bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary structure of pyruvate, orthophosphate dikinase in the dicotyledonous C4 plant Flaveria trinervia
Authors: Rosche Elke , Westhoff Peter
Published: July 2002

We have isolated and characterized cDNA clones encoding the entire precursor for the leafspecific isoform of pyruvate, orthophosphate dikinase (PPDK) from the dicotyledonous C4 plant Flaveria trinervia. The deduced amino acid sequence reveals a high degree of similarity to the corresponding maize protein indicating a common evolutionary basis. However, no significant similarities are apparent upon comparison of the putative transit peptides. The implications of this divergence are discussed with respect to the evolution of PPDK genes.
Gene structure of mouse cathepsin B
Authors: Ferrara Marc , Wojcik Franck , Rhaissi Houria , Mordier Sylvie , Roux Marie-Paule , Béchet Daniel
Published: July 2002

The structure of a genomic DNA fragment encoding mouse cathepsin B was characterized. The genomic insert spans 15 kbp and contains 9 exons encoding the 339 amino acid residues of mouse preprocathepsin B. Intron break‐points are not found at the junctions of the pre‐peptide, pro‐peptide and mature enzyme. Like other cysteine proteinase genes, the region around the cysteinyl active site is split by an intron, but in contrast with cathepsins L and H the intron break‐point is located immediately after the active site.
Complexes between 15 kDa caldesmon fragment and actin investigated by immuno‐electron microscopy
Authors: Harricane Marie-Cécile , Cavadore Claude , Audemard Etienne , Mornet Dominique
Published: July 2002

The regulatory system of smooth muscle thin filaments is thought to involve a major calcium‐calmodulin and actin binding protein: caldesmon. A dissective approach was used to isolate a 35 kDa C‐terminal fragment of the molecule and to produce antibodies reacting against both the intact and the 15 kDa N‐terminal end of this parental fragment. While this purified 15 kDa caldesmon fragment demonstrates a weak actin association, we observed that it cross‐links actin filaments into loose bundles. These structures were labelled with a selective antibody and showed regular periodic striation with repeats of approximately 40 nm. This work brings additional information to previous reports using an actin and calmodulin binding 25 kDa C‐terminal fragment of the caldesmon molecule [(1989) J. Biol. Chem. 264, 2869‐2875]. We demonstrate that a purified fragment corresponding to a sequence smaller than 96 amino acids, which contains no cystein residue, is able to interact with actin at a single site which is not the calmodulin modulated.
Identification of the binding sites of benzyl penicilloyl, the allergenic metabolite of penicillin, on the serum albumin molecule
Authors: Yvon Mireille , Anglade Patricia , Wal Jean-Michel
Published: July 2002

Tryptic digests of fragment A299 585 of penicilloylated serum albumin obtained from two penicillin‐treated patients or prepared by in vitro conjugation, were analyzed by a tandem immunoaffinity reversed‐phase HLPC. Determinations of benzyl penicilloyl groups (BPO) were performed on the different fractions. Three BPO containing peptides were identified by their amino acid sequence and the bound BPO were located on lysines 432, 541 and 545. Six major BPO binding sites were thus identified on the whole albumin molecule. All of them are lysine residues and correspond to a limited number of definite structures in which lysine and serine residues appear to be closely associated.
Analysis of the regulatory domain of yeast plasma membrane H+‐ATPase by directed mutagenesis and intragenic suppression
Authors: Portillo Francisco , Eraso Pilar , Serrano Ramon
Published: July 2002

The yeast plasma membrane H+‐ATPase is activated in vivo by glucose metabolism, and previous deletion analysis has shown the C‐terminus of the enzyme to be involved in this regulation. Site‐directed mutagenesis demonstrates that Arg909 and Thr912 at the C‐terminus are important for the increase in V
max of the ATPase induced by glucose. Other changes in kinetic parameters induced by glucose are largely independent of these ammo acids. Arg909 and Thr912 form a potential phosphorylation site for calmodulin‐dependent multiprotein kinase. A double mutation of Ser911 and Thr912 to Ala results in no cell growth in glucose medium and greatly reduced activation of the ATPase by glucose. Growth and activity are restored by a third mutation (Ala547 → Val) at the catalytic domain, providing genetic evidence for domain interaction.
Two different types of channels are targets for potassium channel openers in Xenopus oocytes
Authors: Honoré Eric , Lazdunski Michel
Published: July 2002

K+ channel openers elicit K+ currents in follicle‐enclosed Xenopus oocytes. The most potent activators are the pinacidil derivatives P1075 and P1060. The rank order of potency to activate K
+ currents in follicle‐enclosed oocytes was: P1075 (K0.5: 5 μM)>P1060 (K0.5: 12 μM)> BRL38227 (lemakalim) (K0.5:77 μM)>RP61419 (K0.5:100 μM)>(−)pinacidil (K0.5:300 μM). Minoxidil sulfate, nicorandil. RP49356 and diazoxide were ineffective. Activation by the K+ channel openers could be abolished by the antidiabetic sulfonylurea glibenclamide. It was not affected by the blocker of the Ca2+‐activated K+ channels charybdotoxin. The various K+ channel openers failed to activate glibenclamide‐sensitive K+ channels in defolliculated oocytes, but BRL derivatives (K0.5 for BRL38226 is 150 μM) and RP61419 inhibited a background current. The channel responsible for this background current is K+ permeable but not fully selective for K+. It is resistant to glibenclamide. It is inhibited by Ba2+, 4‐aminopyridine. Co2+,.Ni2+ and La3+.
Saccharomyces cerevisiae gene SIT4 is involved in the control of glycogen metabolism
Authors: Posas Francesc , Clotet Josep , Ariño Joaquin
Published: July 2002

