Specific Inhibition of Insoluble Glucan Synthase (GTF-I) by Maillard Reaction Products from Casein and Albumins Maillard Reactio

Agric. Biol. Chem., 54 (6), 1417-1424, 1990 1417

Specific Inhibition of Insoluble Glucan Synthase (GTF-I) by Maillard Reaction Products from Casein and Albumins Shunsuke Kobayashi, Kenji Koga, Osamu Hayashida, Yamaji Nakano* and Yasuhiro Hasegawa Central Research Laboratory, Godo-Shusei Co., Ltd., 250, Nakahara, Kamihongo, Chiba 271, Japan Received December 5, 1989

The Maillard reaction was done with several proteins and aldoses (glucose, glyceraldehyde, and glycolaldehyde) under both neutral and alkaline conditions. Maillard reaction products (MRP) from a -casein and albumins (ovalbumin, bovine serum albumin, humanserum albumin, and a-lactalbumin) inhibited adhesive insoluble glucan synthesis by glueosvltransferase (GTF) of Streptococcus mutatis. The magnitude of the inhibitory activity in MRPfrom as-casein and the three aldoses correlated with the reactivity of the aldoses with proteins. The MRPfrom as-casein and albumins inhibited GTF-I and did not affect GTF-S, while their original proteins stimulated GTF-I specifically. These results suggest that the inhibitory activity of these MRPmay be related to the GTF-I stimulating property of their original proteins.

Food chemists have shownthat stored and inhibited GTF-I specifically. The inhibitory heat-treated foods undergo nonenzymatic activity of MRPfrom several proteins, whose browning that crosslinks proteins and reduces pis are about 4.5, was higher than that of their nutritional value (Maillard reaction). MRPfrom other proteins, whose pis are much There are many possible physiological impli- more than 4.5 or much less than 4.5. The cations of the Maillard reaction in foods. problem is why these proteins (pi about 4.5) Besides the decrease in nutritional availability, induced the high activity. On the other hand, Maillard reaction products (MRP)exert other the reaction of glyceraldehyde and glycolal- effects on biological systems. There are anti- dehyde with proteins is similar to the Maillard bacterial compounds,1 ~4) virus inactivating reaction of glucose with proteins, but reacts compounds,5'6) enzyme inhibitors,7~9) muta- more rapidly than that of glucose.16) Then, it is genic inhibitors, lo'n) and nephrotoxic com- also interesting to examine the relationship pounds.12'13* between the reactivity of these aldoses in the Recently, we have discovered that high mo- Maillard reaction and the GTF-I inhibitory lecular weight MRPfrom several proteins and activity of the resulting MRP. glucose inhibit the synthesis of adhesive In this study, we examined MRPfrom these insoluble-glucan (IG) by glucosyltransferase aldoses and proteins of various pis under both (GTF) from Streptococcus mutans.14) S. mu- neutral and alkaline conditions to clarify the tans forms dental plaque by synthesizing ad- problems described above. hesive IG by GTF.15) GTF consists of two types of enzymes; one (GTF-I) synthesizing water-insoluble glucan (IG) and the other Materials and Methods (GTF-S) synthesizing water-soluble glucan Materials. as-Casein, ovalbumin (OVA), bovine serum (SG).15) Adhesive IG is synthesized with the albumin (BSA), human serum albumin (HSA), a-lactal- combination of GTF-I and GTF-S. The MRP bumin from bovine milk (LA), chymotrypsinogen A from

Corresponding author. 1418 S. Kobayashi et al. bovine pancreas, pepsin from stomach mucosa, lysozyme from egg white, fetuin from fetal calf serum, glucose, ribonuclease A from bovine pancreas^ DL-glyceraldehyde, and glycolaldehyde were purchased from Sigma Co., U.S.A.

