Biosynthesis of Stachyose in Phaseolus vulgaris.

Stachyose, a suigar of the raffinose family, is present in a wide variety of plants (3, 5) and sometimes is the most abundant soluble carbohy(lrate including sucrose. The biosynthesis of stachyose is unknowln, although galactose can be transferred from I raffinose molecule for example to ainother one by a-galactosidase giving rise to stachyose (10). a-Galactosidases, however, show little specificity for the donor as well as the acceptor molecule and they transfer with low efficiency as compared to their hydrolytic activity (2, 3, 10). From physiological (lata evidence has acctumulatedl that in a variety of plants stachyose arises by the transfer of galactose from a specific donorgalactoside to raffinose (8, 9). This galactoside has recently been identified as galactinol, a compouind which was first found in sugar beets by Brown and Serro (1) and showin to be 0-a-Dgalacto-pyranosyl-(1l -> I)-myo-inositol (1,6). WVe have now obtained an enzvme from ripening seeds of dwarf beans (Phaseolus zulslgairis) which transfers galactose specifically and with high yield from galactinol to raffinose giving rise to stachyose and mtyo-inositol. In addition it was found that galactinol constitutes the major galactoside in the beans dulring a certain maturation period and preceedes stachyose in this role (Tanner, unpublished). The enzyme was prepare(l as follows: 177g of unripe seeds were homogenized in 250 ml of 0.1 M phosphate buiffer, pH 7.0, with an Ultra Tuirrax at high speed for 2 minutes. The suipernatant fraction ol)tained after centrifugationi at 10,000 X g for 10 mintutes was dialyzed 45 houirs against 0.05 M phosphate btuffer, pH 7.0. The enzyme was puirified 10-fold by ammonitum sulfate precipitation (3556 %) and treatment with calcium phosphate gel. The incubation mixture contained in a total v%oltume of 0.04 ml: 0.5 ,umole raffinose, 2.4 Atmoles sodiuim phosphate buiffer pH 7.0, enzyme as indicated, andl galactinol-14C (40,000 cpm where not indicated otherwise), isolated from Lamium leaves after I hour photosynthesis in "-CO., according to Senser and Kandler (9). Of the total radioactivity, 95.5 % of this galactinol-'4C was located in the galactose moiety. The reaction was run at 32° and(l stoppedl with 0.1 ml 99 % ethainol. The supernataint fractioin and( the wvashings were chromatographe(d in ethyl acetate: blutanol: acetic acid: wvater (3: 4: 2.5: 4). The radioactiv e areas were locatedl with a strip cotnnter, ain(I the radioactivity measuire(l directly on paper with a methane flow coulnter Frieseke and Hoepfner 407 A. The amoulnt of stachyose formed was proportional to the amount of enzxme added and linear with time (table I). This linearity was maintained uintil 35 % of the galactose-14C of galactinol had been trainsferred to raffinose, then the rate decreasedl. II 9 houirs 80 % of the galactose-14C was transferre(I by 0.14 mg proteini. The reaction pi 0dtuct was chromatographically identical to stachvose in 4 solvent systems (88 % phenol: water: I M Na.. EDTA: acetic acidl 840: 160: 1 :10 n-butanol: propionic acid: water 7=50: 352: 498; ethyl acetate etc. see above: ni-btitanol pyridine: water: acetic acid = 60:40:30: 3). \When treatedl with a-galactosidase the only radioactive compoun(d forme(d from the reaction produict was galactose; in the presence of 8-galactosidase n1o radlioactive galactose was split off. Inctubation of the reactioni produtict with invertase yielded 1 radioactive compoulnd with the same RF in ethyl acetate-butanol-acetic acid-water as manninotriose obtaine(l from auithentic stachyose in the same way. The seconi(d reaction pro(ltct was identified as inyo-inositol by chromatography and with the help of inositol-dehydrogeniase prepared from Aerobacter aerogenes accorcling to XNVeisshach (11). In the presence of raffinose as the acceptor