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Inhibition of Na,K-ATPase by cadmium: different mechanisms in different species
E Kinne-Saffran1, M Hülseweh, C Pfaff
1Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Abstract:
The mechanism of action of Cd on Na,K-ATPase was investigated in two "classical" model systems, the shark rectal gland and rabbit kidney outer medulla. In lyophilized plasma membranes from dogfish rectal gland Cd inhibited Na,K-ATPase activity after 30 min of preincubation with an I50 of 1.3 x 10(-5) M. K-Dependent p-nitrophenylphosphatase (pNPPase) activity was inhibited 50% by Cd at 9.4 x 10(-6) M. Neither Na nor K altered the interaction of the enzyme with Cd. Decreasing the ATP concentration, however, lowered the apparent sensitivity of Na,K-ATPase to Cd. The inhibitory effect was also significantly reduced when the Mg concentration present during the preincubation was increased from 0.5 to 6.0 mM. The apparent Cd sensitivity of the K-dependent pNPPase is lower at 10 mM Mg than at 1 mM Mg. In initial rate experiments 4 x 10(-5) M Cd increased the apparent Km of the enzyme for Mg significantly from 0.88 +/- 0.29 mM to 1.73 +/- 0.3 mM whereas the Vmax (167 +/- 32 mumol/min x mg protein compared to 140 +/- 16 mumol/min x mg protein) remained essentially unchanged. In lyophilized rabbit kidney outer medulla, Cd was found to inhibit Na,K-ATPase activity with an I50 of 1.9 x 10(-5) M. K-Dependent pNPPase was inhibited 50% under identical conditions by Cd at a nominal concentration of 2.1 x 10(-4) M. Increasing K concentrations protected the enzyme from the inhibitory action of Cd as indicated by a 10-fold decrease in sensitivity of pNPPase when the K concentration was increased from 1 to 20 mM. K, 20 mM, delayed also the onset of inhibition by about 15 min at 37 degrees C. These studies suggest that the mode of action of Cd on Na,K-ATPase varies in different species. In rectal gland Cd competes with a Mg site (or sites) critically involved in ATP and pNPP hydrolysis, whereas in rabbit renal medulla Cd interacts with a potassium-binding site. Differences in the protein sequence, protein conformation, and/or in the kind of protein membrane-lipid interaction might contribute to this diversity observed in the inhibitory mechanisms.
Insights
Cadmium (Cd) affects sodium-potassium ATPase (Na,K-ATPase) differently in shark rectal glands and rabbit kidneys. In shark glands, Cd competes with magnesium (Mg) for ATP hydrolysis, while in rabbit kidneys, it binds to potassium (K) sites.
Area of Science:
- Biochemistry
- Toxicology
- Cellular Physiology
Background:
- The sodium-potassium ATPase (Na,K-ATPase) is a crucial ion pump involved in maintaining cellular electrochemical gradients.
- Cadmium (Cd), a toxic heavy metal, is known to interfere with various cellular processes, including enzyme activity.
Purpose of the Study:
- To elucidate the distinct mechanisms by which Cadmium (Cd) inhibits Na,K-ATPase activity in different biological systems.
- To compare the inhibitory effects of Cd on Na,K-ATPase and K-dependent p-nitrophenylphosphatase (pNPPase) in shark rectal gland and rabbit kidney outer medulla.
Main Methods:
- Investigated Cd inhibition of Na,K-ATPase and pNPPase in lyophilized plasma membranes from shark rectal gland and rabbit kidney outer medulla.
- Assessed the influence of varying concentrations of ATP, Mg, and K on Cd's inhibitory potency.
- Conducted initial rate experiments to determine the effects of Cd on enzyme kinetics, specifically Km and Vmax for Mg.
Main Results:
- In shark rectal gland, Cd inhibited Na,K-ATPase and pNPPase, with sensitivity modulated by Mg and ATP concentrations, suggesting competition with Mg sites.
- In rabbit kidney outer medulla, Cd inhibited Na,K-ATPase and pNPPase, with K concentrations protecting the enzyme, indicating interaction with K-binding sites.
- Cd increased the apparent Km for Mg in shark rectal gland Na,K-ATPase without significantly altering Vmax.
Conclusions:
- The mechanism of Cd inhibition of Na,K-ATPase differs between species, specifically between the shark rectal gland and rabbit renal medulla.
- In the shark rectal gland, Cd appears to compete with Mg sites essential for ATP and pNPP hydrolysis.
- In the rabbit renal medulla, Cd interacts with a potassium-binding site on the Na,K-ATPase enzyme.