bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid phosphorylation of 28‐kDa heat‐shock protein by treatment with okadaic acid and phorbol ester of BALB/MK‐2 mouse kerationocytes
Authors: KASAHARA Kohji , IKUTA Togo , CHIDA Kazuhiro , ASAKURA Rieko , KUROKI Toshio
Published: March 2005

Protein phosphorylation by okadaic acid and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was examined using quiescent cultures of BALB/MK‐2, a cell line derived from mouse epidermal keratinocytes. Treatment with okadaic acid caused rapid phosphorylation of five proteins with molecular masses of 65, 55, 50 28 and 15 kDa (p65, p55, p50, p28, p15, respectively) while TPA caused rapid phosphorylation of five proteins with molecular masses of 80, 70, 40, 34 and 28 kDa (p80, p70, p40, p34, p28, respectively). In the present study, we examined p28, a common target protein of okadaic acid and TPA. The phosphorylation of p28 increased depending on time of exposure and doses of okadaic acid and TPA. Combined treatment okadaic acid and TPA resulted in an additive effect. Its position on two‐dimensional gel electrophoresis suggested that p28 is the 28‐kDa heat‐shock protein (HSP28). This possibility was confirmed by migration of p28 with HSP28 and comparative peptide mapping of the two proteins. The phosphoamino‐acid residue of phosphorylated HSP28 was serine. In two‐dimensional tryptic peptide maps, the same peptides were phosphorylated after treatment with both okadaic acid and TPA.
Different isoforms and stock‐specific variants of the cell adhesion molecule C‐CAM (cell‐CAM 105) in rat liver
Authors: EDLUND Magnus , GAARDSVOLL Henrik , BOCK Elisabeth , ÖBRINK Björn
Published: March 2005

C‐CAM is a cell adhesion molecule of the immunoglobulin superfamily with homophilic binding properties. Here we used the polymerase chain reaction to isolate clones of C‐CAM from a rat liver cDNA library. Sequence analyses identified two major isoforms, C‐CAM1 and C‐CAM2, which differed in their 3′ ends. C‐CAM2 lacked a sequence of 53 nucleotides that was present in C‐CAM1. This causes a frame shift and new stop codons, which gives rise to cytoplasmic domains of different sizes in the two isoforms (10 versus 71 amino‐acid residues). In addition, all the clones had a different nucleotide and deduced amino‐acid sequence (variant b) in the most N‐terminal of the four immunoglobulin‐like domains, compared to a previously published C‐CAM sequence (variant a). Northern‐blot analyses with specific oligonucleotide probes demonstrated that two different rat stocks expressed either variant a or variant b. Northern‐blot analyses of rat liver and lung also showed that at least five different C‐CAM transcripts are produced. Two major mRNA size classes of 4.0 kb and 6.0 kb, and one minor class of 3.0 kb were found. Both the 4.0‐kb and 3.0‐kb messenger classes reacted with two different probes that could distinguish between C‐CAM1 and C‐CAM2, while the 6.0‐kb population only reacted with the probe selective for C‐CAM1. Taken together these data demonstrate the existence of four different protein‐coding sequences of rat liver C‐CAM (C‐CAM1 a and b, and C‐CAM2 a and b). We suggest that both allelic variation and alternative splicing may contribute to the isoform‐expression pattern of C‐CAM in rats.
The promoter and intron/exon structure of the human and mouse β3‐adrenergic‐receptor genes
Authors: Van SPRONSEN Anke , NAHMIAS Clara , KRIEF Stéphane , BRIEND‐SUTREN Marie‐Madeleine , STROSBERG A. Donny , EMORINE Laurent J.
Published: March 2005

Transcription‐start sites for the mouse and human β3‐adrergic‐receptor mRNA have been localized in a region comprised between 150 and 200 nucleotides 5′ from the ATG translation‐start codon. Motifs potentially implicated in heterologous regulation of β3‐adrenergic‐receptor expression by glucocorticoids and by β‐adrenergic agonists have been identified upstream from these cap sites. In mouse, a second mRNA initiation region is postulated to exist further upstream. Comparison of the nucleotide sequences of the 3′ end of the human and mouse β3‐adrenergic‐receptor genes to those of the corresponding cDNA revealed that in contrast to β1 and β2 adrenergic receptors, the β3‐adrenergic‐receptor genes comprise several exons. A large exon (1.4 kb) encodes the first 402 and 388 amino‐acid residues of the human and mouse β3‐adrenergic‐receptor, respectively. In man, a second exon (700 bp) contains the sequence coding for the six carboxy‐terminal residues of the receptor and the entire mRNA 3′ untranslated region. In mouse, a second exon (68 bp) codes for the 12 carboxy‐terminal residues of the receptor and a third exon contains the β3‐adrenergic‐receptor mRNA 3′ untranslated region. The use of alternate acceptor splice sites generates two forms of exon 3 (600 bp and 700 bp), yielding two β3‐adrenergic‐receptor transcripts which are differentially expressed in white and brown adipose tissues. Human β3‐adrenergic‐receptor transcripts with different 3′ untranslated regions are produced by continuation of transcription beyond termination signals. Together, our results suggest that utilization of alternate promoters and/or 3′ untranslated regions may allow tissue‐specific regulation of β3‐adrenergic‐receptors expression.
NMR and molecular modeling studies of the interaction of berenil and pentamidine with d(CGCAAATTTGCG)2
Authors: JENKINS Terence C. , LANE Andrew N. , NEIDLE Stephen , BROWN David G.
Published: March 2005

