scholarly journals The α-amylase of the beetle Callosobruchus chinensis. Purification and action pattern

1971 ◽  
Vol 121 (2) ◽  
pp. 317-320 ◽  
Author(s):  
H. Podoler ◽  
S. W. Applebaum
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Callosobruchus Chinensis ◽  
Action Pattern ◽  
Iodine Complex ◽  
Co Precipitation

Callosobruchus chinensis larval amylase was isolated and purified in five steps, which included co-precipitation with glycogen and column chromatography on ECTEOLA-cellulose. The enzyme was homogeneous by disc gel electrophoresis on polyacrylamide. The α-amylase nature was evidenced by the action on amylopectin β-amylase limit-dextrin, by the effect on the substrate–iodine complex and by the action pattern on several polysaccharide substrates. These action patterns are compared with those of other α-amylases.

scholarly journals Purification and characterization of histidine decarboxylase from mouse kidney

1986 ◽  
Vol 234 (2) ◽  
pp. 349-354 ◽  
Author(s):  
S A M Martin ◽  
J O Bishop
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Histidine Decarboxylase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Mouse Kidney ◽  
Purification Procedure ◽  
Purification And Characterization ◽  
A Subunit

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.

The Proteinases of Human Gastric Adenocarcinomata: Their Identification, Separation and Sites of Action on the B-Chain of Oxidized Insulin

1972 ◽  
Vol 42 (1) ◽  
pp. 79-90 ◽  
Author(s):  
D. J. Etherington ◽  
W. H. Taylor
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Deae Cellulose ◽  
Intermediate Form ◽  
Agar Gel ◽  
Equivalent Position ◽  
Normal Tissues ◽  
Highly Active ◽  
Agar Gel Electrophoresis ◽  
Sites Of Action

1. Because of the possibility that the proteinases of gastric adenocarcinomata may differ from those of healthy gastric mucosa, an investigation of these enzymes by means of pH-activity studies, agar-gel electrophoresis, column chromatography and the mode of action on the B-chain of oxidized insulin has been undertaken. 2. Agar-gel electrophoresis of extracts at pH 5 revealed a single zone of proteinase activity situated at the equivalent position to the zone 7 of normal gastric mucosal extracts and not activated at pH 2. The typical pepsins of normal mucosal extracts were not present. 3. Agar-gel electrophoresis at pH 8·2 resolved this single zone into three zones. One was located slightly cathodally (proteinase 1) and the other two anodally (a slower proteinase 2A and a faster 2B). 4. Proteinases 2A and 2B were separated by column chromatography and shown to have identical asymmetrical broad pH-activity curves with the maximum at pH 3·3–3·4. 5. Proteinase 1 had a symmetrical narrow pH-activity curve with the maximum at pH 3·7–3·9. Proteinase 1 was purified and resolved into two highly active major components, 1A and 1B, by column chromatography, first on DEAE-cellulose and then on CM-cellulose. 6. The sites of cleavage of the B-chain of oxidized insulin were determined for proteinases 1A and 1B. The same bonds were split by each, with one exception, but several were split at differing rates indicating that the two enzymes had related, but different, modes of action. 7. The tumour proteinases 1 resemble the cathepsins D in certain respects and the pepsins in others. They may represent an enzyme structure of an intermediate form. There is insufficient evidence to indicate whether they are elaborated by partially differentiated cells or whether they are derived from cells which have become dedifferentiated. No enzymes exactly like them have yet been found in normal tissues.

scholarly journals Purification and characterization of biotin-binding protein II from chicken oocytes

1988 ◽  
Vol 256 (3) ◽  
pp. 797-805 ◽  
Author(s):  
L Bush ◽  
T J McGahan ◽  
H B White
Keyword(s):  
Amino Acid ◽  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Chicken Egg ◽  
Biotin Binding ◽  
Cellulose Column

