Pectinesterase tomato

Ray,J.,Knapp,J.,Grierson,D.,Bird,C and Schuch,W. (1988) Identification and sequence determination of a cDNA clone for tomato pectinesterase. European Journal of Biochemistry. 174. 119-124. 

Tucker,G. A.,Robertson,N.G. and Grierson,D. (1982) Purification and changes in activities of tomato pectinesterase isoenzymes. Journal of the Science of Food and Agriculture. 33. 396-400. 

Fig. 1. — Effect47 of Tomato Pectinesterase on the Activity of Pectate Lyase Acting on Highly Esterified Pectin. [Reaction mixtures contained 0.5% of D-galacturonan (broken line) or poly(D-galacturonic acid methyl ester) methyl glycoside (solid lines), 5 mM CaCl2, 33 mM phosphate buflFer (pH 7.0), and 0.25 unit of lyase in a final volume of 3.0 ml. In addition, the reaction mixture represented by the middle curve contained 0.031 unit of tomato pectinesterase in a final volume of 3.3 ml.] Reprinted, with permission, from M. Lee and J. D. Macmillan, Biochemistry, 9, 1930-1934 (1970). Copyright by the American Chemical Society.

In further work on this series, the same methods were used for the examination of the action pattern of highly purified pectinesterase produced by Fusarium oxysporum f. sp. vasinfectum.49 This pectinesterase was found to affect highly esterifled pectin by a mechanism similar to that of tomato pectinesterase, that is, more than half of the enzymic activity occurred at the reducing ends of the molecules, and the rest attacked a different locus or loci of the pectin chains. These conclusions were supported by a comparison of the effect of clostridial lyase on pectin partly de-esterified in an alkaline solution with its effect on pectin partly de-esterified by Fusarium oxysporum pectinesterase. The lyase did not act on the randomly... 

In the action of pectinesterase, evidently, end-product inhibition takes place.57 For highly purified tomato pectinesterase, Lee and Macmillan50 found an inhibition constant Kt = 7 mM D-galac-topyranosiduronate units, the inhibition being competitive (see Fig. 3). 

Much attention has now been devoted to the purification and characterization of tomato pectinesterase, apparently because of the high content thereof in the ripe fruits. 

By means of gel electrophoresis on cross-linked, hydrolyzed starch,99 with simultaneous checking for proteins, lipids, and pectinesterase activity, it was found, however, that the product isolated after the separation on CM-Sephadex C-50 constitutes but one of five multiple forms of tomato pectinesterase, and is the one present in preponderant proportion98 (see Fig. 4). The accompanying lipid and sugar components were separated from this pectinesterase form in the course of the purification procedure. After analysis of the hydro-lyzate of the final product for fatty acids, as well as for carbohydrate components, it was possible to exclude the possibility of a lipoprotein,30 as well as glycoprotein,100 character of this form of tomato pectinesterase. 

Delincee and Radola100 used a commercial preparation, as well as fresh tomatoes, for the preparation, purification, and characterization of tomato pectinesterase. The tomatoes were pressed and then homogenized directly with ammonium sulfate at 70% saturation. The precipitate obtained was extracted with 0.3 M phosphate and repeatedly salted out with ammonium sulfate, and the product was separated on a column of Sephadex G-75. The pattern of separation was similar to that in preceding work.50,97 A detailed study of the size properties of pectinesterase was conducted by gel-filtration and sedimentation analysis.100 By column and thin-layer gel-filtration on Sephadex G-75, the approximate molecular weight of a number of preparations of tomato pectinesterase was determined, values of 24,000 and 27,000 being obtained. A possible interaction of the... 

Fig. 4. — Monitoring of the Multiple Molecular Forms of Tomato Pectinesterase by Starch-gel Electrophoresis.98 [ENZ, detection of pectinesterase activity by paper print with pectin and Bromothymol Blue PROT, protein staining with nigrosin O, origin. Key A, 1 crude tomato extract after ammonium sulfate salting-out, and dialysis 2 pectinesterase fraction from column of DEAE-Sephadex A-50 3 and 4 pectinesterase fractions from column of Sephadex G-75. B, Two parts of the same gel after horizontal slicing 1, 500 fig of the isolated form of pectinesterase from a column of CM-Seph-adex C-50 with 175 mM phosphate-sodium chloride buffer 2, active fraction at 150 mM buffer 4 and 5, 250 fig and 1 mg of the isolated form of pectinesterase, respectively.]...

By use of starch-gel electrophoresis, the total extract of bananas, and the fractions obtained after separation on DEAE-Sephadex A-50, were found to contain six multiple forms of pectinesterase having electrophoretic patterns different from those of tomato pectinesterase.103... 

Nakagawa et al, (76) reported a break in the Arrhenius plot (log tomato pectinesterase rate vs, 1/T) at 31°C. NaCl (O.IM) increased the activation energy and lowered the break point to 27 °C. 

Pressey and Avants (80) separated, by DEAE Sephadex A-50 chromatography, four forms of tomato pectinesterase, with molecular weights 35,500, 27,000, 23,700, and 24,300. The number and relative amounts of these components varied considerably with ripeness and variety of tomato, and no single extract contained all four. The four forms diflFered in heat stability and response to pH and cation concentration. The fourth form, the major species in most tomato extracts, had properties similar to those of the enzyme described by Lee and Macmillan (75), whose molecular weight, however, was estimated on Sephadex G-75 as 27,500 (81),... 

Three multiple forms of pectinesterase have been found in extracts and purified products from carrot. The enzymes (mol. wts. 2.7 x 10 ) were found to possess properties similar to those of tomato pectinesterase. 

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