bluuebunny/crossref_metadata_2025 huggingface dataset:
Two subcellular locations for peripheral‐type benzodiazepine acceptors in rat liver
Authors: O'BEIRNE Gerard B. , WOODS Margaret J. , WILLIAMS D. Clive
Published: March 2005

Subcellular fractionation of rat liver by differential centrifugation showed the mitochondrial fractions to have the greatest enrichment of ‘peripheral‐type’ benzodiazepine acceptor. Two peaks of acceptor sites were found on isopycnic density‐gradient centrifugation, one peak (ϱ= 1.19 g/ml) corresponding to the peak of mitochondria as judged by marker enzyme distribution and by transmission electron microscopy, and the other peak (ϱ= 1.17 g/ml) which is not mitochondrial as judged by the lack of mitochondrial enzyme markers. Whereas the density of the mitochondrial acceptor was sensitive to sonication and was shown to have an outer‐membrane location, the density of the non‐mitochondrial acceptor was insensitive to sonication.

The non‐mitochondrial acceptor was shown not to be associated with Golgi, lysosomes, rough endoplasmic reticular microsomes, peroxisomes, or some type of plasma membranes, as jugged by differences in the distribution of marker activities. No enrichment of benzodiazepine acceptor was found in the purified nuclear fraction.

Both acceptors were shown to be peripheral‐type high‐affinity acceptors as judged by ligand specificities and by photoaffinity labelling.
Identification and characterization of phospholipase A2 inhibitory proteins in human mononuclear cells
Authors: COMéRA Christine , ROTHHUT Bernard , RUSSO‐MARIE Françoise
Published: March 2005

Calcium‐dependent phospholipid binding and phospholipase A2 inhibitory proteins were isolated from human mononuclear cells. Lipocortins I and H were present whereas lipocortin IV (endonexin I) was not. The other proteins were purified to homogeneity and shown to have molecular masses of 35, 36, 32 and 73 kDa. The 36‐kDa and 73‐kDa proteins are related, the smaller appears to be part of the larger. The 73‐kDa protein is related to the 67‐kDa calelectrin and to lipocortin VI; the 32‐kDa protein is different from endonexin I but related to chromobindin 7 and to lipocortin V. The 35‐kDa protein has been identified by tryptic peptide sequencing as lipocortin III. All these proteins inhibit phospholipase A2 activity in vitro the three smaller ones inhibit the [3H]arachidonic acid release from prelabelled monocytes induced of by the calcium iononphore A23187 in a dose‐dependent manner.
A quantum chemical study of the enzymatic deamination of benzoadenine derivatives
Authors: OROZCO Modesto , CANELA Enric I. , FRANCO Rafael
Published: March 2005

A theoretical study is presented, where, by using both ab initio and semi‐empirical methodologies, the properties of benzoadenine derivatives as substrates of adenosine deaminase are discussed. The results suggest that lin‐benzoadenine and lin‐benzoadenosine can be recognized with an affinity similar to that of adenosine, but only if they are introduced about 0.12 nm deeper inside the active site of the enzyme than the natural substrate adenosine. This fact implies the existence of non‐linear hydrogen bonds inside the active site of adenosine deaminase. Ab initio molecular electrostatic potential values suggest that these hydrogen bonds can exist, and have stability similar to that of linear hydrogen bonds. Finally, the great rate of deamination of lin‐benzoadennie, comparable with that of adenosine despite the absence of the ribose, is explained in the context of the hypothesis that the protonation at the N1 atom is the rate‐determining step of the whole deamination reaction.
Purification and properties of two oxidoreductases catalyzing the enantioselective reduction of diacetyl and other diketones from baker's yeast
Authors: HEIDLAS Jürgen , TRESSL Roland
Published: March 2005

The NADPH‐linked diacetyl reductase system from the cytosolic fraction of Saccharomyces cerevisiae has been resolved into two oxidoreductases catalyzing irreversibly the enationselective reduction of diacetyl (2,3‐butanedione) to (S)‐ and (R)‐acetoin (3‐hydroxy‐2‐butanone) [so‐called (S)‐ and (R)‐diacetyl reductases] (EC 1.1.1.5) which have been isolated to apparent electrophoretical purity.

The clean‐up procedures comprising streptomycin sulfate treatment, Sephadex G‐25 filtration, DEAE‐Sepharose CL‐6B columm chromatography, affinity chromatography on Matrex Gel Red A and Superose 6 prep grade filtration led to 120‐fold and 368‐fold purifications, respectively.

The relative molecular mass of the (R)‐diacetyl reductase, estimated by means of HPLC filtration on Zorbax GF 250 and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was 36000, The (R)‐enzyme was most active at pH 6.4 and accepted in addition to diacetyl C5‐, C6‐2,3‐diketones, 1,2‐cyclohexanedione, 2‐oxo aldehydes and short‐chain 2‐ and 3‐oxo esters as substrates. The enzyme was characterized by high enantioselectivity and regiospecificity. The Km values for diacetyl and 2,3‐pentanedione were determined as 2.0 mM.

