bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic activities in thylakoid membranes, which form medium [32P]NDP and [32P]ATP from 32Pi
Authors: FELDMAN Richard I. , SIGMAN David S.
Published: March 2005

Soluble chloroplast coupling factor 1 (CF1) and the ATP synthase complex, under uncoupled conditions, can form bound ATP from tightly bound ADP and medium Pi. This partial reaction is a powerful probe of the mechanism of ATP synthesis. During our study of the synthesis of bound ATP by CF1 other enzyme activities, which generate [32P]nucleotides from 32Pi, were characterized and controlled. Two enzymes present at significant levels in the preparations are polynucleotide phosphorylase and adenylate kinase. Polynucleotide phosphorylase (PNPase) was found both in thylakoid and CF1 preparations and catalyzed the formation of [β‐32P]ADP via its Pi⌆ ADP exchange activity. The formation of [β‐32P]ADP during net photophosphorylation is attributable to adenylate kinase action on the [32P]ATP formed since hexokinase and glucose effectively block its production. In addition, PNPase also degraded RNA present in thylakoid preparations yielding all four [32P]nucleoside diphosphates. PNPase was also shown to catalyze a Pi↔ ATP exchange that is dependent on RNA primers and other cofactors.
An inverse control by phytochrome of the expression of two nuclear genes in barley (Hordeum vulgave L.)
Authors: BATSCHAUER Alfred , APEL Klaus
Published: March 2005

During the light‐dependent transformation of etioplasts to chloroplasts a rapid decrease of the NADPH‐protochlorophyllide oxidoreductase is induced. At the same time the mRNA activity coding for this enzyme protein also declines rapidly under the influence of phytochrome (Pfr). On the other hand the apoprotein of the light‐harvesting chlorophyll a/b protein and its mRNA activity are inversely affected by the same photoreceptor. Cloned cDNA sequences which are complementary to these mRNAs have been used to assess the effect of phytochrome (Pfr) on the concentration of the two transcripts. The phytochrome‐induced changes of the two translatable mRNAs are paralleled by corresponding changes in the steady‐state concentration of the mRNA sequences. This inverse relationship between the light‐dependent regulation of the NADPH‐protochlorophyllide oxidoreductase and the light‐harvesting chlorophyll a/b protein suggests that a single reversible triggering event can increase the transcription of certain genes and decrease the transcription of others.
Immunochemical characterization of the adenine nucleotide translocator
Authors: SCHULTHEISS Heinz Peter , KLINGENBERG Martin
Published: March 2005

Antibodies have been prepared against purified preparations of the heart and kidney nucleotide translocator in the ‘c’‐conformation. The results show organ‐specific antigenic determinants on the translocator proteins isolated from heart, kidney and liver although a partial cross‐reactivity between these three proteins was demonstrable. The organ specificity was observed both with the solubilized and with the membrane‐bound translocator protein indicating organ‐specific determinants on exposed regions of the carrier. An organ‐specific inhibition of the nucleotide transport in heart mitochondria by the heart carboxyatractylate‐protein antiserum leads to the conclusion that the organ specificity is at least partially conditioned by the binding site for the substrate and/or the closely linked gate of the carrier protein. Apart from the organ specificity the results also demonstrate a specificity of the antibodies for the translocational conformations of the carrier: the ‘c’‐conformation stabilized in the carboxyatractylate‐protein complex and the ‘m’‐conformation present in the bongkrekate‐protein complex. However, after denaturation of the carboxytratylate‐protein and bongkrekate‐protein complexes the binding of the anti‐(carboxyatractylate‐protein) antiserum to both inhibitor‐protein complexes was nearly identical. The conformation specificity was also expressed by the inhibition of the conformation transition from the ‘c’‐ to the ‘m’‐state. This side‐specific inhibition of the nucleotide transport and the identical binding activity of the carboxyatractylate‐protein antiserum against the denatured carboxyatractylate‐protein and bongkrekate‐protein complexes suggested that the conformation‐specific antigenic determinants are topographic surface regions which are determined by the chain folding.
Single‐step elongation of oligodeoxynucleotides using terminal deoxynucleotidyl transferase
Authors: SCHOTT Herbert , SCHRADE Herbert
Published: March 2005

Procedures for the stepwise addition of one or more deoxyribonucleotide residues to the 3′ end of an oligodeoxyribonucleoside phosphate acceptor using commercially available terminal deoxynucleotidyl transferase is described. 2–80 nmol of acceptors with a chain length of four, five or nine monomer units were elongated with a single 2′‐deoxyribonucleoside 5′‐triphosphate in yields of 20–30%. The monomers carried no protecting groups and were used both radioactively labelled and unlabelled.

The elongated oligodeoxynucleoside phosphates were isolated by reverse‐phase (Nucleosil C18) high‐performance liquid chromatography or paper chromatography. The isolated products were sequenced by the fingerprint method. Advantages and disadvantages of this new methodology for the enzymatic synthesis of defined oligodeoxynucleotides are discussed.
5′‐Hydroxyl RNA kinase from mouse L cells
Authors: SERTIC‐PRITSOS Karen , VINOCOUR Michelle , WINICOV Ilga
Published: March 2005