The gene SIT4 of S. cerevisiae, which codes for a protein structurally related to the catalytic subunit of mammalian protein phosphatase 2A, was disrupted in vitro. Analysis of glycogen synthase activity ratio in mutant haploid cells indicated that the enzyme was less active than in wild‐type cells. On the contrary, glycogen phosphorylase a activity was much higher. The activation of glycogen synthase observed in wild‐type cells after incubation with lithium ions was not detected in mutant cells. These results suggest that the product of gene SIT4, a putative protein phosphatase, could be involved in the control of glycogen metabolism in yeast cells.
Isolation and characterization of methylated sugars from the tube of the hydrothermal vent tubiculous annelid worm Alvinella pompejana
Authors: Talmont Franck , Fournet Bernard
Published: July 2002

The tube of Alvinella pompejana contains in its carbohydrates fraction, 3 methylated monosaccharides: 2‐mono‐O‐methyl‐L‐fucose. 3‐mono‐O‐methyl‐L‐fucose and 2,4‐di‐O‐methyl‐L‐fucose. The present work appears to be the first report of the occurrence of 2‐mono‐O‐methyl‐L‐fucose and 3‐monoO‐methyl‐L‐fucose in the animal kingdom. Moreover, it is the first time that 2,4‐diO‐methyl‐L‐fucose is found in nature.
Contribution of proline‐14 to the structure and actions of melittin
Authors: Dempsey Christopher E. , Bazzo Renzo , Harvey Timothy S. , Syperek Inge , Boheim Gunther , Campbell Iain D.
Published: July 2002

The structure and dynamic properties of bee venom molittin and a synthetic analogue. [Ala14]‐melittin (melittin P14A), are compared, using high resolution 1H nuclear magnetic resonance (NMR) spectroscopy and amide exchange measurements in methanol. P14A is shown to adopt a regular, stable α‐helical conformation in solution without the flexibility around the Pro‐14 residue found in melittin. P14A has twice the hemolytic activity of melittin but is less able to induce voltage‐dependent ion conductance in planar bilayers. The results indicate that helix flexibility afforded by the Pro‐14 residue promotes the ability of melittin to adopt the transbilayer associates thought to underlie ion translocation.
Dissociation of the hepatic insulin receptor favours its phosphorylation by casein kinase 2
Authors: Trujillo Ramon , Jose Marta , Plana Maria , Molina Elena , Itarte Emilio
Published: July 2002

The αβ heterodimeric form of untreated hepatic insulin receptor was a substrate for casein kinase 2, whereas the α2β2 heterotetramer was not. On the contrary, autophosphorylation was detected only in the heterotetramer. Dissociation of the receptor by treatment with dithiothreitol decreased its autophosphorylation but favoured phosphorylation of its β‐subunit by casein kinase 2.
Primary structure of myohemerythrin from the annelid Nereis diversicolor
Authors: Takagi Takashi , Cox Jos.A.
Published: July 2002

The metal‐free form of Nereis diversicolor myohemerythrin was purified from whole animal extracts by trichloroacetic acid precipitation and ion exchange chromatography. The amino acid sequence of myohemerythrin has been determined. The protein is composed of 120 residues, possesses an unblocked N‐terminus and is devoid of cysteine residues. It bears 62% sequence identity with Themiste zostericola myohemerythrin, the only other member of this subfamily sequenced to date. Within the family of hemerythrins, homology is particularly high in the segments involved in the binding of the two iron atoms and in the β‐turn‐rich N‐terminal segment.
The photocycle of bacteriorhodopsin immobilized in poly(vinyl alcohol) film
Authors: Bryl K. , Varo G. , Drabent R.
Published: July 2002

The kinetics of photoelectric and optical signals were measured on samples containing oriented purple membranes immobilized in a poly(vinyl alcohol) film and on purple membranes introduced into a PVA—H2O mixture. The bacteriorhodopsin photocycle in the PVA—H2O mixture was complete. The only observed changes were the slowing down of the optical and electrical signals in relation to the M412‐O640 and O640‐bRa11‐trans steps. In the PVA film the O640 intermediate disappeared and a negative photoelectric signal appeared.
The bovine α‐lactalbumin promoter directs expression of ovine trophoblast interferon in the mammary gland of transgenic mice
Authors: Stinnakre M.G. , Vilotte J.L. , Soulier S. , L'Haridon R. , Charlier M. , Gaye P. , Mercier J.C.
Published: July 2002

A hybrid construct derived from ovine trophoblastin cDNA and bovine α‐lactalbumin‐encoding gene, was injected into the pronuclei of mouse eggs. In one of the resulting transgenic mouse lines, expression of the hybrid construct was detected and found to be limited to the mammary gland of lactating females which secreted active ovine trophoblastin. This strongly suggests that important cis‐acting DNA sequences involved in tissue‐specific expression of the bovine gene are located within the second half of the 3′ untranslated region, or/and the proximal 5′and 3′ regions flanking the transcriptional unit.
Receptor‐activated cytoplasmic Ca2+ spikes in communicating clusters of pancreatic acinar cells
Authors: Petersen C.C.H. , Petersen O.H.
Published: July 2002

The transmission of receptor‐generated cytoplasmic Ca2+ signals between communicating pancreatic acinar cells has been investigated by comparing patch‐clamp recordings of Ca2+‐dependent Cl− current in internally perfused single cells and small multi‐cell clusters. Acetylcholine (50 nM) generates shortlasting repetitive spikes of Ca2+‐dependent current and these spikes are not transmitted to neighbouring cells. Cholecystokinin octapeptide (5 pM) also generates repetitive spikes, but a significant proportion of these trigger longer and larger pulses of Ca2+‐dependent current and these waves can easily spread from cell to cell. In pancreatic acinar units it is therefore possible to observe both local Ca2+ signals confined to the cell of its origin as well as Ca2+ signals that spread through communicating junctions to all cells in the unit.
Activity and conformational changes of α‐chymotrypsin in reverse micelles studied by spin labeling
Authors: Marzola P. , Forte C. , Pinzino C. , Veracini C.A.
Published: July 2002

