Nitrophenylphosphate as a tool to characterize gill Na+, K+-ATPase activity in hyperregulating Crustacea

被引:15
|
作者
Furriel, RPM
McNamara, JC
Leone, FA [1 ]
机构
[1] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Pret, Dept Quim, BR-14040901 Ribeirao Preto, SP, Brazil
[2] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Pret, Dept Biol, BR-14040901 Ribeirao Preto, SP, Brazil
来源
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR AND INTEGRATIVE PHYSIOLOGY | 2001年 / 130卷 / 04期
基金
巴西圣保罗研究基金会;
关键词
Na+; K+-ATPase; K+-phosphatase; crustacean gill microsomes; ouabain; vanadate; p-nitrophenylphosphate; Macrobrachium olfersii; kinetic characterization; ion transport;
D O I
10.1016/S1095-6433(01)00400-7
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The kinetic properties of a gill Na+, K+-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na+, K+-ATPase hydrolyzed PNPP (K+-phosphatase activity) obeying Michaelis-Menten kinetics with K-M = 1.72 +/- 0.06 mmol l(-1) and V-max = 259.1 +/- 11.6 U mg(-1). ATP was a competitive inhibitor of K+-phosphatase activity with a K-i = 50.1 +/- 2.5 mu mol l(-1). A cooperative effect for the stimulation of the enzyme by potassium (K-0.5 = 3.62 +/- 0.18 mmol l(-1); n(H) = 1.5) and magnesium ions (K-0.5 = 0.61 +/- 0.02 mmol l(-1), n(H) = 1.3) was found. Sodium ions had no effect on K+-phosphatase activity up to 1.0 mmol l(-1), but above 80 mmol l(-1) inhibited the original activity by approximately 75%. In the range of 0-10 mmol l(-1), sodium ions did not affect stimulation of the K+-phosphatase activity by potassium ions. Ouabain (K-i = 762.4 +/- 26.7 mu mol l(-1)) and orthovanadate (K-i = 0.25 +/- 0.01 mu mol l(-1)) completely inhibited the K+-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K+-phosphatase activity of the Na+, K+-ATPase alone and suggest that the use of PNPP as a substrate to characterize K+-phosphatase activity may be a. useful technique in comparative osmoregulatory studies of Na+, K+-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme. (C) 2001 Elsevier Science Inc. All rights reserved.
引用
收藏
页码:665 / 676
页数:12
相关论文
共 50 条
  • [21] Gill Na+, K+-ATPase activity as a function of size and salinity in the blue crab, Callinectes sapidus
    Li, T
    Roer, RD
    AMERICAN ZOOLOGIST, 2001, 41 (06): : 1505 - 1505
  • [22] SOLUBILIZATION OF NA+,K+-ATPASE
    ESMANN, M
    METHODS IN ENZYMOLOGY, 1988, 156 : 72 - 79
  • [23] VANADIUM AND NA+, K+-ATPASE
    MATSUI, H
    SEIKAGAKU, 1981, 53 (04): : 246 - 250
  • [24] Gill Morphology and Na+/K+-ATPase Activity of Gobionellus oceanicus (Teleostei: Gobiidae) in an Estuarine System
    Helena Rachel da Mota Araujo
    Marisa Narciso Fernandes
    André Luis da Cruz
    Biological Trace Element Research, 2019, 187 : 526 - 535
  • [25] INHIBITION OF GILL NA+ K+-ATPASE ACTIVITY IN DRAGONFLY LARVA, PANTALA-FLAVESENS, BY ENDOSULFAN
    YADWAD, VB
    KALLAPUR, VL
    BASALINGAPPA, S
    BULLETIN OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY, 1990, 44 (04) : 585 - 589
  • [26] NA+, K+-ATPASE ISOLATION
    KRAVTSOV, OV
    UKRAINSKII BIOKHIMICHESKII ZHURNAL, 1976, 48 (06): : 769 - 780
  • [27] INHIBITORS OF THE NA+, K+-ATPASE
    MACGREGOR, SE
    WALKER, JM
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-PHARMACOLOGY TOXICOLOGY & ENDOCRINOLOGY, 1993, 105 (01): : 1 - 9
  • [28] REDISTRIBUTED APICAL NA+,K+-ATPASE RETAINS NA+ PUMPING ACTIVITY
    MOLITORIS, BA
    CLINICAL RESEARCH, 1992, 40 (02): : A317 - A317
  • [29] ASTROCYTE NA+ CHANNELS ARE REQUIRED FOR MAINTENANCE OF NA+/K+-ATPASE ACTIVITY
    SONTHEIMER, H
    FERNANDEZMARQUES, E
    ULLRICH, N
    PAPPAS, CA
    WAXMAN, SG
    JOURNAL OF NEUROSCIENCE, 1994, 14 (05): : 2464 - 2475
  • [30] TRANSPORT NA+, K+-ATPASE
    不详
    BIOKHIMIYA, 1974, 39 (01): : 235 - &