蛋白质羧基甲基化调控人体血小板的细胞内pH值。

Protein carboxyl methylation controls intracellular pH in human platelets.

作者信息

Otsuka K, Roullet C M, McDougal P, McCarron D A, Roullet J B

机构信息

Division of Nephrology, Hypertension and Clinical Pharmacology, Oregon Health Sciences University, Portland 97201-3098, USA.

出版信息

J Hypertens. 1998 Sep;16(9):1261-6. doi: 10.1097/00004872-199816090-00006.

DOI:10.1097/00004872-199816090-00006

PMID:9746112

Abstract

OBJECTIVES

Carboxyl methylation is a reversible post-translational event which regulates the function of several cellular proteins. Because the human Na+-H+ antiporter (NHE-1) possesses a C-terminal consensus sequence for carboxyl methylation, we examined the role of protein carboxyl methylation in the regulation of intracellular pH homeostasis.

DESIGN

Experiments were conducted using human platelets and N-acetyl-S-trans,trans-farnesyl-L cysteine (AFC), a specific prenylcysteine methyltransferase inhibitor. The effect of AFC on both basal intracellular pH (pHi) and on the kinetic properties of the Na+-H+ antiporter was characterized.

MATERIALS AND METHODS

pHi was determined in cell suspensions using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein tetraacetoxymethyl ester, a fluorescent pH indicator. The kinetics properties of the Na+-H+ antiporter activity were determined using platelets acidified with nigericin and challenged with varying extracellular concentrations of Na+.

RESULTS

AFC (20 micromol/l) decreased basal pHi significantly (7.047 +/- 0.011 versus 7.133 +/- 0.012 for control, P< 0.001). The acidification was dose-dependent and reached steady state 3 min after AFC addition. In the absence of extracellular Na+, the platelets were acidified to the same extent with AFC or with ethanol (control): 6.530 +/- 0.031 versus 6.532 +/- 0.031 (P= 0.97). However, upon addition of Na+, the platelets treated with AFC showed a significant decrease in the maximal value for initial pHi recovery compared with controls: 0.788 +/- 0.041 versus 0.983 +/- 0.047 pH/min (P< 0.02). AFC also increased the Hill coefficient (2.89 +/- 0.22 versus 2.14 +/- 0.16, P < 0.03), and tended to decrease K0.5, the [Na+] corresponding to half-maximal activation (51.3 +/- 1.8 versus 60.5 +/- 3.9 mmol/l, P = 0.06) of the antiporter.

CONCLUSION

Our data indicate that inhibition of carboxyl methylation reduces basal pHi and alters the kinetic properties of the Na+-H+ antiporter in human platelets, suggesting that carboxyl methylation is implicated in the regulation of intracellular pH homeostasis.

摘要

目的

羧甲基化是一种可逆的翻译后修饰事件，可调节多种细胞蛋白的功能。由于人类钠氢交换体（NHE-1）具有羧甲基化的C端共有序列，我们研究了蛋白质羧甲基化在调节细胞内pH稳态中的作用。

设计

实验使用人类血小板和N-乙酰-S-反，反-法尼基-L-半胱氨酸（AFC），一种特异性异戊烯基半胱氨酸甲基转移酶抑制剂。表征了AFC对基础细胞内pH（pHi）和钠氢交换体动力学特性的影响。

材料与方法

使用荧光pH指示剂2,7-双羧乙基-5(6)-羧基荧光素四乙酰氧基甲酯在细胞悬液中测定pHi。使用尼日利亚菌素酸化并用不同细胞外浓度的Na+刺激的血小板来测定钠氢交换体活性的动力学特性。

结果

AFC（20 μmol/l）显著降低基础pHi（对照组为7.047±0.011，AFC组为7.133±0.012，P<0.001）。酸化呈剂量依赖性，添加AFC后3分钟达到稳态。在无细胞外Na+的情况下，用AFC或乙醇（对照）处理的血小板酸化程度相同：分别为6.530±0.031和6.532±0.031（P=0.97）。然而，添加Na+后，与对照组相比，用AFC处理的血小板初始pHi恢复的最大值显著降低：分别为0.788±0.041和0.983±0.047 pH/分钟（P<0.02）。AFC还增加了希尔系数（分别为2.89±0.22和2.14±0.16，P<0.03），并倾向于降低K0.5，即对应于反转运体半最大激活的[Na+]浓度（分别为51.3±1.8和60.5±3.9 mmol/l，P=0.06）。