Fructose-induced fluorescence generation of reductively methylated glycated bovine serum albumin: Evidence for nonenzymatic glycation of Amadori adducts

Biochhnica et Biophysica Acta, 1075(1991) 12-19 © 1991 ElsevierScience PublishersB.V.0304-4165/91/$03.50 ADONIS 030441659100216J

Fructose-induced fluorescence generation of reductively methylated glycated bovine serum albumin: Evidence for nonenzymatic glycation of Amadori adducts * G e r a r d o S u f i r e z t, J a i m e M a t u r a n a 1, A r n o l d L. O r o n s k y 2 and Carmen Ravent6s-Sufirez 2 t Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, N Y (U.S.A.) and 2 Medical Research Dirision, American Cyanamid Co., Pearl Rit'er, N Y (U.S.A.)

Key words: Fructation; Maillard reaction; Pyrrole compound; Reductive methylation"(Aminogroup);(Aldol condensation)

In vitro glycation of bovine serum albumin by fructose (fructation) induces fluorescence generation about 10-times faster than glucose (G. Sufirez et al. (1989) J. Biol. Chem. 264, 3674-3679). In order to gain further insight into possible mechanisms that would explain this difference, the protein was glycated with either glucose or fructose and then reincubated in the absence of sugars. In contrast to the previous findings, albumin that had been glycated with glucose generated fluorescence at a higher rate during the sugar-free incubation. However, when partially glycated BSA was reincubated with sugars under conditions where de novo glycation was prevented by reductive methylation of amino groups fr.:ctose induced fluorescence to a much larger extent than glucose. These results are consistent with the notion of covalent addition of sugars to Amadori groups, the earliest stable prodqcts of the Maillard reaction. A chemical pathway is proposed where pyrrolic structures result from the double sugar adduets by aldol condensation and dehydration. These structures might be precursors of fluorophores.

Introduction Protein glycation, i.e. the nonenzymatic reaction between reducing sugars and primary amino groups of proteins, is a specific example of the initial step of a multi-reaction pathway that has been generically known as the Maillard reaction. Although the early steps in this reaction sequence have been characterized in a somewhat detailed manner, at least for the case of glucose-initiated glycation, the high degree of complexity of the advanced stages, even at physiological condi* A preliminary formof this work was presented at the 4th International Symposiumon the Maillard Reaction, Lausanne, Switzerland. September, 1989 (SuArez. G., Maturana, J., Oronsky, A.L. and Rajaram, R. (1990) in The Maillard Reaction in Food Processing, Human Nutrition and Physiology(Finot, P.A. et al., eds.), pp. 499-504, Birkhfiuser, Basel). Abbreviations: BSA, bovine serum albumin; fBSA, fructated BSA; gBSA,glucated BSA;NALMA. Na-acetyl-I.-lysineN ' methylamide; RM-fBSA, reduetively methylated fructated BSA; RM-gBSA,reductively methylated glucated BSA; TNBS, trinitro benzene sulphonic acid. Correspondence: G. Sufirez, Department of Biochemistry and Molecular Biology, New York Medical College. Valhalla, NY 111595. U.S.A.

tions, has made their accurate chemical description a formidable problem, so far unsolved. The Schiff base resulting from the initial sugar attachment to the protein primary amino group undergoes Amadori rearrangement (also known as Heyns rearrangement for the case of glycation by fructose) and ketoamine-linked or aldoamine-linked deoxy sugar adducts are generated. These adducts, which we will refer to as Amadori products, undergo further transformations, presumably involving various divergent pathways, whereby a large multiplicity of poorly characterized compounds are produced. (For recent reviews see Refs. 1 and 2). Lately, the Maillard reaction has become the focus of increasing attention for its possible relevance to the complications of diabetes mellitus and aging [3]. Recently, Sufirez et al. [4] proposed that glycation by fructose (fructation), might be a potential mediator of diabetic complications on the basis that the concentration of this sugar increases in tissues as a result of the operation of the sorbitol pathway [5]. In vitro fructation of bovine serum albumin (BSA) leads to fluorescence generation, an indicator of the advanced Maillard production, at a rate that is about 10-times that induced by glycation by glucose (glucation) [6]. In order to gain insight into the chemical basis for the conver-

sion of Arnadori products to the highly fluorescent structures, partially glycated protein was reisolated aod reincubated under conditions that preclude de novo glycation. The results are consistent with the formation of double sugar adduets at protein amino groups, that would transform into fluorophores. Materials and Methods

