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  • 1. Achen, M G
    et al.
    Roufail, S
    Domagala, T
    Catimel, B
    Nice, E C
    Geleick, D M
    Murphy, R
    Scott, A M
    Caesar, C
    Makinen, T
    Alitalo, K
    Stacker, S A
    Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.2000In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 267, no 9Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.

  • 2.
    Andersson, Hans O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Fridborg, Kerstin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Löwgren, Seved
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Alterman, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mühlman, Anna
    Björsne, Magnus
    Garg, Neeraj
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Kvarnström, Ingemar
    Schaal, Wesley
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Classon, Björn
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Ahlsén, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Nillroth, Ulrika
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Vrang, Lotta
    Öberg, Bo
    Samuelsson, Bertil
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors2003In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 8, p. 1746-1758Article in journal (Refereed)
  • 3.
    Blomqvist, M
    et al.
    Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, Sahlgrenska University Hospital/Mölndal, Sweden.
    Bergquist, Jonas
    Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, Sahlgrenska University Hospital/Mölndal, Sweden.
    Westman, A
    Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, Sahlgrenska University Hospital/Mölndal, Sweden.
    Hâkansson, K
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Hâkansson, P
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Fredman, P
    Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, Sahlgrenska University Hospital/Mölndal, Sweden.
    Ekman, R
    Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, Sahlgrenska University Hospital/Mölndal, Sweden.
    Identification of defensins in human lymphocyte nuclei1999In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 263, no 2, p. 312-8Article in journal (Refereed)
    Abstract [en]

    The cell nucleus plays an essential role in all aspects of cell function and regulation. Most of the nuclear proteins/peptides are synthesized in the cytoplasm and transported into the nucleus through the nuclear pore complexes. The nuclear proteins/peptides conjugate with each other and interact in transcriptional activation/inactivation. Several of the high molecular mass transcription factors (> 30 kDa) have been identified and characterized. However, the information on the low molecular mass proteins/peptides of the nucleus is limited. We have investigated these low molecular mass proteins/peptides from the nucleus of human peripheral blood lymphocytes using reversed-phase high-performance liquid chromatography (RP-HPLC). The HPLC fractions were further analysed by matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, electrospray ionization time of flight (ESI-TOF) mass spectrometry and electrospray ionization fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry for mass determination. Using this combination of mass spectrometry techniques and microsequence analysis, we have shown that human lymphocyte nuclei contain defensins, a mixture of human neutrophil granule peptide 1, 2 and 3.

  • 4. Cervenanský, C
    et al.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Karlsson, Evert
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Role of arginine residues for the activity of fasciculin1995In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 229, no 1, p. 270-275Article in journal (Refereed)
    Abstract [en]

    The West African green mamba, Dendroaspis angusticeps, has two toxins, fasciculins, that are non-competitive inhibitors of acetylcholinesterase. Arginine residues of fasciculin 2 were modified with 1,2-cyclohexanedione. Two of these residues, Arg24 and Arg37, reacted very slowly or not at all. Modification of Arg28 reduced the activity only by 13%. Arg11 and Arg27 are unique for fasciculins; a comparison of the sequences of 175 snake toxins homologous to fasciculins showed that no other toxin has arginine in the corresponding positions. Modification of the two unique arginines had a large effect and decreased the activity by 73% (Arg11) and 85% (Arg27). This was apparently not due to structural perturbations, since the modification did not change the circular dichroic spectra. The two arginine residues probably participate in the binding to acetylcholinesterase. They are located on the same side of the toxin molecule and the distance between their alpha-carbons is 2.7 nm. This may indicate binding to sites that are far apart and suggests that fasciculin covers a large area of the enzyme

  • 5.
    Ek, Pia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zetterqvist, Örjan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ek, Bo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Pettersson, Gunilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gong, Feng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase2002In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 269, p. 5016-5023Article in journal (Refereed)
    Abstract [en]