The interaction of two anti‐trypanosomal agents, berenil and pentamidine, with the A+T‐rich dodecamer d(CGCAAATTTGCG)2 has been examined by high‐resolution 1H‐NMR, optical spectroscopy, and molecular modeling. Proton assignments for the free DNA and each DNA‐ligand complex were obtained using nuclear Overhauser enhancement spectroscopy and total correlation spectroscopy. Complexation induces large changes in chemical shift for protons in the DNA minor groove for the A5–T9 segment, and intermolecular NOEs reveal contacts between the DNA bases and each ligand. The asymmetric binding site for berenil indicated by the NMR data suggests that at least two overlapping sites are involved. Rapid exchange between symmetrically‐equivalent binding sites, via dissociative rearrangement, is consistent with retention of twofold degeneracy for both the ligand and the DNA host. Calculations of binding energy confirm that this DNA duplex contains overlapping sites of similar binding affinity. In contrast, the larger pentamidine molecule occupies a site that spans four or five bp, with asymmetric binding to the minor‐groove 5′‐ATTT sequence. The B‐type conformation of the DNA is not altered substantially by either ligand.
Two‐dimensional 1H‐NMR study of the spatial structure of neurotoxin II from Naja naja oxiana
Authors: GOLOVANOV Alexander P. , LOMIZE Andrei L. , ARSENIEV Alexander S. , UTKIN Yuri N. , TSETLIN Victor I.
Published: March 2005

The spatial structure of neurotoxin II from the venom of the central Asian cobra Naja naja oxiana was determined by two‐dimensional 1H‐NMR techniques and computational analysis. Nearly complete proton resonance assignments for 61 amino acid residues have been made using two‐dimensional (2D) homonuclear total correlated spectroscopy, 2D homonuclear double‐quantum‐filtered correlated spectroscopy and 2D homonuclear NOE spectroscopy (NOESY) experiments. The cross‐peak volumes in NOESY spectra spin‐spin coupling constants of vicinal protons NH‐CαH and CαH‐CβH and the observation of slow deuterium exchange of amide protons were used to define local structure and a set of constraints for distance geometry program DIANA. The average root‐mean‐square deviations are 53 pm for backbone heavy atoms and 118 pm for all heavy atoms of 19 final neurotoxin II conformations. The spatial structure is characterized by a short double‐stranded (residues 1–5 and 13–17) and a triple‐stranded (residues 22–30, 33–41 and 50–54) antiparailel β‐sheets.
Secondary structure and temperature behaviour of acetylcholinesterase
Authors: GÖRNE‐TSCHELNOKOW Ute , NAUMANN Dieter , WEISE Christoph , HUCHO Ferdinand
Published: March 2005

The secondary structure of the acetylcholinesterase and its temperature behaviour have been investigated using Fourier‐transform infrared (FTIR) spectroscopy. The data are compared to the structure obtained by X‐ray analysis of the crystalline enzyme. The secondary structure was determined using the spectral features observed in the amide‐I band (H2O buffer) and amide‐I′ band (D2O buffer) at 1600–1700 cm−1, taking advantage of resolution‐enhancement techniques along with least‐squares band‐fitting procedures. The relative amounts of different secondary‐structure elements, 34–36% for α‐helices, 19–25% for β‐sheets, 15–16% for turns and 13–17% for irregular structures, were estimated. These data, obtained with the enzyme in solution, correlate well with X‐ray data of the crystalline protein [Sussman, J. L., Hard, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. & Silman, I. (1991) Science 253, 872–879]. These results are also in good agreement with those obtained by computing the Ψ and φ angels of the peptide backbone using the Kabsch and Sanders method [Kabsch, W. & Sanders, C. (1983) Biopolymers 22, 2577–2637]. In conjunction with the X‐ray data, two bands in the FTIR spectra were assigned to different populations of long and short α‐helices. Until now this phenomenon has only been described by theoretical calculations [Nevskaya, N. A. & Chirgadze, Yu. N. (1976) Biopolymers 15, 637–648]. The relationship between the thermally induced loss of enzyme activity and secondary‐structure changes has also been investigated. The decrease in enzyme activity to zero at 30–40 °C was accompanied only by minor changes in the secondary structure. At 55–60 °C, denaturation of AChE occurs. In this temperature range, all bands assigned to the various secondary‐structure elements abruptly disappear in a co‐operative and irreversible manner, whereas the β‐aggregation bands (at 1622 cm−1 and the corresponding high‐frequency band) increase in intensity at the same rate.
Purification and characterisation of glyoxalase II from human red blood cells
Authors: ALLEN Rosamund E. , LO Theodore W. C. , THORNALLEY Paul J.
Published: March 2005

Glyoxalase II was purified from human red blood cells. The purification factor was 83 300 and the yield was 24% or 1.7 μg/ml red blood cells. The purified protein was a monomer with a molecular mass of 29 200 Da and an isoelectric point of 8.3. The rate of hydrolysis of S‐D‐lactoylglutathione to reduced glutathione and D‐lactate, catalysed by glyoxalase II, followed Michaelis‐Menten kinetics where the Km and kcat values where 146 ± 9 μM and 727 ± 16 s−1, respectively in 50 mM Tris/HCl, pH 7.4 at 37 °C. Other S‐2‐hydroxyacylglutathione derivatives were also acceptable substrates. S‐p‐Nitrobenzoxycarbonylglutathione was a potent competitive inhibitor of glyoxalase II with a Ki value of 1.20 ± 0.21 μM, and the hemithioacetal formed non‐enzymically from the reaction of methylglyoxal with reduced glutathione was a weak competitive inhibitor with a Ki value of 834 ± 98 μM.
The interaction of daunomycin with model membranes
Authors: BAÑUELOS Sonia , ARRONDO Jose Luis R. , CANAVES Jaume M. , FERRAGUT Jose A. , MUGA Arturo
Published: March 2005