BBP-II, the major biotin-binding protein from chicken oocytes, was purified 12,000-fold with a 22% yield. The purification procedure includes butan-1-ol extraction of yolk lipids, phosphocellulose chromatography of the water-soluble proteins, DEAE-cellulose chromatography at pH 7.4 and hydroxyapatite column chromatography. Final purification was obtained by using a second DEAE-cellulose column chromatography at pH 6.0. BBP-I activity separated from BBP-II activity during elution from the first DEAE-cellulose column. Purified BBP-II was homogeneous on both polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis under conditions that would detect a 1% impurity. The subunit Mr determined from SDS/polyacrylamide-gel electrophoresis was 18,200 (72,600 for tetramer), which compares favourably with an Mr value of 17,300 (69,100) calculated from the amino acid analysis. A single precipitin line formed when rabbit antiserum to the protein was directed against a crude chicken egg-yolk sample. BBP-II purified by this procedure lacked carbohydrate and phosphate, was stable indefinitely when frozen, and was quite stable at room temperature. The N-terminal amino acid sequence showed polymorphism at three positions in the first 23 residues and was about 45% identical with the N-terminal 22 residues of avidin. Antiserum to BBP-II cross-reacted with BBP-I and similar proteins in the yolk of eggs from various birds and alligator as judged by immunodiffusion and enzyme-linked immunosorbent assays. No cross-reaction was observed with chicken egg-white by either of these methods.

scholarly journals An Improved Chromatographic Separation of Wheat Gluten Proteins

1965 ◽  
Vol 18 (1) ◽  
pp. 193 ◽  
Author(s):  
CW Wrigley
Keyword(s):  
Column Chromatography ◽  
Chromatographic Separation ◽  
Gel Electrophoresis ◽  
Carboxymethyl Cellulose ◽  
Wheat Gluten ◽  
Starch Gel Electrophoresis ◽  
Gluten Proteins ◽  
Electrophoretic Patterns ◽  
Starch Gel

Gel electrophoresis is at present the best procedure for the analytical fractiona-tion of wheat gluten proteins. Procedures more suitable for preparative studies, such as gel ffitration and column chromatography, have provided only partial fractionation on the basis of gel electrophoresis (Graham 1963; Lee et al. 1963; Wright, Brown, and Bell 1964). Gel electrophoretic patterns of fractions resulting from the carboxymethyl cellulose (CMC) fractionation of Simmonds and Winzor (1961) have been published by Graham (1963) and by Lee et al. (1963). These results showed that each fraction was heterogeneous, with considerable overlapping of electrophoretic bands from neighbouring fractions, and that fractions eluted late in the chromatogram were contaminated because of tailing of earlier fractions. The modification of the Simmonds and Winzor procedure described below results in an improved fractionation of gluten proteins as judged by starch-gel electrophoresis.

Two-dimensional gel electrophoresis can be used to examine chromatographic effluent fractions from displacement column chromatography.

1982 ◽  
Vol 28 (4) ◽  
pp. 998-999 ◽  
Author(s):  
A R Torres ◽  
G G Krueger ◽  
E A Peterson
Keyword(s):  
Human Serum ◽  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Gel Method ◽  
Minor Protein ◽  
Protein Components ◽  
A Minor

Abstract We show how two-dimensional gel electrophoresis can be used to monitor protein components in effluent fractions from a displacement column. A minor protein in human serum, of interest in studies on psoriasis and highly enriched by using carboxymethyldextrans as displacers on DEAE-Sephacel, was easily detected in the effluent fractions with the two-dimensional gel method because its concentration was sufficiently high and there was no interference by the carboxymethyldextrans or salt.

scholarly journals Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity

1976 ◽  
Vol 155 (3) ◽  
pp. 679-687 ◽  
Author(s):  
M J Crabbe ◽  
R D Waight ◽  
W G Bardsley ◽  
R W Barker ◽  
I D Kelly ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Substrate Specificity ◽  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Diamine Oxidase ◽  
Amine Oxidase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Lysine Vasopressin