α‐Chymotrypsin (CT), spin‐labeled at the active site by using an acylating label which constitutes a substrate for this protein, has been investigated in reverse micelles formed by AOT in isooctane. The electron spin resonance spectra provided information on conformation, dynamics and deacylation activity. The dynamics of the label bound to CT appears to be more hindered in reverse micelles than in aqueous solution, probably owing to the effect of the micellar environment on protein conformation. The deacylation rate in reverse micelles does not show the characteristic bell‐shaped dependence on water content which is generally found for CT enzymatic activity.
Developmental expression of G proteins that differentially modulate adenylyl cyclase activity in mouse brain
Authors: Rius R.Adrian , Streaty Richard A. , Loh Y.Peng , Klee Werner A.
Published: July 2002

Changes in the relative abundance of the G protein α subunits were observed during early mouse development Gsa was almost exclusively present as a large form (Gs‐1) in prenatal brain. Postnatally with a substantial increase in Gpp[NH]p stimulated adenylyl cyclase activity, the small form (G?? increased in amount while Gs‐1 decreased. These results suggest that the Gs‐1 may be the more effective cyclase activator and that changes in alternative splicing are developmentally regulated. Gi1 and Go appeared before birth whereas Gi2, developed postnatally. Opiate stimulation of GTPase and inhibition of adenylyl cyclase were fully expressed prenatally.
Alkyllysophospholipid ET‐18‐OCH3 acts as an activator of protein kinase C in HL‐60 cells
Authors: Heesbeen Ellen C. , Verdonck Leo F. , Hermans Stefanie W.G. , van Heugten Hans G. , Staal Gerard E.J. , Rijksen Gert
Published: July 2002

HL‐60 cells are very sensitive to the cytotoxic action of ether lipids. Several hypotheses have been proposed to explain this cytotoxicity. We investigated the influence of the alkylphospholipid ET‐18‐OCH3 on the activity of protein kinase C. HL‐60 cells were incubated with ET‐18‐OCH3 at a concentration of 20 μg/ml for 4 h. After the incubation the membrane fraction of the HL‐60 cells was isolated and the activity of protein kinase C was determined while it was still associated with the membrane, using the synthetic peptide substrate [Ser25]‐protein kinase C (19–31) as a protein kinase C specific substrate. The activity of the membrane‐bound protein kinase C was increased in HL‐60 cells treated with ET‐18‐OCH3 compared to untreated HL‐60 cells. The increase in protein kinase C activity was not a consequence of translocation and appeared to be additive to the effect of the phorbol ester 12‐myristate 13‐acetate. In contrast, solubilized protein kinase C from HL‐60 cells could be inhibited or stimulated in vitro by ET‐18‐OCH3, dependent on the mode of addition of ET‐18‐OCH3 and phospholipids.
Reduced‐bond tight‐binding inhibitors of HIV‐1 protease Fine tuning of the enzyme subsite specificity
Authors: Urban Jan , Konvalinka Jan , Stehliková Jana , Gregorová Eva , Majer Pavel , Souček Milan , Andreánsky Martin , Fábrys Milan , Štrop Petr
Published: July 2002

Truncation of a peptide substrate in the N‐terminus and replacement of its scissile amide bond with a non‐cleavable reduced bond results in a potent inhibitor of HIV‐1 protease. A series of such inhibitors has been synthesized, and S2–S3′ subsites of the protease binding cleft mapped. The S2 pocket requires bulky Boc or PIV groups, large aromatic Phe residues are preferred in P1 and P′ and Glu in P2′. The S3′ pocket prefers Phe over small Ala or Val. Introduction of a Glu residue into the P2′ position yields a tight‐binding inhibitor or HIV‐1 protease, Boc‐Phe‐[CH2‐NH]‐Phe‐Glu‐Phe‐OMe, with a subnanomolar inhibition constant. The relevant peptide derived from the same amino acid sequence binds to the protease with a K
i of 110 nM, thus still demonstrating a good fit of the amino acid residues into the protease binding pockets and also the importance of the flexibility of P1‐P1′ linkage for proper binding. A new type of peptide bond mimetic, N‐hydroxylamine ‐CH2‐N(OH)‐, has been synthesized. Binding of hydroxylamino inhibitor of HIV‐1 protease is further improved with respect to reduced‐bond inhibitor.
β‐lactamase TEM1 of E. coli Crystal structure determination at 2.5 Å resolution
Authors: Jelsch C. , Lenfant F. , Masson J.M. , Samama J.P.
Published: July 2002

The crystal structure of β‐lactamase TEM1 from E. coli has been solved to 2.5 Å resolution by X‐ray diffraction methods and refined to a crystallographic R‐factor of 22.7%. The structure was determined by multiple isomorphous replacement using four heavy atom derivatives. The solution from molecular replacement, using a polyalanine model constructed from the Cα coordinates of S. Aureus PC1 enzyme, provided a set of phases used for heavy atom derivatives analysis. The E. coli β‐lactamase TEM1 is made up of two domains whose topology is similar to that of the PC1 enzyme. However, global superposition or the two proteins shows significant differences.
Endothelin stimulates both cAMP formation and phosphatidylinositol hydrolysis in cultured embryonic bovine tracheal cells
Authors: Oda Kyoko , Fujitani Yasushi , Watakabe Tadashi , Inui Takashi , Okada Toshikazu , Urade Yoshihiro , Okuda-Ashitaka Emiko , Ito Seiji
Published: July 2002

Embryonic bovine tracheal (EBTr) cells were found to possess receptors for endothelin (ET) of ET‐1‐selective (ETA)subtype with a K
d for ET‐1 of 114 pM and a B
max of 12.9 fmol/105 cells. Stimulation of EBTr cells with 100 pM to 100 nM ET‐1 increased the contents of both inositol phosphates and cAMP in a concentration‐dependent manner, indicating that the receptors are coupled to both phosphatidylinositol hydrolysis and cAMP formation in EBTr cells.
Lys‐373 of actin is involved in binding to caldesmon
Authors: Kolakowski Janusz , Makuch Robert , Dąbrowska Renata
Published: July 2002

