19 Specificity and Membrane Properties of Young Rat Brain Sialyltransferases 1
Downloaded by UNIV LAVAL on April 15, 2016 | http://pubs.acs.org Publication Date: July 31, 1980 | doi: 10.1021/bk-1980-0128.ch019
SAI-SUN NG and JOEL A. DAIN Department of Biochemistry and Biophysics, University of Rhode Island, Kingston, RI 02881
Sialyltransferases are a group of soluble or membrane bound enzymes that transfer s i a l i c acid from CMP-sialic acid to acceptor molecules. The acceptor molecules may be low molecular weight oligosaccharides or higher molecular weight glycolipids and glycoproteins. Twelve a c t i v i t i e s typical of sialyltransferases have been described and these a c t i v i t i e s probably represent eight separate and distinct enzymes (1). Our interest in rat brain s i a l y l transferases stemmed from our work on the ganglioside biosynthetic pathways. These studies by us (2,3) and others (4,5) suggested the existence of more than one pathway for the synthesis of polysialogangliosides depending on when a sialyltransferase is brought into action after other sugar residues have been added to a precursor glycolipid. These early studies had also documented that the rat brain sialyltransferases are mainly membrane-bound. This is of interest because the neuraminidases in brain tissues are also membrane bound. The neuraminidases together with gangliosides have been l o calized in the nerve ending structures (6,7). Theoretically the sialylation and desialylation cycle may mediate a cyclic reaction at a very important locale in a nerve synaptic structure. This hypothetical involvement of s i a l i c acid metabolism in synaptic transmission has gained support from several studies which have suggested a synaptic localization of the glycosyltransferases (8, 9,10,11) and from proposed theoretical models in which the sialoglycolipids are considered an important constituent in the functional units of neuronal membranes (12,13,14). It was apparent, however, that the rat brain sialyltransferases have not been sufficiently characterized for the postulation of a biological role for the sialylation-desialylation cycle. Consequently, we concentrated our efforts to characterizing the general behaviors of the sialyltransferases in their membrane environments in the rat brain. What follows is a summary of our 1
Department of Biochemistry, McGill University, Montreal, Quebec H3C 3Gl, Canada. 0-8412-0556-6/80/47-128-345$5.00/ 0 © 1980 American Chemical Society
Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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results on the properties of rat brain membrane-bound sialyltrans ferases, their sub-cellular localization and our initial attempts to solubilize and purify these enzymes. Part of the results have been published previously (15,16).
Downloaded by UNIV LAVAL on April 15, 2016 | http://pubs.acs.org Publication Date: July 31, 1980 | doi: 10.1021/bk-1980-0128.ch019
Methods In all experiments to be described, brains of 11-15 day old albino rats (Sprague-Dawley) were used. Young rats of this age were chosen because rapid accumulation of gangliosides and sialoproteins have been reported to occur around this period (17). For most studies, total brain homogenates were centrifuged at 105,000 g for 60 min and the pellet used as the enzyme source. Details of the conditions for the enzymic assays have been re ported (15,16). The four reactions below were investigated. The abbreviations for gangliosides are those proposed by Svennerholm (18) . (A)
Endogenous glycolipids Cer-Glc-Gal
^ Cer-Glc-Gal (GM) NeuNAc Cer-Glc-Gal-GalNAc-Gal (GM-ι ) Cer-Glc-Gal-GalNAc-Gal (GD ) I > I I NeuNAc NeuNAc NeuNAc (B) Exogenous glycolipid Cer-Glc-Gal-GalNAc-Gal (GM-. ) Cer-Glc-Gal-GalNAc-Gal (GD-, _ ) I ——> ι I NeuNAc NeuNAc NeuNAc (C) Endogenous glycoproteins Glycoproteins ^ Glycoproteins-NeuNAc (D) Exogenous glycoprotein Desialated (DS) fetuin ^ DS-fetuin-NeuNAc Results and Discussion Kinetic Properties of Sialyltransferases. The sialyltransferase activities with the endogenous glycoprotein and glycolipid acceptors in the standard assays (15) were linear with time for at least 60 min, while those with the exogenously added GMi and DS-fetuin were linear with time only for about 30 min (Figure 1). Activities were directly proportional to the amount of enzyme added up to 0.75 mg protein/assay (Figure 2). The enzyme activities, expressed as nmol of NeuNAc incorpor ated per 0.5 mg protein per 30 min at 37C and pH 6.3, were 0.095, 0.039, 0.17 and 0.64 with the endogenous glycolipids, the endo genous glycoproteins, the exogenous GM^ and exogenous DS-fetuin, respectively. Incorporation into the endogenous glycolipids was always higher than the incorporation into the endogenous glyco3
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Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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Rat Brain Sialyltransferase s
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Downloaded by UNIV LAVAL on April 15, 2016 | http://pubs.acs.org Publication Date: July 31, 1980 | doi: 10.1021/bk-1980-0128.ch019
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Figure 1. Effect of incubation time on sialyltransferase activities. Incorporation of NeuNAc into the following substrates was determined: X, endogenous glyco proteins; Δ , endogenous glycoproteins plus exogenous DS-fetuin; Ο , endogenous glycolipids; O, endogenous glycolipids plus exogenous GM\. (15).
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