Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

Abstract

The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a „nerve ending fraction” consisting of at least 85% well‐preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a „synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na+‐K+ ATPase, 5′‐nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the „nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone‐insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5‐ and 0.7‐fold that in the „nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P2 fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the „synaptosomal membrane fraction”, referred to the „nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane‐bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds‐fetuin. The Km values for CMP‐NeuNAc were different using lactosylceramide and ds‐fetuin as acceptor substrates (0.57 and 0.135 mm, respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds‐fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds‐fetuin (and glycoprotein acceptors). Ganglioside GM3 was recognized as the product of synaptosomal membrane‐bound sialyltransferase activity working on lactosylceramide ...

9-O-Acetylation of Exogenously Added Ganglioside GD3

“…However, other studies have claimed to detect certain glycosylation activities on the plasma membrane. Preti et al (51) have characterized a plasma membrane-anchored sialyltransferase that adds sialic acid to lactosylceramide and GM1 to make GM3 and GD1a. Sialidases have also been detected on the cell surface that remove sialic acids from gangliosides (11,52,53).…”

Neobiosynthesis of Glycosphingolipids by Plasma Membrane-associated Glycosyltransferases*

“…The existence of ganglioside sialylation was previously reported in synaptosomal membrane from calf and rat brains and during the development of neuronal cell cultures (47)(48)(49). However, there was concern that cross-contamination of biochemically isolated membrane fractions may have occurred, and progress in this research area is still limited.…”

Modulation of cell functions by glycosphingolipid metabolic remodeling in the plasma membrane

“…Some information is also available about the in situ sialylation of gangliosides at the cell surface. The original report on the existence of a synaptosomal membrane sialyltransferase in calf brain (Preti et al. 1980) has been confirmed by metabolic studies in chicken embryos (Matsui et al.…”

“…About glycosphingolipids, long time ago it has been shown that synaptosomal membranes, a subset of neuronal membranes highly enriched in gangliosides, carry both a sialidase (Schengrund and Rosenberg 1970; Tettamanti et al. 1972, 1973, 1975), and a sialyltransferase (Preti et al. 1980) activity.…”