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See detailCo-regulation and physical association of the 67-kDa monomeric laminin receptor and the alpha6beta4 integrin.
Ardini, E.; Tagliabue, E.; Magnifico, A. et al

in Journal of Biological Chemistry (1997), 272(4), 2342-5

The interactions between tumor cells and laminin or other components of the extracellular matrix have been shown to play an important role in tumor invasion and metastasis. However, the role of the ... [more ▼]

The interactions between tumor cells and laminin or other components of the extracellular matrix have been shown to play an important role in tumor invasion and metastasis. However, the role of the monomeric 67-kDa laminin receptor (67LR) remains unclear. We analyzed the regulation of 67LR expression under different culture conditions with respect to the expression of other well characterized laminin receptors. In A431 cells treated with laminin for different time periods, the regulation of 67LR expression correlated with expression of the alpha6 integrin subunit but not with the expression of other laminin receptors. Moreover, cytokine treatment resulted in down-modulated expression of the alpha6 integrin subunit and the 67LR. Co-regulation of the expression of the two receptors was further suggested by the observation that specific down-modulation of the alpha6-chain by antisense oligonucleotides was accompanied by a proportional decrease in the cell surface expression of 67LR. Biochemical analyses indicated co-immunoprecipitation of 67LR and the alpha6 subunit with an anti-alpha6 but not an anti-beta1 monoclonal antibody. Co-regulation of 67LR and alpha6 subunit expression, together with the physical association between the two receptors, supports the hypothesis that 67LR is an auxiliary molecule involved in regulating or stabilizing the interaction of laminin with the alpha6beta4 integrin. [less ▲]

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See detailSite-directed mutagenesis of glutamate 166 in two beta-lactamases. Kinetic and molecular modeling studies.
Guillaume, Gilliane; Vanhove, M; Lamotte-Brasseur, J et al

in Journal of Biological Chemistry (1997), 272(9), 5438-44

The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the ... [more ▼]

The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G beta-lactamases yielded enzymes forming stable acyl-enzymes with beta-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of Omega-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the deacylation processes. [less ▲]

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See detailEvidence of involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair
Johnson, Robert; Kovvali, G.; Guzder, Sami et al

in Journal of Biological Chemistry (1996), 271(45), 27897-90

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See detailStructural and Functional Aspects of Chloride Binding to Alteromonas Haloplanctis Alpha-Amylase
Feller, Georges ULg; le Bussy, O.; Houssier, C. et al

in Journal of Biological Chemistry (1996), 271(39), 23836-41

Chloride is the allosteric effector of vertebrate pancreatic and salivary alpha-amylases and of the bacterial alpha-amylase from Alteromonas haloplanctis. Activation experiments of A. haloplanctis alpha ... [more ▼]

Chloride is the allosteric effector of vertebrate pancreatic and salivary alpha-amylases and of the bacterial alpha-amylase from Alteromonas haloplanctis. Activation experiments of A. haloplanctis alpha-amylase by several monovalent anions show that a negative charge, not restricted to that of Cl-, is essential for the amylolytic reaction. Engineering of the chloride binding site reveals that a basic residue is an essential component of the site. The mutation K337R alters the Cl--binding properties, whereas the mutation K337Q produces an active, chloride-independent enzyme. Comparison of the Kd values for Cl- in three homologous alpha-amylases also indicates that the binding affinity is dependent on the chloride coordination mode by this basic residue. Analysis of substrate and chloride binding according to the allosteric kinetic model shows that the chloride effector is not involved in substrate binding. By contrast, the pH dependence of activity and experiments of chemical modifications and Ca2+ inhibition show that the chloride ion is responsible for the pKa shift of catalytic groups and interacts with active site carboxyl groups. [less ▲]

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See detailRAD26, the yeast homolog of human Cockayne 's syndrome group B, encodes a DNA dependent ATPase
Guzder, Sami; Habraken, Yvette ULg; Sung, Patrick et al

in Journal of Biological Chemistry (1996), 271(31), 18314-7

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See detailAntagonistic properties of human prolactin analogs that show paradoxical agonistic activity in the Nb2 bioassay
Goffin, Vincent; Kinet, Sandrina; Ferrag, Fatima et al

in Journal of Biological Chemistry (1996), 271(28), 16573-9

Based on the assumption that the prolactin receptor (PRLR) is activated by PRL-induced sequential dimerization, potential human PRL (hPRL) antagonists were designed that sterically interfere with binding ... [more ▼]