Chemicals. Bovine serum albumin (BSA,x. crystallized and fatty acid-free, was from Sigma (Cat. No. 7511) and used with no further purification. D-(+) glucose (mixed anomers) and /3-D(- ) fructose, crystalline, and sodium cyanoborohydride also originated from Sigma. Proteinase K, a proteirase of broad specificity from the mould Tritirachium album, was obtained from Boehringer Mannheim and had a nominal specific activity of 20 u n i t s / m g using hemoglobin as substrate. All other reagents were of the highest analytical grade available. Analytical procedures. Protein concentration was estimated according to the Lowry procedure using the same batch of BSA that was submited to glycation (see below) as a standard. Early glycation products (Schiff bases and Amadori groups) were quanti*.ated on thc basis of formaldehyde release following periodate oxidation as described by Gallop et al. [7]. Reactive amino groups were assayed following the method of Spadaro et al. [8] with N-a-acetyl-L-lysine N'-methyl amide (NALMA) as a standard when assaying intact protein and t.-leucine for the assay of digested protein. Fluorescence measurements were carried out in a LS5-B Perkin-Elmer Luminescence spectrometer at an excitation wavelength of 340 nm and emission of 420 n m (slit-width: 10 rim) and normalized with respect to a single value of both the protein concentration and the electric signal of the spectrofluorometer as monitored with Perkin-Elmer standard No. 4 (tetraphenylbutylenediamine). Incubation with sugars. Mixtures containing 6 m g / m l BSA and 0.5 M glucose or fructose in 0.05 M phosphate, 0.1 M NaCI (pH 7.0) (PBS), were sterilized by passage through 0.45 p, membranes (Acrodisc ~, Gelm a n Sciences, A n n Arbor, MI) and then incubated either for 5 - 7 days (fructose) or for 15-21 days (glucose) at 37 ° C in a humidified atmosphere. A shorter incubation period for fructose was chosen on the assumption that the fluorescence generation from Amadori groups is faster during fructation as compared with glucation [6]. Therefore, a possible generation of fluorophores in a subsequent incubation in the absence of sugars, as was originally hypothesized, would be obscured if saturation levels of fluorescence had been reached after a long first incubation. After incubation the mixtures were dialyzed 6 times over a period of 5 - 7 days at 4 ° C . The dialysis was carried out

against a t00-fold volume cxcess of PBS. Assays were performed after this final dialysis step.

Reincubation of glycated BSA in the absence of sugars. BSA that was previously incubated with sugars and dialyzed as described above, was reincubated under sterile conditions. Aliquots (1.8 ml) of the dialyzed protein were aportioncd onto separate tubes and sets of three tubes were each reincubated for a specified number of days. In this way each time point corresponded to sample triplets and the risk of bacterial contamination was minimized. The protein concentration during the reincubation varied from 3.79 to 5.69 m g / m l . In one series of reincubations small amounts of sugars (10 -~-10 - s M) were added. Reincubations were terminated by freezing. Reductive methylation of glycated BSA. Dialyzed samples that were incubated with sugars were reductively methylated according to the procedure of Jentoft and Dearborn [9]. Glucated BSA (gBSA), obtained following a 16 day incubation with glucose or fructated BSA (fBSA) generated after a 7 day incubation with fructose, were slowly stirred at a concentration of 5.56 m g / m l or 4.26 m g / m l , respectively, with 2 mM formaldehyde and 20 m M sodium cyanoborohydride in PBS for 6 h at 4 ° C. Following dialysis vs. a 30-fold excess of PBS the procedure was repeated three times. Due to dilution by dialysis the protein concentration during the fourth reductive methylation step was 4.27 m g / m l (gBSA) and 3.5 m g / m l (fBSA). Reactive primary amino groups were quantitated after each methylation step as illustrated in Fig. 1.

