Journal of Nourockemisrry. 1977. Vol. 29. pp. 1085-1093. Pergamon Press. Printed in Great Britain.

SIALYLTRANSFERASES I N RAT BRAIN: INTRACELLULAR LOCALIZATION A N D SOME MEMBRANE PROPERTIES S.-S. NG’.’ and J. A. DAIN~ Department of Biochemistry and Biophysics, University of Rhode Island, Kingston, RI 02881, U.S.A. (Received I 1 Fehruary 1977. Accepted 26 M a y 1977)

Abstract-Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes. The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non-ionic detergent mixture, Triton CF-54rween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effectwas dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GMI,, 700; exogenous DS-fetuin, 230. The action of the nonionic detergents appears to be on a hydrophobic segment of the enzyme molecule, bearing the active site, which is buried in the membrane lipid bilayer. This was substantiated by the partial trypsin resistance of the sialyltransferase activities and the abolition of that resistance when trypsinization was performed in the presence of nonionic detergents. Furthermore, the sialyltransferase activities were markedly inhibited by organic solvents; and these inhibitory effects were inversely proportional to the solvent dielectric constants.

of the glycosyltransferases in brain tissue is still a subject of controversy.

THE SUBCELLULAR localization

Several studies have indicated that these enzymes are concentrated in membranes of the synaptic complexes or synaptic vesicles (DICESARE & DAIN,1972; BosMANN, 1972,1973; DENet al., 1975; BROQUET & LoulSOT,1971). In other studies, however, glycosyltransferase activities were found to be more concentrated in the nonsynaptic membranes (KO & RAGHUPATHY, 1972; MORGAN et al., 1972; RAGHUPATHY et a!., 1972). These discrepancies are in need of clarification because the metabolism of glycoproteins and glycolipids are speculated to play important roles in the differentiated functions of neuronal membranes (BURTONet al., 1964; LEHNINGER, 1968; RAHMANNet al., 1976). We believe that the controversy over this issue is due to the fractionation techniques used by different investigators. Avoiding differential centrifugation, we

report here the use of a continuous sucrose gradient technique for the assessment of the intracellular localization of sialyltransferases (CMP-NeuNAc: glycoprotein or glycolipid N-acetylneuraminyltransferase, EC 2.4.99.-) in rat cerebra. We have also examined several properties of these membrane bound enzyme systems. The preceding companion study (NG & D A W , 1977) reports on the kinetics, products and species of these rat brain sialyltransferases. A preliminary report of some of these results has appeared (NG et al., 1976). MATERIALS AND METHODS Materials

The materials used were: [NeuNAc-4-14C]CMPNeuNAc (9 mCi/mM), Liquefluor and Aquasol, from New England Nuclear Corp. (Boston, MA); trypsin, 3 x recrystallized, from Worthington Biochemical Corp. (Freehold, NJ) Triton CF-54, from Rohm and Haas Co. (Philadelphia, PA); Tween 80 and soya bean trypsin inhibitor, from Sigma Chemical Co. (St. Louis, MO); Sephadex G-25 (superfine),from Pharmacia. Fine Chemicals, Inc. (Piscataway, NJ). Inbred Sparague-Dawley rats were maintained on regular chows from Purina Co. Newborn rats were obtained by controlled periodic mating.

Work done in partial fulfillment of the degree of Doctor of Philosophy in Biochemistry, University of Rhode Island. * Present address: Department of Biochemistry, McGill University, Montreal, Quebec H3C 3G1, Canada. To whom correspondence should be addressed. Abbreoiations used: CMP-NeuNAc, cytidine 5’mono- Methods Preparation of substrates. Detailed procedures for the phospho-N-acetylneuraminic acid DS-fetuin, desialylated fetuin; GM,,, galactosyl-N-acetylgalactosaminyl-(N-ace preparation of CMP-NeuNAc, GMI, and DS-fetuin were tyneuraminyl) galactosyl-glucosylceramide (ganglioside reported in the preceding paper (NG & DAIN,1977). Sialyltransferase assay. The cerebra of 11-16 d a y old rats nomenclature is a modification by NG & DAIN(1976) of were used as the enzyme source. Detailed procedures for SVENNERHOLM (1964). 1085

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S.-S. NG and J. A. DAIN

sialyltransferase assays were reported in the preceding paper (NG & DAIN,1977) and are briefly described as follows. A membrane preparation containing 0.5 mg protein, was added to assay mixtures which included I mM-CMPNeuNAc (1.9 mCi/mmol). 10 mM-MgCI,, 100 mM cacodylate-HCI buffer, pH 6.3 in a final vol of 0.1 ml. When required, 1.5 mg DS-fetuin and 50pg GM,, were added. Unless otherwise stated, incubation time for the endogenous and exogenous reactions were 60 and 30 min, respectively. Incorporation of NeuNAc into glycolipids was determined on the chloroform-methanol extracts that were excluded from Sephadex G-25 columns. Incorporation into glycoproteins was determined on the trichloroacetic acid precipitated pellets after sequential chloroform-methanol extraction to remove the glycolipids. When studying the trypsinized enzyme preparations, 1 mg of soya bean trypsin inhibitor was added per mg of trypsin used, either to the trypsinized enzyme preparation or to the reaction mixture before enzyme addition. This amount of trypsin inhibitor was enough to negate tryptic activities. The inhibitor had no detectable NeuNAc acceptor activity. Sucrose density gradient centrifugation. Cerebra from 15-day-old rats were homogenized with 3 vol of 0.32 M-SUCrose and centrifuged at lo00 g for 10 min. The pellet was washed with 3 vol of 0.32 M-sucrose and centrifuged again at 1000 : for 10 min. The supernatants were pooled and 4 ml, corresponding to about 0.6 g wet weight of brain t i s sue (the wet weight per cerebrum was approx 0.9 g), were layered on a preformed linear sucrose density gradient, 0.4 M-I .6 M, total vol 26 ml. with a 2 ml 2.0 M-sucrose cushion. The gradients were centrifuged with SW 25.1 rotor at 24,000 rev./min (60.OOO g) for 2 h with a Beckman L2-65 ultracentrifuge. The above gradient condition was modified from the discontinuous gradient procedures of WHITTAKER (1959). Fractions were collected by puncturing a hole at the bottom of the tubes and collecting 55 drops (about 0.83 ml) per fraction. For selected fractions, 80 pi was used for protein determination (LOWRYet al., 1951). Eighty microlitres were also assayed for the endogenous glycolipid, endogenous glycoprotein and the exogenous glycolipid sialyltransferase activities, and 50 pl aliquots were used for the assay of the exogenous glycoprotein sialyltransferase activities. Incubation in all cases was at 37°C for 30 min. Aliquots of the gradient fractions were also assayed for activities of acetylcholinesterase. (acetylcholinehydrolase, EC 3.1.1.71, succinate dehydrogenase (succinate: NAD oxidoreductase EC 1.3.99.1)and lactate dehydrogenase (L-lactate: NAD oxidoreductase, EC 1.1.1.27).Acetylcholinesterase activities were determined by the method of ELLMAN et al., (1961) with acetylthiocholine as the substrate. Succinate dehydrogenase was assayed with ferricyanide as the artificial electron acceptor (KING,1967). Lactate dehydrogenase was assayed by the oxidation of NADH by pyru& BODANSKY, 1966). All three marker vate (SCHWARTZ enzyme assays were performed at room temperature with a recording spectrophotometer. Electron microscopy of the sucrose gradient fraction. The sucrose gradient fractions were mixed with equal volumes of ice-cold Karnovsky fixative (KARNOVSKY, 1965) for 30 min. The suspensions were centrifuged at 4°C for 10 min with an Eppendorf centrifuge. The pellets were post-fixed for 1 h with 1.33% osmium in collidine buffer and blockstained for 0.5 h in saturated aqueous uranyl acetate. After dehydration with graded ethanol, the pellets were embedded in Vestopal W as previously described (KURTZ, 1961). Sections (3WOnm) were cut on an LKB Ultra-

tome 111 using glass knives. The sections were mounted on copper grids coated with collodion and carbon and stained for I min by Reynold’s lead citrate (PEASE,1964). The grids were examined in a Philips 300 Electron Microscope operated at 60 kV and using a 20 prn or 30 pm objective aperture.

