Identification of porcine follipsin as plasma kallikrein, and its possibleinvolvement in the production of bradykinin within the follicles of porcine ovaries

To determine the identity of porcine follipsin, a plasma kallikrein cDNA cl one was isolated from a porcine liver cDNA library. The clone encoded a pro tein of 643 amino acids, exhibiting identities 79.7, 72.9, and 74.4% homolo gous to human, rat, and mouse plasma prekallikrein, respectively. The amino acid sequences of four internal peptides isolated from the tryptic digest of follipsin were all found in the deduced sequence. Authentic plasma kalli krein was purified from porcine plasma and compared directly with follipsin . Actions on synthetic substrates and behaviors with proteinase inhibitors were indistinguishable between these two enzymes. The cDNA was expressed in COS-7 cells and the recombinant protein was prepared from the culture medi um of these cells. No active enzyme could be obtained, but the expressed pr otein was reacted with anti-porcine plasma kallikrein antibody. The mRNA wa s detected only in the liver in northern blot analysis. RT-PCR analysis of RNAs revealed that porcine testis, in addition to the liver, expressed the corresponding mRNA. In the ovary, plasma kallikrein was detected as a main band of the active form (Mr=85,000) and the band of the minor inactive prec ursor form (Mr=80,000), respectively. In contrast, the liver extract contai ned only the precursor form, incubation of high molecular weight kininogen with follicular fluid plasma kallikrein resulted in an increased production of bradykinin. Further, the fresh fluid of large-sized follicles of porcin e ovaries was found to contain this peptide hormone at a detectable level. These results indicate that porcine follipsin is plasma kallikrein, and tha t the enzyme may be involved in the production of bradykinin within ovarian follicles. (C) 2000 Wiley-Liss, Inc.