Preparation ofMRP. Proteins (40 mg) were dissolved in 5ml of potassium phosphate buffer (200mM, pH 7.4) containing 0 or lOOmM of aldoses. Some reaction solutions were adjusted to pH 10.5 with 6n NaOH.Reaction solutions containing no aldose at pH7.4, no aldose at pH 10.5, glucose at pH 7.4, glucose at pH 10.5, glyceraldehyde at pH 7.4, glyceraldehyde at pH 10.5, glycolaldehyde at pH 7.4, and glycolaldehyde at pH 10.5 were designated as [pH 7.4], [pH 10.5], [Glc, pH 7.4], [Glc, pH 10.5], [GcerA, pH 7.4], [GcerA, pH 10.5], [GcolA, pH 7.4], and [GcolA, pH 10.5], respectively. The Maillard reaction was done in the reaction solutions at 100°C for 5hr. The reaction was monitored by measuring the absorbance at 420nm, the number of free amino groups, and GTFinhibitory activity. At the end of the reaction period, 3ml of reaction solution was adjusted to pH 4.0 with 6n HC1. The resulting precipitates were dissolved in 3ml of 50mM potassium phosphate buffer (pH 6.5). More than 90% of protein or activity wasrecovered in the solution (main or active fraction). Oneml of the solutions wasput on a Bio- Gel A1.5m gel chromatography column to examine the degree of polymerization. Assay of GTFinhibitory activity. Adhesive IG was synthesized in 1.0 ml of 50 mMpotassium phosphate buffer containing sucrose (lOmg/ml), cell-free GTF from S. mutans B13 (4.0jug/ml), and sodium azide (0.2mg/ml). In the reaction mixture, l.O mg/ml of adhesive IG was synthesized at 37°C for 16hr. When adhesive IG synthesis was inhibited by 50% in the reaction mixture, describing below, containing MRP, its inhibitory activity was designated 1 U. GTF-I and GTF-S were prepared from cell- free GTF by the method ofNamiki et a/.17> IG and SG were synthesized by these enzymes as reported previously.l4}

Analytical methods. Free amino groups (s-amino groups of lysyl residues and TV-terminal amino groups) of MRP were measured by the trinitrobenzenesulfonate method.18) Fig. 1. Maillard Reaction (at 100°C) between as-Casein Absorbance at 420nm was measured after 1 : 30 dilution and Glucose, Glyceraldehyde, and Glycolaldehyde. of the reaction solutions with 50mMpotassium phosphate The Maillard reaction was monitored by measuring the buffer. Amino acids were analyzed with a PICO-TAG absorbance at 420 nm (a), free amino groups (b), and GTF amino acid analyzer after hydrolysis at 107°C for 24hr in inhibitory activity (c). Symbols were as follows: [Glc, pH 6n HC1. 7.4], (O); [Glc, pH 10.5], (#); [GcerA, pH 7.4], (å¡); [GcerA pH 10.5], (å ); [GcolA, pH 7.4]; (A); [GcolA, pH 10.5]^ (A). The remaining amino groups were expressed Results relative (%) to the free amino groups of the original as- Maillard reaction between ocs-casein and glu- casein. cose, glyceraldehyde, or glycolaldehyde The Maillard reaction between a -casein and MRPas a GTF Inhibitor 1419 the three aldoses was done under both neutral about 15%, respectively. The order of GTF and alkaline conditions ([Glc, pH 7.4], [Glc, inhibitory activity in the six reaction solutions pH 10.5], [GcerA, pH 7.4], [GeerA, pH. 10.5], was: [GcerA, pH 7.4], [GcolA, pH 7.4]>[Glc, [GcolA, pH 7.4], and [GcolA, pH 10.5]). The pH 10.5], [GcerA, pH 10.5],, [GcolA, pH reaction at 100°C proceeded time-dependently, 10.5]>[Glc, pH 7.4](Fig. lc). Attheend of the resulting in an increase in absorbance at reaction time, the inhibitory activity obtained 420nm and a decrease in the number of free in [GcerA, pH 7.4] and [GcolA, pH 7.4] was amino groups (Fig. la, b). After 5hr of re- about 3 times higher than that in [Glc, pH action, the free amino groups of as-casein in 10.5]. [Glc, pH 7.4] and the other five reaction The polymerization degree (or aggregation solutions were decreased to about 50% and degree) of main or active fractions was exam-