    Protein histidine phosphorylation in eukaryotes has beensparsely studied compared to protein serine/threonine andtyrosine phosphorylation. In an attempt to rectify this byprobing porcine liver cytosol with the phosphohistidinecontainingpeptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide(phosphopeptide I), we observed a phosphataseactivity that was insensitive towards okadaic acid andEDTA. This suggested the existence of a phosphohistidinephosphatase different from protein phosphatase 1, 2Aand 2C. A 1000-fold purification to apparent homogeneitygave a 14-kDa phosphatase with a specific activity of 3lmolÆmin)1Æmg)1 at pH 7.5 with 7 lM phosphopeptide Ias substrate. Partial amino-acid sequence determination ofthe purified porcine enzyme by MS revealed similaritywith a human sequence representing a human chromosome9 gene of hitherto unknown function. Molecularcloning from a human embryonic kidney cell cDNAlibraryfollowed by expression and purification, yielded aprotein with a molecular mass of 13 700 Da, and anEDTA-insensitive phosphohistidine phosphatase activityof 9 lmolÆmin)1Æmg)1 towards phosphopeptide I. Nodetectable activity was obtained towards a set of phosphoserine-,phosphothreonine-, and phosphotyrosine peptides.Northern blot analysis indicated that the humanphosphohistidine phosphatase mRNA was present preferentiallyin heart and skeletal muscle. These resultsprovide a new tool for studying eukaryotic histidinephosphorylation/dephosphorylation.

  • 6.
    Gottschalk, Ingo
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Lundqvist, Andreas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Zeng, Cheng-Ming
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Lagerquist Hägglund, Christine
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Zuo, Shu-sheng
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Brekkan, Eggert
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Eaker, David
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Lundahl, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter2000In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 267, no 23, p. 6875-6882Article in journal (Refereed)
    Abstract [en]

    Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.

  • 7. Henriksson, Gunnar
    et al.
    Nutt, Anu
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Henriksson, Hongbin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Pettersson, Bert
    Ståhlberg, Jerry
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Pettersson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase.1999In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 259, no 1-2, p. 88-95Article in journal (Refereed)
    Abstract [en]

    A 28-kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium strain K3 and named EG 28. It degrades carboxymethylated cellulose and amorphous cellulose, and to a lesser degree xylan and mannan but not microcrystalline cellulose (Avicel). EG 28 is unusual among cellulases from aerobic fungi, in that it appears to lack a cellulose-binding domain and does not bind to crystalline cellulose. The enzyme is efficient at releasing short fibres from filter paper and mechanical pulp, and acts synergistically with cellobiohydrolases. Its mode of degrading filter paper appears to be different to that of endoglucanase I from Trichoderma reesei. Furthermore, EG 28 releases colour from stained cellulose beads faster than any other enzyme tested. Peptide mapping suggests that it is not a fragment of another known endoglucanases from P. chrysosporium and peptide sequences indicate that it belongs to family 12 of the glycosyl hydrolases. EG 28 is glycosylated. The biological function of the enzyme is discussed, and it is hypothesized that it is homologous to EG III in Trichoderma reesei and the role of the enzyme is to make the cellulose in wood more accessible to other cellulases.

  • 8.
    Hjälm, Göran
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Murray, Edward
    Crumley, Gregg
    Harazim, William
    Lundgren, Stefan
    Onyango, Isaac
    Ek, Bo
    Larsson, Mårten
    Juhlin, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Hellman, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Davis, Hughes
    Åkerström, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Rask, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Morse, Buzzy
    Cloning and sequencing of human gp330, a Ca2+ -binding receptor with potential intracellular signaling properties1996In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 239, no 1, p. 132-137Article in journal (Refereed)
    Abstract [en]

    We present here the complete primary structure of human gp330, the human variant of the principal kidney autoantigen causing Heymann membranous glomerulonephritis in rats. The deduced 4655 amino acid residues give a calculated molecular mass of 519636 Da for the mature protein and consists of a probable 25-amino-acid N-terminal signal peptide sequence, an extracellular region of 4398 amino acids, a single transmembrane-spanning domain of 23 amino acids, and an intracellular C-terminal region of 209 amino acid residues. Three types of cysteine-rich repeats characteristic of the low density lipoprotein receptor (LDLR) superfamily are present in human gp330. In the extracellular region, there are a total of 36 LDLR ligand-binding repeats, comprising four distinct domains, 16 growth factor repeats separated by eight YWTD spacer regions, and one epidermal growth factor-like repeat. No consensus cleavage sequence for the processing endoprotease furin is detected in human gp330. The intracellular tail contains not only two copies of the F(X)NPXY coated-pit mediated internalization signal characteristic of LDLR superfamily members, but also intriguing and potentially functional motifs including several Src-homology 3 recognition motifs, one Src-homology 2 recognition motif for the p85 regulatory subunit of phosphatidylinositol 3-kinase, and additional sites for protein kinase C, casein kinase II and cAMP-/cGMP-dependent protein kinase. There is approximately 77% amino acid identity between human and rat gp330 with minor differences between the extracellular and intracellular regions. Recently gp330 has been implicated in Ca2+ regulation in the parathyroid, the placenta, and the renal tubule, but its overall physiological and pathological role still remains uncertain.

