scholarly journals The dissociation of glucose oxidase by sodium n-dodecyl sulphate

1982 ◽  
Vol 203 (1) ◽  
pp. 285-291 ◽  
Author(s):  
M N Jones ◽  
P Manley ◽  
A Wilkinson
Keyword(s):  
Glucose Oxidase ◽  
Specific Binding ◽  
Enzymic Activity ◽  
Sedimentation Equilibrium ◽  
Prolonged Incubation ◽  
Rate Analysis ◽  
Subunit Molecular Weight ◽  
A Subunit ◽  
Glycine Methyl Ester ◽  
Cationic Residues

1. The enzymic activity of glucose oxidase was determined as a function of pH and sodium n-dodecyl sulphate (SDS) concentration. 2. Glucose oxidase is not deactivated by SDS at pH 6 even after prolonged incubation, but is deactivated at pH 4.3 and 3.65. 3. Sedimentation-rate analysis showed that glucose oxidase dissociates into its two subunits at pH 5 and below, and sedimentation-equilibrium experiments in the presence of SDS gave a subunit molecular weight of 73,500. 4. SDS binds to glucose oxidase in acid solutions; specific binding occurs ap pH 3.65, but at pH 6 only co-operative binding was observed. 5. Glucose oxidases in which some of the carboxy groups were blocked with glycine methyl ester were deactivated by SDS at pH 6.0; the rate of deactivation increased with the extent of esterification. 6. Deactivation of esterified glucose oxidases correlated with thermal analysis of the initial SDS interaction, the exothermicity of the interaction increasing with the extent of esterification. 7. The results show that carboxy groups confer resistance to deactivation by SDS on glucose oxidase by screening cationic residues and inhibiting specific interactions that facilitate dissociation into subunits.

scholarly journals Some properties of aldehyde dehydrogenase from sheep liver mitochondria

1977 ◽  
Vol 163 (2) ◽  
pp. 261-267 ◽  
Author(s):  
G J Hart ◽  
F M Dickinson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Liver Mitochondria ◽  
Enzymic Activity ◽  
Sedimentation Equilibrium ◽  
Thiol Groups ◽  
Control Value ◽  
Sheep Liver ◽  
Spectrophotometric Titrations ◽  
A Subunit ◽  
Subunit Size

Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5′-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents.

A rapid purification procedure for pyruvate kinase from the hyphal fungus Aspergillus nidulans

1988 ◽  
Vol 34 (10) ◽  
pp. 1154-1158 ◽  
Author(s):  
Harry C. M. Kester ◽  
Jos H. A. A. Uitzetter ◽  
Leo H. de Graaff ◽  
Jaap Visser
Keyword(s):  
Molecular Weight ◽  
Liquid Chromatography ◽  
Aspergillus Nidulans ◽  
Pyruvate Kinase ◽  
Purification Procedure ◽  
Subunit Molecular Weight ◽  
Highly Active ◽  
A Subunit ◽  
Rapid Purification ◽  
Dye Affinity Chromatography

Pyruvate kinase was purified from the filamentous fungus Aspergillus nidulans with a 45–55% yield. The procedure involved dye-affinity chromatography and fast protein liquid chromatography, resulting in highly active and pure enzyme in milligram quantities within 2 days. The purified enzyme, a tetramer with a subunit molecular weight of 65 000 and an isoelectric point of 4.7, was used to determine the amino acid composition.

