Purification of soluble and membrane-bound proteases with substrate-analogous inhibitors by affinity chromatography.
Specific modified substrate-analogous amino acids and peptides have been used as affinity ligands in the affinity chromatography of proteases. Alanine methyl ketone-Sepharose (AMK-Sepharose) is introduced as affinity support for the purification of a bacterial alanyl aminopeptidase (AAP) from a membrane protein extract and Arginine-Agarose as support for the preparation of a membrane-bound proteinase of myeloma cells (MP-1). Peptidyl methyl ketones as affinity ligands have been used to separate subtilisin enzymes and the cysteine proteases cathepsin B, L, and S. As a new type of ligands, spacer-bound peptidyl chloromethyl ketones are presented for a specific and oriented immobilization of proteinases. Oriented-immobilized cathepsin B was used to isolate antibodies against this enzyme.
Jahreis
G
G
Institute of Physiological Chemistry, Faculty of Medicine, Martin Luther University, Hollystrasse 1, D-06097 Halle (Saale), Germany.
Peters
K
K
Kirschke
H
H
D016954:Acinetobacter calcoaceticus / Q000201:enzymology; D000818:Animals; D002401:Cathepsin B / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D010450:Endopeptidases / Q000302:isolation & purification*; D006041:Goats; D006801:Humans; D007136:Immunoglobulins / Q000302:isolation & purification; D008565:Membrane Proteins / Q000302:isolation & purification*; D009842:Oligopeptides / Q000737:chemistry; D011480:Protease Inhibitors; D011485:Protein Binding; D013381:Subtilisins / Q000302:isolation & purification
D007136:Immunoglobulins; D008565:Membrane Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; D010450:Endopeptidases; D013381:Subtilisins; D002401:Cathepsin B