A continuous spectrophotometric assay for sucrose phosphate synthetase

A continuous spectrophotometric assay for sucrose phosphate synthetase is described. In this assay, the production of UDP is coupled to NADH oxidation by the enzymes nucleoside-5′-diphosphate kinase, pyruvate kinase, and lactate dehydrogenase. The assay could not be used with crude extracts, but was found suitable for use with partially purified sucrose phosphate synthetase from the leaves of spinach, wheat, and maize. It has obvious advantages for kinetic studies.     

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

The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae: Chlorella vulgaris and Scenedesmus 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.     

A cDNA clone from Zea mays endosperm sucrose synthetase mRNA

A cDNA clone for maize endosperm sucrose synthetase of 62o nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A) RNA in pBR322, and identifying the appropriate clone by hybrid-promoter translation. In Southern blotting to genomic BamHI-digested DNA, a single band only of approximately 20 Kb lights up, indicating that the sucrose synthetase gene is unique, or that closely linked copies are located on this DNA fragment.     

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.     

Evidence that the two sucrose synthetase genes in maize are related

Summary Evidence is presented that the sucrose synthetase coding sequence at the Shrunken locus is distantly related to the sequence encoding a second, minor sucrose synthetase present in maize endosperm. Three doubly mutant sh bz strains lacking at least part of the Sh coding sequence produce an antigenically cross-reactive protein having the same electrophoretic mobility as the Sh-encoded, 92-kD sucrose synthetase monomer, but differing in primary structure. An mRNA is present in endosperm of mutants with deletions at the Sh locus that is weakly homologous to the Sh coding sequence and encodes a 92-kD protein precipitable with antiserum to sucrose synthetase. We conclude that the genes encoding the two different proteins are related.     

Studies on the sucrose phosphate synthetase kinetic mechanism

The kinetic properties of wheat germ sucrose phosphate synthetase, which catalyzes the reaction UDP-glucose + fructose 6-phosphate → UDP + sucrose 6-phosphate have been studied. A plot of the reciprocal initial velocity versus reciprocal substrate concentration gave a series of intersecting lines indicating a sequential mechanism. Product inhibition studies showed that UDP was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. A dead-end inhibitor, inorganic phosphate, was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. The results of initial velocity and product and dead-end inhibition studies suggested that the addition of substrates to the enzyme follows an ordered mechanism.     

Genetic control of sucrose synthetase in maize endosperm

Summary Sucrose synthetase activity in endosperm extracts of seven shrunken(sh) mutants of spontaneous origin and three similar mutants due to the association of the controlling element Ds with the Sh locus is examined. A residual level of 3 to 5% as compared to the normal (Sh) endosperm is seen in all the mutants. The residual activity is similar to that of the Sh locus encoded endosperm sucrose synthetase by several criteria including an identical size of polypeptides and a similarity in antigenic properties. These two enzymes are, however, distinguishable by a slight difference in electrophoretic mobility in native gels and a difference in the relative abundance of enzyme molecules. The latter property is a reflection of a marked difference seen in the developmental profile of enzyme activity in the two genotypes. The earlier hypothesis (Chourey and Nelson 1976) that these two sucrose synthetases are encoded by two separate genes is strengthened by: (a) the presence of the residual enzyme in a sh deletion mutant and (b) an electrophoretic demonstration of two proteins, corresponding to the major and minor sucrose synthetase proteins, in the wild type (Sh) genotype. The two sucrose synthetase genes seem to provide a model system in plants for studying the molecular basis of temporal specificity of genes.Cooperative Investigation, United States Department of Agriculture and Institute of Food and Agricultural Sciences, University of Florida, Florida Agricultural Experiment Station Journal Series No. 3288. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Studies on sucrose synthetase. Kinetic mechanism

The kinetic properties of Helianthus tuberosus sucrose synthetase, which catalyzes the reaction UDP-glucose + fructose = UDP + sucrose, have been studied. A plot of the reciprocal initial velocity versus reciprocal substrate concentration gave a series of intersecting lines indicating a sequential mechanism. Product inhibition studies showed that UDP-glucose was competitive with UDP, whereas fructose was competitive with sucrose and uncompetitive with UDP. On the other hand, a dead-end inhibitor, salicine, was competitive with sucrose and uncompetitive with UDP. The results of initial velocity, product, and dead-end inhibition studies suggested that the addition of substrates to the enzyme follows an ordered mechanism.     

