Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. I. Inhibition by N-ethylmaleimide and trypsin.
We have shown previously (Brooker, R.J., and Slayman, C.W. (1982) J. Biol. Chem. 257, 12051-12055; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 222-226) that the plasma membrane [H+]-ATPase of Neurospora crassa is inhibited by N-ethylmaleimide (NEM), which reacts at an essential nucleotide-protectable site on the Mr = 104,000 polypeptide. The present study demonstrates that Mg2+ has a biphasic effect on NEM inhibition. At low concentrations (0.01-0.1 mM, Mg2+ decreases the sensitivity of the enzyme to NEM, while at high concentrations (greater than 1 mM), it enhances sensitivity. These effects are seen in the presence or absence of nucleotides (ATP, ADP). Mg2+ also acts in a concentration-dependent way to influence the degradation of the ATPase by trypsin. Low concentrations of Mg2+ have little or no effect on tryptic inactivation of ATPase activity or on the disappearance of the Mr = 104,000 polypeptide and the stepwise appearance of Mr = 100,000 and 91,000 tryptic fragments. High concentrations of Mg2+ decrease the rate of inactivation, and a new fragment of Mr = 98,000 is seen. Taken together, the NEM and trypsin results indicate that the Neurospora [H+]-ATPase possesses high and low affinity Mg2+ binding sites which affect the conformation of the enzyme. The divalent cation specificity of the sites has also been investigated. Co2+, Mn2+, and (to a lesser extent) Ni2+ mimic the behavior of Mg2+, but Ca2+ has a different effect, at least at the high affinity site. It appears to bind to that site, based on its ability to inhibit ATP hydrolysis (in the presence of Mg2+), but does not offer protection against NEM inhibition. The results suggest a way in which Ca2+ may serve as a physiological regulator of the ATPase.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D015636:Magnesium Chloride; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D006180:Proton-Translocating ATPases; D014357:Trypsin / Q000378:metabolism*
D002413:Cations, Divalent; D010446:Peptide Fragments; D015636:Magnesium Chloride; D000244:Adenosine Diphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D008274:Magnesium; D005033:Ethylmaleimide