RESULTS

When the total particulate preparation from rat cerebra, with nuclei and large debris removed, was put on a continuous sucrose density gradient, a similar distribution pattern of the exogenous glycoprotein and glycolipid sialyltransferaseactivities was observed (Fig. 1a). Significantly, the peak activities around 0.7 Msucrose were distinct from the protein profile. There were small shoulders in the exogenous sialyltransferase activity profile that overlapped with protein peak I. The profile of the endogenous sialyltransferase activities was less defined than the exogenous enzyme activities but all appeared to be distributed over the same range of the gradient. The specific sialyltransferase activities in the gradient fraction 26 were 0.26, 0.09, 1.03 and 4.16 nmol/mg protein/30 min with the endogenous glycolipids, the endogenous glycoproteins, the exogenous glycolipid and the exogenous glycoprotein respectively. The corresponding values with the brain homogenate (with nuclei and debris removed) were 0.056, 0.022, 0.090 and 0.59 nmol/mg protein/30 min. Thus the gradient conditions resulted in an increase in the specific sialyltransferase activities of 4 folds in the endogenous substrates, 11 folds with the exogenous GMl, and 7 folds with exogenous DS-fetuin. There were three peaks of succinate dehydrogenase activities that overlapped with the three protein peaks (Fig. lb). The relative distribution of succinate dehydrogenase in the gradient suggested the protein peaks I, 111 and IV were enriched in myelin, synaptosomes and mitochondria, respectively, as previously described by WHITTAKER (1959). Protein peaks I and 111 were also enriched in acetylcholinesterase activities whose distribution profile was broader and less defined than succinate dehydrogenase. Lactate deh ydrogenase was essentially confined to the soluble phase and did not penetrate into the gradient. The ultrastructural identities of the gradient fractions were further elucidated by electron microscopic examinations which revealed that peak exogenous activities of sialyltransferases were associated with heterogenous membrane structures. These consisted of smooth membrane vesicles and fragments as well as curved structures which appeared to be derivatives of the Golgi complex (Fig. 2B). There were very few synaptosomes while mitochondria and myelin were essentially absent. Gradient fractions corresponding to protein peaks I, 111, and IV were observed to be enriched in myelin fragments (Fig. 2A), synaptosomes (Fig. 2C) and mitochondria (Fig. 2D), respectively.

1087

FIG.2. Electron micrographs of sucrose gradient fractions (see Fig. 1). A, fraction I; B, fraction 11; C. Fraction 111; D. fraction IV.

-

Sialyltransferases in rat brain

1089

1.6-0.4M

Go%

sucrose

sucrose

300

200

-i I

0.

a

100

5 w

c LL 0

a 3

...,..

0

^

10

-

I0

FRACTION

20

30

20

30

NUMBER

FIG.la. The distribution of endogenous sialyltransferase activities on continuous sucrose density gradient. Enzyme assays were performed according to standard conditions using [N~UNAC-~-'~C]-CMPNeuNAc of specific radioactivity of 1.9 mCi/mmol. Volume of gradient used was 80p1 except for the endogenous glycoprotein sialyltransferase assay in which only 50 pl was used. All incubations were at 37°C for 30 min. x - x . endogenous glycoproteins, A-A, exogenous DS-fetuin; o--.., endogenous glycolipids; M, exogenous GM,,; -----, protein. FIG.1b. Marker enzyme activities: A-A, succinate dehydrogenase; x -x , acetylcholinesterase; M, lactate dehydrogenase.

