bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers
Authors: Baykov Alexander A. , Hyytiä Teppo , Turkina Maria V. , Efimova Irina S. , Kasho Vladimir N. , Goldman Adrian , Cooperman Barry S. , Lahti Reijo
Published: March 2003

Catalysis by Escherichia coli inorganic pyrophosphatase (E‐PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand‐binding and catalytic properties of E‐PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg2+‐binding affinity for two of the three sites present in E‐PPase (3.5‐ to 16‐fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20‐ to 40‐fold). By contrast, Mg2+‐binding and substrate conversion in the enzyme‐substrate complex are unaffected by buffer. These data indicate that E‐PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer–trimer interface. Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16‐fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E‐PPase active site.
Identification of cyclin A/Cdk2 phosphorylation sites in B‐Myb
Authors: Bartsch Olaf , Horstmann Sebastian , Toprak Kaan , Klempnauer Karl‐Heinz , Ferrari Stefano
Published: March 2003

B‐myb is a highly conserved member of the myb proto‐oncogene family that encodes a ubiquitously expressed 110‐kDa sequence‐specific DNA‐binding protein. Transactivation of Myb‐inducible promoters by B‐Myb is repressed by a regulatory domain located at the C‐terminus of the protein. Cyclin A/Cdk2‐mediated phosphorylation apparently releases the negative constraint and triggers B‐Myb transactivation potential. Two‐dimensional tryptic phosphopeptide analysis indicated that the majority of the sites phosphorylated in vivo are targeted in vitro by cyclin A/Cdk2. Six sites in B‐Myb fulfil the requirements for recognition by Cdk2. Using point mutation of the phosphorylation sites to nonphosphorylatable amino acids, we show that five of these sites are targets for Cdk2 in vivo. Mutation of one of these residues (T524) to alanine diminished the ability of B‐Myb to promote transcription of a reporter gene, suggesting that phosphorylation of B‐Myb at this site is important for the regulation of its activity by cyclin A/Cdk2.
Molecular cloning, structural characterization and chromosomal localization of human lipoyltransferase gene
Authors: Fujiwara Kazuko , Suzuki Mikio , Okumachi Yasuyo , Okamura‐Ikeda Kazuko , Fujiwara Tsutomu , Takahashi Ei‐ichi , Motokawa Yutaro
Published: March 2003

Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl‐AMP to the lysine residue of the lipoate‐dependent enzymes. We isolated human lipoyltransferase cDNA and genomic DNA. The cDNA insert contained a 1119‐base pair open reading frame encoding a precursor peptide of 373 amino acids. Predicted amino acid sequence of the protein shares 88 and 31% identity with bovine lipoyltransferase and Escherichia coli lipoate‐protein ligase A, respectively. Northern blot analyses of poly(A)+ RNA indicated a major species of about 1.5 kb. mRNA levels of lipoyltransferase were highest in skeletal muscle and heart, showing good correlation with those of dihydrolipoamide acyltransferase subunits of pyruvate, 2‐oxoglutarate and branched‐chain 2‐oxo acid dehydrogenase complexes and H‐protein of the glycine cleavage system which accept lipoic acid as a prosthetic group. The human lipoyltransferase gene is a single copy gene composed of four exons and three introns spanning approximately 8 kb of genomic DNA. Some alternatively spliced mRNA species were found by 5′‐RACE analysis, and the most abundant species lacks the third exon. The human lipoyltransferase gene was localized to chromosome band 2q11.2 by fluorescence in situ hybridization.
Identification of positively charged residues of FomA porin of Fusobacterium nucleatum which are important for pore function
Authors: Kleivdal Hans , Benz Roland , Tommassen Jan , Jensen Harald B.
Published: March 2003

FomA porin is the major outer‐membrane protein of Fusobacterium nucleatum. It exhibits the functional properties of a general diffusion porin, but has no sequence similarity to other porins. According to the proposed topology model, each monomer of this trimeric protein is a β‐barrel consisting of 16 transmembrane segments with eight surface‐exposed loops. Several conserved charged residues are proposed to extend from the β‐barrel wall into the aqueous channel lumen, and may contribute to a transverse electric field similar to that at the pore constriction of porins with known structure. The goal of our study was to identify particular basic residues contributing to such an electric field in FomA. Several arginines and lysines were replaced by negatively charged glutamates or uncharged alanines. The mutated FomA porins were expressed in Escherichia coli, and the effects on pore function were studied in vivo, by assaying the uptake rate of β‐lactam antibiotics, and in vitro after reconstitution of the purified proteins in lipid bilayer membranes. Some of the point mutations had a significant impact on the channel properties. The substitution R92A produced a 130% increased permeability of the zwitterionic β‐lactam cephaloridine, and the cation selectivity of R92E increased by 70%. The effects of the R90E substitution on channel properties were similar. Most of the point mutations had a minor effect on the voltage gating of the FomA channel, resulting in an increased sensitivity, except for K78E, which showed a decreased sensitivity. The latter mutation had no effect on cation selectivity, but the K78A substitution improved the uptake rate of cephaloridine. The results presented here indicate that arginines 90 and 92 are probably part of the constriction zone of the FomA porin, and lysine 78 and arginines 115 and 117 are probably in close proximity to this region as well.
Phosphorylation of the α‐subunits of the Na+/K+‐ATPase from mammalian kidneys and Xenopus oocytes by cGMP‐dependent protein kinase results in stimulation of ATPase activity
Authors: Fotis Heike , Tatjanenko Liliya V. , Vasilets Larisa A.
Published: March 2003

