Year-end Sale % OFF 
Abstract
The lipid cofactor requirement of hepatic monoacylglycerol acyltransferase (MGAT) (EC 2.3.1.22) was studied in Triton X-100/lipid-mixed micelles. Anionic phospholipids and anionic lysophospholipids stimulated MGAT activity, whereas fatty acids and sphingosine inhibited enzyme activity. Phosphatidic acid was a potent activator, stimulating MGAT 11-fold at 4.2 mol %. Kinetic studies revealed that phosphatidic acid, with an apparent Ka of 0.26 mol %, was a better activator than phosphatidylserine, phosphatidylinositol, or cardiolipin. Of the anionic lysophospholipids, lysophosphatidic acid was a better activator than lysophosphatidylserine, stimulating maximally at less than 3 mol %. Oleate was a more potent inhibitor (Ki, 2.4 mol %) than sphingosine (Ki, 18.3 mol %). The dependence of MGAT on sn-2-monoacylglycerol was not cooperative in the absence or presence of anionic phospholipids, oleic acid, or sphingosine. The apparent Km for sn-2-monoC18:1-glycerol was 1.24 mol % in the presence of maximally activating phospholipid and 0.19 mol % when phospholipid was omitted. MGAT's product sn-1,2-diacylglycerol was a weaker activator than the anionic phospholipids, but the effects of diacylglycerol and phospholipid were additive. Activation by sn-1,2-diC18:1-glycerol was highly cooperative with a Hill coefficient of 3.6. Activation was specific for the sn-1,2-stereoisomer; neither 1,3-diacylglycerol nor the ether analogs of sn-1,2- or 1,3-diacylglycerol were activators. Since several of the lipid modulators of MGAT activity are intracellular second messengers, these data suggest the possibility that regulatory links exist between signal transduction and the synthesis of complex lipids via the monoacylglycerol pathway.
Highlights
Lysophosphatidic acid was a better activator than lysophosphatidylserine, stimulating maximally at less than 3 mol %
MGAT Activity inniton X-100-mixed Micelles-Triton X-100-mixed micelles were used to provide an inertsurface in which hydrophobic substrates and potentiallipid activators and inhibitors were dispersed in physically defined amounts, thereby providing a matrix thatmimics the microsomal membrane bilayer
The average number of lipid molecules present in a mixed micelle was estimated from the detergent aggregation number of 140 for Triton X-100 and the mol % concentration of the lipid present [17]
Summary
Dept. of Nutrition, CB 7400, University of North Carolina, Chapel Hill, NC 27599-7400. The solubilized enzymepreparation was stored in aliquots a t -70 "C in abuffer containing 20% glycerol5, %ethylene glycol, 20nm MES, pH 6.5, 1 m~ EDTA, 1 m~ DTT, 0.1 m~ phenylmethylsulfonylfluoride, 1 pg/ml pepstatin, 0.5 m~ benzamidine, 0.2% Triton X-100, and 0.2 M NaCl. Hydroxylapatite-purified MGAT specific activities were1443, 1422, 1423,and 2341 nmol/min/mg fromfour different purified preparations when MGAT was assayed in a 0.2-ml reaction mixture containing 100m~ Tris-C1, pH8 , l mg/ml BSA, 450 pg/mslonicated microsomal lipids, 150p~ sn-2-monoC18:l-glyceroland 300 p~ sn-1,2-diC18:l-glycerol dispersed together in 10pl of acetone, 5 m~ DTT, 2.5m~ EDTA, 25 p~ [3H]palmitoyl-coenzymeA (66 Ci/mol),and 0.1-0.5 pg of protein [6]. MGAT activity was assayed at 23 "C in a 0.2-ml reaction mixture that contained 100nm Tris-C1, pH7.0,0.5mg/ml BSA,150w sn-2-monoC18:lglycerol,0.22% Triton X-100 (3 m~ micelleconcentration): p~ [3Hlpalmitoyl-CoA0, .25-0.5 pg of hydroxylapatite-purifiedprotein, and the indicated concentrations of specific phospholipids. Hill constants were calculated as described [25]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