  • 9. Kirsebom, Leif A
    et al.
    Amons, R
    Isaksson, Leif A
    Primary structure of mutationally altered ribosomal protein L7/L12 and their effects on growth and translational accruracy1986In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 156, p. 669-675Article in journal (Refereed)
  • 10.
    Klovins, Janis
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Haitina, Tatjana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Ringholm, Aneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Löwgren, Maja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Fridmanis, Davids
    Slaidina, Maija
    Stier, Susanne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Schiöth, Helgi B
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Cloning of two melanocortin (MC) receptors in spiny dogfish: MC3 receptor in cartilaginous fish shows high affinity to ACTH-derived peptides while it has lower preference to gamma-MSH2004In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 271, no 21, p. 4320-4331Article in journal (Refereed)
    Abstract [en]

    We report the cloning and characterization of two melanocortin receptors (MCRs) from the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis shows that these shark receptors are orthologues of the MC3R and MC5R subtypes, sharing 65% and 70% overall amino acid identity with the human counterparts, respectively. The SacMC3R was expressed and pharmacologically characterized in HEK293 cells. The radioligand binding results show that this receptor has high affinity for adrenocorticotropic hormone (ACTH)-derived peptides while it has comparable affinity for alpha- and beta-melanocyte stimulating hormone (MSH), and slightly lower affinity for gamma-MSH when compared with the human orthologue. ACTH(1-24) has high potency in a second-messenger cAMP assay while alpha- and gamma-MSH had slightly lower potency in cells expressing the SacMC3R. We used receptor-enhanced green fluorescence protein (EGFP) fusion to show the presence of SacMC3R in plasma membrane of Chinese hamster ovary and HEK293 cells but the SacMC5R was retained in intracellular compartments of these cells hindering pharmacological characterization. The anatomical distribution of the receptors were determined using reverse transcription PCR. The results showed that the SacMC3R is expressed in the hypothalamus, brain stem and telencephalon, optic tectum and olfactory bulbs, but not in the cerebellum of the spiny dogfish while the SacMC5R was found only in the same central regions. This report describes the first molecular characterization of a MC3R in fish. The study indicates that many of the important elements of the MC system existed before radiation of gnathostomes, early in vertebrate evolution, at least 450 million years ago.

  • 11.
    Lindberg, Jimmy
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pyring, David
    Löwgren, Seved
    Rosenquist, Åsa
    Zuccarello, Guido
    Kvarnström, Ingemar
    Zhang, Hong
    Vrang, Lotta
    Classon, Björn
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Samuelsson, Bertil
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Symmetric fluoro-substituted diol-based HIV protease inhibitors: Ortho-fluorinated and meta-fluorinated P1/P1'-benzyloxy side groups significantly improve the antiviral activity and preserve binding efficacy2004In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 271, no 22, p. 4594-4602Article in journal (Refereed)
    Abstract [en]

