Nonspecific lipid transfer protein in the assay of a membrane-bound enzyme CMP-N-acetyl-neuraminate:lactosylceramide sialyltransferase.
CMP-N-acetylneuraminate:lactosylceramide alpha-2,3-sialyltransferase is tightly associated with the luminal side of the Golgi membrane as is its lipid substrate, lactosylceramide. In order to understand the kinetics, properties, and regulation of this enzyme, it is necessary to alter the amount and type of substrate in the membrane while minimizing changes in the membrane environment or in the conformation of the enzyme. Therefore, nonspecific lipid transfer protein, which accelerates the transfer of phospholipids, cholesterol, and glycosphingolipids between membranes was used to study the properties and kinetics of rat liver CMP-N-acetylneuraminate:lactosylceramide sialyltransferase. These results are compared to those obtained in parallel experiments using detergent-solubilized substrate. Enzyme activity was increased four- to fivefold by transfer protein and was consistently higher than the activity measured in the presence of detergents. In contrast to the results obtained with detergents, the enzyme activity increased linearly with both Golgi protein and with incubation time for up to 60 min. The Km values for the water-soluble substrate, CMP-neuraminic acid, were virtually identical when determined in the presence of transfer protein (0.23 mM) or detergents (0.27 mM). On the other hand, the apparent Km values for the lipophilic substrate, lactosylceramide, were markedly different when determined in the presence of transfer protein (47.9 microM) or in the presence of detergents (1.2 microM). These observations suggest that transfer protein is a useful tool to study the properties and kinetics of membrane-bound enzymes when both the enzyme and substrate are components of the same membrane.
Kadowaki
H
H
Department of Medicine, Framingham Union Hospital, MA 01701.
Symanski
L A
LA
Koff
R S
RS
D000818:Animals; D002352:Carrier Proteins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D005732:Gangliosides / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D007790:Lactosylceramides / Q000378:metabolism; D008081:Liposomes / Q000032:analysis; D008099:Liver / Q000201:enzymology; D008297:Male; D010940:Plant Proteins; D051381:Rats; D011919:Rats, Inbred Strains; D012799:Sialyltransferases / Q000032:analysis*
D002352:Carrier Proteins; D003902:Detergents; D005732:Gangliosides; D007790:Lactosylceramides; D008081:Liposomes; D010940:Plant Proteins; C072087:sterol carrier proteins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; C021343:haematoside synthetase