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|Title:||Studies on the Regulation of Hepatic Hmg Coenzyme a Reductase and the Production of Anti-Hmg-Coenzyme a Reductase Monoclonal Antibodies|
|Author(s):||Clark, Robert Earl|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Rat liver and Chang liver cells have been adapted to growth in serum-free chemically defined Higuchi's medium for the purpose of studying the regulation of HMG-CoA reductase.
Change liver cell mutants resistant to 7-ketocholesterol show 4.5-fold to 7.8-fold elevations in reductase activity relative to wild type Change cells. These clones are 5-fold to 35-fold resistant to the killing effects of 7-ketocholesterol.
Four individual cell lines have been isolated from rat liver cells which grow at 10 uM, 15 uM, 20 uM and 25 uM compactin. These clones have reductase levels ranging from 8-fold to 17-fold higher than wild type cells.
The K(,i) for compactin in wild type rat liver cells is 0.69 nM and 0.86 nM in clone ML-9 which grows in 20 uM compactin.
The stability of the mutational event(s) which confer compactin resistance have been demonstrated by removing the selective agent from the culture medium for up to 7 months. Under these conditions no deleterious effects on cell growth and viability were observed following the addition of compactin to the culture medium.
A BALB/c mouse was immunized with a total of 50 ug purified HMG-CoA reductase. The spleen cells were fused with SP-2/0 myeloma cells using 50% PEG. HAT selection was used to isolate hybridomas which were then screened for anti-HMG-CoA reductase activity using a solid phase radioimmunoassay. Four hybridomas were isolated which secrete anti-HMG-CoA reductase monoclonal antibodies. One hybridoma secretes IgM(,1), another secretes IgG(,2B) and the remaining hybridomas secrete IgG monoclonal antibodies.
A solution assay system using solubilized rat liver HMG-CoA reductase has been developed to characterize the ability of these monoclonal antibodies to precipitate reductase from solution. A mixture of these anti-HMG-CoA reductase monoclonal antibodies has been shown to inactivate 95% of the reductase activity in solution.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1981.
|Date Available in IDEALS:||2014-12-14|