Int. J. Biochem. Vol. 23, No. 7/8, pp. 719-726, 1991 Printed in Great Britain. All rights reserved

0020-711X/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press pie

PRESENCE OF A N EXTRACELLULAR GLYCOSYLTRANSFERASE IN H U M A N DENTAL PLAQUE MARIA ALICIA UGARTE and PROVIDENCIARODRIGUEZ* Laboratorio de Trombosis Experimental, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 21827, Caracas 1020A, Venezuela [Tel. 501-5522]

(Received 6 August 1990) Abstract--1. Glycosyltransferase activity incorporating ~4C-radioactivity from [~4C]sucrose into endogenous acceptor was demonstrated in human dental plaque. 2. The enzyme was localized in dental plaque into two forms: (a) associated form to bacteria (pellet 10,000 g) and (b) released as an extracellular form (supernatant 10,000g). 3. The reaction product was insoluble in 95% ethanol, soluble in trichloroacetic acid, and it was a mixture of saccharides with different sizes, as was demonstrated by column chromatography. 4. Exogenous activity with Dextran T-10 as substrate was also demonstrated, and it represented 9% of the total endogenous activity. 5. Characterization of the extracellular glycosyltransferase, and comparative results with glycosyltransferase secreted by oral bacteria in cultures medium are discussed.

INTRODUCTION

It is generally accepted that the synthesis of extracellular polysaccharides from sucrose is necessary for the formation of dental plaque by oral microorganisms. These complex polysaccharides have been considered important for colonization of the bacteria on tooth surfaces ( H a m a d a and Slade, 1980). Fructan is synthetized by the enzymatic action of fructosyltransferase (FTF). Glucan synthesis is catalyzed by the concerned action of two types o f glycosyltransferase (GTF), one G T F - I which produces a water-insoluble glucan (WIG), and another G T F - S which produces a water-soluble glucan (WSG) (Inoue et al., 1982; Furuta et al., 1985). Results obtained from the in vitro growth of oral streptococci on artificial media have indicated that the enzyme G T F is produced in cytoplasm and released into the culture fluid, followed by its participation in glucan formation. In view of these findings, extracellular and cell-associated G T F are considered alternate states of the same enzyme protein ( H a m a d a and Slade, 1979; Janda and Kuramitsu, 1976; Shimamura et al., 1982). Although many studies have been reported concerning purification and characterization of extracellular G T F - S and G T F - I from culture medium of oral bacteria ( H a m a d a and Slade, 1980; McCabe, 1985; H a m a d a and Takehara, 1987; Yamashita et al., 1988; Furutani et al., 1988) very little attention has been focused on its existence in vivo in human dental plaque. Several reports have shown that the biochemical composition of human dental plaque is influenced by both salivary composition and dietary intake (Ashley and Wilson, 1977, 1978). Since the situation in real plaque is definitively different from the artificial media where a single microbial specie has been grown, it was of interest to investigate the

*To whom all correspondence should be addressed.

existence of the enzyme G T F in human dental plaque. In this report, we show the presence of G T F activity in a crude homogenate of dental plaque from children. In addition its characterization is described, and we compare its properties with the extracellular G T F produced in culture medium of oral bacteria. MATERIALS AND

METHODS

Materials [U-14C]Sucrose (671 Ci/mmol) was supplied by New England Nuclear. Sephadex G-25, Sepharose CL6B and Dextran T-10 were obtained from Pharmacia Fine Chemical. Glass fibre filter GF/A was obtained from Whatman. All other chemicals were purchased from Sigma Chemical Co. Plaque collection Pools of dental plaque were obtained 2 hr after eating from 20 caries-active school children between the age of 9 and 12 yr. In each subject, all the available dental plaque which could be collected using a dental instrument was removed and transferred to a glass vial with screw cap placed on an ice bath. Preparation of plaque homogenate The samples of plaque were suspended in 25 vol (w/v) of chilled 50mM phosphate buffer (pH 7.5) and then homogenized using a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 10,000g for 10min, the supernatant was separated and the pellet was resuspended in the same volume of phosphate buffer. All procedures were carried out at 0-5°C. The activity of glycosyltransferase was assayed in bath fractions. All the experiments reported here were conducted with the supernatant. Stored at -20°C both fractions lose practically no enzymatic activity at least not within 4 weeks. The supernatant was chromatographed on Sepharose CL6B. Protein was determined by the method of Peterson (1977) using bovine serum albumin. Lactate dehydrogenase (LDH), was determined in the supernatant as described by Kornberg (1955). For electron microscopy, the pellet fraction was suspended in 2% phosphotungstic acid. 719