Based on the assumption that the prolactin receptor (PRLR) is activated by PRL-induced sequential dimerization, potential human PRL (hPRL) antagonists were designed that sterically interfere with binding site 2. We previously reported the unexpected agonistic properties of these hPRL analogs in the rat Nb2 bioassay (Goffin, V., Struman, I., Mainfroid, V., Kinet, S., and Martial, J. A. (1994) J. Biol. Chem. 269, 32598-32606). In order to investigate whether such paradoxical agonistic behavior might result from characteristic features of the Nb2 assay (e.g. species specificity), we transfected in the same cell system the cDNA encoding the PRLR from rat or human species along with reporter genes containing PRL-responsive DNA sequences. We characterized the agonistic, self-antagonistic and/or antagonistic effects of wild type rat PRL, wild type hPRL, and three hPRL analogs, mutated either at binding site 1 or at binding site 2. Our results clearly show that the agonistic/antagonistic properties of PRLs are species-specific. We thus propose different models of receptor activation, depending on the relative affinities of each hormonal binding site, which is directed by species specificity. Finally, this is the first report of hPRL binding site 2 analogs showing antagonistic properties on human and, to a lesser extent, rat receptors. [less ▲]

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See detailCharacterization of lactogen receptor-binding site 1 of human prolactin
Kinet, Sandrina; Goffin, Vincent; Mainfroid, Véronique et al

in Journal of Biological Chemistry (1996), 271(24), 14353-60

Prolactin (PRL) binds to two molecules of PRL receptor (PRLR) through two regions referred to as binding sites 1 and 2. Although binding site 1 has been generally assigned to the pocket delimited by helix ... [more ▼]

Prolactin (PRL) binds to two molecules of PRL receptor (PRLR) through two regions referred to as binding sites 1 and 2. Although binding site 1 has been generally assigned to the pocket delimited by helix 1, helix 4, and the second half of loop 1, the residues involved in receptor binding have not yet all been precisely identified. In an earlier alanine-scanning mutational study, we identified three major binding determinants in loop 1 of human PRL (hPRL) (Goffin, V., Norman, M. & Martial, J. A.(1992) Mol. Endocrinol. 6, 1381-1392). Here we focus on the two other regions that form binding site 1, namely helices 1 and 4. Putative binding residues, selected on the basis of a three-dimensional model of hPRL constructed in this laboratory, were mutated to alanine, and recombinant hPRL mutants produced in Escherichia coli were tested for their ability to bind to the PRLR and to stimulate Nb2 cell proliferation. We thus identified nine single mutations (three in helix 1 and six in helix 4) whose effect was to reduce both binding and mitogenic activity by more than half as compared with wild-type hPRL, indicating the functional involvement of the corresponding residues. Adding these to the three binding determinants identified in loop 1, we now propose a complete picture of PRLR-binding site 1 of hPRL. As we earlier hypothesized, the binding site 1 determinants of hPRL differ from those of human growth hormone, a hPRL homolog. [less ▲]

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See detailA novel family of developmentally regulated mammalian transcription factors containing the TEA/ATTS DNA binding domain
Jacquemin, Patrick; Hwang, Jung-Joo; Martial, Joseph ULg et al

in Journal of Biological Chemistry (1996), 271(36), 21775-85

We describe the molecular cloning of two novel human and murine transcription factors containing the TEA/ATTS DNA binding domain and related to transcriptional enhancer factor-1 (TEF-1). These factors ... [more ▼]

We describe the molecular cloning of two novel human and murine transcription factors containing the TEA/ATTS DNA binding domain and related to transcriptional enhancer factor-1 (TEF-1). These factors bind to the consensus TEA/ATTS cognate binding site exemplified by the GT-IIC and Sph enhansons of the SV40 enhancer but differ in their ability to bind cooperatively to tandemly repeated sites. The human TEFs are differentially expressed in cultured cell lines and the mouse (m)TEFs are differentially expressed in embryonic and extra-embryonic tissues in early post-implantation embryos. Strikingly, at later stages of embryogenesis, mTEF-3 is specifically expressed in skeletal muscle precursors, whereas mTEF-1 is expressed not only in developing skeletal muscle but also in the myocardium. Together with previous data, these results point to important, partially redundant, roles for these TEF proteins in myogenesis and cardiogenesis. In addition, mTEF-1 is strongly coexpressed with mTEF-4 in mitotic neuroblasts, while accentuated mTEF-4 expression is also observed in the gut and the nephrogenic region of the kidney. These observations suggest additional roles for the TEF proteins in central nervous system development and organogenesis. [less ▲]

