title: Calf thymus alkaline phosphatase. II. Interaction with detergents.
abstract: 1. A number of detergents were used to dissolve calf thymus plasma membranes rich in alkaline phosphatase (orthophosporic-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) activity. 2. The Stokes' radius (r) of alkaline phosphatase in each detergent was measured by gel filtraton. The size of the solubilized enzyme varied from r = 6.2 nm  in sodium cholate to r = 8.3 nm in Berol EMU-043. With N-alkylsulphates, the apparent size increased with alkyl chain length, with r = 6.4 nm (C9) and r = 7.3 nm (C12). Tween 20 failed to solubilise the enzyme. 3. The effect of each detergent on the catalytic activity of alkaline phosphatase was determined. The non-ionic detergents Triton X-100, Nonidet P-40, Berol EMU-043, Tween 20 and the zwitterionic detergent Empigen BB increased V by 10--50% without substantially altering the Km for p-nitrophenylphosphate. The bile salts sodium deoxycholate and sodium cholate decreased V and increased the apparent affinity of the enzyme for nitrophenylphosphate. Inhibition was concentration-dependent up to the critical micellar concentration, above which it remained constant (deoxycholate, 33% cholate, 76%). Alkylsulphates (C8-12) had no significant inhibitory effect during 24 h at 23 degrees C. 4. Exchanging one detergent for another altered alkaline phosphatase activity to a state characteristic for the second detergent, e.g. the activity of cholate-inhibited alkaline phosphatase was restored to normal levels by excess of Triton X-100 and vice versa. The inhibitory effect of deoxycholate and cholate therefore result primarily from interactions between detergent and alkaline phosphate, rather than from selective removal of lipids from the enzyme. 5. Pure lecithin, lysolecithin and an ether-deoxylysolecithin each reactivated cholate-inhibited alkaline phosphatase in a concentration-dependent fashion. Cholesterol had no effect. 6. The half-life (t1/2) of membrane-bound alkaline phosphatase at 55 degrees C was 64 min. With the exception of Berol, solubilisation in non-ionic detergents caused no marked change in this sensitivity. The enzyme became more labile in deoxycholate (t1/2) = 31 min), but less labile in cholate (t1/2 = 99 min). Alkylsulphates, which are strong denaturants, markedly increased the sensitivity of the enzyme to heat-inactivation (C8, t1/2 = 13 min; C9--12, t1/2 less than 2 min). 7. It is concluded that membrane-bound alkaline phosphatase is separated from most if not all of its neighbouring lipid moieties by these detergents, which bind to the solubilised enzyme. The number and character of molecules binding to the enzyme influence its size and shape, its susceptibility to inactivation and its catalytic activity.
mesh_terms: D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002784:Cholesterol / Q000494:pharmacology; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D010743:Phospholipids / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011487:Protein Conformation; D012995:Solubility; D013950:Thymus Gland / Q000201:enzymology
chemical_list: D002793:Cholic Acids; D003902:Detergents; D010743:Phospholipids; D011092:Polyethylene Glycols; D002784:Cholesterol; D000469:Alkaline Phosphatase