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

Journul ( I / Neurochemisfry 35(2):281-2%, August. Raven Press. New York @ 1980 International Society for Neurochemistry

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Occurrence of S ynaptosomal

Augusto Preti, Amelia Fiorilli,

Sialyltransferase Activity in the Membranes Prepared from Calf

Brain Cortex

Adriana Lombardo, Luigi Caimi, and Guido Tettamanti

I n s t i t ~ t ~ ~ di Chimicu Biologica, Facoltd di Medicinu e Chirurgiu, Universitd di Milano, Milan, iraly

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” con- sisting of a t 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; P, 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, VIS, VIr and VIprotein relationships) and detergent requirements of the synaptosomal membrane-bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and os-fetuin. The K , 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 os-fetuin (and glycoprotein acceptors). Ganglioside GM, was rec- ognized as the product of synaptosomal membrane-bound sialyltransferase activity working on lactosylceramide as acceptor substrate.

Received January 4, 1980; accepted January 17, 1980. Address reprint requests to Prof. Guido Tettamanti, Istituto di

Chimica Biologica, FacoltA di Medicina e Chirurgia, Via Saldini, 50, 20133 Milan, Italy. acid.

Abbreviations used: AChE, Acetylcholinesterase; CMP-[4- ‘’C]NeuNAc, N-A~etyl-neuraminyl-[4-~~C]cytidinemono- phosphate; LDH, Lactate dehydrogenase; TCA, Trichloroacetic

281

282 A . PRETl ET A L .

The brain is the tissue having the highest content of sialylglycoconjugates, sialylglycolipids (gangliosides), and sialylglycoproteins (Svennerholm, 1970a,b; Wiegandt, 1971). These compounds are mainly membrane-bound and, particularly gangliosides, are fundamental components of the neuronal plasma membrane (Rahmann et al., 1976; Ledeen, 1978). The carbohydrate chains of glycoconjugates extend away from the lipid bilayer of the plasma membrane toward the external surface (Bretscher, 1973; Yamakawa and Nagai, 1978), and contribute to the chemical characterization of the glycocalix. The negative charge provided by sialic acid residues is an important feature of the glycocalix and any fluctuation of the surface sialic acid concentration can be expected to affect the functional perfor- mances of the membrane, adapting it to specific environmental requirements.

The neuronal plasma membranes contain the enzyme sialidase, which is capable of removing sialic acid from both gangliosides and sialylglycoproteins (Schengrund and Rosenberg, 1970; Tettamanti et al., 1972; 1973~) . Conversely, the presence, in the same membranes, of a sialyltransferase activity is controversial (Van den Eijnden and van Dijk, 1974; Den et al., 1975; Landa et al., 1977; Ng and Dain, 1977b). This is due to the fact that sialyltransferase, as other glycosyltransferases, is definitely carried by intracellular membrane structures, mainly Golgi apparatus (Keenan et al., 1974). Therefore the an- swer as to the occurrence of sialyltransferase also in the plasma membranes, relies on the availability of plasma membranes almost completely devoid of contaminating intracellular membranes.

In the present study this was our approach to the problem. As a source of neuronal plasma membranes we chose nerve endings (synaptosomes) which: (a) are known not to contain Golgi apparatus; (b) can be prepared at a very high degree of homogeneity and with a low contamination of intracellular structures; and (c) provide pure plasma membranes (‘ ‘synaptosomal membranes”) after hypoosmotic shock. The biochemical “markers” we used were: lactate dehydrogenase for intraterminal (“trapped”) cytosol and, therefore, for nerve endings (Mclntosh and Plummer, 1976); Na+-K+ ATPase, 5’-nucleotidase, gangliosides and sialidase for authentic plasma membranes; acetylcholinesterase for either intracellular or plasma membranes (bilocular enzyme); thiamine pyrophosphatase for Golgi apparatus and plasma membranes (bilocular enzyme) (Lane, 1968; Morgan et al., 1972); cerebroside sulphotransferase for Golgi apparatus (Sieg- rist et al., 1979); rotenone-insensitive NADPH cytochrome c reductase and rotenone-insensitive NADH cytochrome c reductase for light endoplasmic reticulum and outer mitochondria1 membranes.

MATERIALS A N D METHODS

Mu teriu 1s

All chemicals were of analytical grade. Solvents were redistilled before use. The water routinely used was freshly redistilled on a glass apparatus. N-Acetyl- neuraminyl-[4-14C]cytidinmonophosphate (CMP-14- ‘TINeuNAc) (the batches having a specific radioactivity higher than 1.5 pCi/pmol were diluted with ‘cold’ CMP- NeuNAc, to give a constant specific radioactivity of 1.5 pCi/pmol) and 3‘-phosphoadenosyl-5’ph0spho-[”~S]sul- phate (PAP-35S) (293 mCi/mmol) were purchased from New England Nuclear Corp. ; unlabelled PAPS, cytidin- triphosphate (CTP), adenosine triphosphate (ATP), N - acetylneuraminic acid (NeuNAc), rotenone, physostigmine, fetuin (from calf fetal serum), ouabain, thiamine pirophosphate, Tween 80, crystalline bovine serum albumin from Sigma Chem. Co. Galactosylceramide (from bovine brain) was a generous gift of Dr. T. Burkart (Dept. of Paediatrics, University of Bern, Switzerland). Triton CF-54 was from Rohm and Haas; Sephadex G 25 (superfine) and Ficoll (dialysed for 2 days at 2°C against redistilled water, in order to remove ion contaminants, then lyophilized) from Pharmacia; Dowex 2-X8, 200-400 mesh (prepared in acetate form according to Sven- nerholm, 1957) from BioRad; nicotinamide-adenine dinucleotide, reduced form (NADH) and nicotinamide- adenine dinucleotide phosphate, reduced form (NADPH), from Boeringher Mannheim; Instagel and Soluene 350 from Packard; Scintimix 3 from Koch-Light; silica gel precoated thin-layer plates from Merck GmbH.

Brain Sampling

Brains from 3-month-old calves were purchased from the slaughterhouse. Immediately after death, the organs were removed and transported on ice to the laboratory. Meninges and vessels were removed and grey matter from brain cortex was grossly dissected, weighed, washed in cold homogenizing solution (see below), and homogenized.

Methods

Preparation of substrates Unlabelled CMP-NeuNAc was prepared according to

Van den Eijnden and van Dijk (1972); desialylated fetuin (DS-fetuin) was prepared by chemical hydrolysis according to Spiro (1964). The preparation used in this study was desialylated over 95% with the liberation of 193 nmol of NeuNAc from each mg of fetuin. Since the molecular weight of fetuin is 45,000 there are about 200 nmol of NeuNAc acceptor sites per mg of os-fetuin. Lactosyl- ceramide was prepared from calf brain gangliosides by partial chemical hydrolysis, according to Ghidoni et al. (1976); GM, ganglioside (containing NeuNAc) was prepared from dog erythrocytes according to Handa and Yamakawa (1964) and further purified by preparative thin-layer chromatography; GM,, GD,,, GDlh, and GTIh individual gangliosides were prepared from calf brain ac-

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SIAL YLTRANSFERASE IN S YNAPTOSOMAL MEMBRANES 283

cording to Tettamanti et al. (1973b)’. The assessment of homogeneity and chemical characterization of all these glycolipids was established by the criteria stated by Ghi- doni et al. (1976). The purity of the final products was, in all cases, over 98%. Sialyllactose (a 2-3 isomer) was prepared from cow colostrum according to Ohman and Hygsted (1968).

Preparation of nerve endings and of nerve ending plasma membranes.

All work was done at 4°C; the g values are average centrifugal forces. See the flowsheet of Fig. 1.

Nrn9r endings. Our primary aim was to obtain a preparation of nerve endings with the lowest contamination of

HOMOGENAIE 10 :I l . rc.-” t 8 ” SPE

NUCLEAR 4 r FRACTION

MITOCCIJNDRIAL FRACTION 4 535009, I h

1050009 I h

FRACTION

SYNAPTOSOMAI FRACTION

a-4 - l o 5 0 0 0 1 7 0 -

r;l S Y h 4P T < i S U M A l

MEMBRANE F R 4C T ION

FIG. 1. Flow sheet of the procedure for the preparation of the synaptosomal membrane fraction from calf brain cortex. Time of centrifugation from beginning of acceleration to beginning of deceleration. SPE: 0.32~-sucrose, 1 mM-potassi- urn phosphate buffer, 0.1 mM-EDTA, pH 7.5; PE: 1 mM-potassium phosphate buffer, 0.1 mM-EDTA, pH 7.5.

