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Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers. II. Estimations of fluxes in vivo and evidence that fructokinase catalyses a near rate-limiting reaction

Author:
Viola, R.
Source:
Planta 1996 v.198 no.2 pp. 186-196
ISSN:
0032-0935
Subject:
Solanum tuberosum, tubers, carbohydrate metabolism, beta-fructofuranosidase, sucrose, glucose, fructose, sucrose synthase, starch, isotope labeling, phosphorylation, glucokinase, fructokinase, enzyme activity, culture media, hexoses
Abstract:
Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179-185). When the discs were incubated with either [U-14C]glucose or [U-14C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-14C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of 13C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-13C]fructose or [2-13C]glucose. The pattern of 13C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 micromole.hexose .gFW-1.h-1 in the absence of exogenous glucose to 3.7 micromole. hexose. gFW-1.h-1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.
Agid:
1403909