bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant
Authors: TANIGUCHI Mitsutaka , SAWAKI Hiromichi , SASAKAWA Hideo , HASE Toshiharu , SUGIYAMA Tatsuo
Published: March 2005

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an cDNA‐malic‐enzyme‐type C4 plant and screened the library for AspAT isozymes. A full‐length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAt are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28‐amino‐acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. Miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.
Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues
Authors: CACCIA Paolo , NITTI Gianpaolo , CLETINI Ornella , PUCCI Piero , RUOPPOLO Margherita , BERTOLERO Federico , VALSASINA Barbara , ROLETTO Fulvia , CRISTIANI Cinzia , CAUET Gilles , SARMIENTOS Paolo , MALORNI Antonio , MARINO Gennaro
Published: March 2005

The production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes. Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability. Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups. Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm‐FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form. Cm‐FGF and unmodified rhbFGF showed similar affinity both for heparin and for high‐affinity receptors. Cm‐FGF was more stable than the unmodified molecule as measured by HPLC and SDS/PAGE analysis. Interestingly, Cm‐FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts. This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications.
In vivo and in vitro studies of the purine‐cytosine permease of Saccharomyces cerevisiae
Authors: BRÈTHES Daniel , CHIRIO Maria‐Chantal , NAPIAS Christian , CHEVALLIER Marie‐Renée , LAVIE Jean Louis , CHEVALLIER Jean
Published: March 2005

The FCY2 gene of the purine‐cytosine permease (PCP) of Saccharomyces cerevisiae and the allele fcy2‐21 have been cloned on the yeast multicopy plasmid pJDB207. The corresponding plasmids were introduced into a S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus. The resulting strains were designated pAB4 and pAB25 respectively. The pAB25 strain, which carries the fcy2‐21 allele, contains four amino acid changes in the open reading frame of the PCP (Weber et al., 1989). The influence of these mutations was studied on cells by determination of the uptake constants of purine bases and cytosine [apparent Michaelis constant of transport (Ktapp) and Vmax] and on plasma‐membrane preparations, by measurements of binding parameters at equilibrium [Kd and maximum amount of binding sites/Bmax)].

For strain pAB4, the Ktapp and Vmax of uptake were almost similar for all solutes considered [1.8—2.6 μM and 8.5—10.2 nmol · min−1· (107 cells)−1]. The main effect of the mutations in strain pAB25 was based on a large increase in Ktapp for all ligands except adenine.

Plasma membranes of each strain displayed one class of specific binding sites. Variations in Kd of 0.4—1 μM were observed for pAB4. These slight variations had no effect on the Ktapp of uptake measured for the corresponding solutes. In contrast, using pAB25 membranes, Kd increased dramatically; 2.6 μM, 40 μM and 96 μM for adenine, cytosine and hypoxanthine, respectively. These increments were correlated to variations in Ktapp of the uptake for cytosine and hypoxanthine.

Therefore, we conclude that modification in the Ktapp of uptake in the strain carrying fcy2‐21 allele is merely due to a modification of the binding ability of the permease for its ligands.
Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase
Authors: BAKRIM Naïma , ECHEVARRIA Cristina , CRETIN Claude , ARRIO‐DUPONT Martine , PIERRE Jean Noël , VIDAL Jean , CHOLLET Raymond , GADAL Pierre
Published: March 2005

The phosphoenolpyruvate (PPrv) carboxylase isozyme involved in C4 photosynthesis undergoes a day/night reversible phosphorylation process in leaves of the C4 plant, Sorghum. Ser8 of the target enzyme oscillates between a high (light) and a low (dark) phosphorylation status. Both in vivo and in vitro, phosphorylation of dark‐form carboxylase was accompanied by an increase in the apparent Ki of the feedback inhibitor L‐malate and an increase in Vmax. Feeding detached leaves various photosynthetic inhibitors, i.e. 3‐(3,4‐dichlorophenyl)‐1, 1‐dimethylurea, gramicidin and DL‐glyceraldehyde, prevented PPrv carboxylase phosphorylation in the light, thus suggesting that the cascade involves the photosynthetic apparatus as the light signal receptor, and presumably has the electron transfer chain and the Calvin‐Benson cycle as components in the signal‐transduction chain. Two proteine‐serine kinases capable of phosphorylating PPrv carboxylase in vitro have been partially purified from light‐adapted leaves. One was isolated on a calmodulin‐Sepharose column; it was calcium‐dependent but did not require calmodulin for activity. The other was purified on a blue‐dextran–agarose column and the only Me2+ required for activity was Mg2+. In reconstituted phosphorylation assays, only the latter caused the expected decrease in malate sensitivity of PPrv carboxylase suggesting that this protein is the genuine PPrv‐carboxylase‐kinase. Desalted extracts from light‐adapted leaves possessed a considerably greater phosphorylation capacity with immunopurified dephosphorylated PPrv carboxylase as substrate than did dark extracts. This light stimulation was insensitive to type 2A protein phosphatase inhibitors, okadaic acid and microcystin‐LR, which suggests that the kinase is a controlled step in the cascade which leads to phosphorylation of PPrv carboxylase. The higher phosphorylation capacity of light‐adapted leaf tissue was nullified by pretreatment with the cytosolic protein synthesis inhibitor, cycloheximide. Thus, protein turnover is involved as part of the mechanism controlling the activity of the kinase purified on blue‐dextranagarose. However, no information is available with respect to the specific nature of the link between the above‐mentioned light transducing steps and the protein kinase that achieves the physiological response. Finally, the in vivo phosphorylation site (Ser8) in the N‐terminal region of the C4 type Sorghum PPrv carboxylase is also present in a non‐photosynthetic form of the Sorghum enzyme (Ser7), as deduced by cDNA sequence analysis.
Isolation, characterization, and sequence analysis of a cDNA clone encoding L‐protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea‐leaf mitochondria
Authors: BOURGUIGNON Jacques , MACHEREL David , NEUBURGER Michel , DOUCE Roland
Published: March 2005

