Role of the enzyme calmodulin-binding domain in membrane association and phospholipid inhibition of endothelial nitric oxide synthase

Richard C Venema, H. S. Sayegh, J. F. Arnal, D. G. Harrison

Research output: Contribution to journalArticle

96 Citations (Scopus)

Abstract

Endothelial nitric oxide synthase (eNOS) is a calmodulin (CaM)-dependent, membrane-associated, myristoylated enzyme, which has an important role in regulation of vascular tone and platelet aggregation. In this study, wild- type and mutant forms of bovine eNOS were overexpressed in a baculovirus/Sf9 insect cell system and examined for interactions with membrane phospholipids. Purified wild-type eNOS binds to pure anionic phospholipid vesicles but not to neutral phospholipid vesicles, demonstrating that eNOS attachment to lipid bilayers requires electrostatic as well as hydrophobic interactions. Moreover, catalytic activity of the enzyme is patently inhibited by anionic phospholipids, notably phosphatidylserine (PS), but not by neutral phospholipids. eNOS activity is also significantly inhibited upon enzyme binding to biological membranes isolated from cultured cells. Binding of eNOS to PS vesicles prevents subsequent binding of the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not affected by site-specific mutation of the myristic acid acceptor site in the enzyme. Deletional mutation of the eNOS CaM-binding domain, however, results in loss of binding capacity of the enzyme not only for CaM-Sepharose but also for PS vesicles. Furthermore, removal of the CaM-binding domain converts eNOS from a membrane to a cytosolic protein when the enzyme is expressed in Sf9 cells. These data demonstrate that electrostatic interactions between anionic membrane phospholipids and basic residues in the eNOS CaM-binding domain are important for enzyme membrane association. Membrane association can thus function to inhibit eNOS catalytic activity by interfering with the interaction of the enzyme with calmodulin.

Original languageEnglish (US)
Pages (from-to)14705-14711
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number24
DOIs
StatePublished - Jan 1 1995

Fingerprint

Nitric Oxide Synthase Type III
Calmodulin
Phospholipids
Membranes
Enzymes
Phosphatidylserines
Sf9 Cells
Static Electricity
Sepharose
Catalyst activity
Biological membranes
Mutation
Lipid bilayers
Baculoviridae
Myristic Acid
Lipid Bilayers
Platelets
Coulomb interactions
Hydrophobic and Hydrophilic Interactions
Platelet Aggregation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Role of the enzyme calmodulin-binding domain in membrane association and phospholipid inhibition of endothelial nitric oxide synthase. / Venema, Richard C; Sayegh, H. S.; Arnal, J. F.; Harrison, D. G.

In: Journal of Biological Chemistry, Vol. 270, No. 24, 01.01.1995, p. 14705-14711.

Research output: Contribution to journalArticle

Venema, Richard C ; Sayegh, H. S. ; Arnal, J. F. ; Harrison, D. G. / Role of the enzyme calmodulin-binding domain in membrane association and phospholipid inhibition of endothelial nitric oxide synthase. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 24. pp. 14705-14711.
@article{41fa1656dfd5428fb1b9386367357675,
title = "Role of the enzyme calmodulin-binding domain in membrane association and phospholipid inhibition of endothelial nitric oxide synthase",
abstract = "Endothelial nitric oxide synthase (eNOS) is a calmodulin (CaM)-dependent, membrane-associated, myristoylated enzyme, which has an important role in regulation of vascular tone and platelet aggregation. In this study, wild- type and mutant forms of bovine eNOS were overexpressed in a baculovirus/Sf9 insect cell system and examined for interactions with membrane phospholipids. Purified wild-type eNOS binds to pure anionic phospholipid vesicles but not to neutral phospholipid vesicles, demonstrating that eNOS attachment to lipid bilayers requires electrostatic as well as hydrophobic interactions. Moreover, catalytic activity of the enzyme is patently inhibited by anionic phospholipids, notably phosphatidylserine (PS), but not by neutral phospholipids. eNOS activity is also significantly inhibited upon enzyme binding to biological membranes isolated from cultured cells. Binding of eNOS to PS vesicles prevents subsequent binding of the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not affected by site-specific mutation of the myristic acid acceptor site in the enzyme. Deletional mutation of the eNOS CaM-binding domain, however, results in loss of binding capacity of the enzyme not only for CaM-Sepharose but also for PS vesicles. Furthermore, removal of the CaM-binding domain converts eNOS from a membrane to a cytosolic protein when the enzyme is expressed in Sf9 cells. These data demonstrate that electrostatic interactions between anionic membrane phospholipids and basic residues in the eNOS CaM-binding domain are important for enzyme membrane association. Membrane association can thus function to inhibit eNOS catalytic activity by interfering with the interaction of the enzyme with calmodulin.",
author = "Venema, {Richard C} and Sayegh, {H. S.} and Arnal, {J. F.} and Harrison, {D. G.}",
year = "1995",
month = "1",
day = "1",
doi = "10.1074/jbc.270.24.14705",
language = "English (US)",
volume = "270",
pages = "14705--14711",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "24",

