Effect of nitrate on pea sucrose synthase

The in vivo and in vitro nitrate effects on pea (Pisum sativum L.) sucrose synthase (SS) were studied. At the period of plant transition from heterotrophic to autotrophic nutrition, exogenous nitrate (14.2 mM) absorbed in the form of KNO₃ and Ca(NO₃)₂ during 10–20 days activated SS in the roots by 22–100% as compared with plants grown on nitrogen-free medium. Such effect was observed only at plant growing under high light (natural illumination up to 25 klx) and thus their sufficient supplement with sucrose. Under low light (climate-controlled chamber, 2.5 klx), nitrate could not activate SS. In the in vitro experiments, nitrate activated SS exponentially by a dose-dependent mode with the plateau at 3–5 mM, where its activity was increased by 50%. It is supposed that there is a second constituent in SS activation by nitrate, and it carries information about plant carbohydrate status. Possible mechanisms of nitrate-induced SS activation are discussed.     

Effect of ammonia on sucrose synthase in pea roots

The effects of ammonium on activity of sucrose synthase (SS) in the roots of pea (Pisum sativum L.) plants were studied. On the medium containing 14.2 mM (NH₄)₂SO₄, SS activity increased by 20–200% for 10–20 days of plant growth as compared with the roots of plants growing without nitrogen. Illuminance affected the degree of effects. Under natural illumination, ammonium affected SS activity not only in sunny days (up to 25 klx) but also in cloudy days (3–6 klx) but to a lower degree. Under stable low light (2.5 klx), ammonium did not affect SS activity. In the in vitro experiments, at (NH₄)₂SO₄ concentrations from 0 to 1 mM, SS activity was suppressed (up to 10%), whereas 1–37.5 mM (NH₄)₂SO₄, it was increased (up to 50%).     

Characterization of two sucrose synthase isoforms in sugarbeet root

Two sucrose synthase isoforms (EC 2.4.1.13) have been identified in developing sugarbeet (Beta vulgaris L.) roots. To aid in understanding the physiological significance of these multiple sucrose synthase isoforms, the two isoforms were partially purified and some of their physical and kinetic properties determined. Both isoforms were tetrameric proteins with native molecular masses of 320 kDa. The isoforms exhibited similar kinetic properties as well as similar changes in activity in response to changes in temperature. The isoforms differed, however, in their subunit composition. Sucrose synthase isoform I (SuSyI) was composed of two 84 kDa subunits and two 86 kDa subunits. Sucrose synthase isoform II (SuSyII) was a homotetramer with a subunit size of 86 kDa. The amino acid composition of the two subunits was similar, although differences in alanine, glycine, isoleucine and lysine content were noted. The activity of the two isoforms differed in response to varying pH conditions. The optimum pH for sucrose cleaving activity was observed at pH 6.0 and 6.5 for SuSyI and SuSyII, respectively. The optimum pH for sucrose synthesizing activity occurred at pH 7.5 and 7.0 for SuSyI and SuSyII, respectively. The observed differences in subunit composition and reactivity at different pH values suggest that multiple isoforms of sucrose synthase may provide a mechanism to regulate sucrose metabolism in sugarbeet root by differential regulation of expression of the two isoforms and modulation of their activity by changes in cellular pH.     

Contribution of sucrose synthase, ADP-glucose pyrophosphorylase and starch synthase to starch synthesis in developing pea seeds

Using genetic variability existing amongst nine pea genotypes (Pisum sativum L.), the biochemical basis of sink strength in developing pea seeds was investigated. Sink strength was considered to be reflected by the rate of starch synthesis (RSS) in the embryo, and sink activity in the seed was reflected by the relative rate of starch synthesis (RRSS). These rates were compared to the activities of three enzymes of the starch biosynthetic pathway [sucrose synthase (Sus), ADP-glucose pyrophosphorylase and starch synthase] at three developmental stages during seed filling (25, 50 and 75% of the dry seed weight). Complete sets of data collected during seed filling for the nine genotypes showed that, for all enzyme activities (expressed on a protein basis), only Sus in the embryo and seed coat was linearly and significantly correlated to RRSS. The contribution of the three enzyme activities to the variability in RSS and RRSS was evaluated by multiple regression analysis for the first two developmental stages. Only Sus activity in the embryo could explain, at least in part, the significant variability observed for both the RSS and the RRSS at each developmental stage. We conclude that Sus activity is a reliable marker of sink activity in developing pea seeds.     

