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.
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