The Mr of the (S)‐diacetyl reductase was determined as 75000 by means of HPLC filtration on Zorbax GF 250. The enzyme decomposed into subunits of Mr 48000 and 24000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The optimum pH was 6.9. the purified (S)‐enzyme reduced stereospecifically a broad spectrum of substrates, comprising 2,3‐, 2,4‐ and 2.5‐diketones, 2‐oxo aldehydes, 1,2‐cyclohexanedione and methyl ketones as well as 3‐, 4‐ and 5‐oxo esters. The 2.3‐ and 2,4‐diketones are transformed to the corresponding (S)‐2‐hydroxy ketones; 2,5‐hexanedione, however, was reduced to (S,S)‐2,5‐hexanediol. The Km values for diacetyl and 2,3‐pentanedione were estimated as 2.3 and 1.5 mM, respectively. Further characterization of the (S)‐diacetyl reductase revealed that it is identical with the so‐called ‘(S)‐enzyme’, involved in the enantioselective reduction of 3‐, 4‐ and 5‐oxo esters in baker's yeast.
Differential scanning calorimetry of lobster haemocyanin
Authors: GUZMÁ‐CASADO Mercedes , PARODY‐MORREALE Antonio , MATEO Pedro L. , SÁNCHEZ‐RUIZ José M.
Published: March 2005

Differential scanning calorimetry has been performed with Palinurus vulgaris haemocyanin monomers and hexamers. The denaturation of the protein is irreversible. Both the temperature of the transition maximum and the enthalpy are lower for the monomer than for the hexamer. A scan rate dependence of the temperature of the maxims if found for both the monomer and the hexamer; for the hexamer at least, this can be explained in terms of a two‐state kinetic model. Some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible scanning calorimetric traces.
Inhibition of UDP‐glucuronosyltransferase activity by possible transition‐state analogues in rat‐liver microsomes
Authors: NOORT Daan , COUGHTRIE Michael W. H. , BURCHELL Brian , van der MAREL Gijs A. , van BOOM Jacques H. , van der GEN Arne , MULDER Gerard J.
Published: March 2005

A series of possible transition state analogues of the glucuronidation reaction catalyzed by UDP‐glucuronosyltransferase were tested for their inhibitory effect on glucuronidation of various substrates in a rat liver microsomal fraction. In general 4‐nitrophenol glucuronidation was more effectively inhibited than that of 1‐naphthol, bilirubin or testosterone. 2‐(1‐Naphthyl)ethyl‐UDP and 2,2,2‐(triphenyl)ethyl‐UDP were the most effective inhibitors. Their inhibitory effect was competitive towards both UDP‐glucuronic acid and 4‐nitrophenol. These compounds were much more effective inhibitors than UDP; therefore addition of a lipophilic group enhances the inhibitory potency of UDP. The various UDP derivatives showed differences in their abilities to inhibit the glucuronidation of the four acceptor substrates, supporting the concept that the different forms of UDP‐glucuronosyl transferase have different active sites.
Localization of phosphoserine residues in the a subunit of rabbit skeletal muscle phosphorylase kinase
Authors: MEYER Helmut E. , MEYER Gerhard F. , DIRKS Herbert , HEILMEYER Ludwig M. G.
Published: March 2005

The α subunit of skeletal muscle phosphorylase kinase, as isolated, carries phosphate at the serine residues 1018, 1020 and 1023. Employing the S‐ethyl‐cysteine method, these residues are found to be phosphorylated partially, i.e. differently phosphorylated species exist in muscle. Serine 1018 is a site which can be phosphorylated by the cyclic‐AMP‐dependent protein kinase. The serine residues 972, 985 and 1007 are phosphorylated by phosphorylase kinase itself when its activity is stimulated by micromolar concentrations of Ca2+. These phosphorylation sites are not identical to those found to be phosphorylated already in the enzyme as prepared from freshly excised muscle. A ‘multiphosphorylation loop’ uniquely present in this but not in the homologous β subunit contains all the phosphoserine residues so far identified in the α subunit.
Purification and properties of uroporphyrinogen decarboxylase from Saccharomyces cerevisiae
Authors: FELIX Françoise , BROUILLET Nicole
Published: March 2005

Uroporphyrinogen decarboxylase (EC 4.1.1.37) was purified about 14000‐fold to homogeneity from the yeast Saccharomyces cerevisiae with a 70% overall yield. The purification included affinity chromatography on uroporphyrin‐I‐Affi‐Gel 102. The specific activity of the final preparation was 1750 nmol coproporphyrinogen formed · h−1 (mg protein)−1 at pH 7.5 and 37°C using 4 μM uroporphyrinogen I as substrate. The purified enzyme has a minimum molecular mass of 38 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 46 kDa by gel filtration, suggesting that yeast uroporphyrinogen decarboxylase is a monomer. Chromatofocusing gave a pI of 6.0. Enzyme activity was inhibited by metals, such as Cu2 +, Zn2 +, Fe2 +, Fe3 + and by sulfhydryl‐specific reagents, but no cofactor requirement could be demonstrated. The optimum pH was pH 5.7 for uroporphyrinogens I and III and heptacarboxylate porphyrinogen I as estimated by coproporphyrinogen formation. The optimum pH for substrate decarboxylation was pH 5.7 for uroporphyrinogen I, but pH 6.8 for the two other substrates. The Km values at pH 5.7 were 10 nM for uroporphyrinogen I.6 nM for uroporphyrinogen III and 7 nM for heptacarboxylate porphyrinogen I as measured by coproporphyrinogen formation. The pattern of accumulation of intermediate and final decarboxylation products and the rates of the successive decarboxylations were determined for the three substrates at different concentrations at pH 5.7 and pH 6.8. The rate‐limiting step at 4 μM substrate concentration was the elimination of the second carboxyl group of uroporphyrinogen III and the fourth carboxyl of uroporphyrinogen I. An antiserum to purified yeast uroporphyrinogen decarboxylase was used to characterize the protein in several mutants.
Phosphorylation of high‐Mr caldesmon by protein kinase C modulates the regulatory function of this protein on the interaction between actin and myosin
Authors: TANAKA Toshihoko , OHTA Hisataka , KANDA Keiko , TANAKA Toshio , HIDAKA Hiroyoshi , SOBUE Kenji
Published: March 2005