L‐protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a λ gt11 pea library encoding the complete L‐protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N‐terminal sequence of the first 18 amino acid residues of the purified L‐protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L‐protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea‐leaf mitochondria by gel‐permeation chromatography followed by gel electrophoresis and Western‐blot analysis, it was shown that polyclonal antibodies raised against the L‐protein of the glycine‐cleavage system recognized proteins with an Mr of about 60 000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northernblot analysis of RNA from different tissues such as leaf, stem, root and seed, using L‐protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H‐protein (a specific protein component of the glycine‐cleavage system) is known to be expressed primarily in leaves. Third, Southern‐blot analysis indicated that the gene coding for L‐protein in pea is most likely to be present in a single copy/haploid genome.
Human‐immundeficiency‐virus‐type‐1‐encoded Vpu protein is phosphorylated by casein kinase II
Authors: SCHUBERT Ulrich , SCHNEIDER Thomas , HENKLEIN Peter , HOFFMANN Klaus , BERTHOLD Evelyn , HAUSER Hansjörg , PAULI Georg , PORSTMANN Tomas
Published: March 2005

Vpu as a human‐immunodeficiency‐virus‐type‐1‐encoded 81‐amino‐acid integral‐membrane protein was expressed in Escherichia coli using the inducible ptrc promoter of an ATG fusion vector. Recombinant Vpu is associated with membranes of E. coli and could be partially solubilized by detergents. Recombinant Vpu was phosphorylated in vitro with purified porcine casein kinase II (CKII) as well as with a CKII‐related protein kinase found in cytoplasmic extracts of human and hamster cells. Recombinant Vpu associated with E. coli membranes has turned out to be the best substrate for in vitro phosphorylation with CKII. This reaction can be inhibited by heparin and the ATP analogue 5,6‐dichloro‐1‐(β‐D‐ribofuranosyl)benzimidazole (DRB), both known to be potent inhibitors of CKII. Radiolabelled γATP and γGTP were used as phosphate donors for in vitro phosphorylation of recombinant Vpu. In vivo phosphorylation of Vpu‐1‐infected H9 cells was also inhibited by DRB. We concluded therefrom that the Vpu protein is phosphorylated by the ubiquitous CKII in HIV‐1‐infected human host cells.

Two seryl residues in the sequence of Vpu (position 52 and 56) correspond to the consensus s/TXXD/E for CKII. These potential phosphorylation sites are located within a well‐conserved dodecapeptide of Vpu (residues 47–58), which is found in different HIV‐1 strains as well as in a Vpu‐like protein of SIVCPZ.

Monoclonal and polyclonal antibodies directed against two different epitopes of Vpu were used for immunoprecipitation of Vpu from HIV‐1‐infected cells and for detection of Vpu in Western blot analyses. Vpu from HIV‐1‐infected cells as well as recombinant Vpu expressed in E. coli were determined by SDS/PAGE using 6 M urea to be 9 kDa, which corresponds to the calculated molecular mass of Vpu.
Purification and characterization of a soluble β‐fructofuranosidase from Daucus carota
Authors: UNGER Christoph , HOFSTEENGE Jan , STURM Arnd
Published: March 2005

Soluble β‐fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N‐glycosylated with high‐mannose‐containing and high‐xylose‐containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68000 from a gelfiltration column. On SDS/PAGE, the protein separated in the presence of SDS and 2‐mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68‐kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43‐kDa and 25‐kDa polypeptides were N‐terminal and C‐terminal proteolytic fragments of the 68‐kDa polypeptide. A comparison of partial amino acid sequences of the soluble β‐fructofuranosidase with the complete sequence of carrot cell‐wall β‐fructofuranosidase showed that their N‐terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.
Amino acid and cDNA sequences of a neutral phospholipase A2 from the long‐nosed viper (Vipera ammodytes ammodytes) venom
Authors: KRIŽAJ Igor , LIANG Ning‐Sheng , PUNGERČAR Jože , ŠTRUKELJ Borut , RITONJA Anka , GUBENŠEK Franc
Published: March 2005

