Expression of human recombinant 72 kDa gelatinase and tissue inhibitor of metalloproteinase-2 (TIMP-2): characterization of complex and free enzyme.
The human 72 kDa gelatinase/type IV collagenase is a metalloproteinase that is thought to play a role in metastasis and angiogenesis. The 72 kDa progelatinase can be isolated from conditioned media as a complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2). To investigate 72 kDa gelatinase-TIMP-2 interactions and to compare the activity of the complex versus that of the free enzyme, we have expressed and purified human 72 kDa progelatinase and TIMP-2 as single proteins in a recombinant vaccinia virus mammalian cell expression system. The recombinant 72 kDa progelatinase was able to bind TIMP-2, and it digested gelatin and collagen type IV after activation by p-aminophenylmercuric acid (APMA). The specific activity of the recombinant free enzyme was 20-fold higher than the activity of an APMA-treated stoichiometric complex of recombinant 72 kDa progelatinase and TIMP-2. Also, TIMP-2 caused an 86% inhibition of activity when added to the activated enzyme at a 1:1 molar ratio. Activation of the free recombinant 72 kDa progelatinase yielded the 62 kDa species and two fragments of 46 and 35 kDa that cross-reacted with monoclonal antibodies to the 72 kDa proenzyme. TIMP-2 inhibited the conversion of the recombinant proenzyme to the 62 kDa species and the appearance of the 45 and 35 kDa bands. These results suggest that TIMP-2 is not only a potent inhibitor of the activated enzyme but also prevents the generation of low-molecular-mass species and full enzymic activity from the zymogen.
Molecular Oncology Inc., Gaithersburg, MD 20878.
R
R
Fridman
R E
RE
Bird
M
M
Hoyhtya
M
M
Oelkuct
D
D
Komarek
C M
CM
Liang
M L
ML
Berman
L A
LA
Liotta
W G
WG
Stetler-Stevenson
T R
TR
Fuerst
D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010434:Pepsin A / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019716:Tissue Inhibitor of Metalloproteinase-2
D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D010434:Pepsin A; D018093:Gelatinases