}

TY - JOUR

T1 - Role of the enzyme calmodulin-binding domain in membrane association and phospholipid inhibition of endothelial nitric oxide synthase

AU - Venema, Richard C

AU - Sayegh, H. S.

AU - Arnal, J. F.

AU - Harrison, D. G.

PY - 1995/1/1

Y1 - 1995/1/1

N2 - Endothelial nitric oxide synthase (eNOS) is a calmodulin (CaM)-dependent, membrane-associated, myristoylated enzyme, which has an important role in regulation of vascular tone and platelet aggregation. In this study, wild- type and mutant forms of bovine eNOS were overexpressed in a baculovirus/Sf9 insect cell system and examined for interactions with membrane phospholipids. Purified wild-type eNOS binds to pure anionic phospholipid vesicles but not to neutral phospholipid vesicles, demonstrating that eNOS attachment to lipid bilayers requires electrostatic as well as hydrophobic interactions. Moreover, catalytic activity of the enzyme is patently inhibited by anionic phospholipids, notably phosphatidylserine (PS), but not by neutral phospholipids. eNOS activity is also significantly inhibited upon enzyme binding to biological membranes isolated from cultured cells. Binding of eNOS to PS vesicles prevents subsequent binding of the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not affected by site-specific mutation of the myristic acid acceptor site in the enzyme. Deletional mutation of the eNOS CaM-binding domain, however, results in loss of binding capacity of the enzyme not only for CaM-Sepharose but also for PS vesicles. Furthermore, removal of the CaM-binding domain converts eNOS from a membrane to a cytosolic protein when the enzyme is expressed in Sf9 cells. These data demonstrate that electrostatic interactions between anionic membrane phospholipids and basic residues in the eNOS CaM-binding domain are important for enzyme membrane association. Membrane association can thus function to inhibit eNOS catalytic activity by interfering with the interaction of the enzyme with calmodulin.

AB - Endothelial nitric oxide synthase (eNOS) is a calmodulin (CaM)-dependent, membrane-associated, myristoylated enzyme, which has an important role in regulation of vascular tone and platelet aggregation. In this study, wild- type and mutant forms of bovine eNOS were overexpressed in a baculovirus/Sf9 insect cell system and examined for interactions with membrane phospholipids. Purified wild-type eNOS binds to pure anionic phospholipid vesicles but not to neutral phospholipid vesicles, demonstrating that eNOS attachment to lipid bilayers requires electrostatic as well as hydrophobic interactions. Moreover, catalytic activity of the enzyme is patently inhibited by anionic phospholipids, notably phosphatidylserine (PS), but not by neutral phospholipids. eNOS activity is also significantly inhibited upon enzyme binding to biological membranes isolated from cultured cells. Binding of eNOS to PS vesicles prevents subsequent binding of the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not affected by site-specific mutation of the myristic acid acceptor site in the enzyme. Deletional mutation of the eNOS CaM-binding domain, however, results in loss of binding capacity of the enzyme not only for CaM-Sepharose but also for PS vesicles. Furthermore, removal of the CaM-binding domain converts eNOS from a membrane to a cytosolic protein when the enzyme is expressed in Sf9 cells. These data demonstrate that electrostatic interactions between anionic membrane phospholipids and basic residues in the eNOS CaM-binding domain are important for enzyme membrane association. Membrane association can thus function to inhibit eNOS catalytic activity by interfering with the interaction of the enzyme with calmodulin.

UR - http://www.scopus.com/inward/record.url?scp=0029003452&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029003452&partnerID=8YFLogxK

U2 - 10.1074/jbc.270.24.14705

DO - 10.1074/jbc.270.24.14705

M3 - Article

C2 - 7540177

AN - SCOPUS:0029003452

VL - 270

SP - 14705

EP - 14711

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 24

ER -