Fig. 1. Electron micrograph of 10,000g pellet from dental plaque homogenate, x 13,000.

720

Extracellular GTF in human dental plaque

721

Table I. Glycosyltransferas¢ activity in fractions from dental plaque Total Specific Activity Protein activity activity in plaque Fraction (rag) (kU) (kU/mg protein) (U/mg plaque) 10,000g supernatant 1.51 19.90 13.18 120 10,000g pellet 8.30 126.75 15.27 732 Each reaction mixture contained 100 #g of protein of the fraction indicated, 20 # M sucrose (20,000 dpm) and 20 mM phosphate buffer (pH 7.5) in a final volume of 500/zl. One unit is the amount of enzyme which transfer 1 dpm of ~4C-radioactivityinto polysaccharide per 60 rain at 37°C.

Assay of glycosyltransferase activity The standard assay of GTF activity was performed in a final volume of 500/~1 containing 20/~M [U-]4C]sucrose (20,000 dpm), 50 mM phosphate buffer (pH 7.5) and 2.5 #g of protein from the plaque preparation. The incubation was carried out at 37°C for 2.5 min and stopped by placing it on an ice bath and adding I ml of 95% chilled ethanol. After 15 min on ice, the total polysaccharides were separated by filtration under vacuum on glass fibre filters (2.4 cm). The precipitate was washed free of ethanol-soluble radioactivity with l-ml portions of 95% ethanol, then the filters were dried in an oven at 60°C for 10 min. For the determination of radioactivity, the filters were counted in 6 ml of Aquasol. Controls with trichloroacetic acid denatured enzyme were run with each incubation and the values observed (usually 2-3%) were substrated from those of the test samples. Enzyme activity was expressed as dpm of ]4C-radioactivity transferred to endogenous acceptor per 2.5 min. Exogenous GTF was measured with the standard assay described for endogenous activity above, but 10 nM Dextran T-10 was added as exogenous substrate. The radioactivity incorporated into the exogenous acceptor was determined by subtracting the value obtained when the assay was performed in the absence of added acceptor.

RESULTS I n c u b a t i o n s o f []4C]sucrose with the s u p e r n a t a n t a n d pellet fraction from h u m a n dental plaque lead to the transfer of radioactivity to a n e n d o g e n o u s acceptor precipitable by 9 5 % ethanol. Enzymatic activities in b o t h fractions are summarized in Table 1. A l t h o u g h a similar specific activity was observed in the two fractions, the enzymatic activity per m g o f plaque present in the pellet is approx. 6 times higher t h a n t h a t o f activity from the supernatant. The electron m i c r o g r a p h o f b o t h fractions showed t h a t the pellet (Fig. l) contains the bacteria existent in the dental plaque, a n d the s u p e r n a t a n t was free of them. D e t e r m i n a t i o n o f L D H activity in the s u p e r n a t a n t d e m o n s t r a t e d only the presence o f 2.3 x 10 -4 I U / m g of plaque. The elution profile o b t a i n e d o n Sephadex G-25 gel filtration (Fig. 2) of the p r o d u c t formed, d u r i n g 8 a n d 24 hr showed the i n c o r p o r a t i o n o f radioactivity into

25

Sepharose CL6B chromatography The 10,000g supernatant (12.8mg of protein) was lyophilized and resuspended in l ml of 0.1 M phosphate buffer, pH 7.5 (standard buffer). This sample was applied to a Sepharose CL6B column (2.5 x 90era) which has been equilibrated with the standard buffer, The column was eluted with the same buffer. Endogenous and exogenous enzymatic activities and absorbance at 220 nm were determined in the fractions.

