Abstract
We describe here an activable neutral cholesteryl esterase (EC 3.1.1.13) in arteries similar to the hormone-sensitive lipase of adipose tissue and adrenal cortex. Maximum enzyme activity in rabbit aorta was given by cholesteryl ester substrates dispersed as a mixed micelle with phosphatidylcholine and Na taurocholate (molar ratio 1:4:2). A quantitative assay of enzymic activity was obtained with the following component concentrations: 6.0 microM cholesteryl [1-14C]oleate, 23.7 microM phosphatidylcholine, 12.5 microM Na taurocholate, 0.04% serum albumin, and 85 mM K phosphate buffer, pH 7.0. The enzymic activity in aortic homogenates was stimulated 2-fold by addition of 5 microM glucagon or 100 microM dibutyryl cAMP. This activation was Mg-ATP dependent. Addition of 50 micrograms/ml of exogenous protein kinase could reverse the action of protein kinase inhibitor on dibutyryl cAMP activation of the neutral cholesteryl esterase. In addition to activation by cAMP-dependent protein kinase, the enzyme could be distinguished from the more active arterial lysosomal cholesteryl esterase by its pH 7.0 optimum, relative stability to preincubation at elevated temperatures, and exclusive localization in the cell cytosol. Subcellular fractionation of lipid-laden arterial foam cells revealed a significant portion of the neutral cholesteryl esterase bound to cytoplasmic cholesteryl ester-rich lipid droplets. Our results suggest that the breakdown of cytoplasmic cholesteryl ester droplets in arterial cells may be under hormonal regulation.
Highlights
We describe here an activable neutral cholesteryl sensitivelipase [2]
Concentration of ATP in homogenate (1.3 g of tissue, wet weight, in 5 ml) after dialysis, 220 p ~be;fore dialysis, 240 p ~A.TP assay by luciferase method [36, 37]. *Neutral cholesteryl esterase activity in homogenate before dialysis measured in the presence of 100 p~ dibutyryl CAMP,50 pg of protein kinase, and 10 I"Mg-ATP 131.1microunits/mg of protein
Previous descriptions of the properties of the arterial neutraclholesteryl esterase havedisagreed considerably; pH optimum ranging from 6.6-8.6 has been given, and localization of the enzyme in particulate and/or soluble fractions has been described [4,5,6,7,8,9,10]. Some of these differences may reflect contamination of crude tissue preparations with the active lysosomal cholesteryl esterase or may be due to differences in the modes of substrate dispersion used by various investigators
Summary
From the DepartmentsofBiochemistry a n d Pathology, Cornell University Medical College a n d the Laboratory of Biochemical Cytology, The Rockefeller University, New York, New York 10021. Maximum enzyme activity in rabbit aorta was given by cholesteryl ester substrates dispersed as a mixed micelle with phosphatidylcholine and Na taurocholate (molar ratio 1:4:2). A quantitative assay of enzymic activity was obtained with the following component concentrations: 6.0 PM cholesteryl [l-'4C]oleate, 23.7 p~ phosphatidylcholine, 12.5 p~ Na taurocholate, 0.04%serum albumin, and 85 mM K phosphate buffer, pH 7.0. A mixed micelle of cholesteryl oleate/phosphatidylcholine/Na taurocholate, prepared by a modification of the procedure described by Vahouny et al [24],yielded the highest enzyme activity. This prepabout 3 times higher than neutral cholesteryl esterase activity. Phosphatidylcholine/Na taurocholate of1:2:1 gave one-third of the activity observed with the optimum ratio of 1:4:2.Of
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