Limited proteolysis of actin with trypsin removes its two or three C‐terminal amino acid residues [Proc. Natl. Acad. Sci. USA 81 (1984) 3680–3684]. Carboxypeptidase B‐treatment of G‐ and F‐actin previously digested with trypsin revealed that in the first case preferential release of three and in the second two C‐terminal amino acid residues takes place. Tryptic removal of three but not two C‐terminal amino acid residues of actin causes weakening of its interaction with caldesmon and lowering of the caldesmon‐induced inhibitory effect on actomyosin ATPase activity. Therefore, it is concluded that the third amino acid residue from the C terminus of actin, Lys‐373, is important for the interaction with caldesmon.
Raman spectroscopic study on the conformation of a peptide fragment representing the DNA‐binding domain of filamentous virus Pf3 coat protein
Authors: Miura Takashi , Takeuchi Hideo , Harada Issei
Published: July 2002

Raman spectra have been measured of a nonapeptide which has an amino acid sequence identical to that of the C‐terminal region of the major coat protein subunit of Filamentous bacteriophage Pf3. The peptide shows a strong tendency to form a β‐sheet structure in aqueous solution. The β‐sheet formation is significantly promoted by complexation with single‐stranded DNA but not with double‐stranded DNA. It is suggested that the C‐terminal region of the Pf3 coat protein binds to the single‐stranded DNA genome in the virion with a β‐sheet conformation, in sharp contrast with the α‐helical binding in other filamentous bacteriophages.
A marine algal Na+‐activated ATPase possesses an immunologically identical epitope to Na+,K+‐ATPase
Authors: Wada Masato , Urayama Osamu , Satoh Shinobu , Hara Yukichi , Ikawa Yoji , Fujii Tadashi
Published: July 2002

Immunological homology was investigated between Heterosigma akashiwo (a marine alga) Na+‐activated ATPase and animal Na+,K+‐ATPase. The former polypeptide [(1989) Plant Cell Physiol. 30, 923‐928] reacted with anti‐serum raised against the amino‐terminal half of the pig kidney Na+,K+‐ATPase α subunit. It is suggested that the Na+,K+‐ATPase epitope within the amino‐terminal region is conserved in the plant Na+‐activated ATPase, and the region containing the epitope may be important for Na ion transport.
Phosphoramidon‐sensitive endothelin‐converting enzyme in vascular endothelial cells converts big endothelin‐1 and big endothelin‐3 to their mature form
Authors: Matsumura Yasuo , Tsukahara Yaeko , Kuninobu Kazuko , Takaoka Masanori , Morimoto Shiro
Published: July 2002

Incubation of big endothelin‐3 (big ET‐31–41) with the membrane fraction obtained from cultured endothelial cells (ECs) resulted in an increase in immunoreactive‐ET (IR‐ET). This increasing activity was markedly suppressed by phosphoramidon, which is known to inhibit the conversion of big ET‐11–39 to ET‐11–21. Reverse‐phase HPLC of the incubation mixture of the membrane fraction with big ET‐3 revealed one major IR‐ET component corresponding to the elution position of synthetic ET‐31–21. When the cultured ECs were incubated with big ET‐3, a conversion to the mature ET‐3, as well as an endogenous ET‐1 generation, was observed. Both responses were markedly suppressed by phosphoramidon. By the gel filtration of 0.5% CHAPS‐solubilized fraction of membrane pellets or ECs, the molecular mass of the proteinase which converts big ET‐1 and big ET‐3 to their mature form was estimated to be 300–350 kDa. Phosphoramidon almost completely abolished both converting activities of the proteinase. We conclude that the above type of phosphoramidon‐sensitive metalloproteinase functions as an ET‐converting enzyme to generate the mature form from big ET‐1 and big ET‐3 in ECs.
Prokaryotic polyprotein precursors
Authors: Thöny-Meyer Linda , Böck august , Hennecke Hauke
Published: July 2002

Polyproteins have been found only recently in prokaryotes. The four known examples of single bacterial genes encoding precursors that are posttranslationally processed into two mature proteins are addressed here with respect to (i) their genomic arrangement, (ii) the sites of proteolytic processing, (iii) the relevant proteases, (iv) their maturation pathway, and (v) the function of the mature proteins. How these polyproteins may have evolved is also discussed.
Xenopus laevis oocyte Gα subunits mRNAs Detection and quantitation during oogenesis and early embryogenesis by competitive reverse PCR
Authors: Oñate Alejandro , Herrera Luisa , Antonelli Marcelo , Birnbaumer Lutz , Olate Juan
Published: July 2002

The expression of mRNAs coding for different Xenopus laevis oocyte Gα subunits was analyzed by the PCR technique. Using the nucleotide sequences of five previously cloned cDNAs for oocyte Gα subunits [FEBS Lett. 244, 188–192, 1989; FEBS Lett. 268, 27–31, 1990] and the highly sensitive reverse PCR reaction we found that Gαo, Gαi‐1, Gαi‐3 and Gαs species are present in oocyte stage VI, Gαo mRNA being the most abundant transcript. Gαo mRNA was further quantitated through oogenesis, unfertilized eggs and early embryogenesis stages by a competitive PCR reaction using an ‘in vitro’ deleted Gαo mRNA as the internal standard. Using this approach we found that Xenopus Gαo mRNA levels were constant during oogenesis and unfertilized eggs at a concentration of 3.5 pg of mRNA/stage (5 × 105 molecules) and diminish gradually during early embryogenesis, reaching a level of 0.3 pg in the gastrula stage. These findings show that oocyte Gαo, and perhaps the rest of the α subunits, are expressed as maternal mRNAs and could play an important role in signal transduction at the beginning of oocyte cell differentiation.
Stoichiometric association of extrinsic cytochrome c550 and 12 kDa protein with a highly purified oxygen‐evolving photosystem II core complex from Synechococcus vulcanus
Authors: Shen Jian-Ren , Ikeuchi Masahiko , Inoue Yorinao
Published: July 2002