The amino acid sequence of a non‐toxic phospholipase A2, ammodytin I2, from the venom of the long‐nosed viper (Vipera ammodytes ammodytes) and its cDNA sequence have been determined. The protein sequence was elucidated by sequencing the peptides generated by CNBr cleavage, mild acid hydrolysis and tryptic digestion of maleylated and non‐maleylated protein. Sequencing of the cDNA showed that the protein is synthesized as an 137‐amino‐acid‐residue precursor molecule consisting of a 16‐residue signal peptide, followed by a 121‐residue mature enzyme. Ammodytin I2 cDNA shows 73% nucleotide and 59% amino acid identities in the mature protein region in comparison to that of ammodytoxin A, the most presynaptically neurotoxic phospholipase A2 from the long‐nosed viper. Identities in the signal‐peptide region are considerably higher, 96% and 100%, respectively.
Identification of a high‐affinity binding protein for a hepta‐β‐glucoside phytoalexin elicitor in soybean
Authors: COSIO Eric G. , FREY Thomas , EBEL Jürgen
Published: March 2005

A putative receptor protein for a hepta‐β‐glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent‐solubilized proteins from soybean root membranes. Incubation of partially purified β‐glucan‐binding proteins with a photolabile 125I‐labeled 2‐(4‐azidophenyl)ethyl‐amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70‐kDa band in SDS/PAGE. Half‐maximal inhibition of the 125I‐labeling of the protein band by underivatized hepta‐β‐glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand‐saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand‐binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside‐binding activity. Photoaffinity labeling of proteins purified by glucan‐affinity chromatography showed the 70‐kDa band as the main component along with weak 125I‐labeling of a 100‐kDa band. The 70‐kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside‐binding proteins in detergent‐solubilized soybean membranes.
The physical state of ubiquinone‐10, in pure form and incorporated into phospholipid bilayers
Authors: CASTRESANA José , ALONSO Alicia , ARRONDO José‐Luis R. , GOÑI Félix M. , CASAL Héctor
Published: March 2005

Long‐chain quinones are essential components of both bacterial and eukaryotic respiratory chains, and some of the main unsolved questions on energy transduction in membranes are complicated by the lack of consistent information on the physical state of the quinones in membrane bilayers. We have recorded, at various temperatures and under different conditions, the infrared spectra of ubiquinone‐10 (the main species in mitochondria) and several analogues. The C = O stretching vibration band located at 1663–1670 cm−1 has been identified as the most sensitive one to phase and environmental changes. Three distinct phases have been characterized in which pure ubiquinone‐10 may exist: crystalline (LC1), isotropic liquid (IL) and liquid crystalline (Lc). The only allowed thermotropic transitions are LC1→ IL, IL→ Lc and Lc→ LC1. Our investigations with pure quinones provide a simpler and more detailed description of their phase changes than any of the previous studies and shed light on their behaviour in membranes. When incorporated into phospholipid bilayers, ubiquinone‐10 appears to be removed from the aqueous environment and is found to exist, in the 4–70°C range, in an isotropic liquid phase, in the form of small aggregates.
NMR and EPR studies on a monoheme cytochrome c550 isolated from Bacillus halodenitrificans
Authors: SARAIVA Lígia M. , DENARIAZ Gerard , LIU Ming‐Y. , PAYNE William J. , Le GALL Jean , MOURA Isabel
Published: March 2005

A c‐type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme–methionine coordination. The mid‐point redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV.

When studied by 1H‐NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H‐NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H‐NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned.

EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax∼ 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax∼ 3.5 converts mainly to a signal at g∼ 2.96. The pKa associated with this change is around 11.3.

The N‐terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.
Subunit interactions of GTP‐binding proteins
Authors: HEITHIER Helmuth , FRÖHLICH Margareta , DEES Christian , BAUMANN Martin , HÄRING Martin , GIERSCHIK Peter , SCHILTZ Emile , VAZ Winchil L. C. , HEKMAN Mirko , HELMREICH Ernst J. M.
Published: March 2005

