Functional sizes of hepatic enzymes of cholesteryl ester metabolism determined by radiation inactivation.

Cellular cholesteryl ester metabolism is regulated largely by the balance between intracellular esterification catalyzed by acyl-CoA:cholesterol acyltransferase and cholesteryl ester hydrolysis catalyzed by the cholesteryl ester hydrolases. The hydrolases include both cytosolic and membrane-associated activities; acidic and neutral activities have been described in both compartments. Esterification via the acyltransferase is membrane-associated. Neither the acyltransferase nor the membrane-associated hydrolases have been purified and characterized, and little is known about their genes. Thus, nothing is known about their sizes or structures. Radiation inactivation was used to determine the functional sizes in situ of acyl-coenzyme A:cholesterol acyltransferase, fatty acyl-CoA hydrolase, and acidic and neutral membrane-associated cholesteryl ester hydrolase activities. The functional M(r) +/- SD of the acyltransferase was 213 +/- 35 kD; for the acidic membrane-associated hydrolase, 48 +/- 2 kD; for the neutral membrane-associated hydrolase, 94 +/- 15 kD; and for the fatty acyl-CoA hydrolase, 65 +/- 15 kD. Monoexponential curves were observed in all cases using radiation exposures that inactivated enzyme activities to < or = 10% of control values. Substrate specificity and inhibition studies suggested that the active sites of the acyltransferase and fatty acyl-CoA hydrolase were different, supporting the concept that the hydrolase is not part of the functional unit required for cholesterol esterification.

Analysis of microsomal cholesteryl ester hydrolases by radiation inactivation.

Radiation inactivation by high energy electrons, a method for determining the size of a protein without prior purification, was used to study the acid and neutral cholesteryl ester hydrolase (CEH) activities of rat liver microsomes. The same preparations were also assayed for the microsomal, "nonspecific" carboxylesterases using o-nitrophenyl acetate as substrate. Non-specific esterase activity surviving radiation could be fit to a single exponential function, the slope of which yielded a target size of 47 +/- 5 kDa (mean +/- S.D., n = 7). Surviving CEH activity assayed at pH 5 could also be fit to a single exponential that yielded a target size of 71 +/- 14 kDa (n = 5). In contrast, the surviving CEH activity assayed at pH 7 was more complex. The data from six experiments were described as the sum of two exponentials, indicating that most of the activity is due to an entity that is three to four times larger and a minor amount to one that is half the size of the pH 5 enzyme. The results are consistent with the suggestion that the acid and neutral microsomal CEH activities are due to distinct enzymes, which are not the "nonspecific" carboxylesterases. Their sizes also differ from those previously determined for lysosomal acid lipase and other lipases in the liver.