Chromatograph); of reaction products Incubation mixture used for the analysis of the products contained the following components: 100 #g of protein; 20/zM of [U-~4C]sucrose (125,000 dpm); 50 mM phosphate buffer (pH 7.5), in a final volume of 200/zl. Incubations were performed at 37°C during 8 and 24 hr, and stopped on an ice bath. Reaction products obtained were run through a Sephadex G-25 column (0.9 x 41 cm) equilibrated and eluted with the standard buffer. The fractions were counted in 2 ml of Aquasol. A larger amount of reaction product was obtained from an incubation mixture containing in 2 ml total volume: 20/zM [U-14C]sucrose (9.5 x 106dpm), 50mM phosphate buffer (pH 7.5), 9.5 mg of protein, in the presence of 0.2% sodium azide. The incubation was carried out at 37°C for 48 hr, and chromatographed on Sepharose CL6B column equilibrated with the standard buffer. The elution of the column and the determination of radioactivity were performed as described above. Absorbance at 220 nm was also determined in the fractions. In other experiments the reaction product obtained from the larger incubation was mixed with 2 ml of 6 M guanidine hydrochloride (GuHC1). After 60min the sample was applied to the Sepharose CL6B column described above. Radioactivity and absorbance at 220 nm were determined in the fractions. BC 23/7tS--F

A

2.5

2.0 A o v

E

0.5 0.1

20

40

60

Frach0n Number

Fig. 2. Sephadex G-25 chromatography of reaction product. Reaction mixture of 100/~ g of protein (10,000 g supernatant fraction) with [14C]sucrose (129,000 dpm) was performed as described in Materials and Methods (standard conditions) at 8 ( 0 Q); and 24 hr (O O) of incubation. The complete assay mixture was subjected to gel filtration on Sephadex G-25 column (0.9 x 41 era) equilibrated and eluted with the standard buffer. Fractions of 0.5 ml were collected, and radioactivity was measured for each fraction.

722

MARIAALICIA UGARII~ and PROWDZ~CXA RODRIOUEZ 25

IV

Ii

2304 "E Ill

c

o

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02

I0

I

II

30

70

IlO

Fraction Number

Fig. 3, Sepharose CL6B chromatography of reactmn product. Incubation of 9.5 mg of protein (10,000g supernatant fraction) with [14C]sucrose (9.5 x l&dpm) in the presence of 0.2% sodium azide was carried out as described under Materials and Methods during 48 hr. The reaction mixture was chromatographed on Sepharose CL6B column (2.5 × 92 cm), equilibrated and eluted with the standard buffer. Fractions of 5 ml were collected. Radioactivity (O O) and protein absorbance at 220 nm (© ©) were determined in the fractions. one compound that was eluted with Vo, with a yield of 0.36 and 8.5% respectively of the total radioactivity at the two times studied. At 24 hr incubation I 90£

A

the graphic showed two maxima in 1/"o, indicating that it is not a single compound. A major peak eluted with Vt (Fig. 1) corresponds to unreacted []4C]sucrose added to the assay mixture and radioactive sugars product of the enzymatic reaction. When the reaction mixture was applied to the Sepharose column (Fig. 3) the proteins were eluted in four peaks and the radioactivity was only present in peak III (fractions 76-82) and peak IV (fractions 87-92). This last peak corresponds with Vt, where [14C]sucrose, [14C]glucose and [14C]fructose standards were eluted. In order to characterize these two radioactive peaks, the fraction of each one of them with the highest radioactivity (79 and 96) were further chromatographed on a Sephadex G-25 column. The fraction 79 (peak III) as shown in Fig. 4(A) yielded 28% of the radioactivity in Vo and the rest 72% in several ~4C containing peaks eluted between Iio and lit. Figure 4(B) shows that most of the radioactivity (96%) from fraction 96 (peak IV) was eluted with V~ and only 4% in Vo. These results are similar to the previous data obtained from Sephadex G-25. As the reaction product peak III (Fig. 3) showed the presence of proteins and radioactivity, to test if it is a dissociable complex the reaction mixture previously treated with GuHCI was applied to the same Sepharose column. The results shown in Fig. 5 demonstrated the absence of peak III, and the appearance of all the radioactivity in peak IV. Efforts to precipitate the radioactive product from the incubation mixture with 10 and 50% trichloroacetic acid were negative (data not shown).

