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[6A8 alpha-mannosidase catalyzes p-nitrophenyl-alpha-D-mannopyranoside]
1Department of Immunology, Institute of Basic Medical Sciences, CAMS, PUMC, Beijing 100005, China.
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.
Conclusion:
The protein encoded by 6A8 cDNA is a kind of alpha-mannosidase, which belongs to type II alpha-mannosidase.