A 5′‐hydroxyl RNA kinase from mouse L cells has been partially purified and characterized. The enzyme transfers the γ‐phosphorus from ATP to 5′‐hydroxyl termini of RNA much more efficiently than DNA substrates, and is virtually inactive on 3′‐CMP. The molecular mass of the predominant kinase activity is estimated to be 93–96 kDa from denaturing and non‐denaturing polyacrylamide gel analyses. A minor band of lower molecular mass has been also observed. The enzyme activity requires Mg2+ and is inhibited by both Mn2+ and Zn2+. Antibodies to small nuclear ribonucleoproteins have no effect on this activity.
Acetylcholine‐like effects of 1‐O‐alkyl‐2–acetyl‐sn‐glycero‐3‐phosphocholine (‘platelet‐activating factor’) and its analogues in exocrine secretory glands
Authors: SÖLING Hans‐Dieter , EIBL Hansjörg , FEST Werner
Published: March 2005

The effect of 1‐O‐hexadecyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (1), 1‐O‐octadecyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (2), 1‐O–hexadecyl‐sn‐glycero‐3‐phosphocholine (3), 1‐O‐octadecyl‐2‐O‐methyl‐sn‐glycero‐3‐phosphocholine (4) and its enantiomer 3‐O‐octadecyl‐2‐O‐methyl‐sn‐glycero‐1‐phosphocholine (5) on the secretion of amylase from guinea pig isolated parotid gland and exocrine pancreatic lobules was examined. Compounds 1, 2 and 4 led to a significant stimulation of amylase release in both systems, effects being already visible between 10–100pM. Maximal stimulation with compounds 1 and 2 occurred at 5nM, with compound 4 at 1 nM. Higher concentrations were less effective and at 0.1 μM stimulation was very low. In contrast, compound 5 showed a continuous increase in activity up to 0.01–0.1 μM without a decrease at higher concentrations. Compound 3 had no effect.

For compound 1, its effects on calcium and lipid metabolism have been analyzed and compared with those of the acetylcholine analogue carbamoylcholine. Compound 1 mimicked in every respect the effects of carbamoylcholine. It stimulated the uptake of 45Ca by isolated parotid gland lobules in a non‐ionophoretic way. In isolated pancreatic lobules it enhanced the incorporation of [32P]phosphate into phosphatidic acid, phosphatidylinositol and poly(phosphoinositide), increased the formation of diacylglycerols and triacylglycerol, led to the same two‐phasic responses of myo‐[3H]inositol‐labeled polyphosphoinositides, and initiated a rapid short‐lasting formation of free inositol triphosphate.

Accordingly, ‘platelet activating factor(s)’ can affect the function of exocrine glands at low concentrations. The effects observed resemble those produced by acetylcholine and result most likely from the interaction of platelet‐activating factor with plasma membrane receptors.
Carotenoid‐deficient maize seedlings fail to accumulate light‐harvesting chlorophyll a/b binding protein (LHCP) mRNA
Authors: MAYFIELD Stephen P. , TAYLOR William C.
Published: March 2005

Yellow leaves of chlorophyll‐deficient seedlings and white leaves of Carotenoid deficient seedlings contain no detectable light‐harvesting chlorophyll a/b binding proteins (LHCP). Chlorophyll‐deficient leaves contain plastids which are arrested in development prior to chloroplast formation [Mascia, P. N. and Robertson, D. S. (1978) Planta (Berl.) 143, 207–211] while carotenoid‐deficient leaves contain plastids which are arrested in development at a rudimentary stage [Bachmann, M. D., Robertson, D. S., Bowen, C. C., and Anderson, I. C. (1967) J. Ultrastruc. Res. 21, 41–60]. Chlorophyll‐deficient leaves have normal levels of nuclear‐encoded LHCP mRNA while carotenoid‐deficient leaves contain only trace amounts of LHCP mRNA. Similar results were obtained with carotenoid deficiences caused by nuclear gene mutations and by treatment with the herbicide norflurazon which blocks carotenoid biosynthesis. We conclude that events at early stages of plastid development influence the accumulation of a nuclear‐encoded mRNA.
Molecular basis of the ‘box effect’
Authors: LABOUESSE Michel , NETTER Pierre , SCHROEDER Renee
Published: March 2005

In the mitochondrial DNA of Saccharoimyces cerevisiae, the genes cob‐box and oxi3, coding for apocytochrome b and cytochrome oxidase subunit I respectively, are split. Several mutations located in the introns of the cob‐box gene s of cytochrome b and cytochrome oxidase subunit I (this is known as the ‘box effect’).‐We have elucidated the molecular basis of this phenomenon: these mutants are unable to excise the fourth intron of oxi3 from the cytochrome oxidase subunit I pre‐mRNA; the absence of a functional bI4 mRNA maturase, a trans‐acting factor encoded by the fourth intron of the cob‐box gene explains this phenomenon. This maturase was already known to control the excision of the bI4 intron; consequently we have demonstrated that it is necessary for the processing of two introns located in two different genes.

Mutations altering this maturase can be corrected, but only partially, by extragenic suppressors located in the mitochondrial (mim2) or in the nuclear (NAM2) genome. The gene product of these two suppressors should, therefore, control (directly or indirectly) the excision of the two introns as the bI4 mRNA maturase normally does.
The complete amino acid sequence of the major α subunit of the lectin from the seeds of Dioclea grandiflora (Mart)
Authors: RICHARDSON Michael , CAMPOS Francisco D. A. P. , MOREIRA Renato A. , AINOUZ Iracaema L. , BEGBIE Robert , WATT William B. , PUSZTAI Arpad
Published: March 2005

The complete amino acid sequence of the major a subunit of the lectin from seeds of Dioclea grandiflora was determined. The sequence was deduced from analysis of peptides derived from the native α subunit by digestion with trypsin, chymotrypsin, the Staphylococcus aureus V8 protease, and pepsin; and from larger peptides produced by digestion of the citraconylated protein with trypsin. The α subunit consists of a single polypeptide chain of 237 amino acids which differs from the sequence of concanavalin in 53 positions. Significant levels of heterogeneity were observed in five positions in the sequence.
Phosphoenolpyruvate‐dependent phosphorylation site in enzyme IIIglc of the Escherichia coli phosphotransferase system
Authors: DÖRSCHUG Michael , FRANK Rainer , KALBITZER Hans Robert , HENGSTENBERG Wolfgang , DEUTSCHER Josef
Published: March 2005