Effect of nonionic detergents on sialyltransferase activities

The nonionic detergent mixture, Triton CF-54/ Tween 80 (2/1, w/w), stimulated all four sialyltransferase activities, the effect being much more pronounced with the exogenous substrates. At 1 mg detergent mixture per 'mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; GMI,, and 700 and DS fetuin, 230 (Fig. 3). Effect of trypsin on sialyltransferase activities

Pretreatment of enzyme preparations with trypsin diminished all four sialyltransferase activities (Fig. 4). The sensitivity to trypsinization was very apparent and the maximum decrease in enzyme activities was

reached with the lowest concentration of trypsin used (0.025%,w/v). Residual activities were about 50% for the endogenous glycoprotein reaction and about 70% for the three other reactions. This greater decrease in endogenous glycoprotein activities was presumably due to a removal of endogenous substrates by trypsin pretreatment. The residual sialyltransferase activities were not due to a compartmentalization effect. Freezing and thawing twice and six times did not change the enzyme activities (Table 1). Furthermore, trypsinization following freezing and thawing resulted in residual activities similar to those with trypsinization alone. However, when the enzyme preparation was pretreated with nonionic detergent followed by trypsinization, nearly all enzyme activities were abolished. The action of the detergent thus appeared to be on the part of the enzyme molecules bearing the active

S.-S. NG and J. A. DAIN

1090

f0

$

o ! F l .7 : 0.2

00

0.4

8

0.6

DETERGENT (mg

FIG.3. E k t s of nonionic detergents on sialyltransferase activities. x -x , endogenous glycoproteins; A-A, exogenous DSfetuin; O----O, endogenous glycolipids; M, exogenous GM,,.

sites. These regions of the enzyme molecules appeared to be masked in the native cell membrane and were thus resistant to tryptic action. The action of the nonionic detergents exposed these sites, making them susceptible to the action of trypsin. EfSect of organic solvents on sialyltransferase activities

The hypothesis that the active sites of sialyltransferases are situated in a hydrophobic environment in native membranes was tested with the use of organic solvents of various dielectric constants. With all the seven organic solvents tested, marked inhibition 0

2.0

10

[SOLVENT] (MI

o.lp-g opxzIL-y 0

0.05

0.1

X

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1.0 [SOLVENT]

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FIG.4. Effect of trypsin pretreatment on sialyltransferase activities. Trypsinization was a t 37°C for 15 min. x -x , endogenous glycoproteins; A-A, exogenous DSfetuin; +o, endogenous glycolipids; O---O, exogenous GMI,.

2.0 (MI

FIG.5. Effect of organic solvents on sialyltransferase activities with endogenous glycolipid (A) and endogenous glycoprotein (B) acceptors. The solvents were methanol (x-x), ethanol (+----O), acetone (A-A), n-pro2-chloro ethanol (A-A), n-butanol panol (%o), ( 0 4 ) and chloroform ( 0 4 ) .

Sialyltransferases in rat brain

TABLE 1. EFFECTOF FREEZING

1091

AND THAWING, DETERGENT, AND TRYPSIN ON SIALYLTRANSFERASE ACTIVITY

NANA Incorporated (nmol/mg protein/30 min) Endogenous GL Endogenous G P Exogenous GM, Exogenous DSF

%

Pre-treatment of enzyme(*)

nmol

control

Control Freezing and thawing, twice Freezing and thawing, six times Trypsin, 0.2% Freezing and thawing, six times + trypsin, 0.2% Detergents, 1.67%((t) trypsin, 0.2%

0.13 0.12

100

%

%

%

nmol

control

nmol

control

92

0.026 0.029

100 112

0.11 0.13

100 118

0.65 0.65

100 100

0.11 0.092

90 74

0.030 0.011

115 42

0.13 0.11

118 100

0.65 0.55

100 85

0.086

69

0.011

42

0.12

109

0.53

82

0.020

16

-(I)

-(I)

0.001

0.9

0.06($)

nmol

control

~~

+

9($)

