Premium
Identification of matrix metalloendoproteinase inhibitor (TIMP) in human parotid and submandibular saliva: partial purification and characterization
Author(s) -
Drouin Louis,
Overall Christopher M.,
Sodek Jaro
Publication year - 1988
Publication title -
journal of periodontal research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.31
H-Index - 83
eISSN - 1600-0765
pISSN - 0022-3484
DOI - 10.1111/j.1600-0765.1988.tb01615.x
Subject(s) - saliva , chemistry , collagenase , ammonium sulfate precipitation , size exclusion chromatography , sepharose , chromatography , biochemistry , microbiology and biotechnology , enzyme , biology
Matrix metalloendoproteinase inhibitor (TIMP) is constitutively expressed by a variety of cells and is present in most connective tissues, and in serum and amniotic fluid. In our previous studies, collagenase inhibitor activity has been identified in saliva (5). To determine the nature of this inhibitor, fresh human parotid saliva was first concentrated by ammonium sulfate precipitation and the 20–60% saturated ammonium sulfate fraction containing the inhibitor activity was chromatographed on an AcA 54 gel filtration column. The inhibitor eluted with an apparent Mr of 29000 and a 42‐fold purification was achieved. For characterization, the inhibitor was further purified by heparin‐Sepharose chromatography. The inhibitor, which bound to heparin‐Sepharose in 0.0 M NaCl at neutral pH and remained bound after a 0.18 M NaCl step elution, was eluted with 0.25 M NaCl. The partially purified inhibitor was characterized by its stability at low pH (pH 2.0) and after heat treatment (60° C and 95° C), and by its resistance to organomercurial (APMA) and trypsin treatments. These properties, together with immunoreactivity with an anti‐human TIMP polyclonal antibody on immunoblots. are consistent with the collagenase inhibitor activity being TIMP. Collagenase in either the active or precursor forms was not delected in parotid or submandibular ductal saliva nor after AcA 54 chromatography of parotid ductal saliva. The presence of TIMP in saliva has important implications. First, it has the potential to suppress the activity of matrix metalloendoproteinases released during periodontal inflammation; and second, in the analysis of collagenolytic enzymes in gingival crevicular fluid, it is clear that care must be taken to avoid contamination of the crevicular fluid with saliva.