I The ganglioside nomenclature of Svennerholm (197Oa) is followed.

intracellular membranes: thus we were ready to accept even a very low final yield. The procedure used for isolating nerve endings was derived from that of Morgan et al. (1971). Briefly, homogenization was performed by 12 up-and-down strokes with a motor-operated Teflon- glass homogenizer (stated radial clearance, 0.25 mm; 800 r.p.m.) using 80-90-g aliquots of grey matter and 4 vol. of the homogenizing solution (0.32 M-sucrose containing I mM-potassium phosphate buffer and 0.1 mM-EDTA, pH 7 .5 ) . The homogenate was diluted to 10% (wlv) in homogenizing solution, filtered through four layers of cheese cloth, and centrifuged at 1000 g for 15 min. The resulting pellet was washed with the same starting volume of homogenizing solution and centrifuged as above. The combined supernates were centrifuged at 11,500 g for 25 min and the pellet was washed four times with 9 vol. (each time) of homogenizing solution and centrifuged ( 1 1,500 g for 25 min). The final pellet is referred to as the “P, fraction”. Samples of the combined supernates were centrifuged at 105,000 g for 1 h and the resulting pellet is called “Microsomes”. Portions of the P2 fraction, containing the material derived from 12-15 g of starting fresh tissue, were suspended with the homogenizing solution (final volume of the mixture, 20 ml) and overlaid on 15 ml of 9% Ficollz and centrifuged at 53,500 g for 1 h (International B-60 ultracentrifuge, 110 SB rotor). The material floating at the gradient surface (constituted by myelin fragments, membranes, and small nerve endings) was collected for further processing. The remaining liquid was carefully as- pirated and the pellet (comprising nerve endings, a few membranes, mitochondria, and lysosomes) was suspended in 5-6 vol. of the homogenizing solution after superficial washing with the same solution. Aliquots of this suspension, which contained the material corresponding to 12- 15 g of starting fresh tissue, were poured on top of a 9% ( 1 1 m1)/16% (12 ml) Ficoll gradient and centrifuged at 53,500 g for 1 h. Three bands were obtained: one floating over 9% Ficoll, one at the 9%/16% interface, and a pellet.

The material floating over 9% Ficoll was combined with that floating in the previous gradient and diluted three times, and a sample, corresponding to 5-6 g of starting fresh tissue, was centrifuged at 105,000 g for 40 min. The pellet was suspended with 4-5 vol. of homogenizing solution and poured on top of a 0.8 M-sucrose solution (containing 1 mwpotassium phosphate buffer, pH 7.5, and 0.1 mM-EDTA) (25 ml) and centrifuged at 53,500 g for 2 h. The material floating at the surface was collected, diluted with 5 vol of homogenizing solution and centrifuged at 105,000 g for 40 min. The resulting pellet, containing the bulk of myelin fragments and some light membranes present in the P, fraction, is referred to as the “Myelin fraction”. The band at the 9%/16% Ficoll interface was accurately collected, diluted with 3 -4 vol. of homogenizing solution, and centrifuged at 105,000 g for 40 min. The pellet, homogeneously suspended with 25 vol. of homogenizing solution, was centrifuged at 11,500g for 25 min; the newly obtained sediment is called the “nerve ending fraction ’ ’ .

* A l l Ficoll solutions contained 0.32 M-sucrose, 1 mM- potassium phosphate buffer 0.1 mM-EDTA, pH 7.5.

J . Neurochem.. Vol. 35, N o . 2 , 1980

284 A . PRETI ET AL.

Nerve en ding plus m ( I me ni h ru ti t s ( ‘ s y n tip t os o rn u I membrunes”). The original procedures of Morgan et al. (1971) and of Gurd et al. (1974) were chosen as reference. The nerve ending fraction was suspended in 10 ml per g f resh starting t issue of 1 mM-potassium phosphate buffer plus 0.1 mM-EDTA, p H 7.5, and submitted to hypoosmotic shock according to Ledeen et al. (1976) under the conditions, specified by Venerando e t al. (1978), yielding the optimum rupturing of particles. The shocked mixture was centrifuged at 11,500 g for 20 min; the pellet was discarded and the supernate centrifuged at 105,000 g for 70 min. The pellet obtained was suspended in 1 mM-potassium phosphate buffer plus 0.1 mM- EDTA, pH 7.5 (0.25 ml/g starting fresh tissue), and centrifuged at 53,500 g for 2 h on the discontinuous sucrose density gradient: 0.4 M (7.5 m1)/0.6 M (7.5 m1)/1.0 M (15 ml). The material floating at the 0.6 ~ i 1 . 0 M interface [corresponding to the F and G subfractions of Morgan et al. (1971)l was collected, diluted with 4-5 vol. of the 1 mM-potassium phosphate buffer, pH 7.5, plus 0.1 mM- EDTA solution, and centrifuged at 105,000 g for 70 min. The pellet, suspended with 20 vol. of 1 mM-potassium phosphate buffer plus 0.1 mM-EDTA, pH 7.5, was centrifuged at 11,500 g for 15 min, the pellet being discarded. The supernate, centrifuged at 105,000 g for 70 rnin, gave a pellet which is called the “synaptosomal membrane fraction”.

An equal aliquot of nerve ending fraction, suspended with homogenizing solution (and not submit ted t o hypoosmotic treatment) was submitted to the same treatment as above and provided a preparation denomi- nated as the “synaptosomal membrane fraction without hypoosmotic treatment”. This fraction contained membranes (behaving as authentic synaptosomal membranes) present in the starting nerve ending fraction and not originated by hypoosmotic treatment.

Sialyltransferase assay The method of Ng and Dain (1977b), with some modifi-

cations, was followed. As exogenous glycolipid and glycoprotein acceptor substrates, lactosylceramide and desialylated fetuin were employed.

Assuy on endogenous uccPptor substrates. Mixtures containing, in a final volume of 100 pl, 0.6-1 .0 mg of enzyme preparation (as protein), 0.1 M-cacodylate buffer of the desired pH, 1.0 mM-CMP-NeuNAc, and a mixture of Triton C F 54 and Tween 80 (2: I w/w; 1.5-fold the amount of protein, as mg) were incubated a t 37°C for a n appropriate time (60 rnin in the routine experiments)’. The incubation was stopped by addition of 2.5 ml of ice- chilled 10% trichloroacetic acid (TCA), vigorous stirring, and centrifugation at 3500 g for 5 min. The pellet was twice washed with the same volume of 10% TCA, the supernates being discarded each time. The final washed pellet was dissolved with 2 ml of 0.01 M-potassium phosphate buffer, the pH being carefully adjusted to 7.0, and was treated with 4 vol. of tetrahydrofuran (four times), according to Tettamanti et al. (1973b). This enabled us to completely separate lipid (and glycolipid) material, in the

Detergents and lactosylceramide, when used, were dissolved in chloroform-methanol, 2: I by vol. A sample of this solution, containing the desired amount of each component, was evaporated to dryness under a stream of nitrogen in the incubation test tube.

supernate, from protein (and glycoprotein) in the pellet. The pellet was allowed to dry, and was then dissolved with 1 ml Soluene 350 (overnight, a t 40°C) and finally submitted to counting after addition of 10 ml of Instagel (“glycoprotein-bound radioactivity”).

The pooled supernates were evaporated to dryness and the residue was dissolved with 3 ml of chloroform/ methanol/water, (60:30:4.5, by vol.). The mixture was poured on a column (diameter, 0.6 cm) containing 1 g of Sephadex G 25 , superfine, previously equilibrated with chloroform/methanol/water (60:30/4.5). The column was twice washed with 1.5 ml (each time) of the same solvent system, and the pooled eluates evaporated to dryness (overnight a t 40°C). The residue, dissolved with 10 ml of Instagel, was submitted to counting (“glycolipid-bound radioactivity”). A mixture, stopped at zero time and processed identically as above, was used as the blank.