Fluorescence energy transfer [cf. Förster, T. (1948) Ann. Phys. 6, 55–75] was tested for its suitability to study quantitative interactions of subunits of Go with each other and these subunits or trimeric Go with the β1‐adrenoceptor in detergent micelles or after reconstitution into lipid vesicles [according to Feder, D., Im, M.‐J., Klein, H. W., Hekman, M., Holzhöfer, A, Dees, C., Levitzki, A., Helmreich, E. J. M. & Pfeuffer, T. (1986) EMBO J. 5, 1509–1514]. For this purpose, αo‐ and βγ‐subunits and trimeric Go purified from bovine brain, the βγ‐subunits from bovine rod outer segment membranes and the β1‐adrenoceptor from the turkey erythrocyte were all labelled with either tetramethylrhodaminmaleimide or fluorescein isothiocyanate under conditions which leave the labelled proteins functionally intact. In the case of αo‐ and βγ‐interactions, specific high‐affinity binding interactions (Kd∼ 10 nM) and nonspecific low‐affinity binding interactions (Kd∼ 1 μM) could be readily distinguished by comparing fluorescence energy transfer before and after dissociation with 10 μM guanosine 5′‐O‐[γ‐thio]triphosphate and 10 mM MgCl2 where only low‐affinity binding interactions remained. Interactions between αo‐ and βγ‐subunits from bovine brain or from bovine retinal transducin did not differ much. The βγ‐subunits from bovine brain were found to bind with high transfer efficiency and comparable affinities to the hormone‐activated and the nonactivated β1‐receptor reconstituted in lipid vesicles: Kd= 100 ± 20 and 120 ± 20 nM, respectively; however, βγ‐subunits from transducin appeared to bind more weakly to the β1‐adrenoceptor than βγ‐subunits from bovine brain. Separated purified homologous αo‐ and βγ‐subunits from bovine brain interfered mutually with each other in binding to the β1‐adrenoceptor presumably because they had a greater affinity for each other than for the receptor. These findings attest to the suitability of fluorescence energy transfer for studying protein – protein interactions of G‐proteins and G‐protein‐linked receptors. Moreover, they supported the previous finding [Kurstjens, N. P., Fröhlich, M., Dees, C., Cantrill, R. C., Hekman, M. & Helmreich, E. J. M. (1991) Eur. J. Biochem. 197, 167–176] that βγ‐subunits can bind to the nonactivated β1‐adrenoceptor.
Synthesis of chlorophyll a regulates translation of chlorophyll a apoproteins P700, CP47, CP43 and D2 in barley etioplasts
Authors: EICHACKER Lutz , PAULSEN Harald , RÜDIGER Wolfhart
Published: March 2005

Accumulation of plastid‐encoded chlorophyll apoproteins and chlorophyll synthesis are controlled by light in angiosperms. An in vitro system utilizing isolated and lysed barley (Hordeum vulgare L.) etioplasts revealed the specific accumulation of P700, CP47, CP43 and D2 triggered by de novo synthesis of chlorophyll. Accumulation rates of radiolabelled chlorophyll apoproteins were linear for about 30 min. Pulse/chase translation assays showed that synthesis of chlorophyll does not result in increased chlorophyll apoprotein stability. Instead turnover rates of chlorophyll apoproteins were higher in the presence than in the absence of chlorophyll. Chlorophyll‐dependent accumulation of chlorophyll apoproteins must therefore be regulated on the level of translation. Translation of chlorophyll apoproteins was blocked to about 50% by addition of 30–50 μM aurintricarboxylic acid or 20μM kasugamycin. The kinetics of chlorophyll‐dependent translation indicated that the in vitro translation system is capable of translation initiation. The capability of translation initiation was lost in lysed etioplasts after preincubation for at least 5 min without chlorophyll synthesis. The results suggest that initiation is involved in chlorophyll‐dependent regulation of translation.
Characterization of the endogenous ADP‐ribosylation of wild‐type and mutant elongation factor 2 in eukaryotic cells
Authors: FENDRICK James L. , IGLEWSKI Wallace J. , MOEHRING Joan M. , MOEHRING Thomas J.
Published: March 2005

Anti‐[ADP‐ribosylated elongation factor 2 (EF‐2)] antiserum has been used to immunoprecipitate the modified form of EF‐2 from polyoma‐virus‐transformed baby hamster kidney (pyBHK) cells [Fendrick, J. L. & Iglewski, W. J. (1989) Proc. Natl Acad. Sci. USA 86, 554–557]. This antiserum also immunoprecipitates a 32P‐labelled protein of similar size to EF‐2 from a variety of primary and continuous cell lines derived from many species of animals. One of these cell lines, chinese hamster ovary CHO‐K1 cells was further characterized. The time course of labelling of ADP‐ribosylated EF‐2 with [32P]orthophosphate was similar in pyBHK cells and in CHO‐K1 cells. The kinetics of labelling were more rapid for cells cultured in 2% serum than 10% serum, with incorporation of 32P reaching a maximum at 6 h and 10 h, respectively. EF‐2 mutants of pyBHK and CHO‐K1 cells resistant to diphtheria‐toxin‐catalyzed ADP‐ribosylation of EF‐2 remain sensitive to cellular ADP‐ribosylation of EF‐2. The 32P‐labelled moiety of ADP‐ribosylated EF‐2 was digested by snake venom phosphodiesterase and the product was identified as AMP. The same 32P‐labelled tryptic peptide was modified by toxin in wild‐type EF‐2 and by the cellular transferase in mutant EF‐2. When purified EF‐2 from pyBHK cells was incubated with [carbonyl‐14C]nicotinamide and diphtheria toxin fragment A, under conditions for reversal of the ADP‐ribosylation reaction, [14C]NAD was generated. The results suggest that cellular ADP‐ribosylated EF‐2 exists in a variety of cell types, and the ribosylated product is identical to that produced by toxin ADP‐ribosylation of EF‐2, except in diphthamide mutant cells. Studies with the mutant cell lines indicate that the toxin and the cellular transferase, however, recognize different determinants at the ADP‐ribose acceptor site in EF‐2. The cellular transferase does not require the diphthamide modification of the histidine ring in the amino acid sequence of EF‐2 for the transfer of ADP‐ribose to the ring. Therefore, we would expect the cellular transferase active site to be similar to, but not identical to, the critical amino acids demonstrated in the active site of diphtheria toxin and Pseudomonas exotoxin A.
Reconstitution in vivo of penicillin G acylase activity from separately expressed subunits
Authors: BURTSCHER Helmut , SCHUMACHER Günter
Published: March 2005

