate decarboxylase from Klebsiella aerogenes was shown to be composed of three different subunits α, β, γ with Mr 65000, 34000 and 12000, respectively. On dodecylsulfate polyacrylamide gels the smallest of these subunits was heavily stained with silver but poorly with Coomassie brilliant blue. All three subunits were resolved and clearly detectable by high‐performance liquid chromatography in a dodecylsulfate‐containing buffer. Biotin was localized exclusively in the α chain.

Freezing and thawing of the isolated membranes in the presence of 1 M LiCl released the α chain which was subsequently purified to near homogeniety by affinity chromatography on monomeric avidin‐Sepharose. No β or γ chain were detectable in this α chain preparation and no oxaloacetate decarboxylation was catalyzed. The isolated α chain, however, was a catalytically active carboxyltransferase as evidenced from the isotopic exchange between [1‐14C]pyruvate and oxaloacetate. The rate of this exchange reaction was about 9 U/mg protein and was completely independent of the presence of Na+ ions. The ease with which the α chain was released from the membrane characterize this subunit as a peripheral membrane protein. The β and γ chain, on the other hand, stick so firmly in the membrane that they are only released by detergents, thus indicating that these are integral membrane proteins.

Limited tryptic digestion of oxaloacetate decarboxylase led to a rapid cleavage of the α chain, yielding a polypeptide of Mr 51000 which was devoid of biotin. Degradation of the β chain required prolonged incubation periods and was markedly influenced by Na+ ions which had a protective effect against proteolysis.

A proton is required in the decarboxylation of oxaloacetate and CO2 arises as primary product. The other alternative, i. e. generation of HCO−3 with H2O as substrate, has been excluded.
NADH oxidation in submitochondrial particles protects respiratory chain activity against damage by adriamycin‐Fe3+
Authors: DEMANT Erland J. F.
Published: March 2005

Oxidative decomposition of polyunsaturated fatty acid moieties of membrane lipid in pig heart submitochondrial particles, as initiated by ferric ion complexes of the antineoplastic drug adriamycin and concomitant inactivation of oxidase activities, is counteracted by EDTA, low oxygen pressure, a phenolic antioxidant and NADH oxidation through the respiratory chain but not by scavengers of reactive oxygen species. Protection by NADH is strengthened by removal of cytochrome c from the submitochondrial particles and by antimycin A but abolished by rotenone.

Inhibition of cytochrome c oxidase activity by the adriamycin‐Fe3+ complex is reversible and activity is recovered upon cholate solubilization of the particles. ADP inhibits binding of the complex to the submitochondrial particles and protects both cytochrome c oxidase activity and membrane lipid.

The results are discussed in relation to the possible role of mitochondrial function in protection against free‐radical‐mediated effects of adriamycin.
On the cytotoxicity of vitamin C and metal ions
Authors: SAMUNI Amram , ARONOVITCH Jacob , GODINGER Dina , CHEVION Mordechai , CZAPSKI Gidon
Published: March 2005

The toxicity of ascorbate towards phage lambda and the phages T2–T7 has been investigated. At room temperature the T‐odd and lambda bacteriophages are highly susceptible to ascorbate‐induced damage, whereas the T‐even phages are practically resistant. The toxicity of ascorbate is dependent on the presence of copper (or iron) and oxygen, although oxygen is not required in the presence of H2O2. Hydrogen peroxide is essential for the ascorbate‐induced phage inactivation and the damage is prevented by catalase. At the concentrations used, most of the copper ions are bound to the phage particles. Chelating agents such as EDTA or histidine fully protect the phages, whereas salicylate only reduces the rate of phage inactivation. OH scavengers such as sucrose, formate, mannitol, tert‐butyl alcohol or poly(ethylene glycol) have no protective effect.

Experiments with DNA labeled phages indicate that both phage adsorption and DNA injection are impaired as a result of the exposure to ascorbate and copper. The failure to express the viral genetic information as a result of single and double‐strand breaks in the DNA, probably also contribute to the loss of the plaque‐forming ability of the phages.

The results are interpreted in terms of a ‘site‐specific’ Fenton mechanism according to which the binding of the transition metal ions to the biological target is a prerequisite for the production of damage. The bound metal ion is reduced either by O−2, ascorbate or other reductants and is subsequently reoxidized by H2O2 yielding OH˙ radicals. This cyclic redox reaction of the metal generates OH˙ radicals which react with vital macromolecules with a high probability of causing ‘multi‐hit’ damage. This ‘site‐specific’ formation of OH˙ radicals, which takes place near the target molecules, accounts both for the high damaging efficiency and for the failure of OH˙ scavengers to protect against it.
β‐Agarases I and II from Pseudomonas atlantica
Authors: MORRICE Lora M. , McLEAN Maitland W. , LONG William F. , WILLIAMSON Frank B.
Published: March 2005

β‐Agarase I and II were characterised by their action on agar‐type polysaccharides and oligosaccharides.