Rehwubation of reductively methylated glycated BSA. After dialysis vs. 100-fold volume excess of PBS reductively methylated gBSA (RM-gBSA, 3.58 m g / m l ) and reductively methylated fBSA (RM-fBSA, 3.04 m g / m l ) were reincubated at 3 7 ° C either in the absence of sugars or in the presence of sugars or NALMA, as indicated. The appropriate mixtures were sterilized by passage through 0.45/zm membranes. To prevent further bacterial contamination reincubation was carried out in separate 1.2-1.5 ml aliquots and each aliquot was incubated for a given number of days and then frozen. Prior to assays, all samples were dialyzed twice in excess of PBS. Digestion with proteinase K. RM-gBSA (2.53 m g / m l ) or RM-fBSA (4.04 mg/ml), were incubated with 12.5 i.tg/ml proteinase K for 16 h at 3 7 ° C in PBS and then boiled. Extent of digestion was ascertained by the increase in amino group reactivity. Following digestion amino groups rose 17-24-fold in RM-gBSA and 2536-fold in RM-fBSA. Results

Rebwubation of glycated BSA hi ihe absence of sugars. After exhaustive dialysis to remove the sugar no appreciable increase of fluorescence was observed in

REDUCTIVE METHYLATIONSTEPS Fig. 1. Extent of blocking of primary amino groups of glycated BSA at consecutive steps to reductive methylation, gBSA was pro.pared by incubating BSA (6 mg/ml) with 0.5 M glucose for 16 days and fBSA by incubating with 0.5 M fructose for 7 days as detailed in the text. (0), gBSA; (e) IBSA.

fBSA w h e n r e i n c u b a t e d (Fig. 2). E a r l i e r studies have also d o c u m e n t e d the lack of fluorescence g e n e r a t i o n following the :~tI:uval uf :,ugh.-, fl'oiil l.,oteins unC,crgoing glycation [10,11]. In contrast, the fluorescence of g B S A rose significantly (80%). Thus, this fluorescence g e n e r a t i n g capacity is in the inverse relationship as c o m p a r e d with the one occurring d u r i n g a c o n t i n u e d incubation in the p r e s e n c e of sugars, i n the course of u n i n t e r r u p t e d in vitro fructation carried out in the conditions of the present study, fluorescence increased by a b o u t 100% from the 7th day to the 32nd day [6]. This t i m e interval c o r r e s p o n d s to the sugar-free stage of the two-stage incubation described here. U n i n t e r r u p t e d in vitro glucation, on the o t h e r hand, p r o c e e d s with a slowly increasing fluorescence [6]. T h e s e a p p a r ently paradoxical results that a p p e a r to conflict with the earlier findings could be explained by a m u c h faster conversion of A m a d o r i - g r o u p derived i n t e r m e d i ates of fBSA to f l u o r o p h o r e s as c o m p a r e d with those of gBSA. D u e to this h i g h e r reactivity, f l u o r o p h o r e s would e m e r g e shortly a f t e r the A m a d o r i r e a r r a n g e m e n t in fBSA at such a rate t h a t all the potentially available groups would b e c o n v e r t e d d u r i n g the first incubation and thus no p r e c u r s o r s would b e left for the second incubation. This c o n t e n t i o n was s u p p o r t e d by the gradual and significant (30%) g e n e r a t i o n of fluo-

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REINCUBATIONTiME (DAYS) Fig. 2. Fluorescence generation upon reincubation of glycated BSA in the absence of sugars. BSA, 6 mg/ml, was incubated with 0.5 M glucose (21 days) or 0.5 M fructose (7 days), dialyzed six times and reincubated at 37 *C in the absence or in the presence of trace concentrations of sugars. Fluorescence: exc., 340 nm; em., 420 nm. Each value is the mean of duplicate determinations on each of three incubation samples. (A) gBSA; (B) fBSA. Circles, in the absence of sugars; triangles, in the presence of 10-s M sugar.

