The beta-mannosyltranserease (BMT) that adds mannose in a beta-1, 4-linkage to dolichyl-PP-G1cNAc-G1cNAc to form Man-beta-GlcNAc-PP-dolichol was solubilized from pig aorta microsomes, and purified about 200 fold from the solubilized fraction. These purification steps included ion exchange on De-52, hydroxylapatite, and High Q chromatography. The enzymatic activity could be kept fairly stable in 20% glycerol containing o.5 mM dithiothreitol. The enzyme showed a pH optimum of about 8.0 in 50 mM Tris buffer. The rate of mannose transfer was linear with time and protein and the Km for GDP-mannose was about 0.06 uM, and for dolichy1-PP-G1cNAc-G1cNAc about 60 uM. At the final purification step, the enzyme showed 5 bands of SDS gels, but at this stage it is not clear which band is the enzyme of interest. The antibiotic, enramycin, was found to be a potent inhibitor of the enzyme and inhibition was shown to be of a competitive nature. In order to continue these studies the following specific aims are proposed: 1) To purify the BMT at to homogeneity and obtain amino acid sequence information from various peptides, 2) To determine the properties of the enzyme and its possible regulation by metabolites, 3) To determine the effects of enramycin and other antibiotics on this enzyme and other enzymes of the lipid-linked oligosaccharide pathway, 4) To clone the BMT to obtain large amounts of enzyme for characterization, 5) To identify the active site of the enzyme. These studies should demonstrate the role of this enzyme in the biosynthetic pathway and its possible sites of regulation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
1K01HL003831-01
Application #
2666239
Study Section
Special Emphasis Panel (ZHL1-CSR-K (F1))
Project Start
1998-05-01
Project End
2003-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Arkansas at Pine Bluff
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Pine Bluff
State
AR
Country
United States
Zip Code
71601