bluuebunny/crossref_metadata_2025 huggingface dataset:
Translocation of proteins into rat liver mitochondria
Authors: ONO Hideyu , TUBOI Syozo
Published: March 2005

The precursor polypeptides of a large subunit of succinate dehydrogenase and ornithine aminotransferase (the enzymes which are located in the mitochondrial inner membrane and matrix respectively) were synthesized as a larger molecular mass than their mature subunits, when rat liver RNA was translated in vitro. These precursor polypeptides were also detected in vivo in ascites hepatoma cells (AH‐130 cells). When the 35S‐labeled precursor polypeptides were incubated with isolated rat liver mitochondria at 30°C in the presence of an energy‐generating system, these two precursors were converted to their mature size and the 35S‐labeled mature‐size polypeptides associated with mitochondria. Furthermore, these mature‐size polypeptides were recovered from their own locations, the inner mitochondrial membrane and the matrix.

The precursor of ornithine aminotransferase incubated with rat liver mitochondria at 0°C was specifically and tightly bound to the surface of the mitochondria even in the presence of an uncoupler of oxidative phosphorylation. This precursor, bound to the mitochondria, was imported into the matrix when the mitochondria were reisolated and incubated at 30°C in the presence of an energy‐generating system, suggesting that a specific receptor may be involved in the binding of the precursor.

The processing enzyme for both precursor polypeptides seemed to be located in the mitochondrial matrix and was partially purified from the mitochondria. A metal‐chelating agent strongly inhibited the processing enzyme and the inhibition was recovered by the addition of Mn2+ or Co2+.
Determinants for protein translocation across mammalian endoplasmic reticulum
Authors: IBRAHIMI Ibrahim M. , CUTLER Daniel , STUEBER Dietrich , BUJARD Hermann
Published: March 2005

The translocation of fragments of prelysozyme lacking varying portions of the COOH terminus of the protein is studied in comparison to full‐length prelysozyme using transcription‐couppled capping of RNA and subsequent translation in a wheat germ cell‐free system. The fragments are generated by restricting cloned lysozyme cDNA at selected sites. We found that fragments of 102 and 74 amino acid residues could still be translocated by mammalian endoplasmic reticulum. Addition of signal‐recognition particles (SRP) to the cell‐free system blocked the nascent chain synthesis. The SRP‐depleted membrane by itself could neither process nor translocate the prepolypeptide chain. The presence of both components was essential for processing and translocation as well as the release of the nascent chain arrest induced by SRP. However, when the size of the fragment was limited to 51 amino acids, the SRP‐induced arrest, the translocation and processing failed to take place. These results define minimum length and structural requirements for translocation of the nascent chain across mammalian endoplasmic reticulum.
Target size analysis of opioid receptors
Authors: OTT Susanne , COSTA Tommaso , WÜSTER Michael , HIETEL Bernhard , HERZ Albert
Published: March 2005

Target size analysis of opioid receptor is complicated by the presence of multi‐exponential inactivation curves. Irradiation of intact frozen tissue proved essential to eliminate such artifacts, due to indirect irradiation effects. Upon irradiation condition, opioid binding activity was inactivated in a single mono‐exponential manner.

Identical inactivation curves were obtained for μ, δ and κ binding activities in brain membranes from rat, guinea‐pig and frog and in NG 108‐15 cells: the molecular mass obtained was 98 ± 2 kDa. However, when opioid binding was assayed in the presence of Na+, Mg2+ and GTP, the molecular mass was found to be only 56 ± 4.4 kDa.

We suggest that the opoid recognition site comprises a unit of 56 kDa and that in the absence of Na+, Mg2+ and GTP an additional membrane component of 40–44 kDa is necessary for high‐affinity opioid binding.
Structure of initiation factor eIF‐3 from rat liver
Authors: BEHLKE Joachim , BOMMER Ulrich‐Axel , LUTSCH Gudrun , HENSKE Annemarie , BIELKA Heinz
Published: March 2005

On the basis of hydrodynamic, electron microscopic and biochemical investigations a new model of the structure of initiation factor eIF‐3 is proposed.

From sedimentation and diffusion coefficients of 16.35 S and 2.13 × 10−7 cm2/s, respectively, as well as from sedimentation equilibrium measurements, a molecular mass of about 650 kDa was determined for isolated eIF‐3. This is in agreement with molecular mass estimations by sodium dodecyl sulphate gel electrophoresis. A partial specific volume of 0.723 cm3/g was determined by means of the amino acid composition and the specific volume increments of the amino acids. From this value and from the molecular mass, a volume of 780 nm3 was calculated for eIF‐3.

In electron micrographs of isolated eIF‐3, images with triangular profiles and side lengths of 14 nm, 16 nm, and 17 nm have been observed. Taking into account the calculated volume and considering the triangular image as one face of the particle, it is suggested that eIF‐3 has the shape of a flat triangular prism with a height of about 7 nm and the above‐mentioned side‐lengths.

This model is in agreement with results of electron microscopic investigations of eIF‐3 in native small ribosomal subunits [Lutsch, G., Benndorf, R., Westermann, P., Bommer, U.‐A. & Bielka, H. (1986) Eur. J. Cell Biol. 40/2, in press]. The high frictional ratio of about 1.7 also supports eIF‐3 to be rather of a flat than of a globular shape.
The inactive subunit of ruminant procarboxypeptidase A‐S6 complexes
Authors: KERFELEC Brigitte , CAMBILLAU Christian , PUIGSERVER Antoine , CHAPUS Catherine
Published: March 2005

Subunit III has so far been found only in the pancreas of ruminants in a non‐covalent association (procarboxypeptidase A‐S6) with two different proteins: the procarboxypeptidase A itself (subunit I) and a C‐type chymotrypsinogen (subunit II). In contrast with these latter two proteins, which are zymogens of pancreatic proteases, subunit III seems to be devoid of any activity towards specific substrates of pancreatic proteases. However, it possesses a weakly functional active site which allows it to hydrolyze a non‐specific ester, p‐nitrophenyl acetate, and to react with several active‐site titrants. The binding of proflavin to subunit III shows that this protein owns a non‐polar binding site with a very high Kd compared to that of chymotrypsin. The comparison of the amino acid sequences of subunit III and some serine proteases showed that subunit III is closely related to an elastase. Models of the tertiary structure of subunit III suggest a conformational modification that affects the substrate binding and could explain the lack of specific enzymatic activity. The presence of subunit III in the ternary complex is not related to an enzymatic function. This protein does not participate in the activation process of subunit I but prevents the denaturation of this subunit at low pH. This may represent its biological role in the acidic environment of the duodenum in ruminants.
α‐Glucan synthesis on a protein primer, uridine diphosphoglucose: protein transglucosylase I
Authors: MORENO Silvia , CARDINI Carlos E. , TANDECARZ Juana S.
Published: March 2005

It was found that the DEAE‐cellulose‐treated UDP‐Glc:protein transglucosylase I catalyzing the first step (reaction 1) in the formation of α‐glucan bound to protein in potato tuber is not only specific for the glucosyl donor but also for the endogenous acceptor. A single radioactive 38‐kDa macromolecular component appeared during denaturing polyacrylamide gel electrophoresis of reaction 1 product. The labeled component is probably the polypeptide subunit of the endogenous acceptor which is being glucosylated. The radioactivity incorporated in reaction 1 product was isolated from a protease digest as a low‐molecular‐mass glucopeptide fraction. A β‐elimination reaction carried out in the presence of a reducing agent demonstrated that only one glucosyl moiety is transferred from UDP‐Glc to the aminoacyl residue, thus forming an O‐glucosidic linkage. 3H‐labeled sodium borohydride showed that serine and threonine are involved in the peptide bond to glucose. Ion‐exchange chromatography on DEAE‐cellulose, affinity chromatography on concanavalin‐A—Sepharose, gel filtration on Sephacryl S‐300 and sucrose density gradient centrifugation failed to separate the enzyme catalyzing reaction 1 from the endogenous acceptor.
Purification and characterization of a neutral processing mannosidase from calf liver acting on (Man)9(GlcNAc)2 oligosaccharides
Authors: SCHWEDEN Jürgen , LEGLER Günter , BAUSE Ernst
Published: March 2005

A processing mannosidase acting on (Man)9(GlcNAc)2 oligosaccharides, Man9 mannosidase, has been purified 2190‐fold from calf liver crude microsomes by a four‐step procedure involving (a) differential salt/detergent extraction, (b) affinity chromatography on AH‐Sepharose 4B with N‐5‐carboxypentyl‐1‐deoxy‐mannojirimycin as ligand, (c) ConA‐Sepharose and (d) DEAE‐Sephacel chromatography. (Man)9 mannosidase has a subunit molecular mass of 56 kDa and does not bind to ConA‐Sepharose, indicating the absence of high‐mannose oligosaccharides. The enzyme has a pH optimum close to pH 6.0 and requires divalent cations for activity, Ca2+ being most effective. It is inhibited by 1‐deoxymannojirimycin (dMM), N‐methyl‐dMM and N‐5‐carboxypentyl‐dMM with Ki= 7 μM, 75 μM, and 140 μM, respectively.

Man9 mannosidase cleaves three of the four α1,2‐linked mannose residues from the (Man)9(GlcNAc)2 oligosaccharide, does not hydrolyse the remaining (Man)6(GlcNAc)2 structure and is not active against aryl α‐mannosides. This pronounced substrate specificity points to the participation of Man9 mannosidase in the N‐linked processing pathway and, in addition, clearly distinguishes this enzyme from the mannosidases reported previously. As Man9 mannosidase appears to act in the processing sequence immediately after the three glucose residues have been removed from the (Glc)3(Man)9(GlcNAc)2 intermediate, we assume that the enzyme is located in the endoplasmic reticulum.
Activation of phosphorylase kinase through autophosphorylation by membrane component phospholipids
Authors: NEGAMI Akira I. , SASAKI Hiromasa , YAMAMURA Hirohei
Published: March 2005

Phosphatidic acid (PtdOH) has been shown not only to stimulate autophosphorylation and autoactivation of phosphorylase kinase of rabbit skeletal muscle but also to decrease the apparent Ka for Ca2+ on autophosphorylation sharply [Negami et al. (1985) Biochem. Biophys. Res. Commun. 131, 712–719]. In this study we investigated the interaction between PtdOH and other phospholipids on autophosphorylation and autoactivation of this enzyme.

Acidic phospholipids, such as phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns) and PtdOH, stimulated this reaction about 2–4‐fold, and the approximate Ka values of this reaction were 10 μg/ml, 6.3 μg/ml and 30 μg/ml respectively.

The molar ratio of PtdIns and PtdSer with maximal effect on autophosphorylation was about 1:1. Under these conditions PtdOH stimulated the initial velocity of autophosphorylation about 5.2‐fold.

When fully autophosphorylated, about 12–13 mol phosphate per tetramer (αβγδ) were incorporated in the presence of mixed acidic phospholipids (PtdOH:PtdIns:PtdSer = 2:1:1), which was about twice as much as values observed without effectors.

In the presence of mixed acidic phospholipids there was a concomitant enhancement of kinase activity, about 30–40‐fold at pH 6.8 and 2.5–3‐fold at pH 8.2.

Mixed acidic phospholipids sharply decreased an apparent Ka for Ca2+ from 4 × 10−5 M to 8 × 10−7 M.

With mixed acidic phospholipids as effectors this autophosphorylation occurred through an intramolecular mechanism. Based on these results, autophosphorylation and autoactivation of phosphorylase kinase in the presence of acidic phospholipids may account for an important regulatory mechanism of glycogenolysis in muscle contraction.
Demonstration of membrane receptors for human natural and recombinant 125I‐labeled tumor necrosis factor on HeLa cell clones and their role in tumor cell sensitivity
Authors: LEHMANN Volker , DRÖGE Wulf
Published: March 2005

Samples of natural and recombinant tumor necrosis factor (TNF) were labelled with 125I by use of Iodobeads and were used to study TNF receptor expression on different HeLa clones. Both labeled preparations retained cytotoxic activity after 125I‐labelling and were indistinguishable from their unlabeled counterparts on sodium dodecylsulfate/polyacrylamide gel electrophoresis. They bound specifically to TNF‐sensitive cells but not to HeLa clones that were resistant to TNF. The binding of 125I‐TNF was competed up to 90% by the addition of non‐radiolabeled TNF. The binding reached a half‐maximal level at 10 pM and saturation at 100 pM. These concentrations approximated those required for cell destruction. Scatchard analysis of the binding data yielded linear plots suggesting that TNF binds to homogeneous TNF receptor sites. The number of receptor sites ranged between 770 and 2200 sites on TNF‐sensitive cells. The surface expression of these TNF receptors appeared to be necessary but not sufficient for the cells to become sensitive against the cytotoxic action of TNF. One of the HeLa clones was found to express the same number of TNF receptors as the most sensitive clone, but was nevertheless resistant to the toxic effect of TNF.
Upstream sequences modulate in vitro transcription from Drosophila yolk protein genes I and II
Authors: VOSS Dieter , PONGS Olaf
Published: March 2005

A Drosophila transcription system was employed to study transcription in vitro from templates carrying yolk polypeptide I and II (YPI, YPII) promoter regions. The transcription start sites of YPI and YPII genes are linked by 1225 base pairs of intergenic DNA and the genes are transcribed in divergent directions. Transcription was studied in vitro from templates carrying YPI or YPII promoter regions separately. We have successively deleted intergenic DNA upstream of the YPI or the YPII transcription start site and have assayed the dependence of transcription efficiency an template concentration. The results indicate that in vitro YPI and YPII transcript synthesis is controlled by separate cis‐acting DNA sequence elements, which are located between 86 and 159 base pairs upstream of the YPI transcription start site and between 161 and 341 base pairs upstream of the YPII transcription start site. These elements stimulate in vitro transcription up to fivefold. Transcription was also studied with templates which contained both YP promoters. The transcription data indicate that the intergenic DNA contains additional DNA motif(s), which appararently modulate in vitro transcription coordinately from both YP promoters in an orientation‐dependent manner.
Structure and expression of ubiquitin genes in higher plants
Authors: GAUSING Kirsten , BARKARDOTTIR Rosa
Published: March 2005

cDNA clones encoding ubiquitin were isolated from a barley leaf cDNA library using a mammalian ubiquitin cDNA clone as probe. The nucleotide sequence of one of the clones codes of 2.2 perfect repeats of the 76‐aminoacid‐long ubiquitin protein with an extra lysine residue at the C‐terminus. The barley ubiquitin amino acid sequence differs from the animal sequence at three positions and from the yeast sequence at two positions. The ubiquitin poly(precursor) are coded by a multigene family with 8 – 10 genes that produce four or five different size messengers between 700 and 2000 nucleotides in length. The large poly(A)‐rich RNAs are constitutively expressed in vegetative tissues whereas the 700‐nucleotide messenger(s) were only detected in tissues containing dividing cells.
Binding of gossypol to purified tubulin and inhibition of its assembly into microtubules
Authors: MEDRANO Francisco Javier , ANDREU Jose Manuel
Published: March 2005

Gossypol is a polyphenolic pigment, which is employed as a male antifertility drug. It inhibits, among other reported effects, the growth of cultured mammalian cells, spermiogenesis, flagellar motility in Trypanosoma and sperm, dynein ATPase and the lactate dehydrogenase X (LDH‐X) isozyme.

We have characterized the non‐covalent binding of gossypol to purified calf brain tubulin in 10 mM phosphate buffer, 0.1 mM GTP pH 7.0 at 25°C. Equilibrium measurements were performed by difference spectroscopy. A peak at 435 nm was produced by the perturbation of gossypol light absorption upon binding to tubulin. The experimental isotherm was fitted by 1.96 ± 0.06 gossypol binding sites per tubulin molecule, with identical apparent equilibrium binding constants of (7.5 ± 1.1) × 104 M−1. The complex formed could be separated from free gossypol by gel chromatography.

Binding of gossypol was independent of the presence of 0.1 mM GTP in the buffer. Gossypol did not affect the binding of ligands to the colchicine site. Gossypol interacted with vinblastine but apparently did not bind to the vinblastine sites of tubulin. Gossypol did not displace anilinonaphthalene sulphonate (ANS) bound to tubulin, but caused a strong (fivefold) quenching of its fluorescence. This indicated that gossypol probably binds in the vicinity of the ANS site of tubulin.

Gossypol inhibited in vitro microtubule assembly at the same concentration range employed in the binding studies. An increase in the critical protein concentration required for polymerisation was observed, most simply interpreted by a stoichiometric mechanism. Gossypol did not induce any noticeable distortion of the microtubules observed under the electron microscope. This compound constitutes a new tubulin ligand and an inhibitor of microtubule assembly in vitro.
Steroid sulfatase
Authors: CONARY Jon , NAUERTH Anette , BURNS Gary , HASILIK Andrej , von FIGURA Kurt
Published: March 2005

Antibodies raised against steroid sulfatase purified from human placenta were used to follow the biosynthesis of this enzyme in human skin fibroblasts. Steroid sulfatase is synthesized as a membrane‐bound Mr‐63 500 polypeptide with asparagine‐linked oligosaccharide chains. Within 2 days, newly synthesized steroid sulfatase is processed to a mature Mr‐61000 form. The decrease in size is due to processing of the oligosaccharide chains, which are cleavable by endoglucosaminidase H in both the early and the mature form of steroid sulfatase. The processing involves mannosidase(s) sensitive to 1‐deoxy‐manno‐nojirimycin. The half‐life of the steroid sulfatase polypeptides is 4 days. Synthesis of steroid‐sulftase‐related polypeptides and steroid sulfatase activity were not detectable in fibroblasts from four patients with X‐linked ichthyosis.
Theoretical analysis of the flux control properties of a substrate cycle
Authors: SORRIBAS Albert , BARTRONS Ramon
Published: March 2005

Substrate cycles are able to increase the flux control of a non‐equilibrium reaction in a wide range of situations related to the effects of the metabolites involved in the cycle on the reaction producing them. No limit exists for that amplification if appropriate conditions are attained if those effects are positive. In all cases, the ratio between the rate of the reverse reaction and the net flux through the pathway plays an important role in defining the final amplification.
Characterization of functional domains in human tissue‐type plasminogen activator with the use of monoclonal antibodies
Authors: HOLVOET Paul , LIJNEN Henri Roger , COLLEN Désiré
Published: March 2005

Two murine monoclonal antibodies (MA‐2G6 and MA‐1C8), secreted by hybridomas obtained by fusion of myeloma cells with spleen cells from mice immunized with human tissue‐type plasminogen activator (t‐PA), inhibited the activity of t‐PA on fibrin plates. MA‐2G6 inhibited the amidolytic activity of t‐PA and did not react with t‐PA in which the active‐site serine was blocked with diisopropylfluorophosphate nor with t‐PA in which the active‐site histidine was alkylated by reaction with d‐Ile‐Pro‐Arg‐CH2Cl. This indicated that MA‐2G6 is directed against an epitope covering the active site of t‐PA. MA‐1C8 did not inhibit the amidolytic activity of t‐PA, but abolished both the binding of t‐PA to fibrin and the stimulatory effect of fibrin on the activation of plasminogen by t‐PA. Thus MA‐1C8 is directed against an epitope which covers the fibrin‐binding site of t‐PA. The A and B chains of partially reduced two‐chain t‐PA were separated by immunoadsorption on immobilized MA‐1C8 and MA‐2G6. The purified B chain reacted with MA‐2G6 but not with MA‐1C8 and activated plasminogen following Michaelis‐Menten kinetics with kinetic constants similar to those of intact t‐PA (Km= 100 μM and kcat= 0.02 s−1). However, fibrin or CNBr‐digested fibrinogen did not stimulate the activation of plasminogen by the B chain.

The purified A chain reacted with MA‐1C8 but not with MA‐2G6. It bound to fibrin with an affinity similar to that of intact t‐PA but did not activate plasminogen.

It is concluded that the active center of t‐PA is located in the B chain and the fibrin‐binding site in the A‐chain. Both functional domains are required for the regulation by fibrin of the t‐PA‐mediated activation of plasminogen.
Sulphation and the vegetative growth of Dictyostelium discoideum
Authors: DAVIS Simon J. , WHELDRAKE John F.
Published: March 2005

The utilization of [35S]sulphate by bacterially grown amoebae of Dictyostelium discoideum strain NP73 was examined in this study. During vegetative growth the sulphation of at least ten macromolecules was observed. These macromolecules had molecular masses less than 66 kDa and isoelectric points below 5. Simple tests indicated that the sulphate linkage was periodate‐sensitive but not acid‐labile which implied that carbohydrate moieties, rather than tyrosine residues, were sulphated. Pulse‐chase experiments indicated that the sulphated macromolecules were extremely stable during vegetative growth, but that secretion occurred on starvation, resulting in the loss of the sulphated macromolecules to the extracellular medium. Incorporation of [35S]sulphate into these macromolecules by amoebae declined rapidly within 2 h of starvation on membrane filters. In contrast, incorporation by amoebae starving in suspension culture continued for 6–8 h. Similar patterns of [35S]sulphate incorporation were observed for two other strains of D. discoideum (strains AX2 and NC4) and for Polysphondylium violaceum. Since in a previous study it was shown that the sulphation inhibitor, sodium selenate, arrests the growth of D. discoideum [Davis, S. J. & Wheldrake, J. F. (1985) FEMS Micro Lett. 30, 353–358], it is suggested that the sulphation of these macromolecules is necessary for the vegetative growth of D. discoideum.
Structure and in vitro transcription of tRNA gene clusters containing the primers of MuLV reverse transcriptase
Authors: RUSSO Tommaso , COSTANZO Francesco , OLIVA Adriana , AMMENDOLA Rosario , DUILIO Angela , ESPOSITO Franca , CIMINO Filiberto
Published: March 2005

Three genes coding for mouse tRNAPro have been isolated from a genomic library and characterized both structurally and functionally. Two of these (tPro52 and tPro53) code for the tRNA primer of reverse transcriptase of MuLV. The third one (tPro51) shows several differences (mutations and deletions) that probably prevent the folding of the matured transcript into the cloverleaf structure, and is therefore a pseudogene. This pseudogene gives rise to a RNA transcription product in vitro. tPro52 is clustered with a tRNALys gene and with a tRNAAla gene, which is strongly homologous to the rat identifier repeated sequence. tPro53 is clustered with a tRNAAsp and a tRNAGly gene. Other tRNA‐hybridizing sequences are present in the λ clones that contain tPro51 and tPro53.
Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii
Authors: TOGURI Toshihiro , MUTO Shoshi , MIYACHI Shigetoh
Published: March 2005

Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall‐less mutant CW‐15, which secretes CA into the culture medium, were pulse‐labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti‐CA serum.

A 42‐kDa polypeptide with isoelectric point (pI) of 7.1–7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)‐rich RNA in a cell‐free reticulocyte lysate system from a rabbit was a 38‐kDa polypeptide. This was cotranslationally converted into the 42‐kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42‐kDa precursor had a high affinity to concanavalin A, it was assumed to have a high‐mannose‐type oligosaccharide. The mature enzyme had a pI of 6.1–6.2 and was composed of more than two isoforms, which had a complex‐type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.
Pectinesterase
Authors: MARKOVIČ Oskar , JÖRNVALL Hans
Published: March 2005

The first primary structure of a pectinesterase has been determined by analysis of the main form of this plant enzyme from tomatoes. The analysis was untraditional in the sense that few data were obtained by frequently used methods. Thus, digestion with trypsin, Glu‐specific protease and several other enzymes gave limited cleavages and highly insoluble products. Instead, the determination was to a large extent based on peptides derived from chemical cleavages with CNBr at Met, N‐chlorosuccinimide at Trp and hydroxylamine at Asn‐Gly, plus an enzymatic cleavage at modified cysteine residues after chemical derivatizations.

The structure shows the protein chain to be 305 residues long, with four half‐cystine residues and five tryptophan residues. The N‐terminus has a free α‐amino group (from isoleucine). Two types of residue, tyrosine and histidine, have been functionally implied in the catalytic activity of the enzyme. Tyrosine is common in the protein (at 18 positions). However, only two histidine residues are found, and both are close to tyrosine residues in the primary structure (His‐128 separated by one intervening residue from Tyr‐126, and His‐269 adjacent to Tyr‐268), defining segment(s) of possible interest in relation to the active site.
The role of binding subsite A in reactions catalyzed by hen egg‐white lysozyme
Authors: FUKAMIZO Tamo , HAYASHI Katsuya , GOTO Sachio
Published: March 2005

The role of binding subsite A, located at the terminal of the six binding subsites of hen egg‐white lysozyme, in substrate binding and catalytic reactions was investigated by kinetic studies using a chemical modification method. Computer simulation showed that, although subsite A participates in the binding of the substrate, a decrease in the affinity of subsite A to the sugar residue does not cause a lowering of the rate of substrate consumption but changes the mode of the reaction by changing the distribution of the products formed. The binding free energies of subsites for Asp‐101‐modified lysozymes were estimated by data‐fitting from the experimental time‐courses. The contribution of Asp‐101 in hen egg‐white lysozyme to the substrate binding at subsite A was estimated to correspond to a binding free energy of about −3 kJ/mol, 30% of the total binding free energy of subsite A. Modification of Asp‐101 affected not only the binding free energy of subsite A but also that of subsite C.
Internalization of low‐density‐lipoprotein‐specific receptors in leukemic guinea pig lymphocytes
Authors: SAINTE‐MARIE Josette , VIDAL Michel , PHILIPPOT Jean R. , BIENVENUE Alain
Published: March 2005

Leukemic guinea pig lymphocytes (L2C) have ten times as many low‐density lipoprotein (LDL) receptors as healthy lymphocytes, but LDL accounts for only 38% of the cholesterol in L2C cells, compared to more than 95% in normal cells. Our data show that LDL fails to regulate cholesterol biosynthesis and that there is a defect in LDL internalization and receptor turnover in L2C cells. We also demonstrate that the degradation of LDL is not a limiting process. By discriminating between binding and internalization, we show that internalization in L2C is much slower than in normal cells and that the decrease in metabolism is related to the slow turnover of the LDL receptors.
Visualization of intermediary transcription states in the complex between Escherichia coli DNA‐dependent RNA polymerases and a promoter‐carrying DNA fragment using the gel retardation method
Authors: HEUMANN Hermann , METZGER Willi , NIEHÖRSTER Marcus
Published: March 2005

DNA‐dependent RNA polymerase in complex with a DNA fragment was analyzed by electrophoresis in non‐denaturing gels as core enzyme, holoenzyme, during initiation and elongation. The DNA fragment carried the promoter A1 of the phage T7. The stoichiometry between holoenzyme and promoter and between σ and core enzyme in complex with DNA was determined. Holoenzyme bound as a monomer to the DNA, whereas core enzyme formed aggregates before binding to the DNA. If the molar ratio of holoenzyme to DNA exceeded 0.5:1 a second holoenzyme molecule interacted with the DNA fragment with diminished affinity. A large difference in the frictional coefficient of the holoenzyme‐promoter and the core enzyme‐DNA complex indicated a drastic conformational difference between the two types of complexes. The stability of the holoenzyme‐promoter complex decreased with decreasing temperature, accompanied by at least partial dissociation of‐holoenzyme into core enzyme and σ factor. Addition of nucleoside triphosphates did not change the electrophoretic mobility of the complex if abortive transcription only was allowed, but increased it after addition of all four nucleoside triphosphates owing to release of the σ factor.
Lipoic acid is the site of substrate‐dependent acetylation of component X in ox heart pyruvate dehydrogenase multienzyme complex
Authors: HODGSON Jeffery A. , DE MARCUCCI Olga G. , LINDSAY John G.
Published: March 2005

The recently characterized Mr‐50000 polypeptide associated with mammalian pyruvate dehydrogenase complex, referred to as component or protein X, was shown to incorporate N‐ethylmaleimide only in the presence of pyruvate or NADH. Component X, modified with N‐ethyl[2,3‐14C]maleimide in the presence of pyruvate, was isolated and subjected to acid hydrolysis. The radioactive products were resolved on an amino acid analyser and these coeluted with products from similarly modified and hydrolysed lipoate acetyltransferase. Preincubation of pyruvate dehydrogenase complex with pyruvate or NADH and acetyl‐CoA resulted in a time‐dependent diminution of incorporation of radiolabelled N‐ethylmaleimide into component X and lipoate acetyltransferase and, correspondingly, in the extent of inhibition of overall complex activity by N‐ethylmaleimide.
Nucleotide sequence of the pntA and pntB genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli
Authors: CLARKE David M. , LOO Tip W. , GILLAM Shirley , BRAGG Philip D.
Published: March 2005

A 3240‐base‐pair DNA fragment spanning the pyridine nucleotide transhydrogenase (pnt) genes of Escherichia coli has been sequenced. The sequence contains two open‐reading frames, pntA and pntB of 1506 and 1386 base pairs, coding for the transhydrogenase α and β subunits, respectively. The coding sequences are preceded by a promoter‐like structure and are most likely co‐transcribed. Each coding sequence is preceded by a Shine‐Dalgarno sequence.

The amino‐terminal amino acid sequences were determined from the purified α and β subunits of the transhydrogenase. These sequences agree with those predicted from the nucleotide sequences of the pntA and pntB genes. The predicted relative molecular masses of 53906 (α) and 48667 (β) are close to the values obtained by analysis of the subunits by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Several hydrophobic regions large enough to span the cytoplasmic membrane were observed in each subunit. These results indicate that transhydrogenase is an intrinsic membrane protein.
Interaction of vasoactive intestinal peptide (VIP) and N‐terminally modified VIP analogs with rat pancreatic, hepatic and pituitary membranes
Authors: ROBBERECHT Patrick , COY David H. , DE NEEF Philippe , CAMUS Jean‐Claude , CAUVIN Annick , WAELBROECK Magali , CHRISTOPHE Jean
Published: March 2005

Six vascoactive intestinal peptide (VIP) analogs inhibited [125I]iodo‐VIP and [125I]iodo‐helodermin binding to high‐affinity VIP receptors in rat hepatic membrances. They also stimulated adenylate cyclase activity through these receptors, their decreasing order of potency being VIP > [d‐Ala4]VIP > [d‐Asp3]VIP > [d‐Ser2]VIP > [d‐His1]VIP > [d‐Phe2]VIP > [d‐Arg2]VIP, with the latter two peptides acting as partial agonists only.

All VIP analogs tested on rat pancreatic membranes were able to stimulate adenylate cyclase, their order of potency being very similar to that observed on hepatic membranes. [d‐Ser2]VIP, [d‐His1]VIP, [d‐Arg2]VIP and [d‐Phe2]VIP were partial agonists with an intrinsic activity of, respectively, 0.8, 0.7, 0.35 and 0.09 as compared to that of VIP = 1.0. [d‐Phe2]VIP competitively and selectively inhibited VIP‐stimulated adenylate cyclase activity (Ki= 0.1 μM).

On male rat anterior pituitary homogenates the order of potency of the peptides was VIP > [d‐Ala4]VIP > [d‐Asp3]VIP > [D‐Ser2]VIP > [D‐His1] and [d‐His1]VIP acted as partial agonists. Besides, [d‐Phe2]VIP and [d‐Arg2]VIP were inactive as well as unable to inhibit VIP‐stimuated adenylate cyclase activity.

These results indicated that (a) the efficacy of VIP receptor/effector coupling depended on the tissue tested; (b) the possibility exists to design a VIP antagonist by appropriate modification in the N‐terminal moiety of the molecule.
The effects of platelet‐derived transforming growth factor β on normal human diploid gingival fibroblasts
Authors: WRANA Jeffrey L. , SODEK Jaro , BER Rebecca L. , BELLOWS Carlton G.
Published: March 2005

