SEARCH

SEARCH BY CITATION

Keywords:

  • pectin methylesterase;
  • pectin methylesterase inhibitor;
  • kiwi fruit

Abstract

  1. Top of page
  2. Abstract
  3. References

The pectin methylesterase inhibitor from kiwi fruit (Actinidia chinensis) was purified by a single-step procedure based on affinity chromatography. Partially purified tomato pectin methylesterase was covalently bound to Sepharose. The affinity resin strongly and selectively binds the inhibitor, which could be eluted in high yield as a single, homogeneous and sharp peak by high salt concentration at pH 9.5 without loss of inhibitory activity. The purified protein possesses a molecular mass of 18 kDa, as estimated by SDS/PAGE, whereas by gel filtration under native conditions, its molecular mass appears to be 25 kDa. The inhibitor interacts with pectin methylesterase, forming a 1:1 complex, as demonstrated by gel-filtration experiments.

The inhibitor was glycosylated. Its glycidic portion can be removed by digestion with N-glycosidase F after protein denaturation and, to a minor extent, by digestion with N-glycosidase H. No glycidic residue could be removed by digesting the native protein with those N-glycosidases. Antibodies against pectin methylesterase inhibitor were raised in rabbits and used to evidence protein expression during fruit ripening. The results showed that the inhibitor is present in the unripe fruit as an inactive precursor with a higher molecular mass (30 kDa) and is transformed into the active protein, most likely by proteinase action, during the course of the ripening process.

Abbreviations
PME

pectin methylesterase

PMEI

pectin methylesterase inhibitor

Enzymes
 

PME (EC 3.1.1.11)

 

polygalacturonase (EC 3.2.1.15)

 

actinidin (EC 3.4.22.14)

References

  1. Top of page
  2. Abstract
  3. References
  • 1
    Ricard, J. & Noat, G. (1986) Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 1. A theory of the ionic control of a complex multi-enzyme system, Eur. J. Biochem. 155, 183190.
  • 2
    Moustacas, A. M., Nari, J., Diamantidis, G., Noat, G., Crasnier, M., Borel, M. & Ricard, J. (1986) Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 2. The role of pectin methyl esterase in the modulation of electrostatic effects in soybean cell walls, Eur. J. Biochem. 155, 191197.
  • 3
    Nari, J., Noat, G., Diamantidis, G., Woudstra, M. & Ricard, J. (1986) Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methylesterase activity and electrostatic effects in soybean cell walls, Eur. J. Biochem. 155, 199202.
  • 4
    Brady, C. J., McGlasson, W. B. & Speirs, J. (1987) The biochemistry of fruit ripening, in Tomato Biotechnology (Nevins, D. & Jones, R., eds) pp. 279288, Alan R. Liss, New York .
  • 5
    Goldberg, R., Bordenave, M., Pierron, M., Prot, R. & Mutaftschiev, S. (1992) Enzymatic processes in growing cell walls: possible control by pectinmethylesterases, in Plant cell walls as biopolymers with physiological functions (Masuda, Y., ed.) pp. 269274, Proceedings of the 32nd Yamada conference, Yamada Science foundation, Osaka , Japan .
  • 6
    Goldberg, R., Morvan, C. & Roland, J.-C. (1986) Composition, properties and localization of pectins in young and mature cells of the mung bean hypocotyl, Plant Cell Physiol. 27, 417429.
  • 7
    Giovane, A., Quagliuolo, L., Castaldo, D., Servillo, L. & Balestrieri, C. (1990) Pectin methyl esterase from actinidia chinensis fruits, Phytochemistry 29, 2821.
  • 8
    Lim, Y. M. & Chung, M. C. M. (1993) Isolation and characterization of pectin methylesterase from papaya, Arch. Biochem. Biophys. 307, 1520.
  • 9
    Fayyaz, A., Asbi, B. A., Ghazali, H. M., Che Man, Y. B. & Jinap, S. (1993) Pectinesterase extraction from papaya, Food Chem. 47, 183185.
  • 10
    Markovic, O. & Jornvall, H. (1986) Pectinesterase. The primary structure of tomato enzyme, Eur. J. Biochem. 158, 455461.
  • 11
    Balestrieri, C., Castaldo, D., Giovane, A., Quagliuolo, L. & Servillo, L. (1990) A glycoprotein inhibitor of pectin methylesterase in kiwi fruit (Actinidia chinensis), Eur. J. Biochem. 193, 183187.
  • 12
    Salvi, G., Giarrizzo, F., De Lorenzo, G. & Cervone, F. (1990) A polygalacturonase-inhibiting protein in the flowers of Phaseolus vulgaris L., J. Plant Physiol. 136, 513518.
  • 13
    Marquis, H. & Bucheli, P. (1994) Inhibition of tomato pectin methylesterase by partially purified kiwi pectin methylesterase inhibitor protein, Int. J. Food Sci. Technol. 29, 121128.
  • 14
    Rillo, L., Castaldo, D., Giovane, A., Servillo, L., Balestrieri, C. & Quagliuolo, I., (1992) Purification and properties of pectin methylesterase from mandarin orange fruit, J. Agric. Food Chem. 40, 591593.
  • 15
    Hagerman, A. E. & Austin, P. J. (1986) Continuous spectrophotometric assay for plant pectin methyl esterase, J. Agric. Food. Chem. 34, 440444.
  • 16
    Pfeiffer, N. E., Torres, C. M. & Wagner, F. W. (1983) Proteolytic activity in soybean root nodules, Plant Physiol. 71, 797802.
  • 17
    Boland, M. J. & Hardman, M. J. (1972). Kinetic studies on the thiol protease from Actinidia chinensis, FEBS Lett. 27, 282284.
  • 18
    Patel, M., Kayani, I. S., Mellor, G. W., Sreedharan, S., Templeton, W., Thomas, E. W., Thomas, M. & Brockleurst, K. (1992) Variation in the P2–S2 stereochemical selectivity towards the enantiomeric N-acetylphenylalanylglycine 4-nitroanilides among the cysteine proteinases papain, ficin and actinidin, Biochem. J. 281, 553559.