Purification and characterization of two sucrose synthase isoforms from Japanese pear fruit

Two isoforms (SS I and SS II) of sucrose synthase (SS; EC 2.54.1.13) were purified from Japanese pear fruit and their properties were compared. SS I mainly appeared in young fruit and SS II mainly in mature fruit. SS I and SS II were purified to the specific activity of 3.37 and 4.26 (units (mg protein)(-1)), respectively. The MW of native and subunit proteins of SS I and SS II were almost the same and both SSs seemed to be a tetramer composed of an 83 kDa polypeptide. However, the ionic charges of the native proteins and the kinetic parameters of SSs were different. Specifically, the Km value for UDP-glucose in SS I was the same as that for UDP, while the Km value for UDP-glucose in SS II was less than that for UDP. SS II easily reacted for sucrose synthesis than sucrose cleavage compared with SS I. Therefore, it is considered that SS I and SS II play different roles in the utilization of carbohydrate in young and mature fruit, respectively.     

Multiple functions of junctin and junctate, two distinct isoforms of aspartyl beta-hydroxylase

The single genomic locus, AbetaH-J-J, encodes three functionally distinct proteins aspartyl beta-hydroxylase, junctin and junctate by alternative splicing. Among these three proteins, junctin and junctate could play important roles in the regulation of intracellular Ca(2+) by regulating either Ca(2+) release from intracellular Ca(2+) stores or Ca(2+) influx in various biological processes. Here we review recent findings concerning the expressional regulations and the proposed functions of junctin and junctate.     

Comparison of a novel tomato sucrose synthase,SlSUS4, with previously described SlSUS isoforms reveals distinct sequence features and differential expression patterns in association with stem maturation

Sucrose synthase (SUS) plays a role in many contexts of sugar metabolism, including low-oxygen and low-ATP respiration and the synthesis of cellulose. In tomato (Solanum lycopersicum), as in many plants, SUS is encoded by genes at several independent loci. Here, we report the isolation of a novel tomato SUS (SlSUS) isoform, SlSUS4, that is homologous to potato SUS isoform 1 (StSUS1) and also shows greater homology to SUS isoforms of other plants than to the other tomato SUS isoforms. All three tomato isoforms are very similar in genomic structure and sequence, yet each is located on a separate chromosome. Real-time expression analysis of the three distinct isoforms revealed widely varying patterns of expression, in terms of both tissue specificity and overall magnitude of expression. Analysis of SlSUS expression along the tomato stem revealed opposing expression gradients for two of the SlSUS isoforms, in apparent correlation with vascular tissue maturation. Western-blot analysis of SlSUS protein showed an increasing SlSUS concentration gradient along the developmental axis of the tomato stem, with the protein concentrated mainly in the vascular tissue of the stem. These gene expression and protein accumulation patterns indicate that each isoform may play a discrete role in the development of tomato plants, most notably in the development of vascular tissue in the stem.     

Involvement of sucrose synthase in sucrose catabolism

Maize scutellum slices incubated in water utilized sucrose at a maximum rate of 0.12,μmol/min per g fr. wt of slices. When slices were incubated in DNP, there was a three-fold increase in the rate of sucrose utilization. Sucrose breakdown in higher plants can be achieved by pathways starting with either invertase or sucrose synthase (SS). Invertase activity in scutellum homogenates was found only in the cell wall fraction, indicating that SS was responsible for sucrose breakdown in vivo. SS in crude scutellum extracts broke down sucrose to fructose and UDPG at 0.39,μmol/min per g fresh wt of slices. The UDPG formed was not converted to UDP + glucose, UMP + glucose-1-P, UDP + glucose-1-P or broken down by any other means by the crude extract in the absence of PPi. In the presence of PPi, UDPG was broken down by UDPG pyrophosphorylase which had a maximum activity of 26 μmol/min per g fr. wt of slices. Levels of PPi in the scutellum could not be measured using the UDPG pyrophosphorylase: phosphoglucomutase: glucose-6-P dehydrogenase assay because they were too low relative to glucose-6-P which interferes in the assay. An active inorganic pyrophosphatase was present in the scutellum extract which could prevent the accumulation of PPi in the cytoplasm. ATP pyrophosphohydrolase, which hydrolyses ATP to AMP and PPi, was found in the soluble portion of the scutellum extract. The enzyme activity was increased by fructose-2,6-bisP and Ca²⁺. In the presence of both activators, enzyme activity was 1.1 μmol/min per g fr. wt of slices, a rate sufficient to supply PPi for the breakdown of UDPG. These results indicate that sucrose breakdown in maize scutellum cells occurs via the SS: UDPG pyrophosphorylase pathway.     

