TY - JOUR
T1 - Purification and characterization of a Co(II)-sensitive α-mannosidase from Ginkgo biloba seeds
AU - Woo, Kwan Kit
AU - Miyazaki, Mitsuhiro
AU - Hara, Shintaro
AU - Kimura, Mariko
AU - Kimura, Yoshinobu
N1 - Funding Information:
* This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Basic Research (B), no. 11556062, 1999–2002). y To whom correspondence should be addressed. Fax: +81-86-251-8388; E-mail: yosh8mar@cc.okayama-u.ac.jp Abbreviations: MES, 2-morpholinoethanesulfonic acid; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl] propanesulfonic acid; 1-dMM, 1-deoxymannojirymycin; PA, pyridylamino; M5A, Manα1–6(Manα1–3)Manα1–6(Manα1–3)Manβ1–4GlcNAcβ1–4GlcNAc–PA; M5A0, Manα1– 6(Manα1–3)Manα1–6(Manα1–3)Manβ1–4GlcNAc–PA; M6B, Manα1–6(Manα1–3)Manα1–6(Manα1–2Manα1–3)Manβ1–4GlcNAcβ1–4GlcNAc– PA; M6B0, Manα1–6(Manα1–3)Manα1–6(Manα1–2Manα1–3)Manβ1–4GlcNAc–PA; SF-HPLC, size-fractionation HPLC; Con A, concanavalin A
PY - 2004/12
Y1 - 2004/12
N2 - An α-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified α-mannosidase was estimated to be 120 kDa by SDS-PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo α-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala-Phe-Met-Lys-Tyr-X-Thr-Thr-Gly-Gly-Pro- Val-Ala-Gly-Lys-Ile-Asn-Val-His-Leu-. The α-mannosidase activity for Man5GlcNAc1 was enhanced by the addition of Co 2+, but the addition of Zn2+, Ca2+, or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man6 GlcNAc1 was significantly faster than that for pyridylaminated Man6GlcNAc2, suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013-1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an α-mannosidase, which is activated by Co2+ and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.
AB - An α-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified α-mannosidase was estimated to be 120 kDa by SDS-PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo α-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala-Phe-Met-Lys-Tyr-X-Thr-Thr-Gly-Gly-Pro- Val-Ala-Gly-Lys-Ile-Asn-Val-His-Leu-. The α-mannosidase activity for Man5GlcNAc1 was enhanced by the addition of Co 2+, but the addition of Zn2+, Ca2+, or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man6 GlcNAc1 was significantly faster than that for pyridylaminated Man6GlcNAc2, suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013-1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an α-mannosidase, which is activated by Co2+ and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.
KW - Co (II)
KW - Free N-glycan metabolism
KW - Ginkgo biloba
KW - Plant glycoprotein
KW - α-mannosidase
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U2 - 10.1271/bbb.68.2547
DO - 10.1271/bbb.68.2547
M3 - Article
C2 - 15618626
AN - SCOPUS:13544261617
SN - 0916-8451
VL - 68
SP - 2547
EP - 2556
JO - Bioscience, Biotechnology and Biochemistry
JF - Bioscience, Biotechnology and Biochemistry
IS - 12
ER -