Specific albumin binding to microvascular endothelium in culture

1988 ◽  
Vol 254 (3) ◽  
pp. H425-H437 ◽  
Author(s):  
J. E. Schnitzer ◽  
W. W. Carley ◽  
G. E. Palade
Keyword(s):  
Specific Binding ◽  
Molecular Transport ◽  
Cell Number ◽  
Dissociation Rate ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Albumin Binding ◽  
Microvascular Endothelium ◽  
Rat Serum ◽  
Rate Analysis

The specific binding of rat serum albumin (RSA) to confluent microvascular endothelial cells in culture derived from the vasculature of the rat epididymal fat pad was studied at 4 degrees C by radioassay and immunocytochemistry. Radioiodinated RSA (125I-RSA) binding to the cells reached equilibrium at approximately 20 min incubation. Albumin binding was a slowly saturating function over concentrations ranging from 0.01 to 50 mg/ml. Specific RSA binding with a moderate apparent affinity constant of 1.0 mg/ml and with a maximum binding concentration of 90 ng/cm2 was immunolocalized with anti-RSA antibody to the outer (free) side of the endothelium. Scatchard analysis of the binding yielded a nonlinear binding curve with a concave-upward shape. Dissociation rate analysis supports negative cooperativity of albumin binding, but multiple binding sites may also be present. Albumin binding fulfilled many requirements for ligand specificity including saturability, reversibility, competibility, and dependence on both cell type and cell number. The results are discussed in terms of past in situ investigations on the localization of albumin binding to vascular endothelium and its effect on transendothelial molecular transport.

scholarly journals Thiol reduction of human α2-macroglobulin. The subunit structure

1972 ◽  
Vol 127 (1) ◽  
pp. 187-197 ◽  
Author(s):  
J. M. Jones ◽  
J. M. Creeth ◽  
R. A. Kekwick
Keyword(s):  
Molecular Weight ◽  
Large Scale ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Experimental Conditions ◽  
A Value ◽  
Α2 Macroglobulin ◽  
A Subunit ◽  
Normal Human ◽  
Ph Range

1. Human α2-macroglobulin was prepared from a fraction obtained during the large-scale separation of normal human plasma proteins for clinical use. 2. Sedimentation-equilibrium measurements indicated a molecular weight of 725000. A value of 18.1S was obtained for s020,w. 3. The dissociation that occurs in the pH range 4.5–2.5 and in the region of neutrality in urea-containing solutions is consistent with a dimeric structure of the molecule. 4. The effects of the thiol reagents mercaptoethanol, mercaptoethylamine and N-acetylcysteine were investigated over a range of experimental conditions. Distinct components having sedimentation coefficients of 15, 12 and 8.5S were identified. 5. Conditions were found under which limited reduction with thiol liberated a subunit with a molecular weight approximately one-quarter of that of the intact molecule. This subunit retains the serological specificity of the whole molecule.

Intracellular Signal Triggered by Cholera Toxin inSaccharomyces boulardii and Saccharomyces cerevisiae

1998 ◽  
Vol 64 (2) ◽  
pp. 564-568 ◽  
Author(s):  
Rogelio L. Brandão ◽  
Ieso M. Castro ◽  
Eduardo A. Bambirra ◽  
Sheila C. Amaral ◽  
Luciano G. Fietto ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cholera Toxin ◽  
Mammalian Cells ◽  
Specific Binding ◽  
Saccharomyces Boulardii ◽  
Yeast Cells ◽  
B Subunit ◽  
Nonfermentable Carbon Source ◽  
A Subunit ◽  
Yeast Surface

ABSTRACT As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.

scholarly journals The effect of proteinases on phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis

1981 ◽  
Vol 199 (3) ◽  
pp. 715-723 ◽  
Author(s):  
H J Gilbert ◽  
G W Jack
Keyword(s):  
Phenylalanine Ammonia Lyase ◽  
Bond Cleavage ◽  
Rhodotorula Glutinis ◽  
Peptide Bond ◽  
Enzymic Activity ◽  
Duodenal Juice ◽  
Prosthetic Group ◽  
Prolonged Incubation ◽  
Peptide Bond Cleavage ◽  
Site Binding

