Molecular weight of starch synthetase from Oryza sativa leaves

Soluble ADP-glucose: α-1,4-glucan-4-glucosyltransferase with primed activity was extracted from rice leaves and purified by (NH₄)₂SO₄ fractionation, gradient elution on DEAE-cellulose and finally by Sephadex G200 gel filtration or amylopectin-cellulose chromatography. The purified enzyme was essentially homogeneous electrophoretically, but exhibited two peaks corresponding to MW of 22 000 and 67 000 on Sephadex G200 chromatography and five distinct bands on sodium dodecyl sulfate gel electrophoresis with MW of 11·5, 20, 35, 50 and 68 × 10³.     

Solubilization of starch synthetase bound to Oryza sativa starch granules

Starch synthetase was solubilized from purified starch granules of ripening grains of rice at the midmilky stage. The procedure consisted of making the granules amorphous and dispersing the amorphous starch by sonication in 75% dimethysulfoxide. A starch synthetase-amylose complex was isolated by discontinuous sucrose density gradient centrifugation, which does not require added primer and can utilize both ADP glucose and UDP glucose. A starch-free protein fraction was obtained by treatment with sodium dodecyl sulfate and β-mercaptoethanol.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.     

Comparison of soluble starch synthases and branching enzymes from developing maize and teosinte seeds

Soluble starch synthases and branching enzymes were purified from developing seeds of the maize inbreds W64A and Ia5125, annual teosintes cv. Galinat's Northern Teosinte and race “Nobogame” and diploid perennial teosinte. Two fractions of starch synthase were obtained by DEAE-cellulose chromatography in all purifications. Starch synthase I fractions had citrate stimulated activity and were most active in primed reactions containing glucogen. Starch synthase II fractions were more active in primed reactions with amylopectine and showed no citrate stimulated activity. Three fractions of branching enzymes were similar kinetically and chromatographically. In addition, antibodies prepared against maize branching enzymes cross reacted with teosinte enzymes. Precipitation lines double diffusion experiments and similar neutralizations of enzymes. precipitation lines of identity in double diffusion experiments and similar neutralization of enzyme activity wiyh increasing levels of antiserum, support the conclusion that maize and teosinte enzymes are highly homologous.     

Starch synthases and starch branching enzymes from Pisum sativum

Concentrations of ADPglucose:α-1,4-glucan-4-glucosyltransferase (starch synthase) and α-1,4 glucan: α-1,4-glucan-6-glycosyltransferase (branching enzyme) from developing seeds of Pisum sativum were measured. Primed starch synthase activity increased from 8 to 14 days after anthesis and decreased by 50 % at 26 days. Citrate-stimulated starch synthase activity was highest at 10 days after anthesis decreasing to low levels by 22 days. Branching enzyme activity increased from 8 to 18 days after anthesis and decreased little by 26 days. Two fractions of starch synthase were recovered by gradient elution from DEAE-cellulose of extracts from 12- and 18-day-old seeds. The two fractions differed in primer specificity, K_m for ADPG and relative amounts of citrate-stimulated activity. A major and minor fraction of branching enzyme were observed in extracts from both 12- and 18-day-old seeds. Marked differences in the relative abilities ofthe two branching enzyme fractions to stimulate phosphorylase and to branch amylose as well as pH optima were found. Although the content of the starch synthase and branching enzyme fractions varied with seed age, little difference was seen in the properties of chromatographically similar fractions. Therefore, the changes in starch synthase and branching enzyme activity during pea seed development resulted from changes in the concentrations of a few enzyme forms, but not the appearance of different enzyme forms.     

Activation of particulate starch synthetase from Zea mays embryo

The effect of spermine on particulate ADP-glucose: starch synthetase from the developing embryo of sweet corn has been studied. Spermine induces a considerable increase of glucose incorporation from ADP-glucose into the starch granules. The change in kinetic constants, the distribution of incorporated glucose between amylose and amylopectin and the pattern of incorporation into starch granules or malto-oligosaccharides has been studied. The data were compared with those obtained with citrate ions.     

Starch and water-soluble polysaccharides from sugary endosperm of sorghum

The starch water-soluble polysaccharides from sugary (su) endosperm of sorghum were isolated and characterized. Starch granule structure and co     

Characterization of starch produced by suspension cell cultures of Indica rice (Oryza sativa L.)

Suspension cultures of rice (Oryza sativa L.), initiated from seed, produced significant amounts of starch. Starch accumulated in the cultured cells throughout the growth phase and reached a maximum of 7% of the cell dry weight at stationary phase. Starch was present in compound granules which were birefringent under polarized light. Suspension culture starch had a higher amylose content and a lower gelatinization temperature than rice grain starch. Additionally, starch branching enzyme, an enzyme involved in starch biosynthesis, was characterized by anion exchange chromatography in culture cells and endosperm. Culture cells had at least one major form of starch branching enzyme which differed from the multiple enzyme forms present in endosperm.     

Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms

The dynamic changes of the activities of enzymes involving in starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthases (SSS), starch branching enzyme (SBE) and starch debranching enzymes (DBE) were studied, and changes of fine structure of amy- lopectin were characterized by isoamylase treatment during rice grain development, using trans anti-waxy gene rice plants. The relationships between the activities of those key enzymes were also analyzed. The amylose synthesis was significantly inhibited in transgenic Wanjing 9522, but the total starch content and final grain weight were less affected as compared with those of non-transgenic Wanjing 9522 rice cultivar. Analyses on the changes of activities of enzymes involving in starch bio- synthesis showed that different enzyme activities were expressed differently during rice endosperm development. Soluble starch synthase is relatively highly expressed in earlier stage of endosperm de- velopment, whilst maximal expression of granule-bound starch synthase (GBSS) occurred in mid-stage of endosperm development. No obvious differences in changes of the activities of AGPase and SBE between two rice cultivars investigated, except the DBEs. Distribution patterns of branches of amy- lopectin changed continually during the development of rice grains and varied between two rice culti- vars. It was suggested that amylopectin synthesis be prior to the synthesis of amylose and different enzymes have different roles in controlling syntheses of branches of amylopectin.     

An analysis of soluble starch synthase isozymes from the developing grains of normal and shx cv. Bomi barley (Hordeum vulgare)

Soluble starch synthase (SSS, EC 2.4.1.21) catalyzes formation of the α-1,4 bonds of amylopectin. It occurs in multiple isozymes which are either type I, primer-independent in the presence of citrate, or type II. always primer-dependent. To analyze the enzyme. a sensitive native gel assay was developed, monitoring ADP-[¹⁴C]glucose incorporation into insoluble α-glucan in the presence of either sodium citrate or glycogen primer or both. Using this system, we observed multiple type I and type II forms in developing grains of barley ( Hordeum vulgare L.) cv. Bomi, the relative activities of which vary with seed development. At least one form comigrates in native gels with starch branching enzyme. Assays of the shx mutant, which is severely reduced in starch accumulation and in type I SSS activity, indicate that one type I isozyme becomes primer-dependent.     

The relationship of starch metabolism to grain size in wheat

The present investigation was aimed at determining the levels of important enzymes of starch metabolism at different stages of grain development in wheats differing in final grain size and starch content per grain, to ascertain whether these enzymes have some relationship with grain size and/or starch content. Active starch synthesis in these varieties started from 14 days onward and continued till 35 days after anthesis. Invertase was active only during initial stages of grain development. Sucrose-UDP-glucosyltransferase had maximum activity at the 14 and/or 21 day stage and was present throughout the period of grain development. UDP- and ADP-glucose pyrophosphorylases and amylase were most active during the period of active starch synthesis and at the same time tended to parallel grain size and starch content at different stages of grain development.     

Lytic bodies from cereals hydrolysing maltose and starch

Protein bodies and spherosomes from sorghum contained carbohydrase activity against maltose, starch and p-nitrophenyl-α-d-glucoside. Maltase activities in sorghum and also in maize lytic bodies were very high; carbohydrase activities of lytic bodies from whole wheat, whole barley, sorghum aleurone, sorghum embryo and maize embryo were considerably lower. The pH response of sorghum lytic bodies was bimodal with an optimum in the range of 3·4–4·2 and a minimum or a shoulder near pH 3·8. Protein bodies from sorghum, maize, wheat and barley reduced the iodine-colouring capacity of soluble starch to give a purple colour typical of a β-limit dextrin. With spherosomes colour reduction was usually more rapid, eventually taking the breakdown of starch beyond the achroic point. The lytic bodies produce both maltose and glucose from starch, except in the case of maize when only glucose was found. The data suggest that protein bodies contain a linked β-amylase-maltase system and that spherosomes contain a linked α-amylase-maltase system.     

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.     

Alkaline inorganic pyrophosphatase from immature wheat grains

Changes in the level of alkaline inorganic pyrophosphatase and ADPG-pyrophosphorylase were monitored in developing wheat grains at weekly intervals aft     

Some structural features of amylomaize starch

Amylomaize starch was sub-fractionated into two components, complexing (C-fraction) and non-complexing (S-fraction) fractions, and properties of the tw     

Grain size,sucrose level and starch accumulation in developing rice grain

Five rices (Oryza sativa L.) differing in final grain size were studied at the midmilky stage to determine if any factor could be identified which might limit rate of starch accumulation. Only UDP glucose pyrophosphorylase activity increased with increasing grain size. Detached rice panicles incubated in liquid medium containing 1% sucrose and 0.1% glutamine, in addition to minerals and vitamins, produced grains similar to those on intact plants. Sucrose level (0–1.5%) in the medium determined the extent of dry matter and starch accumulation and influenced physiological development of the ripening grains. Chemical and enzymic composition of the grain were similar to previously reported levels in grains of intact panicles analysed at regular intervals after anthesis. Addition of 3-P glycerate or K⁺ to the medium did not improve dry matter accumulation in the developing grain.     

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.