The effect of sucrose on the rate of de novo sucrose biosynthesis in leaf protoplasts from spinach, wheat and barley

Protoplasts from the leaves of wheat, spinach, and barley were found to synthesize [14C]sucrose from 14CO2 at rates comparable with those of the parent tissue. CO2 fixation and sucrose biosynthesis ceased virtually immediately when the light was switched off. The effect of sucrose pretreatment on the rate of de novo sucrose biosynthesis was found to vary with leaf age and with plant species. Protoplasts from young wheat and spinach leaves showed an apparent stimulation of the rate of sucrose biosynthesis after sucrose pretreatment. In protoplasts from mature leaves of spinach, sucrose pretreatment produced inhibition. After sucrose pretreatment protoplasts from mature spinach leaves showed low rates of CO2 fixation, and sucrose biosynthesis compared with controls. Conversely, with protoplasts from mature leaves of wheat and barley, the rate of CO2 fixation was unchanged and there was little or no effect on the rate of sucrose biosynthesis after sucrose pretreatment. Preincubation with sucrose had no effect on the activity of sucrose-phosphate synthetase (EC 2.4.1.14), cytoplasmic fructose-1,6-bisphosphatase (EC 3.1.3.11), or UDPglucose pyrophosphorylase (EC 2.7.7.9) from spinach leaves. It was concluded that there is no direct feedback inhibition of sucrose on the sucrose biosynthetic pathway in leaves of spinach, wheat, and barley. The mechanism of inhibition of sucrose biosynthesis by sucrose in spinach remains to be elucidated.     

Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments yielded two additional isozymic peaks, I and III, thereby suggesting its heterodimeric nature. We envisage that the three isozymes in GA3-treated wheat aleurone layers are formed by the random dimerization of two classes of enzyme subunits. The two enzyme subunits which differ in their net charge could be the product of two genes of AdoMet synthetase (SAM1 and SAM2). Based on this assumption, we propose that a single isozyme I in water imbibed control wheat aleurones is the product of SAM1 gene of AdoMet synthetase. The occurrence of three isozymes in GA3-treated aleurones could be ascribed to the expression of an alternate gene of AdoMet synthetase (SAM2 gene).(ABSTRACT TRUNCATED AT 400 WORDS)     

Phenylalanyl-tRNA synthetase of wheat embryos. Purification, molecular weight, structure, properties]

From wheat germ, a phenylalanyl-tRNA synthetase (E.C.6.1.1.20) has been isolated and purified 187 fold by means of ammonium sulfate fractionation (40-50 per cent) followed by Sephadex G-200 gel filtration, chromatographies on DEAE-cellulose and hydroxyapatite. The enzyme appears to be homogeneous on Sephadex G-200 molecular filtration and polyacrylamide gel electrophoresis. Molecular weight determinations by sucrose gradient centrifugation, gel filtration and gel electrophoresis give an average of 250 00 daltons. The enzyme is dissociated in 1 per cent sodium dodecyl sulfate into two different equimolar components of 80 000 and 50 000 daltons ; this result suggests that the phenylalanyl-tRNA synthetase has a subunit structure : alpha2 beta2. Dissociation with sodium dodecyl sulfate and dithiothreitol gives four other components, probably resulting from the breakdown of the subunits. Optima values of pH, Mg2+ and K+ concentrations, effect of SH-compnents, kinetic parameters have been determined in the aminoacylation reaction. Physical and catalytic properties of wheat germ phenylalanyl-tRNA synthetase appear very similar to those of the yeast and E. coli enzymes.     