The sensitivity of the keto and carbonyl infrared bands of daunomycin (DNM) to hydrogen bonding with the solvent, has been used to study the effect of the physical state and lipid composition of the bilayer on drug location. Our results show that penetration of daunomycin into dihexa‐decylphosphatidylcholine (Hxd2GroPCho) or dipalmitoylphatidylcholine bilayers, is dependent on the molecular packing of the lipid. DNM incorporates into the bilayer once the interdigitation of the gel phase of Hxd2GroPCho has been removed, above the pretransition temperature. Melting of the hydrocarbon chains of both lipids, at the main transition temperature, allows a similar and deeper drug penetration into the bilayers. Experiments using liposomes with different lipid compositions suggest that the relative concentration of certain lipids may modulate the location of DNM within the bilayer. Cholesterol, in a concentration‐dependent manner, inhibits incorporation of anthracycline into apolar regions of the bilayer, while the presence of the negatively charged lipid dihexadecyl‐phosphatidic acid is able to prevent the inhibitory effect of the steroid, allowing deeper penetration of the drug. Due to the importance of drug‐membrane interactions in anthracycline cytotoxicity, the relevance of the observed differences in daunomycin location, caused by physical and/or chemical changes in the biological membranes, is discussed.
Purification and biochemical characterization of the hydantoin hydrolyzing enzyme from Agrobacterium species
Authors: RUNSER Serge M. , MEYER Patrick C.
Published: March 2005

A soluble hydantoinase (5,6‐dihydropyrimidine amidohydrolase) was purified to homogeneity from a newly isolated Agrobacterium species. This hydrolase consists of about 578 aminoacyl residues and is a slightly acidic protein with an isoelectric point of 6.5. The first 22 N‐terminal amino acid residues were determined by Edman degradation. Determination of the relative molecular mass of the protein by gel‐filtration chromatography gave an apparent value of 250000. The subunit Mr was 62000, as estimated by analytical SDS/PAGE and 66500, as estimated by denaturing gel‐filtration chromatography. The pure hydantoinase exhibits the following hydrodynamic properties: a sedimentation coefficient of 8.8 S as determined by sedimentation velocity experiments; a Stokes radius of 6.8 nm; a diffusion coefficient of 31.5 μm2·s−1 as determined by analytical gelfiltration chromatography. From these experimental data, the following physical constants could be calculated: a theoretical Mr of 265000, a frictional ratio, f/fo, of 1.59, a maximal axial ratio, a/b, of 3.1; a Perrin shape factor, F, of 1.37. As shown by different Km values, the preferred substrates of this hydrolase were 5‐monosubstituted hydantoins bearing aromatic substituents. 5,5‐Dimethylhydantoin and different thio analogs of the 5‐p‐hydroxyphenylhydantoin molecule are competitive inhibitors of this hydrolase. The classification of this microbial hydantoinase, which exhibits no hydrolytic activity with all the dihydropyrimidines tested, under the systematic name of 5,6‐dihydropyrimidine amidohydrolase, and its putative metabolic role are further discussed.
Metformin decreases gluconeogenesis by enhancing the pyruvate kinase flux in isolated rat hepatocytes
Authors: ARGAUD Doriane , ROTH Hubert , WIERNSPERGER Nicolas , LEVERVE Xavier M.
Published: March 2005

Metformin (dimethylbiguanide) has been used for more than 30 years as an antihyperglycemic agent in the treatment of diabetes mellitus, but its effect on gluconeogenesis is still controversial. In isolated hepatocytes from fasted rats, a significant inhibition of glucose production from lactate/pyruvate (10: 1, mol/mol), fructose, alanine or glutamine, following metformin addition, is observed. Moreover, in hepatocytes perifused with dihydroxyacetone as the gluconeogenic substrate and treated with 0.5 mM metformin, an inhibition of the glucose flux and a simultaneous stimulation of the lactate/pyruvate flux were observed. This enhancement of lactate/pyruvate formation appears to be due to an effect on the pyruvate‐kinase enzyme. A direct effect of metformin on pyruvate kinase cannot explain this result, since pyruvate‐kinase activity was not affected by metformin at this concentration. In contrast, the addition of metformin caused a significant decrease in the cellular ATP concentration, a known allosteric inhibitor of this enzyme. This could explain the stimulation of pyruvate‐kinase activity following metformin addition and thus the inhibition of gluconeogenesis.
Site directed mutagenesis of DNA polymerase I (Klenow) from Escherichia coli
Authors: PANDEY Virendra N. , KAUSHIK Neerja , SANZGIRI Rita P. , PATIL Manohar S. , MODAK Mukund J. , BARIK Sailen
Published: March 2005

We have reported that a domain containing Arg682 in the Klenow fragment of Escherichia coli DNA polymerase I (pol I) is important for the template‐dependent dNTP‐binding function [Pandey, V. N., Kaushik, N. A., Pradhan, D. S. & Modak, M. J. (1990) J. Biol. Chem. 265, 3679–3884]. In order to further define the role of Arg682 in the catalytic process, we have performed site‐directed mutagenesis of this residue. For this purpose the Klenow‐coding region of the DNA‐pol‐I gene was selectively amplitied from the genomic DNA of E. coli and was cloned in an expression vector, pET‐3a. This clone under appropriate conditions overproduces the Klenow fragment in E. coli. Using this clone (pET‐3a‐K) as the template, two mutant polymerase clones were constructed in which arginine has been replaced with either alanine, [R682A] pol I, or lysine [R682K] pol I. Both mutant enzymes showed significantly lower specific activity as compared to the wild‐type enzyme. The Kinetic analyses of the mutant enzymes indicated a 3–4‐fold increase in the Km for the substrate dNTP, a 20–25‐fold decrease in the Vmax and an overall decrease in the processive nature of DNA synthesis in both the mutant enzymes. The reverse mutation of Ala682 to the wild‐type form Arg682 fully restored the processive nature and the polymerase activity of the enzyme. These observations suggest that the positively charged guanidino group in the side chain of Arg682 is catalytically important but not absolutely essential for synthesis of DNA. Furthermore it appears to maintain high processivity of the DNA synthesis catalyzed by the enzyme.
Factors effecting the thermostability of cysteine proteinases from Carica papaya
Authors: SUMNER Ian G. , HARRIS Gillian W. , TAYLOR Mark A. J. , PICKERSGILL Richard W. , OWEN A. Jane , GOODENOUGH Peter W.
Published: March 2005

Thermal denaturation of four Carica papaya cysteine proteinases (papain, chymopapain, papaya proteinases 3 and 4) was studied as a function of pH using high‐sensitivity differential scanning calorimetry.