Penicillin G acylase from Escherichia coli ATCC11105 is synthesized as a precursor polypeptide with a signal sequence for secretion into the periplasm and an endopeptide separating two subunit domains. Proteolytic processing leads to mature, heterodimeric penicillin G acylase. We have shown that the α‐ and β‐subunits of the enzyme, which have no detectable enzymatic activity on their own, can reconstitute enzyme activity when their genes are put into an E. coli host on separate plasmids. Activity is reconstituted in the cytoplasm whereas normally processing and formation of the active heterodimer occurs in the periplasm. Enzyme activity can reach levels close to wild type in the strain used. The activity recovered from a combination of α‐subunit linked to a 54‐amino‐acid endopeptide and β‐subunit was lower than with the subunits alone.
Chemical modification of functional arginyl residues in beef kidney d‐Aspartate oxidase
Authors: TEDESCHI Gabriella , NEGRI Armando , CECILIANI Fabrizio , BIONDI Pier Antonio , SECCHI Camillo , RONCHI Severino
Published: March 2005

Chemical modification of beef kidney d‐aspartate oxidase by phenylglyoxal is a biphasic process involving the transient formation of an enzymatic species with a decreased activity versus dicarboxylic substrates, an increased activity versus d‐proline and a new activity versus other monocarboxylic d‐amino acids which is absent in the native protein. Prolonged incubation with the modifier causes complete inactivation of the enzyme. The presence of the competitive inhibitor l‐tartrate in the incubation mixture prevents enzyme inactivation. Kinetic and structural data suggest that complete loss of activity is paralleled by modification of eight arginine residues, of which two are critical for the specificity and the activity of the enzyme. We propose that the two essential arginine residues are located in the substrate binding site of d‐aspartate oxidase.
Evidence that cellobiose oxidase from Phanerochaete chrysosporium is primarily an Fe(III) reductas
Authors: KREMER Simon M. , WOOD Paul M.
Published: March 2005

Kinetic measurements were made for purified cellobiose oxidase in 100 mM acetate (pH 4.0) at 30°C, with excess cellobiose as substrate and O2 or Fe(III) as acceptor. With O2 at 230 μ as sole electron acceptor, the O2 uptake rate corresponded to a one‐electron turnover number of 0.13 ± 0.01 s−1. Measurements at different O2 concentrations indicated Km(O2) > 120 μM. In separate experiments, the reduction of Fe(III) acetate was monitored at 340 nm in the absence of oxygen. The maximum velocity of Fe(III)‐acetate reduction (Vmax) was 4.5 ± 0.7 s−1, while Km[Fe(III) acetate] was 34 ± 12 μM. With ferricyanide in place of Fe(III) acetate, the corresponding values were 6.9 ± 0.7 s−1 and 23 ± 5 μM. Redox titrations established the potential of the heam prosthetic group of the oxidase at pH 4.0 as + 165 mV. The midpoint potential for Fe(III)/Fe(II) acetate at pH 4.0 is much higher, a value of + 535 mV being obtained with 200 μM Fe. Cellobiose oxidase resembles yeast flavocytochrome b2 and differs from the neutrophil NADPH oxidase in having the potential of its haem group far above the potential for one‐electron reduction of O2 to superoxide (Em,4=−110 mV). A kinetic comparison led to the conclusion that the role of cellobiose oxidase is as an Fe(III) reductase. Fe(II) may have a biological importance as a component of Fenton's reagent [Fe(II)/H2O2]. The concentration of cellobiose oxidase in the growth medium at harvest (0.3 μM) can provide a far higher flux of Fe(II) than a non‐enzymic proposal in the literature.
The C‐terminal region of subunit 4 (subunit b) is essential for assembly of the F0 portion of yeast mitochondrial ATP synthase
Authors: PAUL Marie‐Françoise , GUERIN Bernard , VELOURS Jean
Published: March 2005