Characterization of glycosyltransferase Incorporation of 14C-radioactivity from [J4C]sucrose into endogenous acceptor from dental plaque was proportional to time for at least 5 min, between

i

~o

I

~

B

I

vo

VT

250

60c

g 15.£ "o

3D

5.0

20

40

Fraction Number

60

20

40

60

Fraction Number

Fig. 4. Sephadex G-25 chromatography of peak III and IV (Fig. 3) from Sepharose CL6B column. (A) 200#1 aliquot of fraction 79 eorr~ponding to peak III, was applied on Sephadex G-25 column (0.9× 37.5em) as described in Fig. 2. (13) 200ttl of fraction 96 corresponding to peak IV was chromatographed on Sephadex G-25 column as described in (A).

723

Extraeellular GTF in human dental plaque

'

67-80), and it was approx. 58% lower than the endogenous. On the other hand, Dextran caused a decrease of 43% on the endogenous GTF activity present in peaks 1 and 2.

;,

DISCUSSION

0.50

0.25 ~

50

60

120

Fraction Number

Fig. 5. Sepharose CL6B chromatography of reaction product after GuHC1 treatment. The reaction mixture performed as described in Fig. 3, was mixed with the same volume of 6 M GuHCI during 60 min. Chromatography of the sample and symbols are the same as in Fig. 3. 60 and 100 min of incubation the activity remained constant [Fig. 6(A)]. Glycosyltransferase activity was also dependent on the protein concentration up to 2 #g/ml [Fig. 6(B)]. The extent of glycosylation also increased with substrate concentrations up to 3/zM sucrose [Fig. 6(C)]. As can be seen, a plateau was reached between a substrate level of 3 and 4/~M, the activity was increased proportionally up to 5/z M. At sucrose concentration above this level the glycosylation was decreased and reached a maximum of inhibition of 40% between 1 and 20 mM (data not shown). As indicated in Table 2 the addition of 0.27 and 0.55 mM NaF stimulated the glycosyltransferase activity 1.8 and 0.9%-fold respectively, whereas at 5 mM the enzyme was 16% inhibited. Mg 2+ at 0.8 and 5mM inhibited the activity 88%. Since there exists evidences that extracellular glycosyltransferase from a culture of Streptococcus mutans is inhibited by an water-soluble extract of cocoa (Paolino and Kashket, 1985), we studied its effect on our preparation at 0.75 and 7.5 mg/ml concentrations. Thirtyeight and 72% inhibition was observed, respectively (Table 2).

Heterogeneity of glycosyltransferase activity As shown in Fig. 7 four peaks of enzyme activity were obtained from the 10,000g supernatant on a Sepharose column. Peak 1 (fractions 35-45) and peak 2 (fractions 46--63) showed the highest enzymatic activity, a minor peak 3 (fractions 67-80) and peak 4 (fractions 83-95) with a very low activity of the enzyme. Determination of exogenous GTF activity in the presence of Dextran T-10 demonstrated one maximum of activity with this substrate. This exogenous activity was associated with peak 3 (fractions