β‐Agarase I, an endo‐enzyme, was specific for regions containing a minimum of one unsubstituted neoagarobiose unit [3,6‐anhydro‐α‐l‐galactopyranosyl‐(1 → 3)‐d‐galactose], hydrolysing at the reducing side of this moiety. Yaphe demonstrated that agar was degraded by this enzyme to neoagaro‐oligosaccharides limited by the disaccharide but with a predominance of the tetramer [Yaphe, W. (1957) Can. J. Microbiol. 3, 987–993]. β‐Agarase I slowly degraded neoagarohexaose but not the homologous tetrasaccharide. [1‐3H] Neoagarohexaitol was cleaved to neoagarotetraose and [1‐3H]neoagarobiitol. The highly substituted agar, porphyran was degraded to methylated, sulphated and unsubstituted neoagaro‐oligosaccharides which were invariably terminated at the reducing end by unsubstituted neoagarobiose.

The novel enzyme, β‐agarase II, was shown to be an endo‐enzyme. Preliminary evidence indicated this enzyme was specific for sequences containing neoagarobiose and/or 61‐O‐methyl‐neoagarobiose. It degraded agar to neoagaro‐oligosaccharides of which the disaccharide was limiting and predominant. β‐Agarase II rapidly degraded isolated neoagarotetraose and neoagarohexaose to the disaccharide. With [1‐3H]neoagarohexaitol, exo‐action was observed, the alditol being cleaved to neoagarobiose and [1‐3H]neoagarotetraitol. Neoagarotetraitol was hydrolysed at 4% of the rate observed for the hexaitol. Porphyran was degraded to oligosaccharides, the neutral fraction comprising 24% of the starting carbohydrate. This fraction was almost exclusively disaccharides (22.4%) containing neoagarobiose (7.4%) and 61‐O‐methyl‐neoagarobiose (15%). β‐Agarase II is probably the ‘β‐neoagarotetraose hydrolase’ reported by Groleau and Yaphe as an exoenzyme against neoagaro‐oligosaccharides [Groleau, D. and Yaphe, W. (1977) Can. J. Microbiol. 23, 672–679].
Cell‐wall lipopolysaccharide of the urinary‐tract‐infective Escherichia coli 04:K12:H−
Authors: SCHMIDT M. Alexander , JANN Barbara , JANN Klaus
Published: March 2005

The O‐specific polysaccharide moiety (04 polysaccharide) of the 04 antigen (lipopolysaccharide) from Escherichia coli 04:K12:H− was isolated in pure form by degradation of the lipopolysaccharide and chromatography on Sephadex G‐50. The primary structure of the 04 polysaccharide was elucidated by composition, Smith degradation, nuclear magnetic resonance spectroscopy, methylation and oligosaccharide analysis. Oligosaccharides were obtained by deaminating fragmentation of partially and completely de‐N‐acetylated polysaccharides. The polysaccharide consists of repeating units of the pentasaccharide

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which are joined in the polymer through α‐1, 4‐linkages. The 04 polysaccharide has a mean molecular mass of 13800 Da and consists of the core oligosaccharide and about 14 pentasaccharide repeating units.
Freeze‐fracture analysis of membrane appression and protein segregation in model membranes containing the chlorophyll‐protein complexes from chloroplasts
Authors: RYRIE Ivan J.
Published: March 2005

Cation‐induced membrane appression and lateral segregation of chlorophyll‐protein complexes have been investigated by freeze‐fracture analysis of model membranes containing photosystem 1 and the light‐harvesting complex of photosystem 2. In light‐harvesting complex proteoliposomes, cations caused extensive membrane adhesion and a segregation of protein into appressed regions. A marked flattening of the appressed membranes, sometimes together with a co‐alignment of the particles on the opposing membrane faces, strongly suggests a direct transmembrane attraction between the protein particles. Photosystem 1 membranes were not appressed by cations but some clustering of the particles occurred, together with their exclusion from some regions of the lipid. By incorporating dipalmitoylglycerophosphocholine into the membranes, it is shown that a similar exclusion of the particles can occur due to liquid‐crystalline to gel state transitions of the lipids.

Proteoliposomes containing both the light‐harvesting complex and photosystem 1 displayed cation‐induced membrane appression, but only between regions containing the light‐harvesting complex. Photosystem 1 was largely confined to unappressed membranes. Destacking occurred at low salt concentrations irrespective of whether photosystem 1 was present, showing that in proteoliposomes, and probably in thylakoids, this process does not require the presence of highly charged complexes from unappressed regions.
The identity of conformational states of reconstituted and native histone octamers
Authors: GREYLING H. Johann , SCHWAGER Sylva , SEWELL B. Trevor , von HOLT Claus
Published: March 2005

Histone octamers were reconstituted from the following preparations: (a) natural histone H3‐H4 tetramer and histone H2A‐H2B dimer, either selectively extracted from chromatin with solutions of sodium chloride or prepared by dissociation of the natural octamer; (b) acid‐denatured core histones, either an unfractionated mixture or individually purified proteins.