Assmv o n exogenous mcceptor substrotes. Mixtures containing, in a final volume of 100 pl, 0.1-0.3 mg of enzyme preparation (as protein), 0.1 M-cacodylate buffer, 1 mM-CMP-NeuAc, appropriate amounts of a mixture (2: 1 , by wt.) of Triton C F 54 and Tween 80 and of lactosylceramide (0.2 mM in the routine experiments) or Of DS-fetuin (3.0 mM as acceptor sites, in the routine experiments) were incubated at 37°C for a specific time (generally 60 min).

When lactosylceramide was employed, the incubations were stopped by addition of 2 ml of chloroform-methanol (2: 1 , viv), vigorous stirring ( 1 min twice), and centrifugation at 5000 g for 15 min. The pellet was washed once again with 2 ml of chloroform/methanol/water (60:30:4.5, by vol.); the two pooled supernates were submitted to column chromatography on Sephadex G 25 and the eluates were counted as described above.

When DS-fetuin was employed, the reactions were stopped, the protein separated, and the glycoprotein- bound radioactivity counted as described for the assay on endogenous substrates . The blanks were the corresponding assay mixtures incubated under the same conditions in the absence of added substrates. Radioactivity measurements were done in a Packard TriCarb 3525 spectrometer. In all cases the specific activity of sialyltransferase is expressed as c.p.m. (reaction product)/mg protein/h.

Analysis of glycolipid reaction products This was performed when lactosyl ceramide was used

as acceptor substrate, the expected reaction product being GM, ganglioside. For this purpose, t o the pooled eluates from the Sephadex G 25 column, 30 p g of cold s tandard GM,, ganglioside (dissolved in chloroform- methanol, 2.1, by vol.) was added. The mixture was evaporated t o dryness , the residue dissolved in the minimum volume of n-propanol-water ( ] : I , viv) and an aliquot subjected to thin-layer chromatography, in parallel with a mixture of reference standard gangliosides, under the following experimental conditions: solvent system, chloroform/methanol/water, (60:35:8, by vol.); 2 h run at 20-22°C; detection of the spot by exposure to iodine vapours. The lane corresponding to the incubated sample was then divided into 0.5-cm horizontal strips, and each strip was accurately scraped off, put into a counting vial, dissolved with 10 ml of toluene containing 0.4% Scintimix 3 , and counted for radioactivity.

J . Neiiruchem., Vul. 35, N u . 2 , 1980


Assay of marker enzymes Ouabain-inhibited Na+-K+ ATPase (ATP phosphohy-

drolase, EC 3.6.1.3) was assayed according to Morgan et al. (1971); acetylcholineesterase (AChE) (acetylcholine hydrolase, EC 3.1.1.7), in the presence of 3 x M- physostigmine and 0.25% Triton X- 100, according to Ellman et al. (1961); 5’-nucleotidase (5’-ribonucleotide phosphohydrolase, EC 3.1.3.5) by the method of Touster et al. (1970); thiamine pyrophosphatase (TPPase) (thiamine pyrophosphate pyrophosphohydrolase) (EC 2.5.1.3) according to Seijo et aI. (1970); cerebroside sulphotransferase (CST) 3’-phosphoadenosylsuIphate-galactosylceramide 3’-sulphotransferase, EC 2.8.2.11) according to Siegrist et al. (1977); rotenone-insensitive NADPH: cytochrome c oxidoreductase (EC 1.6.99.1) and rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3) by the method of Sottocasa et al. (1967) as modified by McIntosh and Plummer (1976). Membrane-bound sialidase (EC 3.2.1.18) was assayed as total activity (acting on endogenous and exogenous sialoderivatives) according to Tettamanti et al. (1975) using siaiyllactose as added substrate. Lactate dehydrogenase (LDH) (EC 1.1.1.27) was assayed as kee, total and occluded activity according to McIntosh and Plummer (1976). In its occluded form this enzyme has a function as a marker of the cytoplasm trapped (“occluded”) into nerve endings, in other words, of nerve endings (synaptosomes) themselves. The activity of all marker enzymes is expressed in International Units (pmol transformed substrate/min at 37°C for ATPase, 5‘-nucleotidase, AChE, sialidase, thiamine pyrophosphatase and cerebroside sulphotransferase; at 30°C for the others).

Other methods Protein was determined by the method of Lowry et al.

(1951) with bovine serum albumin as standard. Prior to analysis, proteins were precipitated by addition of 10% (final concentration) TCA, standing for 20 min at room temperature, and centrifugation.

Electron microscopy Pxeparations were centrifuged at 105,000 g for 1 h and

fixed for 12-14 h in 5% glutaraldehyde-0.1 M-sodium phosphate buffer (pH 7.4). After post-fixation with 0.1% osmic acid, the fractions were dehydrated and embedded in Araldite. Sections stained with uranyl acetate and lead citrate were examined in a Siemens Elmiscope I.

RESULTS

Morphological Features and Biochemical Characteristics of the “Synaptosomal

Membrane Fraction”

The “nerve ending fraction”, upon electron mi- croscopic examination, appeared to be constituted largely of well-preserved nerve endings, with very few mitochondria, membranes and myelin fragments (Fig. 2, a). The homogeneity, as nerve endings, was estimated to be higher than 85%. The “synaptosomal membrane fraction” was made up essentially of membranes and membrane vesicles,

with some junctional processes (Fig. 2, b). We carefully examined this fraction at very high magnification looking for structures of vesicular or cis- ternal shape containing granulo-floccular electron- dense material, described (Siegrist et al., 1979) as derivates of the Golgi apparatus in the intact neuronal cell. None of these structures could be detected.

A comparison of the content of significant biochemical parameters in the “nerve ending fraction” and in the “synaptosomal membrane fraction” is set out in Table 1. With reference to the “nerve ending fraction”, the “synaptosomal membrane fraction” carried: (a) practically no occluded LDH activity, excluding the presence of unruptured nerve endings; (b) specific activities of ATPase, 5‘- nucleotidase, sialidase, thiaminepyrophosphatase, and specific concentration of gangliosides , from 3.5- to 5-fold greater, qualifying the fraction as a preparation of highly pure plasma membranes; (c) very low absolute content and lowered specific activities of cerebroside sulphotransferase and NADH and NADPH cytochrome c reductases, indicating a low contamination by intracellular membranous structures, in particular of the Golgi apparatus, in the fraction. As shown in Table 2, about one-sixth (as protein) of the material present in the “synaptosomal membrane fraction” was not derived by hypoosmotic shock from nerve ending rupture, since it was obtained in the absence of hypoosmotic treatment. However, the material exhibited the same biochemical characteristics as the corresponding one collected after hypoosmotic shock; i.e., it had the features of plasma membranes.

Recovery of Nerve Endings and Synaptosomal Plasma Membranes

The content and recovery of nerve endings in the various preparations obtained during subcellular fractionation of brain was established by determining occluded cytosol as occluded LDH. As shown in Table 3 , the P2 fraction contained about 20%, and the nerve ending fraction 9.4% of the occluded LDH present in the starting homogenate rid of debris and nuclei. Thus, assuming LDH to be evenly distributed in the different nerve endings, the recovery of nerve endings in the “nerve ending fraction” was 9.4%. Owing to the high homogeneity, as nerve endings, of the “nerve ending fraction,” the content of authentic plasma membrane markers (ATP-ase, 5‘- nucleotidase, gangliosides and neuraminidase) in this fraction is a record of the plasma membranes present in the same fraction. The “synaptosomal membrane fraction” carried (see Table 1) 10.8% of the ATP-ase, 9.8% of the gangliosides, 13.1% of the 5‘-nucleotidase, and 13.6% of the neuraminidase contained in the starting nerve ending fraction.

J . Neurochem., V o f . 35, No. 2, 1980

286 A . PRETI ET A L .

FIG. 2A. Electron micrograph of the “nerve ending fraction,” ~20,000. NE = nerve ending.

