ANALYSIS OF MICROSOMAL CHOLESTERYL ESTER HYDROLASES BY RADIATION INACTIVATION

Radiation inactivation by high energy electrons, a method for determin ing the size of a protein without prior purification, was used to stud y 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 acet ate as substrate. Nonspecific esterase activity surviving radiation co uld be fit to a single exponential function, the slope of which yielde d 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 tha t yielded a target size of 71 +/- 14 kDa (n = 5). In contrast, the sur viving CEH activity assayed at pH 7 was more complex. The data from si x experiments were described as the sum of two exponentials, indicatin g 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 aci d and neutral microsomal CEH activities are due to distinct enzymes, w hich are not the ''nonspecific'' carboxylesterases. Their sizes also d iffer from those previously determined for lysosomal acid lipase and o ther lipases in the liver.