Regulation of beta-D-galactoside alpha 2----3 sialyltransferase activity. The effects of detergents and lysophosphatidates.
Peptide maps of Form A and Form B of porcine submaxillary gland beta Gal alpha 2----3 sialyltransferase were essentially identical, consistent with the view that the two forms are not different enzyme species but that one, the B form (Mr = 44,000) is derived from the A form (Mr = 49,000). Analysis of the sialyltransferase activity in subcellular fractions from homogenates of porcine submaxillary glands reveals that 85% of the total activity of the transferase is bound to membranes, mostly in the Golgi apparatus, and that the remainder is soluble. The relative amounts of the membrane-bound and soluble forms as well as their response to detergents suggests that they are the cellular counterparts to the A and B forms of the transferase. The activity of Form A and the membrane-bound enzyme is stimulated to similar extents by various detergents. Triton-type detergents are more effective than Brij-type. Lysophosphatidylcholine is a potent stimulator of the activity of Form A but lysophosphatidylethanolamine is without effect and lysophosphatidylserine and lysophosphatidylglycerol are inhibitory. C16-18 acyl derivatives of lysophosphatidylcholine stimulate the activity more extensively than the C14 acyl derivative, and the C12 acyl derivative is without effect. In contrast, Form B is fully active in the absence of all detergents tested although it is inactivated just as Form A by lysophosphatidylglycerol and octylglucoside. Kinetic analysis of Forms A and B reveal that detergents stimulate the activity of Form A by lowering the KD and KM of CMP-NeuAc and increasing the Vmax of the reaction. Form B in contrast, which is fully active in the absence of detergents, has kinetic parameters like those of Form A in the presence of detergent. Taken together, these results suggest that Form A of the sialyltransferase, but not Form B, contains a lipid-binding domain, and that binding of detergents or lipids to the domain modulates the activity of the enzyme.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008246:Lysophospholipids; D008970:Molecular Weight; D017830:Octoxynol; D010712:Phosphatidic Acids / Q000494:pharmacology*; D010715:Phosphatidylglycerols / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013363:Submandibular Gland / Q000201:enzymology*; D013501:Surface-Active Agents / Q000494:pharmacology*; D013552:Swine; D014166:Transferases / Q000378:metabolism*; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D003902:Detergents; D008244:Lysophosphatidylcholines; D008246:Lysophospholipids; D010712:Phosphatidic Acids; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D013501:Surface-Active Agents; C026223:lysophosphatidylglycerol; C025059:lysophosphatidylserine; D011092:Polyethylene Glycols; D017830:Octoxynol; D014166:Transferases; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase