Pullulanase from rice endosperm.

Pullulanase (EC 3.2.1.41) in non-germinating seeds was compared with that in germinating seeds. Moreover, pullulanase from the endosperm of rice (Oryza sativa L., cv. Hinohikari) seeds was isolated and its properties investigated. The pI value of pullulanase from seeds after 8 days of germination was almost equal to that from non-germinating seeds, which shows that these two enzymes are the same protein. Therefore, the same pullulanase may play roles in both starch synthesis during ripening and starch degradation during germination in rice seeds. The enzyme was isolated by a procedure that included ammonium sulfate fractionation, DEAE-cellulofine column chromatography, preparative isoelectric focusing, and preparative disc gel electrophoresis. The enzyme was homogeneous by SDS/PAGE. The molecular weight of the enzyme was estimated to be 100 000 based on its mobility on SDS/PAGE and 105 000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 4.7. The enzyme was strongly inhibited by beta-cyclodextrin. The enzyme was not activated by thiol reagents such as dithiothreitol, 2-mercaptoethanol or glutathione. The enzyme most preferably hydrolyzed pullulan and liberated only maltotriose. The pullulan hydrolysis was strongly inhibited by the substrate at a concentration higher than 0.1%. The degree of inhibition increased with an increase in the concentration of pullulan. However, the enzyme hydrolyzed amylopectin, soluble starch and beta-limit dextrin more rapidly as their concentrations increased. The enzyme exhibited alpha-glucosyltransfer activity and produced an alpha-1,6-linked compound of two maltotriose molecules from pullulan.

Download Full-text

CO2-Responsive CCT Protein interacts with 14-3-3 proteins and controls the expression of starch synthesis-related genes

10.22541/au.161704145.57069338/v1 ◽

2021 ◽

Author(s):

Hiroshi Fukayama ◽

Naoki Shibatani ◽

Hirofumi Miyagawa ◽

Aiko Koudou ◽

Yasuo Yamauchi ◽

...

Keyword(s):

Oryza Sativa L ◽

Large Subunit ◽

Starch Synthesis ◽

Gel Filtration ◽

Parenchyma Cell ◽

Mesophyll Cell ◽

Leaf Sheath ◽

Bimolecular Fluorescence Complementation ◽

Promoter Regions

CO2 responsive CCT protein (CRCT) is a positive regulator of starch synthesis related genes such as ADP-glucose pyrophosphorylase large subunit 1 and starch branching enzyme I particularly in the leaf sheath of rice (Oryza sativa L.). The promoter GUS analysis revealed that CRCT expressed exclusively in the vascular bundle, whereas starch synthesis related genes were expressed in different sites such as mesophyll cell and starch storage parenchyma cell. However, the chromatin immunoprecipitation (ChIP) using a FLAG-CRCT overexpression line and subsequent qPCR analyses showed that the 5’-flanking regions of these starch synthesis-related genes tended to be enriched by ChIP, suggesting that CRCT can bind to the promoter regions of these genes. The monomer of CRCT is 34.2 kDa, however CRCT was detected at 270 kDa via gel filtration chromatography, suggesting that CRCT forms a complex in vivo. Immunoprecipitation and subsequent MS analysis pulled down several 14-3-3-like proteins. A yeast two-hybrid analysis and bimolecular fluorescence complementation assays confirmed the interaction between CRCT and 14-3-3-like proteins. Although there is an inconsistency in the place of expression, this study provide important findings regarding the molecular function of CRCT to control the expression of key starch synthesis-related genes.

Download Full-text

Double repression of soluble starch synthase genes SSIIa and SSIIIa in rice (Oryza sativa L.) uncovers interactive effects on the physicochemical properties of starch

Genome ◽

10.1139/g11-010 ◽

2011 ◽

Vol 54 (6) ◽

pp. 448-459 ◽

Cited By ~ 47

Author(s):

Guoyu Zhang ◽

Zhijun Cheng ◽

Xin Zhang ◽

Xiuping Guo ◽

Ning Su ◽

...

