Characterization and partial purification of the insulin-like growth factor (IGF)-dependent IGF binding protein-4-specific protease from human fibroblast conditioned media.
Insulin-like growth factors (IGFs) are one of the most potent stimulators of cell growth. IGFs are modulated by six high affinity binding proteins (IGFBPs) which are, in turn, regulated through post-translational modifications such as proteolysis. In the conditioned media of human fibroblasts, IGFBP-4 is cleaved by an apparently novel IGFBP-4-specific protease that requires IGF for functional activity. We have used several biochemical manipulations, including size exclusion chromatography, native gel electrophoresis, chaotropic salt precipitation, hydrophobic interaction chromatography, ion exchange chromatography, isoelectric focusing, lectin affinity chromatography, and metal chelating affinity chromatography to both characterize and partially purify the IGF-dependent IGFBP-4 protease. Our results indicate that this protease is a highly glycosylated, Zn+2 binding metalloprotease with a native molecular weight greater than 200 kDa.
Lawrence
J B
JB
Mayo Clinic, Rochester, MN 55905, USA.
Bale
L K
LK
Haddad
T C
TC
Clarkson
J T
JT
Conover
C A
CA
D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011266:Pregnancy-Associated Plasma Protein-A; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D017077:Culture Media, Conditioned; D018974:Insulin-Like Growth Factor Binding Protein 4; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A; D015032:Zinc