scholarly journals Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene

1985 ◽  
Vol 227 (3) ◽  
pp. 719-725 ◽  
Author(s):  
M R Hyman ◽  
P M Wood
Keyword(s):  
Protective Effect ◽  
Gel Electrophoresis ◽  
Ammonia Oxidation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Competitive Inhibitor ◽  
Ion Concentrations ◽  
First Order ◽  
First Order Kinetics ◽  
Labelling Experiment ◽  
Suicide Substrate

Acetylene brings about a progressive inactivation of ammonia mono-oxygenase, the ammonia-oxidizing enzyme in Nitrosomonas europaea. High NH4+ ion concentrations were protective. The inactivation followed first-order kinetics, with a rate constant of 1.5 min-1 at saturating concentrations of acetylene. If acetylene was added in the absence of O2, the cells remained active until O2 was re-introduced. A protective effect was also demonstrated with thiourea, a reversible non-competitive inhibitor of ammonia oxidation. Incubation of cells with [14C]acetylene was found to cause labelling of a single membrane polypeptide. This ran on dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr value of 28 000. It is concluded that acetylene is a suicide substrate for the mono-oxygenase. The labelling experiment provides the first identification of a constituent polypeptide of ammonia mono-oxygenase.

scholarly journals Immunoblot analysis of glutaminase peptides in intact and solubilized mitochondria isolated from various rat tissues

1987 ◽  
Vol 242 (3) ◽  
pp. 743-747 ◽  
Author(s):  
R A Shapiro ◽  
W G Haser ◽  
N P Curthoys
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoblot Analysis ◽  
Brain Mitochondria ◽  
Rat Tissues ◽  
First Order ◽  
First Order Kinetics ◽  
Triton X ◽  
The Brain ◽  
Isolated Rat

Antibodies were prepared against isolated rat renal glutaminase and affinity-purified against the 65 kDa peptide contained in the purified rat brain glutaminase. The affinity-purified IgGs were then used to compare the glutaminase immunoreactive peptides contained in samples that had been subjected to SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose. The purified brain glutaminase and isolated brain mitochondria contain 68 and 65 kDa peptides that exhibit nearly equivalent immunostaining. Partial proteolysis of the isolated 68 and 65 kDa peptides with Staphylococcus aureus V8 proteinase produced an identical pattern of immunoreactive proteolytic fragments. However, digestion of the two peptides with chymotrypsin resulted in similar, but slightly different, patterns. The pattern of immunostaining was unaltered even when the brain mitochondria were solubilized with Triton X-100 and stored for 2 days at 4 degrees C. A very similar pattern was observed when intact renal mitochondria were subjected to immunoblot analysis. However, when renal mitochondria were solubilized, the 68 kDa peptide was rapidly degraded to the 65 kDa form. At 4 degrees C this reaction occurs with apparent first-order kinetics and a t1/2 of 35 min. Degradation of the 65 kDa form of the renal glutaminase occurs with much slower kinetics, but is nearly complete after 24 h. Solubilization of mitochondria isolated from various zones of the kidney indicated that the responsible endogenous proteinase was localized primarily in the cortex. Mitochondria isolated from intestinal or renal papillary tissue contain four glutaminase immunoreactive peptides (Mr 68,000, 65,000, 61,000 and 58,000). The smallest of these peptides is identical in size with the single immunoreactive peptide observed in liver tissue.

scholarly journals Purification and properties of an aryl β-xylosidase from a cellulolytic extreme thermophile expressed in Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 645-650 ◽  
Author(s):  
R C Hudson ◽  
L R Schofield ◽  
T Coolbear ◽  
R M Daniel ◽  
H W Morgan
Keyword(s):  
Escherichia Coli ◽  
Thermal Inactivation ◽  
Competitive Inhibitor ◽  
Coli Strain ◽  
Extreme Thermophile ◽  
First Order ◽  
Optimal Activity ◽  
First Order Kinetics ◽  
Purification And Properties ◽  
Optimum Ph