Phenylalanine ammonia-lyase (EC 4.3.1.5) of the yeast Rhodotorula glutinis was rapidly inactivated by duodenal juice. It was susceptible to chymotrypsin and subtilisin and to a lesser extent trypsin. Initial proteolysis of the enzyme by chymotrypsin and trypsin resulted in cleavage of the monomeric subunit (75 000 Mr) into a large (65 000 Mr) and a small (10 000 Mr) peptide. The small peptide was rapidly degraded. The 65 000-Mr fragment was resistant to prolonged incubation with chymotrypsin, but was degraded by trypsin under the same conditions. Phenylalanine ammonia-lyase was cleaved into several polypeptides by subtilisin, the 65 000-Mr peptide being totally absent. The N-terminal region of the enzyme was contained in the 65 000-Mr fragment, as was the dehydroalanine moiety, the prosthetic group. Active-site-binding ligands protect the enzyme from inactivation by the three proteinases, and peptide-bond cleavage by trypsin and chymotrypsin. Several chemical modifications were performed on phenylalanine ammonia-lyase. Some decreased its antigenicity, and ethyl acetimidate decreased the rate of degradation of the 65 000-Mr peptide by trypsin. The modification did not protect the enzyme from proteolytic inactivation of the enzymic activity. These observations are discussed in terms of the structure of phenylalanine ammonia-lyase and site of action of the proteinases.

scholarly journals Binding kinetics of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets

1984 ◽  
Vol 223 (3) ◽  
pp. 901-909 ◽  
Author(s):  
E Kloprogge ◽  
J W N Akkerman
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Human Platelets ◽  
Equilibrium Analysis ◽  
Binding Studies ◽  
Prolonged Incubation ◽  
Fibrinogen Receptor ◽  
Receptor Aggregation ◽  
Specific Receptors ◽  
High Doses

The binding of [3H]PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human gel-filtered platelets was measured at 22 degrees C. Specific binding reached saturation within 15 min at high doses of [3H]PAF-acether (0.5-0.9 nM), whereas about 90 min were required when low doses (0.02-0.5 nM) were used. Above 1 nM, [3H]PAF-acether non-specific binding increased progressively, which together with the demonstration of a 3H-labelled metabolite suggested uptake and metabolism of [3H]PAF-acether. Equilibrium analysis revealed one class of specific receptors with a Ka of 18.86 +/- 4.82×10(9) M-1 and 242 +/- 64 binding sites per platelet. Non-equilibrium binding revealed a similar Ka (16.87×10(9) M-1). Specific binding became irreversible after prolonged incubation, a process that was enhanced at increasing concentrations of [3H]PAF-acether. Platelets made desensitized to PAF-acether by prior incubation with unlabelled PAF-acether failed to bind a second dose of PAF-acether (3H-labelled), suggesting that desensitization resulted from loss of available binding sites. Under the conditions of the binding studies, PAF-acether induced exposure of the fibrinogen receptor, aggregation (in a stirred suspension) and alterations in (poly)-phosphatidylinositides. These results suggest that PAF-acether initiates platelet responses via receptor-mediated processes.

Common Action of Inducers and Inhibitors of Aggregation on Phosphorylation of Surface Localized Ca-Binding Protein

1975 ◽  
Author(s):  
F. M. Booyse ◽  
J. Marr ◽  
D. Tomlinson ◽  
D. G. Yang ◽  
M. E. Rafelson
Keyword(s):  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Phosphorylated Protein ◽  
Phosphoprotein Phosphatase ◽  
Camp Dependent Protein Kinase ◽  
Subunit Molecular Weight ◽  
Dependent Protein ◽  
A Subunit ◽  
Kinase Phosphorylation ◽  
Common Action

