Lysosomal acid cholesteryl esterase activity in normal and lipid-laden aortic cells.

نویسندگان

  • N J Haley
  • S Fowler
  • C de Duve
چکیده

We have investigated the kinetic properties of acid cholesteryl esterase (EC 3.1.1.13) in preparations of rabbit aortic cells, with the aim of establishing conditions suitable for the quantitative assay of the enzyme in freshly prepared homogenates and subcellular fractions, whether derived from normal cells or from atheromatous cells heavily laden with cholesterol and cholesteryl esters. As substrate we used cholesteryl [ l-'4C]oleate incorporated at a 1 : 100 molar ratio into egg-lecithin liposomes as described by Brecher and co-workers (J. Lipid Res. 1976. 17: 239), and measured the radioactivity remaining in the alkaline buffer phase after organic solvent extraction of unhydrolyzed substrate. When the liposome substrate was used as such, more than 80% of the enzyme activity was latent in fresh homogenates. I t could be released quantitatively without inactivation by addition of digitonin, but this created a requirement for taurocholate, presumably because digitonin disrupts liposomes. The following conditions gave satisfactory linearity with both time of incubation and enzyme concentration, with both normal and atheromatous cell preparations, and were adopted for the assay: 12.7 pM cholesteryl oleate dispersed in 1.27 mM egg lecithin; 50 mM acetate buffer, pH 3.9; 2.0 mM Na taurocholate; and 0.005%' digitonin. The enzyme has a pH optimum of 3.9, and, under our conditions, has an apparent Km of about 1.5 pM. I t is markedly inhibited by salt solutions and is sensitive to freezing, especially at high ionic strength. Subcellular rractionation by sucrose density gradient centrifugation indicates that a considerable part of the enzyme is localized in lysosomes, both in normal and in either moderately or heavily lipid-laden atheromatous aortic cells. The proportion of activity associated with the soluble fraction is however higher for acid cholesteryl esterase than for two acid glycosidases, in all three cell preparations. When assayed under optimal conditions, lipid-laden atheromatous cells displayed up to 3.5 times the acid cholesteryl esterase activity o f normal aortic cells. This finding does not support the hypothesis that lipid overloading of lysosomes in atheromatous arterial cells occurs as a consequence of a relative deficiency o f acid cholesteryl esterase.-Haley, N. J., S. Fowler, and C. de Duve. Lysosomal acid cholesteryl esterase activity in normal and lipid-laden aortic cells. J. Lipid Rr.c.. 1980. 21: 961-969. Supplementary key words tion . lipid storage ' lysosomal lipase . smooth muscle cells atherosclerosis . isopycnic centrifugaaccumulation of lipid that occurs in aortic cells of cholesterol-fed rabbits may be the consequence of a relative deficiency of the digestion of the cholesteryl esters that are taken u p by the cells in association with pinocytized lipoproteins ( 1 -3). Supporting such a mechanism were some preliminary findings suggestive of an inverse correlation between lysosomal lipid storage and cholesteryl esterase activity (2), and the clinical observation that a congenital deficiency of this enzyme does indeed lead t o massive atherosclerosis (4). T h e possibility could not be excluded, horvever, that the low cholesteryl esterase activity observed in the lipid-laden lysosomes was a consequence, rather than the cause, of the engorgement of these

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عنوان ژورنال:
  • Journal of lipid research

دوره 21 8  شماره 

صفحات  -

تاریخ انتشار 1980