Multiple isoforms of porcine aromatase are encoded by three distinct genes

Cytochrome P450 aromatase, a product of the CYP 19 gene and the terminal enzyme in the estrogen biosynthetic pathway, is synthesized by the ovary, endometrium, placenta, and peri-implantation embryos in the pig and other mammals, albeit to varying levels, implying its functional role(s) in pregnancy events. The aromatase produced by the pig tissues exists as three distinct isoforms (type I - ovary, type II - placenta, and type III - embryo), with presumed differences in substrate specificities, expression levels, activity, and mode of regulation. In order to delineate the molecular mechanisms whereby estrogen synthesis is regulated in these diverse tissues, the present study examined if these aromatase isoforms represent products of multiple genes or of a single gene via complex splicing mechanisms. Porcine genomic DNA from a single animal was used as a template in the polymerase chain reaction (PCR) to amplify isoform-specific sequences corresponding to exons 4 and 7, respectively. Nucleotide sequence analysis of the generated fragments revealed the presence of only clones corresponding to the three known aromatase types. Screening a porcine Bacterial Artificial Chromosome (BAC) library for aromatase gene by PCR yielded a single clone approximately 80 kb in length. Southern blot analysis, using probes specific for exons 1A-1B, 2-3, 4-9, and 10 sequences indicated that the BAC genomic clone contains the entirety of the coding exons as well as the proximal promoter region. Sequence analysis of the fragment generated with exon 4 primers determined that this BAC clone contains only the type II gene. The presence and relative orientation of the untranslated 5'- exons 1A and 1B, previously demonstrated for the type III isoform were evaluated in the BAC clone and genomic DNA by PCR. The 265 bp fragment generated from both PCR reactions was confirmed by sequence analysis to contain exons 1A and 1B that are located contiguous to each other and separated by only three bp. A diagnostic procedure for typing aromatase isoforms was developed, based on the presence of specific restriction sites within isoform-specific exons. The use of this protocol confirmed the existence of only three aromatase isoforms in the porcine genome and indicated changes in aromatase types expressed by the uterine endometrium as a function of pregnancy stage. The presence of distinct genes encoding each of the aromatase isoform predicts important differences in the mechanisms underlying the molecular evolution and regulation of porcine aromatase, unique from those of other mammals, and suggests a critical role for P450 aromatase steroidal products in uterine functions related to pregnancy events.     

Invertase,sucrose synthase and sucrose phosphate synthase in lyophilized spruce needles; microplate reader assays

Summary Data on changes of apparent activities of enzymes involved in sucrose metabolism of developing spruce needles are presented. Extractable activities of sucrose phosphate synthase (SPS, sucrose synthesis), and sucrose synthase (SS) and acid invertase (both sucrolysis) were determined in small volumes using a novel microplate reader system which combined high rates of activity with good reproducibility and high sample throughput. During a developmental period of up to 18 months after bud break characteristic changes in SPS and SS occurred. During the first 4 months of needle development SS declined while SPS increased which is indicative of a transition from net import to net export of photoassimilates (sink/source transition). After needle maturation both enzymes exhibited parallel annual changes with increasing rates towards autumn, which was mirrored by the pool sizes of sucrose (possibly due to the acquisition of frost hardiness). Acid invertase activity was comparable to that of SS but showed only marginal seasonal changes. Approximately 70% of its total activity was found to be soluble.     

Light/Dark profiles of sucrose phosphate synthase, sucrose synthase, and Acid invertase in leaves of sugar beets

The activity of sucrose phosphate synthase, sucrose synthase, and acid invertase was monitored in 1- to 2-month-old sugar beet (Beta vulgaris L.) leaves. Sugar beet leaves achieve full laminar length in 13 days. Therefore, leaves were harvested at 2-day intervals for 15 days. Sucrose phosphate synthase activity was not detectable for 6 days in the dark-grown leaves. Once activity was measurable, sucrose phosphate synthase activity never exceeded half that observed in the light-grown leaves. After 8 days in the dark, leaves which were illuminated for 30 minutes showed no significant change in sucrose phosphate synthase activity. Leaves illuminated for 24 hours after 8 days in darkness, however, recovered sucrose phosphate synthase activity to 80% of that of normally grown leaves. Sucrose synthase and acid invertase activity in the light-grown leaves both increased for the first 7 days and then decreased as the leaves matured. In contrast, the activity of sucrose synthase oscillated throughout the growth period in the dark-grown leaves. Acid invertase activity in the dark-grown leaves seemed to be the same as the activity found in the light-grown leaves.     

