A homogeneous multimeric protein isolated from the green alga, Scenedesmus ohliquus, has both latent phosphoribulokinase activity and glyceraldehyde-3-phosphate dehydrogenase activity. The glyceraldehyde-3-phosphate dehydrogenase was active with both NADPH and NADH, but predominantly with NADH. Incubation with 20 mM dithiothreitol and 1 mM NADPH promoted the coactivation of phosphoribulokinase and NADPHdependent glyceraldehyde-3-phosphate dehydrogenase, accompanied by a decrease in the glyceraldehyde-3-phosphate dehydrogenase activity linked to NADH. The multimeric enzyme had a Mr of 560000 and was of apparent subunit composition 8G6R. R represents a subunit of M , 42000 conferring phosphoribulokinase activity and G a subunit of 39000 responsible for the glyceraldehyde-3-phosphate dehydrogenase activity. On SDS-PAGE the Mr-42000 subunit comigrates with the subunit of the active form of phosphoribulokinase whereas that of M,-39 000 corresponds to that of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase.The multimeric enzyme had a szo,w of 14.2 S. Following activation with dithiothreitol and NADPH, sedimenting boundaries of 7.4 S and 4.4 S were formed due to the depolymerization of the multimeric protein to NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (4 G) and active phosphoribulokinase (2 R). It has been possible to isolate these two enzymes from the activated preparation by DEAE-cellulose chromatography.Prolonged activation of the multimeric protein by dithiothreitol in the absence of nucleotide produced a single sedimenting boundary of 4.6 S, representing a mixture of the active form of phosphoribulokinase and an inactive dimeric form of glyceraldehyde-3-phosphate dehydrogenase.Algal thioredoxin, in the presence of 1 mM dithiothreitol and 1 mM NADPH, stimulated the depolymerization of the multimeric protein with resulting coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. Light-induced depolymerization of the multimeric protein, mediated by reduced thioredoxin, is postulated as the mechanism of light activation in vivo. Consistent with such a postulate is the presence of high concentrations of the active forms of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase in extracts from photoheterotrophically grown algae. By contrast, in extracts from the dark-grown algae the multimeric enzyme predominates.There is now considerable evidence that the activities of several enzymes of the Calvin cycle are regulated by light [l]. The first enzyme demonstrated to be activated in vivo by light was NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase [2]. The effect of light was shown to be reversed by a subsequent period of darkness. The stimulatory effect of light was also observed in isolated spinach chloroplasts [3 -41. Incubation of the isolated chloroplast enzyme with a number of effector molecules (e.g. NADPH and ATP) stimulated the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase activity [5 -71. The effect o...
Two forms of phosphoribulokinase from the alga, Scenedesmus obliquus, have been purified to homogeneity by DEAE-cellulose, Ultrogel AcA34 and hydroxyapatite chromatography. An active form of the enzyme is a dimer of identical 42000-M, subunits. A latent form of phosphoribulokinase, requiring incubation with dithiothreitol for activity, is of M , 470000 and apparent subunit composition XsY4. The subunits X and Y are of M , 39000 and 42 000 respectively. The latent form of phosphoribulokinase is lost during DEAE-cellulose chromatography but this is prevented by NAD.Depolymerisation of the latent phosphoribulokinase to give the low-M, form of the enzyme accompanied its activation by dithiothreitol. An algal protein with all the properties of thioredoxin stimulates activation of the latent phosphoribulokinase when incubated with low concentrations of dithiothreitol.The latent form of phosphoribulokinase predominates in the heterotrophically grown algae whilst under photoheterotrophic conditions equal amounts of both enzyme forms are present in algal extracts. This is consistent with the suggestion that light activation of phosphoribulokinase in vivo is also due to depolymerisation of the large-M, latent form of the enzyme.There is considerable evidence that the in vivo activities of some enzymes of the Calvin cycle are stimulated by illumination. When actively photosynthesising algae were subjected to a light-dark transition, the transient changes in the pool sizes of some metabolites could be explained by dark inactivation of the enzymes sedoheptulose bisphosphatase, fructose bisphosphatase and ribulose bisphosphate carboxylase [l]. Despite a rapid decline in the concentration of ribulose bisphosphate, dark inactivation of phosphoribulokinase was not initially postulated as its activity was thought merely to be limited by the supply of ATP. A similar decline in ribulose bisphosphate concentration was observed when vitamin K5 was added to the illuminated algae. Vitamin K5 was considered to prevent the light activation of phosphoribulokinase by partially mimicking dark metabolism, diverting electron flow from the reduction of NADP without affecting ATP synthesis [2].When extracted from illuminated chloroplasts, phosphoribulokinase activity was greater than that extracted from chloroplasts kept in the dark [3-81. It is possible for light to stimulate the activity of an enzyme in two ways: either directly by the modulation of activity as a result of light-promoted alterations of stromal ion and metabolite concentrations or by a redox-mediated change in the enzyme resulting in an increase in its potential maximum activity. In the latter case electrons are transferred by light from the reaction center of Correspondence to R. Powls,
Au algal ch~uroplast protein possessing latent ~ADFH~e~nde~t ~ly~e~a~dehyde-3-phosphate dehydratenase and latent ~osphoribulok~na~ activities has been purified. Both activities were stimulated by incubation with dithiothreitoi and NADPH. The protein had subunit composition 8G6R and on activation depolymerized to discrete NADPH-dependent glyceraldehyde-3-phosphate dehydrogeuase (4G) and phosphoribulokinase (2R). Similar depolymerization promoted by reduced thioredoxin could account for light-dependent activations of phosphoribuioki~~ and ~ARPH-de~nde~~ ~~y~~aldehyde-3-phosphate dehydrogenase observed in algae and higher plants.
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