Thus, assuming again an even distribution of these membrane markers in the different nerve endings, the recovery of synaptosomal plasma membrane, after hypoosmotic treatment of nerve endings, can be calculated to range from 10 to 14%. In conclu- sion, the overall recovery of synaptosomal membranes from the original homogenate to the “synaptosomal membrane fraction” was 10- 14% of 9.4%, that is, 0.94-1.3%.

Sialytransferase activity in the “Synaptosomal Membrane Fraction”

The data concerning the total and specific activity of sialyltransferase, assayed on endogenous and exogenous acceptors in the starting brain homoge-

nate and in the main brain subcellular fractions obtained, are displayed in Table 4. The fractions with the highest sialyltransferase content were the “microsomes” and the R2 fraction. Pudied mitochondria (the pellet obtained after the second centrifugation of the Pz fraction on the 9%/16% Ficoll gradient) were shown to carry an extremely low sialyltransferase activity. The fractions with the highest specific activity of this enzyme were those richest in membranes (intracellular membranes and plasma membranes): the ‘‘microsomes” and the “synaptosomal membrane fraction”. Noteworthily, the greatest enzyme activities were recorded using lactosylceramide and Ds-fetuin as acceptors in agreement with previously reported findings (Ng and Dain, 1977~). However, both the specific activ-

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SIALYLTRANSFERASE IN S YNAPTOSOMAL MEMBRANES 287

FIG. 28. Electron micrograph of the “synaptosomal membrane fraction (magnification: ~11,000). MEMB = plasma membranes.

ity and the distribution in the different subcellular fractions of sialyltransferase activity on lactosylceramide did not exactly parallel that on Ds-fetuin.

As shown in Fig. 3 the subcellular distribution of sialyltransferase, as relative specific activity referred to homogenate, although similar to that of

sialidase and gangliosides, resembled most that of AChE and TPP-ase bilocular enzymes present in the plasma membranes and in intracellular structures (the endoplasmic reticulum AChE, and the Golgi apparatus TPP-ase).

The sialyltransferase activity present in the

TABLE 1. Biochemical churacteristics ofthe “nerve ending fraction” and of the “synaptosomal membrane fraction, ” obtained from carf brain

Nerve ending Synaptosomal membrane fraction activity fraction activity

(or concentration) (or concentration)

Parameter Total Specific Total Specific

“Occluded” LDH ATP-ase 5’-Nucleotidase Gangliosides Sialidase TPP-ase Cerebroside

sulphotransferase

reductase

reductase

NADH-cyt. c

NADPH-cyt. L‘

4.4 * 0.25 1491 ? 110 101 rfr 8.7

171.6 ? 15 3.17 c 0.2

21.45 ? 1.2

3.3 ? 0.3

171.6 r 16

15.18 ? 2.3

0.67 226

15.3 26 0.48 3.25

0.50

26

2.3

0.003 ? 0.0001

13.2 ? 1.6 16.83 ? 1.4 0.43 ? 0.05 2.28 ? 0.15

0.045 * 0.003

161 ’. 12

3.53 c 0.4

0.316 5 0.02

0.016 865.6 71.1 90.5 2.3

12.2

0.24

19.1

1.7

Gangliosides ate expressed as nmol bound N-acetylneuraminic acid; enzyme activities in milli International Units (micro Int. Units in the case of cerebroside sulphotransferase). The data shown, referred to 1 g of starting fresh tissue, are the mean values of six experiments c S.E. For details of method see Materials and Methods.

J . Neurochem., Vol. 35, N o . 2, 1980

288 A . PRETI ET A L .

With Without hypoosmotic hypoosmotic

Parameter shock shock

Protein, mg" Gangliosides 5'-Nucleotidase ATP-ase ACh-esterase Sialidase Thiaminepyrophosphatase NADH-cyt. c reductase NADPH-cyt. o' reductase Cerebroside sulphotransferase

0.186 3.48 4.65 3.83 3.55 4.81 3.77 0.75 0.77 0.48

0.030 3.02 4.56 3.45 3.41 4.63 3.07 0.72 0.75 0.44

For each parameter the specific activity (or concentration) ratio (specific activity-or concentration-in the synaptosomal membrane frac- tionispecific activity-or concentration-in the nerve ending fraction) is given.

'' Referred to 1 g starting fresh tissue.

LDH

Protein (total (m I.U. Preparation (mg) m 1.U.) mg protein)

Homogenate 104.8 f 7 47.1 f 4 0.45

P, fraction 24.8 2 9.9 ? 1 0.40

Nerve ending fraction 6.6 5 0.7 4.4 -+ 0.3 0.67

(without debris and nuclei)

Recovery", % 23.8 20.6

Recovery", 7r 6.3 9.4

The "hornogenate" is the tissue homogenate rid of debris and nuclei. LDH activity is expressed in milli International Units. The data shown, referred to I g starting fresh tissue, are the mean values of six experiments, ~ s . E . For details see Materials and Methods.

I' Referred to homogenate.

TABLE 4. Totirl c m t r t i t and spcw'jic acti1,ity (spec. cic, t . ) ojsinlvltrcrnsfernse in difjerent srrbcellrilor ,fractions oj' brain cortex

Preparation -_ ~

Homogenate Micrdsome P2 fraction Myelin fraction Nerve ending

fraction S ynaptosomal

membrane fraction

Protein ( mg/g t .f. )

116.4 f 10 30.4 ? 3.5 24.8 ? 2 3.45 -c 0.25 6.6 f 0.5

0.186 2 0.015

~ _ _ _ _ _

Endogenous Endogenous Exogenous Exogenous glycoprotein glycolipid lactosyl ceramide Ds-fet ui n

Total" Spec." Total" Spec." Total" Spec.I' Total" Spec."

11524 f 1230 99 12222 r 1150 105 211382 2 15500 1816 184145 rt_ 13500 1582 5077 f 450 167 4104 * 280 135 105306 f 7580 3464 63566 2 5150 2091 2406 f 210 97 3026 -+ 310 122 53469 2 3650 2156 45012 2 3410 1815 210 2 17 61 190 f 15 55 3685 f 325 1068 4382 f 415 1270 172 r 12 26 238 ? 17 36 7194 f 580 1090 5326 f 382 807

20.6 f 2 111 29.6 f 2.3 159 815 2 75 4380 356 t- 28 1912

~ ~ ~-

The enzyme activity was assayed on endogenous glycoprotein and glycolipid acceptors and on exogenous lactosyl ceramide and Ds-fetuin. Total enzyme activity is expressed a s c.p.m./hlg fresh tissue; the specific activity as c.p.m./hlrng protein. The data shown are the mean values of six experiments S . E .

" c.p.m.lh/mg protein; c.p.rn./Wg fresh tissue.

J . Nrurochem., Vo l . 35, N o . 2 , I980


0 m

5 2

g

s 0 1

2

0

c a

> - 4

x u 0

v, a b c d a b c d a b c d a b c d a b c d a b c d a b c d a b c d

FIG. 3. Subcellular distribution of sialyltransferase activity compared with that of several markers of plasma membranes (Na+-K+ ATPase, gangliosides, sialidase), of the Golgi apparatus (cerebroside sulphotransferase, CST), of intracellular and plasma membranes (acetylcholine esterase, AChE) of the Golgi apparatus and plasma membranes (thiamine pyrophosphatase, TPP- ase) in calf brain cortex. For each enzyme the specific activity ratio referred to homogenate (specific activity in the fraction/ specific activity in the starting homogenate) is given; for gangliosides the specific concentration ratio is given. (a) Homogenate; (b) microsomes; (c) P, fraction; (d) nerve ending fraction-sialyltransferase activity on lactosylceramide (Lac-cer) or Ds-fetuin as acceptor substrates.

“synaptosomal membrane fraction” expressed as relative specific activity referred to that in the “nerve ending fraction” (Fig. 4) unequivocally appeared to closely parallel that of authentic plasma membrane markers (ATP-ase, S’nucleotidase, gangliosides, sialidase). However, it was quite different from that of intracellular membrane markers, like NADH and NADPH cytochrome c reductases, and, in particular, from that of CST, the Golgi apparatus marker.