Phosphorylation of Na+/K+‐ATPase by cGMP‐dependent protein kinase (PKG) has been studied in enzymes purified from pig, dog, sheep and rat kidneys, and in Xenopus oocytes. PKG phosphorylates the α‐subunits of all animal species investigated. Phosphorylation of the β‐subunit was not observed. The stoichiometry of phosphorylation estimated for pig, sheep and dog renal Na+/K+‐ATPase is 3.5, 2.2 and 2.1 mol Pi per mol α‐subunit, respectively. Proteolytic fingerprinting of the pig α1‐subunits phosphorylated by PKG using specific antibodies raised against N‐terminus or C‐terminus reveals that phosphorylation sites are located within the intracellular loop of the α‐subunit between the 35 kDa N‐terminal and 27 kDa C‐terminal fragments. Phosphorylation sites within the α1‐subunit of the purified Na+/K+‐ATPase do not appear to be easily accessible for PKG since incorporation of Pi requires 0.2% of Triton X‐100. Administration of cGMP and PKG in the presence of 5 mm ATP, which prevents inactivation of the Na+/K+‐ATPase by detergent, leads to stimulation of hydrolytic activity by 61%. Administration of 50 µm of cGMP or dbcGMP in yolk‐free homogenates of Xenopus oocytes leads to stimulation of ouabain‐dependent ATPase activity by 130–198% and to incorporation of 33P into the α‐subunit without the detergent. Hence, PKG plays regulatory role in active transmembraneous transport of Na+ and K+ via phosphorylation of the catalytic subunit of the Na+/K+‐ATPase.
Interaction of purified human proteinase 3 (PR3) with reconstituted lipid bilayers
Authors: Goldmann Wolfgang H. , Niles John L. , Arnaout M. Amin
Published: March 2003

Proteinase 3 (PR3), the major target autoantigen in Wegener’s granulomatosis is a serine proteinase that is normally stored intracellularly in the primary granules of quiescent neutrophils and monocytes. Upon cell activation, a significant portion of this antigen is detected on the cell surface membrane. The nature of the association of PR3 with the membrane and its functional significance are unknown. We investigated the interaction of purified human PR3 with mixtures of zwitterionic (dimyristoyl‐l‐α‐phosphatidylcholine, DMPC) and anionic (dimyristoyl‐l‐α‐phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers using differential scanning calorimetry and lipid photolabeling, and measured the affinity of this interaction using spectrophotometry. Two other primary granule constituents, human neutrophil elastase (HNE) and myeloperoxidase (MPO) were investigated for comparison. In calorimetric assays, using lipid vesicles of mixed DMPC/DMPG, increasing PR3 concentrations (protein/lipid molar ratio from 0 to 1 : 110) induced a significant decrease of the main chain transition enthalpy and a shift in chain melting temperatures which is indicative of partial insertion of PR3 into the hydrophobic region of the lipid membranes. This was confirmed by hydrophobic photolabeling using liposomes containing trace amounts of the photoactivable [125I]‐labeled phosphatidylcholine analog TID‐PC/16. The molar affinity of PR3, HNE, and MPO to lipid vesicles of different DMPC/DMPG ratios was then determined by spectrophotometry. At a DMPC/DMPG ratio of 1 : 1, molar affinities of PR3, Kd = 4.5 ± 0.3 µm; HNE, 14.5 ± 1.2 µm; and MPO, 50 ± 5 µm (n = 3) were estimated. The lipid‐associated PR3 exhibited two‐fold lower Vmax and Km values, and its enzyme activity was slightly more inhibited (Ki) by the natural α1‐proteinase inhibitor (α1‐PI) or an autoantibody to PR3.
Cloning and structural analysis of leydin, a novel human serine protease expressed by the Leydig cells of the testis
Authors: Poorafshar Maryam , Hellman Lars
Published: March 2003

We present the cloning and structural analysis of a novel member of the large family of trypsin‐related serine proteases. Northern blot analysis shows that this protease, in adult tissues, is expressed almost exclusively in the human testis. In addition, a larger transcript was detected in relatively high abundance in several embryonic tissues, indicating different functions during embryonic and adult life. Sera raised against this protease was used to locate the expression in adult tissues to the testosterone producing cells of the testis, the interstitial Leydig cells. We therefore propose the name leydin for this novel protease. Leydin is clearly distinct from acrosin, the other testis‐specific serine protease which is expressed by the spermatocytes. Leydin is probably a two‐chain protease such as acrosin, prostasin, and coagulation factor XI. The heavy chain consists of 246 amino acids, corresponding to a molecular mass of 27384 Da and a net charge of +10.76. The size of the light chain is between 9 and 18 amino acids depending on the site of proteolytic cleavage, which remains to be determined. The amino‐acid residues surrounding the active site indicate a trypsin‐like cleavage specificity. The presence of two dibasic sequences Arg‐Arg and Lys‐Arg at the N‐terminus of the heavy chain indicate that one or more subtilisin‐like endopeptidases are responsible for the processing of leydin. However, leydin may also be activated by a trypsin‐like enzyme, possibly by auto catalysis.
Characterization of low‐molecular‐mass trypsin isoinhibitors from oil‐rape (Brassica napus var. oleifera) seed
Authors: Ascenzi Paolo , Ruoppolo Margherita , Amoresano Angela , Pucci Piero , Consonni Roberto , Zetta Lucia , Pascarella Stefano , Bortolotti Fabrizio , Menegatti Enea
Published: March 2003

A new low‐molecular‐mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil‐rape (Brassica napus var. oleifera) seed, designated 5‐oxoPro1‐Gly62‐RTI‐III. The 5‐oxoPro1‐Gly62‐RTI‐III isoinhibitor is longer than the Asp2‐Pro61‐RTI‐III and the Ser3‐Pro61‐RTI‐III forms, all the other amino acid residues being identical. In RTI‐III isoinhibitors, the P1‐P1′ reactive site bond (where residues forming the reactive site have been identified as Pn…P1 and P1′…Pn′, where P1‐P1′ is the inhibitor scissile bond) has been identified at position Arg21‐Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5‐Cys27, Cys18‐Cys31, Cys42‐Cys52 and Cys54‐Cys57. The disulphide bridge arrangement observed in the RTI‐III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5‐Cys27, Cys18‐Cys31 and Cys42‐Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H‐NMR data indicates the presence of ααNOEs and 3JαNH coupling constants, typical of the β‐structure(s). These data suggest that the three‐dimensional structure of the RTI‐III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three‐finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI‐III isoinhibitors binding to bovine β‐trypsin and bovine α‐chymotrypsin are 3.3 × 109 m−1 and 2.4 × 106 m−1, respectively, at pH 8.0 and 21.0 °C. The serine proteinase : inhibitor complex formation is a pH‐dependent entropy‐driven process. RTI‐III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI‐2). Therefore, RTI‐III and MTI‐2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.
Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus
Authors: Agalidis Ileana , Othman Samya , Boussac Alain , Reiss‐Husson Françoise , Desbois Alain
Published: March 2003

The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction center was isolated in the presence of octyl β‐d‐thioglucoside by ammonium sulfate precipitation and solubilization at pH 9 in a solution of Deriphat 160. Several biochemical properties of this purified cytochrome were characterized. In particular, it forms small oligomers and its N‐terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate and dithionite, different oxidation/reduction states of the isolated cytochrome were studied by absorption, EPR and resonance Raman spectroscopies. All the data show two hemes quickly reduced by ascorbate, one heme slowly reduced by ascorbate and one heme only reduced by dithionite.

 The quickly ascorbate‐reduced hemes have paramagnetic properties very similar to those of the two low‐potential hemes of the reaction center‐bound cytochrome (gz = 3.34), but their α band is split with two components peaking at 552 nm and 554 nm in the reduced state. Their axial ligands did not change, being His/Met and His/His, as indicated by the resonance Raman spectra. The slowly ascorbate‐reduced heme and the dithionite‐reduced heme are assigned to the two high‐potential hemes of the bound cytochrome. Their α band was blue‐shifted at 551 nm and the gz values decreased to 2.96, although the axial ligations (His/Met) were conserved. It was concluded that the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in gz indicates an increased symmetry of their cooordination spheres. These structural modifications impaired the cytochrome’s essential function as the electron donor to the photooxidized bacteriochlorophyll dimer of the reaction center. In contrast to its native state, the isolated cytochrome was unable to reduce efficiently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the cytochrome, its four hemes are still divided into two groups with a pair of low‐potential hemes and a pair of high‐potential hemes.
Fluorescence measurements detect changes in scallop myosin regulatory domain
Authors: Málnási‐Csizmadia András , Hegyi György , Tölgyesi Ferenc , Szent‐Györgyi Andrew G. , Nyitray László
Published: March 2003

Ca2+‐induced conformational changes of scallop myosin regulatory domain (RD) were studied using intrinsic fluorescence. Both the intensity and anisotropy of tryptophan fluorescence decreased significantly upon removal of Ca2+. By making a mutant RD we found that the Ca2+‐induced fluorescence change is due mainly to Trp21 of the essential light chain which is located at the unusual Ca2+‐binding EF‐hand motif of the first domain. This result suggests that Trp21 is in a less hydrophobic and more flexible environment in the Ca2+‐free state, supporting a model for regulation based on the 2 Å resolution structure of scallop RD with bound Ca2+[Houdusse A. and Cohen C. (1996) Structure4, 21–32]. Binding of the fluorescent probe, 8‐anilinonaphthalene‐1‐sulphonate (ANS) to the RD senses the dissociation of the regulatory light chain (RLC) in the presence of EDTA, by energy transfer from a tryptophan cluster (Trp818, 824, 826, 827) on the heavy chain (HC). We identified a hydrophobic pentapeptide (Leu836–Ala840) at the head–rod junction which is required for the effective energy transfer and conceivably is part of the ANS‐binding site. Extension of the HC component of RD towards the rod region results in a larger ANS response, presumably indicating changes in HC–RLC interactions, which might be crucial for the regulatory function of scallop myosin.
A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1
Authors: Pomper Barbara K. , Vorholt Julia A. , Chistoserdova Ludmila , Lidstrom Mary E. , Thauer Rudolf K.
Published: March 2003

