Tate C A, Bick R J, Chu A, Van Winkle W B, Entman M L
J Biol Chem. 1985 Aug 15;260(17):9618-23.
We previously demonstrated that the hydrolysis of GTP by canine cardiac sarcoplasmic reticulum is not sensitive to calcium and does not support the translocation of calcium and oxalate into the vesicular space. In response to GTP, however, calcium is accumulated into a compartment which is sensitive to pH and ionophore. In the present paper, we further explored the relationship between GTP hydrolysis and GTP-induced calcium accumulation. Both ATP- and GTP-induced calcium accumulation were prevented by the sulfhydryl reagent, N-ethylmaleimide (NEM; I50 = 0.2 mM). In contrast, the sensitivity of NTP hydrolysis to NEM differed markedly; GTPase activity was not affected by NEM, whereas ATPase activity was markedly inhibited. Conversely, although the GTPase was noncompetitively inhibited by the ATP analogue, adenylyl imidodiphosphate (Ki = 8 microM), and was competitively inhibited by the GTP analogue, guanylyl imidodiphosphate (Ki = 60 microM), GTP-induced calcium accumulation was not affected by the NTP analogues at any concentration. Therefore, the GTP-dependent accumulation of calcium into the pH- and ionophore-sensitive compartment of cardiac SR may not require GTP hydrolysis but may be dependent on GTP binding. The previously reported noncompetitive inhibition of the GTPase by ATP was also observed when the calcium-dependent hydrolysis of ATP was prevented by NEM (Ki = 1.2 microM). Along with the noncompetitive inhibition of the GTPase by adenylyl imidodiphosphate, the inhibition of the GTP by ATP in the presence of NEM suggests that ATP binding may be involved in the observed inhibition. The Ki for the noncompetitive inhibition of GTPase activity is compatible with ATP binding to the high affinity catalytic site of the ATPase. Thus, although GTP-induced calcium accumulation differs somewhat from ATP-dependent calcium translocation, the similarities between the two processes (i.e. similar time courses and sensitivity to pH, ionophore, and sulfhydryl modification) suggest that they may be related in some manner.
我们之前证明，犬心脏肌浆网中GTP的水解对钙不敏感，也不支持钙和草酸盐转运到囊泡腔中。然而，在GTP作用下，钙会积累到一个对pH和离子载体敏感的区室中。在本文中，我们进一步探讨了GTP水解与GTP诱导的钙积累之间的关系。巯基试剂N-乙基马来酰亚胺（NEM；I50 = 0.2 mM）可阻止ATP和GTP诱导的钙积累。相反，NTP水解对NEM的敏感性有显著差异；GTP酶活性不受NEM影响，而ATP酶活性则受到明显抑制。相反，尽管GTP酶被ATP类似物腺苷酰亚胺二磷酸非竞争性抑制（Ki = 8 microM），并被GTP类似物鸟苷酰亚胺二磷酸竞争性抑制（Ki = 60 microM），但GTP诱导的钙积累在任何浓度下都不受NTP类似物的影响。因此，GTP依赖性钙积累到心脏肌浆网对pH和离子载体敏感的区室中可能不需要GTP水解，但可能依赖于GTP结合。当NEM（Ki = 1.2 microM）阻止ATP的钙依赖性水解时，也观察到了之前报道的ATP对GTP酶的非竞争性抑制。连同腺苷酰亚胺二磷酸对GTP酶的非竞争性抑制，在NEM存在下ATP对GTP的抑制表明ATP结合可能参与了观察到的抑制作用。GTP酶活性非竞争性抑制的Ki与ATP结合到ATP酶的高亲和力催化位点相符。因此，尽管GTP诱导的钙积累与ATP依赖性钙转运有所不同，但这两个过程之间的相似性（即相似的时间进程以及对pH、离子载体和巯基修饰的敏感性）表明它们可能在某种程度上相关。
