scholarly journals Cathepsin D. Purification of isoenzymes from human and chicken liver

1970 ◽  
Vol 117 (3) ◽  
pp. 601-607 ◽  
Author(s):  
A. J. Barrett
Keyword(s):  
Isoelectric Focusing ◽  
Cathepsin D ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Specific Activities ◽  
Acetone Fractionation ◽  
High Degree

1. The Barrett (1967) assay for cathepsin D was slightly modified. 2. The enzyme was purified from liver of man and chicken by a procedure involving autolysis, acetone fractionation, ion-exchange chromatography and isoelectric focusing. 3. Several isoenzymes of cathepsin D were resolved in the isoelectric-focusing step, and three major forms, α,β and γ, were distinguished for each species. 4. A modified analytical method of isoelectric focusing in polyacrylamide gel indicated a high degree of homogeneity of the purified β and γ isoenzymes from each species, and this was supported by their constant high specific activities. 5. Gel filtration of the isoenzymes in a calibrated column of Sephadex G-100 showed that each had a molecular weight of 45000. 6. Human cathepsin D had a pH optimum of 3.5, and that of chicken enzyme was 3.0, haemoglobin being used as substrate. In each species, the three isoenzymes have the same pH-dependence curve. 7. The purified cathepsin D samples showed very little action on acid-denatured albumin.

scholarly journals The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

1972 ◽  
Vol 130 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Colin H. Self ◽  
P. David J. Weitzman
Keyword(s):  
Molecular Weight ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Low Concentrations ◽  
Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

scholarly journals Salivary apyrase of Rhodnius prolixus. Kinetics and purification

1986 ◽  
Vol 233 (3) ◽  
pp. 885-891 ◽  
Author(s):  
J J F Sarkis ◽  
J A Guimarães ◽  
J M C Ribeiro
Keyword(s):  
Atp Hydrolysis ◽  
Competitive Inhibition ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Rhodnius Prolixus ◽  
Exchange Chromatography ◽  
Phosphate Esters ◽  
Inorganic Pyrophosphatase ◽  
Blood Sucking ◽  
Specific Activities

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5′-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.

Enolase from Lobster (Homarus americanus)

1971 ◽  
Vol 28 (6) ◽  
pp. 879-882 ◽  
Author(s):  
M. John Chapman ◽  
Christopher Chin ◽  
Finn Wold
Keyword(s):  
Heat Treatment ◽  
Ion Exchange ◽  
Ammonium Sulfate ◽  
Catalytic Properties ◽  
Gel Filtration ◽  
Homarus Americanus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Acetone Fractionation

Enolase has been isolated from lobster muscle by acetone fractionation, heat treatment, ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Preliminary characterization of the pure enzyme shows that the catalytic properties are very similar to those of the enolases from rabbit and fish.

The phosphatidyl-myo-inositol-4,5-bisphosphate phosphatase from Crithidia fasciculata

1981 ◽  
Vol 59 (7) ◽  
pp. 469-476 ◽  
Author(s):  
F. B. St. C. Palmer
Keyword(s):  
Cetyltrimethylammonium Bromide ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Phosphate Esters ◽  
Relative Mass ◽  
Crithidia Fasciculata ◽  
Specific Activities ◽  
Triton X

The phosphatase which specifically removes one phosphate group from phosphatidyl-myo-inositol 4,5-bisphosphate was purified up to 6000-fold from the cytosol of the protozoan Crithidia fasciculata. Lipoproteins which interfere with the purification were precipitated by reducing the pH to 4.5. The enzyme was isolated from the supernatant by ammonium sulfate fractionation, gel filtration (Sepharose CL-6B), ion–exchange chromatography (DEAE-Sepharose CL-6B), and hydrophobic chromatography on detergent-saturated phenyl-Sepha-rose CL-4B. The preparations had specific activities of 44–110 μmol∙min−1∙mg protein−1 with phosphatidyl-myo-inositol 4,5-bisphosphate, but were inactive with a variety of lipid and nonlipid phosphate esters. The enzyme was stable in the presence of salt and exhibited a relative mass of 117 000. It formed larger aggregates in the absence of salt and was dissociated into monomers of relative mass 57 000 by sodium dodecyl sulfate.Addition of Triton X-100 to the assay mixture reduced the dependence upon moderation of the charge of the substrate by cetyltrimethylammonium bromide. In the presence of both detergents the Mg2+ dependence of the enzyme was reduced (Km for Mg2+ = 40 μM) while the "apparent" Km for the substrate was unchanged at 240 μM. Substrate precipitation at higher Mg2+ concentrations was eliminated.

scholarly journals Purification and properties of l-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus

1987 ◽  
Vol 248 (3) ◽  
pp. 871-876 ◽  
Author(s):  
M E Hoey ◽  
N Allison ◽  
A J Scott ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Potential Inhibitors ◽  
Triton X

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a ‘wall + membrane’ fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.

scholarly journals Multiple enzyme purifications from muscle extracts by using affinity-elution-chromatographic procedures

1977 ◽  
Vol 161 (2) ◽  
pp. 265-277 ◽  
Author(s):  
R K Scopes
Keyword(s):  
Gel Filtration ◽  
Phosphoglycerate Kinase ◽  
Gradient Elution ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Rabbit Muscle ◽  
Chromatographic Techniques ◽  
Ph Values ◽  
Chicken Muscle ◽  
Specific Activities