Enzyme‐IIIglc is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimurium and is phosphorylated by phosphorenolpyruvate in a reaction requiring enzyme I (phosphorenolpyruvate‐protein phosphotransferase), and the histidine‐containing phospho‐carrier protein HPr. In this paper we report the isolation of IIIglc from E. coli and the characterization of the active center. Alkaline hydrolysis of [32P]P‐IIIglc and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P‐IIIglc of S. typhimurium. Here we present 1H‐NMR measurements of IIIglc and P‐IIIglc from E. coli which further substantiate that the phosphoryl group in P‐IIIglc is linked to the N‐3 position of a histidyl residue. After phosporylation of IIIglc with [32P]Phosphoenolpyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val‐His‐Phe‐Gly‐Ile‐Asp. The lower electrophoretic mobility of P‐IIIglc on dodecylsulfate/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed‐phase column compared to unphosphorylated protein may indicate a structural change following phosphoenolpyruvate‐dependent phosphorylation.
Galactosyl transferases of baby hamster kidney (BHK) cells
Authors: GLEESON Paul A. , FEENEY James , MILLS Gary , HUGHES R. Colin
Published: March 2005

Extracts of BHK (baby hamster kidney) cells catalyse incorporation of galactose from UDP‐galactose into asialo bovine submaxillary gland mucin. The galactosylated oligosaccharide products were released by alkaline‐borohydride treatment and purified by Bio‐Gel P2 chromatography and high‐performance liquid chromatography. The structures of the oligosaccharide sequences synthesised have been identified unequivocally by high resolution 500 MHz 1H‐NMR as galactosyl‐(β→3) N‐acetylgalactosamine and galactosyl (β→4) N‐acetylglucosaminyl (β→3)‐N‐acetylgalactosamine. Characterization of the latter sequence shows the presence in bovine mucin of the type III core sequence N‐acetylglucosamine‐(β→3) N‐acetylgalactosamine. Fractionation of BHK cell extracts on α‐lactalbumin‐Agarose has shown that the (β→4)‐galactosyl transferase responsible for synthesis of the trisaccharide binds to α‐lactalbumin, a modulator of the (β→4)‐galactosyl transferase involved in N‐glycan assembly. The evidence that the same transferase activity may be responsible for galactose transfer to both O‐glycans and N‐glycans is discussed.
Release of the inhibitory action of the natural ATPase inhibitor protein on the mitochondrial ATPase
Authors: BELTRÁN Carmen , de GÓMEZ‐PUYOU Marietta T. , GÓMEZ‐PUYOU Armando , DARSZON Alberto
Published: March 2005

The rate of ATP hydrolysis by submitochondrial particles prepared from bovine heart mitochondria in the presence of Mg2+ and ATP increases from a value of 0.4 μmol min−1 mg−1 to 6–7 μmol min−1 mg−1 upon incubation for 5–6 h at 38 °C. The increase in activity does not occur in particles that have been passed through a Sephadex column. The activation is prevented and partially reversed by ATP. This indicates that the increase in hydrolytic activity is due to abolition of the inhibitory action of the natural ATPase inhibitor protein of Pullman and Monroy [(1963) J. Biol. Chem. 238, 3762–3769]. At maximal activation approximately 50% of the inhibitor protein of the starting preparation remains in the particles as inferred from direct assay of inhibitor protein content and by its interaction with 125I‐labeled antibodies directed against the inhibitor protein. The extent of the activation, which presumably is an index of the equilibrium between active and inactive enzymes, is strictly dependent on salts. The rate of the activation depends on the concentration of salts and is favored by alkaline pH. From results of experiments on the effect of temperature on the rate of activation of the ATPase, it was calculated that the activation energy, ΔH‡ and ΔS‡ of the process were 53.34 kJ/mol, 50.83 kJ/mol and ‐158.99 J mol−1 K−1, respectively. The data indicate that in its native inhibiting state, the interaction of the inhibitor protein with the enzyme involves electrostatic interactions. Also it is concluded that abolition of the inhibitory action of the protein on ATPase activity is not compulsorily linked to release of the protein into the water space.
Uncoupled synthesis of H1°‐like histone H1s during late erythropoiesis in Xenopus laevis
Authors: RUTLEDGE Robert G. , NEELIN James M. , SELIGY Verner L.
Published: March 2005

This study investigated the synthesis of Xenopus histones during erythropoiesis. Although cessation of DNA replication in the mid‐stages of erythroid maturation is accompanied by arrested synthesis of histone H1 and core histones, synthesis of H1s (an H1°‐like histone) was found to continue into late stages of erythropoiesis, as has been reported for avian erythrocyte histone H5. This was accompanied by a threefold increase in the relative amount of Xenopus H1s, similar to the accumulation reported for H5 during avian erythropoiesis and for H1° in some differentiated mammalian cells. The structural and metabolic homologies of avian H5, mammalian H1°, and Xenopus H1s imply that these lysine‐rich histones have closely related functions distinct from those of H1, and thus represent a subclass of lysine‐rich histones.
Properties of receptors for the Ca2+‐channel blocker verapamil in transverse‐tubule membranes of skeletal muscle
Authors: GALIZZI Jean‐Pierre , FOSSET Michel , LAZDUNSKI Michel
Published: March 2005