High‐Mr caldesmon, which is involved in smooth muscle contraction, was phosphorylated by protein kinase C. By chymotryptic digestion, actin‐ and calmodulin‐binding assays and immunoprecipitation with the antibody to the C‐terminal 35‐kDa fragment, we have identified that all phosphate groups are incorporated exclusively into this fragment, which is the functional domain for binding actin and calmodulin. Phosphorylation of high‐Mr caldesmon and its C‐terminal 35‐kDa fragment reduced their binding abilities to both F‐actin and calmodulin. Further, their inhibitory effects on the actin‐activated ATPase activity of gizzard myosin were also reversed in proportion to the degree of phosphorylation. These results suggest that phosphorylation of high‐Mr caldesmon by protein kinase C, which is restricted within the C‐terminal 35‐kDa domain, results in the modulation of its activity in the smooth muscle actin–myosin interaction.
Side chain mobility in bovine calbindin D9k
Authors: RIGLER Rudolf , ROSLUND Jonny , FORSEN Sture
Published: March 2005

The local motion of Tyr13 in wild type and mutant calbindin (Mr 8500, 75 amino acids) was investigated by time‐resolved fluorescence spectroscopy performed at the MAX synchrotron in Lund, Sweden. Two‐dimensional fluorescence spectroscopy (excitation‐emission mapping) was used to characterize the emission of Tyr13 against the background of phenylalanine residues in the presence and absence of Ca2+. Local restricted motion of Tyr13 is observed in wild‐type calbindin with only minor differences between the Ca2+‐saturated and Ca2+‐free forms. In a mutant, where Pro20 is exchanged for Gly and Ala14 and Asn21 are deleted, the local mobility of Tyr13 is enhanced close to values characteristic for free rotational diffusion. An increase of the overall rotational motion in this mutant form by a factor of two and the enhanced local mobility of Tyr13 indicate local and global conformational changes that also affect the Ca2+‐binding properties. Tyr13 occurs in two isomeric species differing in lifetime of the excited state; the major species is populated to 85–90%.
Cholera toxin differentially decreases membrane levels of α and β subunits of G proteins in NG108‐15 cells
Authors: KLINZ Franz‐Josef , COSTA Tommaso
Published: March 2005

Treatment of NG108‐15 neuroblastoma × glioma cells (24 h) with cholera toxin (0.1 – 10 μg/ml) resulted in a concentration‐dependent reduction of the membrane levels of subunits of GTP‐binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Goα and Gβ1 were reduced by 40 – 50%, whereas those of Gαcommon immunoreactivity and Gi2α were only reduced by 10 – 20% following treatment with 10 μg/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01 – 10 ng/ml) and then decreased at high (0.1 – 10 μg/ml) concentrations. Thus, the down regulation of G‐proteins subunits produced by cholera toxin requires its (ADP‐ribosyl)transferase activity but does not result from a cAMP‐mediated mechanism. The toxin‐mediated decrease of Goα in the membrane was correlated with a diminution of opioid‐receptor‐mediated stimulation of high‐affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108‐15 cells.

Northern‐blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for Gβ1 did not change. Thus, the cholera‐toxin‐induced decrease of G‐protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP‐ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Goα and Gβ1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.
Occurrence of tyrosine sulfate in proteins – a balance sheet
Authors: HILLE Annette , HUTTNER Wieland B.
Published: March 2005

The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins.

Upon metabolic labelling of HepG2, Ltk−, AtT20 and PC12 cells with [35S]sulfate or [3H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein‐bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein‐bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of trosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane‐spanning proteins contain potential tyrosine sulfation sites.

In HepG2 cells, three tyrosine‐sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150‐kDa tyrosine‐sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle.

Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes oftrans‐Golgi‐derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine‐sulfated protein or protein class.
The structure of Proteus mirabilis O3 O‐specific polysaccharide containing N‐(2‐hydroxyethyl)‐D‐alanine
Authors: VINOGRADOV Evgeny V. , KACA Wieslaw , SHASHKOV Alexander S. , KRAJEWSKA‐PIETRASIK Danuta , ROZALSKI Antoni , KNIREL Yuriy A. , KOCHETKOV Nikolay K.
Published: March 2005

O‐Specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O3 lipopolysaccharide. The polysaccharide was dephosphorylated with 48% HF to give a linear polysaccharide and an amino acid, N‐(2‐hydroxyethyl)‐D‐alanine. The structure of the polysaccharide was determined by methylation, Smith degradation and computer‐assisted analysis of the 13C‐NMR spectra of original and dephosphorylated polymers and oligomers. The structure of the amino acid was investigated by using 1H and 13C‐NMR spectroscopy and mass spectrometry (applied to the acetylated methyl ester derivative). Its absolute configuration was established by comparison of the optical rotation value and CD spectrum of the natural and synthetic product.

On the basis of the data obtained, it was concluded that the repeating unit of P. mirabilis O3 O‐specific polysaccharide has the following structure:

image

Removal of the amino acid phosphate substituent significantly decreased serological activity of the O‐specific polysaccharide, thus showing the immunodominant role of this group. Serological cross‐reactions between P. mirabilis O3 and O27 were demonstrated and tentatively substantiated.
A hysteretic cycle in glucose 6‐phosphate metabolism observed in a cell‐free yeast extract
Authors: ESCHRICH Klaus , SCHELLENBERGER Wolfgang , HOFMANN Eberhard
Published: March 2005

The dynamics of a partial glycolytic reaction sequence which converts glucose 6‐phosphate to triose phosphates is described. The study was performed with cell‐free extracts from baker's yeast harvested in the logarithmic and stationary growth phases. The experiments are based on a flow‐through reactor supplied with the desalted cell‐free extract as well as glucose 6‐phosphate, ATP and phosphoenolpyruvate.