Soluble isoforms of starch synthase and starch-branching enzyme also occur within starch granules in developing pea embryos

Developing wild-type pea embryos contain two major isoforms of starch synthase and two isoforms of starch-branching enzyme. One of the starch synthases and both starch-branching enzymes occur both in the soluble fraction and tightly bound to starch granules. The other starch synthase, which is very similar to the waxy proteins of other species, is exclusively granule-bound. It is inactive when solubilized in a native form from starch granules, but activity is recovered when the SDS-denatured protein is reconstituted from polyacrylamide gels.
Evidence is presented which indicates that all of these proteins become incorporated within the structure of the granule as it grows. It is proposed that the granule-bound waxy protein is active in vivo at the granule surface, whereas the remaining proteins are active in the soluble fraction of the amyloplast. The proteins become trapped within the granule matrix as the polymers they synthesize crystallize around them, and they probably play no further part in polymer synthesis.     

Purification of tomato sucrose synthase phosphorylated isoforms by Fe(III)-immobilized metal affinity chromatography

The major phosphorylation site of maize sucrose synthase (SuSy) is well conserved among plant species but absent in the deduced peptide sequence of the tomato SuSy cDNA (TOMSSF). In this study, we report the in vitro phosphorylation of 25-day-old tomato fruits SuSy on seryl residue(s) by an endogenous Ca2+-dependent protein kinase activity. Two distinct 32P-labeled peptides detected in the tryptic peptide map of in vitro 32P-radiolabeled tomato fruit SuSy were purified. Amino acid sequencing and phosphoamino acid analysis of the major 32P-labeled peptide revealed the presence of a SuSy isozyme in young tomato fruit having the N-terminus phosphorylation site present in other plant species. By using Fe(III)-immobilized metal affinity chromatography [Fe(III)-IMAC] as a final purification step of tomato fruit SuSy, two 32P-labeled tomato SuSy isoforms were separated from a nonradiolabeled SuSy fraction by using a pH gradient. The major 32P-SuSy isoform was phosphorylated exclusively at the seryl residue related to the phosphorylation site of maize SuSy. The multiphosphorylated state of the second radiolabeled SuSy fraction was indicated by a higher retention during Fe(III)-IMAC and by tryptic peptide mapping analysis. Kinetic analyses of SuSy isoforms purified by Fe(III)-IMAC have revealed that phosphorylation of the major phosphorylation site of tomato fruit SuSy was not sufficient by itself to modulate tomato SuSy activity, whereas the affinity for UDP increased about threefold for the multiphosphorylated SuSy isoform.     

Chloroplastic isoforms of DnaJ and GrpE in pea

The folding and assembly of proteins in cells often requires the assistance of molecular chaperones such as the Hsp70 and Hsp60 heat shock proteins. Hsp70 chaperones cooperate with DnaJ and GrpE homologues to ensure a productive folding cycle. In this study we describe the gene for the first chloroplast localized DnaJ homologue and present evidence that the gene product is at least partially associated with the inner envelope membrane. Immunoblot analysis also provides evidence for the presence of a GrpE homologue in plastids.     

Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled

In previous work, we identified a Saccharomyces cerevisiae glycogen synthase gene, GSY1, which codes for an 85-kDa polypeptide present in purified yeast glycogen synthase (Farkas, I., Hardy, T.A., DePaoli-Roach, A.A., and Roach, P.J. (1990) J. Biol. Chem. 265, 20879-20886). We have now cloned another gene, GSY2, which encodes a second S. cerevisiae glycogen synthase. The GSY2 sequence predicts a protein of 704 residues, molecular weight 79,963, with 80% identity to the protein encoded by GSY1. Amino acid sequences obtained from a second polypeptide of 77 kDa present in yeast glycogen synthase preparations matched those predicted by GSY2. GSY1 resides on chromosome VI, and GSY2 is located on chromosome XII. Disruption of the GSY1 gene produced a strain retaining about 85% of wild type glycogen synthase activity at stationary phase, while disruption of the GSY2 gene yielded a strain with only about 10% of wild type enzyme activity. The level of glycogen synthase activity in yeast cells disrupted for GSY1 increased in stationary phase, whereas the activity remained at a constant low level in cells disrupted for GSY2. Disruption of both genes resulted in a viable haploid that totally lacked glycogen synthase activity and was defective in glycogen deposition. In conclusion, yeast expresses two forms of glycogen synthase with activity levels that behave differently in the growth cycle. The GSY2 gene product appears to be the predominant glycogen synthase with activity linked to nutrient depletion.