Preparation and characterization of recombinant tissue inhibitor of metalloproteinase 4 (TIMP-4).
TIMP-4, a novel human tissue inhibitor of metalloproteinase, was identified and cloned (Greene, J., Wang, M., Raymond, L. A., Liu, Y. E., Rosen, C., and Shi, Y. E. (1996) J. Biol. Chem. 271, 30375-30380). In this report, the production and characterization of recombinant TIMP-4 (rTIMP4p) are described. rTIMP4p, expressed in baculovirus-infected insect cells, was purified to homogeneity by a combination of cation exchange, hydrophobic, and size-exclusion chromatographies. The purified protein migrated as a single 23-kDa band in SDS-polyacrylamide gel electrophoresis and in Western blot using a specific anti-TIMP-4 antibody. Inhibition of matrix metalloproteinase (MMP) activities by rTIMP4p was demonstrated in five MMPs. Enzymatic kinetic studies revealed IC50 values (concentration at 50% inhibition) of 19, 3, 45, 8, and 83 nM for MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, respectively. Purified rTIMP4p demonstrated a strong inhibitory effect on the invasion of human breast cancer cells across reconstituted basement membranes. Thus, TIMP-4 is a new enzymatic inhibitor in MMP-mediated extracellular matrix degradation and may have therapeutic potential in treating cancer malignant progression.
Liu
Y E
YE
Department of Pediatrics, Long Island Jewish Medical Center, Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11040, USA.
Wang
M
M
Greene
J
J
Su
J
J
Ullrich
S
S
Li
H
H
Sheng
S
S
Alexander
P
P
Sang
Q A
QA
Shi
Y E
YE
D001943:Breast Neoplasms / Q000473:pathology / Q000628:therapy; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D009361:Neoplasm Invasiveness; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D019714:Tissue Inhibitor of Metalloproteinases
D006023:Glycoproteins; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases