HYDROLYSIS OF MEMBRANE-BOUND LIVER ALKALINE-PHOSPHATASE BY GPI-PLD REQUIRES BILE-SALTS

Circulating liver plasma membrane fragments (LPMF) were purified from human serum by means of a monoclonal antileucine aminopeptidase antibo dy, AD-1. This was done by immunoaffinity chromatography or by incubat ing the sera with AD-1-coated nitrocellulose disks. Alkaline phosphata se (ALP, EC 3.1.3.1) is bound to these LPMF through a glycosylphosphat idylinositol (GPI) anchor and is referred to as membrane-bound liver A LP (Mem-LiALP). Low concentrations of Triton X-100 or high bile salt c oncentrations released GPI anchor-bearing LiALP (Anch-LiALP) from puri fied LPMF; once released, Anch-LiALP was slowly and progressively conv erted to hydrophilic dimeric LiALP [soluble LiALP (Sol-LiALP)], free f rom its GPI anchor. Low levels of GPI-specific phospholipase D (GPI-PL D) activity were measured in the pure LPMF. Apparently, this membrane- associated GPI-PLD was released by the action of detergents and contri buted to the spontaneous conversion of Anch-LiALP to Sol-LiALP. In the absence of detergents, GPI-PLD had little effect on Mem-LiALP, both i n purified form as well as in serum. In vitro, isolated Anch-LiALP was converted to Sol-LiALP by both GPI-specific phospholipase C and GPI-P LD. Sol-LiALP in serum, however, appeared to be the product of GPI-PLD activity only. Five- to tenfold higher concentrations of Triton X-100 were needed to release Anch-LiALP from LPMF in serum, compared with t hose required in a solution of purified LPMF. In serum, as well as in purified conditions, only a small range of detergent or bile salt conc entrations permitted the conversion of Mem-LiALP to Sol-LiALP. A model is proposed for the release in the circulation of Mem-LiALP, Anch-LiA LP, and Sol-LiALP, involving both LPMF-associated GPI-PLD and liver si nusoid bile salts.