Complexes assembled from these histones elute from exclusion chromatography columns with octamer size as verified by cross‐linking with dimethylsuberimidate. The reconstituted octamers all crystallize in the same form of helical tubes as the natural octamer.
An absolute requirement for the 5′ cap structure for mRNA translation in sea urchin eggs
Authors: WINKLER Matthew M. , BRUENING George , HERSHEY John W. B.
Published: March 2005

Translation of a variety of RNAs was studied in a cell‐free translation system derived from sea urchin eggs. While RNAs such as globin or tobacco mosaic virus are efficiently translated, viral RNAs which do not contain the 5′ cap structure, such as cow pea mosaic virus (CPMV) and poliovirus, are not translated. Mixing experiments with reticulocyte lysates indicated that the lack of translation of uncapped viral RNAs is not due to the presence of a potent inhibitor or the absence of an activating agent. RNA competition experiments between capped and uncapped RNAs indicated that uncapped RNAs do not interact with the sea urchin egg initiation machinery. Proteolytic removal of the 5′ viral protein did not allow the translation of CPMV RNA. However, chemical decapping of vesicular stomatitis virus mRNA completely inhibited the translation of this mRNA in the sea urchin cell‐free system. We conclude that the sea urchin egg lacks the initiation pathway used to initiate uncapped mRNAs in mammalian cells and thus has an absolute requirement for the 5′ cap structure for initiation. In addition we discuss the implications of these findings for the control of protein synthesis after fertilization of the sea urchin egg.
Cloning and characterization of the ribosomal genes of the sea‐urchin Paracentrotus lividus
Authors: PASSANANTI Claudio , FELSANI Armando , GIORDANO Roberto , METAFORA Salvatore , SPADAFORA Corrado
Published: March 2005

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the PstI site of pBR 322 by the dG · dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping.

The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot‐hybridization of the restriction fragments, using as probes nick‐translated 32P‐labelled cloned ribosomal DNA fragments or 18S and 26S sea‐urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 × 103 bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.
Cell‐free synthesis of rat α2‐macroglobulin and induction of its mRNA during experimental inflammation
Authors: NORTHEMANN Wolfgang , ANDUS Tilo , GROSS Volker , HEINRICH Peter C.
Published: March 2005

Poly(A)‐rich RNA was isolated from the livers of acutely inflamed rats by extraction with guanidinium HCl and oligo(dT)‐cellulose chromatography. After translation in a recticulocyte lysate and immunoprecipitation with a specific antiserum to α2‐macroglobulin a polypeptide with an apparent molecular weight of 162000 could be detected. The cell‐free synthesis of α2‐macroglobulin was stimulated 8‐fold by the addition of RNase inhibitor. Full‐length α2‐macroglobulin polypeptide chains appeared after 35 min in the presence of 1.85 mM Mg2+ and 100 mM K+. A nucleotide number of about 5100 was estimated for α2‐macroglobulin by means of sucrose gradient centrifugation of poly(A)‐rich RNA followed by translation in vitro and immunoprecipitation of α2‐macroglobulin. In normal liver α2‐macroglobulin mRNA represented about 0.0007% of total translatable RNA. Acute inflammation generated by intramuscular injection of turpentine led to a 66‐fold increase in translatable α2‐macroglobulin mRNA after 18 h, followed by a rapid decrease. In accordance to the induction of α2‐macroglobulin mRNA serum concentrations of α2‐macroglobulin increased to about 2 mg/ml. Unlike α2‐macroglobulin mRNA serum α2‐macroglobulin levels remained unchanged up to 60 h.
Defective complex formation between single‐stranded DNA and mutant recA430 or recA1 protein of Escherichia coli
Authors: WABIKO Hiroetsu , OGAWA Tomoko , OGAWA Hideyuki
Published: March 2005

The formation of a complex between recA protein and single‐stranded DNA (ssDNA) was studied by filter‐binding assays and sucrose density gradient centrifugation. In the presence of excess ATP, the wild‐type recA protein formed salt‐resistant complexes with ssDNA. One mutant recA430 (lexB30) protein bound to ssDNA slightly less effectively than wild‐type protein, and the complexes had lost the stability to salt. Another mutant recA1 protein did not form complexes with ssDNA. On the other hand, in the absence of ATP, all proteins bound to ssDNA with the same efficiency, but all of the complexes were unstable in the presence of salt. The hybridization reaction in which homologous ssDNA is converted to double‐stranded DNA (dsDNA) catalyzed by the recA430 protein was more sensitive to salt than that catalyzed by the wild‐type protein. No hybridization reaction was found with the recA1 protein.
Sequence‐specific resonance assignments in the 1H nuclear‐magnetic‐resonance spectrum of the Lac repressor DNA‐binding domain 1–51 from Escherichia coli by two‐dimensional spectroscopy
Authors: ZUIDERWEG Erik R. P. , KAPTEIN Robert , WÜTHRICH Kurt
Published: March 2005