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See detailPeptide G, containing the binding site of the 67-kDa laminin receptor, increases and stabilizes laminin binding to cancer cells.
Magnifico, A.; Tagliabue, E.; Buto, S. et al

in Journal of Biological Chemistry (1996), 271(49), 31179-84

We investigated the effect of peptide G, a synthetic peptide derived from the sequence of the 37-kDa laminin receptor precursor, on the interaction of laminin in two tumor cell lines one of which produces ... [more ▼]

We investigated the effect of peptide G, a synthetic peptide derived from the sequence of the 37-kDa laminin receptor precursor, on the interaction of laminin in two tumor cell lines one of which produces laminin and one of which does not. Addition of peptide G to the culture medium induced a significant increase in the amount of endogenous laminin detectable on the cell membrane of both cell lines. Moreover, pretreatment of exogenous laminin with peptide G dramatically increased laminin binding on both cell lines. Kinetics analysis of membrane-bound labeled laminin revealed a 3-fold decrease in the kd of peptide G-treated laminin compared with untreated or unrelated or scrambled peptide-treated laminin. Moreover, the affinity constant of peptide G-treated laminin increased 2-fold, with a doubling of the number of laminin binding sites, as determined by Scatchard analysis. Expression of the VLA6 integrin receptor on the cell membrane increased after incubation with peptide G-treated laminin. However, the lower binding inhibition of peptide G-treated laminin after anti-VLA6 antibody or cation chelation treatment indicates that membrane molecules in addition to integrin receptors are involved in the recognition of peptide G-modified laminin. These "new" laminin-binding proteins also mediated cell adhesion to laminin, the first step in tumor invasion. Together, the data suggest that peptide G increases and stabilizes laminin binding on tumor cells, involving surface receptors that normally do not take part in this interaction. This might explain the abundant clinical and experimental data suggesting a key role for the 67-kDa laminin receptor in the interaction between cancer cells and the basement membrane glycoprotein laminin during tumor invasion and metastasis. [less ▲]

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See detailFusogenic Properties Of The C-Terminal Domain Of The Alzheimer Beta-Amyloid Peptide
Pillot, T.; Goethals, M.; Vanloo, B. et al

in Journal of Biological Chemistry (1996), 271(46), 28757-65

A series of natural peptides and mutants, derived from the Alzheimer beta-amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was ... [more ▼]

A series of natural peptides and mutants, derived from the Alzheimer beta-amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. These peptide domains were identified by computer modeling and correspond to respectively the C-terminal (e.g. residues 29-40 and 29-42) and a central domain (13-28) of the beta-amyloid peptide. The C-terminal peptides are predicted to insert in an oblique way into a lipid membrane through their N-terminal end, while the mutants are either parallel or perpendicular to the lipid bilayer. Peptide-induced vesicle fusion was demonstrated by several techniques, including lipid-mixing and core-mixing assays using pyrene-labeled vesicles. The effect of peptide elongation toward the N-terminal end of the entire beta-amyloid peptide was also investigated. Peptides corresponding to residues 22-42 and 12-42 were tested using the same techniques. Both the 29-40 and 29-42 beta-amyloid peptides were able to induce fusion of unilamellar lipid vesicles and calcein leakage, and the amyloid 29-42 peptide was the most potent fusogenic peptide. Neither the two mutants or the 13-28 beta-amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the alpha-helical content of the two C-terminal peptides at increasing concentrations of trifluoroethanol, which was accompanied by an increase of the fusogenic potential of the peptides. Our data suggest that the alpha-helical content and the angle of insertion of the peptide into a lipid bilayer are critical for the fusogenic activity of the C-terminal domain of the amyloid peptide. The differences observed between the fusogenic capacity of the amyloid 29-40 and 29-42 peptides might result from differences in the degree of penetration of the peptides into the membrane and the resulting membrane destabilization. The longer peptides, residues 22-42 and 12-42, had decreased, but significant, fusogenic properties associated with perturbation of the membrane permeability. These data suggest that the fusogenic properties of the C-terminal domain of the beta-amyloid peptide might contribute to the cytotoxicity of the peptide by destabilizing the cell membrane. [less ▲]

