Abstract
Trimeresurus flavoviridis (Habu snake) venom contains phospholipase A2 (PLA2) isozymes which are rich in disulfide bond and show diverse activities in spite of their highly homologous structures. When reduced form of Tyr(NO2)-67-PLA2, which is a mimic of Asp-49-PLA2 mutant, was oxidized at pH 8.0 in the presence of 5 mM L-cysteine and 5 mM Ca2+, native form of Tyr(NO2)-67-PLA2 was produced as being judged from both activity regeneration and adequate HPLC profile. Reduced forms of two Lys-49-PLA2 isozymes with extremely low lipolytic activity, which are called basic proteins I and II, also generated native forms of proteins when oxidized under the same conditions as above. Protein disulfide isomerase accelerated proper folding of reduced Tyr(NO2)-67-PLA2 at an initial phase of oxidation and was effective for rearrangement of incorrectly paired disulfide bonds of Asp-49-PLA2 to native construction. However, this isomerase failed to convert reduced form of partially active L-fragment, Asp-49-PLA2 lacking N-terminal octapeptide, to the native form, indicating that the entire sequence of protein in a nascent state is primarily required for proper folding. The present data together with the fact that reduced Asp-49-PLA2 folded properly (S. Tanaka et al. J. Biochem., 96, 1443 (1984)) afforded a strong basis for the structure and function study of T. flavoviridis PLA2S by means of in vitro mutagenesis technique.
| Original language | English |
|---|---|
| Pages (from-to) | 2655-2659 |
| Number of pages | 5 |
| Journal | Bulletin of the Chemical Society of Japan |
| Volume | 65 |
| Issue number | 10 |
| DOIs | |
| Publication status | Published - Oct 1 1992 |
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All Science Journal Classification (ASJC) codes
- Chemistry(all)
Cite this
Refolding of trimeresurus flavoviridis phospholipases A2 . / Tsuno, Shinji; Ogawa, Tomohisa; Nakashima, Kinichi; Oda, Naoko; Lee, Sannumu; Shimohigashi, Yasuyuki; Aoyagi, Haruhiko; Ohno, Motonori.
In: Bulletin of the Chemical Society of Japan, Vol. 65, No. 10, 01.10.1992, p. 2655-2659.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Refolding of trimeresurus flavoviridis phospholipases A2
AU - Tsuno, Shinji
AU - Ogawa, Tomohisa
AU - Nakashima, Kinichi
AU - Oda, Naoko
AU - Lee, Sannumu
AU - Shimohigashi, Yasuyuki
AU - Aoyagi, Haruhiko
AU - Ohno, Motonori
PY - 1992/10/1
Y1 - 1992/10/1
N2 - Trimeresurus flavoviridis (Habu snake) venom contains phospholipase A2 (PLA2) isozymes which are rich in disulfide bond and show diverse activities in spite of their highly homologous structures. When reduced form of Tyr(NO2)-67-PLA2, which is a mimic of Asp-49-PLA2 mutant, was oxidized at pH 8.0 in the presence of 5 mM L-cysteine and 5 mM Ca2+, native form of Tyr(NO2)-67-PLA2 was produced as being judged from both activity regeneration and adequate HPLC profile. Reduced forms of two Lys-49-PLA2 isozymes with extremely low lipolytic activity, which are called basic proteins I and II, also generated native forms of proteins when oxidized under the same conditions as above. Protein disulfide isomerase accelerated proper folding of reduced Tyr(NO2)-67-PLA2 at an initial phase of oxidation and was effective for rearrangement of incorrectly paired disulfide bonds of Asp-49-PLA2 to native construction. However, this isomerase failed to convert reduced form of partially active L-fragment, Asp-49-PLA2 lacking N-terminal octapeptide, to the native form, indicating that the entire sequence of protein in a nascent state is primarily required for proper folding. The present data together with the fact that reduced Asp-49-PLA2 folded properly (S. Tanaka et al. J. Biochem., 96, 1443 (1984)) afforded a strong basis for the structure and function study of T. flavoviridis PLA2S by means of in vitro mutagenesis technique.
AB - Trimeresurus flavoviridis (Habu snake) venom contains phospholipase A2 (PLA2) isozymes which are rich in disulfide bond and show diverse activities in spite of their highly homologous structures. When reduced form of Tyr(NO2)-67-PLA2, which is a mimic of Asp-49-PLA2 mutant, was oxidized at pH 8.0 in the presence of 5 mM L-cysteine and 5 mM Ca2+, native form of Tyr(NO2)-67-PLA2 was produced as being judged from both activity regeneration and adequate HPLC profile. Reduced forms of two Lys-49-PLA2 isozymes with extremely low lipolytic activity, which are called basic proteins I and II, also generated native forms of proteins when oxidized under the same conditions as above. Protein disulfide isomerase accelerated proper folding of reduced Tyr(NO2)-67-PLA2 at an initial phase of oxidation and was effective for rearrangement of incorrectly paired disulfide bonds of Asp-49-PLA2 to native construction. However, this isomerase failed to convert reduced form of partially active L-fragment, Asp-49-PLA2 lacking N-terminal octapeptide, to the native form, indicating that the entire sequence of protein in a nascent state is primarily required for proper folding. The present data together with the fact that reduced Asp-49-PLA2 folded properly (S. Tanaka et al. J. Biochem., 96, 1443 (1984)) afforded a strong basis for the structure and function study of T. flavoviridis PLA2S by means of in vitro mutagenesis technique.
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U2 - 10.1246/bcsj.65.2655
DO - 10.1246/bcsj.65.2655
M3 - Article
VL - 65
SP - 2655
EP - 2659
JO - Bulletin of the Chemical Society of Japan
JF - Bulletin of the Chemical Society of Japan
SN - 0009-2673
IS - 10
ER -