Characterization of (Na +, K +)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology Vol. 126; no. 3; pp. 303 - 315
• Main Authors: Furriel, R.P.M., McNamara, J.C., Leone, F.A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01-07-2000
• Subjects: (Na+, K+)-ATPase; Animals; ATP; Crustacean gill; Enzyme Activation; Freshwater shrimp; Gills - enzymology; Gills - ultrastructure; Macrobrachium; Microsomal fraction; Microsomes - enzymology; Ouabain; Palaemonidae - enzymology; Palaemonidae - ultrastructure; Sodium-Potassium-Exchanging ATPase - analysis; Vanadate; BCIP, 5-bromo-4-chloro-3-indole; Ouabain; LDH, lactate dehydrogenase; AMPOL, 2-amino-2-methylpropan-1-ol; PGK, phosphoglycerate kinase; Vanadate; DMSO, dimethylsulfoxide; (Na+, K+)-ATPase; Freshwater shrimp; NBT, nitroblue tetrazolium; Macrobrachium; PK, pyruvate kinase; Microsomal fraction; PEP, phosphoenolpyruvate; ATP; Crustacean gill; GAPDH, glyceraldehyde-3-phosphate dehydrogenase
• ISSN: 1096-4959; 1879-1107
• DOI: 10.1016/S0305-0491(00)00184-X

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 α-subunit isoform of about 110 kDa. The (Na
+, K
+)-ATPase hydrolyzed ATP with Michaelis–Menten kinetics with
K
0.5=165±5 μM and
V
max=686.1±24.7 U mg
−1. Stimulation by potassium (
K
0.5=2.4±0.1 mM) and magnesium ions (
K
0.5=0.76±0.03 mM) also obeyed Michaelis–Menten kinetics, while that by sodium ions (
K
0.5=6.0±0.2 mM) exhibited site–site interactions (
n=1.6). Ouabain (
K
0.5=61.6±2.8 μM) and vanadate (
K
0.5=3.2±0.1 μM) 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
0F
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.