Two forms of a-galactosidase (a-D-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total a-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total a-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000-and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-a-D-galactoside substrate. Km values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, Km values were 10 and 30 millimolar for the 50,000-and 25,000-molecular weight forms, respectively.The present study involved the resolution, partial purification, and characterization of two forms of a-galactosidase in cucumber leaves as a step in understanding the role of a-galactosidase in the breakdown of the transport sugars, stachyose and raffmose (17). Several properties of the two forms were compared, including their mol wt, substrate and inhibitor specificities, activation energies, and their occurrence as a function of leaf age.a-Galactosidase (a-D-galactoside galactohydrolase, E.C. 3.2.1.22) has also been studied in squash (15, 16), another cucurbit that transports stachyose. Thomas and Webb (15,16) Enzyme Extraction. Crude extracts were prepared by homogenizing 25 g of cucumber leaves with 80 ml of 0.1 M Na-acetate buffer (pH 5.2) for 3 min in a VirTis homogenizer. The homogenate was centrifuged at 27,000g for 15 min, and the supernatant was retained and filtered through cheesecloth.Assays for a-Galactosidase. a-Galactosidase activity was monitored routinely using I mm p-nitrophenyl-a-D-galactoside as substrate at pH 4.6 in 0.1 M Na-acetate buffer and at 30 C, as described by Pharr et al.

Galactose-1-phosphate uridyltransferase (EC 2.7.7.10), responsible for the conversion of galactose-1-phosphate (Gal-1-P) to uridine diphosphate galactose (UDPgal) was examined in fruit peduncles of Cucumis sativus L. Two uridyltransferases (pyrophosphorylases), from I and II, were partially purified and resolved on a diethylamino-ethyl-cellulose column. Form I can utilize glucose-1-phosphate (Glc-1-P), while form II can utilize either Gal-1-P or Glc-1-P, with a preference for Gal-1-P. Form I was more heat stable than form II. Both Glc-1-P and Gal-1-P activities of form II were inactivated at the same rate by heating. The finding of a uridyltransferase with preference for Gal-1-P indicates that cucumber may have a Gal-1-P uridyltransferase (pyrophosphorylase) pathway for the catabolism of stachyose in the peduncles. The absence of the enzyme UDP-glucose-hexose-1-phosphate uridyltransferase (EC 2.7.7.12) in this tissue rules out catabolism by the classical Leloir pathway. The incorporation of carbon from UDPglc into Glc-1-P as opposed to sucrose may be regulated by the activities of the uridyltransferases. Pyrophosphate, in the same concentration range, inhibits UDP-gal formation (Ki=0.58±0.10 mM) and stimulates Glc-1-P formation. The ratio of units of pyrophosphatase to units of Gal-1-P uridyltransferase was higher in peduncles from growing fruit than from unpollinated fruit. Modulation of carbon partitioning through a uridyltransferase pathway may be a factor controlling growth of the cucumber fruit.

Leaves of Cucumis sativus L. contained predominantly, non-reducing sugars which included verbascose, stachyose, raffinose and sucrose. Glucose and fructose were also present. The major sugars of fruit were glucose, fructose, and sucrose. Stachyose was present in small fruit (5 to 7 g fresh weight), but no soluble galactose-containing saccharides were found in larger fruit. Other plant parts also contained the raffinose saccharides. The identities of these sugars were established by partial enzymatic hydrolysis and paper chromatographic examination of the hydrolytic products. Four species of lactic acid bacteria from cucumber fermentations were able to ferment stachyose, raffinose, sucrose, melibiose, galactose, glucose, and fructose.