Size of the catalytically active unit of rat hepatic carboxylester lipase in the presence and absence of bile salt.
Carboxylester lipase (CEL) catalyzes the hydrolysis of cholesteryl esters, retinyl esters, and triacylglycerols. CEL monomer has a MW of approximately 70000. Hydrolysis of these esters is stimulated by millimolar trihydroxy bile salts such as cholate, that also induce aggregation. Liver cytosols from 12 rats were frozen and irradiated at -135 degrees C with high energy electrons. In several experiments, paired samples of cytosol were adjusted to 20 mM cholate before irradiation. All samples were assayed for CEL using cholesteryl oleate as substrate. In untreated cytosols, CEL activity surviving radiation exposure could be fit to a single exponential function, the slope of which yielded a target size of 91 +/- 18 kDa. In a subset of these cytosols irradiated in the presence of cholate the calculated target size was 100 +/- 19 kDa, a value indistinguishable from that obtained for untreated cytosols. Some samples were also assayed using retinyl palmitate and triolein as substrates. With retinyl palmitate the mean target sizes were 96 and 108 kDa in the absence and presence of cholate, respectively, approximately the same as those observed when using cholesteryl oleate. When triolein was used as substrate the target sizes in the absence of cholate were smaller than for the other two esters (67 +/- 18 kDa) and closer to the known monomer molecular weight, but again cholate had no significant effect on this size. The structure responsible for CEL activity contains no more than one 70000 MW monomer and the results show that cholate-induced oligomerization is not required for catalytic activity.
Harrison
E H
EH
Department of Biochemistry, Medical College of Pennsylvania and Hahnemann University, Philadelphia, USA. harrison@auhs.edu
Rojas
C J
CJ
Kempner
E S
ES
D000818:Animals; D001647:Bile Acids and Salts / Q000494:pharmacology*; D001665:Binding Sites; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002788:Cholesterol Esters / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000528:radiation effects; D005615:Freezing; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D002787:Sterol Esterase / Q000378:metabolism; D014304:Triolein
D001647:Bile Acids and Salts; D002788:Cholesterol Esters; D014304:Triolein; C014409:cholesteryl oleate; D002265:Carboxylic Ester Hydrolases; C022523:retinyl esterase; D043182:Carboxylesterase; D002787:Sterol Esterase