Systematic separation and purification of elastase, gelatinase (matrix metalloproteinase 9), and collagenase (matrix metalloproteinase 8) from polymorphonuclear leukocytes in dialyzers previously used by patients with renal failure.
We developed a simple and effective method for the systematic separation and purification of human polymorphonuclear leukocyte (PMN) proteinases, elastase, gelatinase (matrix metalloproteinase 9, type IV collagenase), and collagenase (matrix metalloproteinase 8), derived from the extracts of hollow fiber dialyzers that had been utilized in the treatment of patients with renal failure. The fraction containing elastase was grossly separated from that containing gelatinase and collagenase by heparin-Sepharose chromatography and purified in an aprotinin column. The remaining two enzymes were then separated using the gelatin-Sepharose column after gel chromatography following ammonium sulfate precipitation. Gelatinase and collagenase were further purified by gelatin-Sepharose chromatography as a latent form and by collagen-Sepharose chromatography as an activated form. This novel method offers procedural advantages over existing methods that separate PMNs from the whole blood of volunteers for experimental research purposes.
Kobayashi
T
T
Department of Dermatology, Chiba University School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan. tkobayas@derma01.m.chiba-u.ac.jp
Nishikawa
T
T
Hattori
S
S
Yoshida
N
N
Takagi
T
T
Watanabe
H
H
Hori
H
H
Nagai
Y
Y
D000818:Animals; D007611:Aprotinin / Q000378:metabolism; D002457:Cell Extracts; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism; D003956:Dialysis / Q000295:instrumentation*; D004591:Electrophoresis, Polyacrylamide Gel; D005780:Gelatin / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007958:Leukocyte Count; D020784:Matrix Metalloproteinase 8 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D051437:Renal Insufficiency / Q000097:blood*
D002457:Cell Extracts; D005780:Gelatin; D006493:Heparin; D003094:Collagen; D007611:Aprotinin; D010196:Pancreatic Elastase; D020784:Matrix Metalloproteinase 8; D020780:Matrix Metalloproteinase 9