Purification of L-Type Pyruvate Kinase from Human Liver by Affinity Chromatography on Blue-Dextran-Sepharose Column

Enzyme ◽  
1977 ◽  
Vol 22 (6) ◽  
pp. 407-411 ◽  
Author(s):  
Joelle Marie ◽  
Axel Kahn
Keyword(s):  
Affinity Chromatography ◽  
Pyruvate Kinase ◽  
Human Liver ◽  
Blue Dextran ◽  
Sepharose Column

Activation of latent collagenase from polymorphonuclear leukocytes by cathepsin G

1979 ◽  
Vol 44 (10) ◽  
pp. 3177-3182 ◽  
Author(s):  
Mária Stančíková ◽  
Karel Trnavský
Keyword(s):  
Affinity Chromatography ◽  
Trypsin Inhibitor ◽  
Polymorphonuclear Leukocytes ◽  
Soya Bean ◽  
Cathepsin G ◽  
Sepharose Column ◽  
Bean Trypsin Inhibitor ◽  
Human Polymorphonuclear Leukocytes

Cathepsin G was isolated from human polymorphonuclear leukocytes and purified by affinity chromatography on Antilysin-Sepharose column. Purified enzyme activated later collagenase isolated from leukocytes. Activation at 36°C was maximal after 30 min incubation. Inhibitors of cathepsin G - soya-bean trypsin inhibitor, diisopropyl phosphofluoridate and Antilysin were active in inhibiting the activation of latent collagenase by cathepsin G.

A three step purification procedure for human liver ferritin

1980 ◽  
Vol 58 (6) ◽  
pp. 494-498 ◽  
Author(s):  
M. Pagé ◽  
J. Lagueux ◽  
C. Gauthier
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Purification Procedure ◽  
Human Liver Ferritin ◽  
Normal Human Liver ◽  
Normal Human ◽  
Liver Ferritin

We describe a method for the purification of normal human liver ferritin by ultrafiltration, gel filtration on Sephacryl S-300, and affinity chromatography on DEAE-Affi Gel Blue. The purity of the ferritin obtained was verified by immunoelectrophoresis, Ouchterlony immunodiffusion, polyacrylamide gel electrophoresis, and electrofocusing. This rapid method yields 32% of the original ferritin.

scholarly journals Molecular Modelling of Human Red Blood Cell Pyruvate Kinase: Structural Implications of a Novel G1091 to A Mutation Causing Severe Nonspherocytic Hemolytic Anemia

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4987-4995 ◽  
Author(s):  
Wouter W. van Solinge ◽  
Rob J. Kraaijenhagen ◽  
Gert Rijksen ◽  
Richard van Wijk ◽  
Bjarne B. Stoffer ◽  
...  
Keyword(s):  
Blood Cell ◽  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Red Blood Cell ◽  
Molecular Modelling ◽  
Human Liver ◽  
The Other ◽  
Salt Bridges ◽  
Compound Heterozygotes ◽  
Allosteric Properties

Abstract We present a novel G1091 to A mutation in the human liver and red blood cell (RBC) pyruvate kinase (PK) gene causing severe hemolytic anemia. In two families, three children were severely PK-deficient compound heterozygotes exhibiting the G1091 to A mutation and a common G1529 to A mutation on the other allele. In one family, the mother, a G1091 to A heterozygote, later had a second baby with a new husband, also a G1091 to A carrier. The baby was homozygous for the G1091 to A mutation and died 6 weeks after birth from severe hemolysis. Both mutant alleles were expressed at the RNA level. The G1091 to A mutation results in the substitution of a conserved glycine by an aspartate in domain A of RBC PK, whereas the G1529 to A mutation leads to the substitution of a conserved arginine residue with glutamine in the C-domain. Molecular modelling of human RBC PK, based on the crystal structure of cat muscle PK, shows that both mutations are located outside the catalytic site at the interface of domains A and C. The mutations are likely to disrupt the critical conformation of the interface by introducing alternative salt bridges. In this way the Gly364 to Asp and Arg510 to Gln substitutions may cause PK deficiency by influencing the allosteric properties of the enzyme.

Dye affinity chromatography of ricin subunits

1986 ◽  
Vol 6 (12) ◽  
pp. 1035-1040 ◽  
Author(s):  
Simonetta Sperti ◽  
Lucio Montanaro ◽  
Fioretta Rambelli
Keyword(s):  
Affinity Chromatography ◽  
Simple Method ◽  
Blue Dextran ◽  
A Chain ◽  
Dye Affinity Chromatography ◽  
Blue Sepharose

A rapid and simple method for the isolation of the two chains of ricin is described. The method involves two chromatographic runs, the first on blue-Sepharose and the second on blue dextran-Sepharose. It is moreover shown that the A chain of ricin, but not the B chain, binds to poly(U)-Sepharose.

scholarly journals Characterization of human liver α-D-mannosidase purified by affinity chromatography

1976 ◽  
Vol 153 (3) ◽  
pp. 579-587 ◽  
Author(s):  
N C Phillips ◽  
D Robinson ◽  
B G Winchester
Keyword(s):  
Affinity Chromatography ◽  
Concanavalin A ◽  
Human Liver ◽  
Early Stage ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Lysosomal Storage ◽  
Sepharose 4B

Human liver acidic α-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral α-mannosidase did not bind to the concanavalin A-Sepharose so the two types of α-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was α-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that α-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of α-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human α-mannosidase. The purified enzyme completely removed the α-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic α-mannosidase.

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