The role of the C‐terminal part of yeast ATP synthase subunit 4 (subunit b) in the assembly of the whole enzyme was studied by using nonsense mutants generated by site‐directed mutagenesis. The removal of at least the last 10 amino‐acid residues promoted mutants which were unable to grow with glycerol or lactate as carbon source. These mutants were devoid of subunit 4 and of another F0 subunit, the mitochondrially encoded subunit 6. The removal of the last eight amino‐acid residues promoted a temperature‐sensitive mutant (PVY161). At 37°C this strain showed the same phenotype as above. When grown at permissive temperature (30°C) with lactate as carbon source, PVY161 and the wild‐type strain both displayed the same generation time and growth yield. Furthermore, the two strains showed identical cellular respiration rates at 30°C and 37°C. However, in vitro the ATP hydrolysis of PVY161 mitochondria exhibited a low sensitivity to F0 inhibitors, while ATP synthesis displayed the same oligomycin sensitivity as wild‐type mitochondria. It is concluded that, in this mutant, the assembly of the truncated subunit 4 in PVY161 ATP synthase is thermosensitive and that, once a functional F0 is formed, it is stable. On the other hand, the removal of the last eight amino‐acid residues promoted in vitro a proton leak between the site of action of oligomycin and F1.
Sequence specific 1H‐NMR assignments and secondary structure of a carboxy‐terminal functional fragment of apolipoprotein CII
Authors: LYCKSELL Per‐Olof , ÖHMAN Anders , BENGTSSON‐OLIVECRONA Gunilla , JOHANSSON Lennart B.‐Å. , WIJMENGA Sybren S. , WERNIC Dominik , GRÄSLUND Astrid
Published: March 2005

The structural properties of a synthetic fragment of human apolipoprotein CII (apoCII) has been studied by circular dichroism and proton nuclear magnetic resonance. The fragment corresponds to the carboxy‐terminal 30 amino acid residues and retains the ability of apoCII to activate lipoprotein lipase. Like native apoCII, the fragment has a tendency to self‐associate in pure aqueous solution. Addition of 1,1,1,3,3,3‐hexafluoro‐2‐isopropanol to aqueous solvent dissolves the aggregates and leads to an increase in the α‐helical content of the peptide, probably by stabilizing transient helical structures. The resonances in the 1H‐NMR spectrum of the fragment in 35% (CF3)2CHOH were assigned through standard procedures from nuclear Overhauser enhancement spectroscopy, correlated spectroscopy and total correlated spectroscopy experiments. The NMR data indicates the formation of a stable α helix spanning Ile66–Gly77. Another α helical turn may be formed between Lys55 and Ala59 and possibly span even further towards the carboxyl terminus. These structural elements are different from those previously predicted for this part of the sequence of apoCII.
Specificity of the polycation‐stimulated (type‐2A) and ATP, Mg‐dependent (type‐1) protein phosphatases toward substrates phosphorylated by P34cdc2 kinase
Authors: AGOSTINIS Patrizia , DERUA Rita , SARNO Stefania , GORIS Jozef , MERLEVEDE Wilfried
Published: March 2005

p34cdc2 kinase, a critical regulator of the cell cycle, has been shown to recognize the consensus sequence S/TP in proteins such as histone H1, the retinoblastoma gene product RB and the carboxyl‐terminal domain of eukaryotic RNA polymerase II. Using phosphorylated synthetic peptides, representing the p34cdc2 phosphorylation sites in these proteins and histone H1 protein as substrates, we investigated the substrate specificity of the different oligomeric forms of the polycation‐stimulated (PCS/type‐2A) protein phosphatase and the active catalytic subunit of the ATP,Mg‐dependent (AMDc/type 1) protein phosphatase. The results show that the oligomeric structure of the PCS phosphatases is an important determinant for efficient dephosphorylation. The trimeric PCSH1 and PCSM phosphatases are about 10–20‐fold‐better histone H1 phosphatases than the dimeric PCSH2 and PCSL phosphatases and about 100‐fold better than the catalytic subunit (PCSC), suggesting a regulatory role for the 72‐kDa, 65‐kDa and 55‐kDa subunits. The RB peptide = INGS(P)PRT(P)PRRGQNR, is preferred over phosphorylase a (8‐fold) by the PCSH1 phosphatase and is about a 40‐fold and 95‐fold‐better substrate for the PCSH1 phosphatase than for the PCSM and PCSL phosphatases, respectively. The primary structure surrounding the S/T(P)P motif, by itself a strong negative determinant for dephosphorylation, can harbour positive features which relieve the constraint imposed by the carboxyl‐terminal proline. Thus, the RB peptide INGS(P)PRT(P)PRRGQNR, in which the T(P)P configuration is preferred over the S(P)P sequence, is an extremely good and specific substrate for the PCSH1 phosphatase (Km= 10 μM, Vmax= 3882 nmol · min−1· mg−1). The AMDC phosphatase is a poor phosphatase for all the phosphopeptides tested, unless Mn2+ is added. Its histone H1 phosphatase activity is much less sensitive than its phosphorylase a and phosphopeptide phosphatase activity to inhibition by the modulator or inhibitor‐1. The results strongly suggest a role for the trimeric PCSH1 phosphatase in reversing the p34cdc2 phosphorylations.
Assignment of Bacillus thermoamylolique faciens KP1071 α‐glucosidase I to an exo‐α‐1,4‐glucosidase, and its striking similarity to bacillary oligo‐1,6‐glucosidases in N‐terminal sequence and in structural parameters calculated from the amino acid composition
Authors: SUZUKI Yuzuru , YONEZAWA Kazuaki , HATTORI Masafumi , TAKII Yukio
Published: March 2005

α‐Glucosidase I of Bacillus thermoamyloliquefaciens KP1071 (FERM P8477, facultative thermophile) was purified to homogeneity. The relative molecular mass was estimated to be 62 000 Da. From its catalytic properties, the enzyme has been assigned to an exo‐α‐1,4‐glucosidase. The enzyme shares its antigenic groups in part with Bacillus stearothermophilus ATCC12016 (obligate thermophile) exo‐α‐1,4‐glucosidase.

