Bull. Soc. Sea Water Sci. Jpn., 56, (2002) Bulletin of the Society of Sea Water Science, Japan

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1 Bull. Soc. Sea Water Sci. Jpn., 56, (2002) Bulletin of the Society of Sea Water Science, Japan

2 Tablel Effect of NaC1 concentration on PPO activity Each data value in parentheses is relative value to th control as 100. Fig. 1 Zymograms of SDS-PAGE of polyphenol oxidase activity stained in the absence (A) and presence (B) of 0.5 M NaCl. Gel was incubated with H2O or 0.5 M NaC1 solution for 10 min and then stained in 0.1 M phospate buffer (ph 6.8) containing 5 mm catechol and 0.05% (w/v) phenylenediamine at 25t. Lanel:apple PPO, Lane2: potato PPO, Lane3: mung bean PPO

$Table 2 Summary of purification of mung bean PPO Fig. 2 Elution profile of PPO activity and total protein from mung bean sprouts after chromatofocusing. The black bar denotes the fractions (no.$

3 Table 2 Summary of purification of mung bean PPO Fig. 2 Elution profile of PPO activity and total protein from mung bean sprouts after chromatofocusing. The black bar denotes the fractions (no.23-27) pooled in the final purified product. Arrows show the peaks of 6 isozymes of PPO, pi 6.8, pi 6.7 pi 6.3, p1 6.0, pi 5.3 and pi 4.8. Fig. 3 Zymograms of SDS-PAGE of protein samples from different purification steps of PPO. Lane 1, molecular mass standards, Lane 2, xg supernatant fraction, Lane 3, fraction precipitated with 40-70% saturation of ammonium sulfate. Lanes 4 and 5, pooled active fractions after butyl toyopearl column chromatography and chromatofocusing, respectivery. Applied proteins were 150 ng per lane except for lane 5, in which about 15 ng of protein was used. Proteins visualizd by silver staining.

4 Fig.4 Lineweaver-Burk plots of mung bean PPO (pi 6.7) Fig. 5 Inhibition of the activity of mung bean PPO isozymes as a function of NaC1 concentration. Enzyme activity was assayed in the presence of NaCI after incubation with each concentration of NaC1 solution for 15 min at 30 C.

5 Fig. 6 Lineweaver-Burk plots of mung bean PPO in the absence and presence of NaCl.

6 potato tubers", Bio/Technology, 12, pp (1994) 9) M. Murata, M. Nishimura, M. Murai, M. Haruta, S. Homma and Y. Itoh," A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential", Biosci. Biotechnol. Biochem., 65, pp (2001) 10) W. Takeuchi, H. Takahashi and M. Kojima, " Purification and charact erization of the main isozyme of polyphenol oxidase in mung bean (Vigna mungo) seedings", Biosci. Biotech. Biochem., 56, pp (1992) 11) U. K. Laemmli, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature, 227, pp (1970) 12) K. Oba, N. Iwatsuki, I. Uritani, A. M. Alvalez and 1) A. M. Mayer," Polyphenol oxidase in plants-recent V. V. Garsia, `Partial purification and characterization progress", Phytochemistry, 26, pp (1987) of polyphenol oxidase isozymes in banana bud", Biosci. Biotech. Biochem., 56, pp (1992) 13) T. Kuwabara, T. Masuda and S. Aizawa," A 4) M. Tsurutani, M. Murata and S. Homma," Comparison of enzymatic browning of Japanese pear and apple", Food Sci. Technol. Res., 6, pp (2000) 5) T. Shahar, N. Hennig, T. Gutfinger, D. Hareven and E. Lifshitz, "The tomato 66.3-kD polyphenoloxidase gene: Molecular identification and developmental expression", Plant Cell., 4, pp (1992) 6) P. K. Boss, R. C. Gardner, B. J. Janssen and S. P. Ross, "An apple polyphenol oxidase cdna is upregulated in wounded tissues", Plant Mol. Biol., 27, pp (1995) 7) M. Haruta, M. Murata, A. Hiraide, H. Kadokura, M. Yamasaki, M. Sakuta, S. Shimizu and S. Homma, "Cloning genomic DNA encoding apple polyphenol oxidase and comparison of the gene product in esherichia coli and in apple", Biosci. Biotechnol. Biochem., 62, pp (1998) 8) C. W. B. Bachem, G. Speckmann, P. C. G. Linde, F. T. M. Verheggen, M. D. Hunt, J. C. Steffens and M. Zabeau, "Antisense expression of polyphenol oxidase genes inhibits enzymatic browning in dithiothreitol-sensitive tetrameric protease from spinach thylakoids has polyphenol oxidase activity", Plant Cell Physiol., 38, pp (1997) 14) P. W. Thygosen, I. B. Dry and S. P. Robinson, " polyphenol oxidase i n potato. A muligene family that exhibits differential expression patterns", Plant Physiol., 109, pp (1995) 15) A. Janovitz-Klapp, F. Richard and J. Nicolas," Polyphenoloxidase from apple, partial purification and some properties", Phytochemistry, 28, pp (1989) 16) N. J. Rivas and J. R. Whitaker,"Purification and some properties of twe polyphenol oxidase from bartlett pears", Plant Physiol., 52, pp (1973) 17) J. K. Palmer, "Banana polyphenoloxidase preparation and properties", Plant Physiol., 38, pp (1963)

7 Abstract Mechanism of the Suppressive Effect of NaC1 on the Polyphenol Oxidase Activity of Vegetables Kazuko OBA*, Atsuko YA1vIA1vIOTO*, Sachiko ITO*, Ayumi FuJIE * and Wakako TAKEUCHI * NaC1 inhibited the activity of polyphenol oxidase (PPO) isozymes in apples, potatoes and mung bean sprouts. To elucidate the mechanism for the suppressive effect of NaC1 on PPO activity in vegetables, we purified PPO from mung bean sprouts by means of ammonium sulfate fractionation and 2 column chromatographies. Six isozymes of PPO from mung bean sprouts were detected by means of SDSpolyacrylamide gel electrophoresis and chromatofocusing. The PPO isozyme of highest activity was pi 6.7. The PPO isozyme was purified as a single protein and the molecular mass was found to be 40 kda by means of SDSpolyacrylamide gel electrophoresis. The enzyme showed an apparent Km value of 1.3 mm for chlorogenic acid. The enzyme was inhibited non-competitively by NaC1 with a Ki value of 0.22M (1.27%). These results suggest that NaC1 inhibits PPO activity non-competitively, and results in preventing the enzymatic browning of vegetables. Key Words: Sodium chloride, Polyphenol oxidase, Enzymatic browning, Mung bean sprouts, Vegetables * Nagoya Women's University, Depaitment of Food Science and Nutrition, Nagoya , Japan

Purification and Properties of Acid Pyrophosphatase from the Potato Yasuko TANEMURA, Hiroshi WADA, Takashi ITO, Haruhito TSUGE* and Kazuji OHASHI* D

Purification and Properties of Acid Pyrophosphatase from the Potato Yasuko TANEMURA, Hiroshi WADA*, Takashi ITO*, Haruhito TSUGE* and Kazuji OHASHI* Department of Home Economics, Shotoku Gakuen Women's