Enzymes of starch and sucrose metabolism in Zea mays leaves

Mesophyll and bundle sheath cells of maize leaves were separated and enzymes of starch and sucrose metabolism assayed. The starch content and activities of ADPglucose (ADPG) starch synthetase and phosphorylase expressed both on a chlorophyll and a protein basis were much lower in mesophyll cells compared to bundle sheath preparations. Exposure of the leaves to continuous illumination for 2·5 days caused the starch content of mesophyll cells to rise greatly and led to considerable increases in ADPG starch synthetase and phosphorylase activity. In glasshouse grown leaves the bulk of invertase, sucrose phosphate synthetase, sucrose phosphatase, UDPglucose pyrophosphorylase and amylase was situated in the mesophyll layer. Sucrose synthetase, ADPG starch synthetase and phosphorylase were largely confined to the bundle sheath. No enzyme could be completely assigned to one particular cell layer. Upon continuous illumination both ADPG starch synthetase and phosphorylase increased in the mesophyll bythe same relative amount. The mesophyll is likely to be a major site for sucrose synthesis in maize leaves.     

A mini exon in the sucrose:sucrose 1-fructosyltransferase gene of wheat

We previously reported the cloning of a wheat sucrose:sucrose 1-fructosyltransferase (1-SST) cDNA, designated wft2. Wft2 proteins have fructosyltransferase enzyme activity and initiate fructan synthesis (Biosci. Biotechnol. Biochem. 66 (2002) 2297). In the current study, we cloned a genomic DNA fragment carrying the full-length 1-SST gene from winter wheat (Triticum aestivum). The genomic 1-SST gene is 3326 bp in length and contains four exons and three introns. Exon 2 has only 9 bp. This sequence encodes a part of a beta-fructosidase motif (NDPNG), a highly conserved motif found in plant invertases. This is the first report of a mini exon, one of the smallest exons known in plants, being found in a genomic 1-SST gene.     

Chloroplastic methionyl-tRNA synthetase from wheat

Wheat chloroplastic methionyl-tRNA synthetase was isolated and appeared to be a monomer with a molecular weight of 75,000 daltons. Its catalytical properties in the aminoacylation for various isoacceptors tRNA_s^Met from E. coli and wheat germ revealed a recognition of prokaryotic tRNAs and wheat cytoplasmic tRNA_i^Met, but not tRNA_m^Met. Using pI determinations and catalytical properties, it could be detected in non-chloroplastic quiescent wheat germ a form of methionyl-tRNA synthetase having the same properties as the chloroplastic one's.     

Translocation of [C]sucrose in wheat

Flag leaves of wheat plants (Triticum aestivum L. em. Thell. cv `Duke') were supplied with ¹⁴C(glucosyl)sucrose. Translocated [¹⁴C]sucrose was isolated, then hydrolyzed. Label appeared in both the hexose moieties indicating that some randomization of label had occurred. However, near the radioactive front essentially all of the ¹⁴C was in the glucose moiety, suggesting that randomization occurred after unloading, supporting the conclusion that sucrose was taken up intact by phloem and translocated unaltered.     

Chlorophyll synthetase is latent in well preserved prolamellar bodies of etiolated wheat

Membrane fractions containing intact etioplasts, etioplast inner membranes, prolamellar bodies or prothylakoids from wheat ( Triticum aestivum L. cv. Walde) were assayed for chlorophyll synthetase activity. Calculated on a protein basis, the etioplast inner membrane fraction showed a higher activity than the intact etioplasts. The activity was higher in the prolamellar body fraction than in the prothylakoid fraction. However, when the fractions were incubated in isolation medium with 50% (w/w) sucrose and 0.3 m M NADPH, chlorophyll synthetase activity could not be detected in the prolamellar body fraction, while the prothylakoid fraction maintained a high activity. The spectral shift to a shorter wavelength of the newly formed endogenous chlorophyllide was very rapid in the prothylakoid fraction but slow in the prolamellar body fraction. The relation between the spectral shift of chlorophyllide and the esterification activity in the fractions is discussed. Even exogenous short-wavelength chlorophyllide could not be esterified in well preserved prolamellar bodies. This indicates that chlorophyll synthetase is present in an inactive state in the prolamellar body structure. A large-scale method for the synthesis of geranylgeranylpyrophosphate, one of the substrates of the chlorophyll synthetase reaction, is also presented.     