Recently it was found that Methylobacterium extorquens AM1 contains both tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F) as carriers of C1 units. In this paper we report that the aerobic methylotroph contains a methenyl H4MPT cyclohydrolase (0.9 U·mg−1 cell extract protein) and a methenyl H4F cyclohydrolase (0.23 U·mg−1). Both enzymes, which were specific for their substrates, were purified and characterized and the encoding genes identified via the N‐terminal amino acid sequence. The purified methenyl H4MPT cyclohydrolase with a specific activity of 630 U·mg−1 (Vmax = 1500 U·mg−1; Km = 30 µm) was found to be composed of two identical subunits of molecular mass 33 kDa. Its sequence was ≈ 40% identical to that of methenyl H4MPT cyclohydrolases from methanogenic archaea. The methenyl H4F cyclohydrolase with a specific activity of 100 U·mg−1 (Vmax = 330 U·mg−1; Km = 80 µm) was found to be composed of two identical subunits of molecular mass 22 kDa. Its sequence was not similar to that of methenyl H4MPT cyclohydrolases or to that of other methenyl H4F cyclohydrolases. Based on the specific activities in cell extract and from the growth properties of insertion mutants it is suggested that the methenyl H4MPT cyclohydrolase might have a catabolic, and the methenyl‐H4F cyclohydrolase an anabolic function in the C1‐unit metabolism of M. extorquens AM1.
Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA
Authors: Appleby Ruth D. , Porteous William K. , Hughes Gillian , James Andrew M. , Shannon Daniel , Wei Yau‐Huei , Murphy Michael P.
Published: March 2003

Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed ρ° cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in ρ° cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP4–for ADP3–by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete FoF1‐ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin‐permeabilized mammalian ρ° cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in ρ° cells and an incomplete FoF1‐ATP synthase was also required. The maximum value of this potential was ≈110 mV in permeabilized cells and ≈ 67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete FoF1‐ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear‐encoded proteins but ≈ 65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of ρ° mitochondria showed that the azide‐sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete FoF1‐ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete FoF1‐ATP synthase in maintaining the mitochondrial membrane potential in ρ° cells.
Active‐site mutations which change the substrate specificity of the Clostridium stercorarium cellulase CelZ
Authors: Riedel Kathrin , Bronnenmeier Karin
Published: March 2003

CelZ from the cellulolytic thermophile Clostridium stercorarium has been described as a ‘monomeric’ cellulase able to effect both the endoglucanolytic hydrolysis of internal glycosidic linkages and the exoglucanolytic degradation from the chain ends in a processive mode of action. The putative catalytic residues of this family 9 cellulase, Asp84 and Glu447 located within the N‐terminal domain of the modular protein, were replaced by site‐directed mutagenesis. A minimized CelZ derivative (CelZC′) comprising the catalytic domain and the adjacent cellulose‐binding domain (CBD) family IIIc domain C′ was used as target for mutagenesis. Six mutant enzymes and the unmodified CelZC′ protein were purified to homogeneity and compared with respect to thermoactivity, substrate specificity, product profile and synergism. CD studies revealed that no major changes to the overall structure of the proteins had occurred. Replacement of either one or both catalytic residues completely eliminated the ability of CelZ to attack insoluble Avicel preparations indicative of the exo‐activity, whereas the endo‐activity measured via hydrolysis of CM‐cellulose was retained upon substitution of the catalytic base Asp84. Thus, endo‐active CelZ mutants defective in the exo‐activity were available for co‐operativity studies with the C. stercorarium exoglucanase CelY. Synergism was found to be dependent on the endo‐activity of CelZ. Mutants Asp84Gly and Asp84Glu were able to enhance the degradation of crystalline cellulose significantly, although no products could be released from this substrate by individual action of the mutants.
Purification and characterization of the 16‐kDa heat‐shock‐responsive protein from the thermophilic cyanobacterium Synechococcus vulcanus, which is an α‐crystallin‐related, small heat shock protein
Authors: Roy Sanjit K. , Hiyama Tetsuo , Nakamoto Hitoshi
Published: March 2003

A 16‐kDa protein, one of the major proteins that accumulates upon heat‐shock treatment in the thermophilic cyanobacterium Synechococcus vulcanus, was purified to apparent homogeneity. The N‐terminal and internal amino acid sequences of the protein exhibited a homology to the α‐crystallin‐related, small heat shock proteins from other organisms. The protein was designated HspA. Size‐exclusion chromatography and nondenaturing gel electrophoresis demonstrated that HspA formed a large homo‐oligomer consisting of 24 subunits. It prevented the aggregation of porcine malic dehydrogenase at 45 °C and 50 °C and citrate synthase at 50 °C. The activity of the malic dehydrogenase, however, was not protected under these heat‐shock conditions or reactivated after a shift in temperature from 45 or 50 °C to 21 °C. HspA was able to enhance the refolding of chemically denatured rabbit muscle lactate dehydrogenase in an ATP‐independent manner. A homologue to the 16‐kDa protein was also found to be induced upon heat‐shock treatment in the mesophilic cyanobacterium Synechocystis sp. PCC 6803.
Phospholipase C‐δ3 binds with high specificity to phosphatidylinositol 4,5‐bisphosphate and phosphatidic acid in bilayer membranes
Authors: Pawelczyk Tadeusz , Matecki Andrzej
Published: March 2003

