Purification and Characterization of a Co(II)-Sensitive [alpha]-Mannosidase from Ginkgo biloba Seeds

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2004-12, Vol.68 (12), p.2547
• Main Authors: Kit WOO, Kwan, MIYAZAKI, Mitsuhiro, HARA, Shintaro, KIMURA, Mariko, KIMURA, Yoshinobu
• Format: Article
• Language: English
• ISSN: 0916-8451; 1347-6947

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 Co2+, 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 Man6GlcNAc1 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.