Purification and characterization of a co(II)-sensitive alpha-mannosidase from Ginkgo biloba seeds.
An alpha-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified alpha-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 alpha-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 alpha-mannosidase activity for Man(5)GlcNAc(1) was enhanced by the addition of Co(2+), but the addition of Zn(2+), Ca(2+), or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man(6)GlcNAc(1) was significantly faster than that for pyridylaminated Man(6)GlcNAc(2), 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 alpha-mannosidase, which is activated by Co(2+) and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.
Woo
Kwan Kit
KK
Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
Miyazaki
Mitsuhiro
M
Hara
Shintaro
S
Kimura
Mariko
M
Kimura
Yoshinobu
Y
D000595:Amino Acid Sequence; D003035:Cobalt / Q000494:pharmacology*; D020441:Ginkgo biloba / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity; D043323:alpha-Mannosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D009844:Oligosaccharides; D010940:Plant Proteins; D003035:Cobalt; D043323:alpha-Mannosidase