Activity of an enzyme converting single-chain tissue-type plasminogen activator to the two-chain form in preovulatory human follicular fluid.
The biological role of the tissue-type plasminogen activator (tPA)/plasmin system has long been implicated in ovarian function. We have recently shown that the follicular fluid of human ovaries contains an alpha(2)-macroglobulin/protease complex capable of converting single-chain (sc) tPA to the two-chain (tc) enzyme tPA, suggesting the occurrence of its corresponding enzyme in a free form in the fluid. The aim of the current study is therefore to gain further information about the putative sctPA-converting enzyme present in follicular fluid. Incubation of human recombinant sctPA with the fluid brought about the production of tctPA. It was also demonstrated that tctPA production resulted in the activation of endogenous fluid plasminogen. Production of tctPA and plasmin both was strongly inhibited by aprotinin, suggesting that the enzyme is a serine protease. The sctPA-converting enzyme was partially purified from the fluid by column chromatographies. The enzyme preferably hydrolyzed synthetic peptide substrates containing arginine at the P(1) position. The enzyme preparation had a protease inhibitor profile similar to that observed with the crude fluid sample. These results clearly demonstrated that follicular fluid contains an enzyme capable of efficiently converting sctPA to tctPA. Discovery of this sctPA-converting enzyme strongly suggests that the tPA/plasmin system in the preovulatory follicle of human ovaries is operated through the proteolytic conversion of sctPA to tctPA rather than being regulated by a fibrin-dependent mechanism.
Ohnishi
Junji
J
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
Yokota
Jyunko
J
Rajapakse
R G S C
RG
Ohnishi
Eriko
E
Kudo
Takayuki
T
Wada
Shin-Ichiro
S
Takahashi
Takayuki
T
D005260:Female; D005341:Fibrinolysin / Q000378:metabolism; D015571:Follicular Fluid / Q000201:enzymology*; D005498:Follicular Phase; D006801:Humans; D066298:In Vitro Techniques; D009842:Oligopeptides; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism*
D009842:Oligopeptides; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin