Purification and characterization of the plasminogen activator inhibitors PAI-1, PAI-2, and PN-1 from the human glioblastoma U138.
This investigation presents data which indicate that the plasminogen activator inhibitor (PAI) activity secreted from U138 cells is composed of three separate PAIs: PAI-1, PAI-2, and PN-1. It was demonstrated that the U138 PAI-1-like protein had an apparent molecular mass of 50 kilodaltons (kDa) and was purified to apparent homogeneity by elution from an anti-PAI-1 immunoaffinity column. These fractions were also reactive with a second anti-PAI-1 monoclonal antibody using immunoblotting techniques. Northern blot analysis of RNA isolated from unstimulated U138 cells demonstrated positive hybridization with the cDNA specific for human PAI-1. The U138 PAI-2-like protein was adherent to an anti-PAI-2 immunoaffinity column and was demonstrated to be nonadherent to concanavalin A-agarose, heparin-Sepharose, and the anti-PAI-1 immunoaffinity column. The eluted U138 PAI-2-like protein was demonstrated to have an apparent molecular mass of 60 kDa and was also reactive with a second anti-PAI-2 monoclonal antibody using immunoblotting techniques. Further, the cDNA specific for PAI-2 was demonstrated to hybridize to a 2.5-kilobase message from RNA isolated from U138 cells. A third PAI was detected that was nonadherent to concanavalin A-agarose and both of the anti-PAI columns. This 50-kDa PAI was adherent to heparin-Sepharose and thrombin-agarose columns, and was not reactive with any antibodies for either PAI-1 or PAI-2. Northern blot analysis of U138 RNA demonstrated positive hybridization with an oligodeoxynucleotide specific for PN-1. This investigation demonstrates with biochemical, immunological, and molecular data that the U138 glioblastoma constitutively produces three PAIs.(ABSTRACT TRUNCATED AT 250 WORDS)
Murphy
P G
PG
Joint Injury and Arthritis Research Group, University of Calgary Health Sciences Centre, AB, Canada.
Lenz
S P
SP
Dobson
M
M
Arndt
A D
AD
Hart
D A
DA
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D015342:DNA Probes; D005909:Glioblastoma / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000302:isolation & purification*; D017396:Plasminogen Activator Inhibitor 2 / Q000235:genetics / Q000302:isolation & purification*; D012313:RNA / Q000032:analysis; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D015342:DNA Probes; D017395:Plasminogen Activator Inhibitor 1; D017396:Plasminogen Activator Inhibitor 2; D012313:RNA