Spectroscopic and binding studies of azide to type-2-copper-depleted ascorbate oxidase from zucchini

1991 ◽  
Vol 4 (2) ◽  
pp. 81-89 ◽  
Author(s):  
Luigi Casella ◽  
Michele Gullotti ◽  
Gianfranco Pallanza ◽  
Alessandro Pintar ◽  
Augusto Marchesini
Keyword(s):  
Ascorbate Oxidase ◽  
Binding Studies

scholarly journals Azide-binding studies reveal type 3 copper heterogeneity in ascorbate oxidase from the green zucchini squash (Cucurbita pepo)

1988 ◽  
Vol 251 (2) ◽  
pp. 441-446 ◽  
Author(s):  
L Casella ◽  
M Gullotti ◽  
G Pallanza ◽  
A Pintar ◽  
A Marchesini
Keyword(s):  
Cucurbita Pepo ◽  
Ascorbate Oxidase ◽  
Binding Studies ◽  
High Affinity ◽  
Hyperfine Splitting Constant ◽  
Splitting Constant ◽  
A Minor ◽  
Type 3 ◽  
Azide Binding

Titration of native ascorbate oxidase from green zucchini squash (Cucurbita pepo) with azide in 0.1 M-phosphate buffer, pH 6.8, exhibits a biphasic spectral behaviour. Binding of the anion with ‘high affinity’ (K greater than 5000 M-1) produces a broad increase of absorption in the 400-500 nm region (delta epsilon approximately 1000 M-1.cm-1) and c.d. activity in the 300-450 nm region, whereas azide binding with ‘low affinity’ (K approximately 100 M-1) is characterized by an intense absorption band at 420 nm (delta epsilon = 6000 M-1.cm-1), corresponding to negative c.d. activity and a decrease of absorption at 330 nm (delta epsilon = -2000 M-1.cm-1). The high-affinity binding involves a minor fraction of the protein containing Type 3 copper in the reduced state, and the spectral features of this azide adduct can be eliminated by treatment of the native enzyme with small amounts of H2O2, followed by dialysis before azide addition. As shown by e.s.r. spectroscopy, Type 2 copper is involved in both types of binding, its signal being converted into that of a species with small hyperfine splitting constant [12 mT (approximately 120 G)] in the case of the low-affinity azide adduct. The spectral similarities of the two types of azide adducts with the corresponding adducts formed by native laccase, which also exhibits Type 3 copper heterogeneity, are discussed.

scholarly journals Comparative binding of bovine, human and rat insulin-like growth factors to membrane receptors and to antibodies against human insulin-like growth factor-1

1986 ◽  
Vol 233 (1) ◽  
pp. 215-221 ◽  
Author(s):  
L C Read ◽  
F J Ballard ◽  
G L Francis ◽  
R C Baxter ◽  
C J Bagley ◽  
...  
Keyword(s):  
Growth Factors ◽  
Polyclonal Antiserum ◽  
Competitive Binding ◽  
Membrane Receptors ◽  
Binding Studies ◽  
Comparative Binding ◽  
Placental Membranes ◽  
Insulin Like Growth Factors

The immunological properties of human, bovine and rat insulin-like growth factors (IGF) and insulin were compared in competitive binding studies with Tr10 and NPA polyclonal antisera raised in rabbits against human IGF-1. Bovine IGF-1 was 11-19% as effective as human IGF-1 in competing for binding with 125I-labelled human IGF-1, whereas IGF-2 reacted poorly and insulin did not compete. Similar competitive binding curves were obtained with the mouse monoclonal anti-(human IGF-1) antibody 3D1, except that bovine IGF-1 showed a severalfold greater affinity for the monoclonal antibody than for either polyclonal antiserum. Membranes isolated from human placenta, sheep placenta and foetal-human liver were used as sources of cellular receptors. In human placental membranes, most of the binding of IGF-1 tracers could be attributed to a type-1 receptor, because insulin inhibited up to 65% of tracer binding. The other two tissues apparently contain only type-2 receptors, as evidenced by the very low potency of bovine or human IGF-1 in competing for binding with IGF-2 tracers and the absence of any competition by insulin. In competition for binding with labelled bovine or human IGF-1 to human placental membranes, bovine IGF-1 had a similar potency to human IGF-1, whereas bovine IGF-1 was more potent in binding studies with tissues rich in type-2 receptors. Rat IGF-2 was considerably less effective than human IGF-2 in competition for receptors on any of the membrane preparations.

