FUNCTIONAL COUPLING BETWEEN GLYCOLYSIS AND SARCOPLASMIC-RETICULUM CA2+ TRANSPORT
FUNCTIONAL COUPLING BETWEEN GLYCOLYSIS AND SARCOPLASMIC-RETICULUM CA2+ TRANSPORT
复制标题
DOI：
10.1161/01.res.77.1.88
复制
发表时间：
1995-07-01
影响因子：
20.1
通讯作者：
BECKER, LC
中科院分区：
文献类型：
--
作者：
XU, KY;ZWEIER, JL;BECKER, LC
为了研究糖酵解产生的能量是否在功能上与肌浆网（SR）中的Ca 2+主动转运相耦合，我们确定了糖酵解酶是否与SR膜相关，以及通过这些酶的代谢是否能够支持Ca-45转运。采用蔗糖梯度法从兔骨骼肌和心肌中分离出右外SR囊泡。囊泡内Ca-45转运后，加入糖酵解底物和辅因子的具体的每个糖酵解反应正在研究或加入外源性ATP后，并表示为运输敏感的具体的Ca 2 +-ATP酶抑制剂毒胡萝卜素。我们发现，从醛缩酶开始的整个糖酵解酶链，包括醛缩酶、GAPDH、磷酸甘油酸激酶（PGK）、磷酸甘油变位酶、烯醇化酶和丙酮酸激酶（PK），都与心肌和骨骼肌的SR囊泡相关。碘乙酸，GAPDH的抑制剂，消除由果糖-1，6-二磷酸，醛缩酶的底物支持的Ca-45转运，但运输完全恢复磷酸烯醇丙酮酸（PK的底物），表明这两个ATP产生的糖酵解酶，GAPDH/PGK和PK，与SR和功能上能够提供ATP的钙泵。添加可溶性己糖激酶ATP陷阱消除了由外源性ATP提供燃料的Ca-45转运，但对由内源性产生的ATP（通过糖酵解）支持的Ca-45转运的影响明显较小。同样，在非常低浓度的ATP和ADP（10至50 nmol/L），ATP，从ADP和磷酸烯醇式丙酮酸内源性产生的支持15倍以上的Ca-45转运比ATP，在相同的浓度外源性供应。这些结果与糖酵解ATP与Ca 2+转运的功能耦合一致，并支持由SR相关糖酵解酶产生的ATP可能通过驱动SR Ca 2+泵在细胞Ca 2+稳态中发挥重要作用的假设。
To investigate whether the energy derived from glycolysis is functionally coupled to Ca2+ active transport in sarcoplasmic reticulum (SR), we determined whether glycolytic enzymes were associated with SR membranes and whether metabolism through these enzymes was capable of supporting Ca-45 transport. Sealed right-side-out SR vesicles were isolated by step sucrose gradient from rabbit skeletal and cardiac muscle. Intravesicular Ca-45 transport was measured after the addition of glycolytic substrates and cofactors specific for each of the glycolytic reactions being studied or after the addition of exogenous ATP and was expressed as transport sensitive to the specific Ca2+-ATPase inhibitor thapsigargin. We found that the entire chain of glycolytic enzymes from aldolase onward, including aldolase, GAPDH, phosphoglycerate kinase (PGK), phosphoglyceromutase, enolase, and pyruvate kinase (PK), was associated with SR vesicles from both cardiac and skeletal muscle. Iodoacetic acid, an inhibitor of GAPDH, eliminated Ca-45 transport supported by fructose-1,6-diphosphate, the substrate for aldolase, but transport was completely restored by phosphoenolpyruvate (the substrate for PK), indicating that both of the ATP-producing glycolytic enzymes, GAPDH/PGK and PK, were associated with the SR and functionally capable of providing ATP for the Ca2+ pump. Addition of a soluble hexokinase ATP trap eliminated Ca-45 transport fueled by exogenous ATP but had markedly less effect on Ca-45 transport supported by endogenously produced ATP (via glycolysis). Similarly, at very low concentrations of ATP and ADP (10 to 50 nmol/L), ATP that was produced endogenously from ADP and phosphoenolpyruvate supported 15-fold more Ca-45 transport than ATP that was supplied exogenously at the same concentration. These results are consistent with functional coupling of glycolytic ATP to Ca2+ transport and support the hypothesis that ATP generated by SR-associated glycolytic enzymes may play an important role in cellular Ca2+ homeostasis by driving the SR Ca2+ pump.