The absolute content of sialyltransferase in the “synaptosomal membrane fraction” was low. This is not surprising, since the recovery of synaptosomal membranes, as reported above, was of the order of 1%. For this same reason the total content of synaptosomal membrane-bound sialyltransferase in brian is considered to be quite important.

General Properties of Synaptosomal Membrane-Bound Sialyltransferase

The pH optimum of sialyltransferase contained in the “synaptosomal membrane fraction” was 6.0 with lactosylceramide, Ds-fetuin, and endogenous glycoproteins, and 6.6 with endogenous glycolipids (Fig. 5) . The enzyme saturation was attained at 0.15 mM-lactosylceramide (K, = 0.025 mM) and 4.0 mM (as sialyl acceptors sites) Ds-fetuin ( K , = 1.45 mM) (Fig. 6). With the same two acceptor substrates the apparent K,’s for CMP-NeuNAc were different: 0:57 mM and 0.135 mM respectively, as well as the V,,, (2.61 and 0.923 nmoYWmg of protein, respectively) (Fig. 5 ) . At the pH optimum and at saturating substrate concentration the reaction rate was linear with time until 60 min for the endogenous acceptors and until 120 min for exogenous acceptors, and linear with enzymatic protein up to 0.9 and 0.3 mg with endogenous and exogenous acceptors, respectively. The effect of detergents (Triton CF 54 and Tween 80) on the activity of sialyltransferase was very much dependent on the type of acceptor sub-

strate employed (see Fig. 7). In the case of Ds-fetuin, the presence of detergents caused the enzyme activity to increase to a maximum of twofold, reached at 0.5% detergent concentration. This activation was

SPECIFIC ACTIVITY (OR CONCENTRATION) RATIO d N * p L”

I I

I I

I (Na+-K+)ATEase

~~

I I I

I AchE

I I

DS. Fetuin S.T.

I NADH-Cyt. c reductase

I NADPH-Cyt,c reductase

1 C S T 1

FIG. 4. Sialyltransferase and different markers of plasma membranes (sialidase, Na+-K+ ATP-ase, 5’-nucleotidase, gangliosides), of intracellular and plasma membranes (acetylcholine esterase, AChE), of the Golgi apparatus and plasma membranes (thiamine pyrophosphatase, TPP-ase), of the Golgi apparatus (cerebroside sulphotransferase, CST), and of intracellular structures (rotenone-insensitive NADH and NADPH: cytochrome c reductases) in the “synaptosomal membrane fraction” in calf brain cortex. For each enzyme the specific activity ratio referred to the “nerve ending frac- t ion” (specific activity in the synaptosomal membrane fraction/specific activity in the nerve ending fraction) is given; for gangliosides the specific concentration ratio is given. Lac-Cer S.T. = sialyltransferase activity on lactosylceramide; Ds-fetuin S.T. = sialyltransferase activity on DS- fetuin.



I v.nmoles t i '

Chi;- NeuAc,mM

/ I

-+ 8OOpg proteln

300

- 120 yg protein

-+ 8OOpg proteln

""1

56 60 6 4 68 1 2 76 PH

,,oo{ 5004

z . 1000- 2 5 0 "

100 300 500 700 900 PROTEIN, pg

FIG. 5. Effect of CMP-NeuAc concentration (V/S), of pH (V/pH), of enzymatic protein concentration (V/protein), and of incubation time (V/t) on the activity of synaptosornal membrane-bound sialyltransferase, using calf brain cortex. Acceptor substrates for sialyltransferase: *-*-*, lactosylcerarnide; D-B-B, os-fetuin; 0- -0 , endogenous glycoproteins; A-A-A, endogenous glycolipids.

. . 1 Vrnmoles('4C] NeuAc h" rng" protein

V I

1 5 3.0

Km,145 mM

-0.75 -025 0 5 1.0 2 0 DS-Fetuin. mM ( a s acceptor sites)

V= n m o l e s ~ C ] N e u A c h-' mg-' protein

750/ 750.

.c 500- . Km,0025mM

250-

01.

10 io ) /,,- 005 01 015 035 07 -40 - 3 0 -20 -10

Lac Cer, mM 005 -,I

01 01 5 0.35 07 Lac Cer, mM

140 pg protein

0.31

- -

FIG. 6. Effect of exogenous acceptor substrate (lactosylcerarnide, Lac-Cer, and Ds-fetuin) on the activity of synaptosornal membrane-bound sialyltransferase. For each substrate the corresponding saturating concentration of CMP-NeuNAc and pH optimum were employed.


SIALYLTRANSFERASE IN SYNAPTOSOMAL MEMBRANES 291

1

200

200 400 600 800 pg TRiTON C F - 5 4 1 TWEEN 80, 2/1 W/W

FIG. 7. Effect of detergents (Triton CF 54 and Tween 80) on the activity of synaptosomal membrane-bound sialyltransferase with respect to the acceptor substrate employed and to the amount of enzymatic protein present. A-A-A, Lacto- sylceramide; protein: 140 pg; m-m-m, lactosylceramide; protein: 70 p g ; t-*-*, os-fetuin; protein: 250 pg ; e-e-0.. 0s-fetuin; protein: 125 pg.

within a certain range independent of the amount of enzymatic protein present. Conversely, when lactosylceramide was employed, the enhancement of enzyme activity was much higher (- 10-fold), and, interestingly, dependent upon the amount of enzymatic protein present. The optimal detergent/ protein ratio (w/w) was 2.3-3.0.

The thermal stability of the enzyme activity toward Ds-fetuin and endogenous glycoproteins was much lower than that toward lactosylceramide and endogenous glycolipid acceptor (Fig. 8). The addition of lactosylceramide did not inhibit the enzymatic sialylation of Ds-fetuin and vice versa, this excluding any competition between the two acceptor substrates.

A-A- L a c - C e r

e0-0 DS-Fetuin **-* Endogenous Glycolip. )).. Endogenous Glycoprot.

Identification of Reaction Products with Glycolipid Acceptors

This was established only in the case of lactosylceramide used as acceptor. As shown in Fig. 9, more than 90% of the radioactivity incorporated into glycolipid material was carried by a compound having the same chromatographic behaviour as authentic GM3 ganglioside. All the radioactivity incorporated into this compound was completely re- leased, and lost by dialysis, when the same compound was extensively treated with Vibrio cholerae neuraminidase according to Ghidoni et al. (1976). This finding is consistent with the hypothesis that the enzyme product was GM, ganglioside.

DISCUSSION

The membranes surrounding nerve endings (synaptosomal, or synaptic, membranes) are very rich in glycoconjugates, specially sialylglycoconjugates, among which gangliosides are predominant (Breckenridge et al., 1972; Hansson et al., 1977). It is reasonable to assume that the glycocalix plays an important role in synapse formation and functional specialization, and that synapse plasticity depends to a certain extent on the chemical flexibility of the glycocalix. Such flexibility (that is, a controlled modification of the chemical composition of the glycocalix) would be quite feasible if glycosyltransferases and glycosidases are present at the level of the same membranes. Hence the great interest, and heated debate, on the occurrence of these enzymes at cell surfaces in general. With specific regard to sialic acid residues, their involvement in synapse function, which has been suggested by several authors (Lehninger, 1968; Roseman, 1973; Rahman et al., 1976; Svennerholm, 1979), implies that the content of sialic acid at the synaptic membrane surface is controlled by locally operating enzymes, sialidase and sialyltransferase.

The occurrence in synaptosomal membranes of

1 1 3 5 10 4-

PREINCUBATION AT 50° C , rnin

FIG. 8. Thermal stability at 50°C of synaptosomal membrane-bound sialyltransferase with endogenous or exogenous (lactosylceramide, Lac-Cer) glycolipid or endogenous or exogenous (DS-fetuin) glycoprotein acceptor substrates. The incubation was carried out under optimal assay conditions.