Investigation of azide binding to oxidized type 2 copper depleted ascorbate oxidase from Cucurbita pepo

1989 ◽  
Vol 37 (2) ◽  
pp. 105-109 ◽  
Author(s):  
Luigi Casella ◽  
Michele Gullotti ◽  
Alessandro Pintar ◽  
Gianfranco Pallanza ◽  
Augusto Marchesini
Keyword(s):  
Cucurbita Pepo ◽  
Ascorbate Oxidase ◽  
Azide Binding

scholarly journals Electron spin echo spectroscopic studies of type 1 and type 2 copper in Rhus vernicifera laccase and in Cucurbita pepo medullosa ascorbate oxidase

FEBS Letters ◽  
1981 ◽  
Vol 136 (1) ◽  
pp. 80-84 ◽  
Author(s):  
L. Avigliano ◽  
J.L. Davis ◽  
M.T. Graziani ◽  
A. Marchesini ◽  
W.B. Mims ◽  
...  
Keyword(s):  
Electron Spin ◽  
Cucurbita Pepo ◽  
Spin Echo ◽  
Electron Spin Echo ◽  
Spectroscopic Studies ◽  
Ascorbate Oxidase ◽  
Rhus Vernicifera

PDGF receptors in the rat CNS: during late neurogenesis, PDGF alpha-receptor expression appears to be restricted to glial cells of the oligodendrocyte lineage

Development ◽  
1992 ◽  
Vol 115 (2) ◽  
pp. 535-551 ◽  
Author(s):  
N.P. Pringle ◽  
H.S. Mudhar ◽  
E.J. Collarini ◽  
W.D. Richardson
Keyword(s):  
Glial Cells ◽  
Receptor Expression ◽  
Binding Studies ◽  
Temporal And Spatial Distribution ◽  
Inner Limiting Membrane ◽  
Factor Alpha ◽  
The Brain

Using in situ hybridization, we have visualized cells in the rat central nervous system (CNS) that contain mRNA encoding the platelet-derived growth factor alpha receptor (PDGF-alpha R). After embryonic day 16 (E16), PDGF-alpha R mRNA appears to be expressed by a subset of glial cells, but not by neurons. The temporal and spatial distribution of PDGF-alpha R+ cells, together with 125I-PDGF binding studies on subsets of glial cells in vitro, suggests that PDGF-alpha R may be expressed predominantly, or exclusively, by cells of the oligodendrocyte-type-2 astrocyte (O-2A) lineage. This conclusion is supported by the fact that the numbers of PDGF-alpha R+ cells in developing and adult optic nerves correlate well with independent estimates of the number of O-2A progenitor cells in the nerve at equivalent ages. Small numbers of PDGF-alpha R+ cells are present in the brain at E16, at which time they are found outside the subventricular germinal zones, suggesting that these cells do not express PDGF-alpha R until after, or shortly before they start to migrate away from the subventricular layer towards their final destinations. Reduced numbers of PDGF-alpha R+ cells persist in the adult CNS. PDGF-alpha R is also expressed strongly in the meningeal membranes and choroid plexus, and in the inner limiting membrane of the retina.

Functional Characterization of Three VWF- A1 Domain Mutations Causing Type 2 Von Willebrand Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1305-1305
Author(s):  
Maha Othman ◽  
Rouzbeh Chegeni ◽  
Linda M Vickars ◽  
Emmanuel J Favaloro ◽  
David Lillicrap
Keyword(s):  
Functional Characterization ◽  
Von Willebrand Disease ◽  
Site Directed Mutagenesis ◽  
Missense Mutations ◽  
Binding Studies ◽  
Disease Subtype ◽  
Platelet Binding ◽  
The Media ◽  
A1 Domain

Abstract Abstract 1305 Poster Board I-327 Through the Canadian Platelet-Type VWD Project (www.pt-vwd.org), we have identified 3 novel candidate mutations within the A1 domain of VWF gene in 5 patients that were provisionally diagnosed as type 2 VWD. These mutations are: L1460F (2 related patients), Y1363C (1 patient), E1389K (2 related patients). The VWF:Ag ranged from 0.19 – 0.54 IU/mL, VWF:RCo ranged from 0.19- 0.21 IU/mL and platelet count ranged from 206-254 × 103/ mm3. High Molecular weight multimers of VWF were absent in the plasma of the patient with the 1460 aa change. These substitutions have not been previously reported in the literature or in the International VWF Mutation Database. All three mutations were created by site-directed mutagenesis in the full-length huVWF cDNA and transiently transfected in the HEK293T/17 cell line. The VWF protein secreted in the media was collected and quantified by ELISA before proceeding with Functional VWF-platelet binding studies. Quantification of the mutant rVWF proteins in the media and cell lysates and multimer analysis of the proteins showed normal secretion and multimer distributions. To determine the GpIb binding properties of the putative mutants, the VWF recombinant protein (0.25 ug) from each of the WT and three mutants was incubated with lyophilized platelets (1 × 108) for 10 minutes at 25°C with different concentrations of ristocetin between 0-1 mg/mL. The platelets were then pelleted by centrifugation at 1200 g and the resulting supernatant was tested for the remaining unbound VWF by ELISA. Compared to the WT VWF protein, only the L1460F mutant showed increased platelet binding under lower concentration of ristocetin. Ristocetin-induced platelet agglutination (RIPA) assays performed using these recombinant proteins confirmed enhanced ristocetin responsiveness for L1460F, and that Y1363C and E1389K alternatively showed reduced responsiveness. Based on these data, a type 2B VWD diagnosis can only be made with respect the L1460F and not the other two A1 domain missense mutations. These data confirm once again that the assignment of the phenotype on clinical and laboratory basis is useful prior to genetic analysis and mutation identification. However, in some situations both will be ultimately required to understand the clinical presentation and to assign a disease subtype. Disclosures Vickars: Novartis Canada: Honoraria, Research Funding.