Keyword(s):

Oryza Sativa ◽

Oryza Sativa L ◽

Starch Synthesis ◽

Soluble Starch ◽

Degree Of Polymerization ◽

Interactive Effects ◽

Rice Grain ◽

Double Mutation ◽

Grain Starch ◽

Two Hybrid

Soluble starch synthases (SSs) are major enzymes involved in starch biosynthesis in developing rice ( Oryza sativa L.) endosperm. Despite extensive studies of SSs in various plant species including rice, the functional modes of action among multiple SS genes are still not clear. Here, we generated transgenic RNA interference (RNAi) repressed lines for seven of the eight members of the rice SS gene family and studied their effects on starch synthesis and grain formation. Consistent with their expression domains, RNAi repression of genes that encode isozymes SSI, SSIIa, and SSIIIa had strong effects on grain development, whereas no obvious phenotypic changes were observed in transgenic plants with the other SS genes being RNAi repressed, indicating functional redundancies among the genes. To study the potential functional interactions of SS genes, we generated SSIIa/SSIIIa double repression lines whose kernels displayed a chalky kernel appearance and had increased amylose levels, increased pasting temperatures, and decreased viscosities. The double mutation also reduced short (degree of polymerization (DP) 5–6) and long (DP 12–23) amylopectin chain contents in the grain and increased the medium long types (DP 7–11). The nonadditive nature of the double mutation line suggests that SSIIa and SSIIIa interact with each other during starch synthesis. Such interaction may be physical via starch phophorylase as indicated by our pair-wise yeast two-hybrid assays on major starch synthesis enzymes. Collectively, the data showed that SSIIa and SSIIIa play distinctive, but partially overlapping, roles during rice grain starch synthesis. The possibility of extensive redundancy or complementarity among SS isozymes is discussed.

Download Full-text

Purification and Characterization of a Polyextremophilicα-Amylase from an Obligate HalophilicAspergillus penicillioidesIsolate and Its Potential for Souse with Detergents

BioMed Research International ◽

10.1155/2015/245649 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 13

Author(s):

Imran Ali ◽

Ali Akbar ◽

Mohammad Anwar ◽

Sehanat Prasongsuk ◽

Pongtharin Lotrakul ◽

...

Keyword(s):

Specific Activity ◽

Amylase Activity ◽

Gel Filtration ◽

Soluble Starch ◽

Ammonium Sulfate Precipitation ◽

Purification And Characterization ◽

Apparent Homogeneity ◽

Sds Page ◽

Good Potential

An extracellularα-amylase from the obligate halophilicAspergillus penicillioidesTISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 U·mg−1andVmax⁡andKmvalues of 1.05 µmol·min−1·mg−1and 5.41 mg·mL−1, respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80°C, and 300 g·L−1NaCl. The addition of CaCl2at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe2+or Zn2+) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 g·L−1. Accordingly, it has a good potential for use as anα-amylase in a low water activity (high salt concentration) and at high pH and temperatures.

Download Full-text

Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

Biochemical Journal ◽

10.1042/bj3250761 ◽

1997 ◽

Vol 325 (3) ◽

pp. 761-769 ◽

Cited By ~ 64

Author(s):

Isabelle GARCIA ◽

Matthew RODGERS ◽

Catherine LENNE ◽

Anne ROLLAND ◽

Alain SAILLAND ◽

...

Keyword(s):

Nucleotide Sequence ◽

Gel Filtration ◽

Peptide Sequence ◽

Amino Acid Residues ◽

Carrot Cells ◽

Expression Studies ◽

Sds Page ◽

Molecular Masses ◽

The Difference ◽

Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

Download Full-text

QTL-seq identifies cooked grain elongation QTLs near soluble starch synthase and starch branching enzymes in rice (Oryza sativa L.)

Scientific Reports ◽

10.1038/s41598-019-44856-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Siwaret Arikit ◽

Samart Wanchana ◽

Srisawat Khanthong ◽

Chatree Saensuk ◽

Tripop Thianthavon ◽

...

