Fatty Acids and Anionic Phospholipids Alter the Palmitoyl Coenzyme A Kinetics of Hepatic Monoacylglycerol Acyltransferase in Triton X-100 Mixed Micelles
详细信息 查看全文
- 作者：Rosalind A. Coleman ; P. Wang ; and B. Ganesh Bhat
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：July 23, 1996
- 年：1996
- 卷：35
- 期：29
- 页码：9576 - 9583
- 全文大小：728K
- 年卷期：v.35,no.29(July 23, 1996)
- ISSN：1520-4995
文摘
In order to gain a better understanding of the kineticsof activation and inhibition of hepaticmonoacylglycerol acyltransferase (MGAT) (EC 2.3.1.22) by fatty acid, weexamined the effect of fattyacid with respect to MGAT's long-chain acyl-CoA substrate in TritonX-100 mixed micelles. Atconcentrations between 2.5 and 5.3 mol %, oleic acid stimulated MGATactivity 2-fold, whereas oleicacid inhibited MGAT at concentrations higher than 7.5 mol %. Thedependence on palmitoyl-CoA washighly cooperative with a Hill constant of greater than 2.4. Whenpresent at less than 3 mol %, oleic acideliminated the lag in the dependence curve. When concentrations ofoleic acid were higher than 3 mol%, Michaelis-Menton kinetics were observed with an apparentKm value of about 54 
M forpalmitoyl-CoA but with progressively decreasing Vmaxvalues. This effect was not observed with octanoicacid,suggesting that the medium-chain fatty acid is unable to associatestably with the mixed micelle and,thus, cannot substantially alter substrate affinity. When anionicphospholipids were tested, phosphatidicacid, lysophosphatidic acid, phosphatidylserine, andphosphatidylinositol eliminated some of the lag inactivation by palmitoyl-CoA. At high molar concentrations of theanionic lipid activators, apparentKmvalues ranged from 77 M for phosphatidic acid to 196 M forphosphatidylinositol. Zwitterionicphospholipids had no effect, nor did the non-phospholipid activatorsbovine serum albumin or sn-1,2-diacylglycerol. CaCl2, but not neomycin or KCl, couldovercome the inhibitory effect of oleic acid; thus,the inhibitory effect of fatty acid did not appear to occur byelectrostatic interactions. These blockers didnot change the effects observed with the anionic phospholipidactivators or with the inhibitor, sphingosine.An altered Km for palmitoyl-CoA in thepresence of fatty acid or anionic phospholipid suggests thatbothlong-chain fatty acids and phospholipid cofactors may induce aconformational change in MGAT, therebyaltering the enzyme's affinity for its long-chain acyl-CoA substrate.These data further support thehypothesis that the synthesis of glycerolipids via the monoacylglycerolpathway may be highly regulatedvia a variety of lipid second messengers such as phosphatidic acid anddiacylglycerol, as well as by theinflux of fatty acids derived from high-fat diets, or from thehydrolysis of adipocyte triacylglycerol duringfasting or diabetes.
M forpalmitoyl-CoA but with progressively decreasing Vmaxvalues. This effect was not observed with octanoicacid,suggesting that the medium-chain fatty acid is unable to associatestably with the mixed micelle and,thus, cannot substantially alter substrate affinity. When anionicphospholipids were tested, phosphatidicacid, lysophosphatidic acid, phosphatidylserine, andphosphatidylinositol eliminated some of the lag inactivation by palmitoyl-CoA. At high molar concentrations of theanionic lipid activators, apparentKmvalues ranged from 77 M for phosphatidic acid to 196 M forphosphatidylinositol. Zwitterionicphospholipids had no effect, nor did the non-phospholipid activatorsbovine serum albumin or sn-1,2-diacylglycerol. CaCl2, but not neomycin or KCl, couldovercome the inhibitory effect of oleic acid; thus,the inhibitory effect of fatty acid did not appear to occur byelectrostatic interactions. These blockers didnot change the effects observed with the anionic phospholipidactivators or with the inhibitor, sphingosine.An altered Km for palmitoyl-CoA in thepresence of fatty acid or anionic phospholipid suggests thatbothlong-chain fatty acids and phospholipid cofactors may induce aconformational change in MGAT, therebyaltering the enzyme's affinity for its long-chain acyl-CoA substrate.These data further support thehypothesis that the synthesis of glycerolipids via the monoacylglycerolpathway may be highly regulatedvia a variety of lipid second messengers such as phosphatidic acid anddiacylglycerol, as well as by theinflux of fatty acids derived from high-fat diets, or from thehydrolysis of adipocyte triacylglycerol duringfasting or diabetes.