Kang J J, Cunningham H B, Jachec C, Priest A, Dahms A S, Sabbadini R A
Department of Chemistry, San Diego State University, California 92182.
Arch Biochem Biophys. 1991 Oct;290(1):214-23. doi: 10.1016/0003-9861(91)90611-l.
T-tubule membrane vesicles isolated from skeletal muscle contain a very active Mg(2+)-ATPase (EC 3.6.1.34) which is modulated by lectins and is located in the junctional region near the sarcoplasmic reticulum membranes (1). The effects of several prominent lipophilic agents upon the ATPase have led us to evaluate the action of diacylglycerols and phorbol esters upon the enzyme. The ATPase is inhibited by submicromolar levels of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (sn-OAG), with K0.5s of 0.2 and 0.5 microM, respectively. Significantly, 4-alpha-phorbol 12,13-didecanoate (4-alpha-phorbol) the TPA analogue shown to be inactive toward protein kinase C (PKC), inhibited the ATPase with a K0.5 of 0.3 microM, and 1-stearoyl-2-arachidonyl-sn-glycerol, the preferred endogenous activator of PKC, was not inhibitory toward the ATPase. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (a membrane permeant PKC inhibitor) and peptide 19-36 (the highly specific PKC pseudosubstrate inhibitor) were both without effect upon the ATPase and did not affect TPA inhibition. ATPase activity was not altered under phosphorylating conditions in experiments using exogenous rat brain PKC. ConA protected ATPase activity against inhibition by TPA, 4-alpha-phorbol, and sn-OAG. Additionally, phorbol-12,13-dibutyrate binding studies demonstrated that the ATPase was capable of significant phorbol binding with ConA protection. The data are consistent with a direct and specific effect of phorbol esters and diacylglycerols upon the ATPase, without any participation of PKC. We conclude that the transverse tubule (T-tubule) ATPase is an alternate receptor for diacylglycerol and TPA in skeletal muscle and that the mode of action of these agents upon the ATPase (inhibition) is opposite to their mode of action on PKC (activation). The data demonstrate that substantial care must be taken in ascribing either cellular or subcellular effects of phorbol esters and diacylglycerols exclusively to the activation of PKC and that alternate receptors may exist. Criteria are recommended for the demonstration of PKC-independent modulation by phorbols and diacylglycerols.
从骨骼肌中分离出的横管膜囊泡含有一种活性很强的镁离子 - ATP酶（EC 3.6.1.34），该酶受凝集素调节，位于肌浆网膜附近的连接区域（1）。几种重要的亲脂性试剂对该ATP酶的影响促使我们评估二酰基甘油和佛波酯对该酶的作用。该ATP酶受到亚微摩尔浓度的佛波酯12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）和二酰基甘油1 - 油酰 - 2 - 乙酰 - sn - 甘油（sn - OAG）的抑制，其半抑制浓度（K0.5）分别为0.2和0.5微摩尔。值得注意的是，4 - α - 佛波醇12,13 - 二癸酸酯（4 - α - 佛波醇），这种对蛋白激酶C（PKC）无活性的TPA类似物，以0.3微摩尔的K0.5抑制该ATP酶，而1 - 硬脂酰 - 2 - 花生四烯酰 - sn - 甘油，PKC的首选内源性激活剂，对该ATP酶没有抑制作用。1 - （5 - 异喹啉磺酰基） - 2 - 甲基哌嗪二盐酸盐（一种膜渗透性PKC抑制剂）和肽19 - 36（高度特异性的PKC假底物抑制剂）对该ATP酶均无作用，也不影响TPA的抑制作用。在使用外源性大鼠脑PKC的实验中，磷酸化条件下ATP酶活性未改变。刀豆球蛋白A（ConA）可保护ATP酶活性免受TPA、4 - α - 佛波醇和sn - OAG的抑制。此外，佛波醇 - 12,13 - 二丁酸酯结合研究表明，该ATP酶能够大量结合佛波醇，且ConA可提供保护。这些数据与佛波酯和二酰基甘油对ATP酶有直接且特异性的作用一致，而无需PKC的参与。我们得出结论，横管（T管）ATP酶是骨骼肌中二酰基甘油和TPA的另一种受体，并且这些试剂对ATP酶的作用方式（抑制）与其对PKC的作用方式（激活）相反。数据表明，在将佛波酯和二酰基甘油的细胞或亚细胞效应完全归因于PKC激活时必须格外谨慎，可能存在其他受体。推荐了用于证明佛波醇和二酰基甘油不依赖PKC调节的标准。
