Changes in sucrose synthetase activities in aging potato tuber slices.

Abstract

The mechanism by which the reversible conversion of starch ito sucrose takes place in potato tubers exposed to cold is as yet essenitialDly unknown. Early studies on the stubject triedl to correlate this conversion with variations in the activity of related enzymes (10). Those responsible for the synthesis of stucrose and sucrose-6-phosphate (1) are undoubtedily highly relevant. In addition to its role in stucrose 'synthesis, the sucrose synthebase lead to the formation of UDP-gluic'ose and ADP-glucose (2), the glucosyl donors for starch synthesis catalyzed by ithe particullate (6) and the soluble (5) synithetase, respectively. As in sweet corn (4), the sugar nuoleotide-s would thu,s be the 'link between starch and sucrose synthetase in potato tubers. With the aim of obtaining some insighit into the regutlatory mechanism of these enzymes we have carried out experimenits witth aging potato tuiber slices, in view of t;he special properties of the itissue in these condi'tions (3). Potato tubers (Solanum tuiberosum var. Huincul) were uised. Tissue plulgs, 1.5 cm in diameter, were cut from the central parenchima wiith a cork borer. The plulgs were sllicecd with a microtome inito discs, 1 mm in thickness, whiich were aged according to Click and Hackett (3). The liquid 'of 'aging was eeilther water or 'the soilutition indicated in each case. In preliminary experiments, similar result's 'ha'd been obta,ined by addition of calcoiuim sulifate or antibiotics to 'the aging med-ium, or uisling aeration technliques (11 ). For 'the 'assay of stucrose and sucrose-6-P syntheitases, fresh (control) or aged slices were washed wi,th water, 'blotted vwiith filiter paper and grouind in a mortar with 0.01 M mercaptoethanol. The extract was strained 'through cheese-cloth and then centrifuged at 3000 X g in order to separate starch. The supernatant was dialyzed overnight again:st 0.05 m itriis-HCl buffer, pH 7.3, and used as a souirce of the enzyme. The assay was performed as follows: 0.5 jimole of UDP-glIucosse, 0.5 ,umole of fructose or fructo,se-6-P, 5 jvmnles of tris-HCI buffer, pH 7.3, and 5 to 10 ,l of enzyme (equivalent to 0.05-0.1 mg of protein), in a fina,l voolume of 25 ixl, were incubated at 370 for 30 minutes. The reaction was stopped by ad'ding NaOH to a fiinal concentration of 0.25 M and heabing for 15 m,inuites at 1000. This treaitmenit destroyed free fruotose or fructose-6-P, and sucrose or sucrose-6-P co'u;ld be estim,ated with Ithe thiobarhyitturic acid reagent (8)2. One tunit of ac,tivity is the amoulnt of enzyme producing ithe form,ation of 0.01 pmole of suicrose or sucrose-6-P uinder Ithe described assay condiitions, which were Isdlected according to the results of Slabnik, Frydman, and Cardini (unpublished). The reaction was linear both with respect to enzyme concentration andl tibme. Assay of possible interfering pho,sphatases and( inverta,se in these experimental condiitions was performed !in botih enzymatic extracts with negative resulits.