In the reaction system the quasi‐irreversible reactions catalyzed by 6‐phosphofructo‐1‐kinase, pyruvate kinase, and fructose‐1,6‐bisphosphatase are involved. When substrate is supplied continuously, only stable stationary states can be observed. With transient perturbations of the substrate supply, multiple stationary states appear. Cyclic transitions between unique stable stationary states were induced by appropriate changes of the rate of substrate supply. A hysteretic cycle could then be demonstrated when, during reverse transitions, a parameter region of multistability was passed. The presence (in resting yeast) or absence (in growing yeast) of fructose‐1,6‐bisphosphatase did not significantly influence the dynamic capabilities of the investigated reation sequence. The kinetic properties of the cell‐free extracts fit mathematical models developed for in vitro systems reconstituted from purified enzymes.
The Semliki‐Forest‐virus‐specific nonstructural protein nsP4 is an autoproteinase
Authors: TAKKINEN Kristiina , PERÄNEN Johan , KERÄNEN Sirkka , SÖDERLUND Hans , KÄÄRIÄINEN Leevi
Published: March 2005

The Semliki‐Forest‐virus‐specific nonstructural proteins are translated as a large polyprotein (2431 amino acid residues), from which the mature polymerase components nsP1, nsP2, nsP3 and nsP4 are released by proteolytic cleavages. The complete ns polyprotein (P1234) can be cleaved in two alternative ways yielding either P123 (with sequences of nsP1, nsP2 and nsP3) and nsP4 or P12 (nsP1 plus nsP2) and P34 (nsP3 plus nsP4). We studied the possible autoproteolytic role of nsP4 involved in the cleavage between nsP3 and nsP4 in an in vitro transcription‐translation system. cDNAs encoding P34 precursor and shorter precursor protein segments covering the nsP3‐nsP4 cleavage region, were cloned under the T7 RNA polymerase promoter. The mRNAs synthesized in vitro were capped and translated in rabbit reticulocyte lysates. The translational products were analyzed by SDS/PAGE. The precursor proteins containing nsP4 sequences were cleaved yielding the products with expected sizes, indicating that the cleavage took place at the nsP3‐nsP4 junction. By deleting and truncating the cDNA coding for nsP4, the proteolytic activity was mapped within the 102 amino‐terminal amino acids of nsP4. The cleavage between nsP3 and nsP4 can be inhibited by pepstatin A and probably takes place in cis, since exogenously added nsP4 was unable to mediate it.
Spinach cytosolic fructose‐1,6‐bisphosphatase
Authors: LADROR Uri S. , LATSHAW Steven P. , MARCUS Frank
Published: March 2005

Cytosolic fructose‐1,6‐bisphosphatase from spinach leaves was purified to homogeneity and characterized. The pure enzyme has a subunit mass of 38 kDa, its Km values for fructose 1,6‐bisphosphate and Mg2+ are 1.5 μM and 260 mM, respectively, and its Vmax is 110–120 units/mg. It is inhibited by fructose 2,6‐bisphosphate and AMP with Ki values of 0.07 μM and 120 μM, respectively. About 90% of the primary structure of the spinach cytosolic fructose‐1,6‐bisphosphatase has been determined by amino‐acid sequencing. The sequence data demonstrate that the cytosolic enzyme lacks the sequence insert characteristic of chloroplast fructose‐1,6‐bisphosphatase. The data include also the sequences of peptides containing all seven cysteine residues. Only two of the seven cysteines are conserved between the two isozymes, none of which is believed to be involved with the light regulation of the chloroplast enzyme. Sequence comparisons between the spinach cytosolic enzyme and gluconeogenic fructose‐1,6‐bisphosphatases from other species reveal similarity ranging over 47–54%, which is higher than the 40–45% similarity between the chloroplast enzyme and gluconeogenic fructose‐1,6‐bisphosphatases. However, similarity between these isozymes and Escherichia coli fructose‐1,6‐bisphosphatase are 44% and 47% for the cytosolic and chloroplast enzymes, respectively. Similarity between the cytosolic and chloroplast counterparts is 52%, indicating wide divergence between these two fructose‐1,6‐bisphosphatases.
Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus
Authors: VENTUROLI Giovanni , GABELLINI Nadia , OESTERHELT Dieter , MELANDRI B. Andrea
Published: March 2005

The cyclic photosynthetic chain of Rhodobacter capsulatus has been reconstituted incorporating into phospholipid liposomes containing ubiquinone‐10 two multiprotein complexes: the reaction center and the ubiquinol‐cyctochrome‐c2 reductase (or bc1 complex).

In the presence of cytochrome c2 added externally, at concentrations in the range 10–104 nM, a flash‐induced cyclic electron transfer can be observed. In the presence of antimycin, an inhibitor of the quinone‐reducing site of the bc1 complex, the reduction of cytochrome b561 is a consequence of the donation of electrons to the photo‐oxidized reaction center. At low ionic strength (10 mM KCI) and at concentrations of cyctochrome c2 lower than 1 μM, the rate of this reaction is limited by the concentration of cytochrome c2. At higher concentrations the reduction rate of cytochrome b561 is controlled by the concentration of quinol in the membrane, and, therefore, is increased when the ubiquinone pool is progressively reduced. At saturating concentrations of cytochrome c2 and optimal redox poise, the half‐time for cytochrome b561 reduction is about 3 ms.

