534 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /-, No.+*, /-. /.+ (,**0) 8 ao Structure and Stability of a-amylase Inhibitors from the Seeds of Shiroha

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1 534 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /-, No.+, /-./.+ (,0) 8 ao Structure and Stability of a-amylase Inhibitors from the Seeds of Shirohanamame (Phaseolus coccineus L.), Murasakihanamame (Phaseolus coccineus L.), Toramame (Phaseolus vulgaris L.) and Uzuramame (Phaseolus vulgaris L.) Sayuri Sawada, Yuri Yanaga and Misao Tashiro School of Human Environmental Sciences, Mukogawa Women s University, 0.0 Ikebiraki-cho, Nishinomiya, Hyogo 00-2//2 The structure and stability of four a-amylase inhibitors (TAI, UAI, MAI-, and SAI-,) from Toramame (Phaseolus vulgaris L.), Uzuramame (Phaseolus vulgaris L.), Murasakihanamame (Phaseolus coccineus L.) and Shirohanamame (Phaseolus coccineus L.) were examined. The amino acid sequence of SAI-, was the same as the known sequence for MAI-,, but was slightly di#erent from that of TAI and UAI. The sugar chain composition of SAI-, was remarkably di#erent from that of MAI-,. Thermal stability at ph /., 1 indicated that SAI-, and MAI-, have markedly similar stabilities. Likewise, TAI and UAI, which also had identical amino acid sequences, exhibited markedly similar stabilities. However, SAI-, and MAI-, were more stable than TAI and UAI. These results suggest that thermal stability of a-amylase inhibitors depends on a few amino acid substitutions in their primary structures. (Received Feb. 1,,0 ; Accepted Jun. -,,0) ao ao AI +1 AI 23 + AI ph 2 2 AI AI TAI, DAI, MAI-, 00-2//2 0.0 Corresponding author ssawada@mukogawa-u.ac.jp ++ ao UAI TAI +, UAI TAI +, MAI-, +- ao SAI-, AI AI + AI ao TAI UAI MAI-, SAI-, Phaseolus vulgaris L. Phaseolus coccineus L. +-+., AI / mg 0 mol/l / m mol/l ph 2. +./ ml mol/l ph 2.

2 9 : ao 535 AI AI 2 mol / l ph 2. DEAE-Sephacel +. mol/l NaCl gradient a b / m mol/l NH.HCO - PD-+ amersham pharmacia baiotech - AI a b /.1 mol/l HCl ++,. 2-/. Hewlett-Packard,.+ N & C protein sequencer / a MAI-, SAI-, / m mol/l NH. HCO - TPCK-trypsin S. aureus V2 protease : + : / ww -1,,. b TLCK-chymotrypsin / m mol/l NH.HCO - -1 : + : + ww MAI-,, SAI-,,. lysylendopeptidase Tris HCl ph : + : / ww MAI-,, SAI-,,. bovine trypsin type XIII : TPCK treted bovine aochymotrypsin type : TLCK treated Staphylococcus aureus V2 protease type XVII Sigma lysylendopeptidase from Achromobacter lyticus M / TFA TSKgel ODS-2TS../ +/ cm HPLC./ mlmin,./ TFA linear gradient a. - b.2.,, nm 1 PA +/+0 glycopeptidase A ph / PA +1 PA PA PALPAK Type S..0,/ cm, size-fractionation HPLC A : - ph 1.- -/0/ vv B : - ph 1.- // vv 0 B, - min linear gradient. +. mlmin, -, nm. nm PA PA-Sugar Chain M-M3 M- Mana+-0 Mana+- Manb+.GlcNAcb+.GlcNAc-PA ; M/A, Mana+0 Mana+- Mana+0 Mana+- Manb+.GlcNAcb+.GlcNAc-PA ; M0B, Mana+0 Mana+- Mana+0 Mana+,Mana+- Manb +. GlcNAcb +. GlcNAc-PA ; M 1 B, Mana + 0 Mana+- Mana+0 Mana+,Mana+,Mana+- Manb +. GlcNAcb +. GlcNAc-PA ; M 2 C, Mana + 0 Mana+,Mana+- Mana+0 Mana+,Mana+,Mana +- Manb+.GlcNAcb+.GlcNAc-PA ; M3A, Mana+, Mana+0 Mana+,Mana+- Mana+0 Mana+,Mana +,Mana+- Manb+.GlcNAcb+.GlcNAc-PA 2 AI AI ao Type+-A, Sigma DNS./ ml AI / m mol/l NaCl, / m mol/l CaCl,,./ Triton X-+ / m mol/l Na-acetate bu#er ph /../ ml ml / m mol/l NaCl, / m mol/ l CaCl,,./ Triton X-+,/ m mol/l PIPES bu#er ph 0.3.// +. ml + DNS + ml / + ml /, nm ao +l / mmol/l NaCl, / m mol/l CaCl,,./ Triton X-+,/ m mol/l PIPES bu#er ph 0.3. ml,2 nm.+ Triton X-+, + 1 mol/l 3 / m mol/l NaCl, / m mol/l CaCl,,./ Triton X-+ / m mol/l Na-acetate bu#er ph /..- ml,+ 1 mol/l 1 +,,., ml..- ml,+ 1 mol/l./ ml ph / ml.// + ml + DNS