Tyrosyl-tRNA synthetase from wheat germ

Tyrosyl-tRNA synthetase (TyrRS) was purified 5,000-fold from wheat germ extract by ultracentrifugation, precipitation with ammonium acetate, and column chromatography. Under denaturing conditions the enzyme ran as a single band on SDS-polyacrylamide electrophoresis with an apparent Mr of 55,000. The native molecular weight determined by gel filtration was 110,000, suggesting a quaternary structure of an alpha 2 type for native TyrRS. Purified enzyme activity, based on the aminoacylation reaction, was studied in terms of Mg2+, ATP, pH, and KCl dependence. Optimum concentrations were 6 mM Mg2+, 4 mM ATP, and 200 mM KCl at pH 8. The Km values for ATP, tyrosine, and tRNA were 40, 3.3, and 1.5 microM, respectively. The instability of the TyrRS activity and the methods used for stabilizing it are discussed. In wheat germ extract we found a second tyrosylating activity that works with Escherichia coli tRNA, but not with wheat germ tRNA. We believe that this enzyme is the mitochondrial tyrosyl-tRNA synthetase of wheat germ.     

Examination of the mechanism of sucrose synthetase by positional isotope exchange

The mechanism of the sucrose synthetase reaction has been probed by the technique of positional isotope exchange. [beta-18O2, alpha beta-18O]UDP-Glc has been synthesized starting from oxygen-18-labeled phosphate and the combined activities of carbamate kinase, hexokinase, phosphoglucomutase, and uridine diphosphoglucose pyrophosphorylase. The oxygen-18 at the alpha beta-bridge position of the labeled UDP-Glc has been shown to cause a 0.014 ppm upfield chemical shift in the 31P NMR spectrum of both the alpha- and beta-phosphorus atoms in UDP-Glc relative to the unlabeled compound. The chemical shift induced by each of the beta-nonbridge oxygen-18 atoms was 0.030 ppm. Incubation of [beta-18O2, alpha beta-18O]UDP-Glc with sucrose synthetase in the presence and absence of 2,5-anhydromannitol did not result in any significant exchange of an oxygen-18 from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. It can thus be concluded that either sucrose synthetase does not catalyze the cleavage of the scissile carbon-oxygen bond of UDP-Glc in the absence of fructose or, alternatively, the beta-phosphoryl group of the newly formed UDP is rotationally immobilized.     

Transforming wheat vacuolar invertase into a high affinity sucrose:sucrose 1-fructosyltransferase

Vacuolar invertases (VIs) degrade sucrose to glucose and fructose. Additionally, the fructan plant wheat (Triticum aestivum) contains different fructosyltransferases (FTs), which have evolved from VIs by developing the capacity to bind sucrose or fructans as acceptor substrates. Modelling studies revealed a hydrogen bonding network in the conserved WMNDPNG motif of VIs, which is absent in FTs. In this study, the hydrogen bonding network of wheat VI was disrupted by site-directed mutagenesis in the 23WMNDPNG29 motif. While the single mutants (W23Y, N25S) showed a moderate increase in 1-kestose production, a synergistic effect was observed for the double mutant (W23Y+N25S), showing a 17-fold increase in transfructosylation capacity, and becoming a real sucrose:sucrose 1-fructosyltransferase. Vacuolar invertases are fully saturable enzymes, contrary to FTs. This is the first report on the development of a fully saturable FT with respect to 1-kestose formation. The superior kinetics (K(m) approximately 43 mM) make the enzyme useful for biotechnological applications. The results indicate that changes in the WMNDPNG motif are necessary to develop transfructosylating capability. The shift towards smaller and/or more hydrophilic residues in this motif might contribute to the formation of a specific acceptor site for binding of sugar, instead of water.