The verapamil receptor associated with the voltage‐dependent calcium channel of rabbit skeletal muscle transverse tubule membranes has the following properties, (i) This receptor is stereospecific and discriminates between the different stereoisomers of verapamil, gallopamil and diltiazem. (ii) Inorganic divalent cations inhibit the binding of [3H]verapamil to its receptor in an apparently non‐competitive fashion. The rank order of potency is: Ca2+= Mn2+ > Mg2+ > Sr2+ > Ba2+≫ Co2+≫ Ni2+. Ca2+ and Mn2+ have inhibition constants of 0.3 mM. Binding of [3H]verapamil is also sensitive to monovalent cations such as Cs+, K+, Li+ and Na+. The most active of these cations (Cs+ and K+) have inhibition constants in the range of 30 mM. (iii) Binding of [3H]verapamil is pH‐dependent and reveals the presence on the verapamil receptor of an essential ionizable group with a pKa of 6.5. (iv) A low‐affinity binding site for verapamil and for some other Ca2+ channel blockers is detected by studies of dissociation kinetics of the [3H]verapamil receptor in the presence of high concentrations of verapamil, gallopamil, bepridil and diltiazem. (v) GTP and nucleoside analogs change the properties of [3H]verapamil binding to verapamil binding sites. High‐affinity binding sites seem to be transferred into low‐affinity sites. Dissociation constants obtained from inhibition studies of [3H]verapamil binding are in the range of 0.1–0.3 mM for GTP, ATP and Gpp(NH)p.
Encephalomyocarditis virus replication complexes preferentially utilizing nucleoside diphosphates as substrates for viral RNA synthesis
Authors: KOONIN Eugene V. , AGOL Vadim I.
Published: March 2005

Replication complexes (RC) of the encephalomyocarditis (EMC) virus were shown previously to contain components that exhibit marked preference for nucleoside diphosphates over nucleoside triphosphates (NTP) as substrates for viral RNA synthesis [Koonin and Agol (1983), Virology 129, 309–318]. These NDP‐preferring components have now been found to posses the following properties.

When RC preparations were fractionated by sucrose density gradient centrifugation, the fractions containing NDP‐preferring components exhibited a considerably higher nucleotide kinase activity as compared to either the fractions containing NTP‐preferring components or corresponding fractions from mock‐infected cells.

When NDP‐preferring RC were incubated with ADP and three other NTP, very low concentrations of endogenously generated ATP ensured a greater rate of RNA synthesis than did much higher concentrations of exogenous ATP.

When an equimolar mixture of differently labelled UDP and UTP was used as a substrate for NDP‐preferring RC, the label from UDP predominated in the newly synthesized RNA, even though the UDP‐derived UTP constituted a minor portion of the total UTP pool.

When labelled UDP was diluted with unlabelled uridine nucleotides, unlabelled UTP proved to be far less efficient than unlabelled UDP in diminishing the specific radioactivity of UMP incorporated into RNA by NDP‐preferring RC. These data are interpreted in the sense that the NTP generated by the built‐in nucleotide kinase system are not freed into the external milieu but rather form a separate pool preferentially used for synthesis of viral RNA by NDP‐preferring RC. It is suggested that this functional compartmentation of NTP may be significant for the replication of viral RNA in vivo.
Yeast mitochondria contain a linear RNA strand complementary to the circular intronic bI1 RNA of cytochrome b
Authors: HALBREICH Avraham , GRANDCHAMP Claude , FOUCHER Marcelle
Published: March 2005

bIl RNA (excised from the first intron of the long form of the cytochrome b gene of Saccharomyces cerevisiae mitochondria) hybridizes with the two strands of a Bg/II‐MboI DNA segment from this region. This fraction is resistant to digestions by DNase I and RNase T1 and disappears completely upon alkali hydrolysis. Strand‐specific labeling of an intronic DNA fragment, cloned in pBR322 plasmid, was accomplished through the use of a T4 DNA polymerase. The purity of the probes was demonstrated by cloning an exon‐intron fragment and labeling it by the, same procedure; mRNA and pre‐mRNA bands hybridized only with the transcribed DNA strand whereas bI1 RNA hybridized with the two strands under the stringent washing conditions employed (tm+20 °C).

Several experimental results argue against the possibility that the observation of two complementary bI1 RNA strands results from a partial self‐complementarity of the RNA.

a) A pre‐mRNA intermediate from a box8 (G5046) mutant, still containing this intron, hybridizes only with the transcribed DNA strand of the pure intronic probe.

b) The amount of the non‐sense bI1 RNA strand is very low, in cells from two wild‐type strains, relative to the sense RNA strand during the early stages of growth on glucose. It increases as the cells are released from glucose repression.

c) bI1 RNA is resistant to RNase.

d) Very little self‐complementarity is seen by computer analysis of the sequence.

Purified bI1 RNA is seen by electron microscopy under non‐denaturing conditions as a mixture of double‐stranded circular and linear molecules thus confirming the existence of the two complementary strands. The disappearance of all material following alkali hydrolysis demonstrates that these are indeed two RNA strands. Under fully denaturing conditions a mixture of single‐stranded circular and linear molecules is seen as reported previously (Cell, 19, 321–329, 1980). We conclude that yeast mitochondria contain the two complementary bI1 RNA strands, one circular and the other linear. Considering a largely asymmetrical transcription of the mitochondrial genome in yeast and assuming that circularization of some intronic RNAs is part of RNA processing, we do not believe that the two strands are each a mixture of linear and circular molecules.

