Search results for "Isoelectric focusing"
showing 9 items of 39 documents
Secretion of haemolysins and proteases by Aeromonas hydrophila EO63: separation and characterization of the serine protease (caseinase) and the metal…
2004
C . E S T E V E A N D T . H . B I R K B E C K . 2004. Aims: To determine the haemolysins and proteases excreted by the virulent strain EO63 of Aeromonas hydrophila grown in complex media and to then fractionate and characterize them, in particular those with elastolytic activity. Methods and Results: The amount of haemolytic and proteolytic activity in EO63 culture supernatants was dependent on the culture media used. In all media, haemolysins appeared during the phase of active growth and haemolytic activity decreased quickly thereafter, as previously described for aerolysin. In contrast, proteases were mainly released during the stationary phase. Serine protease activity in EO63 culture s…
Purification, isolation and characterization of a phosphoglycolate phosphatase isoenzyme from human erythrocytes.
1982
1. Preparation, purification and characterization of a phosphoglycolate phosphatase (PGP) isoenzyme from human erythrocytes was achieved by DEAE-Sepharose CL-6B chromatography and isoelectric focusing using carrier ampholytes, pH 4-6. 2. The isoenzyme has an isoelectric point of 5.00 +/- 0.05 and could be purified 33,000 fold to a specific activity of 32.7 U/mg of protein. It represents the PGP phenotype 1 consisting of a single isoenzyme. 3. The enzyme is composed of two subunits (mol. wt 35,000) which are identical and not connected by SS-bridges. 4. At 4 degrees C the isoenzyme is more stable in the pH range of 7-9 than at acid pH values. 5. Incubation at 30 and 40 degrees C for 4 hr doe…
Determination of cow’s milk in non-bovine and mixed cheeses by capillary electrophoresis of whey proteins in acidic isoelectric buffers
2000
An improved method for the determination of cow's milk in non-bovine cheese is reported: electrophoresis of whey proteins in acidic, isoelectric buffers. Two background electrolytes (BGEs) have been tested: (i) 50 mM iminodiacetic acid (pH=isoelectric point=2.30 at 25 degrees C), 0.5% hydroxyethylcellulose, 0.1% Tween 20 and 6 M urea (apparent pH 3.1), E=300 V/cm, for the separation of alpha-lactalbumins (alpha-LAs); (ii) a BGE with the same composition, but supplemented with 10% Tween 20, E=450 V/cm, for the fractionation of beta-lactoglobulins (beta-LGs). Surfactants have a discriminating effect on the retention behaviour of the bovine alpha-LA and beta-LG proteins, owing to the different…
Fast detection of bovine milk in Rocquefort cheese with phastsystem by gel isoelectric focusing and immunoblotting
1992
.Summary - A fast procedure for the detection of bovine milk in Roquefort cheese is described. It is based on the separation by rapid isoelectric focusing on Phasteysterne apparatus of 12-caseinsfrom the milk of the 2 species. The presence of bovine milk is also confirmed by the detection in the electrophoretic pattern of a ~-casein derived peptide from bovine milk, during cheese ripening, identified by immunoblotting. By using Ihis procedure, levels of bovine milk as low as 5% were easily delecled in Roquefort cheese ripened for a period varying from 10 days to 5 months.
Cellular and Subcellular Localization of Peroxidase Isoenzymes in Plants and Cell Suspension Cultures from Lupinus polyphyllus
1989
Abstract , leaf protoplasts and cell suspension cultures of Lupinus polyphyllus and isolated vacuoles were studied for cellular and subcellular localization of peroxidase isoenzymes. Isoelectric focusing revealed 16 peroxidase isoenzymes. The basic peroxidase isoenzymes are predominantly localized in the vacuole and, to a minor degree, unbound in the intercellular space. The acidic isoenzymes are cell wall-bound in plants and not detectable in suspension-cultured cells. Large amounts (up to 11.0 U/ml) of a single basic isoenzyme are detectable in the spent medium of cell suspension cultures.
Occurrence and Heterogeneity of Chymotrypsin Inhibitors in Vegetative Tissues of Barley
1977
Inhibitors of chymotrypsin and the alkaline proteinase of Aspergillus oryzae were present in the shoots of barley seedlings and weak activities were also detected in the shoot tops of 6-week-old plants. Treatments which induce inhibitor formation in tomato and potato leaves had no effect when tested on mature leaves, seedlings, or young tillers of barley. Fractionation experiments with isoelectric focusing showed that the barley leaves contained several proteinase inhibitors acting on both chymotrypsin and the Aspergillus proteinase, and one inhibitor which acted only on the Aspergillus enzyme. All of these inhibitors were different from the five Aspergillus proteinase inhibitors which are …
Isolation and characterization of a chlorogenic acid esterase from Aspergillus niger.
1980
Abstract The isolation and characterization of a specific chlorogenic acid esterase is described. The enzyme activity is measured by determination of the hydrolysis product caffeic acid. The enzyme had been concentrated by means of ultrafiltration and column-chromatography. The pH- and temperature optimum were 6.5 and 45 °C respectively. Divalent cations were not required for the enzyme activity. As other esterases, this enzyme is inhibited by di-isopropyl-phosphorofluoridate. The Km-value is 0.70 mᴍ chlorogenic acid, the molecular weight 240000. The described enzyme is specific for chlorogenic acid. On the other hand a typical unspecific esterase like the pig liver esterases does not split…
Methods for Separating Native Enzymes
1994
In the course of electrophoresis the stability of an enzyme depends on such conditions as (a) pH-value, (b) ion strength and ion species, (c) effector molecules, (d) temperature and (e) properties of the separation matrix. These parameters were empirically optimized for starch gel electrophoresis [1–3] and cellulose acetate electrophoresis [4, 5] when analyzing predominantly animal and human specimen. A major advantage of these types of separation media is that practically every buffer system can be used to separate enzymes whereas in disc-gel electrophoresis [6–8] the number of applicable buffer systems is limited. When using isoelectric focusing to separate native enzymes no buffer choice…
Novel acrylamido monomers with higher hydrophilicity and improved hydrolytic stability: II. Properties of N-acryloylaminopropanol
1996
The physico-chemical properties and the electrophoretic behavior of the novel set of monomers reported by (Simo-Alfonso et al., Electrophoresis 1996, 17, 723-731) have been evaluated. Of utmost importance was the combination of high hydrophilicity and extreme hydrolytic stability, most desired properties for, any electrophoretic matrix, especially for protein fractionation. One of these monomers (N-acryloylaminopropanol, AAP) was found indeed to be extremely hydrophilic (with a partition coefficient P of only 0.10, vs. P = 0.13 for N-acryloylaminoethoxyethanol and P = 0.20 for acrylamide) and to possess excellent stability to alkaline hydrolysis. Its hydrolysis constant (0.008 L mol-1 min-1…