The mutual interference between the second-derivative bands of tyrosine and tryptophan in proteins has been evaluated in terms of the ratio r between two peak to peak distances. The r values have been found to be not only related to the tyrosine/tryptophan ratio but also dependent on the polarity of the medium in which tyrosyl residues are embedded. The results obtained on purified proteins have been found consistent with the available X-ray information and with the existing solvent perturbation data.

The structure and dynamics of enigmatic hexa(3,5-di-tert-butylphenyl)ethane was characterized via NMR spectroscopy for the first time.

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The finding of a powerful inhibitor of pectin methylesterase in ripe kiwi fruit is reported. The inhibitor was revealed to be a glycoprotein. It was purified to homogeneity and found to have a molecular mass of about 28 kDa, as estimated by gel filtration chromatography, SDSjPAGE and analytical ultracentrifugation. The sugar portion is composed of galactose, arabinose and rhamnose, the latter being much less represented. The amino acid composition showed a very high content of acidic residues compared to basic ones, which is the reason for the very low isoelectric point of the protein (less than 3.5). The kind of inhibition on kiwi pectin methylesterase was found to be competitive with an apparent Ki of 0.22 pM, using citrus pectin as a substrate. Moreover, the inhibitor is effective in inhibiting pectin methylesterase in the pH range 3.5 -7.5. Kiwi inhibitor appears to be specific for pectin methylesterase, inasmuch as it was found to be ineffective against other polysaccharidedegrading enzymes, such as polygalacturonase and amylase. Conversely, it appears to be completely aspecific as far as the pectin methylesterase source is concerned. In fact, it was found to inhibit this enzyme effectively from all the sources we assayed, i. e. orange, tomato, apple, banana, potato.The enzyme pectin methylesterase (PME) is a ubiquitous plant enzyme believed to play a fundamental role in vegetable cell metabolism [l]. The protein is cell-wall-bound [2] and is involved in the ripening process of fruit [3] as well as in cell wall extension during cell growth. In the ripening mechanism, PME acts by producing pectin with a low degree of methylation which is then subject to further enzymatic cleavage by polygalacturonase [4]. The role played by PME in cell wall extension consists essentially in generating a local pH decrease which, in turn, activates enzymes involved in wall autolysis and extension [5 -71. The control of this growth mechanism, therefore, relies on opposite pH sensitivities of the enzymes involved in the process: the PME, having an optimum pH around neutrality, decreases its activity as a consequence of the pH decrease due to its own action. Conversely, the local low pH activates the glycohydrolases, which produce a limited cell wall autolysis, and the glycosyltransferases, which build up and elongate the cell wall. As a consequence of the cell wall reconstitution, a pH increase occurs that activates PME, and so on.Control of the enzymatic activity of PME is also of prime importance in biotechnological processes which concern the production and storage of fruit juices and purees. In fact, it is well known [4, 81 that PME activity changes the texture of fruit products. For this reason, product storage at very low temperature is required or a pasteurization process at high temperature must be employed to inactivate the PME in indus-

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 retidue 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.keyword^: pectin methylesterase ; pectin methylesterase inhibitor; kiwi fruit.The enzyme pectin methylesterase (PME) hydrolyzes the methylesters of polygalacturonic acid. It is generally present in higher plants and in pathogenic fungi tissues, bound to the cell wall through electrostatic interaction; in fact, it is removed from the cell wall by extraction with high-salt-concentration solutions. PME is believed to be involved in cell-wall extension during cell growth 11-31, as well as in the fruit ripening process [4][5][6]. The biochemical peculiarities of this enzyme from varous sources have been largely investigated [7-91 and the primary structure of the tomato enzyme has been assessed [lo].We have found a protein inhibitor of PME (PMEI) in kiwi fruit, which possesses a high affinity for the enzyme 1111. This inhibitor is non specific with respect to the enzyme sources; It inhibits PME from all of the vegetable source tested, while it is totally inactive in inhibiting other polysaccharide-degrading enzymes such as polygalacturonase or amylase. The inhibitor is a very acidic protein (PI below 3.5) and appears to be glycosylated. The method of inhibition is shown to be through complex formation with PME in a 1 : 1 ratio.The physiological role of this protein is still unclear. It could be invohed in the defence mechanism of plants against pathogens; in fact, in plants, the presence of a polygalacturonaseinhibiting protein is known, which exhibits an activity toward microbial polygalacturonase, which is a polysaccharide-degrading enzyme [12]. PMEI could also be involved in some control Correspondmce to A. Giovane, Diparti...