Comparative Biochemistry and Physiology Part B 126 (2000) 303 – 315 Characterization of (Na + ,K + )-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii R.P.M. Furriel a , J.C. McNamara b , F.A. Leone a, * a Departamento de Quı ´mica, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Uni6ersidade de Sa˜o Paulo, Ribeira˜o Preto 14040 -901, SP, Brazil b Departamento de Biologia, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Uni6ersidade de Sa˜o Paulo, Ribeira˜o Preto 14040 -901, SP, Brazil Received 7 September 1999; received in revised form 10 February 2000; accepted 25 February 2000 Abstract To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na + ,K + )-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na + , K + )-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na + ,K + )-ATPase activity. Western blotting showed that M. olfersii gill contains a single a-subunit isoform of about 110 kDa. The (Na + , K + )-ATPase hydrolyzed ATP with Michaelis – Menten kinetics with K 0.5 =165 95 mM and V max =686.1 924.7 U mg -1 . Stimulation by potassium (K 0.5 =2.4 90.1 mM) and magnesium ions (K 0.5 =0.76 90.03 mM) also obeyed Michaelis – Menten kinetics, while that by sodium ions (K 0.5 =6.0 90.2 mM) exhibited site – site interactions (n =1.6). Ouabain (K 0.5 =61.6 92.8 mM) and vanadate (K 0.5 =3.2 90.1 mM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A 1 . These data suggest that the (Na + ,K + )-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F 0 F 1 - and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na + ,K + )-ATPase activity may be regulated by the Na + concentration of the external medium. © 2000 Elsevier Science Inc. All rights reserved. Keywords: (Na +,K+)-ATPase; Crustacean gill; Ouabain; Vanadate; ATP; Macrobrachium; Freshwater shrimp; Microsomal fraction www.elsevier.com/locate/cbpb 1. Introduction The (Na + ,K + )-ATPase (E.C.3.6.1.37) is a P- type ATPase involved in cation transport by many vertebrate and invertebrate tissues, and is encoded by a multigene family (Skou and Es- mann, 1992; Pressley, 1996; Beauge´ et al., 1997). Although the minimum functional unit of this enzyme is a heterodimer consisting of a catalytic a-subunit and a glycosylated b-subunit (Skou and Esmann, 1992; Pressley, 1996; Beauge´ et al., Abbre6iations: AMPOL, 2-amino-2-methylpropan-1-ol; BCIP, 5-bromo-4-chloro-3-indole; DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; NBT, nitroblue tetrazolium; PEP, phosphoenolpyruvate; PGK, phosphoglycerate kinase; PK, pyruvate kinase. * Corresponding author. Tel.: +55-16-6023668; fax: +55- 16-6338151. E-mail address: fdaleone@ffclrp.usp.br (F.A. Leone) 0305-0491/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII:S0305-0491(00)00184-X