A highly purified, native photosystem II (PS II) core complex was isolated from thylakoids of Synechococcus vulcanus, a thermophilic cyanobacterium by lauryldimethylamine N‐oxide (LDAO) and dodecylβ‐d‐maltoside solubilization. This native PS II core complex contained, in addition to the proteins that have been well characterized in the core complex previously purified by LDAO and Triton X‐100, two more extrinsic proteins with apparent molecular weights of 17 and 12 kDa. These two proteins were associated with the core complex in stoichiometric amounts and could be released by treatment with 1 M CaCl2 or 1 M alkaline Tris but not by 2 M NaCl or low‐glycerol treatment, indicating that they are the real components of Ps II of this cyanobacterium. N‐Terminal sequencing revealed that the 17 and 12 kDa proteins correspond to the apoprotein of cytochrome c
550, a low potential c‐type cytochrome, and the 9 kDa extrinsic protein previously found in a partially purified PS II preparation from Phormidium laminosum, respectively. In spite of retention of these two extrinsic proteins, no homologues of higher plant 23 and 17 kDa extrinsic proteins could be detected in this cyanobacterial PS II core complex.
Cloning and sequencing of the cDNA encoding rice elongation factor 1β′
Authors: Matsumoto Shogo , Oizumi Naoto , Taira Hideharu , Ejiri Shin-ichiro
Published: July 2002

A cDNA clone coding for rice elongation factor 1β′(EF‐1β′) was isolated from a rice anther cDNA library. The clone, named RB′, was 980 bp long and contained a single open reading frame coding for 223 amino acids; the first 31 amino acids, except for the first methionine, which is absent in the mature protein, are identical to those of the purified protein determined with a protein sequencer. The amino acid sequence of rice EF‐1β′ shows homology to the C‐terminal half of Artemia salina EF‐1β (59%) and human EF‐1β (63%), but might not have a phosphorylation site for casein kinase II which has been conserved in Artemia salina EF‐1β and EF‐σ, human EF‐1β and silkworm EF‐1β′.
The light‐harvesting core‐complex and the B820‐subunit from Rhodopseudomonas marina. Part I. Purification and characterisation
Authors: Meckenstock Rainer U. , Brunisholz René A. , Zuber Herbert
Published: July 2002

The BChla‐containing B880‐complex (core‐complex) of Rhodopseudomonas marina (Rhodospirillaceae) was isolated with a new purification method. The isolation of the B880‐complex was performed by solubilisation of the photosynthetic membranes with the detergent LDAO and subsequent fractionated ammonium‐sulfate precipitation with about 50% recovery. The B880‐complex retained its original spectral properties as revealed with absorption, fluorescence and circular dichroism spectroscopy. Furthermore, we dissociated the B880‐complex with the detergent n‐octyl‐β‐glucoside (OG) and purified the developed subcomplex by the method of Miller et al. [1], which showed an absorption maximum at 820 nm (B820). The α‐ to β‐polypeptide ratio and the α‐ or β‐polypeptide to BChla ratio, respectively, were estimated to be 1:1 in both complexes. The molecular weights of the B880 and the B820‐complexes, determined by gel filtration chromatography, were 181 and 32 kDa, respectively. Thus, it appears that the B880‐complex of RP. marina consists of 24 polypeptides and the B820‐complex of four polypeptides. Six B820‐complexes or possible subunits could form the B880‐complex. On the basis of these data we propose a model for the structure or BChla containing core‐complexes.
Uptake and metabolism of fluorescent ceramide analogs by rat oligodendrocytes in culture
Authors: Giudici Maria L. , Vos Jan P. , Marchesini Sergio , Van Golde Lambert M.G. , Lopes-Cardozo Matthijs
Published: July 2002

We studied the metabolism of sphingolipids by oligodendrocytes derived from rat spinal cord by providing lipid vesicles with either N‐lissamine‐rhodaminyl‐ceramide (LRh‐Cer) or N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐ceramide (NBD‐Cer) to the cells cultured in a chemically‐defined medium. With both probes them the fluorescent product turned out to be sphingomyelin (SM). Most or LRh‐SM was not cell‐associated but recovered from the culture medium, probably due to back‐exchange to the lipid vesicles. The accumulation of LRh‐SM, both in the cells and in the medium, was inhibited in the presence of monensin or brefeldin A, whereas the production of NBD‐SM was much less affected by these Golgi perturbing drugs. With LRh‐Cer as substrate, LRh‐labelled fatty acid (FA), galactosyl‐ and sulfogalactosyl‐ceramides (GalCer and SGalCer) were also formed. NBD‐Cer, however, was metabolized to glucosylecramide (GlcCer) and GalCer but not to SGalCer or NBD‐FA. These data demonstrate that chemical modifications of ceramide alter its metabolism in oligodendrocytes and that the metabolites of LRh‐Cer reflect the glycolipid composition of myelin more closely than those of NBD‐Cer.
Effect of site‐directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase
Authors: Cols Neus , Marfany Gemma , Atrian Sílvia , Gonzàlez-Duarte Roser
Published: July 2002

