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

Abstract

During germination and seedling establishment, the total pyrophosphate-dependent phosphofructokinase (PFP) activity in the cotyledons increases. Two types of subunits with molecular weights of 68 (a-subunit) and 65 (,-subunit) kilodaltons are present. The increase in activity coincides with an approximately 10-fold increase in #-subunit and twofold increase in a-subunit content. Different isoforms of PFP are present at all stages of incubation, but the ratio between the isoforms significantly changes. A linear relationship exists between the ratio of the two PFP subunits and the ratio of the two isoforms of the enzyme. The more anionic (peak 2) isoform of the enzyme apparently is favored by a high ratio of total #-subunit to a-subunit content.The is-to a-subunit ratio of the peak 2 isoform is also approximately fivefold higher than that of the peak 1 (less anionic) isoform. It is evident that the two subunits are not coordinately expressed and the level of expression of each subunit appears to be the primary factor determining the molecular form in which the enzyme is present. In some tissues, only the 65 kilodalton polypeptide is expressed in large amounts. The peak I isoform has a higher affinity for pyrophosphate than the peak 2 isoform, while the affinity for fructose-6-phosphate is similar. Both molecular forms are activated by fructose-2,6-bisphosphate.In addition to the ATP-dependent phosphofructokinase, plants also contain a pyrophosphate-dependent phosphofructokinase, PFP,2 that catalyzes the conversion of Fru-6-P to Fru-2,6-P2 in the cytosol. 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).Although the kinetic properties of PFP from several plant species have been studied (4,8,11,19,(24)(25)(26)28) 2Abbreviations: PFP, PPi:D-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90); Fru-6-P, fructose-6-phosphate; Fru-2,6-P2, fructose-2,6-bisphosphate; IgG, immunoglobulin.In several species, increased PFP activity is associated with the tissues in which a net sucrose breakdown occurs (32 and references therein). In certain tissues, like castor bean endosperm (21) and the cotyledons of Citrullus lanatus (7), however, maximum PFP activity during germination coincides with the peak in gluconeogenesis. Whether these changes in PFP activity during specific physiological conditions (6, 1 1, 17, 21, 32) are due to activation of existing enzyme or to induced gene expression is unclear.The increase in PFP activity during germination in the cotyledons of C. lanatus is due to an increase in the PFP concentration (7), presumably by increased gene expression. However, interconversion between different molecular forms, as was reported for the PFP from pea (31) and potato (20), might also contribute to changes in PFP activity. In the latter studies, it was shown that PFP can be reversibly converted from a high to a low molecular form depending on the presence of 31). The enzyme from potato...

Expression of the Genes for the α- and β-Subunits of Pyrophosphate-Dependent Phosphofructokinase in Germinating and Developing Seeds from Ricinus communis

“…There have been many studies of the kinetic and physical properties of PFP from a wide variety of plant species (4,6,7,10,15,16,23,24,28,29). The primary structure of PFP, however, as deduced from cDNA clones for the a-and 1B-subunits, has only recently been established (8), and there are no detailed reports describing patterns or levels of expression of the genes for the two subunits of this enzyme.…”

“…3) may be the result of contamination of the endosperm tissue, which is very small and difficult to harvest (14), with nucellus, a tissue that contains high levels of mRNA for both subunits of PFP. Expression of the two subunits of PFP in developing (6,10,16,30), and also changes in PFP activity that coincide with specific physiological conditions (e.g. 5, 13).…”

“…Alternatively, the a-and (3-subunits may have similar immunoreactivity, as is seen with purified PFP, and the differences observed using crude tuber extracts may result from genuine variation in the amount of each subunit present in this tissue (e.g. 6,10,16,30). The exact relative cross-reactivity of the a-and 13-subunits of PFP in the crude extracts of castor seed endosperm used in this study is not known.…”

“…Proteins were extracted from the same samples of tissue as were used for RNA extraction, and assayed for PFP activity as described (6). Electrophoresis and immunoblotting were performed as in ref.…”

“…The first of these, PFK (ATP:D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), utilizes ATP, catalyzes an irreversible reaction, and is found in the majority of organisms. The second, PFP2 (EC 2.7.1.90), catalyzes a reversible reaction with PPi as the phosphoryl donor and is found in a wide variety of tissues from a number of plant species (1,6,7,9,10,13,16,18,29,30) and a limited number of other organisms (20)(21)(22).…”