Linked Life Data

10551860

Source:http://linkedlifedata.com/resource/pubmed/id/10551860

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
46
pubmed:dateCreated
2000-1-3
pubmed:abstractText
Prostaglandin endoperoxide H synthases 1 and 2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs. Both isozymes are integral membrane proteins but lack transmembrane domains. X-ray crystallographic studies have led to the hypothesis that PGHS-1 and -2 associate with only one face of the membrane bilayer through a novel, monotopic membrane binding domain (MBD) that is comprised of four short, consecutive, amphipathic alpha-helices (helices A-D) that include residues 74-122 in ovine PGHS-1 (oPGHS-1) and residues 59-108 in human PGHS-2 (hPGHS-2). Previous biochemical studies from our laboratory showed that the MBD of oPGHS-1 lies somewhere between amino acids 25 and 166. In studies reported here, membrane-associated forms of oPGHS-1 and hPGHS-2 were labeled using the hydrophobic, photoactivable reagent 3-trifluoro-3-(m-[(125)I]iodophenyl)diazirine, isolated, and cleaved with AspN and/or GluC, and the photolabeled peptides were sequenced. The results establish that the MBDs of oPGHS-1 and hPGHS-2 reside within residues 74-140 and 59-111, respectively, and thus provide direct provide biochemical support for the hypothesis that PGHS-1 and -2 do associate with membranes through a monotopic MBD. We also prepared HelA, HelB, and HelC mutants of oPGHS-1, in which, for each helix, three or four hydrophobic residues expected to protrude into the membrane were replaced with small, neutral residues. When expressed in COS-1 cells, HelA and HelC mutants exhibited little or no catalytic activity and were present, at least in part, as misfolded aggregates. The HelB mutant retained about 20% of the cyclooxygenase activity of native oPGHS-1 and partitioned in subcellular fractions like native oPGHS-1; however, the HelB mutant exhibited an extra site of N-glycosylation at Asn(104). When this glycosylation site was eliminated (HelB/N104Q mutation), the mutant lacked cyclooxygenase activity. Thus, our mutational analyses indicate that the amphipathic character of each helix is important for the assembly and folding of oPGHS-1 to a cyclooxygenase active form.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
12
pubmed:volume
274
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
32936-42
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:10551860-Amino Acid Sequence, pubmed-meshheading:10551860-Animals, pubmed-meshheading:10551860-Azirines, pubmed-meshheading:10551860-Binding Sites, pubmed-meshheading:10551860-COS Cells, pubmed-meshheading:10551860-Cyclooxygenase 1, pubmed-meshheading:10551860-Cyclooxygenase 2, pubmed-meshheading:10551860-Glycosylation, pubmed-meshheading:10551860-Humans, pubmed-meshheading:10551860-Isoenzymes, pubmed-meshheading:10551860-Membrane Proteins, pubmed-meshheading:10551860-Models, Molecular, pubmed-meshheading:10551860-Molecular Sequence Data, pubmed-meshheading:10551860-Peptide Fragments, pubmed-meshheading:10551860-Peroxidases, pubmed-meshheading:10551860-Photoaffinity Labels, pubmed-meshheading:10551860-Prostaglandin-Endoperoxide Synthases, pubmed-meshheading:10551860-Protein Folding, pubmed-meshheading:10551860-Protein Structure, Secondary, pubmed-meshheading:10551860-Sheep, pubmed-meshheading:10551860-Solubility, pubmed-meshheading:10551860-Transfection
pubmed:year
1999
pubmed:articleTitle
The membrane binding domains of prostaglandin endoperoxide H synthases 1 and 2. Peptide mapping and mutational analysis.
pubmed:affiliation
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.