Tyr152 and Lys156 may be functionally important residues in Drosophila ADH as they are conserved in the genus and in all short‐chain dehydrogenases. In addition, unaltered Gly positions could have a crucial role in the building of the structural framework. We have modified Drosophila ADH and expressed the mutant forms in E. coli. Mutation of Tyr152 to Glu or Gin, Lys156 to Ile, Gly184 to Leu, and the double mutant Gly130 to Cys and Gly133 to Ile, all rendered, with different substrates and at different pHs, an inactive enzyme. Results suggest that Tyr152 and Lys156are involved in catalysis and that Gly130, Gly133 and Gly184 contribute substantially to the structure of the active form.
Molecular cloning and expression of a novel truncated form of chicken trkC
Authors: Okazawa Hitoshi , Kamei Masaki , Kanazawa Ichiro
Published: July 2002

The trk family of tyrosine protein kinase genes serves crucial roles for the development of the nervous system and the survival of neurons. The members of this gene family, trk, trkB and trkC, bind a distinct neurotrophin of the nerve growth factor (NGF) gene family, and trigger the intracellular signals which elicit trophic and differentiating effects on neurons. Adding to these neurotrophic receptor kinases, the truncated forms without the tyrosine kinase domain have been cloned and characterized. It has been thought that the existence of truncated forms is limited to trkB; however, very recently the truncated trkC has been cloned in rat [(1993) Neuron 10, 963‐974; (1993) Neuron 10, 975‐990]. We independently approached and molecularly cloned a truncated form which belongs to the chicken trkC. The truncated trkC possesses the binding and the transmembrane domains but not the tyrosine kinase domain. Northern blot analysis shows that the truncated form is preferentially expressed in the adult central nervous system. The truncated form is scarcely expressed during the embryonic stages. The conservation of the truncated trkC beyond species suggests they have specific functions.
Energy transfer processes in Rhodopseudomonas palustris grown under low‐light conditions
Authors: Nishimura Yoshinobu , Shimada Keizo , Yamazaki Iwao , Mimuro Mamoru
Published: July 2002

Excitation energy flow in the purple photosynthetic bacterium Rhodopseudomonas palustris grown under a low‐light intensity was studied by time‐resolved fluorescence spectroscopy in the ps time range. This bacterium synthesized the B824 component under this light condition. Time‐resolved spectra at 20°C indicated the sequential energy flow in the order of B803, B856, B882 and B900, long wavelength antenna. An emission from B803 was not observed. A remarkable feature was the emission from B824 throughout the measuring time. After the excitation pulse of 100 ps, the spectra did not change any further, indicating the establishment of an equilibrium among components. Based on the energy distribution after equilibrium, parallel energy transfer pathways to LH 1 were suggested; one including B824 integrated in the B803‐824‐856 complex, and the other, from the B803‐856 complex to B882. The latter was the dominant energy flow pathway in this bacterium.
A role for protein kinase C‐ϵ in angiotensin II stimulation of phospholipase D in rat renal mesangial cells
Authors: Pfeilschifter Josef , Huwiler Andrea
Published: July 2002

The role of Ca2+ and protein kinase C (PKC) in the regulation of phosphatidylcholine‐hydrolyzing phospholipase D (PLD) was investigated in angiotensin II‐stimulated mesangial cells. Elevation of cytosolic free Ca2+ by the calcium ionophore, A23187, or the Ca2+‐ATPase inhibitor, thapsigargin, slightly increased PLD‐stimulated phosphatidylethanol formation. However, chelation of cytosolic Ca2+ with high concentrations of quin 2 did not attenuate angiotensin II‐indueed phosphatidylethanol production, thus suggesting that Ca2+ is not crucially involved in agonist‐stimulated PLD activation. Stimulation of PKC by phorbol esters increased PLD activity in mesangial cells. Down‐regulation of PKC‐α and ‐δ isoenzymes by 8 h phorbol ester treatment still resulted in full PLD activation. In contrast, a 24 h treatment of mesangial cells with phorbol ester, a regimen that also causes depletion of PKC‐ϵ, abolished angiotensin II‐evoked phosphatidylethanol formation. In addition, the selective PKC inhibitor, calphostin C, attenuated hormone‐induced PLD activity. In summary, these data suggest that angiotensin II stimulation of phospholipase D appears to involve the PKC‐ϵ isoenzyme, activated by DAG derived from phosphoinositide hydrolysis.
The primary structure of carboxypeptidase S1 from Penicillium janthinellum
Authors: Svendsen Ib , Hofmann Theo , Endrizzi Jim , Remington S.James , Breddam Klaus
Published: July 2002

The complete amino acid sequence of carboxypeptidase Sl from Penicillium janthinellum has been determined by N‐terminal sequencing of the reduced and vinylpyridinated protein and of peptides obtained by cleavage with cyanogen bromide, iodosobenzoic acid, hydroxylamine, endoproteinase LysC, endoproteinase AspN and Glu‐specific proteinase from B. licheniformis. The enzyme consists of a single peptide chain of 433 amino acid residues and contains 9 half‐cystine residues and one glycosylated asparagine residue. A comparison to other carboxypeptidases shows that the enzyme is homologous to carboxypeptidase‐Y and carboxypeptidase‐MIII from malt. Specificity and binding of substrates are discussed from a three‐dimensional model based on the known structure of carboxypeptidase‐Y from Saccharomyces cereviciae and carboxypeptidase II from wheat.
Uterine cells other than stromal decidual cells are required for 1,25‐dihydroxyvitamin D3 production during early human pregnancy
Authors: Kachkache Moustapha , Rebut-Bonneton Charlyne , Demignon Josiane , Cynober Evelyne , Garabédian Michèle
Published: July 2002

