Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. I. Inhibition by N-ethylmaleimide and trypsin.

R. J. Brooker, C. W. Slayman

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)8827-8832
Number of pages6
JournalJournal of Biological Chemistry
Volume258
Issue number14
StatePublished - Jul 25 1983

Fingerprint

Neurospora crassa
Ethylmaleimide
Proton-Translocating ATPases
Cell membranes
Trypsin
Cell Membrane
Ions
Adenosine Triphosphatases
Nucleotides
Adenosine Triphosphate
Neurospora
Peptides
Divalent Cations
Enzymes
Adenosine Diphosphate
Conformations
Hydrolysis
Binding Sites
Degradation

Cite this

Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. I. Inhibition by N-ethylmaleimide and trypsin. / Brooker, R. J.; Slayman, C. W.

In: Journal of Biological Chemistry, Vol. 258, No. 14, 25.07.1983, p. 8827-8832.

Research output: Contribution to journalArticle

@article{3d26c491bf0b41fe85df2325a01fc58a,
title = "Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. I. Inhibition by N-ethylmaleimide and trypsin.",
abstract = "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.",
author = "Brooker, {R. J.} and Slayman, {C. W.}",
year = "1983",
month = "7",
day = "25",
language = "English (US)",
volume = "258",
pages = "8827--8832",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "14",

}

TY - JOUR

T1 - Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. I. Inhibition by N-ethylmaleimide and trypsin.

AU - Brooker, R. J.

AU - Slayman, C. W.

PY - 1983/7/25

Y1 - 1983/7/25

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0021112310&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021112310&partnerID=8YFLogxK

M3 - Article

C2 - 6223036

AN - SCOPUS:0021112310

VL - 258

SP - 8827

EP - 8832

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 14

ER -