The ratio of non‐sense to sense bI1 RNA in a cytoplasmic petite mutant, A1B1, also varies according to growth conditions. It changes from 2.4 in galactose‐grown cells to 0.95 in glucose‐grown cells thus demonstrating that the effect of glucose repression on mtRNA metabolism may take place without a contribution from the mitochondrial genome.
Localization of a narrow‐specificity thyroliberin hydrolyzing pyroglutamate aminopeptidase in synaptosomal membranes of guinea‐pig brain
Authors: O'CONNOR Brendan , O'CUINN Gerard
Published: March 2005

In this paper we report the presence of a particulate pyroglutamate aminopeptidase in guinea‐pig brain tissue. This activity appears to reside in the synaptosomal membrane and could be released from the membrane by treatment with papain or Triton X‐100. By contrast with a previously described broad‐specificity soluble pyroglutamate aminopeptidase from guinea‐pig brain tissue, the enzyme released from the synaptosomal membrane preparation removed pyroglutamic acid from thyroliberin, acid thyroliberin and <Glu‐His‐Gly alone of the peptides tested. Unlike the soluble tissue enzyme the present enzyme was inhibited by the presence of EDTA and the activity released from synaptosomal membranes by papain was found to have a relative molecular mass of 230000, almost one order of magnitude greater than that reported for the soluble enzyme.
Isolation and partial purification of a clonidine‐displacing endogenous brain substance
Authors: ATLAS Daphne , BURSTEIN Yigal
Published: March 2005

A new compound, designated clonidine‐displacing substance (CDS), has been isolated from calf brain by ion‐exchange chromatography, zone electrophoresis and high‐performance liquid chromatography. CDS binds specifically to α2‐adrenergic receptors in rat brain and human platelet membranes, as measured in direct binding experiments using [3H]clonidine and [3H]yohimbine respectively. Unlike clonidine or other α2‐agonists, CDS does not affect basal levels of adenylate cyclase in human platelets at the highest concentrations obtainable. The apparent molecular mass of the compound is estimated to be 500 ± 50 Da, as determined by gel‐filtration chromatography on Sephadex G‐15. The new compound is thermostable, not affected by proteolytic enzymes, such as trypsin. chymotrypsin, pronase, papain and pyroglutamase, or by boiling in 0.2 M HCl for 5min. It does not bind to α1receptors in rat brain or to β‐adrenergic receptors in turkey erythrocytes, since it is unable to displace [3H]prazosin and [125I]cyanopindolol from α1 and β‐receptors respectively.
Conformational adaptation of RTEM β‐lactamase to cefoxitin
Authors: CITRI Nathan , KALKSTEIN Abraham , SAMUNI Amram , ZYK Naomi
Published: March 2005

Cefoxitin, a poor substrate of the RTEM β‐lactamase (penicillin amido‐β‐lactam hydrolase, EC 3.5.2.6), induces a reversible change in the conformation of the enzyme. The change is manifested in gradual loss of catalytic activity and increased susceptibility to proteolytic inactivation. It is prevented by antibodies, which stabilize the native conformation. By contrast, divalent cations, which have no effect on the native enzyme, delay recovery from the cefoxitin‐induced state, presumably by reacting with sites made accessible in the partly unfolded enzyme. Prolonged exposure to excess of cefoxitin causes a similar delay.

The kinetic evidence, namely, the initial burst of consumption of cefoxitin and the subsequent gradual recovery of activity with better substrates, appears to be consistent with acylation of the active site by cefoxitin followed by a slower deacylation step [Fisher et al. (1980) Biochemistry 19, 2895–2901]. However, additional evidence leads us to conclude that the kinetics observed reflect deformation of the active site, rather than its blockage, by cefoxitin. Of most significance is the transient change in specificity, i. e. a preferential interaction of the recovering enzyme with substrates which are closest in structure to cefoxitin.
Stepwise dissociation of yeast 60S ribosomal subunits by LiCl and identification of L25 as a primary 26S rRNA binding protein
Authors: EL‐BARADI Tarek T. A. L. , RAUÉ Hendrik A. , DE REGT Victoria C. H. F. , PLANTA Rudi J.
Published: March 2005

Treatment of yeast 60S ribosomal subunits with 0.5 M LiCl was found to remove all but six of the ribosomal proteins. The proteins remaining associated with the (26S + 5.8S) rRNA complex were identified as L4, L8, L10, L12, L16 and L25. These core proteins were split off sequentially in the order (L16 + L12), L10, (L4 + L8), L25 by further increasing the LiCl concentration. At 1.0 M LiCl only ribosomal protein L25 remains bound to the rRNA. Upon lowering the LiCl concentration the core proteins reassociate with the rRNA in the reverse order of their removal. The susceptibility of the ribosomal proteins to removal by LiCl corresponds quite well with their order of assembly into the 60S subunit in vivo as determined earlier [Kruiswijk et al. (1978) Biochim. Biophys. Acta 517, 378–389]. Binding studies in vitro using partially purified L25 showed that this protein binds specifically to 26S rRNA. Therefore our experiments for the first time directly identify a eukaryotic ribosomal protein capable of binding to high‐molecular‐mass rRNA. Binding studies in vitro using a blot technique demonstrated that core proteins L8 and LI6 as well as protein L21, though not present in any of the core particles, are also capable of binding to 26S rRNA to approximately the same extent as L25. About nine additional 60S proteins appeared to interact with the 26S rRNA, though to a lesser extent.
Photoaffinity labelling of dopamine receptors
Authors: WOUTERS Walter , VAN DUN Jacky , LADURON Pierre M.
Published: March 2005