Studies of the effects of transforming growth factor (TGF) β on normal human diploid gingival fibroblasts (HGF) have been carried out to determine possible physiological effects of this growth factor. Responses distinctly different from those characterized using established cell lines were observed. Whether alone, or in combination with EGF (2.5 ng/ml), human platelet‐derived TGF‐β (0.1 ng/ml or 1.0 ng/ml) did not induce anchorage‐independent growth of HGFs in soft agar assays. However, TGF‐β with EGF acted synergistically in promoting a 1.8‐fold increase in anchorage‐dependent proliferation of quiescent HGFs. At the same concentrations TGF‐β alone stimulated the incorporation of [35S]methionine into both cellular (cell‐layer) and matrix (medium) proteins by as much as 3‐fold and 1.7‐fold respectively. Densitometric analysis of fluorographs of radiolabeled media proteins separated by SDS‐PAGE revealed that the TGF‐β‐stimulated protein synthesis was selective. However, synthesis of collagen, the major protein synthesized and secreted by HGFs, was stimulated by TGF‐β to the same extent as the average secreted protein. Protein synthesis and cell proliferation were significantly greater in subconfluent cells compared to confluent and multilayered cells. These effects are likely to reflect physiological activity of platelet‐derived TGF‐β which may act to promote the wound healing response.
Induction of polynucleotide‐protein cross‐linkages by ultraviolet irradiation
Authors: BUDOWSKY Edward I. , AXENTYEVA Maria S. , ABDURASHIDOVA Gulnara G. , SIMUKOVA Natalia A. , RUBIN Leonid B.
Published: March 2005

The efficiency and specificity of RNA‐protein cross‐linking in the 30S subunit ofEscherichia coliribosomes, induced by low‐intensity (1015photons cm−2s−1, 254 nm) and high‐intensity [(1.6–6.8)×1024photons cm−2s−1, 266 nm, pulse duration 10−8s] ultraviolet radiation, are studied. Under the former conditions proteins S4, S7 and S9/S11, and under the latter conditions these proteins together with S3, S18 and S20, are cross‐linked to 16S RNA. Biphotonic processes operate in the latter case. In the presence of 2‐mercaptoethanol cross‐linking occurs either directly, via a higher excited state or via activated intermediates with life‐times less than 25 ns. Crosslinks thus formed are specific, i.e. they are formed between regions of macromolecules which are in contact in the native (non‐disturbed) complex prior to excitation. The efficiency of cross‐linking (per photon absorbed) is 20–100 times higher upon two‐step excitation as compared with single‐step excitation and an analysable number of cross‐links are produced in a single pulse. Only base U‐1239 of 16S RNA is cross‐linked to protein S7 by lowintensity radiation, whereas the adjacent base, G‐1240 is also involved in laser‐induced cross‐linking. A transition from the former to the latter conditions allows one to reduce the duration of irradiation from several minutes to several nanoseconds.
Calcium release from calmodulin and its C‐terminal or N‐terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped‐flow fluorescence with Quin 2 and intrinsic tyrosine
Authors: SUKO Josef , WYSKOVSKY Wolfgang , PIDLICH Johann , HAUPTNER Rudolf , PLANK Brigitte , HELLMANN Gertrude
Published: March 2005

Calcium dissociation from the C‐terminal and N‐terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped‐flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA.

Calcium dissociation from the C‐terminal half of calmodulin, which contains only the two high‐affinity calcium‐binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s−1 and 12.8 s−1, respectively, in the C‐terminal half and 10.5 s−1 and 10.8 s−1, respectively, in intact calmodulin (pH 7.0, 25°C, 100 mM KCl).

Alkylation of the C‐terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s−1 and 0.73 s−1 respectively; tyrosine: kobs 1.65 s−1 and 0.61 s−1 respectively). Alkylation of intact calmodulin resulted in a fourphase calcium dissociation measured with Quin 2 (Kobs 85.3 s−1, 11.1 s−1, 1.92 s−1 and 0.59 s−1); the latter two phases are assumed to represent calcium release from high‐affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s−1 and 0.53 s−1 respectively) and the rate parameters determined in the C‐terminal half. Evidently perturbation of the calcium‐binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high‐affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N‐terminal half resulted in biphasic calcium release with rates (kobs 153 s−1 and 10.9 s−1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin‐melittin or fragment‐melittin complexes, measured with Quin 2, were slower and monophasic in the C‐terminal half (kobs 1.12 s−1), biphasic in the N‐terminal half (Kobs 140 s−1 and 26.8 s−1 respectively) and triphasic in intact calmodulin (kobs 126 s−1, 12.1 s−1 and 1.38 s−1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low‐affinity sites mainly due to a reduced calcium ‘off rate’, presumably because of conformation restrictions.

Phenoxybenzamine‐alkylated calmodulin and calmodulin‐melittin complex inhibit the endogenous calcium/calmodulin‐dependent protein kinase of cardiac sarcoplasmic reticulum. The alkylated calmodulin is actually an activator with lower affinity than the unmodified calmodulin suggesting a perturbation of both the calciumbinding domains as well as the enzyme‐binding domain(s).
Characterization of inverted repeated sequences in Ascaris nuclear DNA
Authors: LANDOLT Peter , TOBLER Heinz
Published: March 2005

The inverted repeated sequences of the chromatin‐eliminating nematode Ascaris lumbricoides var. suum have been examined by electron microscopy and by hydroxyapatite chromatography, both in the germ‐line and in the somatic DNA. 38% of the inverted repeats of the germ‐line DNA analysed in the electron microscope have a single‐stranded loop, in comparison to about 50% of looped structures in the somatic DNA. The loops are on average 2.3 × 103 base pairs (bp) long. The rest of the foldback DNA consists of simple hairpins. The average length of looped and unlooped inverted repeats is of the order of 300–400 bp in the germ‐line and in the somatic DNA. The content of S1‐resistant foldback duplexes isolated by hydroxyapatite chromatography amounts to 1.3% in spermatids, with an average length of 350 bp, and to 1.1% in intestinal or larval cell nuclei, with a length of about 320 bp. We estimate by two different methods that there exist approximately 12500 inverted repeats per haploid germ‐line genome and approximately 8000 in the haploid somatic genome. A statistical analysis of the data indicates that the great majority of the foldback sequences are randomly distributed in the Ascaris genome, with a spacing of about (40–80) × 103 bp, both in the germ‐line and in the somatic DNA.
The light‐dependent accumulation of the P700 chlorophyll a protein of the photosystem I reaction center in barley
Authors: KREUZ Klaus , DEHESH Katayoon , APEL Klaus
Published: March 2005

The light‐dependent accumulation of the P700 chlorophyll a protein of the photosystem I reaction center has been studied in greening barley (Hordeum vulgare L.) seedlings.