1. Isoelectric focusing studies of human placental diamine oxidase showed the pI value of the active enzyme to be 6.5. This information was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH gradient elution and this, together with affinity chromatography on concanavalin A-Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration of diamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultracentrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx. 1.0 and 1.2g-atom respectively/70000mol.wt. unit. 5. The e.s.r. spectral intensity did not decrease nor did the spectral line shape change when excess of p-dimethylaminomethylbenzylamine was added to the enzyme. 6. Addition of K13CN to the enzyme eliminated the copper e.s.r. signal without affecting the manganese signal. 7. The placental enzyme therefore appears to differ from other amine oxidases in terms of its metal cofactor requirement, molecular weight and substrate specificity, and possible roles in vivo for this enzyme are discussed.

scholarly journals Characterization and Identification of Allergen Protein in Shrimp Before and After Heated with SDS-PAGE Method

2019 ◽  
Vol 2 (2) ◽  
pp. 87-93
Author(s):  
Emma Emawati ◽  
Idar Idar ◽  
Resta Ramadiyanti
Keyword(s):  
Molecular Weight ◽  
Anion Exchange ◽  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Food Allergies ◽  
Chromatography Method ◽  
Anion Exchange Column ◽  
Sds Page

Food allergies are one of the most common allergies in Indonesian society. Generally, when children aged 5-6 years food allergies will disappear, except peanut allergies and allergies to seafood, such as fish, shellfish and crustaceans. This study aims to determine the pattern of separation of allergen proteins in shrimp using anion exchange column chromatography method and identify allergen proteins in shrimp using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Protein extraction from shrimp using Phosphate buffer saline (PBS) pH 7.2 and centrifuged at 10,000 rpm for 10 min at 4�C. Protein separation was carried out by anion-exchange column chromatography method, and the fraction obtained was measured at 280nm wavelength. The highest yield at absorbance was identified by using SDS-PAGE. Polyacrylamide gel electrophoresis was used to determine the protein profile and molecular weight of shrimp extract. Coloring of protein bands using silver staining. Data were analyzed descriptively based on the migration value of the sample protein bands compared to the marker protein band (Rf). The results of protein allergen profile analysis on shrimp using SDS-PAGE showed that the shrimp contained a protein band with a molecular weight of 37.77 kDa for cooked shrimp and 37.03 kDa for fresh shrimp.

scholarly journals Purification and chemical characterization of human hexosaminidases A and B

1976 ◽  
Vol 159 (3) ◽  
pp. 535-539 ◽  
Author(s):  
J E S. Lee ◽  
A Yoshida
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Deae Cellulose Column ◽  
Hexosaminidase A ◽  
Cellulose Column

N-Acetyl-β-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with α-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.

ADP-ribosylation of pancreatic histone H1 and of other histones

1980 ◽  
Vol 58 (6) ◽  
pp. 509-515 ◽  
Author(s):  
G. G. Poirier ◽  
P. Savard
Keyword(s):  
Column Chromatography ◽  
Salt Concentration ◽  
Gel Electrophoresis ◽  
Histone H1 ◽  
Adp Ribosylation ◽  
The Core ◽  
Core Histones ◽  
Differential Binding

Incubation of pancreatic nuclei with high NAD concentrations resulted in increased ADP-ribosylation of histone H1. Interaction of [3H]ADP-ribosylated histone H1 with chromatin was significantly different from unmodified histone H1. The presence of a protein which is eluted at a lower salt concentration and which is ADP-ribosylated was also noticed. Pancreatic histones were isolated by column chromatography and their degree of ADP-ribosylation evaluated both by gel electrophoresis and by chromatography: histone H1 was the main acceptor while the core histones H3, H2B, and H2A were lightly labelled. Histones H1 and H10 have a differential binding to pancreatic chromatin and histone H10 is not ADP-ribosylated.

Sign in / Sign up

Export Citation Format

Share Document