The assignment of the 1H nuclear magnetic resonance (NMR) spectrum of the DNA‐binding domain 1–51 of lac repressor from Escherichia coli is described and documented. The assignments are based entirely on the amino acid sequence and on two‐dimensional NMR experiments at 360 MHz and 500 MHz. Individual assignments were obtained at 18 °C for the backbone protons of 44 out of the total of 51 amino acid residues, the exceptions being Met‐1, Lys‐2, Tyr‐7, Arg‐35, Glu‐36, Lys‐37 and Ile‐48. Complete assignments of the non‐labile hydrogen atoms of the side chain were obtained for 33 residues, and for Asn‐46 and Asn‐50 the δ amide protons were also identified. The chemical shifts for the assigned resonances at 18 °C are listed for an aqueous solution at pH 4.9 and at pH 6.8.
Structural studies on the phosphate‐free lipid A of Rhodomicrobium vannielii ATCC 17100
Authors: HOLST Otto , BOROWIAK Dietmar , WECKESSER Jürgen , MAYER Hubert
Published: March 2005

The structure of the free lipid A from Rhodomicrobium vannielii ATCC 17100 was elucidated. It consists of a central β‐1′,6‐linked glucosamine disaccharide which is not substituted by phosphate. About 30% of the disaccharide molecules are substituted with mannopyranose in β‐1,4′‐linkage to the non‐reducing glucosamine. The reducing glucosamine can be directly reduced with NaBH4, indicating either that this glucosamine is not substituted at C1 or its substituent has been removed during the preparation of free lipid A or is removed during reduction with NaBH4. The following formula shows the ‘backbone’ structure of the free lipid A from Rm. vannielii ATCC 17100: β‐Manp(1→4)‐β‐GlcpN(1→6)GlcpN.

3‐(R)‐Hydroxyhexadecanoic acid is linked to the amino group of the reducing glucosamine. The residue at the amino group of the non‐reducing glucosamine has not been identified. The hydroxyl groups of the central disaccharide are acylated with 3‐(tetradecanoyloxy)‐tetradecanoic acid, 3‐hydroxytetradecanoic acid, Δ14‐ docosenoic acid (Δ14‐C22:1) and acetyl groups. The hydroxyl groups of the mannose are not substituted.
Lipopolysaccharides of the cyanobacterium Microcystis aeruginosa
Authors: RAZIUDDIN Syed , SIEGELMAN Harold W. , TORNABENE Thomas G.
Published: March 2005

Lipopolysaccharides (LPS) of two isolates of Microcystis aeruginosa were extracted with phenol/water and purified. Cesium chloride gradient ultracentrifugation of these preparations yielded only one fraction. The LPS contained significant amounts of 3‐deoxy‐d‐manno‐octulosonic acid, glucose, 3‐deoxy sugars, glucosamine, fatty acids, fatty acid esters, hexoses, and phosphate. Heptose, a characteristic sugar component of the polysaccharide moiety of LPS of most gram‐negative bacteria was absent. Lipopolysaccharides and lipid A hydrolysate of LPS preparations were active in mouse lethality and Limulus lysate gelation. The lipid A moiety was slightly less active in toxicity and Limulus lysate gelation assays than the intact LPS. The LPS and lipid A moiety of the two isolates of M. aeruginosa were less active in toxicity in mice and Limulus test than LPS of Salmonella abortus equi.
Distance measurement by energy transfer
Authors: STEINHÄUSER Klaus G. , WOOLLEY Paul , DIJK Jan , EPE Bernd
Published: March 2005

Ribosomal proteins L6, L11 and the complex [(L12)4· L10] were labelled specifically at their respective single thiol groups, either with the acetylaminoethyl‐dansyl or with the acetamidofluorescein fluorophore. The labelled proteins were then reconstituted, singly or in pairs, into ribosomal 50 S subunits; the presence of the label had no observable effect on the composition, shape or activity of the reconstituted subunits.