The ratios of calorimetric enthalpy to Van't Hoff enthalpy suggest that, for all these proteins, denaturation occurs as a non two state process, via an intermediate structure.

Differences in the thermal stabilities of the proteinases; chymopapain > papaya proteinase 3 > papain > papaya proteinase 4, were correlated to their amino acid sequence to explain the observations in terms of mobility and specific residue mutation.

Three‐dimensional structures of papain and papaya proteinase 3 were similarly used to illustrate the influence of atomic mobility on stability.
Purification and characterization ofThermus caldophilusGK24 DNA polymerase
Authors: PARK Jong Hoon , KIM Joong Su , KWON Suk‐Tae , LEE Dae‐Sil
Published: March 2005

A thermostable DNA polymerase fromThermus caldophilusGK24 was purified to near homogeneity by chromatographic methods, including ion‐exchange, gel‐filtration and affinity chromatography. The purified enzyme had a specific activity of 8400 U/mg at 75°C and a molecular mass of 95 kDa, estimated by SDS/PAGE and Superose‐12 gel filtration. Reaction conditions were investigated in terms of pH, metal‐ion concentration and temperature. Experimental results showed thatT. caldophilus(Tca) DNA polymerase had a maximum activity near pH 8.7 at 75°C. The N‐terminal sequence of the enzyme was highly similar to that ofThermus aquaticus(Taq) DNA polymerase, which was consistent with the fact that the enzyme had 5′‐to‐3′ exonuclease activity and no 3′‐to‐5′ exonuclease activity. Gene amplification usingTcaDNA polymerase resulted in longer products than amplification usingTaqDNA polymerase.
Type‐2 plasminogen‐activator inhibitor is a substrate for trophoblast transglutaminase and Factor XIIIa
Authors: JENSEN Poul Henning , LORAND Laszlo , EBBESEN Peter , GLIEMANN Jørgen
Published: March 2005

Plasminogen‐activator inhibitor type‐2 (PAI‐2), a serine‐proteinase inhibitor, suppresses fibrinolysis by blocking both urokinase and tissue‐type plasminogen activators. The 43‐kDa PAI‐2 molecule is an abundant cytosolic protein in certain cell types, but can upon appropriate stimulation be secreted as an approximately 60–70‐kDa glycoprotein. However, in trophoblast membranes PAI‐2 activity is associated with large covalent complexes (Jensen, P. H., Nykjær, P., Andreasen P. A., Lund, L., Åstedt, B. Lecander, I. & Gliemann, J. (1989) Biochim. Biophys. Acta 986, 135–140). This study shows that PAI‐2 can act as a substrate for both tissue transglutaminase and activated plasma factor XIII. In the presence of Ca2+, either of these will catalyze the incorporation of primary amines, such as putrescine, into PAI‐2. Moreover, in reactions with tissue transglutaminase, PAI‐2 homopolymers and, in conjunction with other biological substrates, heteropolymers were observed. As judged by the test of incorporating 125I‐urokinase into SDS‐resistant 125I‐urokinase/PAI‐2 complexes, polymerized PAI‐2 retained its inhibitory activity. Furthermore, syncytiotrophoblast microvillous membranes and trophoblast detergent extracts incorporated 125I‐PAI‐2 into large structures in a reaction inhibited by putrescine and a synthetic inhibitor of transglutaminase. Trophoblast transglutaminase was identified as a tissue transglutaminase by non‐denaturing gel electrophoresis and dansylcadaverine activity staining, fibronectin binding and Western blotting with a specific antibody. The transglutaminase‐catalyzed and Ca2+‐dependent anchoring of PAI‐2 to extracellular membrane structures might have the purpose of focally regulating fibrinolysis.
Transient and cell‐specific expression of tissue‐type plasminogen activator and plasminogen‐activator‐inhibitor type 1 results in controlled and directed proteolysis during gonadotropin‐induced ovulation
Authors: PENG Xiao‐Rong , HSUEH Aaron J. W. , NY Tor
Published: March 2005

