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.

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 Characterization of cDNAs encoding isoforms of hamster 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase

1998 ◽  
Vol 20 (1) ◽  
pp. 99-110 ◽  
Author(s):  
FM Rogerson ◽  
J Courtemanche ◽  
A Fleury ◽  
JG LeHoux ◽  
JI Mason ◽  
...  
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Cdna Libraries ◽  
Sexually Dimorphic ◽  
High Affinity ◽  
Liver And Kidney ◽  
Low Levels ◽  
Type 3

Western blot analyses of various hamster tissues reveal high levels of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in adrenal and liver, and moderate levels of expression in kidney. The expression in liver is sexually dimorphic; very high levels of protein are observed in adult male liver but very low levels are seen in the female liver. Three distinct cDNAs encoding isoforms of 3 beta-HSD were isolated from hamster cDNA libraries. The type 1 isoform is a high-affinity dehydrogenase/isomerase expressed in adrenal and male kidney. The type 2 isoform is also a high-affinity dehydrogenase/isomerase expressed in kidney and male liver. The type 3 enzyme is a 3-ketosteroid reductase expressed predominantly in kidney. Sequencing of the clones showed that all three are structurally very similar, although types 1 and 2 share the greatest degree of similarity. Immunohistochemical staining for 3 beta-HSD in the adrenal was found throughout the adrenal cortex. In the kidney staining was confined to tubules, and in the liver, heavy staining was found in hepatocytes. The cloning of cDNAs for 3 beta-HSD from the liver and kidney should help in elucidating the function of this enzyme in these tissues.

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

scholarly journals An e.p.r. study of the non-equivalence of the copper sites of caeruloplasmin

1986 ◽  
Vol 238 (1) ◽  
pp. 291-295 ◽  
Author(s):  
L Calabrese ◽  
M Carbonaro
Keyword(s):  
Slow Phase ◽  
Copper Binding ◽  
Blue Copper ◽  
Sequence Of Events ◽  
Copper Site ◽  
Hyperfine Splitting Constant ◽  
Type 1 Copper ◽  
Splitting Constant

The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent ‘oxidative’ attack of proteins and lipids.

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.

scholarly journals Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms

1986 ◽  
Vol 240 (2) ◽  
pp. 403-412 ◽  
Author(s):  
E Kloprogge ◽  
J W Akkerman
Keyword(s):  
Platelet Activation ◽  
Binding Sites ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
A Minor

When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [(3H]arachidonate mobilization and thromboxane synthesis) and secretion [(14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding via mutually independent mechanisms.

Survey of Fusarium spp. Associated with Fruit Rot of Pumpkin in Ohio

2010 ◽  
Vol 11 (1) ◽  
pp. 9
Author(s):  
Christian A. Wyenandt ◽  
Richard M. Riedel ◽  
Landon H. Rhodes ◽  
Mark A. Bennett ◽  
Steven G. P. Nameth
Keyword(s):  
Cucurbita Pepo ◽  
Fusarium Species ◽  
Fruit Rot ◽  
Fusarium Spp ◽  
Surface Type ◽  
Race 1 ◽  
Type 3 ◽  
Magic Lantern

From 2000 to 2002 commercial pumpkin (Cucurbita pepo) fields in Ohio were surveyed for Fusarium fruit rot. From six counties in 2000, 2001, and 2002, a total of 43 isolates from eight farms, 84 isolates from nine farms, and 40 isolates from six farms were collected, respectively. Fusarium solani f. sp. cucurbitae race 1 was the most commonly isolated Fusarium species from infected pumpkin fruit in Ohio. Other Fusaria isolated from infected fruit included F. oxysporum, F. graminearum, and F. acuminatum. A survey of infected pumpkin fruit in the field and inoculation studies of mature, healthy pumpkin cultivar Magic Lantern in the laboratory resulted in three types of fruit rot symptoms. Type 1, caused by F. oxysporum and F. acuminatum, resulted in a slow-expanding rot just below the rind surface of the infected fruit. Type 2, caused by F. graminearum, resulted in an expanding, slightly sunken, irregular rot of the rind surface. Type 3, caused by F. solani resulted in expanding, circular sunken lesions on the fruit surface bearing white to tan sporodochia. This is the first report of F. solani f. sp. cucurbitae race 1, F. oxysporum, F. acuminatum, and F. graminearum causing fruit rot of pumpkin in Ohio. Accepted for publication 26 July 2010. Published 27 August 2010.

scholarly journals Dependence on freezing of the geometry and redox potential of type 1 and type 2 copper sites of Japanese-lacquer-tree (Rhus vernicifera) laccase

1981 ◽  
Vol 193 (2) ◽  
pp. 639-642 ◽  
Author(s):  
L Morpurgo ◽  
L Calabrese ◽  
A Desideri ◽  
G Rotilio
Keyword(s):  
Redox Potential ◽  
Hyperfine Splitting ◽  
Room Temperature ◽  
Redox Properties ◽  
Blue Copper ◽  
Hyperfine Splitting Constant ◽  
Splitting Constant ◽  
Rhus Vernicifera

The room-temperature e.p.r. spectrum of the Japanese-lacquer-tree (Rhus vernicifera) laccase shows A parallel (the hyperfine splitting constant) and g parallel values of both the Type 1 and Type 2 Cu appreciably different from those measured at liquid-N2 temperature. The geometry of the sites, as inferred from the room-temperature e.p.r. parameters, is more consistent with their redox properties. A rough correlation is found between A parallel and g parallel values and redox potential of the blue copper in several enzymes.

scholarly journals Pulse-radiolysis studies on the interaction of one-electron-reduced species with ascorbate oxidase in aqueous solution

1983 ◽  
Vol 209 (1) ◽  
pp. 167-174 ◽  
Author(s):  
P O'Neill ◽  
E M Fielden ◽  
A Finazzi-Agrò ◽  
L Avigliano
Keyword(s):  
Aqueous Solution ◽  
Pulse Radiolysis ◽  
Ascorbate Oxidase ◽  
Intramolecular Electron Transfer ◽  
Protein Residues ◽  
Type 1 Copper ◽  
Type 3 ◽  
Reducing Equivalents

The interaction of e-aq., CO2-. and one-electron reduced nitroaromatics (RNO2-.) with ascorbate oxidase (AAO) was studied in aqueous solution at pH 6.0 and 7.5 by using the technique of pulse radiolysis. From observations at 330, 410 and 610 nm, interaction of e-aq. and CO2-. with AAO results in non-specific reduction of the protein followed by reduction of Type 1 Cu in a rate-determining intramolecular step. Only a few per cent of the reducing equivalents ultimately results in reduction of Type 1 Cu. With large excesses of reducing equivalents (e-aq. and CO2-.) with respect to the copper concentration, the amount of Type 1 copper reduced never exceeds 50% of the total amount of Type 1 copper after a single radiation pulse. With less-powerful reducing agents, e.g. RNO2-. reduction of Type 1 Cu occurs via a bimolecular step, and there is no evidence for formation of radicals on protein residues. From observations at 330 nm it is evident that Type 2 and/or Type 3 Cu may also be reduced along with Type 1 Cu. Almost stoichiometric reduction of AAO by RNO2-. was observed, e.g. the protein accepts 6-7 reducing equivalents. It is inferred that the various types of redox couples Cu2+/Cu+ are in equilibrium and that intramolecular electron transfer between the different types of Cu is not rate-determining when using RNO2-. as reducing agent.

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
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