Pyrophosphate Fructose-6-P 1-Phosphotransferase from Tomato Fruit

Abstract

Three forms of pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) were purified from both green and red tomato (Lycopersicon esculentum) fruit: (a) a classical form (designated 02) containing a-(66 kilodalton) and ,B-(60 kilodalton) subunits; (b) a form (01) containing a ,-doublet subunit; and (c) a form (Q0o) that appeared to contain a ,B-singlet subunit. Several lines of evidence suggested that the different forms occur under physiological conditions. Q2 was purified to apparent electrophoretic homogeneity; Q1 and Q0 were highly purified, but not to homogeneity. The distribution of the PFP forms from red (versus green) tomato was: Q2, 29% (90%); 01, 47% (6%); and Q0, 24% (4%). The major difference distinguishing the red from the green tomato enzymes was the fructose-2,6-bisphosphate (Fru-2,6-P2)-induced change in Km for fructose-6-phosphate (Fru-6-P), the 'green forms' showing markedly enhanced affinity on activation (Km decrease of 7-9-fold) and the 'red forms' showing either little change (Q0o, Q1) or a relatively small (2.5-fold) affinity increase (02). The results extend our earlier findings with carrot root to another tissue and indicate that forms of PFP showing low or no affinity increase for Fru 6-P on activation by Fru-2,6-P2 (here 01 and Qo) are associated with sugar storage, whereas the classical form (02), which shows a pronounced affinity increase, is more important for starch storage. from most plant sources is highly stimulated by Fru-2,6-P2 (1, 2, 5, 6, 8-10, 15-18, 20, 21), the enzyme from leaves of CAM plants responds only sluggishly to this activator (5,8,10). In another case (wheat seedlings), PFP activity has been reported to be associated with two structurally different enzyme forms (21). Finally, in a recent study (18), our laboratory has identified in carrot roots a form of PFP that differs both kinetically and structurally from other forms of the enzyme studied to date.As a result of this unusual kinetic and structural diversity, we have considered the possibility that the properties of PFP depend on the functional nature of the parent tissue (18). That is, the form of PFP present in one tissue with a given metabolic function may differ significantly from the enzyme in a tissue with another function. We have put this idea to a further test using pericarp of tomato fruit-a tissue known to change from a starch-storing to a sugar-storing function during ripening (7,14). The results indicate that, while occurring in analogous enzyme forms, the PFPs from green and red fruit show a pronounced difference on activation by Fru-2,6-P2. The activated green forms are characterized by a marked increase in affinity for Fru 6-P, whereas the red forms show either little or no change in this connection. A preliminary account of this work has been published (19

Pyrophosphate Dependent Phosphofructokinase of Citrullus lanatus: Molecular Forms and Expression of Subunits

“…As with the PFP isoforms from tomato fruit (29), these isoforms differ in charge and, therefore, can be separated by ion exchange chromatography. The presence of PFP isoforms was also previously reported in wheat tissue (33).…”

“…In contrast, peak 2 is enriched for the fl-subunit. One of the PFP isoenzymes from wheat (33) and tomato fruit (29) and the PFP from carrot tap root (28) and mung bean (4) apparently only contain the fl-type subunit. It is not possible to state precisely the subunit concentration in C. lanatus cotyledons.…”

“…Since the discovery in 1979 of PFP in pineapple leaves (14), significant levels of this enzyme have been found in a variety of plant tissues (1,3,4,11,17,21,23,24,28,29,33).…”

“…The presence of isoenzymes of PFP in wheat (33) and tomato (29), a novel type of PFP in the tap root of carrot (28), as well as a PFP in Phaseolus mungo (3) and Phaseolus vulgaris (1 1) with kinetic properties distinctly different from other PFPs, suggest that PFP has variable properties when isolated from different tissues. Its properties can also change in the same tissue at different stages of development.…”

Pyrophosphate-dependent Phosphofructokinase: Its Oligomeric Forms in Mature Green Tomato Fruit

“…2 represents the results of fraction III-V. Large and small subunits seems to correspond to the C(-and p-subunits, respectively2), and Wong et al reported the value of 66 kDa and 60 kDa for the tomato enzyme. 13 ) Recent results of nucleotide sequencing showed that C(-and p-subunits from potato tuber had molecular masses of 64 kDa and 57 kDa, respectively. 14) It has been also shown that the small, p-subunit is the catalytic subunit.…”