Isolation and characterization of a 70-kDa metalloprotease (gelatinase) that is elevated in Rous sarcoma virus-transformed chicken embryo fibroblasts.
Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSVCEF) secrete a 70-kDa metallo-gelatinase at elevated levels over that of normal CEF. The 70-kDa enzyme has been purified from RSVCEF conditioned medium and represents 1-3% of the total protein in the RSVCEF conditioned medium. A 22-kDa protein, which appears to be the avian form of the tissue inhibitor of metalloproteases (TIMP), is co-isolated in association with the 70-kDa enzyme and can be separated from the enzyme by gel filtration carried out under denaturing conditions. The isolated 70-kDa species is in the zymogen form. It can be activated by treatment with the organomercurial, p-aminophenylmercuric acetate (APMA), yielding a 62-kDa active species derived by an apparent autoproteolytic cleavage from the 70-kDa proenzyme as determined by both substrate gel analysis and immunoblots using a monospecific antibody to the 70-kDa proenzyme. The proenzyme is poorly activated by trypsin and not activated by plasmin. The APMA-activated enzyme rapidly degrades denatured collagens but under identical conditions is unable to degrade native collagens, including basement membrane type IV collagen. Only at very high enzyme to substrate ratios (1:2) will native type IV collagen be hydrolyzed. Partial N-terminal amino acid sequencing of both the 70-kDa proenzyme and the 62-kDa active enzyme indicates that the avian enzyme is a member of the matrix metalloprotease family (MMP-2). When CEF cultures, infected with a temperature sensitive mutant of RSV, conditional for the expression of the transforming src oncogene, were incubated at the permissive and nonpermissive temperatures, differential levels of the 70-kDa enzyme were produced in direct proportion to the functioning of the src oncogene.
Chen
J M
JM
Department of Anatomy, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Aimes
R T
RT
Ward
G R
GR
Youngleib
G L
GL
Quigley
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002642:Chick Embryo; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D018093:Gelatinases; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009857:Oncogenes; D010434:Pepsin A / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D010434:Pepsin A; D018093:Gelatinases