Identification of the membrane-binding domain of rat liver CTP:phosphocholine cytidylyltransferase using chymotrypsin proteolysis.
Limited chymotrypsin proteolysis of CTP:phosphocholine cytidylyltransferase (CT; EC 2.7.7.15) produced several distinct fragments which were mapped to the N terminus of CT using antibodies directed against the N and C terminus and the conserved central domain. A time course of chymotrypsin proteolysis showed a progression in digestion as follows: 42-->39-->35-->30-->28-->26 kDa. The binding of CT and of the chymotrypsin fragments to lipid vesicles was assessed by floatation analysis. The ability of the fragments to bind to activating lipid vesicles correlated with the presence of a putative amphipathic alpha-helix, helix-1, between residues 236 and 293. Fragments lacking this helix could, however, bind to phosphatidylcholine/sphingosine vesicles, which inhibit CT activity, and were capable of dimer formation. The degree of resistance to chymotrypsin degradation increased when CT was bound to the strongly activating lipid vesicles phosphatidylcholine/oleic acid (1:1) and phosphatidylcholine/phosphatidylglycerol (1:1). Conversion of the 39- and 35-kDa fragments, which contain the intact helix-1, to the 30-, 28-, and 26-kDa bands, which lack helix-1, required longer proteolysis times, suggesting that this helical domain is more shielded from solvent upon membrane binding. These results support the theory that CT has a bipartite tertiary structure composed of a globular N-terminal domain and an extended C-terminal domain and that CT interacts with membranes via its putative amphipathic helix which intercalates into the membrane bilayer of activating phospholipids.
Craig
L
L
Department of Chemistry/Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Johnson
J E
JE
Cornell
R B
RB
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D019916:Choline-Phosphate Cytidylyltransferase; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008081:Liposomes; D008099:Liver / Q000201:enzymology*; D008961:Models, Structural; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D017433:Protein Structure, Secondary; D051381:Rats; D014162:Transfection
D008081:Liposomes; D010446:Peptide Fragments; D010743:Phospholipids; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D002918:Chymotrypsin