At high ionic stength (200 mM KCI), tenfold higher concentrations of cytochrome c2 are required for promoting equivalent rates of cytochrome‐b561 reduction. If the absolute values of these rates are compared with those of the cytochrome‐c2‐reaction‐center electron transfer, it can be concluded that the reaction of oxidized cytochrome c2 with the bc1 complex is rate‐limiting and involves electrostatic interactions.

A significant rate of intercomplex electron transfer can be observed also in the absence of cytochrome c2; in this case the electron donor to the recation center is the cytochrome c1 of the oxidoreductase complex. The oxidation of cytochrome c1 triggers a normal electron transfer within the bc1 complex. The intercomplex reaction follows second‐order kinetics and is slowed at high ionic strength, suggesting a collisional interaction facilitated by electrostatic attraction. From the second‐order rate constant of this process, a minimal bidimensional diffusion coefficient for the complexes in the membrane equal to 3 × 10−11 cm2s−1 can be evaluated.
Vinculin is a permanent component of the membrane skeleton and is incorporated into the (re)organising cytoskeleton upon platelet activation
Authors: ASIJEE Guus M. , STURK Augueste , BRUIN Taco , WILKINSON J. Mike , TEN CATE Jan W.
Published: March 2005

Vinculin, a 130‐kDa protein discovered in chicken gizzard smooth‐muscle cells and subsequently also described in platelets, is believed to be involved in membrane‐cytoskeleton interactions. In this study we investigated vinculin distribution in human blood platelets. Two skeletal fractions and a remaining cytosolic fraction were prepared with a recently described Triton X‐100 lysis buffer causing minimal post‐lysis breakdown by proteolysis. The presence of vinculin was demonstrated in the membrane skeleton and cytosol of resting and thrombin‐activated human platelets. Upon thrombin stimulation vinculin also appeared in the cytoskeleton. This cytoskeletal incorporation was completed during the early stages of platelet aggregation and secretion, when the uptake of myosin, actin‐binding protein and talin was still not maximal. We conclude therefore, that vinculin may play an important role in the structural (re)organisation of the human platelet cytoskeleton upon platelet activation.
Affinity labelling of a partially purified ecdysteroid receptor with a bromoacetylated 20‐OH‐ecdysone derivative
Authors: STRANGMANN‐DIEKMANN Magdalene , KLÖNE Antje , OŻYHAR Andrzej , KREKLAU Frank , KILTZ Hans‐Hermann , HEDTMANN Udo , WELZEL Peter , PONGS Olaf
Published: March 2005

The novel bromoacetyl ecdysteroid IV, (20R,22R)‐2β,3β,14α,20,22,25ξ‐hexahydroxy‐26‐(3‐bromoacetoxypropyl)‐5β‐cholest‐7‐en‐6‐one, BAEIV, has been synthesized by extending the side chain on C26 of 20‐OH‐ecdysone. BAEIV meets all the requirements for an affinity‐labelling reagent. It reacts with the partially purified ecdysteroid receptors of Drosophila melanogaster rapidly and almost quantitatively. Reactions require only micromolar concentrations of BAEIV. The rate of the affinity‐labelling reaction is determined by the association of BAEIV with the ecdysteroid receptor. The value of the apparent reaction rate constant is very similar to that of the association rate constant for the binding of 20‐OH‐ecdysone to the ecdysteroid receptor. Product analysis of the reaction of [14C]BAEIV with the ecdysteroid receptor revealed two labelled peptides having molecular masses 150 kDa and 90 kDa. The smaller peptide is possibly a proteolytic fragment of the larger peptide. The identification of a 150‐kDa peptide by chemical affinity labelling of the ecdysteroid receptor agrees with previously reported photoaffinity‐labelling results from our laboratory. The results also demonstrate that the ecdysteroid receptor of D. melanogaster has a molecular mass higher than all other vertebrate steroid hormone receptors studied so far.
Structural and immunological properties of the phenolic glycolids from Mycobacterium gastri and Mycobacterium kansasii
Authors: GILLERON Martine , VENISSE Anne , FOURNIE Jean‐Jacques , RIVIERE Michel , DUPONT Marie‐Ange , GAS Nicole , PUZO Germain
Published: March 2005

Mycobacterial species‐specific antigens belong to the three following classes: phenolic glycolipids (Phe Gl), acyltrehalose‐containing lipooligosaccharides and polar glycopeptidolipids. These antigens have been chemically defined and alkali‐labile epitopes were found to characterize the lipooligosaccharide antigen type.

In the present study the major Mycobacterium kansasii phenolic glycolipid epitope namely Phe Gl K‐I was delineated as the distal monoacetylated disaccharidic residue: 2,6‐dideoxy‐4‐O‐methyl‐α‐D‐Arbino‐hexopyranosyl‐(1 → 3)‐2‐O‐methyl‐4‐Oacetyl‐α‐L‐fucopyranose. This acetoxy group is required for K‐I epitope recognition demonstrating that alkali‐labile epitopes also occur in the phenolic glycolipid antigen class. Using immunoelectron microscopy, the Phe Gl K‐I epitope was localized around the electron‐transparent layer on the M. Kansasii cell‐wall surface. Furthermore, two new phenolic glycolipids namely Phe Gl K‐III and Phe Gl K‐IV were discovered in minute amounts. They were purified and characterized by their retention time in direct‐phase column HPLC. These molecules are also M. kansasii antigens, whose epitopes differ from that of Phe Gl K‐I. The complete family of phenolic glycolipids Phe Gl K‐I, K‐II, K‐III and K‐IV was found in both rough and smooth variants of both M. kansasii and Mycobacterium gastri species.
Spectroscopic and kinetic aspects of Elephas maximus hemoglobin
Authors: STEPHANOS Joseph J. , ADDISON Anthony W.
Published: March 2005

