PMID- 9449028
OWN - NLM
STAT- MEDLINE
DCOM- 19980320
LR  - 20181113
IS  - 1355-008X (Print)
IS  - 1355-008X (Linking)
VI  - 7
IP  - 1
DP  - 1997 Aug
TI  - Insulin-like growth factor binding protein (IGFBP) substrate zymography. A new 
      tool to identify and characterize IGFBP-degrading proteinases.
PG  - 33-6
AB  - Insulin-like growth factor binding protein (IGFBP) degrading proteinase 
      activities have been described in biological fluids and conditioned media from 
      numerous cell lines. To identify and characterize IGFBP-degrading proteinases, 
      our laboratory has developed IGFBP substrate zymography. Herein, we illustrate 
      how IGFBP substrate zymography can be used both to identify candidate 
      IGFBP-degrading proteinases and characterize their degradative capabilities. For 
      this purpose, human matrix metalloproteinase-3 (MMP-3), a proteinase that 
      degrades IGFBP-3 in human fibroblast cultures, was first electrophoresed through 
      a polyacrylamide gel containing IGFBP-3 as substrate and then analyzed for its 
      ability to degrade the substrate into immunoreactive fragments that were absorbed 
      onto a polyvinylidene difluoride membrane. IGFBP-3 substrate zymography was 
      capable of detecting as little as 20 ng of human MMP-3, demonstrating a 
      sensitivity similar to casein substrate zymography. Using the zymogram as a 
      template, MMP-3 was identified in a standard SDS-polyacrylamide gel run in 
      parallel with the zymogram, and the corresponding area of the gel was excised. 
      Electroelution of the gel slice yielded active MMP-3 when examined by casein 
      substrate zymography. Furthermore, digestion of IGFBP-3 in solution by the 
      electroeluted MMP-3 revealed the same fragmentation pattern of the binding 
      protein as that produced by MMP-3, which had not been electroeluted. Together, 
      these studies demonstrate that IGFBP substrate zymography can be a useful tool 
      for both the identification and the characterization of IGFBP-degrading 
      proteinases.
FAU - Fowlkes, J L
AU  - Fowlkes JL
AD  - Department of Pediatrics, University of Kentucky College of Medicine, Lexington, 
      USA. jlfowlk@pop.uky.edu
FAU - Thrailkill, K M
AU  - Thrailkill KM
FAU - Serra, D M
AU  - Serra DM
FAU - Nagase, H
AU  - Nagase H
LA  - eng
GR  - AR39189/AR/NIAMS NIH HHS/United States
GR  - DK02776/DK/NIDDK NIH HHS/United States
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Endocrine
JT  - Endocrine
JID - 9434444
RN  - 0 (Caseins)
RN  - 0 (Insulin-Like Growth Factor Binding Protein 3)
RN  - 0 (Recombinant Proteins)
RN  - EC 3.4.24.17 (Matrix Metalloproteinase 3)
SB  - IM
MH  - Caseins/metabolism
MH  - Electrophoresis, Polyacrylamide Gel/*methods
MH  - Humans
MH  - Insulin-Like Growth Factor Binding Protein 3/*metabolism
MH  - Matrix Metalloproteinase 3/analysis/isolation & purification/metabolism
MH  - Recombinant Proteins/metabolism
MH  - Sensitivity and Specificity
MH  - Substrate Specificity
EDAT- 1997/08/01 00:00
MHDA- 1998/02/04 00:01
CRDT- 1997/08/01 00:00
PHST- 1997/08/01 00:00 [pubmed]
PHST- 1998/02/04 00:01 [medline]
PHST- 1997/08/01 00:00 [entrez]
AID - 10.1007/BF02778059 [doi]
PST - ppublish
SO  - Endocrine. 1997 Aug;7(1):33-6. doi: 10.1007/BF02778059.