    HIV-1 protease is a pivotal enzyme in the later stages of the viral life cycle which is responsible for the processing and maturation of the virus particle into an infectious virion. As such, HIV-1 protease has become an important target for the treatment of AIDS, and efficient drugs have been developed. However, negative side effects and fast emerging resistance to the current drugs have necessitated the development of novel chemical entities in order to exploit different pharmacokinetic properties as well as new interaction patterns. We have used X-ray crystallography to decipher the structure-activity relationship of fluoro-substitution as a strategy to improve the antiviral activity and the protease inhibition of C2-symmetric diol-based inhibitors. In total we present six protease-inhibitor complexes at 1.8-2.3 A resolution, which have been structurally characterized with respect to their antiviral and inhibitory activities, in order to evaluate the effects of different fluoro-substitutions. These C2-symmetric inhibitors comprise mono- and difluoro-substituted benzyloxy side groups in P1/P1' and indanoleamine side groups in P2/P2'. The ortho- and meta-fluorinated P1/P1'-benzyloxy side groups proved to have the most cytopathogenic effects compared with the nonsubstituted analog and related C2-symmetric diol-based inhibitors. The different fluoro-substitutions are well accommodated in the protease S1/S1' subsites, as observed by an increase in favorable Van der Waals contacts and surface area buried by the inhibitors. These data will be used in the development of potent inhibitors with different pharmacokinetic profiles towards resistant protease mutants.

  • 12.
    Lindholm, Cecilia K
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Henriksson, Maria L
    Hallberg, Bengt
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Shb links SLP-76 and Vav with the CD3 complex in Jurkat T cells2002In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 269, no 13, p. 3279-3288Article in journal (Refereed)
    Abstract [en]

    This study addresses the interactions between the adaptor protein Shb and components involved in T cell signalling, including SLP-76, Gads, Vav and ZAP70. We show that both SLP-76 and ZAP70 co-immunoprecipitate with Shb in Jurkat T cells and that Shb and Vav co-immunoprecipitate when cotransfected in COS cells. We also demonstrate, utilizing fusion protein constructs, that SLP-76, Gads and Vav associate independently of each other to different domains or regions, of Shb. Overexpression of an SH2 domain-defective Shb causes diminished phosphorylation of SLP-76 and Vav and consequently decreased activation of c-Jun kinase upon T cell receptor (TCR) stimulation. Shb was also found to localize to glycolipid-enriched membrane microdomains (GEMs), also called lipid rafts, after TCR stimulation. Our results indicate that upon TCR stimulation, Shb is targeted to these lipid rafts where Shb aids in recruiting the SLP-76–Gads–Vav complex to the T cell receptor ζ-chain and ZAP70.

  • 13. Lu, G
    et al.
    Dobritzsch, Doreen
    Martin-Luther-Universität Halle-Wittenberg.
    Baumann, S
    Schneider, G
    König, S
    The structural basis of substrate activation in yeast pyruvate decarboxylase: A crystallographic and kinetic study2000In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 267, no 3, p. 861-868Article in journal (Refereed)
    Abstract [en]

    The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered. In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer-dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder-order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed.

  • 14. Nilsson, Magnus H. L.
    et al.
    Spurr, Nigel K.
    Saksena, Pushpa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Pathology.
    Busch, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Pathology.
    Nordlinder, Hans
    Peterson, Per A.
    Rask, Lars
    Sundelin, Johan
    Isolation and characterization of a cDNA clone corresponding to bovine cellular retinoic-acid-binding protein and chromosomal localization of the corresponding human gene1988In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 173, no 1, p. 45-51Article in journal (Refereed)
    Abstract [en]

    A bovine adrenal cDNA library was constructed and a clone corresponding to cellular retinoic-acid-binding protein (CRABP) mRNA was isolated and sequenced. The insert of the clone corresponds to 75 bp of the 5′ untranslated portion, the whole translated and the complete 3′ untranslated portion of the bovine CRABP mRNA. A genomic Southern blot, probed with CRABP cDNA, indicated that only one copy of the gene is present in the human genome. Hybridizing bands in restricted chicken and fish DNA were also observed. Using the CRABP cDNA as probe we have located the human CRABP gene to chromosome 3 in hybridizations to mouse-human, hamster-human and rat-human cell hybrids. In situ hybridizations on rat testis cells probed with CRABP and cellular retinol-binding protein antisense mRNA indicate that both proteins are expressed in tubuli cells.

  • 15.
    Parmryd, Ingela
    et al.
    Arrhenius Laboratories for Natural Sciences, Biochemistry Department, Stockholm University, S-106 91 Stockholm, Sweden.
    Shipton, C. A.
    Swiezewska, E.
    Andersson, B.
    Dallner, G.
    Identification of spinach farnesyl protein transferase: Dithiothreitol as an acceptor in vitro1995In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 234, no 3, p. 723-731Article in journal (Refereed)
    Abstract [en]

    Spinach seedlings were found to contain farnesyl protein transferase. The enzyme is activated by Zn2+, but not by Mg2+. The pH optimum is approximately 7.0 and maximal activity is obtained at 40-45 degrees C. The apparent Km for the farnesyl diphosphate substrate is 7 microM. Western blotting of soluble proteins with an antiserum raised against mammalian farnesyl protein transferase demonstrated a specific cross-reactivity with the spinach enzyme. The antiserum preferentially recognises the beta-subunit of the heterodimeric farnesyl protein transferase, and the corresponding spinach polypeptide has a molecular mass of 42 kDa on SDS/PAGE. The enzyme can employ dithiothreitol as an acceptor for the farnesyl moiety and catalyses the formation of a thioether linkage between these substrates. On the basis of this discovery, a new method was developed utilising the hydrophobicity of the reaction product, and its interaction with poly(propylene). During in vivo labelling, the plants took up dithiothreitol, which inhibited the incorporation of [3H]mevalonate metabolites into proteins, indicating that dithiothreitol might be isoprenylated in vivo as well as in vitro. However, isoprenylation of some proteins remains unaffected by dithiothreitol suggesting the existence of different isoprenylation mechanisms. Thus, it is demonstrated that plants possess farnesyl protein transferase, which resembles its mammalian and yeast homologues.

  • 16.
    Ramström, Margareta
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Hagman, Charlotte
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Tsybin, Youri O
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Markides, Karin E
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Salehi, Albert
    Lundqvist, Ingmar
    Håkansson, Rolf
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    A novel mass spectrometric approach to analysis of hormonal peptides in extracts of mouse pancreatic islets2003In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 15, p. 3146-3152Article in journal (Refereed)
    Abstract [en]

    Liquid chromatography mass spectrometry (LC-MS) is a valuable tool in the analysis of proteins and peptides. The combination of LC-MS with different fragmentation methods provides sequence information on components in complex mixtures. In this work, on-line packed capillary LC electrospray ionization Fourier transform ion cyclotron resonance MS was combined with two complementary fragmentation techniques, i.e. nozzle-skimmer fragmentation and electron capture dissociation, for the determination of hormonal peptides in an acid ethanol extract of mouse pancreatic islets. The most abundant peptides, those derived from proinsulin and proglucagon, were identified by their masses and additional sequence-tag information established their identities. Interestingly, the experiments demonstrated the presence of truncated C-peptides, des-(25-29)-C-peptide and des-(27-31)-C-peptide. These novel findings clearly illustrate the potential usefulness of the described technique for on-line sequencing and characterization of peptides in tissue extracts.

  • 17.
    Ramström, Margareta
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Hagman, Charlotte
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Ion Physics.
    Tsybin, Youri O
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Ion Physics.
    Markides, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Ion Physics.
    Salehi, Albert
    Department of Pharmacology, Institute of Physiological Sciences, Lund University.
    Lundquist, Ingmar
    Department of Pharmacology, Institute of Physiological Sciences, Lund University.
    Håkanson, Rolf
    Department of Pharmacology, Institute of Physiological Sciences, Lund University.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    A novel mass spectrometric approach to the analysis of hormonal peptides in extracts of mouse pancreatic islets2003In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 15, p. 3146-3152Article in journal (Refereed)
    Abstract [en]

    Liquid chromatography mass spectrometry (LC-MS) is a valuable tool in the analysis of proteins and peptides. The combination of LC-MS with different fragmentation methods provides sequence information on components in complex mixtures. In this work, on-line packed capillary LC electrospray ionization Fourier transform ion cyclotron resonance MS was combined with two complementary fragmentation techniques, i.e. nozzle-skimmer fragmentation and electron capture dissociation, for the determination of hormonal peptides in an acid ethanol extract of mouse pancreatic islets. The most abundant peptides, those derived from proinsulin and proglucagon, were identified by their masses and additional sequence-tag information established their identities. Interestingly, the experiments demonstrated the presence of truncated C-peptides, des-(25-29)-C-peptide and des-(27-31)-C-peptide. These novel findings clearly illustrate the potential usefulness of the described technique for on-line sequencing and characterization of peptides in tissue extracts.

  • 18.
    Rupp, Eva
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Siegbahn, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Rönnstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    A unique autophosphorylation site in the platelet-derived growth factor alpha receptor from a heterodimeric receptor complex1994In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 225, no 1, p. 29-41Article in journal (Refereed)
    Abstract [en]

    The platelet-derived growth factor (PDGF) alpha and beta receptors undergo dimerization as a consequence of ligand binding. Depending on the PDGF isoform (PDGF-AA, -AB or -BB), homodimers or heterodimers of receptors are formed. In this study, we have used transfected porcine aortic endothelial cells, coexpressing cDNAs for the alpha receptor and the beta receptor at comparable levels, to investigate the properties of the alpha beta-heterodimeric receptor complex. PDGF-AB, which mainly induced alpha beta-heterodimeric complexes, was the most efficient isoform for stimulating mitogenicity. Actin reorganization, in the form of circular membrane ruffling and chemotaxis, was induced by PDGF-AB and PDGF-BB, but not by PDGF-AA, thus indicating that the beta receptor in the homodimeric or heterodimeric configuration was required for induction of motility responses. The molecular basis for the apparent receptor dimer-specific properties was examined by analyzing receptor autophosphorylation and phosphorylation of substrates. The alpha receptor was found to be phosphorylated at an additional tyrosine residue, Tyr754, in the heterodimeric complex as compared to the alpha alpha receptor homodimer. Phosphorylation of this tyrosine residue could permit the binding of a specific signal-tranducing protein. A candidate is a 134,000-M(r) protein, which was shown to associate preferentially with the alpha receptor in the heterodimeric receptor complex. It is possible that phosphorylated Tyr754 in the alpha receptor mediates activation of specific signal-tranducing molecules like the 134,000-M(r) substrate, and thereby initiates signal-tranduction pathways from the alpha beta receptor heterodimer, which are distinct from those initiated via homodimeric receptor complexes.

  • 19.
    Spillmann, Dorothe
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Finne, J
    Identification of a major poly-N-acetyllactosamine-containing cell-surface glycoprotein of mouse teratocarcinoma cells: Appearance on cells induced to primitive endoderm but not parietal endoderm differentiation1994In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 220, no 2, p. 385-394Article in journal (Refereed)
    Abstract [en]

    Mouse teratocarcinoma F9 cells were induced to primitive endoderm differentiation with retinoic acid, and poly-N-acetyllactosamine-containing surface glycoproteins were identified by radiolabelling endo-beta-galactosidase-cleavable glycans with galactosyltransferase and radiolabelled UDP-galactose. One major radiolabelled band with an apparent size of 250-500 kDa was identified which differed from the known poly-N-acetyllactosamine-containing glycoproteins laminin, fibronectin, lysosome-associated membrane protein (LAMP)-1 and LAMP-2. This acidic glycoprotein, resistant to glycosaminoglycan-degrading enzymes and proteases, was purified by extraction and phase partition with Triton X-114, octyl Sepharose and Helix pomatia lectin chromatography. The purified glycoprotein could be digested by endo-beta-galactosidase and glycopeptide N-glycosidase F to an apparent size of 160-240 kDa. During retinoic-acid-induced differentiation into primitive endoderm cells, the glycoprotein showed a several-fold increase and a broadening to an apparent size of 200- > 700 kDa. The glycoprotein was no longer detected in retinoic-acid and dibutyryl-cAMP-treated cells which had undergone further differentiation to parietal endoderm cells, nor in the permanently differentiated parietal endoderm line F9-AC. The results suggest that the glycoprotein is a major carrier of poly-N-acetyllactosamine chains on differentiating teratocarcinoma F9 cells, and that its expression as revealed by the poly-N-acetyllactosamine labelling method is regulated by the stage of cellular differentiation.