In order to acquire an understanding of phospholipase C–δ3 (PLC–δ3) action on substrate localized in lipid membrane we have studied the binding of human recombinant PLC–δ3 to large, unilamellar phospholipid vesicles (LUVs). PLC‐δ3 bound weakly to vesicles composed of phosphatidylcholine (PtdCho) or PtdCho plus phosphatidylethanolamine (PtdEtn) or phosphatidylinositol (PtdIns). The enzyme bound strongly to LUVs composed of PtdEtn + PtdCho and phosphatidylinositol 4,5‐bisphosphate (PtdInsP2). The binding affinity (molar partition coefficient) of PLC‐δ3 to PtdEtn + PtdCho + PtdInsP2 vesicles was 7.7 × 105m−1. High binding of PLC‐δ3 was also observed for LUVs composed of phosphatidic acid (PA). Binding of PLC‐δ3 to phosphatidylserine (PtdSer) vesicles was less efficient. Calculated molar partition coefficient for binding of PLC‐δ3 to PA and PtdSer vesicles was 1.6 × 104 m−1 and 9.4 × 102 m−1, respectively. Presence of PA in the LUVs containing PtdInsP2 considerably enhanced the binding of PLC‐δ3 to the phospholipid membrane. Binding of PLC‐δ3 to phospholipid vesicles was not dependent on Ca2+ presence. In the liposome assay PA caused a concentration‐dependent increase in activity of PLC‐δ3. The stimulatory effect of PA on PLC‐δ3 was calcium‐dependent. At Ca2+ concentrations lower than 1 µm, no effect of PA on the activity of PLC‐δ3 was observed. PA enhanced PLC‐δ3 activity by increasing the Vmax and lowering Km for PtdInsP2. As the mol fraction of PA increased from 0–40 mol% the enzyme Vmax increased 2.3‐fold and Km decreased threefold. Based on the results presented, we assume that PA supports binding of PLC‐δ3 to lipid membranes by interaction with the PH domain of the enzyme. The stimulatory effect of PA depends on calcium‐dependent interaction with the C2 domain of PLC‐δ3. We propose that binding of PLC‐δ3 to PA may serve as a mechanism for dynamic membrane association and modulation of PLC‐δ3 activity.
Biosynthesis of the vacuolar H+‐ATPase accessory subunit Ac45 in Xenopus pituitary
Authors: Holthuis Joost C. M. , Jansen Eric J. R. , Schoonderwoert Vincent Th. G. , Burbach J. Peter H. , Martens Gerard J. M.
Published: March 2003

Vacuolar H+‐ATPases (V‐ATPases) mediate the acidification of multiple intracellular compartments, including secretory granules in which an acidic milieu is necessary for prohormone processing. A search for genes coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of Xenopus intermediate pituitary led to the isolation of a cDNA encoding the complete amino‐acid sequence of the type I transmembrane V‐ATPase accessory subunit Ac45 (predicted size 48 kDa). Comparison of Xenopus and mammalian Ac45 sequences revealed conserved regions in the protein that may be of functional importance. Western blot analysis showed that immunoreactive Ac45 represents a ≈ 40‐kDa product that is expressed predominantly in neuroendocrine tissues; deglycosylation resulted in a ≈ 27‐kDa immunoreactive Ac45 product which is smaller than predicted for the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an N‐glycosylated protein of ≈ 60 kDa; the nonglycosylated, newly synthesized form is ≈ 46 kDa which is similar to the predicted size. Immunocytochemical analysis showed that in Xenopus pituitary, Ac45 is highly expressed in the biosynthetically active melanotrope cells. We conclude that the regionally conserved Xenopus Ac45 protein is synthesized as an N‐glycosylated ≈ 60‐kDa precursor that is intracellularly cleaved to an ≈ 40‐kDa product and speculate that it may assist in the V‐ATPase‐mediated acidification of neuroendocrine secretory granules.
Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins
Authors: Corti Chantal , LeClerc L’Hostis Estelle , Quadroni Manfredo , Schmid Holger , Durussel Isabelle , Cox Jos , Dainese Hatt Paola , James Peter , Carafoli Ernesto
Published: March 2003

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3′‐5′‐cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+‐ATPase, Ca2+‐CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+‐calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+‐ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased theVmax. For MLCK, however, neitherVmaxnorKactwere affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM‐binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.
Grass group I allergens (β‐expansins) are novel, papain‐relatedproteinases
Authors: Grobe Kay , Becker Wolf‐Meinhard , Schlaak Max , Petersen Arnd
Published: March 2003