The observations summarized in the preceding section demonstrated that dental plaque contains an appreciable amount of GTF activity capable of incorporate glycosyl residue from sucrose to an endogenous acceptor. These results constitute the first evidence for the existence of GTF activity in human dental plaque in vivo. Under our conditions, the enzymatic activity corresponds to the two enzymes that transfer both residues from []4C]sucrose. These GTF should be the enzymes responsible for the synthesis of the extracellular polysaccharides (glucans and fructans) constituent of the matrix of human dental plaque reported previously (Gibbons and Banghart, 1967). The results obtained in the electron microscopy indicate that the enzymatic activities found in the pellet and supernatant fractions (Table 1) correspond to the bacteria associated and extracellular GTF, respectively. Also, the low level of LDH present in the supernatant support the view that the cytosolic fraction from the bacteria existent in the plaque does not contribute significantly in the extracellular GTF activity. Similar data have been found in cultures of oral bacteria. Results obtained from cultures of Strep. mutans had demonstrated that the cells grown in sucrose-free medium produce mostly extracellular GTF; whereas they produce mainly GTF associated to the cell when grown in sucrose-containing medium (McCabe and Smith, 1973; Hamada and Tori, 1978). According to our results, in real plaque (Table 1) the distribution of the GTF activity occurs as in the last condition, and reflects that much of the enzyme is in the matrix of the plaque associated to the cell in an active form. The synthesis of a polysaccharide is judged from its insolubility in 95% ethanol and solubility in trichloroacetic acid. As the reaction product was excluded from Sephadex G-25 (Fig. 2) we can ascribe that it has a molecular mass above 5000 Da. Its heterogeneity shown in Vo at 24-hr incubation suggests that the synthesis of polysaccharides takes place with different sizes. The results obtained from Sepharose column after 48 hr of incubation (Fig. 3) indicate that the reaction product reaches a high molecular weight as it is eluted near to Vt. Posterior analysis of peak III in Sephadex G-25 [Fig. 4(A)] confirmed that the reaction products were a mix of polysaccharides (mol. wt > 5000 Da) and several intermediates oligosaccharides (mol. wt 500--5000 Da). These observations are consistent with previous studies of polysaccharides synthesis by oral bacteria in culture. Thus it is well documented that the extracellular glucans produced by Strep. mutans are very polydisperse and they can be subdivided into fractions with different molecular sizes and chemical structures (Inoue and Koga, 1979). On the basis of the results obtained from the exposure of the reaction mixture to GuHCi and

724

M A R I A ALICIA U G A R T E and PROVIDENCIA RODI~JGUEZ

followed by Sepharose column (Fig. 5), it seems likely that the polysaccharide is able to form a dissociable complex with the protein eluted in peak III (Fig. 3). The dissociation of the complex apparently makes the individual molecules elute in Vt (Fig. 3). Also the fact that the reaction product was not precipitable by trichloroacetic acid shows that the glycosyl chain is not covalently bound to the protein. It is interesting to note that the protein associated to the polysaccharide (peak III, Fig. 3) did not show significantly G T F activity. This view is supported by the elution profile obtained from our preparation on Sepharose column (Fig. 7) where it is observed that the most enriched fractions with enzymatic activity are eluted in the first part of the column (peaks 1 and 2), and there is very low activity in the region where the product is eluted (peak III, Fig. 3). In this aspect 15

~

..g

i

I

I

Table 2. Effect of ions and cocoa extract on G T F from dental plaque

Addition

-0.27 0.55 5.00 0.80 5.00 0.75 7.5

100 136 ll4 84 12 13 62 28

None (control) NaF

MgCI 2 Cocoa extract

The enzymatic activity was assayed as described under Materials and Methods, with 10,000 g supernatant fraction. The concentration of NaF and MgCI 2 are in mM in the incubation mixture, and mg/ml for the cocoa extract.

the extracellular GTF in the dental plaque differs from the GTF released into culture fluid of Strep. mutans, which occurs associated to the water-insoluble l

:3.5

A

Concentration

Glycosyltranfferase activity (% of control)

B

J

J

I0

1.5

E ¢,1 5

0.5 I

20

I

40 Time (rain)

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60

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15

//

I00

Protein concentration(IxJ/ml) l

I

b~ .c

E

IC

I

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2.0

4.0

Sucrose

cor~entmtion

6,0 (pMI

Fig. 6. Glyeosyltransferase activity as a function of (A) incubation time, (B) protein concentrations and (C) sucrose concentration. Incubations were performed under standard assay conditions described in Materials and Methods, except with the variations indicated. Activities were measured with the I0,000 g supernatant.

