Solubilization and partial purification of neonatally expressed rat hepatic microsomal monoacylglycerol acyltransferase.
Rat hepatic microsomal monoacylglycerol acyltransferase (MGAT) is a developmentally expressed enzyme that catalyzes the formation of sn-1,2-diacylglycerol from sn-2-monoacylglycerol and fatty acyl-CoA. Treatment of suckling rat liver microsomes with various detergents showed that 0.3% Triton X-100, a nonionic detergent, solubilized a maximum amount of both protein (66%) and MGAT activity (56%). After solubilization with Triton X-100, MGAT was then purified 205-fold by sequential chromatography on QAE-Sephadex, CM-Sepharose (Fast Flow), and hydroxylapatite. Addition of phospholipids to the reaction mixture stimulated the purified enzyme activity more than 1.8-fold. sn-1,2-DiC18: 1-glycerol activated purified MGAT activity. Purified MGAT activity was specific for sn-2-monoacylglycerol; the activity with rac-1-monoC18:1-glycerol and rac-1- and sn-2-monoC18:1-glycerol ethers was less than 4% of the activity with sn-2-monoC18:1-glycerol. The purified MGAT had an isoelectric point of 9.7. The apparent Km and Vmax values of the purified enzyme for sn-2-monoC18:1-glycerol were 21 microM and 1036 nmol/min/mg, respectively. The apparent Km value for palmitoyl-CoA was 6.5 microM. Purified MGAT activity acylated sn-2-monoC18:2-glycerol and sn-2-monoC18:3-glycerol in preference to sn-2-monoC18:1-glycerol, consistent with a role for the monoacylglycerol pathway in retaining essential fatty acids.
Department of Nutrition, University of North Carolina, Chapel Hill 27599.
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Coleman
D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003902:Detergents; D017886:Durapatite; D006882:Hydroxyapatites; D007525:Isoelectric Focusing; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D013379:Substrate Specificity
D003902:Detergents; D006882:Hydroxyapatites; D010171:Palmitoyl Coenzyme A; D017886:Durapatite; D000217:Acyltransferases; C021802:2-acylglycerol O-acyltransferase