* All pretreated enzyme preparations, including the control, were pre-incubated for 15 min at 37“ before they were assayed. f Detergent mixture composed of Tween 8O/rriton CF-54, 2/1, w/w. $ Data not available. $ Approximation, assuming the contribution of endogenous activity negligible. tled controversy. Some of the enzymes have been reported to be present in all major subcellular struc& CAPUTID, tures (ARCEet al., 1966, 1971; DUFFARD 1972; HILDERBRAND et al., 1970). Several studies have indicated that these enzymes are concentrated in the membranes of the synaptic complexes or synaptic & DAN, 1972; BOSMANN, 1972, vesicles (DICESARE 1973; DEN et a[., 1975; BROQUET & LOUISOT, 1971). In other studies, however, the glycosyltransferase activities were found to be relatively low in the synaptic et al., 1972). Both galactosyl membranes (MORGAN and N-acetylgalactosaminyl transferases have since DISCUSSION been shown to be localized in microsomal membranes (KO & RAGHUPATHY,1971, 1972). The non-synaptic The subcellular localization of the glycosyltransferlocalization of the glycosyltransferases is further s u p ases in brain tissues has been a subject of still unsetported by the following observations: 1. Both neuronal and glial cell bodies were shown to possess C sialyltransferase activities (GIELEN& HINZEN,1974); 2. different regions of the brain with different degrees of synaptic densities were found to have comparable specific activities of the enzymes (VANDEN EIJNDEN & VAN DIJK, 1974); and 3. high glycoprotein sialyltransferase activities were observed in the newborn rat brain even though the synaptic structures were not developed. In fact, the sialyltransferase level was 40 found to decrease during postnatal development when active synaptic formation took place (VAN DEN EIJNDENet al., 1975). The controversy over this issue appears to arise Ch from the fractionation techniques used. In all the - I reported studies on cellular fractionation, differential I I 10 20 30 40 centrifugations were made followed by discontinuous sucrose density gradients. The pelleting and resuspenSOLVENT DIELECTRIC CONSTANT sion procedures may cause unnecessary artifacts in FIG.6. Correlation of solvent inhibitory effects on sialylassessing the subcellular localization of the glycosyltransferase activity and their dielectric constants. Solvent designations are Me (methanol), Et (ethanol), Ac (acetone), transferases. In this report, rat cerebral homogenate, Pr (n-propanol),Ce (2-chloroethanol),Bu (n-butanol), and with nuclei and large debris removed, was applied C1 (chloroform. 0 , endogenous glycolipid sialyltransferase directly onto a continuous sucrose density gradient. activities; x , endogenous glycoprotein sialyltransferase Sialyltransferase activities were assayed in the fracactivities. tions collected.

towards the endogenous glycolipid and glycoprotein sialyltransferase activities was observed (Fig. 5a and 5b). The inhibitory effect of the alcohols clearly increased with the chain lengths of the homologs. Chloroform, the solvent with the lowest dielectric constant tested, was the most potent inhibitor. Chloroethanol had an effect intermediate between n-butanol and n-propanol. The effect of acetone is similar to ethanol. In general, the inhibitory effects of the organic solvents were inversely related to their dielectric constants (Fig. 6).