The distances between the labelled thiol groups were measured by a fluorescence energy transfer method detailed elsewhere [Epe, B. et al. (1983) Proc. Natl Acad. Sci. USA, 80, 2579–2583] and were found to be: for L6–L10, 60 Å (6.0 nm); for L6–L11, 46 Å (4.6 nm); for L10–L11, 56 Å (5.6 nm). Reversal of the direction of energy transfer by exchanging labels gave duplicate distances which differed, on average, by about 4%. The distance between the fluorescent labels on L10 and L11 in the [23 S‐RNA · L10 · L11 · (L12)4] ribonucleoprotein complex was the same as in the 50S subunit, but all three distances were greater in 50S subunits which had been reconstituted without the final activation step (incubation at 50°C). This suggests a tightening of the L6/L10/L11 domain of the 50S subunit during the activation step.
Conformational studies of 13 trinucleoside bisphosphates by 360‐MHz 1H‐NMR spectroscopy
Authors: LEE Che‐Hung
Published: March 2005

The ribose protons of 13 trinucleoside bisphosphates (trimers) were studied, using 360‐MHz proton nuclear magnetic resonance spectroscopy. Complete assignments and analyses of the NMR signals of these protons were carried out by the methods of homonuclear decoupling and computer line‐shape simulations. It was shown that the trinucleotides preferred the anti, 3′endo, γ+, βt and ɛt/ɛ− conformations for the glycosidic torsions, the ribose rings, the C4′–C5′ bonds, the C5′–O5′ bonds, and the C3′–O3′ bonds, respectively. It was also found that the trimers, especially those which had noticeable population of ‘bulged’ structures, did not necessarily have a higher population of these preferred local conformations than their component dimers. The overall conformations of the trinucleotides are classified into two categories. The conformations in the first category involve the nearest‐neighbor interactions. Each dinucleotide moiety can assume one of the four stable conformations (I, I′, II and III) or the open forms of dinucleoside monophosphates. However, due to steric hindrance, there are only four cases in which both dinucleotide moieties can assume one of the four stable conformations at the same time. These four combinations of conformations are I‐I,I′‐I′,I‐II and III‐I′, where the first Roman numeral represents the conformation of the NpN′p‐moiety and the second one, that of the ‐pN′pN″ moiety of the trimers. Among them, I‐I and I′‐I′ are helical structures, capable of forming a double helix. The second category contains conformations with bulged structures which have the two dinucleotide moieties in open forms (i.e. no nearest‐neighbor interactions) and the bases of the two terminal residues stacking on each other while the middle residue is bulged out. These bulged conformations may serve as structural models for frame‐shift mutations.
Identification of the α and β subunits of the chloroplast coupling factor one in Chlamydomonas reinhardi
Authors: MERCHANT Sabeeha , SELMAN Bruce R.
Published: March 2005

The α and β subunits of the Chlamydomonas reinhardi coupling factor one have been identified by an immunochemical method and by the reaction of dicyclohexylcarbodiimide with the β subunit. Antibodies raised against the C. reinhardi subunit with the highest apparent molecular weight react with the α subunit of spinach coupling factor one and antibodies raised against the β subunit of the spinach chloroplast enzyme cross‐react with the C. reinhardi subunit of lower apparent molecular weight. Dicyclohexylcarbodiimide also reacts with this subunit. We conclude therefore that the two subunits of highest apparent molecular weight can be named α and β in order of decreasing apparent molecular weight, in contrast to the nomenclature suggested by Piccioni, R. G., Bennoun, P. and Chua, N.‐H. [(1981) Eur. J. Biochem. 117, 93–102].
Analysis of competition for substrate sites in an allosteric enzyme with co‐operative kinetics
Authors: MASTRANTONIO Stefania , NUCCI Roberto , VACCARO Carlo , ROSSI Mosè , WHITEHEAD Edward P.
Published: March 2005

Hypotheses about the interactions of effectors with conformations of allosteric enzymes having co‐operative kinetics can be tested simply and exactly without knowledge of the nature of the intersubunit co‐operativity by using a linkage approach to the analysis of steady‐state kinetics. Applying this approach to competition for substrate sites in the allosteric enzyme donkey spleen dCMP aminohydrolase, we show that the kinetics are consistent with the hypothesis that the substrate dCMP and the competitor dAMP, as well as the allosteric activator dCTP, bind exclusively to the same conformation of the enzyme subunits. The linkage test can be applied in the presence of other effectors without knowledge of how these interact with the enzyme. Our tests showed that dCMP and dAMP are still bound exclusively to this same conformation in the presence of the product dUMP or of the allosteric inhibitor dTTP. We give evidence that dUMP binds to the same conformation as dCMP, but that it is also bound to other conformation(s). The advantages of the linkage approach, and some general problems in steady‐state kinetics of allosteric enzymes, are discussed.
Modification of ribosomal proteins in sea urchin eggs following fertilization
Authors: TAKESHIMA Kazuhito , NAKANO Eizo
Published: March 2005

Analysis of the ribosomal proteins in sea urchin eggs by two‐dimensional polyacrylamide gel electrophoresis revealed postfertilization changes in the proteins of both the small and the large subunits. Five egg‐ribosomal proteins (S7, S16, S19, L19, L31) appeared to undergo rapid changes to the corresponding embryo‐specific proteins. These changes were completed within 30 min after fertilization, and identical electrophoretic patterns were observed among the different developmental stages of embryos. One of the five proteins, S7, showed an increase in the phosphorylated form. The remainder showed qualitative shifts to the corresponding embryo‐specific proteins; however, peptide map analyses revealed the existence of common structural units between the corresponding proteins.