J . Neurochem., Vol. 35, No. 2, 1980


- - GTlb.

a b C GO ’

FIG. 9. Identification of GM, ganglioside as the reaction product of synaptosomal membrane-bound sialyltransferase activity on lactosylceramide. Separation of gangliosides on silica gel-precoated thin-layer plates (solvent: chloroform/methanol/water, 60/35/8 by vol.; 2-h run; 18-20°C; detection of the spots by exposure to iodine vapours). a: standard gangliosides; b: standard GM, ganglioside; c: incubation mixture with added ‘cold’ GM, ganglioside. Incubation under optimal conditions with 0.4 mg of enzymatic protein

sialidase has been definitely assessed (Schengrund and Rosenberg, 1970; Tettamanti et al., 1972). This enzyme also affects the endogenous sialylglycoconjugates (Heijlman and Roukema, 1972; Preti et al., 1973; Tettamanti et al., 1975). Conversely, the presence of sialyltransferase at the synaptic level is, at present, controversial. Den et al. (1975), working on embryonic chicken brain, reported a high sialyltransferase activity in isolated nerve endings. Other authors (Van den Eijnden and Van Dijk, 1974; Landa et al., 1977; Ng and Dain, 1977b; Maccioni et al., 1978), using brains of young rats, observed much higher sialyltransferase activity in brain subfractions rich in intracellular membranes. This led them to consider that the whole sialyltransferase activity detectable in the nerve endings was due to contamination by intracellular membranes. Of course the fact that sialyltransferase, as well as other glycosyltransferases, is linked to intracellular structures, namely the Golgi apparatus, is definite. How- ever, increasing evidence is being reported on the presence of plasma membrane-bound sialyltransferase activity in a number of cells and tissues (Grimes, 1973; Datta, 1974; Shur and Roth, 1975; Porter and Bernacki, 1975; Colombino et al., 1978; Painter and White, 1976; Verbert et al., 1977). Thus the possibility that the Golgi apparatus is not the only site of sialyltransferase activity, and that the plasma membranes (namely the synaptosomal membranes) may contain this enzyme, cannot be excluded and is surely worth checking.

We approached the problem as follows: (a) to prepare synaptosomal membranes at the highest degree of homogeneity, ascertaining, with the use of proper markers, the contamination by intracellular membranes; (b) to assay sialyltransferase activity on these synaptosomal membranes comparing the behaviour of sialyltransferase with that of authentic markers of plasma membranes, of intracellular membranes, of the Golgi apparatus, of the plasma

membranes and of intracellular structures (the Golgi apparatus included) (acetylcholine esterase, thiamine pirophosphatase), through the whole preparation procedure. Our efforts were greatly facilitated by the very recent report (Siegrist et al., 1979) that cerebroside sulphotransferase is specifi- cally located on the Golgi complex in brain tissue, also.

The procedure we adopted for preparing synaptosomal membranes was derived from those of Morgan et al. (1971) and of Gurd et al. (1974), following more recent suggestions and recommenda- tions (McIntosh and Plummer, 1976; Cohen et al., 1977). Essentially, this procedure, aimed at preparing nerve endings with the highest degree of pres- ervation and of homogeneity, was upheld by the following rationale: (a) to use, as the nerve ending source, the 1000- 11,500g sediment (Pz fraction) from brain homogenate, which recovered less than 40% of the nerve endings present, but avoided mas- sive contamination by microsomal membranes and particles; (b) to wash the preparation several times to get rid of contaminating membranes, specially those of intracellular origin; (c) to remove myelin fragments and light membranes by a first centrifugation on 9% Ficoll, causing nerve endings to pre- cipitate, together with mitochondria, into the pellet, and then to purify nerve endings by a second centrifugation on a 9%/16% Ficoll gradient, causing the same particles to float at the gradient interface. As a matter of fact the resulting “nerve ending fraction” proved, when submitted to careful electromicro- scopic examination, to be composed of at least 85% well-preserved nerve endings, which constitutes a substantial improvement on similar available methods. In particular, light membranes and structures of possible intracellular origin, and myelin fragments were very rare. Of course, but as expected, the recovery of nerve endings, assessed by measuring “occluded” LDH (McIntosh and Plum-

J . Neurochem., Vol. 35, No. 2. 1980

293 SIALYLTRANSFERASE IN SYNAPTOSOMAL MEMBRANES

mer, 1976) was less than 10% in the “nerve ending fraction”, with respect to the initial homogenate, and significantly lower than that provided by other procedures.

After hypoosmotic treatment of the “nerve ending fraction” the material floating in the density gradient centrifugation over 1 .O M-sucrose (corresponding to the F plus G fractions of Morgan et al., 1971) and then differentially centrifuged, provided the “synaptosomal membrane fraction”. This material, which morphologically appeared as membranous organelles of different sizes with some junctional complexes, but with no derivates of the Golgi complex, as those described by Siegrist et al. (1979), did not contain unruptured nerve endings, as “occluded” LDH activity was practically absent. In addition it featured a four- to fivefold enrichment (with respect to the “nerve ending fraction”) of authentic plasma membrane markers (ATP-ase, 5’-nucleotidase, sialidase, gangliosides). Such an enrichment is con- idered (Mclntosh and Plummer, 1976) an acceptable proof of the plasma membrane nature of the examined material. Moreover, it presented very low absolute activities and lowered specific activity ratios-about 0.7-(with respect to the “nerve ending fraction”) of typical intracellular membrane markers (rotenone-insensitive NADH and NADPH: cyt. c reductases), and a still lower-about 0.5- specific activity ratio of an authentic marker of the Golgi apparatus, cerebroside sulphotransferase.

This is a demonstration of the extremely low contamination by intracellular membranes and, in particular, of derivates of the Golgi complex. This situation is not surprising since, according to Fishman (1974), such membranes, including Golgi particles, are expected to float at a lower sucrose concentration. Finally, at least 85% of the material constituting the “synaptosomal membrane fraction” originated by hypoosmotic rupture of nerve endings. The proof was provided by parallel experiments in which a sample of the nerve ending fraction, not submitted to hypoosmotic shock, was processed exactly as the shocked sample. Also noteworthy, the same experiments showed that the material floating over 1 .O M-sucrose and obtained from the non-hypoosmotically shocked sample, featured biochemical characteristics of plasma membranes.

In summary, the morphological and biochemical evidences obtained enable us to draw the following conclusions with respect to the nature of the membranes constituting the “synaptosomal membrane fraction”: (a) the bulk of the membranes are the plasma membranes surrounding nerve endings; (b) the contamination by intracellular membranes ac- companying nerve endings in the “nerve ending fraction”, or by intraterminal structures originated from ruptured nerve endings, is very low. In prin- ciple, one possibility cannot be excluded: the above

membranes are, at least partly, of glial origin; in other words, are derived from glial cells adjacent to the synaptic terminals. However, the strictly parallel behaviour of gangliosides (definitely a neuronal marker-Hansson et al., 1977; Ledeen, 1978) and of ATP-ase, 5’-nucleotidase, and sialidase, at the present time, makes this hypothesis unlikely.

With respect to sialyltransferase, the “nerve ending fraction” was found to carry remarkable amounts of enzyme, in agreement with the data reported by Den et al. (1975) in embryonic chicken brain. Still more interestingly, the “synaptosomal membrane fraction” carried a fairly measurable sialyltransferase activity. In both preparations the enzyme was ac- tive on endogenous (glycoproteins, glycolipids) and exogenous (lactosylceramide and Ds-fetuin) acceptors. The behaviour of sialyltransferase upon subcellular fractionation, up to the level of nerve endings, evaluated as specific activity ratio (referred to the initial homogenate) closely paralleled that of the bilocular enzymes used: thiamine pyrophosphatase, present in the plasma membranes and in the Golgi apparatus (Lane, 1968; Morgan et al., 1972), and acetylcholine esterase, present in the plasma membranes and in intracellular membranes. Moving to the “synaptosomal membrane fraction”, the specific activity ratio (referred to the “nerve ending fraction”) of the sialyltransferase here contained, was practically the same as that of ATP-ase, 5’-nucleotidase, gangliosides, sialidase, and thiamine pyrophosphatase, and quite different from that of NADH and NADPH: cyt. c reductases and, particularly, from that of cerebroside sulphotransferase. Thus the only hypothesis consistent with collected evidence is that in calf brain one sialyltransferase is linked to intracellular structures, probably the Golgi apparatus, and one to plasma membranes, namely, synaptosomal plasma membranes. The total amount of sialyltransferase in the “synaptosomal membrane fraction” was low-as low as that of ATP-ase, gangliosides, and sialidase. However, we should consider that the recovery of synaptosomal membranes was only about 1%; the same is probable for sialyltransferase. As a consequence, in brain the total content of synaptosomal membrane-bound sialyltransferase can be evaluated to be about 100-fold more than that recorded in the “synaptosomal membrane fraction.” The resulting figure is quite revealing.

