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Bullfrog plasma cysteine proteinase inhibitor (CPI)
Immunopharmacolooy Immunopharmacology32 (1996) 91-93
ELSEVIER
Bullfrog plasma cysteine proteinase inhibitor(CPI) Hiroshi Mashiko, Hidenobu Takahashi
*
Division of Chemistry of Hygiene, Meiji College of Pharmacy, 1-35-23 Nozawa, Setagaya-ku, Tokyo 154. Japan
Abstract Papain is inhibited by bullfrog plasma. CPI was isolated from bullfrog plasma by two step column chromatography; Cm-papain agarose column chromatography followed by FPLC Mono Q column chromatography with addition of benzamidine. Isolated CPI gave a single band on SDS-PAGE under reducing condition, and the molecular weight of reduced CPI was estimated to be over 200 kDa. When the isolated CPI was stored at 4°C in the absence of benzamidine, the CPI was cleaved and yielded a protein consisting of heavy chain (60 kDa) and light chain (54 kDa). Both chains were linked with disulfide bond(s). The cleaved form of CPI was also isolated from the plasma in the absence of benzamidine. Intact and cleaved form of CPI inhibited papain and ficin, but not trypsin. Kevwords: Bullfrog; Cysteine proteinase inhibitor; Kininogen; Plasma
1. Introduction CPIs are widely distributed in mammalian tissues and body fluids. These inhibitors are classified into the cystatin superfamily. The major mammalian plasma CPIs belonging to the superfamily are kininogens. Several mammalian plasma kininogens have been purified and characterized as CPI (Sueyoshi et al., 1985; Higashiyama et al., 1986; Yamamoto, 1987; Mashiko and Takahashi, 1992). However, little is known about non-mammalian plasma kininogen. Recently, CPIs have been purified
Abbreviations: AMC, 7-amino-4-methylcoumarin;Boc, Tertbutyloxycarbonyl;Cm-, S-carboxymethylated-;CPI, Cysteine proteinase inhibitor: HK, High molecular weight kininogen; ICso, 50% inhibitory concentration; LK, Low molecular weight kininogen; MCA, 4-methylcoumaryl-7-amide;SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Z, Benzyloxycarbonyl Corresponding author.
from avian plasma (Kimura et al., 1987) and reptilian plasmas (Chudzinski et al., 1989; Araujo et al., 1992). Among these CPIs, avian plasma CPI was also a kininogen while reptilian plasma CPIs were kininogen-like proteins. As a pan of this comparative study of plasma CPIs, we investigated amphibian CPI.
2. Materials and methods Bullfrog blood was collected from heart with a polyethylene syringe containing an anticoagulant solution. Plasma was obtained by centrifugation at room temperature. Sources of other materials were: Cm-papain agarose from Takara Shuzo Co., Ltd., Japan; Mono Q column t¥om Pharmacia LKB Biotechnology, Japan; benzamidine-HCl from Tokyo Kasei Kogyo, Japan; kit of molecular weight markers (MW-SDS-200) from Sigma Chemical Co., USA; Z-Phe-Arg-MCA, Boc-Gln-Ala-Arg-MCA and AMC
0162-3109/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 I 62-3 109(95)00()60-7
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11. Mashiko, H. Takahashi / lmmunopharmacology 32 (1996) 91-93
from Peptide Institute Inc., Japan; silver staining kit from Kanto Chemical Co., Inc., Japan, TPCK-trypsin from Worthington Biochemical Corp., USA and papain and ficin from Boehringer Mannheim Yamanouchi, Japan. Inhibition of papain and ficin was determined by our method (Mashiko et al., 1995), using 5 ng of enzymes. Inhibition of trypsin was determined by our method (Mashiko and Takahashi, 1994), using 0.5 ng enzyme and Boc-Gln-Ala-Arg-MCA (Kawabata et al., 1988) as a substrate. Column chromatographies were performed by the modified method of Kimura et al. (1987). SDS-PAGE was performed according to the method of Laemmli (1970). Proteins were stained with silver staining kit.
