Sucrose metabolism in green algae I. The presence of sucrose synthetase and sucrose phosphate synthetase

Summary The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae:Chlorella vulgaris andScenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.Dedicated toLuis F. Leloir on his seventieth birthday.     

Sucrose metabolism in cold-stored potato tubers with decreased expression of sucrose phosphate synthase

Transfer of potato tubers to low temperature leads after 2–4 d to a stimulation of sucrose synthesis, a decline of hexose-phosphates and a change in the kinetic properties, and the appearance of a new form of sucrose phosphate synthase (SPS). Antisense and co-suppression transformants with a 70–80% reduction in SPS expression have been used to analyse the contribution of SPS to the control of cold sweetening. The rate of sucrose synthesis in cold-stored tubers was investigated by measuring the accumulation of sugars, by injecting labelled glucose of high specific activity into intact tubers, and by providing 50 mol m^–3 labelled glucose to fresh tuber slices from cold-stored tubers. A 70–80% decrease of SPS expression resulted in a reproducible but non-proportional (10–40%) decrease of soluble sugars in cold-stored tubers, and a non-proportional (about 25%) inhibition of label incorporation into sucrose, increased labelling of respiratory intermediates and carbon dioxide, and increased labelling of glucans. The maximum activity of SPS is 50-fold higher than the net rate of sugar accumulation in wild-type tubers, and decreased expression of SPS in the transformants was partly compensated for increased levels of hexose-phosphates. It is concluded that SPS expression per se does not control sugar synthesis. Rather, a comparison of the in vitro properties of SPS with the estimated in vivo concentrations of effectors shows that SPS is strongly substrate limited in vivo . Alterations in the kinetic properties of SPS, such as occur in response to low temperature, will provide a more effective way to stimulate sucrose synthesis than changes of SPS expression.     

Glycolate metabolism in green algae

Using ¹⁴C-labelled substrates, the succession of the single steps in the glycolate metabolism was investigated in Mougeotia scalaris and Eremosphaera viridis , which, within the group of green algae, are representatives of the evolutionary lines of Charophyta and Chlorophyta , respectively. In both algae the same metabolites are formed as in higher plants, although in Eremosphaera , which in contrast to Mougeotia does not possess leaf peroxisomes, all reactions are exclusively mitochondrial. Concomitant with the oxidation of glycolate, the synthesis of ATP was demonstrated in Eremosphaera . Formation of tartronic semi-aldehyde or other products different from those in land plants could not be demonstrated in either of these algae. Excretion of glycolate by Mougeotia and Eremosphaera is enhanced by decreasing the CO₂ concentration as well as by increasing the light intensity, but is completely stopped about 14 h later. Whereas increasing enzyme activities of the glycolate pathway apparently reduces glycolate excretion in Mougeotia , activation of CO₂ pumps seems to be the dominant reaction to prevent glycolate excretion in Eremosphaera . Mesostigma viride is one of the phylogenetically oldest algae in the group of Charophyceae . As this alga has already been demonstrated to contain microbodies with enzymes of leaf peroxisomes, the peroxisomal glycolate pathway must have originated at a very early stage. Surprisingly, the organelles from Mesostigma contain also the glyoxysomal marker enzyme isocitrate lyase suggesting these microbodies to be prototypes from which both glyoxysomes and leaf peroxisomes evolved.     

Sucrose phosphate synthase and sucrose accumulation at low temperature

The influence of growth temperature on the free sugar and sucrose phosphate synthase content and activity of spinach (Spinacia oleracea) leaf tissue was studied. When plants were grown at 25°C for 3 weeks and then transferred to a constant 5°C, sucrose, glucose, and fructose accumulated to high levels during a 14-d period. Predawn sugar levels increased from 14- to 20-fold over the levels present at the outset of the low-temperature treatment. Sucrose was the most abundant free sugar before, during, and after exposure to 5°C. Leaf sucrose phosphate synthase activity was significantly increased by the low-temperature treatment, whereas sucrose synthase and invertases were not. Synthesis of the sucrose phosphate synthase subunit was increased during and after low-temperature exposure and paralleled an increase in the steady-state level of the subunit. The increases in sucrose and its primary biosynthetic enzyme, sucrose phosphate synthase, are discussed in relation to adjustment of metabolism to low nonfreezing temperature and freezing stress tolerance.     

Sucrose phosphate synthase and other sucrose metabolizing enzymes in fruits of various species

Recent reports have suggested that sucrose phosphate synthase (EC 2.4.1.14), a key enzyme in sucrose biosynthesis in photosynthetic “source” tissues, may also be important in some sucrose accumulating “sink” tissues. These experiments were conducted to determine if sucrose phosphate synthase is involved in sucrose accumulation in fruits of several species. Peach (Prunus persica NCT 516) and strawberry (Fragaria x ananassa cv. Chandler) fruits were harvested directly from the plant at various stages of fruit development. Kiwi (Actinidia chinensis), papaya (Carica papaya), pineapple (Ananas comosus) and mango (Mangifera indica) were sampled in postharvest storage over a period of several days. Carbohydrate concentrations and activities of sucrose phosphate synthase, sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. All fruits contained significant activities of sucrose phosphate synthase. Moreover, in fruits from all species except pineapple and papaya, there was an increase in sucrose phosphate synthase activity associated with the accumulation of sucrose in situ. The increase in sucrose concentration in peaches was also associated with an increase in sucrose synthase activity and, in strawberries, with increased activity of both sucrose synthase and neutral invertase. The hexose pools in all fruits were comprised of equimolar concentrations of fructose and glucose, except in the mango. In mango, the fructose to glucose ratio increased from 2 to 41 during ripening as sucrose concentration more than doubled. The results of this study indicate that activities of the sucrose metabolizing enzymes, including sucrose phosphate synthase, within the fruit itself, are important in determining the soluble sugar content of fruits of many species. This appears to be true for fruits which sweeten from a starch reserve and in fruits from sorbitol translocating species, raffinose saccharide translocating species, and sucrose translocating species.     

Respiratory metabolism during cold storage of apple fruit. I. Sucrose metabolism and glycolysis

This study provides the first report on the occurrence of the respiratory climacteric during cold storage of apple fruit ( Malus domestica Borkh. cv. Reinette du Canada). The respiratory pattern at 4°C was very similar to that observed during postharvest ripening at room temperature, except that shelf life was considerably extended and the onset of the climacteric delayed. Increasing the calcium content of the apple fruit significantly reduced loss of firmness during cold storage, but showed no effect on respiration or on the other parameters determined. A gradual accumulation of soluble sugars occurred during the first 60 days after harvest and was effectively completed before the climacteric peak was reached. This increase in sugars correlated with an increase in the activity of sucrose-phosphate synthase (EC 2.4.1.14), and a marked change in the kinetic properties of the enzyme was observed after sucrose accumulation ceased. Changes in the hexose-phosphate pool and in glycolytic and gluconeogenic activities indicated an initial increase in the gluconeogenic flow at early stages of the climacteric, followed by activation of glycolysis, with the carbon flow being most likely regulated at the reversible phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate (mostly via pyrophosphate:fructose-6-phosphate phosphotransferase, EC 2.7.1.90) and at the pyruvate kinase (EC 2.7.1.40) steps. The results presented indicate that the respiratory climacteric does not occur to accommodate extra ATP requirements during sucrose synthesis nor can it be a consequence of an increased supply of respiratory substrate.     

Enzymic mechanism of starch breakdown in germinating rice seeds V. Sucrose phosphate synthetase in the scutellum

Some enzymic Properties of a partially purified preparationof sucrose phosphate synthetase (E.C.2.4.1.14) from germinatingrice seed scutella were studied. Examination of the reactionkinetics revealed that the rate of synthesis of sucrose phosphatefollows the Michaelis-Menten equation at an optimum PH of 7.5,having K_m of 25 mM for UDP-glucose, and of 4.9 mM for fructose6-phosphate. UDP inhibited the enzyme reaction competitively;K₁ of 3.3 mM. Fe⁺⁺ and Fe⁺⁺⁺ activated the enzyme reaction about2-fold; K_a, 0.3 mM and 2.0 mM, respectively. Co⁺⁺, Co(NH₃)₆⁺⁺⁺,Mg⁺⁺ and Mn⁺⁺ also activated the enzyme reaction. At high concentrationK⁺ activated the enzyme reaction with the maximum activationof 24% at 400 mM. The molecular weight and S_20,w value of theenzyme were determined as 4.5 ? 10⁵ and 10.4S, respectively. ¹Part IV of this series is Ref. (5). ²California Foundation for Biochemical Research Fellow (1973). (Received December 20, 1973; )     

玉米蔗糖磷酸合成酶(SPS)基因的克隆及表达载体的构建

利用RT-PCR方法从玉米幼苗叶片总RNA中克隆出玉米的蔗糖磷酸合成酶(SPS)基因的全长cDNA片段。该片段与文献报道的序列具有99%的同源性。并分别构建了以双CaMV35S为启动子,以Tnos为终止子的植物双元表达载体PBISPS和以Pcab为启动子,以T35S为终止子的植物表达载体PBSPS,其中PBISPS含有NPTⅡ选择标记基因,PBSPS不含选择标记基因。     

Comparative studies on the regulation of DAHP synthetase activity in blue-green and green algae

Summary Regulation of DAHP synthetase activity was investigated in autotrophically grown blue-green and green algae. Members of the class of blue-green algae possess an enzyme, the activity of which is regulated by l-tyrosine and l-phenylalanine, whereby l-tyrosine is effective in 100 fold lower concentrations. DAHP synthetases of two organisms, Anabaena and Anacystis, were shown to belong to the V-type of allosteric enzymes.In contrast to the DAHP synthetase of blue-green algae regulation of this enzyme could not be demonstrated in two green algae, Ankistrodesmus and Maesotaenium. However, Euglena gracilis, both under conditions of mixotrophic and autotrophic growth, exhibits very effective regulation of this key enzyme; again, the inhibitors are tyrosine and phenylalanine. DAHP synthetase activity of Euglena has been purified about 40 fold; during this enrichment no separation of the enzyme activity inhibited by tyrosine and that by phenylalanine could be observed.     

Occurrence of sucrose and sucrose metabolizing enzymes in achlorophyllous algae

The presence of sucrose and the enzymes related to sucrose metabolism, i.e. sucrose synthase (SS) (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13), sucrose phosphate synthase (SPS) (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was demonstrated in Prototheca zopfii, a colorless alga. The levels of enzyme activities were lower than those obtained in Chlorella vulgaris, which is generally considered the photosynthetic counterpart of P. zopfii. Whem enzyme activities were measured in bleached cells of C. vulgaris, the levels were of the same order than those found in P. zopfii. These results would indicate that the sucrose metabolizing enzymes are not related to the algae ability to carry on photosynthesis.     

The interaction of cardiolipin with rat liver carbamoyl phosphate synthetase I

A selective interaction of rat liver carbamoyl phosphate synthetase I with cardiolipin, and other anionic phospholipids, has been demonstrated. The enzymatic activity of the synthetase is inhibited by cardiolipin and, to a lesser extent, by phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine. This group of anionic phospholipids also induced a conformational change in the synthetase, yielding a species with increased exposure of the linkages between independently folded domains of the enzyme, as determined by limited proteolysis under nondenaturing conditions. The interaction of cardiolipin with carbamoyl phosphate synthetase I was a fairly slow process, with complex kinetics, and was apparently irreversible. The inclusion of Mg2+ or of MgATP in the incubation mixture prevented the cardiolipin effects. The zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine had negligible effects on the structure and activity of the synthetase. This interaction between cardiolipin and carbamoyl phosphate synthetase I potentially constitutes one of the mechanisms by which the synthetase forms its loose association with the inner mitochondrial membrane. Multiple mechanisms, including synthetase conformational changes, cardiolipin phase changes, and ATP/ADP binding site involvement, are possibly involved in the phospholipid/synthetase interaction and the resulting potential regulatory mechanism(s) for urea cycle activity.