Proteolytic activity generated by the plasminogen‐activator system (PA system) is associated with many biological processes. However, it is not known how the proteolytic activity is regulated in vivo in order to obtain directed proteolysis while, at the same time, protecting unrestrained tissue destruction. Using gonadotropin‐induced ovulation as a model, we have studied how two components of the PA system, tissue‐type plasminogen activator (tPA) and plasminogen‐activator‐inhibitor type 1 (PAI‐1), are regulated temporally and spatially by gonadotropins, leading to the initiation and termination of a well‐directed proteolytic process. In‐situ hybridization and in‐situ zymography were used to analyze the expression of tPA and PAI‐1 mRNA and PA‐activity in specific ovarian cell types. Both tPA and PAI‐1 were found to be regulated and to have a distinct expression pattern in different ovarian compartments. tPA was expressed in both granulosa and thecal‐interstitial cells; the highest levels of tPA mRNA were found in the granulosa cells of preovulatory follicles, just prior to ovulation. Consistent with a role for luteinizing hormone/chorionic gonadotropin (LH/CG) in triggering ovulation, the cells and follicles that actively expressed tPA also contained high levels of LH‐receptor mRNA while cumulus cells that contain undetectable amounts of tPA mRNA were devoid of LH‐receptor expression. The highest levels of PAI‐1 mRNA were found about 6 h before ovulation and mainly in the thecal‐interstitial cells and ovarian stroma tissue which encapsulate the follicle. Preovulatory follicies, protruding onto the surface of the ovary with less surrounding stroma tissue, expressed less PAI‐1 compared to small non‐ovulatory follicles embedded in inner part of the ovary. In‐situ zymography also revealed that the PA activity was colocalized to the surface of the ovary just prior to ovulation. Our studies suggest that a proteolytic activity provided by tPA and modulated by PAI‐1 is responsible for a controlled and directed proteolysis leading to rupture of selected follicles during ovulation.
Cannabinoid‐receptor expression in human leukocytes
Authors: BOUABOULA Monsif , RINALDI Murielle , CARAYON Pierre , CARILLON Christine , DELPECH Bruno , SHIRE David , LE FUR Gérard , CASELLAS Pierre
Published: March 2005

Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS), probably through the cannabinoid receptor, which has recently been cloned in rat and human. While numerous reports have also described effects of cannabinoids on the immune system, the observation of both mRNA and cannabinoid receptor has hitherto been exclusively confined to the brain, a reported detection in the testis being the sole example of its presence at the periphery. Here we report the expression of the cannabinoid receptor on human immune tissues using a highly sensitive polymerase‐chain‐reaction‐based method for mRNA quantification. We show that, although present in a much lower abundance than in brain, cannabinoid receptor transcripts are found in human spleen, tonsils and peripheral blood leukocytes. The distribution pattern displays important variations of the mRNA level for the cannabioid receptor among the main human blood cell subpopulations. The rank order of mRNA levels in these cells is B cells > natural killer cells ≥ polymorphonuclear neutrophils ≥ T8 cells > monocytes > T4 cells. Cannabinoid‐receptor mRNA, which is also found in monocytic, as well as T and B leukemia cell lines but not in Jurkat cells, presents a great diversity of expression on these cells as well, B‐cell lines expressing a much higher level than T‐cell lines. The cannabinoid receptor PCR products from leukocytes and brain are identical both in size and sequence suggesting a strong similarity between central and peripheral cannabinoid receptors. The expression of this receptor was demonstrated on membranes of the myelomonocytic U937 cells using the synthetic cannabinoid [3H]CP‐55940 as ligand. The Kd determined from Scatchard analysis was 0.1 nM and the Bmax for membranes was 525 fmol/mg protein. The demonstration of cannabinoid‐receptor expression at both mRNA and protein levels on human leukocytes provides a molecular basis for cannabinoid action on these cells.
Molecular cloning of silkworm (Bombyx mori) antichymotrypsin
Authors: NARUMI Hideki , HISHIDA Tomoaki , SASAKI Takuji , FENG Da‐Fei , DOOLITTLE Russell F.
Published: March 2005

The cDNA of silkworm (Bombyx mori) antichymotrypsin (sw‐Achy) was cloned from larval fat body and its nucleotide sequence was determined. The deduced amino acid sequence of mature sw‐Achy begins with Phe1 and ends with Phe384, with a preceding 16‐amino‐acid signal peptide. The amino‐acid sequence similarities of sw‐Achy with the serine‐proteinase inhibitors (serpins) silkworm antitrypsin, tobacco hornworm alaserpin, human α‐1‐antitrypsin and human α‐1‐antichymotrypsin were 29.6%, 30.3%, 26.1%, and 25.0%, respectively. The highly conserved amino acids in other serpins are also conserved in sw‐Achy. sw‐Achy is thought to be a new member of the serpin family. Multiple alignment of sw‐Achy with 23 other kinds of serpin by the progressive method produced a phylogenetic tree in which all four insect serpins are grouped separately within one branch. The reactive site of sw‐Achy with α‐chymotrypsin was identified as Thr343‐Ser344 by direct amino‐acid sequence analysis of cleaved and purified protein.
Hyperammonemia decreases protein‐kinase‐C‐dependent phosphorylation of microtubule‐associated protein 2 and increases its binding to tubulin
Authors: FELIPO Vicente , GRAU Eugenio , MIÑANA María‐Dolores , GRISOLÍA Santiago
Published: March 2005

Hyperammonemia increases the polymerization of brain microtubules, which is controlled by the binding of microtubule‐associated protein (MAP) 2; binding of MAP‐2 is, in turn, regulated by phosphorylation. We have found that the binding of MAP‐2 to tubulin is greatly increased by hyperammonemia, however, the brain content of MAP‐2 is not affected. Microtubules isolated from hyperammonemic rats contained approximately twice the MAP‐2/mg microtubular protein that of microtubules isolated from control animals. MAP isolated from brain microtubules of hyperammonemic rats stimulated the polymerization of tubulin more than MAP isolated from control animals. This appears to be due to the increased content of MAP‐2. In vitro phosphorylation, using brain homogenates, showed that protein‐kinase‐C‐dependent phosphorylation of MAP‐2 was markedly decreased in hyperammonemic rats. Hyperammonemia also affected the intracellular distribution of brain protein kinase C; its content in the cytosol increased about 23%, while in membranes it decreased by 46%. The possible role of decreased protein‐kinase‐C‐dependent phosphorylation on the increased binding of MAP‐2 to tubulin and in the increased polymerization of microtubules in the brain of hyperammonemic rats is discussed.
Binding of bovine pancreatic trypsin inhibitor to heparin binding protein/CAP37/azurocidin
Authors: PETERSEN Lars C. , BIRKTOFT Jens J. , FLODGAARD Hans
Published: March 2005