These exo‐α‐1,4‐glucosidases strikingly resemble oligo‐1,6‐glucosidases from B. thermoamyloliquefaciens KP1071 and from Bacillus cereus ATCC7064 in the molecular properties tested, including relative molecular mass, N‐terminal sequence of 15 residues, amino acid composition and structural parameters calculated from amino acid composition. We have suggested that bacillary exo‐α‐1,4‐glucosidases take the same folded conformation, i.e. an (α/β)8‐barrel super‐secondary structure in its N‐terminal domain, as bacillary oligo‐1,6‐glucosidases.
Chick heart cells with high intracellular calcium concentration have a higher affinity for cardiac glycosides than those with low intracellular calcium concentration, as revealed by affinity labelling with a digoxigenin derivative
Authors: AHLEMEYER Barbara , WEINTRAUT Horst , ANTOLOVIĆA Roberto , SCHONER Wilhelm
Published: March 2005

Digital‐imaging fluorescence microscopy with fura‐2 allows the determination of intracellular calcium concentration ([Ca2+]i) in single cells. At a cell density of 105 cells/petri dish 44% of the chick embryo heart cells had a high [Ca2+]i of 99.4 ± 7.1 nM and 56% of the cells a low [Ca2+]i of 27.8 ± 4.4 nM (mean ± SE). This laboratory previously reported that high‐[Ca2+]i and low‐[Ca2+]i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca2+]i to reach a new steady state [Ahlemeyer, B., Weintraut, H., Seibold, G. & Schoner, W. (1991) in The sodium pump: recent developments (Kaplan, J. H. & De Weer, P., eds) pp. 653–656, Rockefeller University Press, New York]. This time we used N‐hydroxysuccinimidyl digoxigenin‐3‐O‐methylcarbonyl‐ε‐aminocaproate (HDMA) which binds irreversibly to amino groups of the Na+/K+‐ATPase, and sheep anti‐digoxigenin Fab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside‐binding sites. Half‐maximal labelling of high‐[Ca2+]i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low‐[Ca2+]i cells. Specific labelling of the cells by HDMA was 91% and 80% in high‐[Ca2+]i and low‐[Ca2+]i cells, respectively, as revealed by competition experiments with a 1000‐fold excess of ouabain. HDMA half‐maximally elevated the [Ca2+]i of high‐[Ca2+]i cells at a concentration of 50 pM and that of low‐[Ca2+]i cells at 8.0 nM. Concentrations higher than 0.1 μM produced signs of intoxication. When the labelled cells were subjected to a SDS/PAGE, a 100‐kDa band was found to contain HDMA. The electrophoretic mobility of a protein labelled at 10 nM HDMA was slightly higher than that of a protein labelled at 1.0 μM. The data suggest that different isoforms of the α‐subunit of Na+/K+‐ATPase may exist in low‐[Ca2+]i and high‐[Ca2+]i cells of chick embryo heart.
Transforming growth factor‐β induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts
Authors: WESTERGREN‐THORSSON Gunilla , SCHMIDTCHEN Artur , SÄRNSTRAND Bengt , FRANSSON Lars‐Åke , MALMSTRÖM Anders
Published: March 2005

Human embryonic skin fibroblasts were pretreated with transforming growth factor‐β (TGF‐β) for 6 h and then labeled with [35S]sulphate and [3H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density‐gradient centrifugation, followed by gel, ion‐exchange and hydrophobic‐interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains.

The results showed that TGF‐β increased the production of several different 35S‐labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400–500 kDa) increased 5–7‐fold and a small dermatan sulphate proteoglycan (PG‐S1, also termed biglycan, with a core protein of 43 kDa) increased 3–4‐fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG‐S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF‐β treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected.

To investigate whether TGF‐β also influences the glycosaminoglycan (GAG) chain‐synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF‐β‐treated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF‐β treated cultures contained a larger proportion of D‐glucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4‐ and 6‐sulphation of the GAG chains. By the use of p‐nitrophenyl β‐d‐xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF‐β‐treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF‐β‐treated fibroblasts contained a larger proportion of D‐glucuronosyl residues than those of the control.