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Fig. 3. Fluorescence generation during dialysisat 4°C of glycation mixtures. BSA was incubated with 0.5 M glucose(16 days) or 0.5 M fructose (7 days) and then dialyzedsix times over a 4 day period at 4"C. Fluorescence, measured at each dialysisstep, was normalized with respect to protein concentration.(o), gBSA.(e) fBSA. resccnce during dialysis at 4 ° C of fBSA, but not of gBSA (Fig. 3). At 4 ° C the residual fluorophore generation in gBSA would be too slow to be observable

during dialysis, but it would become apparent at 37 ° C. During dialysis fluorescence appears to be generated from intermediate adducts and not as a result of the reinitiatien of the Mai!!ard reaction hy residual suzars, because incubation of BSA with sugars at 4 ° C failed to induce fluorescence. Amadori groups may not be the immediate precursors for fluorophores during the Maillard reaction, as suggested by the lack of fluorescence increase when glycated protein were incubated in the absence of sugars. It is possible, instead, that a sugar-dependent generation of intermediates at a more advanced stage in the Maillard reaction, would be necessary for fluorescence development. To ascertain further this notion glyeated protein had to be reincubated with sugars and the reinitiation of the MaiUard reaction prevented by reductive methylation. In this way, primary amino groups become unavailable for sugar attachment. By using cyanoborohydride instead of the earlier method employing borohydride [12], reduction of the carbonyl groups in Amadori adducts was prevented. Reduction of these groups would stop the MaiUard reaction sequence. The lack of reduction of Amadori carbonyls in our study was documented by the absence of an increase in the molar yield of formaldehyde upon periodate oxidation [7]. Another advantage of reductive methylation is that it does not affect the protein charge as does acylation of amino groups with succinic anhydride [13]. Reincubation of RM-gBSA as well as RMt'BSA in the absence of sugars resulted in an initial sharp decrease of the fluorescence followed by a grad-

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Fig. 5. Fluorescenceof proteinase K digestsof reincubated reductively methylated glycated BSA. Effectsof sugarsand NALMA. RM-gBSAand RM-fBSA were reincubated at 37 °C with sugarsor NALMA, dialyzed and then digested with proteinase K. Fluorescence was read at 340 nm emission and 420 nm excitation. Additions during reineubation (zx) none; (o), 0.1 M glucose; (e) 0.1 M fructose; (n) 2 mM NALMA. (A) RM-gBSA;(B) RM-fBSA.

ual stabilization of the fluorescence (Fig. 4). No evidence for proteolytic degradation as judged by polyacrylamide gel electrophoresis or increase in amino group reactivity was detected. This ruled out the possibility of detachment of fluorophore-bearing fragments. To explain these results, we hypothesized that the extensive amino group blocking by reductive methylation would lead to a destabilization of the protein tertiary structure. As a consequence, Maillard fluorophores would come into close proximity with quenching groups located in other domains of BSA. This view was further supported by the finding of diminished tryptophan fluorescence in reincubated RM-glycated protein (data not shown). Earlier studies have afforded evidence for intramolecular quenching of Maillard fluorophores [14]. Based on this notion, fluorescence generation was assessed by measurements on proteinase K digests of the previously reductively methylated glycated protein. Proteolytic digestion would abolish approximation effects that would lead to fluorescence quenching. In Fig. 5A is shown that reineubation of RM-gBSA with 0.1 M fructose induced a large increase of the fluorescence, as assayed in its digests. Upon addition of 0.1 M glucose smaller, but still significant, fluorescence developed. Low concentrations of NALMA failed to induce fluorescence when incubated with RM-gBSA. Analo-