3. Results and discussion
Bullfrog plasma inhibited papain activity with an IC50 value of 14.0 nl/ml. This result indicates that bullfrog plasma contains CPI. CPI was isolated by two step column chromatography in the presence of benzamidine. Plasma CPI was adsorbed to Cm-papain agarose column and eluted mainly by 6M guanidineHCI. The affinity of bullfrog plasma CPI to Cmpapain agarose was higher than the affinity of mammalian plasma CPI (LK) to Cm-papain (Mashiko and Takahashi, 1992). Plasma CPI was adsorbed to FPLC Mono Q column, and eluted by 0.15M-0.20M NaC1 concentration. The isolated CPI gave a single band on SDSPAGE under reducing condition, and its molecular weight of reduced CPI was estimated to be over 200 kDa. Isolated CPI showed a heterogeneity under non-reducing conditions similar to mammalian plasma CPIs (HKs) (Higashiyama et al., 1986; Yamamoto, 1987; Mashiko and Takahashi, 1992; Mashiko et al., 1993), specifically, a major band with higher molecular weight and minor band with lower molecular weight were detected. However, their molecular weights were estimated to be over 200 kDa which is much greater than those of mammalian CPIs (HKs). When the isolated CPI was stored at 4°C in the absence of benzamidine, the stored CPI gave two bands on SDS-PAGE under non-reducing conditions, the molecular weights being the same as the non-stored CPI. However, the
band with higher molecular weight became minor, and the band with lower molecular weight became major. Stored CPI also gave two bands on SDSPAGE under reducing conditions. Their molecular weights were 60 kDa (heavy chain) and 54 kDa (light chain), respectively. Therefore, both chains appear to be linked by disulfide bond(s). These results are in good agreement with mammalian HK (Higashiyama et al., 1986; Yamamoto, 1987). The cleaved form of CPI was also isolated by the same method as described above from the plasma in the absence of benzamidine. Intact CPI inhibited papain and ficin activities in a dose dependent manner, with their IC50 values being 0.5 ~ g / m l and 0.62 /~g/ml, respectively. The cleaved form of CPI also inhibited papain and ficin activities, with ICs0 values almost the same as those of intact CPI. The intact and cleaved form of CPI did not inhibit trypsin activity. From these results, it became clear that isolated CPI is a specific proteinase inhibitor. Recently, it was reported that incubation of heat-denatured bullfrog plasma with either glass beads, porcine pancreatic kallikrein or trypsin did not generate bradykinin-like immunoreactivity (Conlon and Yano, 1995). Now, we report the isolation of CPI with high molecular weight from bullfrog plasma. Further work will be needed to determine whether or not this CPI is a precursor to the kinins.
References Araujo, M.S., Andreotti R., Chudzinski A.M., Sampio, C.A.M., Sampio M.U. Caiman kininogen-like cysteine proteinase inhibitor. Agents Actions Suppl. 1992; 38: 322-330. Chudzinski, A.M., Sampio M.U., Oliva M.L.V., Sampio C.A.M. A Bothrops jararaca plasma cysteine-proteinase inhibitor related to mammalian kininogen. Braz. J. Med. Biol. Res. 1989; 22: 945-948. Conlon, J.M., Yano K. Kallikrein generates angiotensin II but not bradykinin in the plasma of the urodele, Amphiuma tridactvlure. Comp. Biochem. Physiol. 1995; 110C: 305-311. Higashiyama S., Ohkubo I., Ishiguro H., Kunimatsu M., Sawaki K., Sasaki M. Human high molecular weight kininogen as a thiol proteinase inhibitor: Presence of the entire inhibition capacity in the native form of heavy chain. Biochemistry 1986; 25: 1669-1675. Kawabata, S., Miura T., Morita T., Kato H., Fujikawa K., lwanaga S., Takada K., Kimura T., Sakakibara S. Highly sensitive peptide 4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin. Eur. J. Biochem. 1988; 172: 17-25.
H. Mashiko, H. Takahashi / Immunopharmacology 32 (1996) 91-93 Kimura, M., Sueyoshi T., Takada K., Tanaka K., Morita T., lwanaga S. Isolation and characterization of ornitho-kininogen. Eur. J. Biochem. 1987; 168: 493-501. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685. Mashiko H., Takahashi H. Purification and some properties of kininogens in canine plasma. Agents Actions Suppl. 1992; 38: 249-256. Mashiko H., Miyamoto K., Takahashi H. Improved method for purification of porcine high molecular weight kininogen and cleavage of the kininogen by the action of porcine plasma kallikrein. Kinin '93 BRAZIL, Guaruja, Brazil 1993; pp. 50. Mashiko H., Takahashi H. Factor XI: Purification from porcine plasma by affinity chromatography and some properties of
93
factor XI and activated factor X1. Biol. Chem. Hoppe-Seyler 1994; 375: 481-484. Mashiko H., Tachibana T., Chou E., Takahashi H. Distribution of cysteine proteinase inhibitor in snake venoms and isolation of the inhibitor from Bitis arietans venom, l l5th of Annual Meeting of Pharmaceutical Society of Japan 1995: 3: pp. 125. Sueyoshi T., Enjyoji K., Shimada T., Kato H., lwanaga S., Bando Y., Kominami E., Katunuma N. A new function of kininogens as thiol proteinase inhibitors: inhibition of papain and cathepsins B, H and L by bovine, rat and human plasma kininogens. FEBS Lett. 1985; 182: 193-195. Yamamoto T. Characterization of guinea-pig high-molecularweight kininogen as multi-functional molecule. Biochim. Biophys. Acta 1987; 914: 259-274.
