Identification of porcine follipsin as plasma kallikrein, and its possible involvement in the production of bradykinin within the follicles of porcine ovaries.
To determine the identity of porcine follipsin, a plasma kallikrein cDNA clone was isolated from a porcine liver cDNA library. The clone encoded a protein of 643 amino acids, exhibiting identities 79.7, 72. 9, and 74.4% homologous 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 kallikrein 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 medium of these cells. No active enzyme could be obtained, but the expressed protein was reacted with anti-porcine plasma kallikrein antibody. The mRNA was 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 precursor form (Mr = 80,000), respectively. In contrast, the liver extract contained 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 porcine ovaries was found to contain this peptide hormone at a detectable level. These results indicate that porcine follipsin is plasma kallikrein, and that the enzyme may be involved in the production of bradykinin within ovarian follicles.
Kimura
A
A
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
Kihara
T
T
Okimura
H
H
Hamabata
T
T
Ohnishi
J
J
Moriyama
A
A
Takahashi
K
K
Takahashi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001920:Bradykinin / Q000096:biosynthesis*; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D015870:Gene Expression; D006801:Humans; D007610:Kallikreins / Q000097:blood / Q000145:classification / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D006080:Ovarian Follicle / Q000378:metabolism*; D012333:RNA, Messenger; D051381:Rats; D012697:Serine Endopeptidases / Q000235:genetics*; D013552:Swine; D014018:Tissue Distribution
D018076:DNA, Complementary; D012333:RNA, Messenger; D007610:Kallikreins; D012697:Serine Endopeptidases; C088241:follipsin; D001920:Bradykinin