Preliminary Studies indicate that several membrane proteins, i.e., alkaline phosphatase, acetylcholinesterase, 5'-nucleotidase, Thy-1 antigen and variant surface glycoprotein of trypanosomes, are covalently modified by phosphatidylinositol and that this modification is responsible for the attachment of these proteins to the membrane. However, since this type of covalent modification has not previously been described, detailed information on its structure and biosynthesis is not available. The proposed studies using mammalian alkaline phosphatase as a model system will provide this information. We will purify alkaline phosphatase from mammalian tissues (human placenta, bovine intestine and pig kidney) in two forms (i) a soluble form from which the diacylglycerol has been removed by treatment with a purified phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, (ii) in a hydrophobic form by a cold butanol extraction which leaves the diacylglycerol still attached to the protein. These purified proteins will be used to determine the structure of the domain on the protein which is covalently modified by phosphatidylinositol. This will be achieved by selective chemical or proteolytic cleavage of the bond between inositol and the protein, purification of the inositol containing products by HPLC and analysis by GC, GC/MS or Fast Atom Bombardment mass spectrometry. We will also develop a tissue culture labelling system specific for phosphatidylinositol covalently bound to alkaline phosphatase. This will enable us to study the biosynthesis of this unusual post-translational modification and its relationship to protein synthesis and the other types of Co- and post-translational processing (e.g., proteolysis and glycosylation) previously reported for alkaline phosphatase. The proposed studies will not only substantiate the hypothesis that phosphatidylinositol is covalently attached to a variety of membrane proteins but will also provide detailed information on its structure and biosynthesis. Since the proposed modification by phosphatidylinositol appears to be involved in the attachment of these proteins of the membrane, this information will be of fundamental importance to our understanding of membrane structure.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM035873-01
Application #
3289211
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1986-02-01
Project End
1989-01-31
Budget Start
1986-02-01
Budget End
1987-01-31
Support Year
1
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Oklahoma Medical Research Foundation
Department
Type
DUNS #
937727907
City
Oklahoma City
State
OK
Country
United States
Zip Code
73104
Du, Xiaohan; Cai, Jiewei; Zhou, Jian-zhong et al. (2002) Tolerance of glycosylphosphatidylinositol (GPI)-specific phospholipase D overexpression by Chinese hamster ovary cell mutants with aberrant GPI biosynthesis. Biochem J 361:113-8
Du, X; Low, M G (2001) Down-regulation of glycosylphosphatidylinositol-specific phospholipase D induced by lipopolysaccharide and oxidative stress in the murine monocyte- macrophage cell line RAW 264.7. Infect Immun 69:3214-23
Low, M G; Stutz, P (1999) Inhibition of the plasma glycosylphosphatidylinositol-specific phospholipase D by synthetic analogs of lipid A and phosphatidic acid. Arch Biochem Biophys 371:332-9
Li, J Y; Low, M G (1999) Studies of the role of the integrin EF-hand, Ca2+-binding sites in glycosylphosphatidylinositol-specific phospholipase D: reduced expression following mutagenesis of residues predicted to bind Ca2+. Arch Biochem Biophys 361:142-8
Xie, M; Low, M G (1995) Streptolysin-O induces release of glycosylphosphatidylinositol-anchored alkaline phosphatase from ROS cells by vesiculation independently of phospholipase action. Biochem J 305 ( Pt 2):529-37
Xie, M; Low, M G (1994) Identification and characterization of an ecto-(lyso)phosphatidic acid phosphatase in PAM212 keratinocytes. Arch Biochem Biophys 312:254-9
Misra, K B; Kim, K C; Cho, S et al. (1994) Purification and characterization of adipocyte heparan sulfate proteoglycans with affinity for lipoprotein lipase. J Biol Chem 269:23838-44
Xie, M; Low, M G (1994) Expression and secretion of glycosylphosphatidylinositol-specific phospholipase D by myeloid cell lines. Biochem J 297 ( Pt 3):547-54
Sillence, D J; Low, M G (1994) Hydrolysis of cell surface inositol phospholipid leads to the delayed stimulation of phosphatidylinositol synthesis in bovine aortic endothelial cells. Biochim Biophys Acta 1224:247-54
Wong, Y W; Low, M G (1994) Biosynthesis of glycosylphosphatidylinositol-anchored human placental alkaline phosphatase: evidence for a phospholipase C-sensitive precursor and its post-attachment conversion into a phospholipase C-resistant form. Biochem J 301 ( Pt 1):205-9

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