Extracellular GTF in human dental plaque I

I

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50I

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4.0 -

2.0

m"-

b

i

~

3£ -

_= 2.0-

t,O-

" 30

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Fraction Number Fig. 7. Sepharos¢ CL6B chromatography o f 10,000 g supernatant. The sample (T2.8mg) was chromatographed on

Sepharose CL6B column as described in Fig. 3. The fractions were assayed for endogenous glycosyltransferas¢ activity (O 0 ) ; exogenous glycosyltransferase activity (A A); and protein absorbance at 220 nm (O O). Exogenous activity was measured with 10 nm Dextran T-10 as exogenous acceptor. F,nzyme activities were determined

with 2.5 mg of protein. polysaccharide product of the enzyme (Smith et al., 1979), and the formation of aggregated forms of the GTF in the presence of Dextran T-2000 has been described (Furutani et al., 1988). Also a specific structural domain of GTF from Strep. sobrinus was found to bind to glucan product (Kobayashi et al., 1989). In consequence in the dental plaque, the polysaccharide is bounded to other proteins present in the matrix. Our results from Sepharose chromatography demonstrate the separation of glycosyltransferase from dental plaque into different fractions with its correspondent endogenous acceptor (Fig. 7). The inhibition of the endogenous glycosyltransferase (peaks 1 and 2, Fig. 7) by Dextran T-10, suggests a competence of both substrates for the enzyme. Since the enzymatic activity in peak 3 was increased with Dextran T-10, it supports the amount of endogenous acceptor present was limiting only in these fractions, however in peaks 1 and 2 the GTF has enough endogenous substrate for its activity. These facts appear to reflect the existence in the matrix of dental plaque of different kinds of GTF with its appropriate endogenous substrate which may account for the heterogenity of the product polysaccharides synthetized. The effect of Dextran resembles to those data reported in cultures of oral streptococci where several kinds of GTF have been purified. Some of them are primer-independent and other ones are primer-dependent enzymes (Ciardi et al., 1977; McCabe, 1985; Tsumori et al., 1989; Yamashita et al., 1988).

725

The saturation of GTF activity by low sucrose and protein concentrations (Fig. 6) indicates a high affinity for donor and acceptor substrates and may reflect its critical importance for the polysaccharide synthesis in dental plaque. Its high affinity for sucrose is in striking contrast to the GTF purified from the culture supernatant of oral bacteria which shows Ks is significantly higher. The Km values reported are in the range of 1-145 mM (Guggenheim and Newbrun, 1969; Tsumori et al., 1985; Yamashita et al., 1988). The finding of a biphasic curve with sucrose (Fig. 6) could be explained in part by the differences in the catalysis of the polymerization of glycosyl units by the various GTF activities present in our preparation (Fig. 7), which contribute to the synthesis of saccharides with distinct sizes and structures. Also the biphasic curve could be the consequence of the formation of oligosaccharides of low molecular weight that serve as substrate for the synthesis of polysaccharides of higher molecular mass. The inhibition of GTF from our preparation with the extract of cocoa was higher than that observed on the enzyme from culture of Strep. mutans. However, at this stage of our work we do not know the composition of the water-soluble extract of cocoa. Our report provides evidence that GTF activity could be modulated in intact dental plaque by F +, Mg 2+, and an extract of cocoa. This fact suggests a physiological significance, since the oral cavity is exposed to effectors present in foods which can regulate the enzyme. However, recently it was reported that NaF at comparable concentrations significantly enhanced the production of extracellular GTF by Strep. mutans (Sato et al., 1989) without the influence of GTF activity itself (Treasure, 1981). The modulation of mammalian GTF by metal ions and phospholipids effectors has been well documented (Wilson et al., 1987; Smaal et al., 1987; Fern/mdez-Briera et al., 1988). In conclusion, according to our data there are various properties of GTF which may make the situation in real plaque in vivo differ from the singlestrain model system reported in the bibliography. SUMMARY