Expansins are a family of proteins that catalyse long‐term extension of isolated plant cell walls due to an as yet unknown biochemical mechanism. They are divided into two groups, the α‐expansins and β‐expansins, the latter group consisting of grass group I allergens and their vegetative homologs. These grass group I allergens, to which more than 95% of patients allergic to grass pollen possess IgE antibodies, are highly immunologically crossreactive glycoproteins exclusively expressed in pollen of all grasses. Alignments of the amino‐acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function. The major grass group I allergen from timothy grass (Phleum pratense), Phl p 1, was chosen as a model glycoprotein and expressed in the methylotrophic yeast Pichia pastoris to obtain a post‐translationally modified and functionally active allergen. The recombinant allergen exhibited proteolytic activity when assayed with various test systems and substrates, which was also subsequently demonstrated with the natural protein, nPhl p 1. These observations are confirmed by amino‐acid alignments of Phl p 1 with three functionally important sequence motifs surrounding the active‐site amino acids of the C1 (papain‐like) family of cysteine proteinases. Moreover, the significantly homologous α‐expansins mostly share the functionally important C1 sequence motifs. This leads us to propose a C1 cysteine proteinase function for grass group I allergens, which may mediate plant cell wall growth and possibly contributes to the allergenicity of the molecule.
Effect of gel re‐organization and tensional forces on α2β1 integrin levels in dermal fibroblasts
Authors: Jenkins Gail , Redwood Katherine L. , Meadows Lisa , Green Martin R.
Published: March 2003

Mechanical forces are known to play an important role in regulating cell function in a wide range of biological systems. This is of particular relevance to dermal fibroblast function, given that the skin is known to be held under an intrinsic natural tension. To understand more about the generation of force by dermal fibroblasts and their ability to respond to changes in it, we have studied the role of the β1 integrin receptors expressed by dermal fibroblasts in their ability to generate tensional forces within a collagen type I matrix and the effect of altered tensional force on integrin expression by dermal fibroblasts.

 Using a purpose‐built culture force monitor, function‐blocking antibodies directed towards the β1 receptors dramatically reduced the tensional forces generated by dermal fibroblasts in a 3D collagen I matrix. However, the specific involvement of α1 or α2 subunits could not be demonstrated.

 Analysis of cellular response demonstrated that cells isolated from contracting collagen gels expressed fourfold higher levels of α2 mRNA than cells isolated from fully restrained gels. The levels of β1 messenger RNA were relatively unaffected by reductions in force. Cells exposed to single reductions in force, however, did not exhibit alterations in either α1 or β1 mRNA levels. We propose, therefore that α2β1 integrin receptor levels in dermal fibroblasts are not altered in response to single reductions of gel tension, but do change following a continual change in force and associated matrix re‐organization
Two forms of cytotoxin II (cardiotoxin) from Naja naja oxiana in aqueous solution
Authors: Dementieva Daria V. , Bocharov Eduard V. , Arseniev Alexander. S.
Published: March 2003

1H‐NMR spectroscopy data, such as NOE intraprotein and (bound water)/protein contacts, 3J coupling constants and deuterium exchange rates were used to determine the in‐solution spatial structure of cytotoxin II from Naja naja oxiana snake venom (CTII). Exploiting information from two 1H‐NMR spectral components, shown to be due to cis/trans isomerization of the Val7–Pro8 peptide bond, spatial structures of CTII minor and major forms (1 : 6) were calculated using the torsion angle dynamics algorithm of the dyana program and then energy refined using the fantom program. Each form, major and minor, is represented by 20 resulting conformers, demonstrating mean backbone rmsd values of 0.51 and 0.71 Å, respectively. Two forms of CTII preserve the structural skeleton as three large loops, including two β‐sheets with bend regions, and demonstrate structural differences at loop I, where cis/trans isomerization occurs. The CTII side‐chain distribution constitutes hydrophilic and hydrophobic belts around the protein, alternating in the trend of the three main loops. Because of the Ω‐shaped backbone, formed in participation with two bound water molecules, the tip of loop II bridges the tips of loops I and III. This ensures the continuity of the largest hydrophobic belt, formed with the residues of these tips. Comparison revealed pronounced differences in the spatial organization of the tips of the three main loops between CTII and previous structures of homologous cytotoxins (cardiotoxins) in solution.
Colocalization of sterol isomerase and sigma1 receptor at endoplasmic reticulum and nuclear envelope level
Authors: Dussossoy Danielle , Carayon Pierre , Belugou Suzanne , Feraut Danièle , Bord Annie , Goubet Catherine , Roque Claude , Vidal Hubert , Combes Thérèse , Loison Gérard , Casellas Pierre
Published: March 2003

SR31747A is a sigma ligand previously described as having original immunosuppressive properties. Two SR31747A targets were recently identified and termed sigma1 or SR‐BP‐1 (SR31747A‐binding protein‐1) and hSI (human sterol isomerase). In order to characterize these proteins further, we examined their expression and localization at the subcellular level. Based on the amino acid sequence deduced from the cloned hSI, anti‐hSI polyclonal antibody was raised against the N‐terminal fragment of the protein. Using this antibody, we performed Western‐blot experiments to demonstrate the presence of hSI in various B and T cell lines, and hSI expression was quantified in these cell lines by flow cytometry and estimated at 15 000–30 000 sites per cell. Subcellular localization studies by both confocal and electron microscopy, performed on THP1 cells with anti‐hSI antibody and with the previously described anti‐(SR‐BP‐1) monoclonal antibody, demonstrated that: (a) hSI was colocalized with SR‐BP‐1; (b) hSI and SR‐BP‐1 were associated with the endoplasmic reticulum and with the outer and inner membranes of the nuclear envelope; (c) both proteins were delocalized during the cell cycle at the mitosis step when the nuclear membranes disappeared. Taken together our results suggest that both SR31747A‐binding proteins not only play a role in sterol metabolism but indirectly affect lipoprotein functions.
Nitrile hydratase involved in aldoxime metabolism from Rhodococcus sp. strain YH3‐3
Authors: Kato Yasuo , Tsuda Takashi , Asano Yasuhisa
Published: March 2003