In comparison with myoglobin and human and Glycera dibranchiata hemoglobins, the heme distal side amino acid exchanges within the heme environment of elephant tetrameric hemoglobin (Hbe) only slightly affect the electronic and ESR spectra of Hbe(III) and Hbe(II) derivatives, several of which were prepared and characterized by optical and ESR spectroscopy. Addition of 2,3‐bisphosphoglycerate [Gri(2,3)P2] or inositol hexakisphosphate to Hbe(II)NO causes tension in the Fe‐N(proximal His) bond, although the behaviour differs in detail from that of HbA(II)NO. There are two equilibrium states of Hbe having significantly different kinetics for the Hbe(III) Hbe(II) reaction of Hbe(III)NO. This autoreduction occurs in the form of two parallel processes, which collapse into one intermediate rate in the presence of Gri(2,3)P2. The temperature dependences of the rates enable deduction of H0 and S0 for the linked equilibrium, and yield liner Eyring plots for Hbe(III)NO, from which activation parameters were estimated on the basis of a previously described mechanism.
The cDNA and protein sequences of mouse lactate dehydrogenase B
Authors: HIRAOKA B. Yukihiro , SHARIEF Farida S. , YANG Yau‐Wen , LI Wen‐Hsiung , LI Steven S.‐L.
Published: March 2005

Mouse lactate dehydrogenase‐B cDNAs were isolated from cDNA libraries of macrophage (ICR strain) and thymus (F1 hybrid of C57BL/6 and CBA strains), and their nucleotide sequences determined. The lactate dehydrogenase‐B cDNA insert of thymus clone mB188 consists of the protein‐coding sequence (1002 nucleotides), the 5′ (46 nucleotides) and 3′ (190 nucleotides) non‐coding regions, and poly(A) tail (19 nucleotides), while macrophage clone mB168 contains a partial lactate dehydrogenase cDNA insert from codon no. 55 to the poly (A) tail. Seven silent nucleotide substitutions at codon no. 142, 143, 186, 187, 241, 285 and 292, as well as a single nucleotide change in the 3′ non‐coding region, were found between these different strains of mice. The predicted sequence of 333 amino acids. excluding initiation methionine, was confirmed by sequencing and/or compositional analyses of a total of 103 (31%) amino acids from tryptic peptides of mouse lactate dehydrogenase‐B protein. The nucleotide sequence of the mouse coding region for lactate dehydrogenase B shows 86% identity with that of the human isoenzyme, and only eight of the 139 nucleotide differences resulted in amino acid substitutions at residues 10, 13, 14, 17, 52, 132, 236 and 317. The rates of nucleotide substitutions at synonymous and nonsynonymous sites in the mammalian lactate dehydrogenase genes are calculated. The rates of synonymous substitutions for lactate dehydrogenase genes A (muscle) and B (heart) are considerably higher than the average rate computed from human and rodent genes. The rates of nonsynonymous substitutions for lactate dehydrogenase genes A (muscle) and B (heart), particularly the latter, are highly conservative. The rats of synonymous and nonsynonymous substitutions for the lactate dehydrogenase‐C gene are about the same as the average rates for mammalian genes. A phylogenetic tree of vertebrate lactate dehydrogenase protein sequences is constructed. In agreement with the previous results, this analysis further indicates that lactate dehydrogenase‐C gene branched off earlier than did lactate dehydrogenase‐A and lactate dehydrogenase‐B genes.
The effect of von Willebrand factor on activation of factor VIII by factor Xa
Authors: KOEDAM Johannes A. , HAMER Rob J. , BEESER‐VISSER Nel H. , BOUMA Bonno N. , SIXMA Jan J.
Published: March 2005

Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 U/ml) with 0.1 μg/ml factor Xa resulted in a 1.6‐fold activation followed by a decay of coagulant activity. In the presence of 10 μg/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 μg/ml), von‐Willebrand‐factor‐bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light‐chain‐derived fragments of molecular mass 66/68 kDa and 60 kDa and heavy‐chain‐derived fragments of 40 and 42 kDa. Also minor products of 12, 20/21, 23, 27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa.
Targetting of protein phosphatase 1 to the sarcoplasmic reticulum of rabbit skeletal muscle by a protein that is very similar or identical to the G subunit that directs the enzyme to glycogen
Authors: HUBBARD Michael J. , DENT Paul , SMYTHE Carl , COHEN Philip
Published: March 2005