3 536 /- +, ml /, nm AI + SAI-, SAI-, MAI-, N SAI-, a b N N a N b N N a N /- b.3 MAI-, Table + MAI-, SAI-, a b MAI-, SAI-, SAI-, MAI-, a b a V2 V2 Fig. + b a Fig., SAI-, MAI-, Table + Amino acid composition of subunits of MAI-, and SAI-, (mol ) MAI-, SAI-, Amino Acid a b a b Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg , /4/ 24, / /4- +04, 14/ +,4/ 242, / -41 / , /4. 143,4/ -4, 142 / /4, +/4/ 14- +, / , / ,4. Fig. + Peptide maps for SAI-,a (A) and MAI-,a (B). Staphylococcus protease digests of a-subunits of SAI-, and MAI-, were separated by HPLC. : absorbance at,, nm ; : acetonitrile concentration.

4 11 : ao 537 Fig., Peptide maps of SAI-,b (A) and MAI-,b (B). Chymotrypsin digests of SAI-, and MAI-, b-subunits were separated by HPLC. : absorbance at,, nm ; : acetonitrile concentration. a b SAI-, Fig. - a N V2 Fig. + V. V0 V2 V3 V++ N MAI-,a V3 V. XKT XFT NXST X Pro +, 0/ a 10 MAI-,a b N Fig., C/ C0 C + C++ C+- C+0 C+2 C+3 C,/ N MAI-,b C/ C+0 XSS XVS 0-2- b +-3 MAI-,b SAI-, MAI-, Fig. +, Fig., SAI-, MAI-, a b PA HPLC Fig.. a a +, No. + +, HPLC SAI-, MAI-, SAI-, No. +, PA M3 -., No / +/.1 MAI-, M0 No. 1,..1 No. +, M No./ M/ +-.- No.. +. b b 2 No. + 2 b HPLC SAI-, No., MAI-, No., ,,.2 SAI-, No. / 2 +0., +0.+ SAI-, SAI-, MAI-, a b

5 538 /- +,0 + 12,. AI TAI UAI, MAI-, SAI-,. 1 ph../ ph /. Fig. / MAI-, SAI-, TAI UAI MAI-, SAI-, + 1, / TAI, UAI + -, + Fig. - Comparison of the primary sequences from MAI-, and SAI-,. The asparagine residues with CHO denote N-glycosylation sites. Uppercase V and C in peptide alignments indicate peptides generated by digestions with S. aureus V2 protease and TLCK-chymotrypsin, respectively.

6 13 : ao 539 Fig.. Fractionation of PA-oligosaccharides from a-subunits (a) and b-subunits (b) from SAI-, and MAI-, by size-fractionation HPLC. M-, M/, M0, M1, M2 and M3 were authentic PA-oligosaccharides. Fig. / Thermal stability of a-amylase inhibitors at 1C, ph /.., TAI ;, UAI ;, MAI-, ;, SAI-,. TAI, UAI MAI-, SAI-, SAI-, MAI-, +- SAI-, HPLC. TAI, UAI, MAI-,, SAI-, TAI, MAI-, a +-3 b, AI TAI aai-+ cdna +2 Phaseolus coccineus ao AJ2./310,. MAI-, b 03 TAI MAI-, a, Asn +, Asn 0/ M0 B M3A b, Asn 0- Asn 2- M-X M -FX SAI-, MAI-, SAI-, MAI-, SAI-, MAI-, SAI-, MAI-, AI AI AI AI TAI, UAI, MAI-,, SAI-,. AI SAI-, MAI-, TAI UAI SAI-, MAI-,