Nitrile hydratase responsible for aldoxime metabolism from the E‐pyridine‐3‐aldoxime degrading bacterium, Rhodococcus sp. strain YH3‐3 was purified and characterized. Addition of cobalt ion was necessary for the formation of enzyme. The enzyme activity was highly induced not only by nitriles and amides but also by several aldoxime compounds. The enzyme was purified ≈ 108‐fold with a 16% yield from the cell‐free extract of the strain. The native enzyme had a Mr of ≈ 130 000 and consisted of two subunits (α‐subunit, 27 100; β‐subunit, 34 500). The enzyme contained approximately 2 mol cobalt per mol enzyme; it showed a maximum activity at 60 °C and at 40 °C under the rate assay and end‐point assay conditions, respectively, and was stable over a wide range of pH (pH 2.5–11.0). The enzyme had a wide substrate specificity: it acted on aliphatic saturated and unsaturated as well as aromatic nitriles. The N‐terminus of the β‐subunit showed good sequence similarities with those of other nitrile hydratases. Nitrile hydratase is part of the metabolic pathway for aldoximes in microorganisms.
Dynamin and Rab5 regulate GRK2‐dependent internalization of dopamine D2 receptors
Authors: Iwata Ken , Ito Kazumitsu , Fukuzaki Atsushi , Inaki Kouichiro , Haga Tatsuya
Published: March 2003

Dopamine D2 receptors (D2Rs; short form, which is one of the alternative splicing variants) expressed in COS‐7 cells are internalized in an agonist‐dependent manner only when G protein‐coupled receptor kinase 2 (GRK2) is coexpressed [Ito, K., Haga, T., Lameh, J. & Sadée, W., (1999) Eur. J. Biochem.260, 112–119]. We have examined the effects of coexpression of dynamin, a small molecular mass GTP‐binding protein, rab5A, and their mutants on the internalization of D2Rs in the presence of both dopamine (10 or 100 µm) and GRK2. The rate and extent of D2R internalization was increased or decreased by coexpression of dynamin I or a dominant‐negative form of dynamin I (dynamin I K44E), respectively. The effects of coexpressing these two dynamins were more prominent at 10 µm dopamine than at 100 µm. In the presence of 10 µm dopamine, internalization of D2R was completely suppressed when dynamin I K44E was coexpressed, and the half‐life (t½) of D2R internalization decreased relative to cells not expressing dynamin from 82 to 29 min when dynamin I was coexpressed. Internalization of D2Rs was facilitated or suppressed by coexpression of a constitutively active form of rab5A (rab5A Q79L) or a dominant‐negative form of rab5A (rab5A S34N), respectively. The t½ of D2R internalization at 10 µm dopamine decreased from 82 to 16 min in cells coexpressing rab5A Q79L. The effect of coexpression of rab5A S34N was more apparent at 100 µm dopamine than at 10 µm; the t½ of D2R internalization at 100 µm dopamine increased from 20 to 56 min and the proportion of internalized D2Rs after 120 min decreased from 53 to 28%. These results indicate that the internalization of D2Rs is dependent on the action of dynamin as well as GRK2, and is regulated by the action of rab5A.
A region between −141 and −61 bp containing a proximal AP‐1 is essential for constitutive expression of urokinase‐type plasminogen activator receptor
Authors: Dang Jinjun , Boyd Douglas , Wang Heng , Allgayer Heike , Doe William F. , Wang Yao
Published: March 2003

An 8.5‐kb 5′‐flanking region of the human urokinase‐type plasminogen activator receptor (uPAR) gene was cloned and the detailed uPAR promoter region defined in an 188‐bp fragment between bases −141 and +47 relative to the transcription‐start site. 5′‐Deletion to −100 and −60 in the region abolished its promoter activity, indicating that an 81‐bp segment between −141 and −61, which contains a proximal AP‐1 site at position −70, is required for uPAR promoter activity. Nuclear extracts from HCT116 cells contain proteins that specifically bind to the AP‐1 site. Mutation of the AP‐1 motif reduced uPAR promoter activity in comparison with the wild‐types. Induction of uPAR expression by phorbol ester requires this AP‐1 motif in colon cancer cells. Cotransfection with the c‐jun and c‐fos expression vectors stimulated the uPAR promoter activity four‐ to fivefold. These results demonstrate that the proximal AP‐1 motif is responsible for ≈ 50% of the basal expression of the uPAR gene.
Separate roles for H‐Ras and Rac in signaling by transforming growth factor (TGF)‐β
Authors: Yamamoto Hiroaki , Atsuchi Nobuhiko , Tanaka Haruki , Ogawa Wataru , Abe Mayumi , Takeshita Akira , Ueno Hikaru
Published: March 2003