A new compound, azapride, i.e. 4‐azido‐5‐chloro‐2‐methoxy‐

N‐[1‐(phenylmethyl)‐4‐piperidinyl]benzamide, the azide derivative of the dopamine antagonist clebopride, was synthesized and tested for its usefulness as a photoaffinity probe for dopamine receptors. Without photolysis the azide behaved as a very selective dopamine antagonist. When a microsomal preparation of dog striatum was pretreated with the azide and subsequently irradiated with ultraviolet light, dopamine receptors became irreversibly blocked. This reaction was dependent on both azide concentration and time of irradiation. The irradiation‐induced inhibition was quite selective for the dopamine receptor; various other receptor systems were not affected. Moreover the irreversible inhibition of dopamine receptors from dog striatum could be prevented by dopamine antagonists as well as by a dopamine agonist, but not by a serotonin antagonist. The irradiation‐induced inactivation by azapride remained unchanged in the presence of the scavenger p‐aminobenzoic acid.

These findings provide evidence that azapride labels dopamine receptors specifically and irreversibly and by a true photoaffinity mechanism. It should therefore be of great value for further molecular characterization and purification of dopamine receptors.
l‐Lactate dehydrogenase from Thermus caldophilus GK24, an extremely thermophilic bacterium
Authors: TAGUCHI Hayao , MATSUZAWA Hiroshi , OHTA Takahisa
Published: March 2005

Heat‐stable and fructose‐1,6‐bisphosphate‐activated l‐lactate dehydrogenase (EC 1.1.1.27) has been purified from an extremely thermophilic bacterium, Thermus caldophilus GK24 [Taguchi, H., Yamashita, M., Matsuzawa, H. and Ohta, T. (1982) J. Biochem. (Tokyo) 91, 1343–1348]. N‐terminal sequence analysis of the first 34 amino acids of the enzyme indicates that the N‐terminal arm region (first 1–20 residues) known for the vertebrate l‐lactate dehydrogenases is completely missing in the T. caldophilus enzyme, while there is a high homology of sequence between the regions which are considered to be part of the NAD‐binding domain. The C‐terminal amino acid of the enzyme was phenylalanine. Analysis of the amino acid composition showed that T. caldophilus enzyme contained much more arginine and fewer lysine than other bacterial and vertebrate l‐lactate dehydrogenases.

On modification reaction with 2,3‐butanedione in the presence of NADH and oxamate, an enhanced activity of the T. caldophilusl‐lactate dehydrogenase was obtained independently of fructose 1,6‐bisphosphate, and the modified enzyme was desensitized to fructose 1,6‐bisphosphate. Amino acid analysis indicated that such a desensitization in the active state was caused by the modification of only one arginine residue per the enzyme subunit. Desensitization of the enzyme was inhibited in the presence of fructose 1,6‐bisphosphate. A similar desensitization was observed using 1,2‐cyclohexanedione instead of 2,3‐butanedione. The enzyme was irreversibly modified with 2,3‐butanedione and characterized. The irreversibly modified enzyme also showed an enhanced activity independently of fructose 1,6‐bisphosphate, and its pyruvate saturation curve was similar to that of the native enzyme measured in the presence of fructose 1,6‐bisphosphate. Fructose 1,6‐bisphosphate, which increases the thermostability of the native enzyme, did not affect that of the modified enzyme, while thermostability of the modified enzyme slightly decreased. Amino acid analysis indicated that only the arginine content was decreased by the modification. These results show that arginine residue(s) exist in the binding site for fructose 1,6‐bisphosphate on the enzyme, and that the arginine residue(s) play some important role in the allosteric regulation of the enzyme activity.
The nature of the interaction of eukaryotic initiation factor 2 with double‐stranded RNA
Authors: ITAMAR Daliah , GONSKY Rivkah , LEBENDIKER Mario , KAEMPFER Raymond
Published: March 2005

In addition to binding messenger RNA molecules at specific sequences, eukaryotic initiation factor 2 (eIF‐2) also binds to double‐stranded RNA (dsRNA). The dsRNA is a powerful inhibitor of initiation of eukaryotik translation, causing the inactivation of eIF‐2, but in the presence of certain mRNA templates, dsRNA fails to establish inhibition. Such mRNA templates bind to eIF‐2 with higher affinty than does dsRNA, while globin mRNA, a template sensitive to inhibition, binds with lower affinity.

Here, the nature of the interaction between dsRNA and eIF‐2 was studied by examining both the binding of eIF‐2 to Penicillium chrysogenum dsRNA molecules carrying 32P label at their 5′ ends, and the ability of eIF‐2 to protect such label against pancreatic ribonuclease digestion. The results reveal binding sites for eIF‐2 at the 5′ ends, as well as throughout internal regions of the dsRNA molecule. At least 15 molecules of eIF‐2 can be accommodated on a 3000‐base molecule of P. chrysogenum dsRNA eIF‐2 protects a 105‐base‐pair 5′‐terminal fragment in dsRNA against digestion, but exhibits no noticeable preference for the 5′ ends. By contrast, eIF‐2 fails to protect label at the 5′ ends of denatured dsRNA molecules, even though it binds to them at internal sites more avidly than to native dsRNA.

Binding of eIF‐2 to dsRNA is not restricted to specific sequences: eIF‐2 binds with equal affinity to the synthetic dsRNA sequence, poly(rI·rC). The data support the interpretation that eIF‐2 recoginzes the A conformation in dsRNA rather than sequence. Apparently binding of eIF‐2 at sites spaced 200 base pairs apart prevents relaxation of the intervening length of the double helix, thereby stabilizing the dsRNA molecule against ribonuclease attack.

