Document Detail


Biosynthesis and palmitoylation of endothelial nitric oxide synthase: mutagenesis of palmitoylation sites, cysteines-15 and/or -26, argues against depalmitoylation-induced translocation of the enzyme.
MedLine Citation:
PMID:  7547976     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The presence of myristoylated endothelial nitric oxide synthase (eNOS) in cytosolic fractions of bovine aortic endothelial cells (BAEC) suggests that N-myristoylation of eNOS is not sufficient for membrane localization. Therefore, we examined if posttranslational palmitoylation was another molecular signal for the membrane attachment of eNOS. Metabolic labeling showed incorporation of [3H]palmitic acid into the membrane-bound, but not the cytosolic, form of eNOS. Fatty acid analysis demonstrated the labeled fatty acid incorporated into eNOS was palmitate, linked via a hydroxylamine-labile thioester bond. Biosynthesis and turnover studies show that the turnover of palmitate is much faster than the protein itself. However, the rates of palmitoylation and depalmitoylation were not affected by bradykinin or ionomycin treatment of BAEC. To examine the contribution of palmitoylation to the membrane association of eNOS, we mutated cysteine-15 and -26. Both mutations markedly diminished palmitoylation of eNOS, but did not significantly alter its membrane association. Additionally, [3H]palmitic acid was not incorporated into nonmyristoylated mutant eNOS (G2A eNOS), suggesting that myristoylation is necessary for subsequent palmitoylation of the enzyme. Taken together, our data imply that palmitoylation does not play a major role in membrane association of eNOS and other amino acid sequences, in conjunction with N-myristoylation, are necessary and sufficient for membrane association of the enzyme.
Authors:
J Liu; G García-Cardeña; W C Sessa
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  34     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1995 Sep 
Date Detail:
Created Date:  1995-11-06     Completed Date:  1995-11-06     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  12333-40     Citation Subset:  IM    
Affiliation:
Molecular Cardiobiology Program, Yale University School of Medicine, New Haven, Connecticut 06536-0812, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Base Sequence
Biological Transport
Blotting, Western
Bradykinin / pharmacology
Calcium / metabolism
Cattle
Cell Fractionation
Cysteine / genetics
Cytosol / enzymology
Endothelium, Vascular / enzymology*
Ionomycin / pharmacology
Membranes / metabolism
Molecular Sequence Data
Mutagenesis, Site-Directed
Myristic Acid
Myristic Acids / metabolism
Nitric Oxide Synthase / metabolism*
Palmitic Acid
Palmitic Acids / metabolism*
Protein Binding
Protein Processing, Post-Translational*
Recombinant Proteins / metabolism
Signal Transduction / drug effects
Structure-Activity Relationship
Transfection
Grant Support
ID/Acronym/Agency:
F32-HL09224/HL/NHLBI NIH HHS; HL51948/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/Myristic Acids; 0/Palmitic Acids; 0/Recombinant Proteins; 52-90-4/Cysteine; 544-63-8/Myristic Acid; 56092-81-0/Ionomycin; 57-10-3/Palmitic Acid; 58-82-2/Bradykinin; 7440-70-2/Calcium; EC 1.14.13.39/Nitric Oxide Synthase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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