These modifications were observed in the three species of sea urchin studied (Pseudocentrotus depressus, Hemicentrotus pulcherrimus and Anthocidaris crassispina), except in the case of one protein (L31). Purification of ribosomes by different procedures based on high‐salt treatment gave the same results with respect to the egg‐specific and embryo‐specific proteins.
Synergistic inhibition of [3H]muscimol binding to calf‐brain synaptic membranes in the presence of l‐homocysteine and pyridoxal 5′‐phosphate
Authors: GRIFFITHS Roger , WILLIAMS David C. , O'NEILL Cora , DEWHURST Ian C. , EKUWEM Clement E. , SINCLAIR Crawford D.
Published: March 2005

1. l‐Homocysteine and pyridoxal 5′‐phosphate (PxyP) inhibited [3H]muscimol binding to freeze‐thawed, Triton‐treated calf brain membranes (containing high‐affinity muscimol‐binding sites: Kd 9.5 ± 0.6nM, Bmax 5.2 ± 0.2 pmol/mg protein). The homocysteine–pyridoxal‐5′‐phosphate (Hcy‐PxyP) thiazine complex had no effect.

2. l‐Homocysteine was found to be a partially competitive inhibitor, thus demonstrating an allosteric inhibition with Ki value of 1.96 mM for free receptor and Ki of 13 mM for receptor‐muscimol complex.

3. PxyP was shown to be a two‐site pure competitive inhibitor of [3H]muscimol binding with cooperativity of PxyP binding such that Ki values for PxyP of 20 mM and 2.1 mM were found.

4. l‐Homocysteine and PxyP when added simultaneously to binding assays, caused a greater degree of inhibition than that observed at the same total specific concentration of either inhibitor alone. This synergistic inhibitory effect was shown to be due to a homocysteine‐induced increase in the affinity of PxyP‐binding sites.

5. Three alternative models are suggested to explain the observed synergistic effects whereby it is proposed that PxyP and [3H]muscimol binding is mutually exclusive, while l‐homocysteine with PxyP and l‐homocysteine with muscimol, exhibit non‐exclusivity.

6. Non‐linear regression analysis of binding data was undertaken in order to substantiate conclusions drawn from graphical procedures and in an attempt to ascertain which mathematical model best fitted the experimental data describing the synergistic inhibitory effects of l‐homocysteine and PxyP.

7. This synergistic inhibitory effect of PxyP and l‐homocysteine on the post‐synaptic γ‐aminobutyric acid receptor may provide a basis for explanation of the mechanism of homocysteine‐induced seizures.
Resolution of the discrepancy between a gene translation — termination codon and the deduced sequence for release of the encoded polypeptide
Authors: ATKINS John F. , GESTELAND Raymond F.
Published: March 2005

The translation‐termination codon of the synthetase gene of the RNA phage MS2 has been determined, by nucleotide sequencing and suppression studies in vitro, to be UAG. However in one of the only two studies on the signals for polypeptide chain release at the end of genes, Capecchi and Klein [(1970) Nature (Lond.) 226, 1029–1033] deduced that the synthetase of an almost identical phage, R17, is released at UAA. Here we show that under certain conditions the synthetase is released at the UAG terminator but that this UAG is especially prone to read‐through with resulting release at the downstream UAA codon. The possible significance of the UAG being in a context prone to leakiness is discussed but is unresolved.
Modulatory effects on proteinase kinetics caused by association of both enzyme and substrate to heparin
Authors: PETERSEN Lars Chr.
Published: March 2005

Heparin is shown to produce modulatory effects on the amidolytic activity of trypsin, thrombin and plasmin with various synthetic peptide substrates. Simple Michaelis‐Menten kinetics are observed in the absence of heparin. In its presence an enhancement effect is observed at low substrate concentrations, and an inhibitory effect is observed at high substrate concentrations. Other polyanions like dextran sulphate, phosvitin and inositol hexakisphosphate produces a similar effect. The modulatory effect of heparin is abolished when it binds cations. Co‐binding of both substrate and enzyme to heparin seems to be a necessary requirement for the effect to occur. A model is proposed which can account semiquantitatively for the kinetics observed. It is suggested that the mechanism, which involves co‐binding of substrate and enzyme in an competitive manner to a macromolecular structure, may be of primary importance as a regulatory mechanism in blood coagulation and fibrinolysis.
Classification of phospholipases A2 according to sequence. Evolutionary and pharmacological implications
Authors: DUFTON Mark J. , HIDER Robert C.
Published: March 2005

The sequences of 32 phospholipases A2 (EC 3.1.1.4) were systematically compared on the basis of polypeptide chain length and similarity at selected amino acid positions around the active site. Two difference matrices were constructed and the various groupings present in the data were expressed in dendrogram form. The two methods of comparison yielded different results, and this is seen as a consequence of separate aspects of phospholipase evolution being highlighted in each case.