The amount of protein phosphatase 1 (PP1) activity in rabbit skeletal muscle associated with membranes (predominantly sarcoplasmic reticulum) is similar to that bound to glycogen‐protein particles. Membrane‐vesicleassociated (sarcovesicular) PP1 can be solubilised with 0.5% Triton X‐100 (but not 0.5 M NaCl) and is complexed to a protein that is structurally and functionally very similar or identical to the G subunit which targets PP1 to glycogen‐protein particles. This conclusion is based on immunoblotting and immunotitration experiments using two different preparations of G‐subunit‐specific antibodies, binding of Triton‐solubilised sarcovesicular enzyme to glycogen, stimulation of phosphorylase phosphatase activity by glycogen, phosphorylation of the same tryptic peptides by cyclic‐AMP‐dependent protein kinase (A‐kinase) and release of catalytic subunit following phosphorylation by A‐kinase. Membrane‐association is not mediated via glycogen because sarcovesicular PP1 is (1) not released by digestion with α‐amylase or at dilutions which fully dissociate the glycogen‐bound enzyme, and (2) is solubilised by Triton X‐100 (whereas glycogen‐associated PP1 is not). These findings demonstrate that sarcovesicular PP1 is highly homologous to, or the same as, glycogen‐associated PP1G and raises the possibility that a common targetting subunit may direct PP1 to different subcellular locations.
Electron microscopy of DNA · helicase‐I complexes in the act of strand separation
Authors: WESSEL Rainer , MÜLLER Holger , HOFFMANN‐BERLING Hartmut
Published: March 2005

Electron microscopy was used to characterize the DNA‐unwinding reaction catalysed by Escherichia coli DNA helicase I. Linear DNA with 5′‐protruding strands as well as single‐stranded gaps was incubated, under unwinding assay conditions, with the helicase. E. coli single‐stranded‐DNA‐binding protein (SSB) was added to order the denatured DNA. Up to 70% of the sites of SSB‐complexed DNA were observed as forks. The position of the strand‐separating enzyme was indicated by a gap in the complex between fork and SSB on that arm which initially provided the binding site. The complex between DNA and helicase varied in length although in all cases it was long enough to comprise several helicase I molecules. A mutant helicase I (helicase I del29) which, unlike the wild‐type enzyme, fails to show cooperative DNA‐binding behaviour was found to prevent an abnormally short stretch of DNA near the fork from binding SSB. Apparently, one or very few helicase molecules would be sufficient for the opening of a DNA duplex although, typically, the fork is shifted by a tract of helicase I molecules. SSB displaces helicase I from single‐stranded DNA but fails to do so from a fork or a single‐strand/double‐strand junction. The difference is consistent with the observation that SSB does not inhibit the unwinding reaction despite its rapid association with the separated strands. Helicase I unwinds in the 5′–3′ direction of the bound strand. Observations so far indicate that the enzyme exploits the single strand at the initial DNA‐binding site for orienting its action, and not the complementary, completely base‐paired strand.
Type 5 acid phosphatase
Authors: LORD Deirdre K. , CROSS Nicholas C. P. , BEVILACQUA Maria A. , RIDER Susan H. , GORMAN Patricia A. , GROVES Ann V. , MOSS Donald W. , SHEER Denise , COX Timothy M.
Published: March 2005

The purple acid phosphatases and uteroferrin belong to a diverse multifunctional class of binuclear iron‐containing proteins that includes haemerythrin and ribonucleotide reductase. In the pig, uteroferrin has been implicated in the delivery or iron to the foetus, but the role of the related human type 5 acid phosphatase that is principally found in resident tissue macrophages is not yet clear. To define further the function of this metalloenzyme, we have isolated and sequenced a cDNA clone for type 5 acid phosphatase and investigated expression of its gene in human tissues. The phosphatase clone contains an open reading frame of 975 bp and encodes a protein of 325 amino acids, including a signal peptide of 19 residues and two potential sites for N‐glycosylation. The type 5 acid phosphatase gene mapped to the short arm of human chromosome 19 and was found to have a restriction fragment length polymorphism on digestion with XbaI.

Expression of phosphatase mRNA was restricted to mononuclear phagocytes and the enzyme was induced > 20‐fold on transformation of normal human monocytes to macrophages by culture in serum‐supplemented medium. Type 5 acid phosphatase thus represents a tightly regulated system for the study of molecular events in the differentiation programme of the normal macrophage.
Presence and characterization of glycolipid sulfotransferase in human cancer serum
Authors: GASA Shinsei , ČASL Martin‐Tino , MAKITA Akira , SAKAKIBARA Naoyuki , KOYANAGI Tomohiko , ATSUTA Tomoyoshi
Published: March 2005

Sulfotransferase, which catalyzes sulfation of the carbohydrate of galactosylceramide (GalCer) and is localised in the Golgi membrane of cells, was assayed for activity in human serum. To do this, an organic solvent was added to the incubated reaction mixture containing GalCer as an acceptor and phosphoadenosine phospho[35S]‐sulfate as a donor of sulfate to dissociate the synthesized sulfolipid from serum protein. This was followed by isolation of the sulfolipid on an anion‐exchange column. Through this procedure, human serum was found to contain sulfotransferase activity. The serum enzyme was activated by Mn2+. Km values of the enzyme for GalCer and ‘active sulfate’ were 4.6 μM and 5.2 μM, respectively. The enzyme activity was assayed in sera of cancer patients. The serum activity (mean ± SE, 0.27 ± 0.027 pmol ±μl−1· h−1) in renal cell carcinoma patients, whose activity has been demonstrated to be elevated, was significantly (P < 0.005) increased compared to that of the normal control (mean ± SE, 0.18 ± 0.0014 pmol ±μl−1· h−1) and of other urological tumors examined.
Bacteriophage‐associated glycan hydrolases specific for Escherichia coli capsular serotype K12
Authors: ALTMANN Friedrich , CHRISTIAN Rudolf , CZERNY Thomas , NIMMICH Wolfgang , MÄRZ Leopold
Published: March 2005

Four bacteriophages were identified, which carry glycan hydrolases specific for the Escherichia coli K12 capsular polysaccharide. All these glycanases catalyze the hydrolysis of the α‐l‐rhamnosyl‐1,5‐β‐3‐deoxy‐d‐manno‐2‐octulosonic acid linkage as demonstrated with a special thiobarbituric acid assay procedure, which discriminates between the C5 substituted and unsubstituted 3‐deoxy‐d‐manno‐2‐octulosonic acid (dOclA). This assay, together with gel filtration, 1H‐NMR and 13C‐NMR spectroscopy showed that depolymerization led to the dimer of the K12 repeating unit, (,5‐β‐dOcl1Ap‐2,3‐α‐lRhap‐1,2‐αlRhap1,)2, as the primary degradation product.

