Purified tetrameric alkaline phosphatase: The effect of treatments with phosphatidylinositol phospholipase C and sodium dodecyl sulfate

Ectoenzymes in Prorocentrum minimum

“…Detergent extraction was required to solubilize the P. minimum AP for purification, suggesting that it is a membraneassociated ectoenzyme (Dyhrman and Palenik, 1997). AP is often attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor (Hawrylak et al, 1989;Raymond et al, 1992;Morita et al, 1996). However, a GPI anchor was not identified in the case of P. minimum AP (Dyhrman and Palenik, 1997).…”

Properties of Amphiphilic and Hydrophilic Forms of Alkaline Phosphatase from Human Liver

“…The enzyme is anchored to the plasma membrane by a glycosyl-phosphati- the enzyme has an apparent molecular weight of approximately twice that of the hydrophilic dimer without the PI [ 1,3]. Such apparent high molecular weights have also been reported with other isoenzymes of ALP from human fetal intestinal and placental tissues [8,9]. The hydrophobicity of the attached PI causes the enzyme to adsorb to octyl-Sepharose and be incorporated into synthetic liposomes [7,8], The amphiphilic form of the enzyme is converted to a hydro philic dimer by Pi-specific phospholipase C (PI-PLC) [3].…”

“…Such apparent high molecular weights have also been reported with other isoenzymes of ALP from human fetal intestinal and placental tissues [8,9]. The hydrophobicity of the attached PI causes the enzyme to adsorb to octyl-Sepharose and be incorporated into synthetic liposomes [7,8], The amphiphilic form of the enzyme is converted to a hydro philic dimer by Pi-specific phospholipase C (PI-PLC) [3]. In this report we compare sev eral catalytic and physical properties of the amphiphilic and hydrophilic forms of liver ALP, and examine the quaternary structure of the amphiphilic form.…”

“…However, our attempts to demonstrate such an association were unsuc cessful. Previously we reported that low con centrations of SDS seemed to cause a disso ciation of apparent aggregates of amphi philic dimers [8], However, these experi ments are probably best interpreted as the SDS binding to the amphiphilic form in such a way as to negate the effect of the hydrophobic PI on the electrophoretic migration and gel permeation elution volume such that it behaves similarly to the hydrophilic form. We were also unable to detect any significant amount of 3H-labelled Triton X-100 binding to amphiphilic ALP.…”

“…Hybridization of Amphiphilic Placental and Liver ALP Amphiphilic placental enzyme was purified as de scribed [8], and then labelled with [125I]TID as fol lows. Triton X-100 was removed from the pure en zyme by recovering the aqueous layer from a 10-min butanol extraction of the enzyme [1], [125I]TID (5 pi, containing 50 pCi, diluted in 45 pi of 50 mmol/1 Tris, 0.25 mol/1 NaCl, 1.0 mmol/1 MgCL, 0.1 mmol/1 ZnCL, pH 7.5) was added to 1 ml of the enzyme at approximately 60,000 U/l.…”