It appears that, although Elapid snake venom phospholipases are very similar in terms of overall conformation, the area around their active sites distinguishes them into two major groups, namely the Asian Elapids and the marine/Australasian Elapids. Further, the Asian Elapids seem to have active‐site vicinities which are closer to those in the mammalian pancreatic phospholipases. The relevance of the classifications to structure/activity relationships (especially β‐neurotoxicity) and phospholipase evolution is discussed.
lacZ − ‐ Y+ fusions in Escherichia coli
Authors: BOCKLAGE Hubertus , MÜLLER‐HILL Benno
Published: March 2005

The nucleotide sequence of three lacZ−‐Y+ fusions found among spontaneous lacY+ revertants of the lacY− mutant MAB16 in Escherichia coli is reported. MAB16 is a frameshift mutation in codon 6 of the lacY gene. DNA sequence analysis of the fusions shows the first eight N‐terminal codons of the lacY gene can be replaced by 3, 39 or 804/805 N‐terminal codons of the lacZ gene without impairing lac permease activity qualitatively.
Characterization of β‐galactosidase–lactose‐permease chimaeras of Escherichia coli
Authors: GRIESSER Hans‐Werner , MÜLLER‐HILL Benno , OVERATH Peter
Published: March 2005

Escherichia coli strains have been isolated in which 3, 39 or 805 5′‐end codons of lacZ, the gene for the cytoplasmic enzyme β‐galactosidase are fused to codon 9 of lac Y, the gene for lactose permease. Lactose‐permease‐deficient cells, carrying the lacZ‐ Y fusions on F lac pro episomes, are phenotypically positive on eosin/methylene blue/lactose or on melibiose plates, demonstrating that the β‐galactosidase ‐lactose‐permease chimaeras transport lactose and melibiose in vivo.

The apparent affinity for β‐d‐galactopypanosyl 1‐thio‐β‐d‐galactopyranoside (GalSGal) in cells is similar to that of the wild‐type gene product. The maximum velocity of active GalSGal transport is reduced in all three fusion strains. Both lactose and p‐nitrophenyl α‐d‐galactopyranoside inhibit GalSGal uptake.

As demonstrated by immunoblot experiments the chimaeras cross‐react with polyclonal antibodies directed against native lactose permease and they are present in the cell envelope fraction of homogenates. Their apparent molecular weights upon electrophoresis in NaDodSO4/polyacrylamide gels correspond to those expected from their respective primary sequences, taking into account the migration properties of wild‐type lactose permease.−

It is proposed that substitution of eight N‐terminal lactose permease residues by N‐terminal β‐galactosidase residues neither prevents membrane incorporation of permease nor completely impairs the ability to transport galactosides actively. Alternative interpretations of the experimental results are discussed.
Identification of the gene for DNA helicase II of Escherichia coli
Authors: TAUCHER‐SCHOLZ Gisela , HOFFMANN‐BERLING Hartmut
Published: March 2005

Using a modification of the solid‐phase radioimmune assay of Broome and Gilbert [Proc. Natl Acad. Sci. USA, 75, 2746 (1978)] to screen the plaques of λ recombinant phages for the presence of an elevated level of helicase‐II‐specific antigen, we have identified the gene for helicase II in a library of Escherichia coli DNA. The DNA selected was subcloned from λ into plasmid vectors; restriction analysis located the DNA region encoding helicase II in a PvuII fragment identical in size (2900 base pairs) and restriction pattern to that which contains the uvrD gene. Plasmids carrying this DNA fragment complemented the increased sensitivity to ultraviolet irradiation and the mutator phenotype of uvrD mutants. Furthermore, uvrD502 mutant cells were found to liberate no helicase II activity upon extraction. Following transformation with the cloned DNA, active helicase II was recovered from the mutant cells. These results support the view that helicase II is encoded by uvrD.
Cytochrome c1 from Paracoccus denitrificans
Authors: LUDWIG Bernd , SUDA Kitaru , CERLETTI Nico
Published: March 2005

Cytochrome c1 was purified from the bacterium Paracoccus denitrificans. It is an acidic, hydrophobic polypeptide with an apparent molecular weight of around 65000 and a single, covalently attached heme; it crossreacts immunologically with cytochrome c1 from yeast mitochondria. The amino acid sequence of the tryptic heme peptide of the bacterial cytochrome c1 shows extensive homology to the corresponding region of beef heart cytochrome c1 [Wakabayashi, S. et al. (1982) J. Biol. Chem. 257, 9335–9344]. Positive evidence for a stable association of the Paracoccus cytochrome c1 with other polypeptides and b‐type heme components (‘bc1 ‐complex’) has not yet been obtained.
Chemical modification of the lactose carrier of Escherichia coli by plumbagin, phenylarsinoxide or diethylpyrocarbonate affects the binding of galactoside
Authors: NEUHAUS Jean‐Marc , WRIGHT J. Keith
Published: March 2005

