Tomatoes (Lycopersicon escadentum L.) contained a high level of aigalactosidase activity which was due to three forms of the enzyme. During tomato ripening, the sum of their activities remained relatively constant, but the levels of the individual forms of 8-galactosidase changed markedly. The three enzymes were separated by a combination of chromatography of DEAE-Sephadex A-50 and Sephadex G-100. During ripening of tomatoes, /8-galactosidases I and III levels decreased but the P-galactosidase II level increased more than 3-fold. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. However, the enzymes differed in molecular weight, Km value with p-nitrophenyl-flgalactoside, and stability with respect to pH and temperature. /3-Galactosidase II was the only enzyme capable of hydrolyzing a polysaccharide that was isolated from tomatoes and that consisted primarily of a-1, 4-linked galactose. The ability of tgalactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening.During fruit ripening, the most apparent cell-wall change is an increase in water-soluble polyuronide (6)(7)(8)15). The solubilization of polyuronide is generally attributed to the action of polygalacturonase, which appears in many fruits near the onset of ripening (6,15,19). Another change that occurs, at least in apples, strawberries, and tomatoes, is a pronounced loss of galactose from the cell walls. This process does not appear to be related to cell-wall degradation by polygalacturonase (9,17), but possibly to a galactanase. Bartley (2) found a f8-galactosidase in apples that degraded galactan and increased in activity during apple ripening. Tomatoes also contain ,B-galactosidase, but it has been concluded that this enzyme is not responsible for the hydrolysis of cell wall galactans (5). Furthermore, Gross and Wallner (5) rate at which it hydrolyzes p-nitrophenyl-,B-galactoside (Sigma). The reaction mixture consisted of 0.5 ml of 0.1 M citrate (pH 4.0), 0.4 ml of 0.1% BSA, 0.1 ml of diluted enzyme, and 0.4 ml of 13 mm substrate. After 15 min at 37°C, the reactions were terminated by the addition of 2 ml of 0.2 M sodium carbonate, and the liberated p-nitrophenol was measured at 415 nm. One unit of ,8-galactosidase was defined as the amount that hydrolyzed 1 ,umol ofp-nitrophenyl-,f-galactoside/ 15 mi.Galactanase was assayed by measuring the release of reducing sugars from a galactose-rich polysaccharide isolated from tomatoes (see below). The reaction mixture consisted of 0.4 ml of 0.1 M sodium acetate (pH 4.0), 0.4 ml of enzyme solution diluted with 0.2% BSA, and 0.2 ml of 1% polysaccharide. After 1 h at 37°C, the reaction was terminated by heating in boiling water, and the solution was analyzed for reducing groups by the arsenomolybdate method (13). The reaction rates in the ,B-galactosidase and galactanase assays were linear with respect to both enzyme concentration and incubation time.T...

The susceptibility of isolated cell walls to solubilization by polygalacturonase and the effect of pectinesterase on the solubilization were examined. The two polygalacturonases in ripe tomatoes were purified to remove pectinesterase. Both polygalacturonases solubilized uronic acid from pectinesterase‐free tomato cell walls most rapidly at about pH 3.5, well below p H 4.5, the pH optimum for their hydrolysis of pectic acid. At p H 3.5, very low levels of pectinesterase increased cell wall solubilization by the polygalacturonases seueralfold, whereas high concentrations of pectinesterase completely inhibited solubilization. A t pH 5, pectinesterase also increased cell wall solubilization, but higher concentrations were required than at pH 3.5 and high levels were not inhibitory. The materials solubilized at pH 3.5 were galacturonan with a molecular weight of about 110,000 and two fractions of much higher molecular weights consisting primarily of neutral sugars. Galactose accounted for about two‐thirds of the monosaccharides in the neutral polysaccharides.

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Two polygalacturonases (PG I and PG II) have been separated from extracts of ripe peaches (Prunus persica) by chromatography on Sephadex G-100. PG I hydrolyzes polygalacturonic acid from the nonreducing ends of the molecules, releasing galacturonic acid as the product. It functions optimally at pH 5.5, requires Ca2+ for activity, and hydrolyzes low molecular weight substrates most rapidly. In contrast, PG II cleaves the molecular chain of the substrate randomly with a pH optimum at about 4. This enzyme is most reactive with substrates of intermediate molecular weight. It catalyzes the release of watersoluble, but 70% ethanol-insoluble, pectin from washed peach cell walls.Changes in polysaccharides leading to the destruction of the cell wall structure are probably responsible for the marked softening of fruits during ripening. The softening of freestone peaches, for example, has long been associated with the conversion of protopectin to soluble forms (13,(19)(20)(21). Although changes in other polysaccharides may be involved, the solubilization of pectin has received the most attention because of the occurrence of this polysaccharide in the middle lamella, as well as in the cell wall. Elucidation of the mechanism of pectin solubilization has been hindered by the complexity of protopectin and by the failure to identify the enzymes involved. In a recent study (16), we found that polygalacturonase was not detectable in unripe peaches, but activity appeared when the fruit began to soften and then increased sharply as ripening proceeded. Development of the enzyme paralleled the formation of water-soluble pectin. Furthermore, the molecular weights of the solubilized pectin decreased during fruit ripening. The results suggested a key role for polygalacturonase in pectin solubilization and, therefore, in textural changes accompanying peach ripening.The very low level of polygalacturonase in peaches, compared to that in tomatoes, presents problems in studying this system. Because the activity is associated with the particulate fraction of homogenates, we originally conducted our assays with aliquots of washed cell fragments incubated for long periods. We have now developed a procedure for solubilizing and concentrating the polygalacturonase activity in ripe peaches. This paper describes the resolution of the activity into two components and some of their properties. MATERIALS AND METHODSSubstrates. Pectic acid, purchased from Sigma Chemical Company,' was purified by precipitating a 1% solution in water at pH 5 with 2 volumes of 95% ethanol. The precipitate was collected on Miracloth, dissolved in water, and precipitated with ethanol. This procedure was repeated three times, and the pectic acid was finally homogenized with 95% ethanol and acetone and dried under vacuum.The polygalacturonic acids were prepared by enzymatic hydrolysis of pectic acid as described earlier (15). PGA I is a fraction that is insoluble at pH 2 and 25 C, while PGA II is insoluble at pH 2 and 3 C. PGA III is soluble at pH 2 and 3 C, but its ...

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