Purification of progelatinases A and B by continuous-elution electrophoresis.
Progelatinases A and B were purified from HT1080-conditioned culture medium using a continuous-elution electrophoresis. Initially cell culture medium was ammonium sulfate precipitated. The concentrated proteins were affinity chromatographed on gelatin-Sepharose column. The bound gelatinases were eluted with electrophoresis sample buffer and subjected to continuous-elution electrophoresis. In a single run, under the standardized working conditions, the obtained fractions contained four purified enzymes--progelatinase A (M(r) 72,000), its activated forms (M(r) 62,000 and M(r) 59,000), and progelatinase B (M(r) 92,000). Moreover, the continuous-elution electrophoresis allowed the enzymes separation from their respective inhibitors--TIMP-1 (M(r) 28,500) and TIMP-2 (M(r) 21,000). The purified progelatinases A and B demonstrated high specific activities (150-200 U/micrograms).
Laboratory of Cellular Biology, University of Liège, Belgium.
A G
AG
Remacle
E N
EN
Baramova
U H
UH
Weidle
H W
HW
Krell
J M
JM
Foidart
D000645:Ammonium Sulfate; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D018093:Gelatinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D019716:Tissue Inhibitor of Metalloproteinase-2; D019714:Tissue Inhibitor of Metalloproteinases; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D004792:Enzyme Precursors; D006023:Glycoproteins; D011480:Protease Inhibitors; D011506:Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase; D000645:Ammonium Sulfate