An aryl beta-xylosidase was purified to homogeneity from an Escherichia coli strain containing a recombinant plasmid carrying a beta-xylosidase (EC 3.2.1.37) gene from the extremely thermophilic anaerobic bacterium isolate Tp8T6.3.3.1 (‘Caldocellum saccharolyticum’). It has a pI of 4.3 and shows optimal activity at pH 5.7. The enzyme is highly specific, acting on o- and p-nitrophenyl beta-D-xylopyranosides and minimally on p-nitrophenyl alpha-L-arabinopyranoside. It does not act on xylobiose. The Km for p-nitrophenyl beta-D-xylopyranoside at the optimum pH for activity is 10 mM, and at pH 7.0 is 6.7 mM. Xylose is a competitive inhibitor with Ki 40 mM. Thermal inactivation follows first-order kinetics at 65 and 70 degrees C with t1/2 values of 4.85 h and 40 min respectively. The t1/2 at 70 degrees C is increased 3-fold and 4-fold by the addition of 0.5 mg of BSA/ml and 2 mM-dithiothreitol respectively.

scholarly journals Properties of antithrombin-thrombin complex formed in the presence and in the absence of heparin

1983 ◽  
Vol 213 (2) ◽  
pp. 345-353 ◽  
Author(s):  
A Danielsson ◽  
I Björk
Keyword(s):  
Rate Constants ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dissociation Rate ◽  
Exchange Chromatography ◽  
Stable Complex ◽  
Intermediate Step ◽  
First Order ◽  
Activity Measurements ◽  
Dissociation Rate Constants

Purification of antithrombin-thrombin complex by ion-exchange chromatography on DEAE-agarose resulted in predominantly monomeric complex, whereas purification on matrix-linked heparin produced large amounts of aggregated complex. Monomeric antithrombin-thrombin complexes formed in the presence and in the absence of heparin had similar conformations and heparin affinities. Moreover, the first-order dissociation rate constants, measured by thrombin release, of these complexes were similar, 2.3 × 10(-6)-3.4 × 10(-6)S-1, regardless of whether newly formed or purified complex was analysed. Similar dissociation rate constants were also obtained for purified complex formed with or without heparin, from analyses by dodecyl sulphate/polyacrylamide-gel electrophoresis of the release of modified antithrombin, cleaved at the reactive-site bond. No dissociation of intact antithrombin from the complex was detected by activity measurements or by gel electrophoresis. Aggregation of the complex was found to be accompanied by a decrease in apparent dissociation rate. The similar properties of antithrombin-thrombin complexes formed with or without heparin support the concept of a catalytic role for the polysaccharide in the antithrombin-thrombin reaction. Furthermore, the results indicate that the reaction between enzyme and inhibitor involves the rapid formation of an irreversible, kinetically stable, complex that dissociates into active thrombin and modified, inactive, antithrombin by a first-order process with a half-life of about 3 days. The inhibition thus resembles a normal proteolytic reaction, one intermediate step of which is very slow.

scholarly journals Purification and properties of Cellvibrio gilvus cellobiose phosphorylase

1983 ◽  
Vol 209 (3) ◽  
pp. 803-807 ◽  
Author(s):  
T Sasaki ◽  
T Tanaka ◽  
S Nakagawa ◽  
K Kainuma
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Competitive Inhibitor ◽  
Polyacrylamide Gel ◽  
Protamine Sulphate ◽  
Purification And Properties ◽  
Cellobiose Phosphorylase ◽  
Optimum Ph

The cellobiose phosphorylase (EC 2.4.1.20) of Cellvibrio gilvus, which is an endocellular enzyme, has been purified 196-fold with a recovery of 11% and a specific activity of 27.4 mumol of glucose 1-phosphate formed/min per mg of protein. The purification procedure includes fractionation with protamine sulphate, and hydroxyapatite and DEAE-Sephadex A-50 chromatography. The enzyme appears homogeneous on polyacrylamide-gel electrophoresis, and a molecular weight of 280 000 was determined by molecular-sieve chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a single band and mol.wt. 72 000, indicating that cellobiose phosphorylase consists of four subunits. The enzyme had a specificity for cellobiose, requiring Pi and Mg2+ for phosphorylation, but not for cellodextrin, gentibiose, laminaribiose, lactose, maltose, kojibiose and sucrose. The enzyme showed low thermostability, an optimum pH of 7.6 and a high stability in the presence of 2-mercaptoethanol or dithiothreitol. The Km values for cellobiose and Pi were 1.25 mM and 0.77 mM respectively. Nojirimycin acted as a powerful pure competitive inhibitor (with respect to cellobiose) of the enzyme (Ki = 45 microM). Addition of thiol-blocking agents to the enzyme caused 56% inhibition at 500 microM-N-ethylmaleimide and 100% at 20 microM-p-chloromercuribenzoate.

