Characterization of an ecto-phosphatase activity in malpighian tubules of hematophagous bug Rhodnius prolixus
Characterization of an ecto-phosphatase activity in malpighian tubules of hematophagous bug Rhodnius prolixus
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DOI：
10.1002/arch.20013
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发表时间：
2004-09-01
影响因子：
2.2
通讯作者：
Meyer-Fernandes, JR
中科院分区：
文献类型：
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作者：
Ferraro, RB;Sousa, JL;Meyer-Fernandes, JR
我们对长红猎蝽完整马氏管外表面存在的一种磷酸酶活性进行了表征。这种磷酸酶以3.38 ± 0.07纳摩尔 Pi×毫克⁻¹×分钟⁻¹的速率水解底物对硝基苯磷酸酯。从pH 6.4到7.6，随着pH值升高，磷酸酶活性降低，在此pH范围内，马氏管细胞完整性至少能维持1小时。酸性磷酸酶的经典抑制剂，如钼酸铵、氟化物、钒酸盐、mpV - PIC和bpV - PHEN，引起了不同的抑制模式。这种外磷酸酶对p - NPP的表观Km为1.67 ± 0.34毫摩尔，Vmax为5.71 ± 0.37纳摩尔 Pi×毫克⁻¹×分钟⁻¹。氯化锌抑制了78.2%的外磷酸酶活性，Ki为0.35毫摩尔。这种抑制可通过与半胱氨酸和谷胱甘肽（GSH）一起孵育而逆转，但与二硫苏糖醇（DTT）、丝氨酸和氧化型谷胱甘肽（GSSG）一起孵育则不能，这表明半胱氨酸残基对酶活性很重要。吸血3天后磷酸酶活性增加141%，2天后恢复到基础水平。这些结果表明，外磷酸酶活性可能参与一种利尿机制，这在吸血后的最初几天对长红猎蝽体内稳态的控制至关重要。（C）2004威利 - 利斯公司
We have characterized a phosphatase activity present on the external surface of intact Malpighian tubules in Rhodnius prolixus. This phosphatase hydrolyses the substrate p-nitrophenyl phosphate at a rate of 3.38 +/- 0.07 nmol Pi x mg(-1) x min(-1). Phosphatase activity decreased with the increase of the pH from 6.4 to 7.6 pH, a range in which tubules cellular integrity was maintained for at least 1 h. Classical inhibitors of acid phosphatase, such as ammonium molybdate, fluoride, vanadate, mpV-PIC, and bpV-PHEN, caused different patters of inhibition. The ecto-phosphatase present an apparent K-m of 1.67 +/- 0.34 mM and V-max of 5.71 +/- 0.37 nmol Pi x mg(-1) x min(-1) for p-NPP. Zinc chloride inhibited 78.2% of ecto-phosphatase activity, with Ki of 0.35 mM. Such inhibition was reversed by incubation with cysteine and GSH, but not DTT, serine, and GSSG, showing that cysteine residues are important for enzymatic activity. Phosphatase activity increased 141% three days after blood meal, and returned to basal levels 2 days later. These results suggest that ecto-phosphatase activity could be involved in a diuretic mechanism, essential in the initial days after a blood meal for the control of Rhodnius homeostasis. (C) 2004 Wiley-Liss, Inc.