ELSEVIER
Bullfrog plasma cysteine proteinase inhibitor(CPI) Hiroshi Mashiko, Hidenobu Takahashi
*
Division of Chemistry of Hygiene, Meiji College of Pharmacy, 1-35-23 Nozawa, Setagaya-ku, Tokyo 154. Japan
Abstract Papain is inhibited by bullfrog plasma. CPI was isolated from bullfrog plasma by two step column chromatography; Cm-papain agarose column chromatography followed by FPLC Mono Q column chromatography with addition of benzamidine. Isolated CPI gave a single band on SDS-PAGE under reducing condition, and the molecular weight of reduced CPI was estimated to be over 200 kDa. When the isolated CPI was stored at 4°C in the absence of benzamidine, the CPI was cleaved and yielded a protein consisting of heavy chain (60 kDa) and light chain (54 kDa). Both chains were linked with disulfide bond(s). The cleaved form of CPI was also isolated from the plasma in the absence of benzamidine. Intact and cleaved form of CPI inhibited papain and ficin, but not trypsin. Kevwords: Bullfrog; Cysteine proteinase inhibitor; Kininogen; Plasma
1. Introduction CPIs are widely distributed in mammalian tissues and body fluids. These inhibitors are classified into the cystatin superfamily. The major mammalian plasma CPIs belonging to the superfamily are kininogens. Several mammalian plasma kininogens have been purified and characterized as CPI (Sueyoshi et al., 1985; Higashiyama et al., 1986; Yamamoto, 1987; Mashiko and Takahashi, 1992). However, little is known about non-mammalian plasma kininogen. Recently, CPIs have been purified
Abbreviations: AMC, 7-amino-4-methylcoumarin;Boc, Tertbutyloxycarbonyl;Cm-, S-carboxymethylated-;CPI, Cysteine proteinase inhibitor: HK, High molecular weight kininogen; ICso, 50% inhibitory concentration; LK, Low molecular weight kininogen; MCA, 4-methylcoumaryl-7-amide;SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Z, Benzyloxycarbonyl Corresponding author.
from avian plasma (Kimura et al., 1987) and reptilian plasmas (Chudzinski et al., 1989; Araujo et al., 1992). Among these CPIs, avian plasma CPI was also a kininogen while reptilian plasma CPIs were kininogen-like proteins. As a pan of this comparative study of plasma CPIs, we investigated amphibian CPI.
2. Materials and methods Bullfrog blood was collected from heart with a polyethylene syringe containing an anticoagulant solution. Plasma was obtained by centrifugation at room temperature. Sources of other materials were: Cm-papain agarose from Takara Shuzo Co., Ltd., Japan; Mono Q column t¥om Pharmacia LKB Biotechnology, Japan; benzamidine-HCl from Tokyo Kasei Kogyo, Japan; kit of molecular weight markers (MW-SDS-200) from Sigma Chemical Co., USA; Z-Phe-Arg-MCA, Boc-Gln-Ala-Arg-MCA and AMC
0162-3109/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 I 62-3 109(95)00()60-7
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11. Mashiko, H. Takahashi / lmmunopharmacology 32 (1996) 91-93
from Peptide Institute Inc., Japan; silver staining kit from Kanto Chemical Co., Inc., Japan, TPCK-trypsin from Worthington Biochemical Corp., USA and papain and ficin from Boehringer Mannheim Yamanouchi, Japan. Inhibition of papain and ficin was determined by our method (Mashiko et al., 1995), using 5 ng of enzymes. Inhibition of trypsin was determined by our method (Mashiko and Takahashi, 1994), using 0.5 ng enzyme and Boc-Gln-Ala-Arg-MCA (Kawabata et al., 1988) as a substrate. Column chromatographies were performed by the modified method of Kimura et al. (1987). SDS-PAGE was performed according to the method of Laemmli (1970). Proteins were stained with silver staining kit.
3. Results and discussion
Bullfrog plasma inhibited papain activity with an IC50 value of 14.0 nl/ml. This result indicates that bullfrog plasma contains CPI. CPI was isolated by two step column chromatography in the presence of benzamidine. Plasma CPI was adsorbed to Cm-papain agarose column and eluted mainly by 6M guanidineHCI. The affinity of bullfrog plasma CPI to Cmpapain agarose was higher than the affinity of mammalian plasma CPI (LK) to Cm-papain (Mashiko and Takahashi, 1992). Plasma CPI was adsorbed to FPLC Mono Q column, and eluted by 0.15M-0.20M NaC1 concentration. The isolated CPI gave a single band on SDSPAGE under reducing condition, and its molecular weight of reduced CPI was estimated to be over 200 kDa. Isolated CPI showed a heterogeneity under non-reducing conditions similar to mammalian plasma CPIs (HKs) (Higashiyama et al., 1986; Yamamoto, 1987; Mashiko and Takahashi, 1992; Mashiko et al., 1993), specifically, a major band with higher molecular weight and minor band with lower molecular weight were detected. However, their molecular weights were estimated to be over 200 kDa which is much greater than those of mammalian CPIs (HKs). When the isolated CPI was stored at 4°C in the absence of benzamidine, the stored CPI gave two bands on SDS-PAGE under non-reducing conditions, the molecular weights being the same as the non-stored CPI. However, the
band with higher molecular weight became minor, and the band with lower molecular weight became major. Stored CPI also gave two bands on SDSPAGE under reducing conditions. Their molecular weights were 60 kDa (heavy chain) and 54 kDa (light chain), respectively. Therefore, both chains appear to be linked by disulfide bond(s). These results are in good agreement with mammalian HK (Higashiyama et al., 1986; Yamamoto, 1987). The cleaved form of CPI was also isolated by the same method as described above from the plasma in the absence of benzamidine. Intact CPI inhibited papain and ficin activities in a dose dependent manner, with their IC50 values being 0.5 ~ g / m l and 0.62 /~g/ml, respectively. The cleaved form of CPI also inhibited papain and ficin activities, with ICs0 values almost the same as those of intact CPI. The intact and cleaved form of CPI did not inhibit trypsin activity. From these results, it became clear that isolated CPI is a specific proteinase inhibitor. Recently, it was reported that incubation of heat-denatured bullfrog plasma with either glass beads, porcine pancreatic kallikrein or trypsin did not generate bradykinin-like immunoreactivity (Conlon and Yano, 1995). Now, we report the isolation of CPI with high molecular weight from bullfrog plasma. Further work will be needed to determine whether or not this CPI is a precursor to the kinins.
References Araujo, M.S., Andreotti R., Chudzinski A.M., Sampio, C.A.M., Sampio M.U. Caiman kininogen-like cysteine proteinase inhibitor. Agents Actions Suppl. 1992; 38: 322-330. Chudzinski, A.M., Sampio M.U., Oliva M.L.V., Sampio C.A.M. A Bothrops jararaca plasma cysteine-proteinase inhibitor related to mammalian kininogen. Braz. J. Med. Biol. Res. 1989; 22: 945-948. Conlon, J.M., Yano K. Kallikrein generates angiotensin II but not bradykinin in the plasma of the urodele, Amphiuma tridactvlure. Comp. Biochem. Physiol. 1995; 110C: 305-311. Higashiyama S., Ohkubo I., Ishiguro H., Kunimatsu M., Sawaki K., Sasaki M. Human high molecular weight kininogen as a thiol proteinase inhibitor: Presence of the entire inhibition capacity in the native form of heavy chain. Biochemistry 1986; 25: 1669-1675. Kawabata, S., Miura T., Morita T., Kato H., Fujikawa K., lwanaga S., Takada K., Kimura T., Sakakibara S. Highly sensitive peptide 4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin. Eur. J. Biochem. 1988; 172: 17-25.
H. Mashiko, H. Takahashi / Immunopharmacology 32 (1996) 91-93 Kimura, M., Sueyoshi T., Takada K., Tanaka K., Morita T., lwanaga S. Isolation and characterization of ornitho-kininogen. Eur. J. Biochem. 1987; 168: 493-501. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685. Mashiko H., Takahashi H. Purification and some properties of kininogens in canine plasma. Agents Actions Suppl. 1992; 38: 249-256. Mashiko H., Miyamoto K., Takahashi H. Improved method for purification of porcine high molecular weight kininogen and cleavage of the kininogen by the action of porcine plasma kallikrein. Kinin '93 BRAZIL, Guaruja, Brazil 1993; pp. 50. Mashiko H., Takahashi H. Factor XI: Purification from porcine plasma by affinity chromatography and some properties of
93
factor XI and activated factor X1. Biol. Chem. Hoppe-Seyler 1994; 375: 481-484. Mashiko H., Tachibana T., Chou E., Takahashi H. Distribution of cysteine proteinase inhibitor in snake venoms and isolation of the inhibitor from Bitis arietans venom, l l5th of Annual Meeting of Pharmaceutical Society of Japan 1995: 3: pp. 125. Sueyoshi T., Enjyoji K., Shimada T., Kato H., lwanaga S., Bando Y., Kominami E., Katunuma N. A new function of kininogens as thiol proteinase inhibitors: inhibition of papain and cathepsins B, H and L by bovine, rat and human plasma kininogens. FEBS Lett. 1985; 182: 193-195. Yamamoto T. Characterization of guinea-pig high-molecularweight kininogen as multi-functional molecule. Biochim. Biophys. Acta 1987; 914: 259-274.