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See detailStructure-specific nuclease activity in yeast nucleotide excision repair protein RAD2
Habraken, Yvette ULg; Sung, Patrick; Prakash, Louise et al

in Journal of Biological Chemistry (1995), 270(50), 30194-8

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See detailReconstitution of yeast nucelotide excision repair with purified RAD proteins, replication protein A and transcription factor TFIIH
Guzder, Sami; Habraken, Yvette ULg; Sung, Patrick et al

in Journal of Biological Chemistry (1995), 270(22), 12973-6

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See detailTranscription factor NF-kB is activated by photosensitization generating oxidative DNA damages
Legrand-Poels, Sylvie ULg; Bours, Vincent ULg; Piret, Bernard et al

in Journal of Biological Chemistry (1995), 270(12), 6925-6934

Reactive oxygen intermediates like hydrogen peroxide (H2O2) have been shown to serve as messengers in the induction of NF-kappa B and, then, in the activation and replication of human immunodeficiency ... [more ▼]

Reactive oxygen intermediates like hydrogen peroxide (H2O2) have been shown to serve as messengers in the induction of NF-kappa B and, then, in the activation and replication of human immunodeficiency virus (HIV)-1 in human cells. Because H2O2 can be converted into the highly reactive OH. at various locations inside the cells, we started to investigate the generation of Reactive oxygen intermediates by photosensitization. This technique is based on the use of a photosensitizer which is a molecule absorbing visible light and which can be located at various sites inside the cell depending on its physicochemical properties. In this work, we used proflavine (PF), a cationic molecule having a high affinity for DNA, capable of intercalating between DNA base pairs. Upon visible light irradiation, intercalated PF molecules oxidize guanine residues and generate DNA single-strand breaks. In lymphocytes or monocytes latently infected with HIV-1 (ACH-2 or U1, respectively), this photosensitizing treatment induced a cytotoxicity, an induction of NF-kappa B, and a reactivation of HIV-1 in cells surviving the treatment. NF-kappa B induction by PF-mediated photosensitization was not affected by the presence of N-acetyl-L-cysteine while strong inhibition was recorded when the induction was triggered by H2O2 or by phorbol 12-myristate 13-acetate. Another transcription factor like AP-1 is less activated by this photosensitizing treatment. In comparison with other inducing treatments, such as phorbol 12-myristate 13-acetate or tumor necrosis factor alpha, the activation of NF-kappa B is slow, being optimal 120 min after treatment. These kinetic data were obtained by following, on the same samples, both the appearance of NF-kappa B in the nucleus and the disappearance of I kappa B-alpha in cytoplasmic extracts. These data allow us to postulate that signaling events, initiated by DNA oxidative damages, are transmitted into the cytoplasm where the inactive NF-kappa B factor is resident and allow the translocation of p50/p65 subunits of NF-kappa B to the nucleus leading to HIV-1 gene expression. [less ▲]

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See detailCharacterization and partial amino acid sequencing of a 107-kDa procollagen I N-proteinase purified by affinity chromatography on immobilized type XIV collagen.
Colige, Alain ULg; Beschin, A.; Samyn, B. et al

in Journal of Biological Chemistry (1995), 270(28), 16724-30

Procollagen I N-proteinase (EC 3.4.24.14), the enzyme that specifically processes type I and type II procollagens to collagen, was isolated from extracts of fetal calf skin. After two chromatographic ... [more ▼]

