Raymond, Frank D; (1995) Inositol specific phospholipase D in health and disease a GPI-anchor cleaving enzyme. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In recent years a novel mechanism by which proteins are anchored to cell membranes has been elucidated. In excess of 100 molecules of differing structure and function are known to be linked to the cell surface by these glycan phosphatidyl inositol anchors (GPI-anchors). These include cell adhesion molecules, complement regulatory factors, lymphocyte differentiation antigens and ectoenzymes. The relatively recently discovered circulating enzyme; inositol specific phospholipase D (PIPLD) is now known to be capable of cleaving GPI anchors. The complete elucidation of the mechanisms of action and physiological function of PIPLD will therefore have important scientific and clinical implications. In this work; the activity of PIPLD has been investigated. In addition to established methods of analysis, novel analytical systems called aqueous polymer phase systems were developed and adapted for this use. Proteins, membrane particles and cells partition in these phases on the basis of their relative hydrophobicity. Alkaline phosphatase (ALP) was used as a model substrate representative of GPI-anchored molecules to investigate the activity of PIPLD in health and disease. The mechanism of action of PIPLD was studied by conducting in vitro experiments aimed at investigating the effect of PIPLD contained in serum on cultured cells, on prepared membrane fractions and on solubilised GPI-anchor containing molecules. The serum ALP in hepatobiliary and bone disease is measured only as a raised activity however evidence shows that it is present in a range of molecular forms probably related to the specific disease and to mechanisms of production and release. The various isoforms and the mechanisms underlying their formation were investigated. The enzyme was characterised and assay systems developed to examine its physiological function and its activity in disease. Patients with cholestasis and infections were investigated. The range of PIPLD activity was measured in sera from apparently healthy subjects and shown to vary considerably with age and disease. Evidence has been provided which indicates that its principal site of synthesis is the liver and it has been shown to be of probable use as a marker of liver synthetic reserve. In addition PIPLD activity was shown to act as an acute phase reactant. These studies further emphasise the important role PIPLD plays in the metabolism of the groups of molecules linked to the cell surface by GPI anchors.

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