Human decidual cells are known to produce 1,25‐(OH)2D3 at the end of pregnancy, the present study evaluates this capacity, and the part played by stromal decidual cells, in early pregnancy. Cells were obtained from nine human decidua by aspiration or curettage during early pregnancy (7–10 weeks), separated on Ficoll‐Paque and plastic adherence, and incubated for 1h with 25‐(OH)D3. Incubation medium and cells were extracted and chromatographed on two successive HPLC systems. The cells examined were of both physiological and pathological (ectopic pregnancy) origin. Endometrial cells obtained in four non‐pregnant situations (myomas) were also studied to determine whether the l,25‐(OH)2D3 synthesis by the uterus is associated with the appearance of decidual cells. Results show that human decidual cells from early pregnancy convert 25(OH)D3 (2.5 nM or 2.5 μM) into a metabolite with the physicochemical characteristics of synthetic 1,25‐(OH)2D3. This ability is shared by cells isolated during early pregnancy, whether physiological or ectopic (tubal pregnancy). Non‐adherent cells, which include mainly stromal decidual cells, are less able to produce 1,25‐(OH)2D3 than are the adherent cells, suggesting that macrophages, granulocytes or as yet unidentified cell types are required for the 1,25‐(OH)2D3 production by decidual tissue during early human pregnancy. In addition, one out of four experiments with non‐pregnant endometrial cells could produce 1,25‐(OH)2D3 suggesting that, although not the rule in the non‐pregnant state, in vitro production of 1,25‐(OH)2D3 by uterine cells can be found in the absence of decidual cells.
Glutathione, but not glutamine, is detected in 13C‐NMR spectra of perchloric acid extracts from C6 glioma cells
Authors: Portais Jean-Charles , Martin Magali , Canioni Paul , Merle Michel
Published: July 2002

Glutamine, which is expected to be produced by C6 glioma cells, is not detected in both amino‐acid analyses and 13C‐NMR spectra of perchloric acid extracts of cells incubated for 4 h with [1‐13C]glucose in the absence of extracellular glutamine. However, the resonances of a glutamate‐linked product are observed in these spectra. The analysis of the pH dependence of chemical shifts from various glutamate‐derived compounds shows that the observed resonances came from glutathione. Glutamine and glutathione signals are in close proximity on the frequency scale, leading to possible misinterpretation of the spectra.
Reduced content of the quinone acceptor QA in photosystem II complexes isolated from thylakoid membranes after prolonged photoinhibition under anaerobic conditions
Authors: Koivuniemi Antti , Swiezewska Ewa , Aro Eva-Mari , Styling Stenbjörn , Andersson Bertil
Published: July 2002

The plastoquinone content of photosystem II complexes isolated from spinach photosystem II‐enriched membranes subjected to strong photoinhibitory illumination under anaerobic conditions was determined by HPLC. A pronounced decrease of the plastoquinone content was found in the photoinactivated complexes. These results corroborate earlier models in which photoinhibitory illumination is suggested to eventually lead to release of doubly reduced and protonated Q
A
from its site on the D2‐protein.
Orientation and nearest neighbor analysis of psbI gene product in the photosystem II reaction center complex using bifunctional cross‐linkers
Authors: Tomo Tatsuya , Enami Isao , Satoh Kimiyuki
Published: July 2002

Two distinct cross‐linked products containing psbI gene product were generated in the photosystem II reaction center complex from spinach by treatment with bifunctional reagents directed to amino groups. The first product, which was generated by a 3,3'‐dithiobis(succinimidyl propionate) treatment, is deduced to be formed between the ϵ‐amino group of Lys3 of the psbI gene product, and a side‐chain amino group present on the stromal extension of the D2 protein. The CNBr cleavage analysis of the cross‐linked product predicted that the amino group of the D2 protein engaged in the cross‐linking is either one of the three lysine residues on the N‐terminal fragment (from N‐terminus to Met19) or the Lys268 on the 6th fragment (from Val248 to Met275). The second product, which was also generated on the stromal side by a 1,6‐hexamethylene diisocyanate treatment preferentially in alkaline conditions, is predicted to be formed between the ϵ‐amino group of Lys3 of the psbI gene product and the N‐terminal α‐amino group of the α‐subunit of cytochrome b
559.
Thermal stability of lipid‐depleted purple membranes at neutral and low pH values
Authors: Taneva Stefka G. , Koynova Rumiana , Tenchov Boris
Published: July 2002

Differential scanning calorimetry was used to compare the thermal behavior of native and delipidated purple membrane fragments at pH values corresponding to purple, blue and acid‐purple forms. At neutral pH, delipidation results in a 2.5‐ to 3‐times increase in the cooperativity of the denaturational transition, accompanied by a minor increase in its temperature. At pH values below 5 the delipidated membranes exhibit considerably higher thermal stability than the native membranes. The reversible predenaturational transition observed in the native state is not detectable upon delipidation. There is no strict correlation between color changes upon acidification and deionization of either native or delipidated purple membranes and their thermal stability.
Rat brain Na+ channel mRNAs in non‐excitable Schwann cells
Authors: Oh Youngsuk , Black Joel A. , Waxman Stephen G.
Published: July 2002

The expression of rat brain voltage‐sensitive Na+ channel mRNAs in Schwann cells was examined using in situ hybridization cytochemistry and RT‐PCR. The mRNAs of rat brain Na+ channel subtype II and III, but not subtype I, were detected in cultured Schwann cells from sciatic nerve and in intact sciatic nerve, which contains Schwann cells but not neuronal cell bodies. These results indicate that rat brain Na+ channel mRNAs, which have been considered as mainly neuronal‐type messages, are also expressed in glial cells in vitro and in vivo.
CGRP is expressed in primary cultures of human hepatocytes and in normal liver
Authors: Bracq S. , Clement B. , Pidoux E. , Moukhtar M.S. , Jullienne A.
Published: July 2002

We recently reported that human liver and primary cultures of hepatocytes express calcitonin. We therefore studied the expression of calcitonin gene related peptide (CGRP), the alternative splicing product of the calcitonin gene, in hepatocytes and liver. We used polymerase chain reaction amplification with specific primers to detect the presence of CGRP I and II messengers and a specific radioimmunoassay to measure the peptide. We report here that CGRP is synthesized by primary cultures of hepatocytes and in liver. As liver also possesses specific receptors for CGRP in non‐parenchymal cells, a paracrine system could be involved in liver metabolism.
Immunoglobulin‐type domains of titin are stabilized by amino‐terminal extension
Authors: Politou A.S , Gautel M , Joseph C , Pastore A
Published: July 2002