Fig. 2. Gel Chromatography of Active Fractions of as-Casein in MRPin [Glc, pH 7.4], [Glc, pH 10.5], [GcerA, pH 7.4], [GcerA, pH 10.5], [GcolA, pH 7.4], and [GcolA, pH 10.5]. One ml of active fractions was put on a Bio-gel A1.5 m column (1.5 x 42cm) bufferized with 50mMpotassium phosphate buffer (pH 6.5). Gel chromatography was done at a flow rate of 7.0ml/hr with the same buffer (3.5ml/fraction). An absorbance at 280nm (a) and GTF inhibitory activity (% inhibition) at 10/il/ml of reaction solution (b) were measured. Collected main fractions were fractions 10-1 8 ([GcerA, pH 7.4], [GcolA, pH 7.4]), and fractions 13-18 ([Glc, pH 7.4], [Glc, pH 10.5], [GcerA, pH 10.5], and [GcolA, pH 10.5]). Symbols were the same as in Fig. 1. 1420 S. Kobayashi et al. ined by gel chromatography (Fig. 2). MRP under alkaline conditions, while MRPfrom from [Glc, pH 7.4] was not highly polymerized, glucose exhibited more higher activity under while other MRPwere highly polymerized as alkaline conditions than that under neutral reported previously.14) The order of polymeri- conditions. We then examined the inhibitory zation degree of active fractions from the activity of MRPfrom other proteins and the reaction solutions was: [GcolA, pH 7.4]> three aldoses in [Glc, pH 10.5], [GcerA, pH [GcerA, pH 7.4]>[Glc, pH 10.5], [GcolA, 7.4], and [GcolA, pH 7.4] (Table I). In the pH 10.5], [GcerA, pH 10.5]>[Glc, pH 7.4]. three reaction solutions, the inhibitory activity This order was almost the same as that of in MRPfrom as-casein and albumins (BSA, their inhibitory activity. The major active frac- HSA, OVA, and LA), whose pis are near 4.5 tions of each MRPwere collected and their (4.0-5.2), was much higher than that of MRP amino acid compositions were examined. The from pepsin, fetuin, ribonuclease, chymotryp- contents of lysyl plus arginyl residues of as- sinogen A and lysozyme, whose pis are 1.2, casein and MRP in [Glc, pH 7.4], [Glc, pH 3.2-3.8, 9.5, 9.6, and 12.0, respectively. In the 10.5], [GcerA, pH 7.4], [GcerA, pH 10.5], case of chymotrypsinogen A and lysozyme, [GcolA, pH 7.4], and [GcolA, pH 10.5] were their MRPprecipitated in both [GcolA, pH 14.6, 9.6, 6.5, 5.8, 5.9, 4.2, and 4.9(%), re- 7.4] and [GcerA, pH 7.4]. Wealso examined spectively. The order of decrease in the lysyl the inhibitory activity of the original proteins and arginyl residues was correlated to the and those heated 100°C for 5 hr in [pH 7.4] and order of increase in the inhibition activity [pH 10.5]. These proteins had no clear effects (Figs, lb and lc). on adhesive IG synthesis. These original pro- We also examined as-casein heated at 100°C teins except pepsin, fetuin, as-casein, and LA for 5hr in [pH 7.4] and [pH 10.5]. The two precipitated in [pH 7.4] or [pH 10.5] at 100°C heated proteins, which were not polymerized, for 5hr. The results (Table I) suggest that the did not inhibit adhesive IG synthesis (see pi of the original protein is important for Table I). preparing MRPwith high activity. Inhibition of adhesive IG synthesis by MRP Effects of MRPand their original proteins on from several proteins and the three aldoses GTF-I and GTF-S As shown in Fig. lc, as-casein MRPfrom Since as-casein MRPfrom [Glc, pH 10.5] glyceraldehyde and glycolaldehyde had a high- inhibited GTF-I specifically,14) the effects of

Activity in reaction solutions (U//il) Original proteins [pH 7.4] [pH 10.5] [Glc, pH 10.5] [GcerA, pH 7.4] [GcolA, pH 7.4] 0 0 0 Pepsin 1.0 1.1 0 0 Fetuin 2.0 2.0 5.1 0 0 as-Casein 12.1 17.8 21.1 0 0 a-Lactalbumin ll.2 13.9 13.6 0 Ovalbumin 21.8 24.5 22.5 0 18.0 33.1 38.8 Bovine serum albumin 0 Humanserumalbumin 18.1 19.0 28.2 Ribonuclease A 0 0.2 0.2 Chymotrypsinogen A 0.5 Lysozyme

The Maillard reaction was done at 100°C for 5hr. -, activity was not measured (precipitates were formed). MRPas a GTF Inhibitor 1421

Fig. 3. Effects of MRPfrom as-Casein and the Three Aldoses on GTF-I (a) and GTF-S (b). Symbols were the same as in Fig. 1. Synthesized IG was measured by the phenol-sulfuric acid method and SG was measured by the same method after precipitation with 75% ethanol. The results were plotted on semilogarithmic graph paper.