These novel findings indicate that TGF‐β affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAG‐synthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, which can affect cell growth and migration as well as organisation of matrix fibres.
Mitogen‐activated protein kinases phosphorylate nuclear lamins and display sequence specificity overlapping that of mitotic protein kinase p34cdc2
Authors: PETER Matthias , SANGHERA Jasbinder S. , PELECH Steven L. , NIGG Erich A.
Published: March 2005

Members of the mitogen‐activated protein (MAP) kinase family are implicated in mediating entry of cells into the cell cycle, as well as passage through meiotic M phase. These kinases have attracted much interest because their activation involves phosphorylation on both tyrosine and threonine residues, but little is known about their physiological targets. In this study, two distinct members of the MAP kinase family (p44mpk and p42mapk) are shown to phosphorylate chicken lamin B2 at a single site identified as Ser16. Moreover, these MAP kinases cause depolymerization of in‐vitro‐assembled longitudinal lamin head‐to‐tail polymers. Ser16 was previously shown to be phosphorylated during mitosis in vivo, and to be a target of the mitotic protein kinase p34cdc2in vitro. Accordingly, lamins were proposed to be direct in vivo substrates of p34cdc2. This proposal is supported by quantitative analyses indicating that lamin B2, when assayed in vitro, is a substantially better substrate for p34cdc2 than for MAP kinases. Nevertheless, a physiological role of MAP kinases in lamin phosphorylation is not excluded. The observation that members of the MAP kinase family display sequence specificities overlapping that of p34cdc2 raises the possibility that some of the purported substrates of p34cdc2 may actually be physiological substrates of MAP kinases.
Processing of interleukin‐1 in cells of monocytic lineage is differentiation‐dependent
Authors: CARINCI Valeria , GUIDA Serena , FONTANA Maria Rita , PALLA Emanuela , ROSSINI Mara , MELLI Marialuisa
Published: March 2005

The interleukin‐1 (IL‐1) α and β precursor proteins are processed and released from several cell types in the absence of a canonical signal peptide. To gain some insight into the mechanisms that allow the production of IL‐1α and β, we have investigated by immunoprecipitation the synthesis, their release and processing in a promieloblastic cell line of tumoral origin, U937, and in peripheral blood monocytes.

We show that U937 monocytic cells, on induction with a tumor‐promoting agent, synthesize and release into the culture medium proIL‐1β but do not process it. Similarly, peripheral blood monocytes left in adherence for 24 h or longer, prior to addition of lipopolysaccharide, synthesize and release proIL‐1α and β without detectable processing of either cytokine.

Processing and release of IL‐1α and β by peripheral blood monocytes can be observed when monocytes are left to adhere for periods less than 15 h before lipopolysaccharide addition. IL‐1α and β show similar kinetics of release from the cells, suggesting the existence of a common mechanism regulating their secretion. Since peripheral blood monocytes left in adherence in the presence of lipopolysaccharide differentiate into macrophages, we conclude that release and processing of IL‐1 can occur independently and that processing depends on the stage of differentiation of monocytes, i.e. only the monocytes at an early stage of differentiation produce 17‐kDa IL‐1α and β.
Placental alkaline phosphatase has a binding site for the human immunoglobulin‐G Fc portion
Authors: MAKIYA Ricardo , STIGBRAND Torgny
Published: March 2005

Affinity chromatography of human plasma on placental‐alkaline‐phosphatase–Sepharose columns (placental alkaline phosphatase, PLAP) yielded consistently a pure protein which was identified as IgG on the basis of electrophoretical and immunological comparisons with authentic human IgG. SDS/PAGE of the protein revealed, under reducing conditions, two polypeptides of 55 kDa and 25 kDa. The N‐terminal amino acid sequence (12 residues) of the 55‐kDa subunit presented high similarity (83–100%) with known sequences of immunoglobulin gamma chains. The IgG binds by its Fc portion to a fully exposed domain in the plasma‐membrane‐anchored PLAP. Scatchard analysis of the interaction gave a dissociation constant of 3.68 γM, a value close to those found for haematopoietic cells and syncytiotrophoblast Fc receptors. The latter was affinity purified from human placenta as the major IgG‐binding component and presented cross‐immunoreactivity with anti‐PLAP antibodies, indicating that PLAP and the putative placental Fc receptor could be identical molecules.
Propolypeptide and mature portions of von Willebrand factor of bovine origin recognize different sites on type‐I collagen obtained from bovine tendon
Authors: USUI Tomoko , FUJISAWA Tomoyuki , TAKAGI Junichi , SAITO Yuji
Published: March 2005

We compared the binding of propolypeptide and mature portions of von Willebrand factor of bovine origin to fibrillar type‐I collagen obtained from bovine tendon. The propolypeptide (pp‐vWF) and the mature portion (m‐vWF) of human origin consist of 741 and 2050 amino acids, respectively, and are rather large proteins. The collagen‐binding properties of the two proteins of bovine origin were similar in that both bound more avidly to native collagen than to heat‐denatured collagen. Bindings was affected similarly by ionic strength but was not modified either by divalent cations or a synthetic peptide containing Arg‐Gly‐Asp. However, the binding sites in the fibrillar type‐I collagen molecule for pp‐vWF and m‐vWF seem to be different: the two proteins did not effectively compete with each other for binding to collagen. Furthermore, pepsin treatment of fibrillar type‐I collagen resulted in a drastic decrease in the binding of pp‐vWF, while only a moderate decrease in the binding of m‐vWF was observed after the treatment.