We have recently suggested that similarly folded titin modules located at different sarcomeric regions have distinct molecular properties and stability. Could our selection of module boundaries have potentially influenced our conclusions? To address this question we expressed amino‐terminally extended versions of the same modules and determined, with the use of CD and Fluorescence techniques, key thermodynamic parameters characterizing their stability. We present here our results which confirm our previous observations and show that, while amino‐terminal extension has a profound effect on the stability of individual modules, it does not affect at all their folding pattern or their relative stabilities. Moreover, our data suggest that the selection of module boundaries can be of critical importance for the structural analysis of modular proteins in general, especially when a well‐defined intron—exon topography is absent and proteolytic methods are inconclusive.
Expression of the sarcoplasmic reticulum Ca2+‐ATPase in yeast
Authors: Centeno Francisco , Deschamps Stéphane , Lompré Anne-Marie , Anger Marielle , Moutin Marie-Jo , Dupont Yves , Palmgren Michael Gjedde , Villalba Jose Manuel , Møller Jesper Vuust , Falson Pierre , le Maire Marc
Published: July 2002

We describe here an easy system for the production of mg amounts of the rabbit Ca2+‐ATPase SERCA 1a in the yeast S. cerevisiae. The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation. It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca2+‐dependent manner. Using a temperature‐sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.
Structure and stability of pertussis toxin studied by in situ atomic force microscopy
Authors: Yang Jie , Mou Jianxun , Shao Zhifeng
Published: July 2002

Pertussis toxin, both complete and the B‐oligomer, were imaged by atomic force miroscopy (AFM), using specimens prepared by simple surface adsorption on mica without further manipulation. The spatial arrangement of the subunits of the B‐oligomer was clearly resolved, representing the first protein quaternary structure obtained by AFM in situ. The results suggest that the B‐oligomer is a flat pentamer with the two large subunits located next to each other, and the catalytic A‐subunit situated at the center above. We found that the B‐pentamer was structurally stable for temperatures up to 60°C and within the pH range of 4.5–9.5. It is also demonstrated that the AFM was capable of resolving features down to 0.5 nm on the B‐oligomers, indicating its great potential for structural determination.
Phospholipase D activity of isolated rat brain plasma membranes
Authors: Kanfer Julian N. , McCartney Douglas
Published: July 2002

With [14C]oleate‐labeled phosphatidylcholine as a substrate for phospholipase D the hydrolytic activity was measured by phosphatidic acid formation and the transphosphatidylation activity was measured by the phosphatidylethanol formed in the presence of ethanol. The pH optimum was 6.5 with dimethylglutarate as the buffer. EGTA inhibited the transphosphatidylation activity to a greater extent than the hydrolytic activity. In contrast CaCl2, BaCl2, MgCl2 and SrCl2 stimulated the hydrolytic activity without effecting the transphosphatidylation activity. BeCl2 another member of the group IIa transition metals was a very potent inhibitor of both the hydrolytic and transphosphatidylation activity. GTPγS, an activator of G protein‐mediated events, was an inhibitor of both activities.
The small G‐protein ARF1GDP binds to the Gtβγ subunit of transducin, but not to GtαGDP‐Gtβγ
Authors: Franco Michel , Paris Sonia , Chabre Marc
Published: July 2002

AlF4
− activates heterotrimeric G‐proteins Gα subunits but not small GDP/GTP‐binding proteins like ARF1. On retinal membranes containing holotransducin (Gt
α
GDP‐Gt
βγ and incubated with ARFGDP, AlF4
− induced Gt
α
GDP‐AlF4 release and ARFGDP binding, probably to the remaining membrane‐attached Gt
βγ. On phospholipid vesicles reconstitued with Gt
βγ, ARFGDP bound in proportion to Gt
βγ, and was released upon subsequent GtαGDP addition. Thus ARFGDP competes with GtαGDP for binding to Gtβγ, probably through a conserved motif in the ‘α2 helix’ of Gtα and ARF. This motif is found in the C‐terminal helix of PH domains that bind to Gβγ.
A molecular map of titin/connectin elasticity reveals two different mechanisms acting in series
Authors: Gautel Mathias , Goulding David
Published: July 2002

In the I‐band of skeletal muscle sarcomeres, the elastic region of titin consists of immunoglobulin (1g) domains, and non‐modular regions rich in proline, hydrophobic, and charged residues (PEVK). Using immunoelectron microscopy with sequence‐assigned monoclonal antibodies, we demonstrate that extension of the Ig regions in M. psoas occurs largely at sarcomere lengths between 2 and 2.8 μm, decreasing in slope towards higher lengths. The Ig domains do not unfold. Above 2.6 μm, length changes are increasingly due to the PEVK‐rich regions. We therefore propose that rubber‐like properties of the PEVK‐rich regions are mainly contributing to skeletal titin elasticity.
A structural moDAl for the membrane‐integral domain of succinate:quinone oxidoreductases
Authors: Hägerhäll Cecilia , Hederstedt Lars
Published: July 2002

Many succinate:quinone oxidoreductases in bacteria and mitochondria, i.e. succinate:quinone reductases and fumarate reductases, contain in the membrane anchor a cytochrome b whose structure and function is poorly understood. Based on biochemical data and polypeptiDA sequence information, we show that the anchors in different organisms are related DAspite an apparent diversity in polypeptiDA and heme composition. A general structural moDAl for the membrane‐integral domain of the anchors is proposed. It is an antiparallel four‐helix bundle with a novel arrangement of hexa‐coordinated protoheme IX. The structure can be applied to a larger group of membrane‐integral cytochromes of b‐type and has evolutionary and functional implications.