The effects of chemical modification of the lactose carrier of Escherichia coli on galactoside binding (in overproducing strains) and on transport was examined. Both the modifying reagents diethylpyrocarbonate and rose bengal and the thiol reagents phenylarsinoxide and plumbagin can completely inhibit the binding of the substrate p‐nitrophenyl α‐d‐galactopyranoside to the carrier. If care is taken to inhibit galactoside binding only partially, the loss of transport is found to parallel the loss of binding sites. The modified carrier molecules are completely inactive, while the remaining active carrier molecules evince normal transport and binding parameters. The binding of galactoside protects the carrier partially against these forms of chemical modification.

In view of these observations, the results of previous chemical modification studies [Padan, E., Patel, L. and Kaback, H. R. (1979) Proc. Natl Acad. Sci. USA, 76, 6221–6225; Konings, W. N. and Robillard, G. T. (1982) Proc. Natl Acad. Sci. USA, 79, 5480–5484] must be re‐interpreted. Our results stress the utility of studying substrate binding, the first step in the transport cycle.
Effect of interferon treatment on blockade of protein synthesis induced by poliovirus infection
Authors: MUÑOZ Alberto , CARRASCO Luis
Published: March 2005

Treatment of HeLa cells with lymphoblastoid interferon leads to a drastic inhibition of infective poliovirus. Even relatively high concentrations of human lymphoblastoid interferon HuIFN‐α (Ly) (400 IU/ml) do not prevent destruction of the cell monolayer after most of the cells have been infected with poliovirus. Analysis of macromolecular synthesis in a single step growth cycle of poliovirus in interferon‐treated cells detected no viral protein synthesis. In spite of this inhibition of viral translation, the shut‐off of host protein synthesis in interferon‐treated cells is apparent when they are infected both at low and high multiplicities. Although viral RNA synthesis is inhibited considerably in cells treated with interferon, a certain amount is detected, suggesting that some viral replication takes place. Analysis of membrane permeability after poliovirus infection shows a leakage to 86Rb+ ions and modification of membrane permeability to the translation inhibitor hygromycin B at the moment when the bulk of virus protein synthesis occurs. These changes are delayed and even prevented if cells are pretreated with interferon. A situation is described in which host protein synthesis is shut‐down with no major changes in membrane permeability, as studied by the two tests mentioned above. Prevention of viral gene expression by inactivation with ultraviolet light of the input virus or by treatment with cycloheximide blocks the shut‐off of protein synthesis. This does not occur in the presence of 3 mM guanidine. These observations are in agreement with the idea that some poliovirus protein synthesis takes place in interferon‐treated cells and this early gene expression is necessary to block cellular protein synthesis.
Interferon‐inducing, pyrogenic and proclotting enzyme of horseshoe crab activation activities of chemically synthesized lipid A analogues
Authors: MATSUURA Motohiro , KOJIMA Yasuhiko , HOMMA J. Yuzuru , KUBOTA Yoshiyuki , SHIBUKAWA Nobuyuki , SHIBATA Masatoshi , INAGE Masaru , KUSUMOTO Shoichi , SHIBA Tetsuo
Published: March 2005

Interferon‐inducing, pyrogenic and proclotting enzyme of horseshoe crab activation activities of chemically synthesized lipid A analogues were investigated and compared with the same activities of a natural lipid A. These analogues are nonphosphorylated, C‐1 or C‐4′ monophosphorylated and C‐1,4′ bisphosphorylated derivatives of β‐1,6‐linked d‐glucosamine disaccharide possessing both ester‐bound and amide‐bound fatty acid substituents. Fatty acid substituents of the analogues are tetradecanoyl (C14), (R)‐3‐hydroxytetradecanoyl (C14‐OH) or (R)‐3‐tetradecanoyloxytetradecanoyl [C14‐O‐(C14)] groups.

The biological activities of the samples were assayed after solubilization with triethylamine and complexing with bovine serum albumin. Interferon‐inducing activity was exhibited by both the C‐1 monophosphorylated compounds examined. Ester‐bound and amide‐bound fatty acid substituents of these compounds are both C14 or C14 and C14‐OH, respectively. Nonphosphorylated, C‐4′ monophosphorylated and C‐1,4′ bisphosphorylated compounds possessing the same fatty acid substituents as those of the C‐1 monophosphorylated compounds showed no detectable interferon‐inducing activity. C‐4′ monophosphorylated compounds possessing C14‐OH as ester‐bound and C14‐OH or C14‐O‐(C14) as amide‐bound fatty acid substituents exhibited interferon‐inducing activity, but nonphosphorylated compounds possessing the same fatty acid substituents did not. None of the analogues exhibited significant pyrogenicity nor proclotting enzyme of horseshoe crab activation activity under the conditions employed in this study.