  • 20.
    Trojanek, Joanna
    et al.
    Polish academy of Science.
    Ek, Pia
    Scoble, Judith
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Muszynska, Grazyna
    Polish Academy of Science.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Phosphorylation of plant proteins and the identification of protein - tyrosine kinase activity in maize seedlings1996In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 235, no 1-2, p. 338-344Article in journal (Refereed)
    Abstract [en]

    Phosphotyrosine was found to be 0.5% of the total phosphoamino acids labelled with [32P]orthophosphate in endogenous maize seedlings proteins. Two peaks of protein kinase activity towards phosphorylation of synthetic peptide poly (Glu80, Tyr20) were obtained after chromatography of protein extract of dark-grown etiolated maize seedlings on phosphocellulose. The phosphorylation of synthetic peptide as well as endogenous proteins was strongly stimulated by Mn2+. At least three endogenous proteins with molecular masses in the range of 40-65 kDa were predominantly phosphorylated. This phosphorylation was resistant to alkali treatment. Chemical, immunological and enzymatic data indicated the presence of tyrosine kinase activity and also phosphotyrosine in proteins of maize seedlings. The plant enzyme(s) is reminiscent known mammalian cytosolic tyrosine kinase(s).

  • 21.
    Tsuchida, Kazunori
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lind, Thomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kitagawa, H
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sugahara, K
    Lidholt, Kerstin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Purification and characterization of fetal bovine serum beta-N-acetyl-D-galactosaminyltransferase and beta-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis1999In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 264, no 2, p. 461-467Article in journal (Refereed)
    Abstract [en]

    beta-N-Acetylgalactosaminyltransferase II and beta-glucuronyltransferase II, involved in chondroitin sulfate biosynthesis, transfer an N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) residue, respectively, through beta-linkages to an acceptor chondroitin oligosaccharide derived from the repeating disaccharide region of chondroitin sulfate. They were copurified from fetal bovine serum approximately 2500-fold and 850-fold, respectively, by sequential chromatographies on Red A-agarose, phenyl-Sepharose, S-Sepharose and wheat germ agglutinin-agarose. Identical and inseparable chromatographic profiles of both glycosyltransferase activities obtained through the above chromatographic steps and gel filtration suggest that the purified enzyme activities are tightly coupled, which could imply a single enzyme with dual transferase activities; beta-N-acetylgalactosaminyltransferase and beta-glucuronyltransferase, reminiscent of the heparan sulfate polymerase reaction. However, when a polymerization reaction was performed in vitro with the purified serum enzyme preparation under the polymerization conditions recently developed for the chondroitin-synthesizing system, derived from human melanoma cells, each monosaccharide transfer took place, but no polymerization occurred. These results may suggest that the purified serum enzyme preparation contains both beta-N-acetylgalactosaminyltransferase II and beta-glucuronyltransferase II activities on a single polypeptide or on the respective polypeptides forming an enzyme complex, but is different from that obtained from melanoma cells in that it transfers a single GalNAc or GlcA residue but does not polymerize chondroitin.

  • 22.
    Väljamäe, Priit
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Pettersson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mechanism of substrate inhibition in cellulose synergistic degradation2001In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, no 16, p. 4520-4526Article in journal (Refereed)
    Abstract [en]

    A comprehensive experimental study of substrate inhibition in cellulose hydrolysis based on a well defined system is presented. The hydrolysis of bacterial cellulose by synergistically operating binary mixtures of cellobiohydrolase I from Trichoderma reesei and five different endoglucanases as well as their catalytic domains displays a characteristic substrate inhibition. This inhibition phenomenon is shown to require the two-domain structure of an intact cellobiohydrolase. The experimental data were in accordance with a mechanism where cellobiohydrolases previously bound to the cellulose by means of their cellulose binding domains are able to find chain ends by lateral diffusion. An increased substrate concentration at a fixed enzyme load will also increase the average diffusion distance/time needed for cellobiohydrolases to reach new chain ends created by endoglucanases, resulting in an apparent substrate inhibition of the synergistic action. The connection between the binding properties and the substrate inhibition is encouraging with respect to molecular engineering of the binding domain for optimal performance in biotechnological processes.