Fig. 4. Effects of Original Proteins on GTF-I (a) and GTF-S (b). as-Casein (å ), LA (A), OVA (æf), BSA (#), HSA (V), chymotrypsinogen A (O), lysozyme (V),

inFig.3.ribonuclease A (A), fetuin (o), and pepsin (O) were used (0 to 30 jUg/ml). Synthesized glucan was measured as 1422 S. Kobayashi et al, dehyde on two types of GTFwere examined. that the activity of MRPfrom aldoses cor- These MRPinhibited GTF-I and not GTF-S relates with the reactivity of the aldoses in the as as-casein MRPfrom glucose (Fig. 3). MRP Maillard reaction. Consequently, the MRP from the albumins also inhibited GTF-I with higher activity showed a much higher specifically, while MRPfrom pepsin, fetuin, degree of polymerization and a lower content chymotrypsinogen A, ribonuclease A and ly- of lysyl and arginyl residues than the MRP sozymehad no clear effect on the two enzymes with lower activity. When the Maillard re- (data not shown). These results (Fig. 3) sug- action was done at 37°C for 200hr, a similar gested us the examination of the effects of the relationship was observed (data not shown). original proteins on GTF-I and GTF-S (Fig. In this study, the proteins heated in the 4). as-Casein, LA, OVA, BSA, and HSA absence ofaldose ([pH 7.4], [pH 10.5]) did not stimulated GTF-I, but not GTF-S. Similarly, inhibit adhesive IG synthesis (Table I). This these proteins heated at 100°C for 5hr in [pH suggests that denaturation of original proteins 7.4] or [pH 10.5] stimulated GTF-I but not ascribed to heating did not induce GTF in- GTF-S (data not shown). OVA, BSA, and hibitory activity. Ikura et al. showed.that HSAheated in [pH 7.4] were not examined caseins were polymerized by the transgluta- since the three proteins precipitated under this minase from guinea pig liver.24) The as-casein condition. On the other hand, pepsin, fetuin, polymerized by the enzyme showed a wide ribonuclease A, chymotrypsinogen A, and ly- molecular weight distribution and low content sozyme did not clearly stimulate either GTF-I of lysyl residues as MRPfrom as-casein, but nor GTF-S. no inhibitory effect on adhesive IG synthesis These results suggest that the specific in- (data not shown). This shows that the polym- hibition of GTF-I by MRPfrom as-casein and erization of original proteins is essential but albumins may be attributed to the GTF-I not sufficient to induce the inhibitory activity specific stimulation property of the original in the resultant MRP. proteins. MRPfrom as-casein and albumins in [Glc, pH 10.5], [GcerA, pH 7.4], and [GcolA, pH Discussion 7.4] showed high activity, while MRPfrom pepsin, fetuin, ribonuclease A, chymotrypsi- Our previous study has indicated GTFin- nogen A, and lysozyme showed muchlower hibitory activity of high molecular weight activity under the three reaction conditions MRPfrom some proteins and glucose.14) High (Table I). This indicates that the property of activity was obtained in MRPfrom as-casein the original protein is most important for the and albumins only under extreme conditions induction of the activity. Interestingly, as- (pH 10.5, at 100°C). These original proteins, casein and albumins (pi about 4.5) stimulated whosepis are about 4.5, are important as food GTF-I specifically, but pepsin, chymotrypsi- proteins, and many food chemists have studied nogen A, and lysozyme (pl»4.5 or pl«4.5) the Maillard reaction between these proteins showed clear effects on neither GTF-I nor and glucose, an aldohexose.19~23) GTF-S (Fig. 3). In addition, we previously Recently, Acharya et al.16) showed that demonstrated that MRP, having the GTF-I RNase A was easily cross-linked by glyceral- inhibitory activity, formed a complex with the dehyde, an aldotriose, or glycolaldehyde, an enzyme.14) These results suggest that GTF-I aldodiose under physiological conditions (pH binding property of MRPmight be attributed 7.4, at 37°C). Therefore we compared GTF to the GTF-I specific interaction of its original inhibitory activity of MRPfrom as-casein and protein, and it might be the Maillard reaction these aldoses. The activity induced by glyceral- that brings about the appearance of the in- dehyde and glycolaldehyde was higher than hibitory activity. that induced by glucose (Fig. 1), indicating Harlander and Schachtele showed that ly- MRPas a GTF Inhibitor 1423 sophosphatidylcholine (LPC) and other phos- References phoglycerides stimulated GTF-I and GTF-S H. Einarsson, B. G. Snygg and C. Eriksson, J. Agric. from S. mutans 6715.25) LPC stimulated the Food Chem., 31, 1043 (1983). GTF-I 6.5-fold and the GTF-S 2.1-fold. The T. R. Anderson, D. J. Robbins and H. F. Erbersdobler, Nutr. Rep. Int., 30, 493 (1984). results suggested that GTFcan be activated by N. Kato and I. Shibasaki, /. Ferment. Techno/., 52, binding of phosphoglyceride molecules to a 177 (1974). site on the enzyme that is distinct from either S. T. Leite, H. Imasato and P. A. Bobbino, Rev. the glucosyl donor or glucosyl acceptor (pri- Microbiol, 10, 100 (1980). mer) binding sites. In addition, Burckhardt J. Morita, N. Kashimura and T. 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