Hatch G M
Department of Internal Medicine, University of Manitoba, Winnipeg, Canada.
Biochem J. 1994 Jan 1;297 ( Pt 1)(Pt 1):201-8. doi: 10.1042/bj2970201.
The pathway for the biosynthesis of new cardiolipin was investigated in the isolated perfused intact rat heart. Isolated rat hearts were perfused in the Langendorff mode for up to 60 min with Krebs-Henseleit buffer containing 0.1 microM [U-14C]glycerol. Analysis of radioactivity incorporated into phospholipids in the organic phase revealed an increase in radioactivity incorporated into phosphatidylglycerol, cardiolipin and other phospholipids with time of perfusion. This was associated with a loss of radioactivity from phosphatidic acid. In contrast, perfusion of hearts for up to 60 min with 0.1 mM [1,(3)-3H]glycerol in the perfusate revealed an increased radioactivity associated with phosphatidic acid as well as cardiolipin, phosphatidylglycerol and other phospholipids. Perfusion of hearts for up to 60 min with [32P]Pi in the perfusate revealed a time-dependent increase in radioactivity associated with all phospholipids. Perfusion of hearts for up to 60 min with 0.1 microM or 0.1 mM glycerol in the perfusate did not affect the concentration of phosphatidic acid, cardiolipin or phosphatidylglycerol. To determine the rate-limiting step of cardiolipin biosynthesis, hearts were pulsed for 5 min with 0.1 microM [1,(3)-3H]glycerol and chased for up to 60 min with 0.1 microM glycerol in the perfusate. Radioactivity was maximum at the start of the chase in phosphatidic acid (and 1,2-diacylglycerol), and was subsequently chased into phosphatidylinositol, phosphatidylglycerol and cardiolipin (and other phospholipids). Significant radioactivity in phosphatidylglycerol phosphate was not detected. Radioactivity in CDP-sn-1,2-diacylglycerol remained constant throughout the chase. The activities of the enzymes of the Kennedy pathway for cardiolipin biosynthesis in the heart were determined. On the basis of continuous-pulse and pulse-chase labelling studies it is postulated that the cardiac polyglycerophospholipids phosphatidylglycerol and cardiolipin are actively synthesized from newly synthesized phosphatidic acid via the Kennedy pathway. In addition, the results suggest that the rate-limiting step of cardiolipin biosynthesis in the intact heart is probably the conversion of phosphatidic acid into CDP-sn-1,2-diacylglycerol.
在离体灌注的完整大鼠心脏中研究了新的心磷脂生物合成途径。将离体大鼠心脏以Langendorff模式用含有0.1微摩尔[U-14C]甘油的Krebs-Henseleit缓冲液灌注长达60分钟。对有机相中掺入磷脂的放射性分析显示，随着灌注时间的延长，掺入磷脂酰甘油、心磷脂和其他磷脂中的放射性增加。这与磷脂酸中放射性的损失有关。相反，在灌注液中用0.1毫摩尔[1,(3)-3H]甘油灌注心脏长达60分钟，结果显示与磷脂酸以及心磷脂、磷脂酰甘油和其他磷脂相关的放射性增加。在灌注液中用[32P]Pi灌注心脏长达60分钟，结果显示与所有磷脂相关的放射性随时间增加。在灌注液中用0.1微摩尔或0.1毫摩尔甘油灌注心脏长达60分钟，不影响磷脂酸、心磷脂或磷脂酰甘油的浓度。为了确定心磷脂生物合成的限速步骤，用0.1微摩尔[1,(3)-3H]甘油对心脏进行5分钟脉冲，然后在灌注液中用0.1微摩尔甘油追踪长达60分钟。在追踪开始时，磷脂酸（和1,2-二酰基甘油）中的放射性最高，随后被追踪到磷脂酰肌醇、磷脂酰甘油和心磷脂（以及其他磷脂）中。未检测到磷脂酰甘油磷酸中的显著放射性。在整个追踪过程中，CDP-sn-1,2-二酰基甘油中的放射性保持恒定。测定了心脏中心磷脂生物合成的肯尼迪途径中酶的活性。基于连续脉冲和脉冲追踪标记研究，推测心脏中的多甘油磷脂磷脂酰甘油和心磷脂是通过肯尼迪途径由新合成的磷脂酸积极合成的。此外，结果表明完整心脏中心磷脂生物合成的限速步骤可能是磷脂酸转化为CDP-sn-1,2-二酰基甘油。