Procollagen I N-proteinase (EC 3.4.24.14), the enzyme that specifically processes type I and type II procollagens to collagen, was isolated from extracts of fetal calf skin. After two chromatographic steps on concanavalin A-Sepharose and heparin-Sepharose, the semi-purified preparation was used to produce monoclonal antibodies. One reacting antibody was found to recognize not the enzyme itself but type XIV collagen on which the enzyme was bound. This binding, highly sensitive to ionic conditions (plH, salt concentrations) but not affected by non-ionic detergents, was used for affinity chromatography that strongly improved the purification procedure. The enzyme is extensively characterized: 1) it has a molecular mass of 107 kDa as determined by polyacrylamide gel electrophoresis in presence of SDS and of about 130 kDa when estimated by gel filtration on a Sephacryl-S300; 2) in standard assay (pH 7.5, 0.2 M NaCl, 35 degrees C), the activation energy for reaction with amino procollagen type I was 17,000 calories per mole. In the same conditions, Km and Vmax values were, respectively, 435 and 39 nM per hour but varied strongly with pH and salt concentration; 3) the enzyme cleaved the NH2-terminal propeptide of type I procollagen at the specific site, the Pro-Gln bond in the alpha 1 type I procollagen chain; 4) the enzyme contained a high proportion of Gly, Asx, and Glx residues but no Hyp or Hyl; 5) partial amino acid sequences obtained from internal peptides of the enzyme displayed no significant homology with known sequences. The association of procollagen I N-proteinase with a FACIT (fibril-associated collagens with interrupted triple helices) collagen as found here might be of physiological significance. [less ▲]

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See detailIdentification of structural determinants controlling human and mouse stromelysin-3 proteolytic activities
Noël, Agnès ULg; Santavicca, M.; Stoll, I. et al

in Journal of Biological Chemistry (1995), 270

Matrix metalloproteinases (matrixins) constitute a group of extracellular proteinases belonging to the metzincin superfamily. They are involved in both physiological and pathological tissue remodeling ... [more ▼]

Matrix metalloproteinases (matrixins) constitute a group of extracellular proteinases belonging to the metzincin superfamily. They are involved in both physiological and pathological tissue remodeling processes, including those associated with cancer progression. Stromelysin-3, which is expressed in most invasive human carcinomas, is a matrix metalloproteinase with unusual functional properties. In particular, its mature form does not cleave any of the major extracellular matrix components. To define critical structural determinants involved in controlling stromelysin-3 proteolytic activity, we have used site-directed mutagenesis. We show that the deletion of at least 175 C-terminal amino-acids is sufficient to endow mouse stromelysin-3 with activities against casein, laminin, and type IV collagen. In the case of the human enzyme, however, a further and single Ala-235 Pro substitution is necessary to observe similar activities. Ala-235, which characterizes human stromelysin-3 among matrixins, is located immediately after the C terminus of the “Met-turn,” which forms a hydrophobic basis for the catalytic zinc atom in the metzincin family. We conclude that human stromelysin-3 has gained specific functional properties during evolution by amino acid substitution in the catalytic zinc environment, and that it represents an attractive target for specific inhibitors that may be used to prevent cancer progression. [less ▲]

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See detailCloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter.
Sheridan, P. L.; Schorpp, M.; Voz, Marianne ULg et al

in Journal of Biological Chemistry (1995), 270(9), 4575-87

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The ... [more ▼]

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase. [less ▲]

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See detailEvidence for a Second Receptor Binding Site on Human Prolactin
Goffin, Vincent; Struman, Ingrid ULg; Mainfroid, V. et al

in Journal of Biological Chemistry (1994), 269(51), 32598-606

The existence of a second receptor binding site on human prolactin (hPRL) was investigated by site-directed mutagenesis. First, 12 residues of helices 1 and 3 were mutated to alanine. Since none of the ... [more ▼]

The existence of a second receptor binding site on human prolactin (hPRL) was investigated by site-directed mutagenesis. First, 12 residues of helices 1 and 3 were mutated to alanine. Since none of the resulting mutants exhibit reduced bioactivity in the Nb2 cell proliferation bioassay, the mutated residues do not appear to be functionally necessary. Next, small residues surrounding the helix 1-helix 3 interface were replaced with Arg and/or Trp, the aim being to sterically hinder the second binding site. Several of these mutants exhibit only weak agonistic properties, supporting our hypothesis that the channel between helices 1 and 3 is involved in a second receptor binding site. We then analyzed the antagonistic and self-antagonistic properties of native hPRL and of several hPRLs analogs altered at binding site 1 or 2. Even at high concentrations (approximately 10 microM), no self-inhibition was observed with native hPRL; site 2 hPRL mutants self-antagonized while site 1 mutants did not. From these data, we propose a model of hPRL-PRL receptor interaction which slightly differs from that proposed earlier for the homologous human growth hormone (hGH) (Fuh, G., Cunningham, B. C., Fukunaga, R., Nagata, S., and Goeddel, D. V., and Well, J. A. (1992) Science 256, 1677-1680). Like hGH, hPRL would bind sequentially to two receptor molecules, first through site 1, then through site 2, but we would expect the two sites of hPRL to display, unlike the two binding sites of hGH, about the same binding affinity, thus preventing self-antagonism at high concentrations. [less ▲]