0/I

/

I

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S.4.NG and J. A. DAIN

As indicated in Fig. la the distribution of all four short- and long-range order of the glycosyltransferase sialyltransferase activities did not correspond to pro- systems. Studies on concanavalin A receptor topotein peaks 111 and IV which, according to marker graphy on the surface of several cell types strongly 1959; DE- suggest the existence of intracellular agents that bind enzyme activities (Fig. Ib; WHITTAKER, ROBERTISet al., 1962; MCINTOSH & PLUMMER, 1976) to membrane components and restrict their tendency and electron microscopic examinations (Figs. 2C and to attain a random and uniform distribution (BERLIN 2D), were enriched in synaptosomes and mitochon- et al., 1974). The stimulatory effect of nonionic detergents (Fig. dria, respectively. All four sialyltransferase activities occupy the same range in the gradient, overlapping 3) on the sialyltransferase reactions m a y reflect an with protein peak I which was enriched in myelin interaction of the of the hydrophobic environments fragments (Fig. 2A). The exogenous sialyltransferase of the active sites with the detergents, possibly by activities showed peak activities at about 0.7 M-suc- the insertion of the latter into the lipid bilayer surrose (Fig. 1a). Electron microscopic examination rounding the enzymes or by the formation of deterrevealed that the exogenous sialyltransferase peak ac- gent-enzyme complexes, thus inducing more active tivity fraction (11) was associated with an enrichment enzyme conformations (SIMONSet al., 1973; UTER1974). The effect in smooth membrane fragments and vesicles, some MANN & SIMONS,1974; COLEMAN, of which resembled structures of Golgi complexes. of nonionic detergents m a y be similar to the preThere were very few synaptosomes and essentially no viously reported effects of phosphatidyl ethanolamine et a\., 1968), CDP-choline and lysolecithin mitochondria and myelin in that fraction. From these (KAUFMAN et al., 1972; MOOKERJEA & YUNG, 1974), observations, the conclusion can be drawn that sialyl- (MOOKERJEA transferases are concentrated in membrane structures phospho-diglycerol and cardiolipid (KEENANet al., which are not related to synaptosomes and mitochon- 1974). The finding that most of the enzyme activities were dria. These sialyltransferase-enriched structures are presumably derived from the endoplasmic reticulum, resistant to trypsin (Fig. 4) provides the evidence for the Golgi complexes and the plasma membrane. The the integral nature of the proteins (MARCHES et al., sialyltransferase activity associated with the myelin- 1972) with the active sites within the lipid bilayer. enriched fractions (protein peak I) are believed to be This masking effect of a hydrophobic environment due to the light microsomal membranes derived from was not due to compartmentalization effect and it the same structures which also gave rise to the was abolished in the presence of nonionic detergents enzyme peak 11. In addition, the profile of the endo- which made the enzymes completely susceptible to genous sialyltransferase activities reflects a simul- the action of trypsin (Table 1). This membrane orientaneous Occurrence of the sialyltransferases and the tation of sialyltransferases was further indicated by the inhibitory action of organic solvents on the endoendogenous substrates. The gradient pattern is highly reproducible. We genous enzyme activities (Fig. 5). With the four prihave since demonstrated that UDP-galactose: GM, mary alcohols used, the inhibitory effects increased galactosyltransferase is similarly located in this gra- with the length of the alkyl groups. With all the seven dient peak (DAIN& HITCHENER, 1977). The peak organic solvents tested, the extent of inhibition was galactosyltransferase activities also corresponded to inversely related to the solvent dielectric constants peaks of UMP-ase (5’-ribonucleotide phosphohydro- (Fig. 6). It is conceivable that the more lipophilic sollase, EC 3.1.3.5)and UDP-phosphohydrolase (nucleo- vents, with lower dielectric constants, are more sidediphosphate phosphohydrolase, EC 3.6.16) activi- powerful perturbing agents on the hydrophobic enties which are marker enzymes for the endoplasmic vironments of the enzyme molecules. reticulum. We have no information on the relative contribu- Acknowledgements-The technical assistance of Mr. L. (Department of Biochemistry, McGill Univertion of the various cell types in the observed sialyl- GULUZIAN transferase activities. Comparable enzyme activities sity) in electron microscopy is gratefully acknowledged. This work was supported in part by NIH Grant have been reported in the neuronal and glial cell preparations (GIELEN & HINZEN,1974). More defini- NS-05104. tive work will necessarily involve autoradiographic studies as initiated by some investigators (PORTER & REFERENCES BERNACKI, 1975). The mechanisms which are responsible for the H. F. & CAPUIIUR. (1966) Archs Bioshort- and long-range order (SINGER& NICOLSON, ARCEA., MACCIONI chem. Biophys. 116, 52-58. 1972) of the glycosyltransferases and their substrates ARCE A., MACCIONI H. F. & CAPUTIY) R. (1971) Biochem. and products within specific cellular structures are J . 121, 483493. open to speculation. Results in this report suggest BERLINR. D., OLVERJ. M., UKENA T. E. & YIN H. H. that the sialyltransferases are integral proteins with (1974) Nature 247, 45-46. their active sites deeply embedded in the lipid bilayer BOSMANNH. 9. (1972) J . Neurochem. 19, 763-778. of cell membrane. Thus factors located either external BOSMANN H. 9. (1973) J . Neurochem. 20, 1037-1049. or internal to the lipid bilayer can bring about the BROQUETP.& LoulsoT P. (1971) Biochimie 53, 921-927.

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