Immunoblot analysis of total cellular protein fractions and immunogold labelling of the P700 chlorophyll a protein in ultrathin sections of Lowicryl‐embedded leaf tissue revealed that the concentration of this chlorophyll‐binding protein in plastids of dark‐grown barley seedlings is below the limit of detection. Upon illumination with white light, a rapid accumulation of this protein is induced. This light effect seems not to be regulated at the level of transcription. The gene for the P700 chlorophyll a protein has been mapped within the large single‐copy region of the plastid DNA of barley. High levels of transcripts of this gene are present already in dark‐grown seedlings and remain fairly constant throughout an extended illumination period. Polysomes were isolated from etioplasts and chloroplasts. The same high relative concentration of mRNA encoding the P700 chlorophyll a protein was present in both polysome fractions. This result suggests that the light‐dependent accumulation of the P700 chlorophyll a protein during chloroplast formation in barley seedlings is regulated at the translational, or posttranslational, level.
The alternative oxidase of Candida parapsilosis
Authors: GUÉRIN Martine , CAMOUGRAND Nadine
Published: March 2005

The yeast Candida parapsilosis is able to grow on a glycerol medium, supplemented with antimycin A, due to a secondary mitochondrial pathway, able to reoxidize specifically cytoplasmic NADH. It is antimycin‐A‐insensitive, but inhibited by salicylhydroxamic acid and high cyanide concentrations. This pathway involves the participation of a specific pool cytochrome c, reducible by NADH but not by ascorbate/N,N,N′,N′‐tetramethyl‐p‐phenylenediamine, and a cytochrome 590 named cytochrome a1 in bacteria. In C. parapsilosis, both oxidases aa3 and a1 are implicated in the electron transfer pathway.
Oral immunization with a synthetic peptide of cholera toxin B subunit
Authors: GUYON‐GRUAZ Anne , DELMAS Agnès , PEDOUSSAUT Sylvie , HALIMI Hubert , MILHAUD Gerard , RAULAIS Daniel , RIVAILLE Pierre
Published: March 2005

The ability of free synthetic fragments of the cholera toxin (CT), administered by parenteral or oral route, without adjuvant, to induce antibodies cross‐reacting with CT was tested. Two peptides corresponding to the sequences 30–50 and 50–75 of the CT β chain were selected and synthesized. Both free peptides, given intraperitoneally or orally, without adjuvant, elicited seric antibodies cross‐reacting with CT. The anti‐(P50–75) antibodies were able to neutralize the CT activity. Our results show that protection against a toxin at the systemic level can be obtained with a synthetic peptide even when administered by an oral route.
Structure predictions and surface charge of nitrogenase flavodoxins from Klebsiella pneumoniae and Azotobacter vinelandii
Authors: DRUMMOND Martin H.
Published: March 2005

A first approximation to the tertiary structure of the nitrogenase flavodoxins of Klebsiella pneumoniae and Azotobacter vinelandii can be obtained by superimposing their amino acid sequences upon the crystallographically determined structure of the long‐chain flavodoxin from Anacystis nidulans. This procedure is validated by secondary structure predictions based on the sequence alone and by the distribution of polar and hydrophobic residues. It reveals, among other things, a distinctive distribution of surface charge peculiar to the nitrogenase flavodoxins, which is probably important in determining the kinetics of electron transfer with their physiological redox partners. The most likely positions of the phosphodiester bridge which has been described in the A. vinelandii molecule can also be assessed.
β‐Alanine transport in synaptic plasma membrane vesicles from rat brain
Authors: AGULLO Luis , JIMENEZ Benilde , ARAGÓN Carmen , GIMÉNEZ Cecilio
Published: March 2005

The efflux and exchange of β‐alanine were studied in synaptic plasma membrane vesicles from rat brain. The mechanism of β‐alanine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution‐induced efflux requires the simultaneous presence of internal sodium and chloride ions while influx is dependent on the presence of these two ions on the outside [Zafra, F., Aragón, M. C., Valdivieso, F. and Giménez, C. (1984) Neurochem Res. 9, 695–707]. These data show that the release of β‐alanine occurs via the carrier system and that it is cotransported with sodium and chloride ions. β‐Alanine efflux from the membrane vesicles is stimulated by external β‐alanine. This exchange does not require external sodium and chloride but it is dependent on the external concentration of β‐alanine. Half‐maximal stimulation is obtained at a β‐alanine concentration similar to the Km for β‐alanine influx. Results of the direct measurements of the coupling of sodium and chloride to the transport of β‐alanine by using a kinetic approach allow us to propose a stoichiometry for the translocation cycle catalyzed by the β‐alanine transporter of three sodium ions and one chloride ion per β‐alanine zwitterion. To account for all the observed effects of external ions, β‐alanine concentrations and membrane potential on β‐alanine influx and efflux, a kinetic model of the Na+/Cl−/β‐alanine cotransport system is discussed.