Heparin‐binding protein (HBP; also known as CAP37 or azurocidin) is a member of the serine proteinase family. Evolution, however, has reverted this protein into a non‐proteolytic form by mutation of two of the three residues of the active‐site triad. Although proteolytically inactive, the human heparin‐binding protein (hHBP) is still capable of binding bovine pancreatic trypsin inhibitor (BPTI). This was demonstrated by affinity chromatography to BPTI immobilized on a solid matrix and by studies on plasmin inhibition kinetics. hHBP competes with plasmin for BPTI and this effect on plasmin inhibition has been analyzed in terms of a kinetic model. A dissociation constant, Kd= 0.1 μM, was found for the interaction between BPTI and hHBP. The hHBP provides an example of a serine proteinase which has lost its catalytic function by reverting residues of the active center while still preserving its capability of specific interactions with Kunitz inhibitors. pHBP, the porcine counterpart to hHBP, on the other hand, was incapable of BPTI binding. The structural basis for the BPTI binding to the human protein and the species difference is discussed in terms of putative three‐dimensional structures of the proteins derived by comparative molecular modelling methods.
Efficient secretion of attacin from insect fat‐body cells requires proper processing of the prosequence
Authors: GUNNE Hans , STEINER Håkan
Published: March 2005

The attacins constitute a group of immune proteins induced after bacterial infection in the moth Hyalophora cecropia. They are synthesized as preproproteins and undergo post‐translational modification during transport to the hemolymph. The processing and transport rates of attacin were studied in its natural host as a response to infection. Monensin totally inhibited the processing from proattacin to attacin and the radiolabeled proattacin remained intracellular. This observation indicated that the prosequence is removed at or after the trans‐Golgi compartment. It is also suggested that the processing of the prosequence does not occur in acidic vesicles, as the process was not inhibited by the weak base chloroquine. To study prosequence function, the attacin gene and genes with mutations in the prosequence were cloned into the baculovirus Autographa californica nuclear polyhedrosis virus. The processing of proattacin and the transport of attacin were studied by pulsechase experiments with fat body isolated from Trichoplusia ni larvae. The rate of secretion from fat body was lowest for proattacin, which could not be processed to attacin, intermediate for attacins lacking the prosequence and highest for natural attacin. We could not detect any biological activity for proattacin.
Heterologous expression in Escherichia coli of an intact multienzyme component of the erythromycin‐producing polyketide synthase
Authors: ROBERTS Gareth A. , STAUNTON James , LEADLAY Peter F.
Published: March 2005

6‐Deoxyerythronolide B synthase 3 (DEBS 3) is proposed to catalyse the fifth and sixth condensation cycles in the assembly of the polyketide 6‐deoxyerythronolide B, the first isolatable intermediate in the biosynthesis of erythromycin A by Saccharopolyspora erythraea. The gene encoding DEBS 3 has previously been cloned and sequenced, and the deduced product is predicted to house nine fatty acid synthase‐like activities on a 330‐kDa polypeptide chain. The gene has been engineered into a pT‐7‐based expression system for over‐expression in Escherichia coli. Recombinant DEBS 3 was found to constitute, after induction, 1 – 2% of soluble intracellular protein. DEBS 3 was purified from extracts of the recombinant E. coli to apparent homogeneity, and was found not to be modified by covalent attachment of the prosthetic group 4′‐phosphopantetheine. Incubation with (R,S)‐methylmalonyl‐CoA, the presumed source of extension units for polyketide chain assembly, led to hydrolysis of the thioester, implying that the methylmalonyl‐CoA:ACP acyltransferase domains in DEBS 3 are correctly folded and able to catalyse this side‐reaction. During this reaction, DEBS 3 became transiently radiolabelled, consistent with the intermediacy of an acylenzyme. The native molecular mass of the protein by gel filtration chromatography was 668 kDa which corresponds either to a dimer or to a highly asymmetric monomer.
The structural characterization and bilirubin‐binding properties of albumin Herborn, a [Lys240→Glu] albumin mutant
Authors: MINCHIOTTI Lorenzo , GALLIANO Monica , ZAPPONI M. Carla , TENNI Ruggero
Published: March 2005

We report the molecular defect of albumin Herborn, a new genetic variant of human serum albumin which has been found in Germany. Isoelectric focusing analysis of CNBr fragments from the purified variant allowed us to localize the mutation in fragment CNBr 3 (residues 124–298). This fragment was isolated on a preparative scale and subjected to tryptic and V8 protease digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution Lys240→Glu. The ‐2 charge change of albumin Herborn, which is probably due to a A→G transition in the first position of the corresponding codon in the structural gene, has no significant effect on its electrophoretic mobility under non‐denaturating conditions. Therefore we have assumed that residue 240, which has been implicated in the bilirubin primary binding site (Jacobsen, C. (1978) Biochem. J. 171, 453–459), is buried. The binding of bilirubin and biliverdin by albumin Herborn was quantified using the fluorescence quenching method. The apparent equilibrium association constants (Ka±SD) and the number of high‐affinity binding sites (n) of the defatted variant for bilirubin and biliverdin were Ka= 1.03 ± 0.18 × 108 M−1, n= 1.07; and Ka= 7.48 ± 1.10 × 106 M−1, n= 1.01, respectively. The Ka values are about 93.3% and 99.1% of the values found for the normal protein under the same conditions. These results strongly suggest that Lys240 of human serum albumin is not the basic residue involved in ion pairing with one of the carboxylate groups of bilirubin at its high‐affinity site.
Oligomerization of the Nef protein from human immunodeficiency virus (HIV) type 1
Authors: KIENZLE Norbert , FREUND Jens , KALBITZER Hans Robert , MUELLER‐LANTZSCH Nikolaus
Published: March 2005