These results show that, even though dsRNA and mRNA compete in their binding to eIF‐2, the structural features recognized by eIF‐2 in these RNA species are distinct.
RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor
Authors: MIKULÍK Karel , JANDA Ivan , WEISER Jaroslav , ŠŤASTNÁ Jana , JIRÁŇOVÁ Anna
Published: March 2005

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time‐ordered events. RNA and protein synthesis started during the first 5 min and net DNA syntesis at 60–70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4–5‐S RNA were synthesized.

Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C‐labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.
Protease inhibitors from the parasitic worm Parascaris equorum
Authors: CONCETTI Antonio , FIORETTI Evandro , BARRA Donatella , ASCOLI Franca
Published: March 2005

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 109M–1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are ∼ 3.3 × 108M–1 (inhibitor I) and ∼ 2 × 106M–1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka∼ 5 × 107M–1), while inhibitor II is inactive towards this enzyme.
Kinetic studies on surface‐mediated activation of bovine factor XII and prekallikrein
Authors: SUGO Teruko , KATO Hisao , IWANAGA Sadaaki , TAKADA Katsumi , SAKAKIBARA Shunpei
Published: March 2005

The kaolin‐mediated reciprocal activation of bovine factor XII and prekallikrein was divided into the following two reactions: the activation of factor XII by plasma kallikrein (reaction 1) and the activation of prekallikrein by factor XIIa (reaction 2). The effects of high‐Mr kininogen and kalin surface on the kinetics of these activation reactions were studied. High‐Mr kininogen markedly enhanced the rate of reactions 1 and 2 in the presence of kaolin, and the enhancements were highly dependent on the concentrations of the protein cofactor and amount of kaolin surface. For the activation of factor XII by plasma kallikrein (reaction 1), high‐Mr kininogen was required when a low concentration of factor XII and kaolin was used. The molar ratio of the protein cofactor to factor XII for optimal activation was found to be approximately 1:1. The apparent Km value and the kcat/Km value for plasma kallikrein on factor XII were calculated to be 4 nM and 5.2 − 107 s−1· M−1, respectively.

The activation of prekallikrein by factor XIIa, (reaction 2) proceeded even in the absence of high‐Mr kininogen and kaolin. The addition of the protein cofactor and surface to the reaction mixture remarkably accelerated the reaction, and the apparent Km value for factor XIIa on prekallikrein was reduced from 1 μM to 40 nM. Moreover, the kcat/Km value was altered from 7.3 − 104 to 1.1 − 106 s−1· M−1. These results suggest that high‐Mr kininogen accelerates the surface‐mediated activation of factor XII and prekallikrein by enhancing the susceptibility of factor XII to plasma kallikrein, on the one hand, and the affinity of factor XIIa for prekallikrein, on the other hand. Kaolin may play an important role in the concentration and organization of these components on the negatively charged surface.
The effects of terbium (III) on the Ca‐activated, K channel found in the resealed human erythrocyte membrane
Authors: WOOD Phillip G. , MUELLER Hannelore
Published: March 2005

Incorporated terbium, Tb3+, activates the Ca‐activated K channel found in the resealed erythrocyte ghost membrane and allows the net efflux of K. As in the case of Ca activation, low levels of external K stimulate the net efflux of K. The action of incorporated Tb3+ seems to be analogous to that of incorporated Ca.

Externally applied Tb3+, however, inhibits the net efflux of K after either Ca or Tb3+ activation. The net‐efflux of K can be inhibited by Tb3+ even after partial digestion of the channel by low levels of incorporated trypsin. Furthermore, the channel when incorporated into planar bilayers, can be inhibited by Tb3+. Externally applied Tb3+ does not seem to inhibit net K efflux indirectly via inhibition of the pathways for the co‐transported anions. In addition because of the low concentrations of Tb3+ required for inhibition, it seems unlikely that an alteration of surface potential is responsible for the observed effects.
The core proteins of 35 S hnRNP complexes
Authors: WILK Hans‐Erich , WERR Helmut , FRIEDRICH Doris , KILTZ Hans Hermann , SCHÄFER Klaus P.
Published: March 2005

Ribonucleoprotein complexes (hnRNP) containing fragments of heterogeneous nuclear (hn)RNA and sedimenting at 35–40 S were isolated from the nuclei of HeLa S3 cells using the pH 8.0/diffusion technique. These hnRNP complexes are thought to be part of the hnRNA processing apparatus. The major protein components (core proteins) were identified by their constant ratios in native particles and in 35S hnRNP particles reconstituted in vitro. All of the core proteins, with one exception, show an increase in Mr on sodium dodecylsulfate (NaDodSO4)/polyacrylamide gels containing 8 M urea, indicative of secondary structure elements resistant to denaturation by NaDodSO4. The nine core proteins found by us are: A1 [Mr(NaDodSO4) 31 − 103/Mr (urea) 38 − 103, apparent isoelectric point, pIapp 9.3], A2 (32.5 − 103/39 − 103, 8.4), B1a (35.5 − 103/41 − 103, 8.8), B1b (35.5 − 103/44 − 103, 8.3), B1c (35.5 − 103/43 − 103, 5.7) B2 (37 − 103/42 − 103, 9.15), C1 (39 − 103/46 − 103, 9.2). C2 (40.5 − 103/45 − 103, 5.55) and C3 (38.5 − 103/37 − 103, 4.8). Individual proteins were electroeluted from two‐dimensional gels and their amino acid composition determined. Difference indices were calculated and show a group of closely related basic proteins (A1, A2, B1a, B1b, B2, C1), two related slightly acidic proteins (B1c, C2) and a distinct acidic member (C3). Two‐dimensional analysis of tryptic fragments and one‐dimensional separation of peptides after V8 protease treatment support these data. Peptide mapping of the proteins A1 and A2 from bovine and human cells yields identical fragments indicating a high degree of cross‐species conservation. An additional protein (D4: 44 − 103/55 − 103, >9.5) was found, which preferentially associates with heavier. oligomeric hnRNP structures. Only traces of actin are present in the 35S hnRNP fraction. All core proteins are modified by charge. A large part of the charge isomers arises by phosphorylation, which has been shown by labeling with 32PO4in vivo and with [γ‐32P]ATP in vitro. the phosphate transfer is mediated by an endogenous protein kinase associated with the 35S hnRNP complexes. The major core protein A1 exists in two conformeric forms (A1 and A1x) of which only A1x serves as phosphate acceptor in vivo.
4‐Aminobutyrate: 2‐oxoglutarate aminotransferase of Streptomyces griseus:
Authors: YONAHA Kazuo , SUZUKI Koji , TOYAMA Seizen
Published: March 2005