7 540 /- +, AI AI SAI-, MAI-, TAI UAI a 1 +,- TAI, UAI MAI-, SAI-, b TAI, UAI - MAI-, SAI-, AI +-+3,+ AI,,,. AI AI + ao SAI-, MAI-, MAI-, MAI-, SAI-, SAI-, a b N SAI-, MAI-, AI SAI-, MAI-, AI a b a V2 b N AI SAI-, MAI-, AI, 1 ph /. TAI, UAI, SAI-,, MAI-,. AI SAI-, MAI-, TAI UAI SAI-, MAI-, TAI, UAI AI + Powers, J.R. and Whitaker, J.R., Purification and Some Physical and Chemical Properties of Red Kidney Bean (Phaseolus vulgaris) a-amylase Inhibitor. J. Food Biochem., +,,+1, , Wilcox, E.R. and Whitaker, J.R., Structural Features of Red Kidney Bean a-amylase Inhibitor Important in Binding with aoamylase. J. Food Biochem., 2, +23, Lajolo, F.M. and Finardi Filho, F., Partial Characterization of the Amylase Inhibitor of Black Beans (Phaseolus vulgaris), Variety Rico,-. J. Agric. Food Chem., --, +-, /. Moreno, J., Altabella, T. and Chrispeels, M. J., Characterization of a-amylase-inhibitor, a Lectin-Like Protein in the Seeds of Phaseolus vulgaris. Plant Physiol., 3,, / Yamaguchi, H., Isolation and Characterization of the Subunits of Phaseolus vulgaris a-amylase Inhibitor. J. Biochem., ++, 12/ Kasahara, K., Hayashi, K., Arakawa, T., Philo, J.S., Wen, J., Hara, S. and Yamaguchi, H. Complete Sequence, Subunit Structure, and Complexes with Pancreatic a- Amylase of an a-amylase Inhibitor from Phaseolus vulgaris White Kidney Beans. J. Biochem., +,, Berre-Anton, V.L., Bompard-Gilles, C., Payan, F. and Rougé, P., Characterization and Functional Properties of the aoamylase Inhibitor (a-ai) from Kidney Bean (Phaseolus vulgaris) Seeds. Biochim. Biophys. Acta., +-.-, Puls, W. and Keup, U., Influence of an a-amylase Inhibitor (BAY d 113+) on Blood Glucose, Serum Insulin and NEFA in Starch Loading Tests in Rats, Dogs and Man. Diabetologia, 3, 31+, Layer, P., Carlson, G.L. and Dimagno, E.P., Partially Purified White Bean Amylase Inhibitor Reduces Starch Digestion In Vitro and Inactivates Intraduodenal Amylase in Humans. Gastroenterology, 22, +23/+3, +32/ + ao Sawada, S., Takeda, Y. and Tashiro, M., Primary Structures of a- andb-subunits of a-amylase Inhibitors from Seeds of Three Cultivars of Phaseolus Beans. J. Protein Chem.,,+, 3+1,, +, ao / 2/23,,

8 15 : ao ao.2 +2,+22,+ +. ao.0 213, /, pp , +0,- pp., Hase, S., Ikenaka., T. and Matsushima, Y., Structure analysis of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound. Biochem. Biophys. Res. Commun., 2/,,/1, Ho#man, L.M., Ma, Y. and Barker, R.F., Molecular Cloning of Phaseolus vulgaris Lectin mrna and use of cdna as a Probe to Estimate Lectin Transcript Levels in Various Tissues. Nucleic Acids Research,,-, ,2 +32, +3 Frels, J.M. and Rupnow, J.H., Purification and Partial Characterization of Two a-amylase Inhibitors from Black Bean (Phaseolus vulgaris). J. Food Biochem., 2,,2+,,+,,,-, Cinco, F. J., Frels, J.M., Holt, D.L. and Rupnow, J.H., Determination of the Number and Heat Stability of a-amylase Inhibitors in White and Red Kidney Bean (Phaseolus vulgaris). J. Food Sci., /, +/+0 +32/ Yamaguchi, H., Isolation and Characterization of the Subunits of a Heat-labile aoamylase Inhibitor from Phaseolus vulgaris White Kidney Bean. Biosci. Biotech. Biochem., /1,,31-, +33- Kern, G., Schülke, N., Schmid, F.X. and Jaenicke, R., Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast. Protein Sci., +, +, , Wang, C., Eufemi, M., Turano, C. and Giartosio, A., Influence of the Carbohydrate Moiety on the Stability of Glycoproteins. Biochem., -/, 1, Maruyama, N., Salleh, M.R.M., Takahashi, K., Yagasaki, K., Goto, H., Hontani, N., Nakagawa, S. and Utsumi, S., The e#ect of the N-Linked Glycans on Structural Features and Physicochemical Functions of Soybean b- Conglycinin Homotrimers. JAOCS, 13, ,, +2,

520 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /0, No. +, /, /,2 (,**3) 20 * * Comparison of Breaking Properties of Products Baked with Rice Powders,

520 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /0, No. +*, /,* /,2 (,**3) 20 * * Comparison of Breaking Properties of Products Baked with Rice Powders, Wheat Powders and their Starches Atsuko Higo : and