scholarly journals Partial purification of goat kidney β-mannosidase

1988 ◽  
Vol 249 (3) ◽  
pp. 871-875 ◽  
Author(s):  
J I Frei ◽  
K T Cavanagh ◽  
R A Fisher ◽  
R P Hausinger ◽  
M Dupuis ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Cation Exchange ◽  
Anion Exchange ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Fast Protein Liquid Chromatography ◽  
Partial Purification

1. Goat kidney beta-mannosidase was purified 8500-fold to a specific activity of 65,000 nmol/h per mg of protein with a 6% yield by using multiple steps including cation-exchange and anion-exchange fast protein liquid chromatography. This is the first description of a highly purified preparation from goat tissue; however, it was not homogeneous, as judged by silver-stained SDS/polyacrylamide-gel electrophoresis. 2. The enzyme exhibited microheterogeneity when analysed by isoelectric focusing (pI 5.5-6.5). 3. Purified beta-mannosidase hydrolysed the terminal beta-(1→4)-linkage of oligosaccharides that accumulate in beta-mannosidosis.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

scholarly journals Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 389-397 ◽  
Author(s):  
D A Wiginton ◽  
M S Coleman ◽  
J J Hutton
Keyword(s):  
Molecular Weight ◽  
Adenosine Deaminase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Periodic Acid Schiff ◽  
Apparent Homogeneity

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.

Rapid separation of bovine caseins by mass ion exchange chromatography

1989 ◽  
Vol 56 (3) ◽  
pp. 391-397 ◽  
Author(s):  
K. F. Ng-Kwai-Hang ◽  
J. P. Pélissier
Keyword(s):  
Liquid Chromatography ◽  
Ion Exchange ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Rapid Separation ◽  
Rapid Isolation ◽  
Time Required

SummaryThe rapid isolation of major bovine caseins in gram quantities was investigated. Whole casein was precipitated from individual cow's milk by adjusting the pH to 4·6 and the precipitated casein was suspended in 4·5 M urea (pH 8·0) containing 0·02 M imidazole and 0·03 M β-mercaptoethanol, and bound on a QAE Zeta Prep 250 cartridge. Stepwise elution with the urea/imidazole β-mercaptoethanol buffer and varying amounts of NaCl gave five well resolved peaks, which were identified by polyacrylamide gel electrophoresis and fast protein liquid chromatography to be pure γ-casein, κ-casein. β-casein, β-casein and αs-casein, respectively. The ion exchange cartridge was regenerated by flushing with buffer containing 0·50 Μ-NaCl followed by equilibration with starting buffer before separation of next sample. The time required to run each sample including cartridge regeneration and equilibration was 4 hours.

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