We are fully aware that cell fractionation studies, because of their intrinsic limitations, cannot provide definite answers to such general problems as that concerning the presence of glycosyltransferases and glycosidases at the cell surface. However the study presented here provides clear and uncontroversial evidence. This will stimulate, in our opinion, the search for confirmation by new, complementary experimental approaches.

Our results, while in agreement with those of Den


294 A. PRETI ET A L .

et al. (1975), contrast with those of other authors (Van den Eijnden and van Dijk, 1974; Landa et al., 1977; Ng and Dain, 19776) with reference to the presence of sialyltransferase in the nerve endings. This contradiction may partially be explained by animal species or age differences. We believe, however, that the contrast arises essentially from the different approach to the problem, from either the theoretical or methodological point of view. The above authors started from the assumption that sialyltransferase has only one location. Therefore, once it was observed that the subcellular fraction richest in the enzyme was that carrying derivates of the Golgi complex, they concluded that the activity present in other subfractions was a contamination from Golgi structures and, by consequence, did not feel any need for studies with very homogeneous subfractions. Conversely, we started from the working hypothesis that sialyltransferase may have more than one subcellular location; and, for as- sessing it, we did need extremely pure and very well characterized subcellular fractions.

The study of the general properties and kinetics of synaptosomal membrane-bound sialyltransferase was, in the present investigation, limited to the assessment of the characteristics necessary for set- ting up a reliable assay method. The optimum pH, saturating concentrations of CMP-NeuNAc and added acceptor substrates, requirement for detergents and thermal stability, were, in general, close to those reported in previous works (Van den Eijnden and van Dijk, 1972; Ng and Dain, 1977a) for sialyltransferase activity assayed in much cruder preparations.

An observation which diffzrentiates synaptosomal membrane-bound sialyltransferase from previously described brain sialyltransferases is the K , value for CMP-NeuNAc, which was different when the enzyme acted upon lactosylceramide and Ds-fetuin. This finding, together with the thermal resistance of the enzyme, which was, too, different with glycolipid and glycoprotein acceptor substrates, and the absence of competition between lactosylceramide and Ds-fetuin, substantiates the evidence for the presence in synaptosomal membranes of at least two different sialyltransferase activities: one acting on glycolipids, one working on glycoproteins. All this gives further support to the concept, clearly proposed by Ng and Dain (1977), that sialyltransferase occurs in brain in different forms.

To conclude, the presence in synaptosomal membranes of sialyltransferase together with sialidase, poses a serious problem which is worth discussing. In fact, these two enzymes, from a strictly metabolic point of view, may be responsible for a “sialylation-desialylation cycle” implicating the sialylglycoconjugates occurring in the same membranes. In the case of gangliosides several authors (Maccioni et al., 1971; Holm and Sven-

nerholm, 1972; Ledeen et al., 1976) looked in vivo for evidence of partial turnover of gangliosides, but the results indicated that the individual gangliosides appear to be metabolized as a unit. These investigations, extending the classical one on the biosynthesis of gangliosides (Suzuki, 1964), followed, after a single pulse of injected radioactive precursor, the course of radioactivity incorporation into the individual saccharide residues of the gangliosides in their final site, the neuronal plasma membranes. That is, they showed that, as the sum of all possible steps of metabolism, each ganglioside has a different pattern. This simply means, in our opinion, that in determining the metabolic individuality of each ganglioside, the process of biosynthesis prevailing on local turnover (if occurring) and on degradation.

Current investigations in our laboratory are deal- ing with two situations of primary importance for understanding the role of sialyltransferases in brain: the “sidedness” of the plasma membrane-bound enzyme, and the behavioral differences, and possible links, between the enzyme located at the level of the cell surface and of the Golgi apparatus.

ACKNOWLEDGMENT

This work was supported by a grant from the C.N.R. (Consiglio Nazionale delle Ricerche), Italy.

We have pleasure in thanking Dr. Burkart (De- partment of Pediatrics, University of Berne, Swit- zerland) for his advice and help in the determination of cerebroside sulphotransferase activity.

REFERENCES

Breckenridge W. C., Gombos G. , and Morgan I . G. (1972) The lipid composition of adult rat brain synaptosomal plasma membranes. Biochim. Biophys. Acta 266, 695-707.

Bretscher M. S. (1973) Membrane structure: Some general prin- ciples. Science 181, 622-629.

Cohen R . S . , Blomberg F., Berzins K. , and Siekevitz P. (1977) The structure of postsynaptic densities isolated from dog cerebral cortex. J. Crll Biol. 74, 181-203.

Colombino L. F., Bosmann H . B . , and McLean R . J . (1975) Cell surface localization of the sialyltransferase ectoenzyme system during the Chlamydomonas mating reaction. E x p .

Datta P. (1974) Labeling of the external surface of hamster and mouse fibroblasts with [14C]sialic acid. Biochemistry 13,

Den H. , Kaufman B. , McGuire E. J., and Roseman S. (1975) The sialic acids. XVIII-Subcellular distribution of seven glycosyltransferases in embryonic chicken brain. J . B i d . Chem. 250, 739-746.

Ellman G. , Courtney D. , Andres V. , Jr., and Featherstone R . M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochern. Phnrmncol. 7, 88-95.

Fishman P. H . (1974) Normal and abnormal biosynthesis of gangliosides. Chem. Phys. Lipids 13, 305-326.

Ghidoni R . , Sonnino S. , Tettamanti G . , Wiegandt H. , and Zam- botti V. (1976) On the structure of two new gangliosides from beef brain. J . Nrurochem. 27, 511-515.

Grimes W. J. (1973) Glycosyltransferase and sialic acid levels of normal and transformed cells. Biochemistry 12, 990-996.

Gurd J . M., Jones L. R., Mahler H. R. , and Moore W . J . (1974)

Cc,/l R ~ J . 112, 25-30.

3987 -3991.

J . Neurochem.. Vol. 32, N o . 2. I980


Isolation and partial characterization of rat brain synaptic plasma membranes. J . Neurochem. 22, 281 -291.

Handa S. and Yamakawa T. (1964) Chemistry of lipids of post- hemolytic residue or stroma of erythrocytes-Xll. Chemical structure and chromatographic behaviour of hematosides obtained from equine and dog erythrocytes. J p n . J . E-rp.

Hansson U . A., Holmgren J., and Svennerholm L. (1977) U1- trastructural localization of cell membrane GMl ganglioside by cholera toxin. Proc. Nail. Acad. Sci USA 74,3782-3786.

Heijlman J. and Roukema P. A. (1972) The action of calf brain sialidase on gangliosides, sialoglycoproteins and sialo- glycopeptides. J . Neurochem. 19, 2567-2575.

Holm H. and Svennerholm L. (1972) Biosynthesis and biodegra- dation of rat brain gangliosides studied in v i v a . J . Neurochem. 19, 609-622.

Keenan T. W., Morrk D. J . , and Basu S. (1974) Ganglioside biosynthesis. Concentration of glycosphingolipid glycosyltransferases in Golgi apparatus from rat liver. J . B i d . Chem.

Landa C. A., Maccioni H. J . F., Arce A., and Caputto R. (1977) The biosynthesis of brain gangliosides. Separation of membranes with different ratios of ganglioside sialylating activity to gangliosides. Biochem. J . 168, 325-332.

Lane N. J. (1968) Distribution of phosphatases in the Golgi re- gion and associated structures of the thoracic ganglionic neurons in the grasshopper Melanoplus diffrrentialis. J . Cell

Ledeen R. W. (1978) Ganglioside structures and distribution: Are they localized at the nerve ending?J. Supmmol. Strircr.