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See detailA conserved 5' to 3' exonuclease activity in yeast and human nucleotide excision repair protein RAD2 and XPG
Habraken, Yvette ULg; Sung, Patrick; Prakash, Louise et al

in Journal of Biological Chemistry (1994), 269(50), 31342-5

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See detailCold Adaptation of Proteins. Purification, Characterization, and Sequence of the Heat-Labile Subtilisin from the Antarctic Psychrophile Bacillus Ta41
Davail, S.; Feller, Georges ULg; Narinx, E. et al

in Journal of Biological Chemistry (1994), 269(26), 17448-53

The gene of subtilisin S41, an alkaline protease secreted by the psychrophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids residues. The nucleotide sequence and NH2- and COOH-terminal ... [more ▼]

The gene of subtilisin S41, an alkaline protease secreted by the psychrophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids residues. The nucleotide sequence and NH2- and COOH-terminal amino acid sequencing of the purified enzyme indicate that the mature subtilisin S41 is composed of 309 residues with a predicted M(r) = 31,224. Subtilisin S41 shares most of its properties with mesophilic subtilisins (structure of the precursor, 52% amino acid sequence identity, alkaline pH optimum, broad specificity, Ca2+ binding) but is characterized by a higher specific activity on macromolecular substrate, by a shift of the optimum of activity toward low temperatures, and by a low thermal stability. The enzyme also differs by an acidic pI (5.3) and the presence of one disulfide bond. It is proposed that the psychrophilic enzyme possesses a more flexible molecular structure when compared to mesophilic and thermophilic subtilases in order to compensate for the reduction of reaction rates at low temperatures. The model of subtilisin S41 indeed reveals several features able to induce a more flexible, heat-labile conformation: the occurrence of four extended surface loops, a very hydrophilic surface through 11 extra Asp residues, and the lack of several salt bridges and aromatic-aromatic interactions. The low affinity of the Ca1 calcium binding site (Kd(app) = 10(-6) M), resulting possibly from one chelating side chain substitution and the stacking of Gly residues, also reflect a less compact conformation. The difference of free energy of stabilization between subtilisin S41 and a mesophilic subtilisin suggests that the balance of exo- and endothermically formed weak bonds is critical for the enzyme flexibility. [less ▲]

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See detailMechanism of Thiamine Transport in Neuroblastoma Cells. Inhibition of a High Affinity Carrier by Sodium Channel Activators and Dependence of Thiamine Uptake on Membrane Potential and Intracellular Atp
Bettendorff, Lucien ULg; Wins, Pierre

in Journal of Biological Chemistry (1994), 269(20), 14379-14385

Nerve cells are particularly sensitive to thiamine deficiency. We studied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [14C]thiamine was taken up through a ... [more ▼]

Nerve cells are particularly sensitive to thiamine deficiency. We studied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [14C]thiamine was taken up through a saturable high affinity mechanism (Km = 35 nM). This was blocked by low concentrations of the Na+ channel activators veratridine (IC50 = 7 +/- 4 microM) and batrachotoxin (IC50 = 0.9 microM). These effects were not antagonized by tetrodotoxin and were also observed in cell lines devoid of Na+ channels, suggesting that these channels are not involved in the mechanism of inhibition. At high extracellular concentrations, thiamine uptake proceeds essentially via a low affinity carrier (Km = 0.8 mM), insensitive to veratridine but blocked by divalent cations. In both cases, the uptake was independent on external sodium, partially inhibited (10-35%) by depolarization and sensitive to metabolic inhibitors. A linear relationship between the rate of thiamine transport and intracellular ATP concentration was found. When cells grown in a medium of low thiamine concentration (6 nM) were exposed to 100 nM extracellular thiamine, a 3-fold increase in intracellular thiamine diphosphate was observed after 2 h while the concomitant increase in intracellular free thiamine was barely significant. These data suggest a secondary active transport of thiamine, the main driving force being thiamine phosphorylation rather than the sodium gradient. [less ▲]

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