The nef genes, derived from two different human immunodeficiency‐virus‐type‐1 (HIV‐1) strains, were expressed in procaryotic cells (Escherichia coli) and in eucaryotic cells (insect cells infected with nef‐containing baculovirus). The oligomerization of recombinant Nef protein was studied by NMR spectroscopy and immunoblotting under various experimental conditions. 1H‐NMR spectroscopy shows that native folded protein has the tendency to polymerize under low‐salt conditions. These oligomers become covalently linked by disulfide bonds after decreasing the reduction potential, a process which is fully reversible. Cross‐linking studies with bis(sulfo‐succinimidyl)suberate and alkylation with iodoacetic acid under non‐reducing and reducing conditions document for the first time that Nef can also form homomeric structures including monomers, dimers, trimers and tetramers in cell lysates and intact cells. We found disulfide‐linked as well as non‐covalently associated oligomers. Since the Nef molecules are not exclusively found in the cytoplasm of HIV infected cells and since the reduced glutathione concentration in lymphocytes of virus infected persons is known to be unusually low, it might be possible that these Nef oligomers have a biological function in vivo as well.
γ‐Phosphate‐linked ATP‐Sepharose for the affinity purification of protein kinases
Authors: HAYSTEAD Clare M. M. , GREGORY Peter , STURGILL Thomas W. , HAYSTEAD Timothy A. J.
Published: March 2005

Recently, Sowadski and colleagues [Knighton, D. R., Zheng, J., Eyck, L. F. T., Ashford, V. A., Xuong, N., Taylor, S. S. & Sowadski, J. M. (1991) Science 407, 407–420] reported the structure of a ternary complex of the catalytic subunit of cAMP‐dependent protein kinase (cyclic A kinase), MgATP and a 20‐residue inhibitor peptide, at a resolution of 0.27 nm. This structure has since been refined to 0.2‐nm resolution and the orientation of the nucleotide and interactions of MgATP with numerous conserved residues at the active site defined [Zheng, J., Knighton, D. R., Eyck, L. F. T., Karlsson, R., Xuong, N., Taylor, S. S. & Sowadski, J. M. (1993) Biochemistry, in the press]. These studies revealed that the adenosine portion of ATP is buried deep within the catalytic cleft, with the α, β and γ phosphates protruding towards the opening of the cleft. The unique spatial positioning of MgATP within the catalytic cleft of cyclic A kinase and its interactions with conserved amino acids found in all protein kinases, led us to reconsider the use of ATP as an affinity ligand for the purification of these enzymes. In this paper, we describe a straightforward method for the synthesis of [γ‐32P]adenosine‐5′‐(γ‐4‐aminophenyl)triphosphate for the covalent linkage of ATP to Sepharose through its γ phosphate. In the presence of 20 μM ATP, adenosine‐5′‐(γ‐4‐aminophenyl)triphosphate exhibited apparent Ki values of 103.6, 75.18, 176.28 and 120.00 μM against cyclic A kinase, mitogen‐activated protein kinase (p42mapk), mitogen‐activated protein kinase kinase and p60c‐src, respectively. To illustrate the effectiveness of adenosine‐5′‐(γ‐4‐aminophenyl)triphosphate–Sepharose as an affinity column for protein kinases, we have used the resin to purify rabbit skeletal muscle mitogen‐activated protein kinase kinase over 19000‐fold to homogeneity.
Isolation, characterization and N‐terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii
Authors: HAPPE Thomas , NABER J. Dirk
Published: March 2005

Hydrogenase from Chlamydomonas reinhardtii was purified to homogeneity by five column‐chromatography steps under strict anaerobic conditions. The cells were disrupted by mild treatment with detergent. The enzyme was purified 6100‐fold, resulting in a specific activity for H2 evolution of 935 μmol · min−1· mg protein−1 at 25°C, using reduced methyl viologen as electron donor. The optimal temperature for hydrogen evolution is 60°C, the optimal pH value is 6.9. The Km value for methyl viologen is 0.83 mM, for ferredoxin, 35 μM. From SDS/PAGE gels, the protein was judged to be pure. On non‐denaturing gels, run under nitrogen, a single band was detected after activity staining. This band corresponded to the single band observed on denaturing SDS gels, which had an apparent molecular mass of 48 kDa. If the band was cut out of the native gel and incubated with reduced methyl viologen, hydrogen evolution could be measured. The purified enzyme contains 4 Fe atoms/mol. The amino acid composition and the N‐terminal amino acid sequence (24 residues) of the protein were determined. No significant amino acid sequence homologies could be found to any sequences from prokaryotic hydrogenases.
Paracatalytic self‐inactivation of fructose‐1,6‐bisphosphate aldolase
Authors: GUPTA Sharad , HOLLENSTEIN Roger , KOCHHAR Sunil , CHRISTEN Philipp
Published: March 2005

Oxidation of enzyme‐substrate carbanion intermediates by extrinsic oxidants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose‐1,6‐bisphosphate aldolase from rabbit muscle the polypeptide chain had been found to be crosslinked at active‐site Lys229 (Schiff base forming with substrate) and Lys146 by a phosphorylated three‐carbon moiety [Lubini, D. G. E. and Christen, P. (1979) Proc. Natl Acad. Sci. USA 76, 2527–2531]. In the present study, the structure of this crosslink was elucidated by instrumental analysis. Aldolase was paracatalytically modified in the presence of fructose 1,6‐bisphosphate and hexacyanoferrate(III). The completely inactivated enzyme was digested with pronase. The crosslinked peptide was isolated by gel filtration and reverse‐phase HPLC. Mass spectroscopy, 1H‐ and 13C‐NMR showed that a derivative of dihydroxyacetone phosphate forms an amidine with the ɛ‐amino groups of the two lysine residues:

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Characterisation of the ATP‐dependent taurocholate‐carrier protein (gp110) of the hepatocyte canalicular membrane
Authors: BECKER Andreas , LUCKA Lothar , KILIAN Christiane , KANNICHT Christoph , REUTTER Werner
Published: March 2005

The canalicular domain‐specific glycoprotein gp110, which recently has been shown to function as an ATP‐dependent taurocholate transporter, has been purified 1800‐fold from rat liver plasma membranes. gp110 has been characterised as an integral plasma membrane protein with Mr of 100000–115000 and pI of 2.5–3.5 and possesses a highly glycosylated and negatively charged extra‐cellular domain. The broad range of Mr and pI values results from the existence of numerous glycoforms composed of sialylated N‐glycans. After deglycosylation, the polypeptide has Mr 48000 and pI 5.0. In primary cultures of rat hepatocytes, gp110 is synthesised with Mr 110000, while in the presence of tunicamycin the non‐glycosylated form has Mr 48000. In the presence of 1‐deoxymannojirimycin, two forms of Mr 83000 and Mr 91000 were found, which were converted by endo‐β‐N‐acetylglucosaminidase H into a single 52000‐Mr band, indicating the existence of two basic glycoforms at the ollgomannosyl stage of biosynthesis. gp110 was phosphorylated at serine residues in primary cultures of hepatocytes. The sequences of ten internal peptides of gp110 were identical to the sequence of the high‐Mr form of ecto‐ATPase, but ecto‐ATPase activity from plasmamembrane extracts was not depleted by anti‐(gp110) serum. In contrast, Fab fragments of these antibodies inhibit the aggregation of freshly isolated hepatocytes.
Aminotransferases: demonstration of homology and division into evolutionary subgroups
Authors: MEHTA Perdeep K. , HALE Terence I. , CHRISTEN Philipp
Published: March 2005

A total of 150 amino acid sequences of vitamin B6‐dependent enzymes are known to date, the largest contingent being furnished by the aminotransferases with 51 sequences of 14 different enzymes. All aminotransferase sequences were aligned by using algorithms for sequence comparison, hydropathy patterns and secondary structure predictions. The aminotransferases could be divided into four subgroups on the basis of their mutual structural relatedness. Subgroup I comprises aspartate, alanine, tyrosine, histidinol‐phosphate, and phenylalanine aminotransferases; subgroup II acetylornithine, ornithine, ω‐amino acid, 4‐aminobutyrate and diaminopelargonate aminotransferases; subgroup III D‐alanine and branched‐chain amino acid aminotransferases, and subgroup IV serine and phosphoserine aminotransferases. (N‐1) profile analysis, a more stringent application of profile analysis [Gribskov, M., McLachlan, A. D. and Eisenberg, D. (1987) Proc. Natl Acad. Sci. USA 84, 4355–4358], established the homology among the enzymes of each subgroup as well as among all subgroups except subgroup III. However, similarity of activesite segments and the hydropathy patterns around invariant residues suggest that subgroup III, though most distantly related, might also be homologous with the other aminotransferases. On the basis of the comprehensive alignment, a new numbering of amino acid residues applicable to aminotransferases (AT) in general is proposed. In the multiply aligned sequences, only four out of a total of about 400 amino acid residues proved invariant in all 51 sequences, i.e. Gly(314AT)197, Asp/Glu(340AT)222, Lys(385AT)258 and Arg(562AT)386, the number not in parentheses corresponding to the structure of porcine cytosolic aspartate aminotransferase. Apparently, the aminotransferases constitute a group of homologous proteins which diverged into subgroups and, with some exceptions, into substrate‐specific individual enzymes already in the universal ancestor cell.
Functional reconstitution of the Trypanosoma brucei plasma‐membrane D‐glucose transporter
Authors: SEYFANG Andreas , DUSZENKO Michael
Published: March 2005

The D‐glucose transporter of Trypanosoma brucei was solubilized from the plasma membrane and reconstituted into proteoliposomes. Using the reconstitution of D‐glucose transport as the assay and non‐specific L‐glucose uptake as control, we have purified a membrane protein fraction from T. brucei bloodstream‐form ghosts by EDTA/alkali treatment and solubilization with the detergents octylglucoside or octylthioglucoside. Upon removal of the detergent by dialysis, the solubilized protein fraction was reconstituted in sonicated liposomes by a freeze/thaw‐sonication step. The reconstituted transporter catalyzed specific D‐glucose uptake and was compared in several characteristics with the native facilitated‐diffusion transporter as present in live trypanosomes [Seyfang, A. & Duszenko, M. (1991) Eur. J. Biochem. 202, 191–196]. As in vivo, the uptake is time dependent and Na+ independent. Transporter substrate affinity and inhibitor specificity are completely retained and it is inhibited by mercuric ions, phloretin and cytochalasin B, but only partially inhibited by phlorizin. The reconstituted transporter also demonstrates trans‐stimulation properties indicative of the carrier‐mediated transport of D‐glucose. In contrast to the human erythrocyte‐type glucose transporter, in T. brucei D‐fructose uptake was also catalyzed by the same reconstituted protein fraction and specific D‐glucose or D‐fructose transport were mutually competitive. Both the inhibitor studies and the fructose transport capacity in the reconstituted system are in good agreement with the native transport in live trypanosomes. The specific activity of D‐glucose transport was 1.9±0.3 nmol·min−1· mg protein−1 at 0.2 mM D‐glucose and the yield was about 0.8% of total ghost protein after removal of the variant‐surface‐glycoprotein coat. The successful functional reconstitution of a protozoan glucose transporter represents an important step towards its purification and detailed characterization. This is especially interesting since bloodstream‐form trypanosomes depend entirely upon glycolysis for their ATP production.