1. Starting with (NH4)2SO4 fractions of muscle extracts, procedures for purifying four to six separate enzymes from each fraction by using affinity-elution-chromatographic techniques are described. 2. Schemes for purifying 12 separate enzymes from rabbit muscle, and eight from chicken muscle extracts, are included. In nearly all cases the overall procedure involves three steps: the initial (NH4)2SO4 fractionation, the ion-exchange chromatography with affinity elution of the enzyme, and gel filtration. The specific activities of the enzymes so purified are comparable with the highest values in the literature. 3. The five schemes described include illustrations of affinity elution of the separate enzymes at different pH values, at different ionic strengths and in combination with conventional gradient elution. They also include stepwise adsorption on columns at different pH values. 4. Separation of two electrophoretically differing forms of phosphoglycerate kinase was achieved by gradient affinity elution from CM-cellulose. The lower-pI form was eluted by a lower concentration of substrate than the higher-pI form.

Isolation and characterization of cathepsin D from human gastric mucosa

1981 ◽  
Vol 46 (13) ◽  
pp. 3302-3313 ◽  
Author(s):  
Jan Pohl ◽  
Ladislav Bureš ◽  
Karel Slavík
Keyword(s):  
Gastric Mucosa ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Human Gastric Mucosa ◽  
Ph Optimum ◽  
Isolation And Characterization

The molecular weight of the enzyme, purified by ion-exchange chromatography and affinity chromatography, was determined by gel filtration on Sephadex G-100 as 49 000. After treatment with 2-mercaptoethanol, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate resolved the enzyme into two chains, of molecular weights 33 000 and 18 000. This shows that in the native state the enzyme is composed of one light and one heavy chain. Isoelectric focusing in polyacrylamide gel gave four bands, the isoelectric points being 5.5, 6.1, 6.5 and 7.1. The optimum protein substrate (pH optimum 3.2-3.6) was haemoglobin. The best synthetic substrate was methyl ester of pyroglutamyl-histidyl-phenylalanyl-phenylalanyl-alanyl-leucine. The protease was inhibited by the inhibitor of cathepsin D from the potato tubers. It is concluded that the enzyme is cathepsin D from gastric mucosa.

Separation and Characterization of Two Fibrinolytic Inhibitors from Human Placenta

1971 ◽  
Vol 25 (03) ◽  
pp. 580-589 ◽  
Author(s):  
M Uszynski ◽  
U Abildgaard
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Isoelectric Focusing ◽  
Basic Protein ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Coagulation Inhibitor ◽  
Tissue Thromboplastin

SummaryProcedures for the separation of two inhibitors of the activation of plasminogen to plasmin by urokinase are described. Tissue thromboplastin was removed by adsorption to Al(0H)3 gel followed by ultracentrifugation. Plasminogen, plasminogen activator, a coagulation inhibitor and hemoglobin were removed by ion exchange chromatography (CM- or DEAE-Sephadex with NaCl gradients). The minor UK inhibitor is a relative basic protein with a pI of about 5.8. The major inhibitor was purified further by isoelectric focusing, preparative electrophoresis in polyacrylamide gel, and gel filtration. This inhibitor has α1-motility, the pI is about 5.2, and the molecular weight about 100,000. It inactivates urokinase progressively, but does not inhibit streptokinase, plasmin or thrombin.

Caractéristiques fonctionnelles des activités peroxydasiques des feuilles et cals d'une plante à métabolisme acide crassulacéen, Pedilanthus tithymaloides variegatus, Euphorbiaceae

1984 ◽  
Vol 62 (9) ◽  
pp. 901-907 ◽  
Author(s):  
Pierre Bricage
Keyword(s):  
Ion Exchange ◽  
Physicochemical Properties ◽  
Affinity Chromatography ◽  
Enzyme Activities ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Leaf Tissues ◽  
Specific Activities ◽  
Isoenzyme Patterns

Peroxidases (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7) from leaf tissues and calli were extracted and compared in terms of their specific activities at different pH and temperatures, their isoenzyme patterns and physicochemical properties. Three groups of enzyme activities were detected and their purification was performed by conventional methods (ammonium sulfate fractionation, ion-exchange chromatography, gel filtration, affinity chromatography).

scholarly journals Purification and properties of human kidney-cortex hexosaminidases A and B

1977 ◽  
Vol 165 (1) ◽  
pp. 49-53 ◽  
Author(s):  
J E Wiktorowicz ◽  
Y C Awasthi ◽  
A Kurosky ◽  
S K Srivastava
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Human Kidney ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Kidney Cortex ◽  
Enzyme Replacement ◽  
Purification And Properties ◽  
Hexosaminidase A ◽  
Specific Activities

Hexosaminidases (EC 3.2.1.30) A and B from human kidney cortex were purified to homogeneity by using concanavalin A affinity chromatography, ion-exchange chromatography and gel filtration. The yield of homogeneous isoenzymes improved approx. 20-fold, giving preparations of hexosaminidases A and B with specific activities of about 200 and 325 units/mg of protein respectively. The kinetic and structural properties of kidney hexosaminidase isoenzymes were studied and compared with the hexosaminidase isoenzymes from human placenta. The amino acid composition of hexosaminidase A was significantly different from that of hexosaminidase B. In the event of success in developing enzyme-replacement therapy for Tay-Sachs and Sandhoff's diseases, this modified procedure can furnish larger amounts of homogeneous isoenzymes.

Sign in / Sign up

Export Citation Format

Share Document