Keyword(s):

Oryza Sativa ◽

Oryza Sativa L ◽

Soluble Starch ◽

Starch Synthase ◽

Starch Branching Enzymes ◽

Branching Enzymes

Download Full-text

Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

Biochemical Journal ◽

10.1042/bj20041053 ◽

2005 ◽

Vol 387 (1) ◽

pp. 271-280 ◽

Cited By ~ 58

Author(s):

Seonghun KIM ◽

Sun Bok LEE

Keyword(s):

Molecular Mass ◽

Gel Filtration ◽

Maximal Activity ◽

Genome Database ◽

Sds Page ◽

Key Enzyme ◽

Bivalent Metal Ions ◽

Ammonium Sulphate Fractionation ◽

Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

Download Full-text

Editing of Rice Endosperm Plastidial Phosphorylase Gene OsPho1 Advances Its Function in Starch Synthesis

Rice Science ◽

10.1016/j.rsci.2020.10.001 ◽

2021 ◽

Vol 28 (3) ◽

pp. 209-211

Author(s):

Liu Song ◽

Shao Gaoneng ◽

Jiao Guiai ◽

Zhu Maodi ◽

Wu Jiamin ◽

...

Keyword(s):

Starch Synthesis ◽

Rice Endosperm

Download Full-text

A metalloproteinase extracellularly released byCrithidia deanei

Canadian Journal of Microbiology ◽

10.1139/w03-081 ◽

2003 ◽

Vol 49 (10) ◽

pp. 625-632 ◽

Cited By ~ 15

Author(s):

Claudia Masini d'Avila-Levy ◽

Rodrigo F Souza ◽

Rosana C Gomes ◽

Alane B Vermelho ◽

Marta H Branquinha

Keyword(s):

Molecular Mass ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Residual Activity ◽

Major Protein ◽

Motile Cells ◽

Sds Page ◽

Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The incorporation of gelatin into SDSPAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Download Full-text

Crotalin, a vWF and GP Ib Cleaving Metalloproteinase from Venom of Crotalus atrox

Thrombosis and Haemostasis ◽

10.1055/s-0037-1616755 ◽

2001 ◽

Vol 86 (12) ◽

pp. 1501-1511 ◽

Cited By ~ 20

Author(s):

Wen-Bin Wu ◽

Hui-Chin Peng ◽

Tur-Fu Huang

Keyword(s):

Platelet Adhesion ◽

Early Stage ◽

Gel Filtration ◽

Platelet Glycoprotein ◽

Antithrombotic Effect ◽

Binding Motifs ◽

Sds Page ◽

Von Willebrand ◽

Willebrand Factor

SummaryBinding of von Willebrand factor (vWF) to a variety of extracellular matrix (ECM) components and to platelet glycoprotein (GP) Ib-IX-V complex is important in mediating platelet adhesion and aggregation in the early stage of hemostasis. We previously purified a potent antithrombotic protein, named crotalin, functionally acting as a GP Ib antagonist (1). In this study, we further characterized crotalin as a P-I metalloproteinase with a molecular mass of 25 kDa as determined by gel filtration and two-dimensional SDS-PAGE. Crotalin is a vWF binding and cleaving metalloproteinase. In addition, crotalin cleaved platelet GP Ib as judged by flow cytometry and Western blotting. The multiple effects of crotalin on vWF and platelet GP Ib antagonized ristocetin-, but not collagen and thrombin-induced platelet aggregation, suggesting that its effect is specific. We also found that crotalin auto-proteolytically degraded to ~14 and ~10 kDa fragments in the presence of SDS. Interestingly, both degradation fragments, intact and reduced crotalin were able to bind vWF, suggesting the binding of crotalin to vWF is conformation-independent. In conclusion, the results presented further explain the potent antithrombotic effect of crotalin in vivo. In addition, the multiple effects of crotalin may be used as a tool to determine the binding motifs that are responsible for the vWF-ECMs or vWF-GP Ib interaction.

Download Full-text