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INCUBATION TIME (DAYS)

Fig. 7. Amino group consumptionupon reincubation RM-mSA with sugars or NALMA. RM-mSA, prepared as described in the text, was reincubated in the presence of 0.1 M glucose(0), 0.1 M fructose (e), or 2 mM NALMA(El). gous results were obtained on reincubation of RMtBSA with the sugars (Fig. 5B). The Amadori group content of RM-gBSA, as judged by formaldehyde release upon periodate oxidation, decreased upon reincubation with fructose as well as with glucose. The rate of decrease was maximal when N A L M A was reineubated with RM-gBSA (Fig. 6). The number of reactive amino groups did not vary during any of the reincubations of RM-glycated proteins with sugars (Fig. 7). Discussion

The data presented in this article are consistent with the notion that glycated proteins bearing a sugar-derived adduet, possibly an Amadori product, are capable of reacting further with another sugar molecule to form a double adduct. The reaction entails the addition of a sugar-reactive carbonyl group to the secondary amine that results from the Amadori rearrangement of the initial glycation. Since fructose occurs in solution in the linear open form to a much larger extent in comparison with glucose, a higher rate for the attachment of fructose to the secondary amine would be expected. This might explain, in part, the greater rate of fluorescence generation upon reincubation with fructose. The initial adduct may be derived from glucose or fructose. The reactivity of fructose as a secondary glycating agent would resemble that of formal-

dehyde which goes through at least two additional cycles during reductive methylation [9]. An earlier ,tu(!y revealed tile iu vitro f o ~ a t i o n of double adducts on incubation of protein with appropriate proportions of sugars [15]. In that study, further transformation of the adducts was prevented by sodium borohydride reduction. Another study, which was based on proton and 13C-NMR spectra, concluded that a di-hexosyl adduct resulted from the reaction of glucuronic acid with an a-amino acetylated lysine-containing tripeptide [16]. Finally, studies based on mass spectra of products of sulfite-inhibited glycated protein revealed formation of compounds having 2 mol of carbohydrate per lysine amino group [17]. The evidence presented here is indirect and relies on the assumption that amino groups have been blocked by methylation and thus have become unavailable as initial sites of glycation. The major limitations of this assumption is the lack of assurance that the TNBS reaction detects quantitatively all the free primary amino groups of BSA. This concern becomes especially warranted by our detection of only about 30 amino groups in BSA, which is half the value of 60 that would be expected on the basis of the amino acid composition [18]. On the other hand, in this study the TNBS reaction was carried out under conditions that do not promote unfolding of BSA, i.e. in the absence of detergents or denaturing agents. Therefore, only the amino groups that are located at the surface of the protein would react with the reagent. Recent studies have supported the view that polar residues such as lysine might be located both in the surface and the interior of the protein [19]. This would explain the earlier observation that the reaction of TNBS with BSA does not go to completion [20]. On the basis of this, it is also unlikely that a reaction of sugars with primary amino groups that are located in the interior of the protein could occur. This presumption is further supported by our finding that further blocking of primary amino group during the reineubation of RM-glycated BSA with sugars apparently did not occur. Also, the reinitiation of the MaiUard reaction at sites that were not methylated, would have entailed the generation of Amadori groups during the reincubation. Instead, we found that these groups were consumed. Based on these findings, we are tempted to propose a model, at this stage highly speculative, which might be useful for future explorations (see Fig. 8). Fructose would add, via a nucleophilic reaction, to a secondary amino group bearing the Amadori moiety in a glucated or fructated protein. Subsequently, C-1 of the newly added fructose would react, by way of aldol condensation, with the carbonyl group of the original Amadori adduct, resulting in ring closure. The kinetics of this step would be favorable if one realizes that it is an intramolecular addition. Approximation effects have

