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Titolo:
Nitrophenylphosphate as a tool to characterize gill Na+, K+-ATPase activity in hyperregulating Crustacea
Autore:
Furriel, RPM; McNamara, JC; Leone, FA;
Indirizzi:
Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Pret, Dept Quim, BR-14040901 Ribeirao Preto, SP, Brazil Univ Sao Paulo Ribeirao Preto SP Brazil BR-14040901 BCo Preto, SP, Brazil Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Pret, Dept Biol, BR-14040901 Ribeirao Preto, SP, Brazil Univ Sao Paulo Ribeirao Preto SP Brazil BR-14040901 BCo Preto, SP, Brazil
Titolo Testata:
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR AND INTEGRATIVE PHYSIOLOGY
fascicolo: 4, volume: 130, anno: 2001,
pagine: 665 - 676
SICI:
1095-6433(200111)130:4<665:NAATTC>2.0.ZU;2-T
Fonte:
ISI
Lingua:
ENG
Soggetto:
SHRIMP MACROBRACHIUM-OLFERSII; LOBSTER HOMARUS-GAMMARUS; PHOSPHATASE-ACTIVITY; CALLINECTES-SAPIDUS; OSMOTIC REGULATION; SODIUM-PUMP; WATER; SALINITY; OSMOREGULATION; ACCLIMATION;
Keywords:
Na+; K+-ATPase; K+-phosphatase; crustacean gill microsomes; ouabain; vanadate; p-nitrophenylphosphate; Macrobrachium olfersii; kinetic characterization; ion transport;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
38
Recensione:
Indirizzi per estratti:
Indirizzo: Leone, FA Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Pret, Dept Quim, BR-14040901 Ribeirao Preto, SP, Brazil Univ Sao Paulo Ribeirao Preto SP Brazil BR-14040901 BCP, Brazil
Citazione:
R.P.M. Furriel et al., "Nitrophenylphosphate as a tool to characterize gill Na+, K+-ATPase activity in hyperregulating Crustacea", COMP BIOC A, 130(4), 2001, pp. 665-676

Abstract

The kinetic properties of a gill Na+, K+-ATPase from the freshwater shrimpMacrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) asa 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 magnesiumions (K-0.5 = 0.61 +/- 0.02 mmol l(-1), n(H) = 1.3) was found. Sodium ionshad 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 moll(-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 theNa+, 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.

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Documento generato il 29/10/20 alle ore 03:25:03