Human dental plaque has been shown to contain GTF, that catalyzes the transfers of [14C]glycosyl from [14C]sucrose to an endogenous acceptor. The extracellular enzyme (lO,O00g supernatant fraction) was extracted from the dental plaque with phosphate buffer (pH 7.5), but in the precipitate the activity remained associated to the bacteria 6 times greater than that of the enzyme extracted. The reaction product formed was precipitated with 95% ethanol and soluble in trichloroacetic acid like a polysaccharide. Gel filtration on Sephadex G-25, and Sepharose CL6B column permitted its separation from the substrate [14C]sucrose, and it was demonstrated as a mixture of several saccharides with different molecular sizes, which showed the presence of protein. Its treatment with 6 M GuHC1 followed by gel filtration demonstrated that the polysaccharide was released from the protein. The extracellular GTF showed dependence with respect to time, and both protein and substrate concentration. It was strongly inhibited

726

MARIAALICIA.UGARTEand PROVtDENCIAROORtGUEZ

by M g 2+ at 0.8 and 5.0 m M concentrations. Ion F + at 5.0 m M concentration inhibited 16%, but at lower levels was an activator. C h r o m a t o g r a p h y of the extracellular G T F on Sepharose CL6B showed the separation o f four fractions with endogenous enzymatic activity. Only one o f the fractions exhibited exogenous activity with Dextran T-10 as acceptor. Acknowledgements--M. A. Ugarte was supported by a fellowship from the Consejo de Desarrollo Cientifico y Humanistico, and the cost of [14C]sucrose was defrayed by the Facultad de Odontologia, Universidad Central de Venezuela. We thank Dr F. Gil, IVIC, who made the electron microscopy work, Mrs Lilian Palacios for her secretarial work and Mrs Dhuwya Otero for drawing the figures.

REFERENCES

Ashley F. P. and Wilson R. F. (1977) The relationship between dietary sugar experience and the quantity and biochemical composition of dental plaque in man. Archs oral Biol. 22, 409-414. Ashley F. P. and Wilson R. F. (1978) The relationship between calcium and phosphorous concentrations of human saliva and dental plaque. Archs oral Biol. 23, 69-73. Ciardi J. E., Beaman A. J. and Wittenberger C. L. (1977) Purification, resolution and interaction of the glycosyltransferases of Streptoccus mutans 6715. Infect. Immun. 18, 237-246. Fern~indez-Briera A., Luisot P. and Morelis R. (1988) Characterization of a glycosyltransferase activity in liver plasma membrane: modulation by cations and lipidic effectors. Int. J. Biochem. 20, 951-958. Furuta T., Koga T., Nisizawa T., Okahashi N. and Hamada S. 0985) Purification and characterization of glucosyltransferase from Streptococcus mutans. J. gen. Microbiol. 131, 285-293. Furutani M., Iwaki M., Yagi T., Iida M., Horike K. and Nozaki M. (1988) A simple purification method for a glucosyltransferase complex from Streptococcus mutans OMZ 176 with a high yield. Int. J. Biochem. 20, 1327-1332. Gibbons R. J. and Banghart S. B. (1967) Synthesis of extracellular dextran by cariogenic bacteria and its presence in human dental plaque. Archs oral Biol. 12, I 1-24. Guggenheim B. and Newbrun E. (1969) Extracellular glucosyltransferase activity of an HS strain of Streptococcus mutans. Helv. odont. Acta 13, 84-97. Hamada N. and Takehara T. (1987) (1-,3)-2