Abstract
It has previously been shown that when the molecular species specificity of rat liver Golgi CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase was determined, using as the substrate lactosylceramide (LacCer) incorporated into liposomes prepared with rat liver Golgi lipids, the enzyme showed a pronounced variation in activity towards the various molecular species of LacCer (J. Lipid Res. 1989. 30: 1789-1797). In this paper, -the LacCer molecular species specificity of sialyltransferase from neuroblastoma NB2a cells was examined using five naturally occurring and three synthetic molecular species of LacCer. The enzyme activity was determined by following the formation of [14C]GM3 from CMP-[14C]neuraminic acid and individual molecular species of LacCer incorporated into liposomes. Nonspecific lipid transfer protein was included in the enzyme assay to facilitate the transfer of LacCer and other lipids between the liposomes and the membrane where sialyltransferase is located. In these enzyme assays the liposomes contained approximately 10 times more lipid phosphorus than either the microsomal fraction of NB2a cells or the Golgi fraction of rat liver. Thus, in the presence of nonspecific lipid transfer protein, the lipid composition of the membrane where sialyltransferase is located was modified to resemble the lipid composition of the liposomes. When the molecular species specificity of NB2a cell sialyltransferase was determined with LacCer incorporated into liposomes prepared with NB2a cell lipids, the enzyme showed no specificity towards the various molecular species of LacCer. However, when the molecular species specificity of NB2a cell sialyltransferase was determined with LacCer incorporated into liposomes prepared with rat liver Golgi lipids, the enzyme showed a variation in activity towards the various LacCer molecular species similar to that observed with the liver Golgi enzyme using liposomes prepared with liver Golgi lipids. Likewise, when the molecular species specificity of rat liver Golgi sialyltransferase was determined with LacCer incorporated into liposomes prepared with NB2a cell lipids, the liver enzyme then showed no specificity towards the various molecular species of LacCer. These results indicate that the lipid environment of the membrane can alter the molecular species specificity of sialyltransferase towards its lipid substrate, LacCer.
Highlights
In this paper, the LacCer molecular speciespecificity of sialyltransferase from neuroblastoma NB2a cells was examined using five naturally occurring and three synthetic molecular species of LacCer
The liposomes were incubated with the NB2a cell microsomal fraction and CMP-[14C]NeuAcin the presence of nonspecific lipid transfer protein which causes a rapid equilibration of all lipids between liposomes and Kndowaki, Gmnt, and William Molecular species specificity of sialyltransferase 907
When the sialyltransferase activity of the NB2a cell microsomal fraction was determined with each of the molecular species of LacCer incorporated into liposomes prepared with NB2a cell lipids, there was no difference in the rate of N-acetylneuraminic acid (NeuAc) a2-3Galfll-4Glc-ceramide (GM3) synthesis among the various molecular species of LacCer (Fig. 1A)
Summary
In the same buffer and centrifuged at 8,000 g for 10 min. The supernatants were combined and centrifuged at 105,000g for 60 min. 2,4-Dinitrophenylhydrazine tween 0.7 M and 1.3 M sucrose layers This fraction was hydrochloride was prepared according to the method of diluted with 50 mM Tris buffer (pH 7.4) to a sucrose concentration of approximately 0.3 hi and collected by centrifugation at 105,000g for 60 min. Lipids were extracted from rat liver Golgi-rich fraction prepared as described above. The brain lipids in the Folch lower-phase were further separated into individual phospholipid classes on a silica column by HPLC [14] and each class was quantitated by phosphorus analysis [13]. The incubation mixture was prepared on ice with 10 nmol of LacCer in liposomes (200 nmol of lipid phosphorus), 50 pg of protein from sonicated NB2a cell microsomes or sonicated rat liver Golgi, 25-40 pg of nonspecific lipid transfer protein, and sodium cacodylate buffer. Protein was quantitated by the method of Lowry et al [19] with bovine serum albumin as standard
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