The phages (φ12‐W, φ12‐S, φ82‐W1, φ82‐W2) were tested for their ability to infect Escherichia coli strains Su65‐42 (O4:K12:H−) and CDC63‐57 [O139:K82(12):H1]. φ12‐W and φ12‐S, respectively, infected strain Su65‐42 only, φ82‐W2 CDC63‐57 only, and φ82‐W1 both bacterial strains. These distinct host specificities cannot be explained by differences in the action of the glycanases, which depolymerisze the capsules of both strains.
4‐Phenyl‐3‐butenoic acid, an in vivo inhibitor of peptidylglycine hydroxylase (peptide amidating enzyme)
Authors: BRADBURY Alan F. , MISTRY Jashu , ROOS Bernard A. , SMYTH Derek G.
Published: March 2005

The ability of a series of non‐peptide carboxylic acids to act as substrates or inhibitors of the peptide‐amidating enzyme (peptidyl‐glycine hydroxylase) was assessed by determining their ability to reduce the rate of enzymic conversion of d‐tyrosyl‐valyl‐glycine or d‐tyrosyl‐phenylalanyl‐glycine to the corresponding dipeptide amide. The inclusion of a phenyl substituent in a position distal to the carboxyl group promoted the inhibitory action. The inhibition was found to be irreversible when an olefinic double bond, α or β to the carboxyl group, was present in the molecule; the inhibition appeared to be associated with a covalent interaction between the amidating enzyme and the inhibitor. With 4‐phenyl‐3‐butenoic acid the inhibitory properties were manifest only in the presence of cofactors of the enzyme. When 4‐phenyl‐3‐[2‐14C]butenoic acid was used, the radioactivity was shown to be incorporated into protein that co‐chromatographed with active enzyme.

Incubation of rat thyroid carcinoma CA 77 cells in the presence of 4‐phenyl‐3‐butenoic acid led to a decrease in the levels of intracellular amidating activity and of thyrotropin‐releasing hormone, an amidated peptide produced by these cells. The inhibitory effects reached a maximum at approximately 15 h after which the enzyme levels returned to the control values even though the concentration of 4‐phenyl‐3‐butenoic acid in the cells remained unchanged. The results indicate that a mechanism exists in these cells for regulation of amidating activity.
Binding of ADP and adenosine 5′‐[β,γ‐imido]triphosphate to insect flight muscle fibrils
Authors: BIOSCA Josep A. , EISENBERG Evan , REEDY Mary C. , REEDY Michael K.
Published: March 2005

We have studied the binding of ADP and adenosine 5′‐[β,γ‐imido]triphosphate (AdoPP[NH]P) to insect flight muscle fibrils. We find that 25% of the myosin heads, presumably those which do not interact with actin, bind AdoPP[NH]P with a binding constant greater than 3 × 106 M−1, similar to the binding constant of the same compound to the rabbit myosin heads which do not overlap with actin. The remaining heads in insect myofibrils bind AdoPP[NH]P with an association constant of 8 × 103 M−1, which is eight times stronger than the affinity of this compound for rabbit myosin heads in overlap with actin. Therefore, in contrast to the situation with rabbit myofibrils where AdoPP[NH]P binds much more weakly than ADP, with insect myofibrils these two adenosine phosphates bind with almost equal affinity. This is consistent with the numerous structural studies on insect flight muscle which were interpreted on the basis that most of the actomyosin sites were saturated with nucleotide at an AdoPP[NH]P concentration of 1 mM.
Activation of multiple signal transduction pathways by endothelin in cultured human vascular smooth muscle cells
Authors: RESINK Thérèse J. , SCOTT‐BURDEN Timothy , BÜHLER Fritz R.
Published: March 2005

Cellular responses to the vasoconstrictor peptide, endothelin, have been investigated in quiescent cultured human vascular smooth muscle cells (hVSMC). Endothelin caused intracellular alkalinization and activation of the protein synthetic enzyme S6‐kinase, but such responses were not associated with any mitogenic effects of endothelin on hVSMC. Inmyo‐[3H]inositol‐prelabelled hVSMC endothelin elicited a rapid increase in inositol bis‐and tris‐phosphates and concomitant hydrolysis of polyphosphoinositol lipids. In [3H]arachidonate‐prelabelled hVSMC endothelin promoted production of diacylglycerol, the early kinetics of which parallelled poly‐phosphoinositol lipid hydrolysis. Such phospholipase C activation by endothelin was sustained in hVSMC with accumulation of inositol polyphosphates being markedly protracted and the decay of diacylglycerol slow. Endothelin promoted extracellular release of [3H]arachidonate‐labelled material from hVSMC which derived via deacylation of both phosphatidylinositol and phosphatidylcholine. This process was inhibited by phospholipase A2 and lipoxygenase inhibitors, but insensitive to phospholipase C and cyclooxygenase inhibitors. Endothelin‐induced activation of phospholipase C and phospholipase A2 signal transduction pathways (EC50∼ 5–8 nM for both) in hVSMC apparently proceed in an independent parallel manner rather than a sequential one.