CH2--NH-- P ._?Y. . . . ?-o. ~=o -- -P HO--C--H Ho--C--H "J¢" .o ? ,-. ,,oomo~ ,.o-c-. 1 HO--C--H N t H--C--OH . - ,c-o. . _ ~H: / /~ \ H--C--OH ~: I H--C--OH CHzOH o=cc.~o. ~=o c'.+o. AMADOm GROUP FRUCTOSE HO--C--H H--C--OH H_I--oH

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H H Fig. 8. Hypothetical reaction pathway initiated by the addition of fructose to Amadori groups.The pathwayis depicted for the case of an Amadori group resulting from glucatlon. Fructose adds to the secondary amine. Subsequently, C-I of the newly added fructose. reacts, via aldol condensation,with the carbonylgroupof the original Amadori, resulting in ring closure. Double dehydration leads to a substituted pyrrole. been known to enhance rate constants by several orders of magnitude in analogous intramolecular reaction models of enzyme catalysis [21]. Finally, a substituted pyrrole would be generated following a double dehydration step. Pyrrolic structures have been demonstrated as intermediates of the Maillard reaction and thought to be precursors of fluorophorcs [22]. According to our reaction pathway model hybrid moieties would emerge in proteins when fructose reacts with a glucose-derived Amadori group. Fluorescence would evolve from an intermediate generated after the double adduct formation step, which consumes Amadori groups, at rates that are much higher during fructation, in which ease it would even proceed at low temperature. This notion is consistent with three major observations of this study: (1) comparable rates of consumption of Amadori groups during reincubation of RMgBSA with either glucose or fructose (Fig. 6); (2) the smaller rate of fluorescence generation following reincubation with glucose and (3) the rise in fluorescence while fBSA is being dialysed at 4 ° C (Fig. 3). Reaction of a carbonyl of an Amadori group with an E-amino group of lysine, which has been proposed as a

basis for crosslinking [23], do not appear to be a fluorogenic pathway, because of the failure of NALMA to induce fluorescence when incubated with RM-gBSA (Fig. 5) while the level of Amadori groups dropped significantly (Fig. 6). The addition of fructose to an Amadori group secondary amine might be the initial step of a reaction sequence leading to fluorophores in vivo and might explain an apparent paradoxical observation that an antibody that was raised against a Maillard pyrrolic epitope, pyrraline, was found to be much more immunoreactive with protein from tissues than against proteins previously incubated with glucose at a concentration that was 40-times the one usually prevailing in diabetic tissues [24]. In vivo, fructose would be a powerful fluorogenic pyrrole precursor, absent during the in vitro incubation of the above mentioned study and its addition to the glycation sites of proteins might proceed in organs and tissues with an active sorbitol pathway. In principle, fructose could form additional products with glucose or fructose-derived Amadori groups, generating in the first case hybrid adducts. A definitive proof of this reaction pathway should be based on the isolation and structural characterization of the predicted intermediate pyrroles.

Acknowledgements This work was supported by American Heart Association grant No. 82-946 and Lederle Laboratories. We thank Ms. Marie Black for assistance in the typing of the manuscript.

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19 Brown, E.M., Pfeffer, P.E., Kumosinski, T.F. and Greenberg, R. (1988) Biochemistry 27, 5601-5610. 20 Goldfarb, A R . (1966) Biochemistry 5, 2574-2578. 21 Jencks, W.P. (1969) Catalysis in Chemistry and Enz-Jmology, pp. 7-41, McGra'~-HilL New York. 22 Njoroge, F.G., Sayre, L.M. and Monnier, V.M. (1987) Carbohydr. Res. 167, 211-220. 23 Pongor, S.. Ulrich, P.C., Bencsath, F.A. and Cerami, A. (1984) Proc. Natl. Acad. Sci. USA 81, 2684-2688. 24 Hayasc, F., Nagaraj, R.H., Miyata, S., Njoroge, F.G. and Monnier, V.M. (1989) J. Biol, Chem. 263, 3758-3764.