PURIFICATION AND CHARACTERIZATION OF TWO TOMATO POLYGALACTURONASEISOENZYMES

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Abstract
The properties of tomato polygalacturonases at two ripening stages were investigated. Two isoenzymes, PG I and II, were isolated from underripe fruits with an orange skin color. Fully ripe fruits contained only polygalacturonase II. PG I and II were purified by chromatography on DEAE-Sephadex A-50, Sephacryl S-200 and CM-agarose chromatography. PG I had a Mr of 199,500 as determined by Sephacryl S-300 gel filtration and was 50% inactivated at 66.5°C and pH 4.5 after incubation for 5 min. It had an activation energy (Ea) of 16.8 Kcallmol (70.3 times 103 Jlmol), Vmax of 27.7 units/mg protein and Km value of 7.5 times 10−2 mM polygalacturonic acid. PG II had a Mr of 45,700 and was 50% inactivated at 58°C under the same conditions. Both isozymes had a pH optimum of 4.6. PG II had an Ea value of 14.8 Kcallmol (61.9 times 103 Jlmol), Vmax value of 58.8 units/mg protein and Km value of 3.8 times 10−2 mM polygalacturonic acid. PGI gave rise to only one band during electrophoresis in polyacrylamide gels, whereas PG II showed one major and one minor band both with PG activity. Gel electrophoresis in the presence of sodium dodecyl sulfate resulted in two major bands (Mr= 47,500 and 41,000) for PG I and only one major band (Mr= 47,500) for PG II. PG I is composed of several subunits, all of which are glycoproteins.