Analysis of intact 32P-labeled platelets by SDS-polyacrylamidegel electrophoresis shows the presence of only a single phosphorylated protein. This phosphoprotein has been isolated, has a subunit molecular weight of 11,100, is surface localized (lactoperoxidase-125 Iodine labeling) and binds 1 mole of Ca2+ per mole of phosphorylated subunit. The γ-32ऩ from (γ-32P) ATP is specifically incorporated as 0-phosphoserine by a cAMP-dependent protein kinase.The phosphorylated state and hence the Ca-binding of this protein is maintained by membrane associated cAMP-dependent protein kinase phosphorylation and phosphoprotein phosphatase dephosphorylation. Thrombin, collagen, histone and ADP competitively inhibit phosphorylation and Ca-binding by serving as protein kinase acceptors. Epinephrine, serotonin and ristosetin induce dephosphorylation and abolish Ca-binding by activating the phosphoprotein phosphatase. PGEl5 ATP, cAMP increase phosphorylation and Ca-binding by activating the protein kinase ; aspirin and EDTA increase phosphorylation and Ca-binding by inhibiting the phosphoprotein phosphatase. In 3 cases of Glanzmann’s thrombasthenia, increased phosphorylation and Ca-binding was due to decreased phosphoprotein phosphatase activity-activity was restored the addition of ristosetin. A common phosphorylation-dephosphorylation mechanism for the action of inducers and inhibitors on Ca-mobilization and platelet activation will be presented.

scholarly journals Physicochemical properties of Planorbis corneus erythrocruorin

1975 ◽  
Vol 149 (2) ◽  
pp. 437-445 ◽  
Author(s):  
E J Wood ◽  
L J Mosby
Keyword(s):  
Molecular Weight ◽  
High Speed ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Alkaline Conditions ◽  
Planorbis Corneus ◽  
A Subunit ◽  
Partial Dissociation ◽  
Equilibrium Ultracentrifugation ◽  
Intermediate Value

The erythrocruorin from the snail Planorbis corneus had a sedimentation coefficient, s020,w, of 33.5 ± 0.31S, and a molecular weight of 1.65 × 10(6) ± 0.04 × 10(6) by high-speed sedimentation-equilibrium ultracentrifugation. The amino acid composition and absorption spectrum of the protein are reported. A very low number of half-cystine residues was found, corresponding to 0.4 residue per haem group. The haem content was 2.76 ± 0.22%, corresponding to a protein molecular weight of about 22300. Under both acid and alkaline conditions partial dissociation took place to yield mixtures of products that could not be identified. A subunit corresponding to that containing one haem group was not obtained under any of the dossociating conditions tried. Electron microscopy revealed a ring-shaped molecule about 12.2 ± 0.5 nm in diameter. The native erythrocruoerin bound O2 co-operatively, the intermediate value of h in Hill plots having values between 1.7 and 3.4 depending on the conditions.

scholarly journals The effect of vitamin A deficiency and dietary α-tocopherol on the stability of rat-liver lysosomes

1965 ◽  
Vol 97 (2) ◽  
pp. 353-359 ◽  
Author(s):  
OA Roels ◽  
M Trout ◽  
A Guha
Keyword(s):  
Vitamin A ◽  
Vitamin A Deficiency ◽  
Enzymic Activity ◽  
Alpha Tocopherol ◽  
Enzyme Release ◽  
Normal Amount ◽  
Prolonged Incubation ◽  
Liver Lysosomes ◽  
The Stability ◽  
Rat Liver Lysosomes

1. Vitamin A-deficient rats and pair-fed controls were maintained on either normal or raised amounts of dietary alpha-tocopherol. 2. Their livers were fractionated and; free' and; total' lysosomal phosphatase were determined in the various fractions. The rate of release of this enzyme was determined in the mitochondria-lysosome-rich fraction during incubation at pH5 and 37 degrees . 3. The deficient livers showed increased enzymic activity. 4. Prolonged incubation caused more rapid enzyme release from the mitochondria-lysosome-rich fraction of the vitamin A-deficient rats receiving the normal amount of dietary alpha-tocopherol than from the equivalent fraction of their pair-fed controls receiving vitamin A. Raised dietary alpha-tocopherol reversed this phenomenon.

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