Characterization of a B2-bradykinin receptor in human glomerular podocytes

1996 ◽  
Vol 271 (3) ◽  
pp. F754-F761 ◽  
Author(s):  
N. Ardaillou ◽  
V. Blaise ◽  
K. Costenbader ◽  
Y. Vassitch ◽  
R. Ardaillou
Keyword(s):  
Inositol Phosphate ◽  
Ion Concentration ◽  
Scatchard Analysis ◽  
Ribonuclease Protection Assay ◽  
Dependent Manner ◽  
Binding Studies ◽  
Intracellular Calcium Ion ◽  
Hoe 140 ◽  
Ribonuclease Protection

We characterized bradykinin (BK) receptors in a human line of glomerular visceral epithelial cells (hGVEC) transfected by the SV40 virus. [3H]BK bound specifically in a manner consistent with a single high-affinity site. Scatchard analysis yielded dissociation constant and maximum binding values of 0.28 +/- 0.04 nM and 76.6 +/- 4.9 fmol/mg, respectively. Competition binding studies with selective BK type 2 (Hoe-140) receptor antagonist and type 1 ([des-Arg9]BK) receptor agonist showed that hGVEC only expressed type 2 receptors, and this was confirmed by reverse transcriptase-polymerase chain reaction and ribonuclease protection assay. BK stimulated intracellular calcium ion concentration ([Ca2+]i) release in a dose-dependent manner with a threshold at 1 nM. Hoe-140, in contrast with [des-Arg9]BK, abolished this effect. [Ca2+]i stimulation was also inhibited by thapsigargin, an inhibitor of Ca(2+)-adenosinetriphosphatase. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid attenuated but did not suppress the [Ca2+]i peak. These results associated with the stimulatory effect of BK on inositol phosphate production indicated that [Ca2+]i stimulation was produced both by [Ca2+] mobilization from its intracellular stores and by [Ca2+] entry into the cells. In conclusion, hGVEC express specific type 2 BK receptors that enable specific BK-induced responses.

scholarly journals Monoclonal antibodies against protease-sensitive pneumococcal antigens can protect mice from fatal infection with Streptococcus pneumoniae.

1984 ◽  
Vol 160 (2) ◽  
pp. 386-397 ◽  
Author(s):  
L S McDaniel ◽  
G Scott ◽  
J F Kearney ◽  
D E Briles
Keyword(s):  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Minimum Amount ◽  
Binding Studies ◽  
Fatal Infection ◽  
Rough Strain ◽  
Type 3

Monoclonal antibodies were raised against surface determinants of Streptococcus pneumoniae by hyperimmunizing X-linked immunodeficient (xid) CBA/N mice with the heat-killed rough strain R36A. 17 hybridomas produced antibody that bound intact R36A and did not cross-react with phosphocholine, an antigen common in the cell wall of all S. pneumoniae. The antibody produced by at least two of these hybridomas, Xi64 (IgM) and Xi126 (IgG2b), could protect mice from a lethal intravenous challenge of type 3 S. pneumoniae strains WU2 and A66 and of the type 2 strain D39. The minimum amount of antibody required to protect xid mice from 100 WU2 was 4.5 micrograms/mouse for Xi64 and 2.6 micrograms/mouse for Xi126,. Free phosphocholine, C-polysaccharide, and type 3 capsular polysaccharide all failed to inhibit the binding of Xi64 or Xi126 to R36A. These antibodies appeared to bind surface polypeptides, since treatment of R36A with either pepsin or trypsin, or of R36A lysate with trypsin, effectively eliminated the ability of Xi64 and Xi126 to bind antigens in these preparations. Binding studies indicated that these two antibodies recognized different epitopes that were expressed on several but not all serotypes of pneumococci.

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