Active-site-directed inactivation of Schizophyllum commune cellulase by 4′, 5′-epoxypentyl-4-D-(β-D-glucopyranosyl)-β-D-glucopyranoside

1988 ◽  
Vol 66 (8) ◽  
pp. 871-879 ◽  
Author(s):  
Anthony John Clarke
Keyword(s):  
Active Site ◽  
Schizophyllum Commune ◽  
Competitive Inhibitor ◽  
Irreversible Inhibitor ◽  
Inhibitor Molecule ◽  
Enzyme Active Site ◽  
First Order ◽  
First Order Kinetics ◽  
Pseudo First Order ◽  
Inactivation Process

4′,5′-Epoxypentyl-4-D-(β-D-glucopyranosyl)-β-D-glucopyranoside (4) was synthesized by a Koenigs–Knorr reaction of 4-penten-1-ol and acetobromcellobiose, promoted by silver trifluoromethanesulfonate and N,N′-tetramethylurea, and tested as a potential active-site-directed irreversible inhibitor of the Schizophyllum commune cellulase. Incubation of the S. commune cellulase with 4 resulted in a time-dependent irreversible inactivation of the enzyme. The inactivation process obeyed pseudo-first-order kinetics and the hyperbolic plot of kobs as a function of inhibitor concentration provided values for Kd and k2 of 150 mM and 2.0 × 10−4 s−1, respectively, at pH 5.5 and 25 °C. The binding of a competitive inhibitor, cellobiose, to the cellulase prior to incubation with 4 protected the enzyme from rapid inactivation, suggesting that the inactivation is due to attack at the active site. The dependence of the inactivation on pH is consistent with the participation of carboxyl groups. Treatment of the affinity-labeled enzyme with [14C]methoxyamine resulted in the near stoichiometric formation of a stable radiolabelled adduct, suggesting that one inhibitor molecule binds per enzyme active site of the enzyme.

The Effect of Staphylocoagulase on the Subunit Structure of Human Fibrin

1974 ◽  
Vol 31 (01) ◽  
pp. 052-062
Author(s):  
M Tager ◽  
M. J. S King
Keyword(s):  
Calcium Ion ◽  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Activate Factor ◽  
Subunit Structure ◽  
Ion Concentrations ◽  
Alpha Beta ◽  
Polypeptide Chains ◽  
Calcium Lead

SummaryFibrin clots resulting from the action of coagulase thrombin were subjected to Polyacrylamide gel electrophoresis in the presence of hirudin or of heparin to exclude the action of biothrombin.Alpha, beta, and gamma polypeptide chains were resolved not differing from those induced by the action of thrombin on fibrinogen.No evidence was found of any further degradation of any of the polypeptide chains.Although coagulase thrombin fails to activate factor XIII at calcium ion concentrations effective for the cross-linking of fibrin by thrombin, higher concentrations of calcium lead to the activation of factor XIII by coagulase thrombin, resulting in gamma dimer formation.The data support the evidence that coagulase thrombin, like biothrombin, acts as a limited proteolytic agent on fibrinogen.

scholarly journals Complexes of rat α1-macroglobulin and subtilisin are endocytosed by parenchymal liver cells

1985 ◽  
Vol 226 (1) ◽  
pp. 75-84 ◽  
Author(s):  
J Bergsma ◽  
M K Boelen ◽  
A M Duursma ◽  
W G Schutter ◽  
J M Bouma ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Large Subunit ◽  
Subcellular Fractionation ◽  
Liver Cells ◽  
First Order ◽  
First Order Kinetics ◽  
Parenchymal Liver ◽  
Rapid Clearance ◽  
Parenchymal Cells ◽  
Compact Conformation

Rat alpha 1-macroglobulin was isolated from plasma. Gel electrophoresis of the denatured and reduced protein showed two bands, with Mr values of 163 000 and 37 000. The large subunit contained an autolytic site. This subunit was also split after reaction of the macroglobulin with trypsin. Electron microscopy showed that the macroglobulin changed towards a more compact conformation after reaction with this proteinase. Subtilisin, or alpha 1-macroglobulin, was labelled with a sucrose-containing radio-iodinated group that stays in lysosomes after endocytosis and breakdown of the tagged protein. After intravenous injection into rats, alpha 1-macroglobulin was cleared from plasma with first-order kinetics, showing a half-life of about 9 h, whereas complexes of alpha 1-macroglobulin and subtilisin were cleared with half-lives of only 3 min. Liver contained about 60% of the label at 30 min after injection of complexes. About 90% of the liver radioactivity was found in parenchymal cells isolated after perfusion of the liver with a collagenase solution. Subcellular fractionation indicated a lysosomal localization of the complexes. We conclude that endocytosis by parenchymal liver cells is the major cause of the rapid clearance of alpha 1-macroglobulin-proteinase complexes from plasma.

