Native polyacrylamide gel electrophoresis of membrane proteins: Glutaminase detection afterin situ specific activity staining

1993 ◽  
Vol 14 (1) ◽  
pp. 88-93 ◽  
Author(s):  
Juan C. Aledo ◽  
Simón Gómez-Biedma ◽  
Juan A. Segura ◽  
Manuel Molina ◽  
Ignacio Núñez de Castro ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Activity Staining ◽  
Native Polyacrylamide Gel Electrophoresis

scholarly journals Isolation of a human hepatic 60 kDa aspartylglucosaminidase consisting of three non-identical polypeptides

1989 ◽  
Vol 262 (1) ◽  
pp. 189-194 ◽  
Author(s):  
M Baumann ◽  
L Peltonen ◽  
P Aula ◽  
N Kalkkinen
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Inherited Disease ◽  
Ph Optimum ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Molecular Masses ◽  
Polypeptide Chains

We have characterized the properties of human aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which is deficient in the human inherited disease aspartylglucosaminuria. The purification procedure from human liver included affinity chromatography, gel filtration, strong-anion- and strong-cation-exchange h.p.l.c., chromatofocusing and reverse-phase h.p.l.c. In a denaturing SDS/polyacrylamide-gel electrophoresis, the 6600-fold purified enzyme was shown to be composed of three non-identical inactive polypeptide chains of molecular masses 24, 18 and 17 kDa. In a native polyacrylamide-gel electrophoresis, these polypeptide chains ran as one active enzyme complex. As judged from the elution position of the native enzyme in a Biogel P-100 gel filtration, the approximate molecular mass of this complex was 60 kDa. The enzyme had a pI of 5.7, a pH optimum at 6, of 0.48 mM and a specific activity of 200,000 nkat for the substrate 2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-D-glucose. The enzyme showed a 57% loss of activity at 60 degrees C after 45 h but was practically inactive after incubation at 72 degrees C for a few minutes. The molecular structure, Km and specific activity as well as the thermostability of the enzyme described here are different from those reported previously for human aspartylglucosaminidase.

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.

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