Ledeen R. W., Skrivanek J. A., Tirri L. J . , Margolis R. K . , and Margolis R. U . (1976) Gangliosides of the neuron: Localiza- tion and origin Adv. Exp. Med. B i d . 71, 83- 104.

Lehninger A. L. (1968) The neuronal membrane. Proc. Natl. Acad. Sci . U S A 60, 1069-1080.

Lowry 0. H . , Rosebrough N. J., Farr A. L., and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J . Biol. Chem. 193, 265-275.

Maccioni H. J . , Arce A., Caputto R. (1971) The biosynthesis of gangliosides-Labelling of rat brain gangliosides in v ivo . Biochern. J . 125, 1131-1137.

Maccioni H. J . F., De Filipo S. S. , Landa C. A., and Caputto R. (1978) The biosynthesis of brain gangliosides-Ganglioside- glycosylating activity in rat brain neuronal perikarya fraction. Biochem. J . 174, 673-680.

McIntosh C. H. S. and Plummer D. T. (1976) The subcellular localization of acetylcholinesterase and its molecular forms in pig cerebral cortex. J . Neurocliem. 27, 449-457.

Morgan I. G., Wolfe L. S. , Mandel P., and Gombos G. (1971) Isolation of plasma membranes from rat brain. Biochim. Biophys. Acra 241, 737-751.

Morgan I. G., Reith M., Marinari U., Breckenridge W. C., and Gombos G. (1972) The isolation and characterization of synaptosomal plasma membranes, in Glycolipids, Glyco- proteins and Mucopolysaccharides of ihe Nervous Sysiem. (Zambotti V., Tettamanti G . , and Arrigoni M. G., eds) pp. 209-228. Plenum Publishing Corporation, New York.

Ng S. S. and Dain J. A. (1977~) Sialyltransferases in rat brain: Reaction kinetics, product analyses and multiplicities of enzyme species. J . Nrurochem. 29, 1075- 1083.

Ng S . S. and Dain J . A. (19776) Sialyltransferases in rat brain: Intracellular localization and some membrane properties. J . Neurochern. 29, 1085- 1093.

Ohman R. and Hygstedt 0. (1968) The isolation of sialyllac- tosides with the aid of gel filtration. Anal. Biochern. 23,

Painter R. G. and White A. (1976) Effect of concanavalin A on expression of cell surface sialyltransferase activity of mouse thymocytes. Proc. Nail. Acad. Sci. USA 73(3), 837-841.

Porter C . W. and Bernacki R. J . (1975) Ultrastructural evidence for ectoglycosyltransferase systems. Nature 256, 648-650.

Preti A., Lombardo A., Tettamanti G., and Zambotti V. (1973)

Med. 34, 293-304.

249, 310-31.5.

Biol. 37, 89- 104.

8, 1-17.

391 -402.

Removal of N-acetylneuraminic acid from particulate sialylglycoproteins by endogenous membrane bound neuraminidase in calf brain. J . Neurochem. 21, 1559- 1562.

Rahmann H., Rosner H., and Breer H. (1976) A functional model of sialoglycomacromolecules in synaptic transmission and memory formation. J . Theor. Biol. 57, 231-237.

Roseman S. (1973) The Neurosciences, Third Study Program (Schmitt F. 0. and Worden F. G., eds), pp. 795-804. M.I.T. Press, Cambridge, (Massachusetts.)

Schengrund C. L. and Rosenberg A. (1970) Intracellular location and properties of bovine brain sialidase. J . Biol. Chem. 245,

Seijo L. and Rodriguez de Lores Arnaiz G. (1970) Subcellular distribution of thiamine pirophosphatase in rat cerebral cortex Biochim. Biophys. Acia 211, 595-598.

Shur B. D. and Roth S. (1975) Cell surface glycosyltransferase. Biochim. Biophys. A c t a 415, 423-512.

Siegrist H. P., Jutzi H . , Burkart T., Wiesmann U . , and Herschkowitz N. (1977) Age dependent modulation of 3’- phosphoadenosine-5‘-phosphosulfate-galactosyl ceramide sulfotransferase by lipids extracted from the microsomal membranes and artificial lipid mixtures. Biochim. Biophys. Acra 489, 58-63.

Siegrist H. P., Burkart T., Wiesman U. N., Herschkowitz N. N., and Spycher M. A. (1979) Ceramide-galactosyltransferase and cerebroside sulfotransferase localisation in Golgi membranes isolated by a continuous sucrose gradient of mouse brain microsomes. J . Neurochem. 33, 497-504.

Sottocasa G. L., Kuylenstierna B., Ernster L., and Bergstrand A. (1967) An electron transport system associated with the outer membrane of liver mitochondria: A biochemical and morphological study. J . Cell Biol. 32, 415-438.

Spiro R. G . (1964) Periodate oxidation of the glycoprotein fetuin. J . Biol. Chem. 239, 567-573.

Suzuki K . and Korey S . R. (1964) Study on ganglioside metabolism-1. Incorporation of ~-[U-’~C]glucose into individual gangliosides. J . Neurochem. 11, 647-653.

Svennerholm L. (1957) Quantitative estimation of sialic acid-11. A colorimetric resorcinol-hydrochloric acid method. Biochim. Biophys. Acta 24, 604-61 1.

Svennerholm L . ( 1 9 7 0 ~ ) Gangliosides, in Handbook of Neurochemisiry (Lajtha A . , ed), vol. 3 , pp. 425-452, Plenum Press, New York.

Svennerholm L . (1970b) Ganglioside metabolism, in Com- prehensive Biochemistry (Florkin M. and Stotz E. H., eds), vol. 18, pp. 201-215. Pergamon Press, Oxford.

Svennerholm L. (1979) Structure and function of gangliosides, Proc. Ini . Symp. Le Bishenberg, Plenum Press, New York and London (in press).

Tettamanti G., Morgan 1. G., Gombos G., Vincendon G., and Mandel P. (1972) Sub-synaptosomal localization of brain particulate neuraminidase. Brain Res. 47, 515-518.

Tettamanti G., Preti A , , Lombardo A., Bonali F., and Zambotti V. (1973~) Parallelism of subcellular location of major particulate neuraminidase and gangliosides in rabbit brain cortex. Biochim. Biophys. Acta 306, 466-477.

Tettamanti G., Bonali F. , Marchesini S . , and Zambotti V. (l973b) A new procedure for the extraction, purification and fractionation of brain gangliosides. Biochim. Biophys. Acta

Tettamanti G . , Preti A., Lombardo A., Suman T., and Zambotti V. (1975) Membrane-bound neuraminidase in the brain of different animals: Behaviour of the enzyme on endogenous sialoderivatives and rationale for its assay. J . Neurochem.

Touster O., Aronson N. N., Dulaney J. T., and Hendrickson H. (1970) Isolation of rat liver plasma membranes. Use of nu- cleotide pyrophosphatase and phosphodiesterase I as marker enzymes. J . Cell B i d . 47, 604-618.

Van den Eijnden D. H. and van Dijk W. (1972) A convenient method for the preparation of cytidine 5’-monophospho-N- acetylneuraminic acid. Z . Physiol. Chem. 353, 1817- 1820.

Van den Eijnden D. H. and van Dijk W. (1974) Properties and

6 196- 6200.

296, 160- 170.

25, 451-456.


A . PRETI ET A L .

regional distribution of cerebral CMP-N-acetylneuraminic acid: Glycoprotein sialyltransferase. Biockirn . BiophyA.

Venerando B., Preti A , , Lombardo A, , Cestaro B., and Tet- tarnanti G. (1978) Studies on brain cytosol neurarninidase- 11. Extractibility, solubility and intraneuronal distribution of the enzyme in pig brain. Riochim. Biophys. Act 527,

Act0 362, 136-149.

17-30.

Verbert A., Cacan R., Debeire P., and Montreuil J. (1977) Pecu- liar behaviour of ectosialyl transferase toward exogenous substrates. FEBS Lrrt. 74, 234-238.

Wiegandt H. (1971) Glycosphingolipides. Ad,, . Lipid Ras. 9,

Yamakawa T. and Nagai Y. (1978) Glycolipids at the cell surface and their biological functions. Trends Biochern. Sci. 3,

249-289.

128-131.

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

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