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|Title:||Apolipoprotein E Activation and Phosphatidylcholine Substrate Requirements of Lecithin Cholesterol Acyl Transferase|
|Author(s):||Zorich, Nora Lee|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The apolipoprotein activation of lecithin cholesterol acyl transferase (LCAT) by human apolipoprotein E (apo E) was investigated by incorporating apo E into discoidal complexes of egg phosphatidylcholine (egg-PC) and cholesterol by the cholate dialysis method. These complexes were systematically compared to apo A-I complexes synthesized under the same reaction conditions. Apo E was found to be 18% as effective as apo A-I in activating purified human LCAT. Concentration and temperature-dependence experiments on the velocity of the lecithin cholesterol acyl transferase reaction revealed differences in apparent K(,m) values and small differences in apparent V(,max) with very similar activation energies (18-20 kcal/mol). These observations suggest that differences in LCAT activation by apo A-I and apo E are primarily a result in different affinities of the enzyme for the particles. Addition of free apo A-I to apo E complexes resulted in the exchange of bound for free apolipoprotein causing an increase in the reactivity of the enzyme when the unbound apolipoprotein was removed by ultracentrifugation and reisolated complexes were assayed.
The phospholipid substrate specificity of LCAT was examined by analyzing the kinetics and reaction products of LCAT with apo A-I complexes containing a variety of phosphatidylcholines (PCs) differing in acyl chain length and degree of unsaturation. Concentration and temperature-dependence experiments on the velocity of the LCAT reaction with a total of fifteen apo A-I complexes differing in their PC component revealed differences in the apparent V(,max) values and small differences in the apparent K(,m) values. The activation energies were found to vary markedly, 8 to 40 kcal/mol, and to be correlated with the overall degree of unsaturation of the PC acyl chains.
Three pairs of positional isomers of PC were studied to test the currently held concept that the enzyme is specific for the transfer of the sn-2 acyl group to cholesterol. Analysis of cholesterol esters generated by the action of LCAT revealed that the sn-2 fatty acyl chain was favored for transfer to cholesterol when the fatty acyl chain was 18:2 or 18:1 but not if the acyl chain was 18:0. When the sn-2 acyl chain was 18:0, it is possible that transfer of 18:1 or 18:2 from the sn-1 position occurred. In this case it would appear that the enzyme does not show absolute specificity for the acyl group at the sn-2 position.
These results are consistent with a reaction mechanism in which the active site of the enzyme accommodates the entire PC molecule substrate rather than the head group and glycerol backbone portion of the PC molecules present in the bilayer.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.
|Date Available in IDEALS:||2014-12-15|