4‐Aminobutyrate: 2‐oxoglutarate aminotransferase of Streptomyces griseus was purified to homogeneity on disc electrophoresis. The relative molecular mass of the enzyme was found to be 100000 ± 10000 by a gel filtration method. The enzyme consists of two subunits identical in molecular mass (Mr 50000 ± 1000). The transaminase is composed of 486 amino acids/subunit containing 10 and 12 residues of half‐cystine and methionine respectively. The NH2‐terminal amino acid sequence of the enzyme was determined to be Thr‐Ala‐Phe‐Pro‐Gln. The enzyme exhibits absorption maxima at 278 nm, 340 nm and 415 nm with a molar absorption coefficient of 104000, 11 400 and 7280 M−1 cm−1 respectively. The pyridoxal 5′‐phosphate content was calculated to be 2 mol/mol enzyme. The enzyme has a maximum activity in the pH range of 7.5–8.5 and at 50°C. The enzyme is stable at pH 6.0–10.0 and at temperatures up to 50°C. Pyridoxal 5′‐phosphate protects the enzyme from thermal inactivation. The enzyme catalyzes the transamination of ω‐amino acids with 2‐oxoglutarate; 4‐aminobutyrate is the best amino donor. The Michaelis constants are 3.3 mM for 4‐aminobutyrate and 8.3 mM for 2‐oxoglutarate. Low activity was observed with β‐alanine. In addition to ω‐amino acids the enzyme catalyzes transaminiation with ornithine and lysine; in both cases the D isomer is preferred. Carbonyl reagents and sulfhydryl reagents inhibit the enzyme activity. Chelating agents, non‐substrate l and d‐2‐amino acids, and metal ions except cupric ion showed no effect on the enzyme activity.
Structure determination of oligosaccharides isolated from Cad erythrocyte membranes by permethylation analysis and 500‐MHz 1H‐NMR spectroscopy
Authors: HERKT Françoise , PARENTE José Paz , LEROY Yves , FOURNET Bernard , BLANCHARD Dominique , CARTRON Jean‐Pierre , van HALBEEK Herman , VLIEGENTHART Johannes F. G.
Published: March 2005

Alkaline borohydride reductive cleavage (β‐elimination) of glycophorin A isolated from one individual of the rare blood group Cad, resulted in the release of six acidic oligosaccharide‐alditols which were separated by high‐performance liquid chromatography (HPLC) on an alkyl amine silicagel column. The structure of four of them has been determined by the application of methanolysis analysis and 1H‐NMR spectroscopy at 500 MHz. The structures and relative amounts were as follows: oligosaccharide 1: NeuAc(α2–3)Gal(β1 – 3)GalNAcol (3.5%); oligosaccharide 3: GalNAc(β1–4)[NeuAc(α2–3)]Gal(β1–3)GalNAc‐ol (10.5%); oligosaccharide 5: NeuAc(α2–3)Gal(β1–3)[NeuAc(α2–6)]GalNAc‐ol (10.4%); oligosaccharide 6: GalNAc(β1–4)[NeuAc(α2–3)]Gal(β1–3)[NeuAc(α2–6)]GalNAc‐ol (71.2%). The two other oligosaccharides (2 and 4) were obtained in very low amount. The major pentasaccharide (oligosaccharide 6) carries the blood group Cad determinant and is a potent inhibitor of human anti‐Sda antibody.
Formation of carbon monoxide from CO2 and H2 by Methanobacterium thermoautotrophicum
Authors: EIKMANNS Bernhard , FUCHS Georg , THAUER Rudolf K.
Published: March 2005

Cell suspensions of Methanobacterium thermoautotrophicum were found to reduce CO2 with H2 to CO at a maximal rate of 100 nmol − min−1− mg protein−1. Half‐maximal rates were obtained at a H2 and a CO2 concentration in the gas phase of 10% and 30%, respectively. The CO concentration in the gas phase surpassed the equilibrium concentration by a factor of more than 15 which indicates that CO2 reduction with H2 to CO was energy‐driven. This was substantiated by the observation that the cells only formed CO when they also generated methane and that CO formation was completely inhibited by uncouplers.

CO formation by cell suspensions and by growing cells was inhibited by cyanide. Neither methane formation nor the electrochemical proton potential were affected by this inhibitor. Cyanide was shown to inactivate specifically the carbon monoxide dehydrogenase present in M. thermoautotrophicum. It is therefore concluded that reduction of CO2 to CO is catalyzed by this enzyme.

CO production by growing cells was 5–10‐times slower than by resting cells. This is explained by effective CO assimilation in growing cells; when CO assimilation was inhibited by propyl iodide the rate of CO production immediately increased more than tenfold.