A New Fibrinogen Variant With Abnormal Gamma Chain : Fibrinogen Haifa

1981 ◽  
Author(s):  
J Soria ◽  
C Soria ◽  
G Palareti ◽  
M Tavori ◽  
M Samama ◽  
...  
Keyword(s):  
Protective Effect ◽  
Gel Electrophoresis ◽  
Arterial Thrombosis ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gamma Chain ◽  
Peripheral Arterial ◽  
Fibrin Monomers ◽  
Fibrinogen Molecule

A congenital abnormal fibrinogen was detected in a 30 year old woman presenting several episodes of peripheral arterial thrombosis.The abnormality in fibrin formation is located in the fibrin monomers aggregation, since the fibrinopeptides release and the fibrin stabilization were normal.The SDS polyacrylamide gel electrophoresis of plasmic degradation products, obtained either in the presence or in the absence of calcium, revealed an absence of protective effect of calcium for plasmin degradation of Fibrinogen Haifa. Since, Ca++ protects against further plasmin degradation of the gamma chain, we suggest that the anomaly is located in this part of the fibrinogen molecule.Because the anomaly was discovered at Haifa, Israel, we suggest to call this abnormal fibrinogen : “Fibrinogen Haifa”.

scholarly journals Protein and glycoprotein antifreezes in the intestinal fluid of polar fishes

1982 ◽  
Vol 98 (1) ◽  
pp. 429-438
Author(s):  
S. M. O'Grady ◽  
J. C. Ellory ◽  
A. L. DeVries
Keyword(s):  
North Atlantic ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Low Molecular Weight ◽  
Intestinal Fluid ◽  
Melting Points ◽  
Ion Concentrations ◽  
Freezing Points ◽  
Antarctic Fishes ◽  
The Antarctic

Measurements of ion concentrations, freezing points and melting points of intestinal fluid were made for several Antarctic fishes and two North Atlantic species. These measurements indicated that plasma and intestinal fluid are nearly isosmotic. Freezing points of intestinal fluid were approximately 0.9 degrees C below the melting points, suggesting the presence of glyco-protein antifreeze within the intestinal fluid of the Antarctic fishes. Polyacrylamide gel electrophoresis and specific immunoprecipitation with glyco-protein antifreeze antibody confirmed the presence of appreciable quantities of antifreeze and showed that the major antifreeze fractions present in the intestinal fluid are low molecular weight glycopeptides.

Purification and Characterization of Nitrate Reductase from the Halophile Archaebacterium Haloferax mediterranei

1992 ◽  
Vol 47 (9-10) ◽  
pp. 670-676 ◽  
Author(s):  
María C. Alvarez-Ossorio ◽  
Francisco J. G. Muriana ◽  
Francisco F. de la Rosa ◽  
Angel M. Relimpio
Keyword(s):  
Nitrate Reductase ◽  
Salt Concentration ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Chromatography ◽  
Haloferax Mediterranei ◽  
Ammonium Sulphate Precipitation ◽  
First Order Kinetics

Nitrate reductase is induced in cells of Haloferax mediterranei by the presence of nitrate upon anaerobic conditions. This enzyme was purified more than 35-fold with a yield of 49%. Densitograms of polyacrylamide gel electrophoresis show the preparation to be 85% purity. The best enzyme preparation has a specific activity of 13.6 U /m g protein. It is the first halophilic nitrate reductase that has been purified near to homogeneity. The purification consists of five steps: an ammonium sulphate precipitation and four successive gel chromatographies with Sepharose CL-4 B, calcium phosphate, DEAE-Sephacel and Sephacryl S-200. An average Mr of 170,000 was estimated by gel chromatography and non-denaturing gel electrophoresis. Effectiveness of electron donors, cofactors and inhibitors are reported. At low salt concentration the halophilic nitrate reductase was inactivated following first-order kinetics. The Km for nitrate depends on salt concentration and shows values in the range from 2.5 to 6.7 mM.

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