Recombinant rat protein C: comparative studies of structure, function and synthesis with plasma protein C.
In order to elucidate the role of protein C (PC) in the rat, we expressed, purified, and characterized recombinant rat PC. The purified recombinant rat PC was 70-90% two-chain (41 kDa heavy chain; 22 and 23 kDa light chain) and 10-30% single-chain (61 kDa). Amino acid analysis confirmed the presence of 10 moles of gamma-carboxyglutamic acid residues per mol of protein. For comparison, plasma rat PC was purified from a barium citrate precipitate using similar method. Plasma rat PC was a two-chain form (41 kDa heavy chain; 22 kDa light chain) with no detectable single-chain nor 23 kDa light chain. For determination of the in vitro secreted species, primary cultured rat hepatocytes were incubated for 6 h with methionine-free MEM containing vitamin K1, aprotinin, and [35S]methionine. The supernatant was immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. Approximately 90% of the PC radioactivity migrated as a two-chain molecule. These results indicate that rat PC is secreted mainly as a two-chain molecule from the liver. PROTAC-activated forms of recombinant rat PC, plasma rat PC, and plasma human PC hydrolyzed the S-2366 chromogenic substrate at the same rate. Recombinant rat PC was also activated by the thrombin-thrombomodulin complex at a rate similar to plasma rat PC. The anticoagulant activities of the three activated PCs were examined in rat plasma. Both recombinant and plasma rat PC prolonged the activated partial thromboplastin time in a dose-dependent manner, but plasma human PC was less effective.(ABSTRACT TRUNCATED AT 250 WORDS)
Ono
M
M
Molecular Biology Research Laboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan.
Fujiwara
H
H
Okafuji
T
T
Enjoh
T
T
Nawa
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D008099:Liver / Q000166:cytology; D008297:Male; D008969:Molecular Sequence Data; D011486:Protein C / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry*
D000596:Amino Acids; D011486:Protein C; D011993:Recombinant Fusion Proteins