[6A8 alpha-mannosidase catalyzes p-nitrophenyl-alpha-D-mannopyranoside].
OBJECTIVE
To confirm the alpha-mannosidase nature of the protein encoded by 6A8 cDNA.


METHODS
1) To construct a full-length 6A8 cDNA based on the three cloned DNA fragments by means of gene recombinant technique; 2) To insert the 6A8 cDNA into eukaryotic expression vector pCDI; 3) To transfect the recombinant pCDI-6A8 into COS-7 cells; 4) To characterize the nature of the protein encoded by 6A8 cDNA by means of enzymic activity assay and Western blotting assay.


RESULTS
The constructed 6A8 cDNA was the right cDNA in sequence. The enzymetic activity of the homogenate of COS-7 cells transfected with pCDI-6A8 was 3-4 times higher than that of the cells transfected with the mock or the wild cells. The enzymetic reaction could not be inhibited by swainsonine. Western blot showed a band of 120,000 recognized by mAb 6A8. The band in the cells transfected with pCDI-6A8 cDNA was much darker than that in the cells transfected with the mock or in the wild cells.


CONCLUSIONS
The protein encoded by 6A8 cDNA is a kind of alpha-mannosidase, which belongs to type II alpha-mannosidase.
Wang
Z Z
ZZ
Department of Immunology, Institute of Basic Medical Sciences, CAMS, PUMC, Beijing 100005, China.
Li
L
L
Shi
G X
GX
Liu
Y
Y
Zhao
F T
FT
Zhu
L P
LP
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D008358:Mannose / Q000378:metabolism; D014162:Transfection; D043323:alpha-Mannosidase / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*
D018076:DNA, Complementary; D043323:alpha-Mannosidase; D008358:Mannose