The signaling components located downstream of the transforming growth factor (TGF)‐β receptor are poorly understood. We constructed adenoviral vectors expressing a dominant‐negative form of either H‐Ras (AdCARasY57) or Rac (AdCARacN17), and used them to examine the roles of H‐Ras and Rac in TGF‐β signaling using arterial endothelial cells in primary culture, and several established cells including a mink lung epithelial cell line (Mv1Lu). The rapid activation of p42/44 MAP kinase (MAPK) by TGF‐β1 was eliminated completely, and transcriptional activation by TGF‐β1 of the plasminogen activator inhibitor‐1 gene was reduced by 50% in both endothelial cells and Mv1Lu when they were infected with AdCARasY57. However, the antiproliferative effect of TGF‐β, as assessed by the induction of the mRNA for Cdk4/6‐specific inhibitor p15INK4B and by DNA synthesis, was not affected in AdCARasY57‐infected cells. A MAPK kinase (MEK)1/2 inhibitor, U0126 also abolished MAPK activation and partially inhibited transcriptional activation by TGF‐β, suggesting that MAPK may be partially involved in this pathway. MAPK activation, transcriptional activation and growth suppression by TGF‐β were all unaffected in cells infected with AdCARacN17, although the DNA synthesis elicited by serum mitogens was suppressed completely in the infected cells. Our data indicate that H‐Ras is essential for mitogen‐activated protein kinase activation, partly required for transcriptional activation by TGF‐β, but not critically involved in the signaling that exerts the antiproliferative effect of TGF‐β. The results also suggest that Rac may not serve as an essential molecule in signaling by TGF‐β in the cells tested.
Purification and characterization of a cysteine‐rich 11.5‐kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas
Authors: Relf Juliet M. , Chisholm June R. S. , Kemp Graham D. , Smith Valerie J.
Published: March 2003

Extracts of the granular haemocytes of Carcinus maenas were subjected to ion‐exchange chromatography and reverse‐phase (RP)‐HPLC to investigate the presence of an antibacterial protein of ≈ 11 kDa. This protein was isolated, characterized and subjected to partial amino acid sequence analysis. It was found by mass spectrometry to have a molecular mass of 11 534 Da, to be cationic and hydrophobic and active only against marine Gram‐positive bacteria. In addition its activity is stable after heating to 100 °C and is retained at concentrations as low as 10 µg·mL−1. It has an unusual amino acid sequence, unlike any known antibacterial peptide described in the literature but bears a consensus disulphide domain signature, indicating that it might be a member of the four‐disulphide core proteins. Partial cDNA sequence data has been obtained.
Different promoter usage and multiple transcription initiation sites of the interleukin‐1 receptor‐related human ST2 gene in UT‐7 and TM12 cells
Authors: Iwahana Hiroyuki , Yanagisawa Ken , Ito‐Kosaka Akiko , Kuroiwa Kenji , Tago Kenji , Komatsu Norio , Katashima Rumi , Itakura Mitsuo , Tominaga Shin‐ichi
Published: March 2003

The ST2 gene encodes receptor‐like molecules that are very similar to the type I interleukin‐1 receptor. Two distinct types of the ST2 gene products, ST2 (a soluble secreted form) and ST2L (a transmembrane form) are produced by alternative splicing. Here we demonstrate that the human ST2 gene has two alternative promoters followed by distinct noncoding first exons, which are located more than 8 kb apart and are spliced to the common exon 2 containing the translation initiation site. Within 1001 bp upstream of the transcription initiation site of the cloned distal promoter, there are four GATA‐1. The main promoter used for the expression of the ST2 gene in UT‐7, a human leukaemic cell line, is distinct from that in TM12, a human fibroblastic cell line. Although UT‐7 cells use both distal and proximal promoters, the distal promoter is used dominantly for expression of both ST2 and ST2L mRNA. On the other hand, almost all transcription in TM12 cells starts from the proximal promoter. These results contrast with those of former studies on the rat system, in which ST2 and ST2L mRNA were generated by use of the proximal and distal promoters, respectively. Furthermore, UT‐7 cells use multiple transcription initiation sites in both the proximal and distal promoters, whereas the transcription of the ST2 gene in TM12 cells starts at a unique site. Intriguingly, these results suggest that ST2 and ST2L proteins have distinct functions in different cells within different biological systems, such as those of growth control, differentiation and immunological responses.
Post‐translationally modified neuropeptides from Conus venoms
Authors: Craig A. Grey , Bandyopadhyay Pradip , Olivera Baldomero M.
Published: March 2003

Predatory cone snails (genus Conus) comprise what is arguably the largest living genus of marine animals (500 species). All Conus use complex venoms to capture prey and for other biological purposes. Most biologically active components of these venoms are small disulfide‐rich peptides, generally 7–35 amino acids in length. There are probably of the order of 100 different peptides expressed in the venom of each of the 500 Conus species [1,

2]. Peptide sequences diverge rapidly between Conus species, resulting in a distinct peptide complement for each species. Thus, the genus as a whole has probably generated ≈ 50 000 different peptides, which can be organized into families and superfamilies with shared sequence elements [3]. In this minireview, we provide a brief overview of the neuropharmacological, molecular and cell‐biological aspects of the Conus peptides. However, the major focus of the review will be the remarkable array of post‐translational modifications found in these peptides.