氚同位素效应在大鼠肝脏甘油磷脂生物合成中甘油磷酸和磷酸二羟丙酮途径测量中的应用

Tritium isotope effects in the measurement of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver.

作者信息

Manning R, Brindley D N

出版信息

Biochem J. 1972 Dec;130(4):1003-12. doi: 10.1042/bj1301003.

DOI:10.1042/bj1301003

PMID:4656790

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1174550/

Abstract

Rat liver slices were employed to study the relative rates of incorporation of a mixture of [2-(3)H]- or [1,3-(3)H]-glycerol and [1-(14)C]glycerol into lipids. 2. With 0.1mm-glycerol approx. 82% of the newly synthesized lipid, calculated from (14)C incorporation, was present as neutral lipid, 13% as phosphatidylcholine and 5% as phosphatidylethanolamine. Increasing the glycerol concentration to 40mm caused a decrease in the percentage of neutral lipid to 59% and a corresponding increase in the percentage of phosphatidylcholine to 36% of the newly synthesized lipid. 3. The (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in glycerolipid was considerably higher than that in precursor glycerol throughout the range of experimental conditions. In contrast the incorporation of a mixture of [1,3-(3)H]glycerol and [1-(14)C]glycerol into lipid occurred with little or no change in the (3)H/(14)C ratio. 4. Respiring rat liver mitochondria were found to oxidize a mixture of sn-[2-(3)H]- and sn-[1-(14)C]-glycerol 3-phosphate with a resultant increase in the (3)H/(14)C ratio of the remaining sn-glycerol 3-phosphate. This increase is due to a (3)H isotope effect of the mitochondrial sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5), which discriminates against sn-[2-(3)H]glycerol 3-phosphate during oxidation. 5. A method is described for the simultaneous determination of the relative contributions of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver slices. The method involves measurement of the (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in both sn-glycerol 3-phosphate and glycerolipid after incubation of rat liver slices with a mixture of [2-(3)H]glycerol and [1-(14)C]glycerol for various times. 6. By using this method it was shown that 40-50% of the glycerol incorporated into lipid by rat liver slices proceeded via the sn-glycerol 3-phosphate pathway and 50-60% was incorporated via dihydroxyacetone phosphate.

摘要

采用大鼠肝脏切片来研究[2-(3)H]-或[1,3-(3)H]-甘油与[1-(14)C]甘油的混合物掺入脂质的相对速率。2. 当甘油浓度为0.1mmol时，根据(14)C掺入量计算，新合成脂质中约82%为中性脂质，13%为磷脂酰胆碱，5%为磷脂酰乙醇胺。将甘油浓度提高到40mmol时，中性脂质的百分比降至59%，而磷脂酰胆碱在新合成脂质中的百分比相应增加至36%。3. 在整个实验条件范围内，甘油酯中(2-(3)H的每分钟衰变数)/(1-(14)C的每分钟衰变数)的比值显著高于前体甘油中的该比值。相比之下，[1,3-(3)H]甘油和[1-(14)C]甘油的混合物掺入脂质时，(3)H/(14)C比值几乎没有变化。4. 发现呼吸状态的大鼠肝脏线粒体可氧化sn-[2-(3)H]-和sn-[1-(14)C]-甘油3-磷酸的混合物，导致剩余sn-甘油3-磷酸的(3)H/(14)C比值升高。这种升高是由于线粒体sn-甘油3-磷酸脱氢酶（EC 1.1.99.5）的(3)H同位素效应，该酶在氧化过程中对sn-[2-(3)H]甘油3-磷酸有歧视作用。5. 描述了一种同时测定大鼠肝脏切片中甘油磷酸和磷酸二羟丙酮途径在甘油脂质生物合成中相对贡献的方法。该方法包括在大鼠肝脏切片与[2-(3)H]甘油和[1-(14)C]甘油的混合物孵育不同时间后，测量sn-甘油3-磷酸和甘油脂质中(2-(3)H的每分钟衰变数)/(1-(14)C的每分钟衰变数)的比值。6. 通过使用该方法表明，大鼠肝脏切片掺入脂质的甘油中40 - 50%通过sn-甘油3-磷酸途径进行，50 - 60%通过磷酸二羟丙酮掺入。