  • 23.
    Väljamäe, Priit
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Sild, Veljo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Nutt, Anu
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Pettersson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Acid hydrolysis of bacterial cellulose reveals different modes of synergistic action between cellobiohydrolase I and endoglucanase I1999In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 266, no 2, p. 327-334Article in journal (Refereed)
    Abstract [en]

    Intact and partially acid hydrolyzed cellulose from Acetobacter xylinum were used as model substrates for cellulose hydrolysis by 1,4-β-d-glucan-cellobiohydrolase I (CBH I) and 1,4-β-d-endoglucanase I (EG I) from Trichoderma reesei. A high synergy between CBH I and EG I in simultaneous action was observed with intact bacterial cellulose (BC), but this synergistic effect was rapidly reduced by acid pretreatment of the cellulose. Moreover, a distinct synergistic effect was observed upon sequential endo–exo action on BC, but not on bacterial microcrystalline cellulose (BMCC). A mechanism for endo–exo synergism on crystalline cellulose is proposed where the simultaneous action of the enzymes counteract the decrease of activity caused by undesirable changes in the cellulose surface microstructure.

  • 24. Väljamäe, Priit
    et al.
    Sild, Veljo
    Pettersson, Göran
    Johansson, Gunnar
    The initial kinetics of hydrolysis by cellobiohydrolases I and II  is consistent with a cellulose surface-erosion model1998In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 253, p. 469-475Article in journal (Refereed)
  • 25. Väljamäe, Priit
    et al.
    Sild, Veljo
    Pettersson, Göran
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    The Initial Kinetics of Hydrolysis by Cellobiohydrolases I and II is Consistent with a Cellulose Surface-Erosion Model1998In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 253, p. 469-475Article in journal (Refereed)
  • 26. Wang, RG
    et al.
    Lee, SY
    Cerenius, Lage
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus2001In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, no 4, p. 895-902Article in journal (Refereed)
    Abstract [en]

    The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzing the conversion of prophenoloxidase to an active phenoloxidase, has a molecular mass of about 36 kDa in its active form. This protein was cloned from a blood cell cDNA library and its corresponding cDNA of 1736 base pairs encodes a zymogenic protein (proppA) of 468 amino acids. An antibody raised against a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the β-1,3-glucan triggered activation of prophenoloxidase in vitro. The C-terminal half of the proppA is composed of a typical serine proteinase domain, with a sequence similar to other invertebrate and vertebrate serine proteinases. The N-terminal half contains a cationic glycine-rich domain, a cationic proline-rich domain and a clip-domain, in which the disulfide-bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian β-defensins. Antibodies made against both the C- and the N-terminal halves recognize two proppAs under reducing conditions. However, under nonreducing conditions only the anti-C antibody recognized the two proppAs, which suggests that a conformational change takes place upon reduction that allows the anti-N to react with the N-terminal half of proppA. The recombinant clip-domain in crayfish proppA was overexpressed in Escherichia coli and the resulting peptide exhibited antibacterial activity against Gram-positive bacterial strains such as Micrococcus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory concentrations of 1.43 µm and 17.9 µm, respectively. These results suggest that the clip-domains in proppAs may function as antibacterial peptides.

  • 27. Ylinenjärvi, K.
    et al.
    Widersten, Mikael
    Björk, Ingemar
    Hydrophobic sequences can substitute for the wild-type N-terminal sequence of cystatin A (stefin A) in tight binding to cysteine proteinases. Selection of high-affinity N-terminal region variants by phage display1999In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 261, p. 682-688Article in journal (Refereed)
  • 28.
    Åström, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Åström, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    A simple procedure for isolation of eukaryotic mRNA polyadenylation factors1991In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 202, no 3, p. 765-773Article in journal (Refereed)
    Abstract [en]

    We have devised a simple chromatographic procedure which isolates five polyadenylation factors that are required for polyadenylation of eukaryotic mRNA. The factors were separated from each other by fractionation of HeLa cell nuclear extract in two consecutive chromatographic steps. RNA cleavage at the L3 polyadenylation site of human adenovirus 2 required at least four factors. Addition of adenosine residues required only two of these factors. The fractionation procedure separates two components that are both likely to be poly(A) polymerases. The candidate poly(A) polymerases were interchangeable and participated during both RNA cleavage and adenosine addition. They were discriminated from each other by chromatographic properties, heat sensitivity and divalent cation requirement. We have compared our data with published information and have been able to correlate the activities that we have isolated to previously identified polyadenylation factors. However, we have not been able to assign one of the candidate poly(A) polymerases to a previously identified poly(A) polymerase. This simple fractionation procedure can be used for generating an in vitro reconstituted system for polyadenylation within a short period of time.

1 - 28 of 28
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